State and Role of Src Family Kinases in Replicatio
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病菌学杂志 2006年第7期
The Marjorie B. Kovler Viral Oncology Laboratories, University of Chicago, 910 East 58th Street, Chicago, Illinois 60637
ABSTRACT
An earlier report showed that infected cell protein no. 0 (ICP0) of herpes simplex virus 1 (HSV-1) interacts with the SH3 domains of a recently discovered adaptor protein, CIN85. Here, we report the following. (i) ICP0 also interacts with other SH3 domain-containing proteins and, in particular, with nonneuronal members of the Src kinase family. (ii) HSV-1 infection enhanced the activating phosphorylation of Tyr416 of the members of the Src kinase family, modestly enhanced the kinase activity of Src, and posttranslationally modified at least one additional member of the Src kinase family by phosphorylation in a manner dependent on the viral gene products ICP0, unique short 3 (US3), and unique long 13 (UL13). (iii) To define the roles of Src kinase family members, we examined the accumulation of viral proteins, DNA, and mRNA and virus yields from wild-type mouse embryo fibroblasts and sibling cells lacking Src, Fyn, and Yes (SYF–); a mutant cell line, +Src, in which Src was restored to SYF– cells; and the mutant cell line (CSK–) lacking the negative regulator Csk gene of the Src kinase family. Representative , , and 2 proteins accumulated in the largest amounts in SYF– cells and the smallest amounts in +Src compared to wild-type cells. The CSK– cells yielded smaller amounts of the 2 protein and at least 10-fold less virus than wild-type cells. We conclude that HSV-1 proteins regulate the activities of Src family kinases to achieve optimal viral yields in the course of viral replication.
INTRODUCTION
Elsewhere, this laboratory reported that the infected cell protein no. 0 (ICP0) of herpes simplex virus 1 (HSV-1) contains multiple SH3 (Src homology 3) domain binding motifs (23). We reported that these motifs resemble the binding motif for CIN85—an adapter protein that interacts with Cbl and endophilin to negatively regulate receptor tyrosine kinases. We also reported that in cells transiently expressing ICP0, signaling via a representative receptor tyrosine kinase, the epidermal growth factor receptor, was disrupted and that in cells transiently expressing ICP0 and in cells infected with wild-type (WT) virus, the receptor turned over more rapidly than in mock-transfected or mock-infected cells (23). To expand our knowledge of the interactions of ICP0 with SH3 domain-containing cellular proteins, we examined the interaction of ICP0 with members of the Src family kinases (SFKs). The following points are relevant to this report.
First, ICP0 is a multifunctional 775-residue protein made immediately after viral infection. The protein is essential for viral replication following exposure of cells to a low ratio of PFU/cell, but not after high-multiplicity infection. In transfected cells, ICP0 acts as a promiscuous transactivator—it enhances the transcription of genes delivered to the cell by both viral infection and transient transfection (reviewed in reference 12). This key property of ICP0 may reflect its ability to block silencing of the viral genome by cellular-transcription repression machineries, one of which consists of the REST-CoREST-HDAC1/2 complexes, following the release of viral DNA into the nucleus by infecting virus. Thus, in a recent report, ICP0 was shown to interact with CoREST and to dissociate HDAC1/2 from the CoREST-REST complex. Subsequently, other viral gene products mediate the phosphorylation of CoREST and HDAC1/2 and export of these components to the cytoplasm, thus potentially alleviating their repressive effects on gene transcription in the nucleus (11). ICP0 also interacts with several other proteins and exhibits multiple functions, including those of E3 ubiquitin ligases, as reviewed elsewhere in detail (12). ICP0 also blocks the interferon pathway by degrading the ND10 nuclear structures and interfering with the function of IRF3, a potent transcriptional coactivator of specific interferon-induced genes (8, 28).
Second, as perhaps the best-characterized member of the growing family of protein interaction modules, SH3 domains are widely distributed among signaling proteins and play critical roles in a wide variety of biological processes, ranging from regulation of enzymes by intramolecular interactions to increasing the local concentration or altering the subcellular localization of components of signaling pathways and mediating the assembly of large multiprotein complexes. They share a common structure, but individual SH3 domains exhibit different binding specificities mediated both by amino acid sequences within the RT loop of the SH3 domain and sequences flanking the polyproline motifs of the binding protein (reviewed in reference 27). For example, the three SH3 domains of the newly discovered adaptor protein CIN85 bind to a proline- and arginine-rich motif, "Px(P/A)xxR" (21), or "PxxxPR" as predicted by another study (20), rather than the conventional proline-rich sequence with a "PxxP" motif at its core.
Third, SFKs regulate numerous cell-signaling pathways and play key roles in cell morphology, motility, proliferation, and survival (24, 37). SFK includes ubiquitously expressed resident members (e.g., Src, Yes, Fyn, and Yrk), as well as members whose expression is restricted to mainly hematopoietic cell lineages (e.g., Blk, Fgr, Hck, Lck, and Lyn). SFKs have very similar domain organizations: from the amino to the carboxyl terminals, all SFK members contain an amino-terminal 14-carbon myristoyl group, a unique segment, an SH3 domain, an SH2 domain, an SH2-kinase linker, a protein-tyrosine kinase domain, and a carboxyl-terminal regulatory tail containing a conserved tyrosine 527, which is phosphorylated by Csk (carboxyl-terminal Src kinase) and which plays a critical role in the regulation of the kinase activity. Under normal conditions, SFKs are kept in an inactive conformation, which is stabilized mainly through the intramolecular interactions between the SH3 domain and a polyproline type II left-handed helix located in the linker region, as well as a salt bridge formed between the SH2 domain and phosphorylated tyrosine 527. Relevant to this report, the kinase activity of SFKs is regulated by phosphorylation; the chief phosphorylation sites include tyrosine 416, which results in activation from autophosphorylation, and tyrosine 527, which results in inhibition of phosphorylation by Csk (reviewed in references 3 and 34).
Numerous reports have documented the fact that viruses that establish chronic infections target SFK members to interfere with cell signaling (reviewed in reference 9). Viral-protein interactions with SFK members are mediated predominantly by two types of molecular-recognition events. The first results from the interaction of phosphotyrosine residues with SH2 domains, for example, the interaction of Epstein-Barr virus LMP2A protein with the Lyn kinase (29). The second is mediated by the interaction of polyproline motifs with SH3 domains, as exemplified by the association of human immunodeficiency virus type 1 Nef with Hck, Lck, Lyn, Fyn, or Src (2, 35). The consequences of the interaction of viral proteins with SFK members may be kinase dependent. In a recent study, using transient transfections and Huh-7 cells harboring a persistently replicating subgenomic hepatitis C virus replicon, Macdonald et al. (25) demonstrated that NS5A bound to native SFK members in vivo and differentially modulated kinase activity, inhibiting Hck, Lck, and Lyn but activating Fyn. In another case, polyoma virus middle T binds to the catalytic domains of Src, Yes, and Fyn; binding to Src and Yes activates kinase activity, whereas middle T has no effect on Fyn (reviewed in reference 13).
The studies described in this report began with the demonstration that ICP0 interacts with members of the SFKs. Its focus, however, is on the roles, if any, of the SFK members in the course of HSV replication. We report that HSV infection enhanced the activating phosphorylation of Tyr416 of SFKs and modified SFKs through phosphorylation in a manner dependent on ICP0 and on the unique short 3 (US3) and unique long 13 (UL13) viral protein kinases. The ultimate roles of the modified proteins in specific steps in viral replication are still unclear. We also report that cells in which the negative regulator Csk had been deleted yielded lower virus titers and accumulated less late (2) proteins than wild-type sibling cells. These results suggest that HSV-1 regulates the activities of at least two members of the SFKs in order to induce optimal viral replication in infected cells.
MATERIALS AND METHODS
Cells and viruses. HEK293, HEp-2, and Vero cell lines were obtained from the American Type Culture Collection (Manassas, Va). Telomerase-transformed diploid human embryonic lung (HEL) fibroblasts (1) were obtained from Thomas Shenk (Princeton University, Princeton, N.J.). The cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (HEL and HEK293) or 5% newborn calf serum (Vero and HEp-2). HSV-1 strain F [HSV-1(F)], a limited-passage clinical isolate, was the prototype HSV-1 strain used in our laboratory (10).
Recombinant viruses containing mutations on an HSV-1(F) background, R7910 (0; ICP0 null) (15), R2621 (UL41 or vhs) (30), R7356 (UL13), R7041 (US3), and R7353 (UL13/US3) were reported elsewhere (31-33).
Mutant mouse embryo fibroblast (MEF) lines were obtained from A. Imamoto (Ben May Institute, University of Chicago). Specifically, in the SYF– line (19), all three widely expressed members of Src family kinases (Src, Fyn, and Yes) were knocked out; in the +Src line (19), the Src gene was restored by retroviral transduction of the SYF– line; in the CSK– line (14), the negative regulator of Src, the Csk gene, was knocked out, and thus, Src was constitutively activated; in the WT line (14), normal MEFs from same-stage embryos of littermates were developed without the introduction of genetic alterations. All MEFs were immortalized by transformation with simian virus 40 large T antigen.
Antibodies. The antibodies used in these studies were anti-CIN85 (Calbiochem; no. 231006), anti-glutathione S-transferase (GST) (Santa Cruz Biotechnology), anti-Src (Upstate Biotechnology; clone GD11, no. 05-184), anti-panSrc (Santa Cruz Biotechnology; sc-18), anti-Src-pY416 (Sigma; no. S1940), anti-Src-pY527 (Sigma; no. S2065), and rabbit polyclonal anti-thymidine kinase [TK] and monoclonal anti-ICP4, anti-ICP27, anti-ICP0, and anti-US11 (Goodwin Cancer Research Institute, Plantation, FL).
Plasmids. Mouse cDNA clones of SFK members were from the following authors: nonneuronal Src, Tony Hunter (the Salk Institute, CA); neuronal Src (NSrc), David Baltimore (26); Fyn (nonthymic form; FynB), Phil Soriano (Fred Hutchinson Cancer Research Center, WA); and Yes, Joseph Bolen (16). The human FGR (Gardner-Rasheed feline sarcoma virus [v-fgr] oncogene homolog) cDNA clone was purchased from the American Type Culture Collection (ATCC 10437429).
GST fusion proteins. The SH3 domains of SFK members Src, neuronal Src, Fyn, Yes, and Fgr were PCR amplified from appropriate cDNA clones, inserted in frame to vector pGEX-4-T1 or pGEX-4-T2, and verified by DNA sequencing at the University of Chicago Cancer Research Center DNA Sequencing Facility. GST-ICP0 constructs (amino acids 1 to 19, 20 to 241, 245 to 395, 245 to 510, or 543 to 768) were described previously (23). Competent Escherichia coli strain BL-21 cells were transformed with the GST fusion constructs and induced by isopropyl -D-thiogalactoside, and GST fusion proteins were immobilized to glutathione-Sepharose beads and purified according to the manufacturer's protocol (Amersham Pharmacia).
GST pull-down assays. Cells were harvested in cold phosphate-buffered saline (PBS), lysed by brief sonication in RIPA buffer (100 mM Tris, pH 8.0, 140 mM NaCl, 1% Triton X-100, 0.1% sodium dodecyl sulfate [SDS], 1% deoxycholic acid, 0.5 mM EDTA, protease and phosphatase inhibitors), and clarified by centrifugation in a Sorvall Biofuge Pico microcentrifuge at 16,000 x g for 20 min at 4°C. Total cell lysate was diluted in an equal amount of pull-down buffer (50 mM Tris, pH 7.5, 100 mM NaCl, 0.1% Nonidet P-40, 1 mg/ml bovine serum albumin [BSA]) to 1 ml and reacted overnight at 4°C with GST fusion proteins bound to glutathione-Sepharose beads. The GST beads were rinsed extensively in pull-down buffer and resuspended in an equal volume of 1x SDS loading buffer (50 mM Tris, pH 6.8, 2.75% sucrose, 5% 2--mercaptoethanol, 2% SDS). The solubilized proteins were boiled and electrophoretically separated in a denaturing 10% polyacrylamide gel.
Immunoblots. Electrophoretically separated proteins were electrically transferred to a nitrocellulose membrane, blocked at room temperature with 5% nonfat dry milk in PBS, and reacted with primary antibody diluted in PBS-1% BSA (anti-CIN85, 1:500; anti-Src, 1:1,000; anti-panSrc, 1:500; anti-GST, 1:2,000; anti-ICP0, 1:2,000; anti-ICP4,1:2,000; anti-ICP27, 1:2,000; anti-US11, 1:1,000; anti-TK, 1:500), followed by an appropriate secondary antibody conjugated to either peroxidase (Sigma) or alkaline phosphatase (Bio-Rad). Reactive protein bands were visualized with either enhanced chemiluminescence (Amersham Pharmacia) or 5-bromo-4-chloro-3-indolylphosphate-nitroblue tetrazolium (Denville Scientific, Metuchen, NJ), according to the manufacturer's instructions.
Src in vitro kinase assay. HEL cells in T75 flasks were exposed to 10 PFU/cell of HSV-1(F) or mock infected for 0, 3, 6, 9, or 12 h and then harvested and lysed in 400 μl RIPA buffer supplemented with protease and phosphatase inhibitors. Three hundred micrograms of clarified cell lysate was immune precipitated with 2 μg anti-Src for 3 h at 4°C. Fifty percent of each immune precipitate was resuspended in 20 μl Src kinase reaction buffer (100 mM Tris-HCl, pH 7.2, 125 mM MgCl2, 25 mM MnCl2, 2 mM EGTA, 250 μM orthovanadate, 2 mM dithiothreitol), mixed with 10 μg acid-denatured enolase (Boehringer Mannheim no. 104647; diluted in 10 μl Src kinase reaction buffer) and 10 μCi [-32P]ATP (Amersham; diluted in 10 μl Mn-ATP cocktail buffer [75 mM MnCl2, 500 μM ATP, 20 mM MOPS {morpholinopropanesulfonic acid}, pH 7.2, 25 mM -glycerol phosphate, 5 mM EGTA, 1 mM Na3VO4, 1 mM dithiothreitol]), and incubated at 30°C for 10 min, and the reaction was stopped by adding 20 μl of 4x SDS disruption buffer and boiling the mixture at 100°C for 10 min. Phosphorylated enolase was resolved in a 10% denaturing polyacrylamide gel, transferred to a nitrocellulose membrane, scanned, and quantified with a Storm 860 phosphorimager (General Dynamics, Falls Church, VA). The other 50% of the immune precipitate was solubilized, electrophoretically separated, transferred to a nitrocellulose membrane, and reacted with anti-Src antibody to assess the total amount of Src protein used in the previous in vitro kinase assays. Finally, the Src kinase specific activity from each sample was determined by normalizing the phosphorylation level of enolase against the total amount of Src protein.
Detection of the phosphorylation status of Tyr416 and Tyr527 of SFKs. HEK293 or HEL cells were exposed to 10 PFU/cell of HSV-1(F) or relevant deletion mutants or mock infected for the appropriate times. The cells were harvested and lysed in RIPA buffer supplemented with protease and phosphatase inhibitors with brief sonication. Equal amounts of clarified total cell lysate were separated by 10% denaturing polyacrylamide gel, transferred to a nitrocellulose membrane, and reacted with anti-Src-pY416 (1:1,000 dilution) and anti-Src-pY527 (1:1,000 dilution). To detect tyrosine phosphorylation specific to Src kinase, 600 μg of whole-cell lysate was immune precipitated with anti-Src for 3 h at 4°C, and one-third of the immune precipitates were solubilized, electrophoretically separated, transferred to a nitrocellulose membrane, and reacted with anti-Src-pY416 and anti-Src-pY527 as described above. The blots were stripped and reprobed with anti-Src to determine the total amount of Src protein immune precipitated. The reactive protein bands were quantified by phosphorimager or exposed to X-ray film.
Alkaline phosphatase digestion of cell lysates. HEL cells were mock infected or infected with 10 PFU/cell HSV-1(F) for 9 h and lysed in RIPA buffer without phosphatase inhibitors. An aliquot was saved, and phosphatase inhibitors were added immediately to serve as input controls before the treatment. Four hundred micrograms of lysates was mock digested or digested with 20 μl alkaline phosphatase (Boehringer Mannheim) in a total volume of 150 μl at 30°C for 40 min. One hundred micrograms of total protein was electrophoretically separated, transferred to a nitrocellulose membrane, and reacted with anti-panSrc or anti-Src.
Replication of HSV-1 in MEFs. Mutant MEFs in T25 flasks were exposed to 0.1 PFU of HSV-1(F) per cell in 199V medium (Sigma) supplemented with 1% calf serum for 2 h at 37°C. The inoculum was then removed, and the cell monolayers were rinsed with 199V medium to remove the unabsorbed virus. The cells were overlaid with complete medium and incubated at 37°C for an additional 24 h. The cells and medium were subjected to three cycles of freeze-thaw plus brief sonication, and the viral yield from each MEF was titered in Vero cells.
Determination of viral DNA accumulation in MEFs. Mutant MEFs in T25 flasks were infected for 24 h with 0.1 PFU of HSV-1(F) per cell as described above. The infected cells were harvested, resuspended in 400 μl T10E50 buffer (10 mM Tris-HCl, 50 mM EDTA, pH 8.0, 0.5% NP-40), digested sequentially with 2 μl RNase (10 mg/ml) on ice for 15 min and with 10 μl of protease K (10 mg/ml) plus 20 μl of 20% SDS at 37°C for 2 h, and extracted with phenol-chloroform twice. The total cellular DNA was precipitated with ethanol, resuspended in 50 μl distilled water, and digested with BamHI overnight. Equal volumes of digested DNA were separated on a 0.8% agarose gel, alkaline blotted to a zeta-probe nylon membrane (Bio-Rad) with 0.5 M NaOH overnight, and hybridized with 32P-labeled BamHI S fragment of HSV-1 DNA (nick translation kit; Promega) in hybridization buffer (1 mM EDTA, 40 mM Na3PO4, 7% SDS) at 65°C overnight. After hybridization, the blot was rinsed three times at 65°C with rinsing buffer (1 mM EDTA, 40 mM Na3PO4, 5% SDS) and quantified by phosphorimager or exposed to X-ray film for an appropriate time. The yield of viral DNA from each MEF line was normalized against the amount of total cellular DNA loaded and expressed as a ratio to the WT line.
Determination of viral mRNA accumulation in MEFs. Mutant MEFs in T25 flasks were infected for 8 or 12 h with 0.1 PFU of HSV-1(F) per cell as described above. Total RNA was extracted with the aid of TRIzol Reagent (GIBCO-BRL; no. 15596-018) according to the manufacturer's instructions. For Northern blot analysis, 10 μg of total RNA was resolved on a 1% denaturing formaldehyde agarose gel and transferred onto a "Brighstar-Plus" charged membrane (Ambion; no. 10102). The membrane was prehybridized for 2 h at 42°C in 12 ml ULTRAhyb buffer (Ambion; no. 8670) supplemented with denatured salmon sperm DNA (200 μg/ml; Stratagene) and then hybridized overnight after the addition of 32P-labeled DNA probes (Promega; Prime-a-Gene labeling system; no. U1100) specific to the viral genes ICP4 (NruI-XmnI fragment; residues 307 to 464), ICP0 (MluI-HindIII fragment; residues 543 to 775), and ICP27 (NcoI-StuI fragment; residues 189 to 408). The membrane was then rinsed two times with 2x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-0.1% SDS for 5 min and three times with 0.1x SSC-0.1% SDS for 20 min, scanned, and quantified with a General Dynamics Storm 860 phosphorimager.
RESULTS
ICP0 physically interacts with SFK members. Earlier studies indicated that an uncharted region of ICP0 (amino acids 245 to 510) that is relatively proline rich physically interacts with the SH3 domains of CIN85, an adaptor protein that is involved in negative regulation of receptor tyrosine kinases (23). In light of this observation, it was of interest to determine whether ICP0 can also interact with the prototypical SH3 domains of the SFK members of nonreceptor protein tyrosine kinases. To test this hypothesis, the SH3 domains of the SFK members Src, NSrc, Fyn, Yes, and Fgr were PCR amplified from the relevant cDNA clones and fused in frame with GST. The GST-SH3 fusion proteins were made in E. coli and reacted with lysates of HSV-1(F)-infected HEp-2 cells. As shown in Fig. 1A, the negative control (lane 3) consisted of the same DNA fragment of the Fgr SH3 domain inserted in the opposite orientation downstream of GST. Thus, a GST fusion protein of the same molecular weight but with a completely irrelevant amino acid sequence did not pull down ICP0 from the infected-cell lysate. The other four GST-SH3 fusions, i.e., those that included Src, Fyn, Yes, and Fgr, pulled down ICP0. It is noteworthy that the SH3 domain of neuronal Src, which contains a six-amino-acid (RKVDVR) insertion because of an alternative splicing event (7, 22, 26), did not interact with ICP0. This observation suggests the potentially interesting possibility that ICP0 does not interact with Src in neurons or neuroblastoma cells in which NSrc is predominantly expressed. NSrc is expressed during neuronal differentiation (4) and has a two- to fourfold-higher activity than that of c-Src (5, 9), possibly because the additional six amino acids destabilize the autoinhibitory conformation of Src, thereby enabling enhanced activity (36). It should be noted that the mobility of ICP0 in lane 2 was slightly different from that of the input control in lane 1, but this mobility change was not reproducible in other experiments.
The objective of the second series of experiments was to verify the expectation that the domain of ICP0 that interacts with Src is the same one that interacts with CIN85, i.e., the polypeptide containing residues 245 to 510. To address this objective, different GST-tagged regions of ICP0 were reacted with uninfected HEK293 cell lysate, and the proteins pulled down by GST-ICP0 chimeric proteins were electrophoretically separated in denaturing gels, transferred to a nitrocellulose membrane, and reacted with anti-Src monoclonal antibody. As shown in Fig. 1B, Src was pulled down by the two GST-ICP0 chimeric fusions (lanes 5 and 7) containing residues 245 to 510 of ICP0 but not by chimeric proteins containing other domains of ICP0 or by GST alone.
Taken together, these results indicate that ICP0 physically interacts with the Src family of tyrosine protein kinases through sequences contained between residues 245 and 510 and the SH3 domains of SFK members.
Amounts and activities of SFKs in the course of HSV-1 replication. The objective of the experiments described below was to determine whether HSV infection alters the stability or function of Src family protein tyrosine kinases. In the first series of experiments, replicate cultures of HEK293 or HEL cells were mock infected or exposed to 10 PFU of HSV-1(F), ICP0, or vhs mutant virus per cell. The cells were harvested and lysed 4, 8, or 12 h after infection. Equal amounts of cell lysates were electrophoretically separated in denaturing gels, transferred to a nitrocellulose membrane, and reacted with anti-Src antibody. As shown in Fig. 2, over the interval of 12 h from the time of infection, Src did not change significantly either in quantity or in electrophoretic mobility in a denaturing polyacrylamide gel.
The objective of the second series of experiments was to determine if HSV-1 infection alters the specific kinase activity of Src measured by in vitro kinase assays. Briefly, HEL cells were exposed to 10 PFU of HSV-1 per cell, harvested, and lysed 0, 3, 6, 9, or 12 h after infection. Equal amounts of cell lysates from each sample were immune precipitated with anti-Src monoclonal antibody. The immune complexes were collected, and one-half of the immune precipitates from each sample was reacted with 10 μg of acid-denatured enolase and 10 μCi [-32P]ATP. The enolase was resolved in a denaturing polyacrylamide gel, transferred to a nitrocellulose membrane, and quantified with the aid of a General Dynamics Storm 860 phosphorimager. The other half of the immune precipitates was electrophoretically separated in a denaturing polyacrylamide gel, transferred to a nitrocellulose membrane, and reacted with anti-Src antibody to determine the amount of Src protein present in the in vitro kinase assays. The "specific kinase activity" of Src was expressed as the amount of phosphorylated enolase divided by the amount of Src protein immune precipitated from each sample, normalized to mock infection at 0 h after infection, and plotted against the number of hours after infection. As shown in Fig. 3, the specific activity of Src in infected cells was slightly elevated with respect to that of mock-infected cells. The activity peaked between 6 and the 9 h after infection (e.g., the normalized values were 1.4 ± 0.4 for 6-h mock-infected cells versus 2.1 ± 0.5 for 6-h infected cells and 1.1 ± 0.2 for 9-h mock-infected cells versus 2.0 ± 0.9 for 9-h infected cells) and decreased to mock infection levels in the 12-h sample. This experiment was repeated three times with similar results.
The tyrosine kinase activity of SFK members is also reflected in the phosphorylation statuses of two key tyrosine residues that are conserved among the SFK members. Thus, phosphorylation of Tyr416 is stimulatory while phosphorylation of Tyr527 is inhibitory (reviewed in references 3 and 34). In the third series of experiments, we addressed the question of whether the phosphorylation status of SFKs was altered in the course of HSV-1 replication and what viral genes were involved. First, replicate cultures of HEL cells were either mock infected or exposed to 10 PFU of HSV-1(F), ICP0, US3, UL13, or US3/UL13 mutant viruses per cell. The cells were harvested 4, 8, or 12 h after infection and processed as described in Materials and Methods, and equal amounts of total cell lysates harvested at each time point were separated in denaturing gels, transferred to a nitrocellulose sheet, and reacted with anti-Src-pY416 or anti-Src-pY527 antibody. Since Tyr416 and Tyr527 are conserved among all SFK members, anti-phospho-Tyr antibodies raised against these two residues of Src should react with all other SFK members that are expressed (e.g., Fyn and Yes in the case of HEK293 and HEL cells). As shown in Fig. 4, the results were as follows. (i) At all three time points tested in mock-infected cells, phospho-Tyr416 was barely detectable, consistent with the current evidence that under normal conditions SFK members are held in an inactive, autoinhibitory conformation. (ii) Except in cells infected with the ICP0 mutant, phosphorylation of Tyr416 was enhanced by HSV infection. This was observed in the 8- and 12-h samples but not in the 4-h sample. The failure to detect phosphorylation of Tyr416 in lysates of mock-infected cells or cells infected with the ICP0 virus mutant (lanes 1, 3, 7, 9, 13, and 15) was not due to the reduced levels of SFK members, because the reaction of the same samples with the anti-panSrc antibody showed that they contained comparable amounts of proteins of SFK members. These results indicate that in HSV-1-infected HEL cells, ICP0 is required to enhance the activating phosphorylation of Tyr416 of at least one SFK member. (iii) At 8 h after infection, the band reactive with the anti-Src-pY416 formed by lysates of cells infected with wild-type virus migrated more slowly than the corresponding bands in lysates of cells infected with viral mutants lacking either or both viral protein kinases (Fig. 4, top, compare lane 8 with lanes 10, 11, and 12). However, at 12 h after infection, all bands reactive with the anti-Src-pY416 migrated at the same lower rate (lanes 16 to 18). These results suggest that at least one SFK member is posttranslationally modified further in the course of HSV infection and that this modification is delayed in cells infected with the viral-kinase-minus mutant viruses. (iv) The levels of the inhibitory phospho-Tyr527 in lysates of infected cells were similar to those of mock-infected cells. However, at 8 h and 12 h after infection (lanes 8 and 14), only the wild-type virus, but not any of the mutants tested, altered the electrophoretic mobility of at least one SFK member in denaturing polyacrylamide gels, as detected by both anti-panSrc antibody and anti-Src-pY527, which also recognizes all SFK members. This observation indicates that HSV further modifies SFK members in a manner dependent on ICP0, US3, and UL13 products. Because at 12 h after infection this modification was seen only in lysates of cells infected with wild-type virus but not in lysates of cells infected with mutant viruses, we can surmise that this SFK member is not phosphorylated at Tyr-416. (v) The cell lysates were also reacted with antibody to actin as a loading control and with antibodies against ICP0 and viral TK. The decreased levels of TK, a protein, in lysates of HEL cells infected with the ICP0 mutant virus may reflect the requirement for ICP0 for optimal progression of viral replication and accumulation of viral gene products (6).
The phosphotyrosine-specific antibodies used in the experiments described above do not differentiate Src from other SFK members. To specifically detect the tyrosine phosphorylation status of just Src, HEK293 cells were mock infected or infected with HSV-1(F) or the ICP0 mutant virus. The lysates of cells harvested at time zero or 4, 8, or 12 h after infection were reacted with anti-Src antibody first. The immune precipitates were then solubilized, subjected to electrophoresis in denaturing gels, and reacted with the two anti-phosphotyrosine antibodies (Fig. 5A and C) mentioned above. The two blots were then stripped and reprobed with anti-Src to determine the total amount of Src protein immune precipitated in each case (Fig. 5B and D). The reactive protein bands were quantified with the aid of the General Dynamics phosphorimager. As shown in Fig. 5, normalization of the tyrosine phosphorylation level to the total Src protein level in each sample with respect to mock infection at time zero showed that activating phosphorylation (Y416) specific to Src increased as early as 4 h after viral infection and onwards, whereas the inhibitory phosphorylation (Y527) remained basically unchanged, consistent with the results shown in Fig. 3, where starting from 6 h after infection and onwards, in in vitro kinase assays, the kinase activity of Src was modestly higher in HSV-1-infected than mock-infected cells. However, in contrast to the experiment whose results are shown in Fig. 4, when all SFKs were indiscriminately examined by pan-Src antibodies, we could not detect altered electrophoretic mobility in denaturing gels when immune-precipitated Src was reacted with these two anti-phospho-Tyr antibodies, suggesting that Src itself is likely not the target of the further modification by HSV-1 infection observed in Fig. 4.
To verify the results shown in Fig. 5 regarding the effects of HSV infection on the phosphorylation status of Tyr416 and Tyr527 specific to Src in a different cell line, and to test if we could detect enhanced phosphorylation of Tyr416 of SFK members earlier than 8 h after infection, we repeated the above-mentioned experiments with HEL cells. Briefly, HEL cells mock infected or exposed to 10 PFU of HSV-1(F) or mutant viruses per cell were harvested and lysed 6 h after infection. The cell lysates were either electrophoretically separated in a denaturing polyacrylamide gel, transferred to a nitrocellulose membrane and reacted with the above-mentioned two anti-phospho-Tyr antibodies to directly assess the tyrosine phosphorylation statuses of all SFKs expressed in HEL cells, or first immune precipitated with anti-Src and then probed with anti-phospho-Tyr antibodies to detect phosphorylation of Tyr416 and Tyr527 specific to Src. The results shown in Fig. 6 were basically the same as those shown in Fig. 4 and in HEK293 cells (Fig. 5). Specifically, they were as follows.
First, as shown in the top gel of Fig. 6, the antibodies against pY416 and pY527 reacted with all phosphorylated members of SFK. As shown in the lower gel, these antibodies detected tyrosine phosphorylation specific to Src alone. The results shown in the upper gel indicated that as early as 6 h after infection, at least one member of the SFKs was phosphorylated at Tyr416 in all lysates except that obtained from mock-infected cells or cells infected with the ICP0 mutant virus. As in the results shown in Fig. 4, the antibody to phospho-Tyr527 reacted with two relatively sharp bands in all lysates except that of cells infected with wild-type virus. In this instance, the upper of the two bands migrated more slowly than the corresponding band formed by other lysates, suggesting that the protein present in this band was subjected to additional posttranslational modification and that this modification required functions dependent on ICP0, US3, and UL13. Moreover, consistently, this additional modification could also be detected by the other panSrc anti-pY416 antibody, as reflected by the slower-migrating band formed by lysates of HSV-1(F)-infected cells but not by those of mutant-virus-infected cells (upper gel, compare lane 2 with lanes 4, 5, and 6).
Second, the lower panel shows the phosphorylation status of immune-precipitated Src. Anti-Src-pY416 recognized a faint band in lysates of infected but not mock-infected HEL cells at 6 h after infection, whereas anti-Src-pY527 detected strong bands in infected and mock-infected cells. This observation indicates that only a small fraction of Src was phosphorylated at Tyr416 compared to the amount of Src phosphorylated at Tyr 527. Moreover, the phosphorylation at Tyr416 was stronger in the case of bands formed by lysates of mutant-virus-infected cells than in cells infected with wild-type virus. In addition, the lower band formed by lysates of cells infected with wild-type virus (lower gel, lane 8) was much broader and was partially retarded in its electrophoretic mobility, suggesting that some of the Src underwent additional modifications that were not observed in mutant-virus-infected cells.
Posttranslational modification of Src. The results of the experiment shown in the second gel of Fig. 4 indicated that panSrc antibody reacted with a diffuse slower-migrating band formed by lysates of cells infected with wild-type virus harvested 8 or 12 h after infection. This diffuse band was not formed by lysates of mutant virus infected cells and, in particular, the viral protein kinase mutants. This observation prompted us to further examine if the nature of this modification was phosphorylation. For this purpose, HSV-1(F)-infected or mock-infected HEL cell lysates were digested with alkaline phosphatase or mock digested and then electrophoretically separated in a denaturing polyacrylamide gel, transferred to a nitrocellulose membrane, and reacted with anti-panSrc antibody as described in the legend to Fig. 7. As expected, electrophoretically separated lysates of cells infected with wild-type virus formed two bands reactive with the anti-panSrc antibody. The upper band was sharp in mock-infected cell lysates but diffuse in lysates of cells infected with wild-type virus. After alkaline phosphatase digestion, the diffuse upper band formed in infected-cell lysates was replaced by a faster-migrating sharp band. Inasmuch as the diffuse band was detected by panSrc but not by Src-specific antibody, the results indicate that it was a member of SFK but not Src itself. These results are consistent with the conclusion that a member of the SFKs was phosphorylated in a UL13- and US3-dependent manner.
In summary, we conclude the following from these studies. (i) We noted a modest increase in the activity specific to Src (Fig. 3). This activity corresponds to a modest enhancement of phosphorylation of pY416 of Src (Fig. 6). Overall, members of the SFK family are phosphorylated at Tyr416 at some point between 4 and 8 h after infection. These members undergo a second set of modifications that take place earlier in cells infected with wild-type virus (8 h) than in mutant-virus-infected cells (12 h). (ii) The phosphorylation of Tyr527 appears to be relatively constant throughout the period of observation in all virus-infected or mock-infected cell cultures (Fig. 4 to 6). (iii) A member of the SFKs other than Src is posttranslationally phosphorylated in a manner dependent on ICP0, US3, and UL13. This modification was apparent in assays with panSrc antibodies (Fig. 4, lane 2) and with anti-pTyr527 antibody (Fig. 6). The results suggest that this phosphorylation occurs on the background of phosphorylated Tyr 527 protein. (iv) The role of ICP0 in mediating posttranslational modifications of members of the SFKs is not clear. It is conceivable that in ICP0 mutant-infected cells, the progression of viral protein synthesis was delayed or that ICP0 binds and translocates SFK members to specific subcellular compartments. It should also be noted that UL13 phosphorylates US3 and that conceivably the phosphorylated form of US3 is required for the posttranslational modification of SFK members.
Enhanced Src activity repressed viral replication at a low multiplicity of infection. To determine whether alterations in Src kinase activity affected HSV-1 gene expression and replication, we took advantage of four MEF lines described in Materials and Methods and in detail elsewhere (14, 19). Thus, in the SYF– line, all three widely expressed SFK members (Src, Fyn, and Yes) were knocked out. In the +Src line, the Src gene was restored and overexpressed by transduction of the SYF– line with a retroviral vector. In the CSK– line, the Csk gene, the negative regulator of Src, was knocked out, and thus, SFKs were constitutively activated. The control cell line (WT) was derived from the genetically unaltered same-stage embryos of the littermates. In all, the following series of studies were performed.
In the first series of experiments, we verified the accumulation of Src in the MEF lines. In brief, 300 μg of total protein extracted from each cell line was immune precipitated with 2 μg of anti-Src antibody. The immune precipitates were solubilized, electrophoretically separated, and probed with anti-Src antibody. As expected (Fig. 8), Src proteins were overexpressed in the +Src cell line compared to the amount recovered from wild-type cells but were not detected in lysates of the SYF– cell line. The amount of Src detected in the CSK– cell line was slightly lower than that detected in WT MEFs.
In the second series of experiments, we exposed the four MEF lines to 0.1 PFU of HSV-1(F) per cell. The cells were harvested 24 h after infection, and virus yields were measured in Vero cells. The results (Table 1) showed that virus yields decreased about 8-fold in +Src cells and >10-fold in the CSK– line relative to the yield from WT controls. However, the increase in SYF– cells was barely twofold, suggesting that while enhanced Src activity was detrimental, the absence of Src did not enhance viral replication to a significant level. In other studies (unpublished data), when the multiplicity of infection was higher (1 PFU/cell or 10 PFU/cell), there was no significant difference in virus yields from the four MEF lines.
In the third series of experiments, replicate cultures of the four MEF lines were exposed to 0.1 PFU/cell. The cells were harvested 24 h after infection, and the total cellular DNA was extracted, digested to completion with BamHI, electrophoretically separated in an agarose gel, transferred to a nylon membrane, and hybridized with 32P-labeled BamHI S fragment of HSV-1 genomic DNA to measure the accumulation of viral DNAs. The results shown in Fig. 9 were reproducible and showed that viral DNA accumulated in smaller amounts in infected +Src or CSK– cells than in infected WT cells. SYF– cells accumulated approximately half of the total DNA detected in infected WT cells
In the fourth series of experiments, we examined the accumulation of viral proteins in the four MEF lines exposed to 0.1 PFU of HSV-1(F) per cell. The cells were harvested 4, 8, 12, or 16 h after infection, and 100 μg of the solubilized total cellular protein was subjected to electrophoresis in denaturing gels, transferred to nitrocellulose sheets, and probed with antibodies to ICP4, ICP27, TK, and US11. The results, shown in Fig. 10, were as follows: the amounts of accumulating viral proteins were highest in SYF– cells, while the lowest levels were observed in +Src cells. Of particular interest is the observation that in infected CSK– cells there was a reduction in the 2 protein US11 but not in (ICP27 and ICP4) or (TK) proteins. In contrast, infected +Src cells accumulated reduced amounts of all viral proteins surveyed in this experiment, whereas the SYF– cell line accumulated more US11 protein than the other cell lines.
The reductions in the protein levels of ICP27 and ICP4 in the infected +Src cells prompted us to examine the levels of mRNAs in the four cell lines. In these experiments, the replicate cultures of the four cell lines were exposed to 0.1 PFU of HSV-1(F) per cell. The cells were harvested 8 or 12 h after infection. Total RNA was extracted from one set of cultures, electrophoretically separated under denaturing conditions, and probed with 32-P-labeled DNA probes specific to ICP4, ICP0, or ICP27. The second set of cultures were processed as described in Materials and Methods, subjected to electrophoresis in denaturing gels, and probed with antibodies to relevant viral proteins as in Fig. 10. The results, shown in Fig. 11, were as follows.
As predicted by the results of protein analysis shown in Fig. 10, the infected +Src cell line accumulated smaller amounts of ICP4, ICP0, or ICP27 mRNA than other cell lines. The amounts of HSV-1 mRNAs detected in CSK– or SYF– cell lines were also smaller than those detected in cells infected with wild-type virus.
The overall protein profile was similar to that shown in Fig. 10. Thus, the SYF– cells accumulated the largest amounts of the representative proteins assayed in this experiment, whereas the +Src cells accumulated the smallest amounts. Of the proteins tested, the only one accumulating in significantly smaller amounts in the CSK– cell line was US11.
Based on these observations, we conclude the following. (i) The SYF– cell line lacking Src, Yes, and Fyn kinases accumulated the largest amounts of representative , , and 2 proteins and yielded amounts of virus at least equivalent to those obtained in infected wild-type MEFs. (ii) The +Src cell line accumulated the smallest amounts of proteins, consistent with the smallest amounts of accumulated mRNAs. In this cell line, only Src of the SFK members was restored. (iii) Removal of CSK, the negative regulator of SFK members, reduced viral yields but did not have a significant effect on the accumulation of gene mRNAs or the accumulation of the representative or proteins. Rather, the major discernible defect was in the accumulation of the 2 protein, US11.
DISCUSSION
In an earlier report, we showed that ICP0 contains multiple sequences capable of binding the SH3 domain of the adaptor protein CIN85. The initial objective of the studies described in this report was to determine whether ICP0 binds other proteins containing SH3 domains and, in particular, whether ICP0 interacts with members of the SFKs. Once an interaction was observed, the focus of the studies was to determine the role of the Src protein in the course of viral infection. Since the discovery of SFKs, a growing literature has documented the roles of these proteins in the regulation of a vast array of cellular functions, including cell cycle progression, adhesion and spreading, focal adhesion formation, and disassembly, migration, apoptosis, differentiation, and gene transcription (24, 37). Moreover, it has been reported that some viruses manage to manipulate the activities of SFKs so as to benefit from some of the cell-signaling events regulated by SFKs. For example, the HBx gene of the hepadnaviruses activates Src kinases, while activated Src kinases in turn upregulate hepatitis B virus (HBV) reverse transcription and stimulate HBV and woodchuck hepatitis virus replication in established liver cell lines by 5- to 20-fold. Conversely, inhibition of Src kinases reduced the yields of HBV or woodchuck hepatitis virus replication by 10- to 15-fold (17, 18). A central objective of these studies was to survey the roles of the Src proteins in the course of HSV-1 infection. The salient features of this report and their significance are as follows.
First, ICP0 physically interacted with Src, Fyn, Yes, and Fgr, members of the SFKs. Furthermore, the ICP0 domains interacting with these proteins were the same as those that interacted with CIN85. It is noteworthy that NSrc did not interact with ICP0. NSrc differs from Src by the addition of six residues within its SH3 domain. The significance of this observation is uncertain. The data suggest that not all proteins with SH3 domains interact with ICP0 and, conversely, that the interaction of ICP0 with SFK has functional relevance. It is also noteworthy that the levels of Src protein in at least two cell lines remained unchanged over a period of 12 h after infection with wild-type or mutant viruses, which argues that degradation of Src is not an outcome of its interaction with viral gene products.
The role of the interaction of ICP0 with SFK members remains to be elucidated. There is no evidence that ICP0 targets Src for degradation. It is conceivable that ICP0 vectors Src or other members of SFK to specific subcellular compartments. The function of ICP0 with respect to the function of SFK remains to be determined.
Second, the increase in specific activity illustrated in Fig. 3 is modest, as is the phosphorylation of Tyr416. The positive activation of Src must be balanced with the obvious negative effects of overexpressed Src in the +Src mouse embryo fibroblast line and with the evidence that in the SYF– cell line the yield of virus could not be differentiated from that of cells infected with wild-type virus. Conceivably, Src exerts a negative effect on viral replication, and the modest activation reflects this possibility.
Third, we have not measured the activities of other members of the SFKs overall, but the activating phosphorylation of SFK was more robust. We also noted that in infected cells, at least one SFK member undergoes a posttranslational modification in addition to the phosphorylation of Tyr416. We have not been able to identify this member with available antibodies to Yes and Fyn (data not shown).
Fourth, the role of SFK in the course of viral infection may be deduced in part from the studies of the four MEF lines. In essence, overexpression of Src in the absence of Fyn and Yes members appeared to be deleterious to viral replication, whereas deletion of Src, Fyn, and Yes actually increased the amounts of viral proteins accumulating in infected cells. Comparison of +Src and wild-type sibling MEFs suggests that the presence of either Yes or Fyn or both kinases ameliorates the negative effects. The identity and functions of the kinase that confers a positive effect on viral replication is unclear. As noted above, the results presented in Fig. 5, 6, and 7 indicate that at least one member of the SFKs other than Src is modified by phosphorylation in a manner dependent on US3 and UL13 protein kinases. The data obtained in those experiments suggest that either both viral kinases or a US3 kinase modified by UL13 mediates the phosphorylation of a member of the SFKs. Neither of these two viral kinases would be expected to accumulate in HEL cells exposed to ICP0 at low multiplicities of infection. The role of this member of the SFKs in balancing the negative effect of Src remains to be determined.
Finally, it is noteworthy that the absence of the negative regulator of SFK in CSK– MEFs produced an effect very different from that observed in +Src or SYF– MEFs. Whereas in +Src MEFs, the negative effect correlated with a decreased accumulation of gene transcripts, in CSK– cells, the decrease in the synthesis of infectious progeny appears to be correlated with decreased yields of US11 and, potentially, with a decrease in the accumulation of other late or 2 proteins. These results suggest a mechanism of action that takes place late in infection rather than during the transcription of genes. In essence, constitutive activation of SFKs in the absence of Csk gene products differs from overexpression of Src only; it suggests that while Yes and Fyn are essential to block the negative effects of activated Src on viral-gene transcription, they or other members have a negative effect late in infection and must be deactivated for optimal synthesis of late proteins.
To our knowledge, this is the first report of the statuses and roles of SFK members in viral replication. We have opened a Pandora's box. It is clear that SFKs play significant roles, positive and negative, in HSV-1 replication and that HSV alters the functions of these proteins. The roles of individual members of the SFKs remain to be determined.
ACKNOWLEDGMENTS
We thank Akira Imamoto (Ben May Institute, University of Chicago) for invaluable reagents and discussion. We thank Brunella Taddeo and Weiran Zhang for advice.
These studies were aided by National Cancer Institute Grants CA87661, CA83939, CA71933, CA78766, and CA88860.
REFERENCES
Bresnahan, W. A., G. E. Hultman, and T. Shenk. 2000. Replication of wild-type and mutant human cytomegalovirus in life-extended human diploid fibroblasts. J. Virol. 74:10816-10818.
Briggs, S. D., E. C. Lerner, and T. E. Smithgall. 2000. Affinity of Src family kinase SH3 domains for HIV Nef in vitro does not predict kinase activation by Nef in vivo. Biochemistry 39:489-495.
Brown, M. T., and J. A. Cooper. 1996. Regulation, substrates and functions of src. Biochim. Biophys. Acta 1287:121-149.
Brugge, J. S., P. C. Cotton, A. E. Queral, J. N. Barrett, D. Nonner, and R. W. Keane. 1985. Neurons express high levels of a structurally modified, activated form of pp60c-src. Nature 316:554-557.
Brugge, J., P. Cotton, A. Lustig, W. Yonemoto, L. Lipsich, P. Coussens, J. N. Barrett, D. Nonner, and R. W. Keane. 1987. Characterization of the altered form of the c-src gene product in neuronal cells. Genes Dev. 1:287-296.
Cai, W., T. L. Astor, L. M. Liptak, C. Cho, D. M. Coen, and P. A. Schaffer. 1993. The herpes simplex virus type 1 regulatory protein ICP0 enhances virus replication during acute infection and reactivation from latency. J. Virol. 67:7501-7512.
Cartwright, C. A., R. Simantov, P. L. Kaplan, T. Hunter, and W. Eckhart. 1987. Alterations in pp60c-src accompany differentiation of neurons from rat embryo striatum. Mol. Cell. Biol. 7:1830-1840.
Chee, A. V., P. Lopez, P. P. Pandolfi, and B. Roizman. 2003. Promyelocytic leukemia protein mediates interferon-based anti-herpes simplex virus 1 effects. J. Virol. 77:7101-7105.
Collette, Y., and D. Olive. 1997. Non-receptor protein tyrosine kinases as immune targets of viruses. Immunol. Today 18:393-400.
Ejercito, P., E. D. Kieff, and B. Roizman. 1968. Characterization of herpes simplex virus strains differing in their effects on social behavior of infected cells. J. Gen. Virol. 2:357-364.
Gu, H., Y. Liang, G. Mandel, and B. Roizman. 2005. Components of the REST/CoREST/histone deacetylase repressor complex are disrupted, modified, and translocated in HSV-1-infected cells. Proc. Natl. Acad. Sci. USA 102:7571-7576.
Hagglund, R., and B. Roizman. 2004. Role of ICP0 in the strategy of conquest of the host cell by herpes simplex virus 1. J. Virol. 78:2169-2178.
Ichaso, N., and S. M. Dilworth. 2001. Cell transformation by the middle T-antigen of polyoma virus. Oncogene 20:7908-7916.
Imamoto, A., and P. Soriano. 1993. Disruption of the csk gene, encoding a negative regulator of Src family tyrosine kinases, leads to neural tube defects and embryonic lethality in mice. Cell 73:1117-1124.
Kawaguchi, Y., C. Van Sant, and B. Roizman. 1997. Herpes simplex virus 1 alpha regulatory protein ICP0 interacts with and stabilizes the cell cycle regulator cyclin D3. J. Virol. 71:7328-7336.
Klages, S., D. Adam, E. Eiseman, J. Fargnoli, S. M. Dymecki, S. V. Desiderio, and J. B. Bolen. 1993. Molecular cloning and analysis of cDNA encoding the murine c-yes tyrosine protein kinase. Oncogene 8:713-719.
Klein, N. P., and R. J. Schneider. 1997. Activation of Src family kinases by hepatitis B virus HBx protein and coupled signaling to Ras. Mol. Cell. Biol. 17:6427-6436.
Klein, N. P., M. J. Bouchard, L. H. Wang, C. Kobarg, and R. J. Schneider. 1999. Src kinases involved in hepatitis B virus replication. EMBO J. 18:5019-5027.
Klinghoffer, R. A., C. Sachsenmaier, J. A. Cooper, and P. Soriano. 1999. Src family kinases are required for integrin but not PDGFR signal transduction. EMBO J. 18:2459-2471.
Kowanetz, K., I. Szymkiewicz, K. Haglund, M. Kowanetz, K. Husnjak, J. D. Taylor, P. Soubeyran, U. Engstrom, J. E. Ladbury, and I. Dikic. 2003. Identification of a novel proline-arginine motif involved in CIN85-dependent clustering of Cbl and down-regulation of epidermal growth factor receptors. J. Biol. Chem. 278:39735-39746.
Kurakin, A. V., S. Wu, and D. E. Bredesen. 2003. Atypical recognition consensus of CIN85/SETA/Ruk SH3 domains revealed by target-assisted iterative screening. J. Biol. Chem. 278:34102-34109.
Levy, J. B., T. Dorai, L. H. Wang, and J. S. Brugge. 1987. The structurally distinct form of pp60c-src detected in neuronal cells is encoded by a unique c-src mRNA. Mol. Cell. Biol. 7:4142-4145.
Liang, Y., A. Kurakin, and B. Roizman. 2005. Herpes simplex virus 1 infected cell protein 0 forms a complex with CIN85 and Cbl and mediates the degradation of EGF receptor from cell surfaces. Proc. Natl. Acad. Sci. USA 102:5838-5843.
Lowell, C. A., and P. Soriano. 1996. Knockouts of src-family kinases: stiff bones, wimpy T-cells and bad memories. Genes Dev. 10:1845-1857.
Macdonald, A., K. Crowder, A. Street, C. McCormick, and M. Harris. 2004. The hepatitis C virus NS5A protein binds to members of the Src family of tyrosine kinases and regulates kinase activity. J. Gen. Virol. 85:721-729.
Martinez, R., B. Mathey-Prevot, A. Bernards, and D. Baltimore. 1987. Neuronal pp60c-src contains a six-amino acid insertion relative to its non-neuronal counterpart. Science 237:411-415.
Mayer, B. J. 2001. SH3 domains: complexity in moderation. J. Cell Sci. 114:1253-1263.
Melroe, G. T., N. A. DeLuca, and D. M. Knipe. 2004. Herpes simplex virus 1 has multiple mechanisms for blocking virus-induced interferon production. J. Virol. 78:8411-8420.
Miller, C. L., A. L. Burkhardt, J. H. Lee, B. Stealey, R. Longnecker, J. B. Bolen, and E. Kieff. 1995. Integral membrane-protein 2 of Epstein-Barr virus regulates reactivation from latency through dominant-negative effects on protein-tyrosine kinases. Immunity 2:155-166.
Poon, A. P., and B. Roizman. 1997. Differentiation of the shutoff of protein synthesis by virion host shutoff and mutant gamma (1)34.5 genes of herpes simplex virus 1. Virology 229:98-105.
Post, L. E., and B. Roizman. 1981. A generalized technique for deletion of specific genes in large genomes: gene 22 of herpes simplex virus is not essential for growth. Cell 25:227-232.
Purves, F. C., R. M. Longnecker, D. P. Leader, and B. Roizman. 1987. Herpes simplex virus 1 protein kinase is encoded by open reading frame US3, which is not essential for growth in cell culture. J. Virol. 61:2896-2901.
Purves, F. C., W. O. Ogle, and B. Roizman. 1993. Processing of the herpes simplex virus regulatory protein 22 mediated by the UL13 protein kinase determines the accumulation of a subset of mRNAs and proteins in infected cells. Proc. Natl. Acad. Sci. USA 90:6701-6705.
Roskoski, R., Jr. 2004. Src protein-tyrosine kinase structure and regulation. Biochem. Biophys. Res. Commun. 324:1155-1164.
Saksela, K., G. Cheng, and D. Baltimore. 1995. Proline-rich (PxxP) motifs in HIV-1 Nef bind to SH3 domains of a subset of Src kinases and are required for the enhanced growth of Nef+ viruses but not for down-regulation of CD4. EMBO J. 14:484-491.
Sicheri, F., and J. Kuriyan. 1997. Structures of Src-family tyrosine kinases. Curr. Opin. Struct. Biol. 7:777-778.
Thomas, S. M., and J. S. Brugge. 1997. Cellular functions regulated by src family kinases. Annu. Rev. Cell Dev. Biol. 13:513-609.(Yu Liang and Bernard Roiz)
ABSTRACT
An earlier report showed that infected cell protein no. 0 (ICP0) of herpes simplex virus 1 (HSV-1) interacts with the SH3 domains of a recently discovered adaptor protein, CIN85. Here, we report the following. (i) ICP0 also interacts with other SH3 domain-containing proteins and, in particular, with nonneuronal members of the Src kinase family. (ii) HSV-1 infection enhanced the activating phosphorylation of Tyr416 of the members of the Src kinase family, modestly enhanced the kinase activity of Src, and posttranslationally modified at least one additional member of the Src kinase family by phosphorylation in a manner dependent on the viral gene products ICP0, unique short 3 (US3), and unique long 13 (UL13). (iii) To define the roles of Src kinase family members, we examined the accumulation of viral proteins, DNA, and mRNA and virus yields from wild-type mouse embryo fibroblasts and sibling cells lacking Src, Fyn, and Yes (SYF–); a mutant cell line, +Src, in which Src was restored to SYF– cells; and the mutant cell line (CSK–) lacking the negative regulator Csk gene of the Src kinase family. Representative , , and 2 proteins accumulated in the largest amounts in SYF– cells and the smallest amounts in +Src compared to wild-type cells. The CSK– cells yielded smaller amounts of the 2 protein and at least 10-fold less virus than wild-type cells. We conclude that HSV-1 proteins regulate the activities of Src family kinases to achieve optimal viral yields in the course of viral replication.
INTRODUCTION
Elsewhere, this laboratory reported that the infected cell protein no. 0 (ICP0) of herpes simplex virus 1 (HSV-1) contains multiple SH3 (Src homology 3) domain binding motifs (23). We reported that these motifs resemble the binding motif for CIN85—an adapter protein that interacts with Cbl and endophilin to negatively regulate receptor tyrosine kinases. We also reported that in cells transiently expressing ICP0, signaling via a representative receptor tyrosine kinase, the epidermal growth factor receptor, was disrupted and that in cells transiently expressing ICP0 and in cells infected with wild-type (WT) virus, the receptor turned over more rapidly than in mock-transfected or mock-infected cells (23). To expand our knowledge of the interactions of ICP0 with SH3 domain-containing cellular proteins, we examined the interaction of ICP0 with members of the Src family kinases (SFKs). The following points are relevant to this report.
First, ICP0 is a multifunctional 775-residue protein made immediately after viral infection. The protein is essential for viral replication following exposure of cells to a low ratio of PFU/cell, but not after high-multiplicity infection. In transfected cells, ICP0 acts as a promiscuous transactivator—it enhances the transcription of genes delivered to the cell by both viral infection and transient transfection (reviewed in reference 12). This key property of ICP0 may reflect its ability to block silencing of the viral genome by cellular-transcription repression machineries, one of which consists of the REST-CoREST-HDAC1/2 complexes, following the release of viral DNA into the nucleus by infecting virus. Thus, in a recent report, ICP0 was shown to interact with CoREST and to dissociate HDAC1/2 from the CoREST-REST complex. Subsequently, other viral gene products mediate the phosphorylation of CoREST and HDAC1/2 and export of these components to the cytoplasm, thus potentially alleviating their repressive effects on gene transcription in the nucleus (11). ICP0 also interacts with several other proteins and exhibits multiple functions, including those of E3 ubiquitin ligases, as reviewed elsewhere in detail (12). ICP0 also blocks the interferon pathway by degrading the ND10 nuclear structures and interfering with the function of IRF3, a potent transcriptional coactivator of specific interferon-induced genes (8, 28).
Second, as perhaps the best-characterized member of the growing family of protein interaction modules, SH3 domains are widely distributed among signaling proteins and play critical roles in a wide variety of biological processes, ranging from regulation of enzymes by intramolecular interactions to increasing the local concentration or altering the subcellular localization of components of signaling pathways and mediating the assembly of large multiprotein complexes. They share a common structure, but individual SH3 domains exhibit different binding specificities mediated both by amino acid sequences within the RT loop of the SH3 domain and sequences flanking the polyproline motifs of the binding protein (reviewed in reference 27). For example, the three SH3 domains of the newly discovered adaptor protein CIN85 bind to a proline- and arginine-rich motif, "Px(P/A)xxR" (21), or "PxxxPR" as predicted by another study (20), rather than the conventional proline-rich sequence with a "PxxP" motif at its core.
Third, SFKs regulate numerous cell-signaling pathways and play key roles in cell morphology, motility, proliferation, and survival (24, 37). SFK includes ubiquitously expressed resident members (e.g., Src, Yes, Fyn, and Yrk), as well as members whose expression is restricted to mainly hematopoietic cell lineages (e.g., Blk, Fgr, Hck, Lck, and Lyn). SFKs have very similar domain organizations: from the amino to the carboxyl terminals, all SFK members contain an amino-terminal 14-carbon myristoyl group, a unique segment, an SH3 domain, an SH2 domain, an SH2-kinase linker, a protein-tyrosine kinase domain, and a carboxyl-terminal regulatory tail containing a conserved tyrosine 527, which is phosphorylated by Csk (carboxyl-terminal Src kinase) and which plays a critical role in the regulation of the kinase activity. Under normal conditions, SFKs are kept in an inactive conformation, which is stabilized mainly through the intramolecular interactions between the SH3 domain and a polyproline type II left-handed helix located in the linker region, as well as a salt bridge formed between the SH2 domain and phosphorylated tyrosine 527. Relevant to this report, the kinase activity of SFKs is regulated by phosphorylation; the chief phosphorylation sites include tyrosine 416, which results in activation from autophosphorylation, and tyrosine 527, which results in inhibition of phosphorylation by Csk (reviewed in references 3 and 34).
Numerous reports have documented the fact that viruses that establish chronic infections target SFK members to interfere with cell signaling (reviewed in reference 9). Viral-protein interactions with SFK members are mediated predominantly by two types of molecular-recognition events. The first results from the interaction of phosphotyrosine residues with SH2 domains, for example, the interaction of Epstein-Barr virus LMP2A protein with the Lyn kinase (29). The second is mediated by the interaction of polyproline motifs with SH3 domains, as exemplified by the association of human immunodeficiency virus type 1 Nef with Hck, Lck, Lyn, Fyn, or Src (2, 35). The consequences of the interaction of viral proteins with SFK members may be kinase dependent. In a recent study, using transient transfections and Huh-7 cells harboring a persistently replicating subgenomic hepatitis C virus replicon, Macdonald et al. (25) demonstrated that NS5A bound to native SFK members in vivo and differentially modulated kinase activity, inhibiting Hck, Lck, and Lyn but activating Fyn. In another case, polyoma virus middle T binds to the catalytic domains of Src, Yes, and Fyn; binding to Src and Yes activates kinase activity, whereas middle T has no effect on Fyn (reviewed in reference 13).
The studies described in this report began with the demonstration that ICP0 interacts with members of the SFKs. Its focus, however, is on the roles, if any, of the SFK members in the course of HSV replication. We report that HSV infection enhanced the activating phosphorylation of Tyr416 of SFKs and modified SFKs through phosphorylation in a manner dependent on ICP0 and on the unique short 3 (US3) and unique long 13 (UL13) viral protein kinases. The ultimate roles of the modified proteins in specific steps in viral replication are still unclear. We also report that cells in which the negative regulator Csk had been deleted yielded lower virus titers and accumulated less late (2) proteins than wild-type sibling cells. These results suggest that HSV-1 regulates the activities of at least two members of the SFKs in order to induce optimal viral replication in infected cells.
MATERIALS AND METHODS
Cells and viruses. HEK293, HEp-2, and Vero cell lines were obtained from the American Type Culture Collection (Manassas, Va). Telomerase-transformed diploid human embryonic lung (HEL) fibroblasts (1) were obtained from Thomas Shenk (Princeton University, Princeton, N.J.). The cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (HEL and HEK293) or 5% newborn calf serum (Vero and HEp-2). HSV-1 strain F [HSV-1(F)], a limited-passage clinical isolate, was the prototype HSV-1 strain used in our laboratory (10).
Recombinant viruses containing mutations on an HSV-1(F) background, R7910 (0; ICP0 null) (15), R2621 (UL41 or vhs) (30), R7356 (UL13), R7041 (US3), and R7353 (UL13/US3) were reported elsewhere (31-33).
Mutant mouse embryo fibroblast (MEF) lines were obtained from A. Imamoto (Ben May Institute, University of Chicago). Specifically, in the SYF– line (19), all three widely expressed members of Src family kinases (Src, Fyn, and Yes) were knocked out; in the +Src line (19), the Src gene was restored by retroviral transduction of the SYF– line; in the CSK– line (14), the negative regulator of Src, the Csk gene, was knocked out, and thus, Src was constitutively activated; in the WT line (14), normal MEFs from same-stage embryos of littermates were developed without the introduction of genetic alterations. All MEFs were immortalized by transformation with simian virus 40 large T antigen.
Antibodies. The antibodies used in these studies were anti-CIN85 (Calbiochem; no. 231006), anti-glutathione S-transferase (GST) (Santa Cruz Biotechnology), anti-Src (Upstate Biotechnology; clone GD11, no. 05-184), anti-panSrc (Santa Cruz Biotechnology; sc-18), anti-Src-pY416 (Sigma; no. S1940), anti-Src-pY527 (Sigma; no. S2065), and rabbit polyclonal anti-thymidine kinase [TK] and monoclonal anti-ICP4, anti-ICP27, anti-ICP0, and anti-US11 (Goodwin Cancer Research Institute, Plantation, FL).
Plasmids. Mouse cDNA clones of SFK members were from the following authors: nonneuronal Src, Tony Hunter (the Salk Institute, CA); neuronal Src (NSrc), David Baltimore (26); Fyn (nonthymic form; FynB), Phil Soriano (Fred Hutchinson Cancer Research Center, WA); and Yes, Joseph Bolen (16). The human FGR (Gardner-Rasheed feline sarcoma virus [v-fgr] oncogene homolog) cDNA clone was purchased from the American Type Culture Collection (ATCC 10437429).
GST fusion proteins. The SH3 domains of SFK members Src, neuronal Src, Fyn, Yes, and Fgr were PCR amplified from appropriate cDNA clones, inserted in frame to vector pGEX-4-T1 or pGEX-4-T2, and verified by DNA sequencing at the University of Chicago Cancer Research Center DNA Sequencing Facility. GST-ICP0 constructs (amino acids 1 to 19, 20 to 241, 245 to 395, 245 to 510, or 543 to 768) were described previously (23). Competent Escherichia coli strain BL-21 cells were transformed with the GST fusion constructs and induced by isopropyl -D-thiogalactoside, and GST fusion proteins were immobilized to glutathione-Sepharose beads and purified according to the manufacturer's protocol (Amersham Pharmacia).
GST pull-down assays. Cells were harvested in cold phosphate-buffered saline (PBS), lysed by brief sonication in RIPA buffer (100 mM Tris, pH 8.0, 140 mM NaCl, 1% Triton X-100, 0.1% sodium dodecyl sulfate [SDS], 1% deoxycholic acid, 0.5 mM EDTA, protease and phosphatase inhibitors), and clarified by centrifugation in a Sorvall Biofuge Pico microcentrifuge at 16,000 x g for 20 min at 4°C. Total cell lysate was diluted in an equal amount of pull-down buffer (50 mM Tris, pH 7.5, 100 mM NaCl, 0.1% Nonidet P-40, 1 mg/ml bovine serum albumin [BSA]) to 1 ml and reacted overnight at 4°C with GST fusion proteins bound to glutathione-Sepharose beads. The GST beads were rinsed extensively in pull-down buffer and resuspended in an equal volume of 1x SDS loading buffer (50 mM Tris, pH 6.8, 2.75% sucrose, 5% 2--mercaptoethanol, 2% SDS). The solubilized proteins were boiled and electrophoretically separated in a denaturing 10% polyacrylamide gel.
Immunoblots. Electrophoretically separated proteins were electrically transferred to a nitrocellulose membrane, blocked at room temperature with 5% nonfat dry milk in PBS, and reacted with primary antibody diluted in PBS-1% BSA (anti-CIN85, 1:500; anti-Src, 1:1,000; anti-panSrc, 1:500; anti-GST, 1:2,000; anti-ICP0, 1:2,000; anti-ICP4,1:2,000; anti-ICP27, 1:2,000; anti-US11, 1:1,000; anti-TK, 1:500), followed by an appropriate secondary antibody conjugated to either peroxidase (Sigma) or alkaline phosphatase (Bio-Rad). Reactive protein bands were visualized with either enhanced chemiluminescence (Amersham Pharmacia) or 5-bromo-4-chloro-3-indolylphosphate-nitroblue tetrazolium (Denville Scientific, Metuchen, NJ), according to the manufacturer's instructions.
Src in vitro kinase assay. HEL cells in T75 flasks were exposed to 10 PFU/cell of HSV-1(F) or mock infected for 0, 3, 6, 9, or 12 h and then harvested and lysed in 400 μl RIPA buffer supplemented with protease and phosphatase inhibitors. Three hundred micrograms of clarified cell lysate was immune precipitated with 2 μg anti-Src for 3 h at 4°C. Fifty percent of each immune precipitate was resuspended in 20 μl Src kinase reaction buffer (100 mM Tris-HCl, pH 7.2, 125 mM MgCl2, 25 mM MnCl2, 2 mM EGTA, 250 μM orthovanadate, 2 mM dithiothreitol), mixed with 10 μg acid-denatured enolase (Boehringer Mannheim no. 104647; diluted in 10 μl Src kinase reaction buffer) and 10 μCi [-32P]ATP (Amersham; diluted in 10 μl Mn-ATP cocktail buffer [75 mM MnCl2, 500 μM ATP, 20 mM MOPS {morpholinopropanesulfonic acid}, pH 7.2, 25 mM -glycerol phosphate, 5 mM EGTA, 1 mM Na3VO4, 1 mM dithiothreitol]), and incubated at 30°C for 10 min, and the reaction was stopped by adding 20 μl of 4x SDS disruption buffer and boiling the mixture at 100°C for 10 min. Phosphorylated enolase was resolved in a 10% denaturing polyacrylamide gel, transferred to a nitrocellulose membrane, scanned, and quantified with a Storm 860 phosphorimager (General Dynamics, Falls Church, VA). The other 50% of the immune precipitate was solubilized, electrophoretically separated, transferred to a nitrocellulose membrane, and reacted with anti-Src antibody to assess the total amount of Src protein used in the previous in vitro kinase assays. Finally, the Src kinase specific activity from each sample was determined by normalizing the phosphorylation level of enolase against the total amount of Src protein.
Detection of the phosphorylation status of Tyr416 and Tyr527 of SFKs. HEK293 or HEL cells were exposed to 10 PFU/cell of HSV-1(F) or relevant deletion mutants or mock infected for the appropriate times. The cells were harvested and lysed in RIPA buffer supplemented with protease and phosphatase inhibitors with brief sonication. Equal amounts of clarified total cell lysate were separated by 10% denaturing polyacrylamide gel, transferred to a nitrocellulose membrane, and reacted with anti-Src-pY416 (1:1,000 dilution) and anti-Src-pY527 (1:1,000 dilution). To detect tyrosine phosphorylation specific to Src kinase, 600 μg of whole-cell lysate was immune precipitated with anti-Src for 3 h at 4°C, and one-third of the immune precipitates were solubilized, electrophoretically separated, transferred to a nitrocellulose membrane, and reacted with anti-Src-pY416 and anti-Src-pY527 as described above. The blots were stripped and reprobed with anti-Src to determine the total amount of Src protein immune precipitated. The reactive protein bands were quantified by phosphorimager or exposed to X-ray film.
Alkaline phosphatase digestion of cell lysates. HEL cells were mock infected or infected with 10 PFU/cell HSV-1(F) for 9 h and lysed in RIPA buffer without phosphatase inhibitors. An aliquot was saved, and phosphatase inhibitors were added immediately to serve as input controls before the treatment. Four hundred micrograms of lysates was mock digested or digested with 20 μl alkaline phosphatase (Boehringer Mannheim) in a total volume of 150 μl at 30°C for 40 min. One hundred micrograms of total protein was electrophoretically separated, transferred to a nitrocellulose membrane, and reacted with anti-panSrc or anti-Src.
Replication of HSV-1 in MEFs. Mutant MEFs in T25 flasks were exposed to 0.1 PFU of HSV-1(F) per cell in 199V medium (Sigma) supplemented with 1% calf serum for 2 h at 37°C. The inoculum was then removed, and the cell monolayers were rinsed with 199V medium to remove the unabsorbed virus. The cells were overlaid with complete medium and incubated at 37°C for an additional 24 h. The cells and medium were subjected to three cycles of freeze-thaw plus brief sonication, and the viral yield from each MEF was titered in Vero cells.
Determination of viral DNA accumulation in MEFs. Mutant MEFs in T25 flasks were infected for 24 h with 0.1 PFU of HSV-1(F) per cell as described above. The infected cells were harvested, resuspended in 400 μl T10E50 buffer (10 mM Tris-HCl, 50 mM EDTA, pH 8.0, 0.5% NP-40), digested sequentially with 2 μl RNase (10 mg/ml) on ice for 15 min and with 10 μl of protease K (10 mg/ml) plus 20 μl of 20% SDS at 37°C for 2 h, and extracted with phenol-chloroform twice. The total cellular DNA was precipitated with ethanol, resuspended in 50 μl distilled water, and digested with BamHI overnight. Equal volumes of digested DNA were separated on a 0.8% agarose gel, alkaline blotted to a zeta-probe nylon membrane (Bio-Rad) with 0.5 M NaOH overnight, and hybridized with 32P-labeled BamHI S fragment of HSV-1 DNA (nick translation kit; Promega) in hybridization buffer (1 mM EDTA, 40 mM Na3PO4, 7% SDS) at 65°C overnight. After hybridization, the blot was rinsed three times at 65°C with rinsing buffer (1 mM EDTA, 40 mM Na3PO4, 5% SDS) and quantified by phosphorimager or exposed to X-ray film for an appropriate time. The yield of viral DNA from each MEF line was normalized against the amount of total cellular DNA loaded and expressed as a ratio to the WT line.
Determination of viral mRNA accumulation in MEFs. Mutant MEFs in T25 flasks were infected for 8 or 12 h with 0.1 PFU of HSV-1(F) per cell as described above. Total RNA was extracted with the aid of TRIzol Reagent (GIBCO-BRL; no. 15596-018) according to the manufacturer's instructions. For Northern blot analysis, 10 μg of total RNA was resolved on a 1% denaturing formaldehyde agarose gel and transferred onto a "Brighstar-Plus" charged membrane (Ambion; no. 10102). The membrane was prehybridized for 2 h at 42°C in 12 ml ULTRAhyb buffer (Ambion; no. 8670) supplemented with denatured salmon sperm DNA (200 μg/ml; Stratagene) and then hybridized overnight after the addition of 32P-labeled DNA probes (Promega; Prime-a-Gene labeling system; no. U1100) specific to the viral genes ICP4 (NruI-XmnI fragment; residues 307 to 464), ICP0 (MluI-HindIII fragment; residues 543 to 775), and ICP27 (NcoI-StuI fragment; residues 189 to 408). The membrane was then rinsed two times with 2x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-0.1% SDS for 5 min and three times with 0.1x SSC-0.1% SDS for 20 min, scanned, and quantified with a General Dynamics Storm 860 phosphorimager.
RESULTS
ICP0 physically interacts with SFK members. Earlier studies indicated that an uncharted region of ICP0 (amino acids 245 to 510) that is relatively proline rich physically interacts with the SH3 domains of CIN85, an adaptor protein that is involved in negative regulation of receptor tyrosine kinases (23). In light of this observation, it was of interest to determine whether ICP0 can also interact with the prototypical SH3 domains of the SFK members of nonreceptor protein tyrosine kinases. To test this hypothesis, the SH3 domains of the SFK members Src, NSrc, Fyn, Yes, and Fgr were PCR amplified from the relevant cDNA clones and fused in frame with GST. The GST-SH3 fusion proteins were made in E. coli and reacted with lysates of HSV-1(F)-infected HEp-2 cells. As shown in Fig. 1A, the negative control (lane 3) consisted of the same DNA fragment of the Fgr SH3 domain inserted in the opposite orientation downstream of GST. Thus, a GST fusion protein of the same molecular weight but with a completely irrelevant amino acid sequence did not pull down ICP0 from the infected-cell lysate. The other four GST-SH3 fusions, i.e., those that included Src, Fyn, Yes, and Fgr, pulled down ICP0. It is noteworthy that the SH3 domain of neuronal Src, which contains a six-amino-acid (RKVDVR) insertion because of an alternative splicing event (7, 22, 26), did not interact with ICP0. This observation suggests the potentially interesting possibility that ICP0 does not interact with Src in neurons or neuroblastoma cells in which NSrc is predominantly expressed. NSrc is expressed during neuronal differentiation (4) and has a two- to fourfold-higher activity than that of c-Src (5, 9), possibly because the additional six amino acids destabilize the autoinhibitory conformation of Src, thereby enabling enhanced activity (36). It should be noted that the mobility of ICP0 in lane 2 was slightly different from that of the input control in lane 1, but this mobility change was not reproducible in other experiments.
The objective of the second series of experiments was to verify the expectation that the domain of ICP0 that interacts with Src is the same one that interacts with CIN85, i.e., the polypeptide containing residues 245 to 510. To address this objective, different GST-tagged regions of ICP0 were reacted with uninfected HEK293 cell lysate, and the proteins pulled down by GST-ICP0 chimeric proteins were electrophoretically separated in denaturing gels, transferred to a nitrocellulose membrane, and reacted with anti-Src monoclonal antibody. As shown in Fig. 1B, Src was pulled down by the two GST-ICP0 chimeric fusions (lanes 5 and 7) containing residues 245 to 510 of ICP0 but not by chimeric proteins containing other domains of ICP0 or by GST alone.
Taken together, these results indicate that ICP0 physically interacts with the Src family of tyrosine protein kinases through sequences contained between residues 245 and 510 and the SH3 domains of SFK members.
Amounts and activities of SFKs in the course of HSV-1 replication. The objective of the experiments described below was to determine whether HSV infection alters the stability or function of Src family protein tyrosine kinases. In the first series of experiments, replicate cultures of HEK293 or HEL cells were mock infected or exposed to 10 PFU of HSV-1(F), ICP0, or vhs mutant virus per cell. The cells were harvested and lysed 4, 8, or 12 h after infection. Equal amounts of cell lysates were electrophoretically separated in denaturing gels, transferred to a nitrocellulose membrane, and reacted with anti-Src antibody. As shown in Fig. 2, over the interval of 12 h from the time of infection, Src did not change significantly either in quantity or in electrophoretic mobility in a denaturing polyacrylamide gel.
The objective of the second series of experiments was to determine if HSV-1 infection alters the specific kinase activity of Src measured by in vitro kinase assays. Briefly, HEL cells were exposed to 10 PFU of HSV-1 per cell, harvested, and lysed 0, 3, 6, 9, or 12 h after infection. Equal amounts of cell lysates from each sample were immune precipitated with anti-Src monoclonal antibody. The immune complexes were collected, and one-half of the immune precipitates from each sample was reacted with 10 μg of acid-denatured enolase and 10 μCi [-32P]ATP. The enolase was resolved in a denaturing polyacrylamide gel, transferred to a nitrocellulose membrane, and quantified with the aid of a General Dynamics Storm 860 phosphorimager. The other half of the immune precipitates was electrophoretically separated in a denaturing polyacrylamide gel, transferred to a nitrocellulose membrane, and reacted with anti-Src antibody to determine the amount of Src protein present in the in vitro kinase assays. The "specific kinase activity" of Src was expressed as the amount of phosphorylated enolase divided by the amount of Src protein immune precipitated from each sample, normalized to mock infection at 0 h after infection, and plotted against the number of hours after infection. As shown in Fig. 3, the specific activity of Src in infected cells was slightly elevated with respect to that of mock-infected cells. The activity peaked between 6 and the 9 h after infection (e.g., the normalized values were 1.4 ± 0.4 for 6-h mock-infected cells versus 2.1 ± 0.5 for 6-h infected cells and 1.1 ± 0.2 for 9-h mock-infected cells versus 2.0 ± 0.9 for 9-h infected cells) and decreased to mock infection levels in the 12-h sample. This experiment was repeated three times with similar results.
The tyrosine kinase activity of SFK members is also reflected in the phosphorylation statuses of two key tyrosine residues that are conserved among the SFK members. Thus, phosphorylation of Tyr416 is stimulatory while phosphorylation of Tyr527 is inhibitory (reviewed in references 3 and 34). In the third series of experiments, we addressed the question of whether the phosphorylation status of SFKs was altered in the course of HSV-1 replication and what viral genes were involved. First, replicate cultures of HEL cells were either mock infected or exposed to 10 PFU of HSV-1(F), ICP0, US3, UL13, or US3/UL13 mutant viruses per cell. The cells were harvested 4, 8, or 12 h after infection and processed as described in Materials and Methods, and equal amounts of total cell lysates harvested at each time point were separated in denaturing gels, transferred to a nitrocellulose sheet, and reacted with anti-Src-pY416 or anti-Src-pY527 antibody. Since Tyr416 and Tyr527 are conserved among all SFK members, anti-phospho-Tyr antibodies raised against these two residues of Src should react with all other SFK members that are expressed (e.g., Fyn and Yes in the case of HEK293 and HEL cells). As shown in Fig. 4, the results were as follows. (i) At all three time points tested in mock-infected cells, phospho-Tyr416 was barely detectable, consistent with the current evidence that under normal conditions SFK members are held in an inactive, autoinhibitory conformation. (ii) Except in cells infected with the ICP0 mutant, phosphorylation of Tyr416 was enhanced by HSV infection. This was observed in the 8- and 12-h samples but not in the 4-h sample. The failure to detect phosphorylation of Tyr416 in lysates of mock-infected cells or cells infected with the ICP0 virus mutant (lanes 1, 3, 7, 9, 13, and 15) was not due to the reduced levels of SFK members, because the reaction of the same samples with the anti-panSrc antibody showed that they contained comparable amounts of proteins of SFK members. These results indicate that in HSV-1-infected HEL cells, ICP0 is required to enhance the activating phosphorylation of Tyr416 of at least one SFK member. (iii) At 8 h after infection, the band reactive with the anti-Src-pY416 formed by lysates of cells infected with wild-type virus migrated more slowly than the corresponding bands in lysates of cells infected with viral mutants lacking either or both viral protein kinases (Fig. 4, top, compare lane 8 with lanes 10, 11, and 12). However, at 12 h after infection, all bands reactive with the anti-Src-pY416 migrated at the same lower rate (lanes 16 to 18). These results suggest that at least one SFK member is posttranslationally modified further in the course of HSV infection and that this modification is delayed in cells infected with the viral-kinase-minus mutant viruses. (iv) The levels of the inhibitory phospho-Tyr527 in lysates of infected cells were similar to those of mock-infected cells. However, at 8 h and 12 h after infection (lanes 8 and 14), only the wild-type virus, but not any of the mutants tested, altered the electrophoretic mobility of at least one SFK member in denaturing polyacrylamide gels, as detected by both anti-panSrc antibody and anti-Src-pY527, which also recognizes all SFK members. This observation indicates that HSV further modifies SFK members in a manner dependent on ICP0, US3, and UL13 products. Because at 12 h after infection this modification was seen only in lysates of cells infected with wild-type virus but not in lysates of cells infected with mutant viruses, we can surmise that this SFK member is not phosphorylated at Tyr-416. (v) The cell lysates were also reacted with antibody to actin as a loading control and with antibodies against ICP0 and viral TK. The decreased levels of TK, a protein, in lysates of HEL cells infected with the ICP0 mutant virus may reflect the requirement for ICP0 for optimal progression of viral replication and accumulation of viral gene products (6).
The phosphotyrosine-specific antibodies used in the experiments described above do not differentiate Src from other SFK members. To specifically detect the tyrosine phosphorylation status of just Src, HEK293 cells were mock infected or infected with HSV-1(F) or the ICP0 mutant virus. The lysates of cells harvested at time zero or 4, 8, or 12 h after infection were reacted with anti-Src antibody first. The immune precipitates were then solubilized, subjected to electrophoresis in denaturing gels, and reacted with the two anti-phosphotyrosine antibodies (Fig. 5A and C) mentioned above. The two blots were then stripped and reprobed with anti-Src to determine the total amount of Src protein immune precipitated in each case (Fig. 5B and D). The reactive protein bands were quantified with the aid of the General Dynamics phosphorimager. As shown in Fig. 5, normalization of the tyrosine phosphorylation level to the total Src protein level in each sample with respect to mock infection at time zero showed that activating phosphorylation (Y416) specific to Src increased as early as 4 h after viral infection and onwards, whereas the inhibitory phosphorylation (Y527) remained basically unchanged, consistent with the results shown in Fig. 3, where starting from 6 h after infection and onwards, in in vitro kinase assays, the kinase activity of Src was modestly higher in HSV-1-infected than mock-infected cells. However, in contrast to the experiment whose results are shown in Fig. 4, when all SFKs were indiscriminately examined by pan-Src antibodies, we could not detect altered electrophoretic mobility in denaturing gels when immune-precipitated Src was reacted with these two anti-phospho-Tyr antibodies, suggesting that Src itself is likely not the target of the further modification by HSV-1 infection observed in Fig. 4.
To verify the results shown in Fig. 5 regarding the effects of HSV infection on the phosphorylation status of Tyr416 and Tyr527 specific to Src in a different cell line, and to test if we could detect enhanced phosphorylation of Tyr416 of SFK members earlier than 8 h after infection, we repeated the above-mentioned experiments with HEL cells. Briefly, HEL cells mock infected or exposed to 10 PFU of HSV-1(F) or mutant viruses per cell were harvested and lysed 6 h after infection. The cell lysates were either electrophoretically separated in a denaturing polyacrylamide gel, transferred to a nitrocellulose membrane and reacted with the above-mentioned two anti-phospho-Tyr antibodies to directly assess the tyrosine phosphorylation statuses of all SFKs expressed in HEL cells, or first immune precipitated with anti-Src and then probed with anti-phospho-Tyr antibodies to detect phosphorylation of Tyr416 and Tyr527 specific to Src. The results shown in Fig. 6 were basically the same as those shown in Fig. 4 and in HEK293 cells (Fig. 5). Specifically, they were as follows.
First, as shown in the top gel of Fig. 6, the antibodies against pY416 and pY527 reacted with all phosphorylated members of SFK. As shown in the lower gel, these antibodies detected tyrosine phosphorylation specific to Src alone. The results shown in the upper gel indicated that as early as 6 h after infection, at least one member of the SFKs was phosphorylated at Tyr416 in all lysates except that obtained from mock-infected cells or cells infected with the ICP0 mutant virus. As in the results shown in Fig. 4, the antibody to phospho-Tyr527 reacted with two relatively sharp bands in all lysates except that of cells infected with wild-type virus. In this instance, the upper of the two bands migrated more slowly than the corresponding band formed by other lysates, suggesting that the protein present in this band was subjected to additional posttranslational modification and that this modification required functions dependent on ICP0, US3, and UL13. Moreover, consistently, this additional modification could also be detected by the other panSrc anti-pY416 antibody, as reflected by the slower-migrating band formed by lysates of HSV-1(F)-infected cells but not by those of mutant-virus-infected cells (upper gel, compare lane 2 with lanes 4, 5, and 6).
Second, the lower panel shows the phosphorylation status of immune-precipitated Src. Anti-Src-pY416 recognized a faint band in lysates of infected but not mock-infected HEL cells at 6 h after infection, whereas anti-Src-pY527 detected strong bands in infected and mock-infected cells. This observation indicates that only a small fraction of Src was phosphorylated at Tyr416 compared to the amount of Src phosphorylated at Tyr 527. Moreover, the phosphorylation at Tyr416 was stronger in the case of bands formed by lysates of mutant-virus-infected cells than in cells infected with wild-type virus. In addition, the lower band formed by lysates of cells infected with wild-type virus (lower gel, lane 8) was much broader and was partially retarded in its electrophoretic mobility, suggesting that some of the Src underwent additional modifications that were not observed in mutant-virus-infected cells.
Posttranslational modification of Src. The results of the experiment shown in the second gel of Fig. 4 indicated that panSrc antibody reacted with a diffuse slower-migrating band formed by lysates of cells infected with wild-type virus harvested 8 or 12 h after infection. This diffuse band was not formed by lysates of mutant virus infected cells and, in particular, the viral protein kinase mutants. This observation prompted us to further examine if the nature of this modification was phosphorylation. For this purpose, HSV-1(F)-infected or mock-infected HEL cell lysates were digested with alkaline phosphatase or mock digested and then electrophoretically separated in a denaturing polyacrylamide gel, transferred to a nitrocellulose membrane, and reacted with anti-panSrc antibody as described in the legend to Fig. 7. As expected, electrophoretically separated lysates of cells infected with wild-type virus formed two bands reactive with the anti-panSrc antibody. The upper band was sharp in mock-infected cell lysates but diffuse in lysates of cells infected with wild-type virus. After alkaline phosphatase digestion, the diffuse upper band formed in infected-cell lysates was replaced by a faster-migrating sharp band. Inasmuch as the diffuse band was detected by panSrc but not by Src-specific antibody, the results indicate that it was a member of SFK but not Src itself. These results are consistent with the conclusion that a member of the SFKs was phosphorylated in a UL13- and US3-dependent manner.
In summary, we conclude the following from these studies. (i) We noted a modest increase in the activity specific to Src (Fig. 3). This activity corresponds to a modest enhancement of phosphorylation of pY416 of Src (Fig. 6). Overall, members of the SFK family are phosphorylated at Tyr416 at some point between 4 and 8 h after infection. These members undergo a second set of modifications that take place earlier in cells infected with wild-type virus (8 h) than in mutant-virus-infected cells (12 h). (ii) The phosphorylation of Tyr527 appears to be relatively constant throughout the period of observation in all virus-infected or mock-infected cell cultures (Fig. 4 to 6). (iii) A member of the SFKs other than Src is posttranslationally phosphorylated in a manner dependent on ICP0, US3, and UL13. This modification was apparent in assays with panSrc antibodies (Fig. 4, lane 2) and with anti-pTyr527 antibody (Fig. 6). The results suggest that this phosphorylation occurs on the background of phosphorylated Tyr 527 protein. (iv) The role of ICP0 in mediating posttranslational modifications of members of the SFKs is not clear. It is conceivable that in ICP0 mutant-infected cells, the progression of viral protein synthesis was delayed or that ICP0 binds and translocates SFK members to specific subcellular compartments. It should also be noted that UL13 phosphorylates US3 and that conceivably the phosphorylated form of US3 is required for the posttranslational modification of SFK members.
Enhanced Src activity repressed viral replication at a low multiplicity of infection. To determine whether alterations in Src kinase activity affected HSV-1 gene expression and replication, we took advantage of four MEF lines described in Materials and Methods and in detail elsewhere (14, 19). Thus, in the SYF– line, all three widely expressed SFK members (Src, Fyn, and Yes) were knocked out. In the +Src line, the Src gene was restored and overexpressed by transduction of the SYF– line with a retroviral vector. In the CSK– line, the Csk gene, the negative regulator of Src, was knocked out, and thus, SFKs were constitutively activated. The control cell line (WT) was derived from the genetically unaltered same-stage embryos of the littermates. In all, the following series of studies were performed.
In the first series of experiments, we verified the accumulation of Src in the MEF lines. In brief, 300 μg of total protein extracted from each cell line was immune precipitated with 2 μg of anti-Src antibody. The immune precipitates were solubilized, electrophoretically separated, and probed with anti-Src antibody. As expected (Fig. 8), Src proteins were overexpressed in the +Src cell line compared to the amount recovered from wild-type cells but were not detected in lysates of the SYF– cell line. The amount of Src detected in the CSK– cell line was slightly lower than that detected in WT MEFs.
In the second series of experiments, we exposed the four MEF lines to 0.1 PFU of HSV-1(F) per cell. The cells were harvested 24 h after infection, and virus yields were measured in Vero cells. The results (Table 1) showed that virus yields decreased about 8-fold in +Src cells and >10-fold in the CSK– line relative to the yield from WT controls. However, the increase in SYF– cells was barely twofold, suggesting that while enhanced Src activity was detrimental, the absence of Src did not enhance viral replication to a significant level. In other studies (unpublished data), when the multiplicity of infection was higher (1 PFU/cell or 10 PFU/cell), there was no significant difference in virus yields from the four MEF lines.
In the third series of experiments, replicate cultures of the four MEF lines were exposed to 0.1 PFU/cell. The cells were harvested 24 h after infection, and the total cellular DNA was extracted, digested to completion with BamHI, electrophoretically separated in an agarose gel, transferred to a nylon membrane, and hybridized with 32P-labeled BamHI S fragment of HSV-1 genomic DNA to measure the accumulation of viral DNAs. The results shown in Fig. 9 were reproducible and showed that viral DNA accumulated in smaller amounts in infected +Src or CSK– cells than in infected WT cells. SYF– cells accumulated approximately half of the total DNA detected in infected WT cells
In the fourth series of experiments, we examined the accumulation of viral proteins in the four MEF lines exposed to 0.1 PFU of HSV-1(F) per cell. The cells were harvested 4, 8, 12, or 16 h after infection, and 100 μg of the solubilized total cellular protein was subjected to electrophoresis in denaturing gels, transferred to nitrocellulose sheets, and probed with antibodies to ICP4, ICP27, TK, and US11. The results, shown in Fig. 10, were as follows: the amounts of accumulating viral proteins were highest in SYF– cells, while the lowest levels were observed in +Src cells. Of particular interest is the observation that in infected CSK– cells there was a reduction in the 2 protein US11 but not in (ICP27 and ICP4) or (TK) proteins. In contrast, infected +Src cells accumulated reduced amounts of all viral proteins surveyed in this experiment, whereas the SYF– cell line accumulated more US11 protein than the other cell lines.
The reductions in the protein levels of ICP27 and ICP4 in the infected +Src cells prompted us to examine the levels of mRNAs in the four cell lines. In these experiments, the replicate cultures of the four cell lines were exposed to 0.1 PFU of HSV-1(F) per cell. The cells were harvested 8 or 12 h after infection. Total RNA was extracted from one set of cultures, electrophoretically separated under denaturing conditions, and probed with 32-P-labeled DNA probes specific to ICP4, ICP0, or ICP27. The second set of cultures were processed as described in Materials and Methods, subjected to electrophoresis in denaturing gels, and probed with antibodies to relevant viral proteins as in Fig. 10. The results, shown in Fig. 11, were as follows.
As predicted by the results of protein analysis shown in Fig. 10, the infected +Src cell line accumulated smaller amounts of ICP4, ICP0, or ICP27 mRNA than other cell lines. The amounts of HSV-1 mRNAs detected in CSK– or SYF– cell lines were also smaller than those detected in cells infected with wild-type virus.
The overall protein profile was similar to that shown in Fig. 10. Thus, the SYF– cells accumulated the largest amounts of the representative proteins assayed in this experiment, whereas the +Src cells accumulated the smallest amounts. Of the proteins tested, the only one accumulating in significantly smaller amounts in the CSK– cell line was US11.
Based on these observations, we conclude the following. (i) The SYF– cell line lacking Src, Yes, and Fyn kinases accumulated the largest amounts of representative , , and 2 proteins and yielded amounts of virus at least equivalent to those obtained in infected wild-type MEFs. (ii) The +Src cell line accumulated the smallest amounts of proteins, consistent with the smallest amounts of accumulated mRNAs. In this cell line, only Src of the SFK members was restored. (iii) Removal of CSK, the negative regulator of SFK members, reduced viral yields but did not have a significant effect on the accumulation of gene mRNAs or the accumulation of the representative or proteins. Rather, the major discernible defect was in the accumulation of the 2 protein, US11.
DISCUSSION
In an earlier report, we showed that ICP0 contains multiple sequences capable of binding the SH3 domain of the adaptor protein CIN85. The initial objective of the studies described in this report was to determine whether ICP0 binds other proteins containing SH3 domains and, in particular, whether ICP0 interacts with members of the SFKs. Once an interaction was observed, the focus of the studies was to determine the role of the Src protein in the course of viral infection. Since the discovery of SFKs, a growing literature has documented the roles of these proteins in the regulation of a vast array of cellular functions, including cell cycle progression, adhesion and spreading, focal adhesion formation, and disassembly, migration, apoptosis, differentiation, and gene transcription (24, 37). Moreover, it has been reported that some viruses manage to manipulate the activities of SFKs so as to benefit from some of the cell-signaling events regulated by SFKs. For example, the HBx gene of the hepadnaviruses activates Src kinases, while activated Src kinases in turn upregulate hepatitis B virus (HBV) reverse transcription and stimulate HBV and woodchuck hepatitis virus replication in established liver cell lines by 5- to 20-fold. Conversely, inhibition of Src kinases reduced the yields of HBV or woodchuck hepatitis virus replication by 10- to 15-fold (17, 18). A central objective of these studies was to survey the roles of the Src proteins in the course of HSV-1 infection. The salient features of this report and their significance are as follows.
First, ICP0 physically interacted with Src, Fyn, Yes, and Fgr, members of the SFKs. Furthermore, the ICP0 domains interacting with these proteins were the same as those that interacted with CIN85. It is noteworthy that NSrc did not interact with ICP0. NSrc differs from Src by the addition of six residues within its SH3 domain. The significance of this observation is uncertain. The data suggest that not all proteins with SH3 domains interact with ICP0 and, conversely, that the interaction of ICP0 with SFK has functional relevance. It is also noteworthy that the levels of Src protein in at least two cell lines remained unchanged over a period of 12 h after infection with wild-type or mutant viruses, which argues that degradation of Src is not an outcome of its interaction with viral gene products.
The role of the interaction of ICP0 with SFK members remains to be elucidated. There is no evidence that ICP0 targets Src for degradation. It is conceivable that ICP0 vectors Src or other members of SFK to specific subcellular compartments. The function of ICP0 with respect to the function of SFK remains to be determined.
Second, the increase in specific activity illustrated in Fig. 3 is modest, as is the phosphorylation of Tyr416. The positive activation of Src must be balanced with the obvious negative effects of overexpressed Src in the +Src mouse embryo fibroblast line and with the evidence that in the SYF– cell line the yield of virus could not be differentiated from that of cells infected with wild-type virus. Conceivably, Src exerts a negative effect on viral replication, and the modest activation reflects this possibility.
Third, we have not measured the activities of other members of the SFKs overall, but the activating phosphorylation of SFK was more robust. We also noted that in infected cells, at least one SFK member undergoes a posttranslational modification in addition to the phosphorylation of Tyr416. We have not been able to identify this member with available antibodies to Yes and Fyn (data not shown).
Fourth, the role of SFK in the course of viral infection may be deduced in part from the studies of the four MEF lines. In essence, overexpression of Src in the absence of Fyn and Yes members appeared to be deleterious to viral replication, whereas deletion of Src, Fyn, and Yes actually increased the amounts of viral proteins accumulating in infected cells. Comparison of +Src and wild-type sibling MEFs suggests that the presence of either Yes or Fyn or both kinases ameliorates the negative effects. The identity and functions of the kinase that confers a positive effect on viral replication is unclear. As noted above, the results presented in Fig. 5, 6, and 7 indicate that at least one member of the SFKs other than Src is modified by phosphorylation in a manner dependent on US3 and UL13 protein kinases. The data obtained in those experiments suggest that either both viral kinases or a US3 kinase modified by UL13 mediates the phosphorylation of a member of the SFKs. Neither of these two viral kinases would be expected to accumulate in HEL cells exposed to ICP0 at low multiplicities of infection. The role of this member of the SFKs in balancing the negative effect of Src remains to be determined.
Finally, it is noteworthy that the absence of the negative regulator of SFK in CSK– MEFs produced an effect very different from that observed in +Src or SYF– MEFs. Whereas in +Src MEFs, the negative effect correlated with a decreased accumulation of gene transcripts, in CSK– cells, the decrease in the synthesis of infectious progeny appears to be correlated with decreased yields of US11 and, potentially, with a decrease in the accumulation of other late or 2 proteins. These results suggest a mechanism of action that takes place late in infection rather than during the transcription of genes. In essence, constitutive activation of SFKs in the absence of Csk gene products differs from overexpression of Src only; it suggests that while Yes and Fyn are essential to block the negative effects of activated Src on viral-gene transcription, they or other members have a negative effect late in infection and must be deactivated for optimal synthesis of late proteins.
To our knowledge, this is the first report of the statuses and roles of SFK members in viral replication. We have opened a Pandora's box. It is clear that SFKs play significant roles, positive and negative, in HSV-1 replication and that HSV alters the functions of these proteins. The roles of individual members of the SFKs remain to be determined.
ACKNOWLEDGMENTS
We thank Akira Imamoto (Ben May Institute, University of Chicago) for invaluable reagents and discussion. We thank Brunella Taddeo and Weiran Zhang for advice.
These studies were aided by National Cancer Institute Grants CA87661, CA83939, CA71933, CA78766, and CA88860.
REFERENCES
Bresnahan, W. A., G. E. Hultman, and T. Shenk. 2000. Replication of wild-type and mutant human cytomegalovirus in life-extended human diploid fibroblasts. J. Virol. 74:10816-10818.
Briggs, S. D., E. C. Lerner, and T. E. Smithgall. 2000. Affinity of Src family kinase SH3 domains for HIV Nef in vitro does not predict kinase activation by Nef in vivo. Biochemistry 39:489-495.
Brown, M. T., and J. A. Cooper. 1996. Regulation, substrates and functions of src. Biochim. Biophys. Acta 1287:121-149.
Brugge, J. S., P. C. Cotton, A. E. Queral, J. N. Barrett, D. Nonner, and R. W. Keane. 1985. Neurons express high levels of a structurally modified, activated form of pp60c-src. Nature 316:554-557.
Brugge, J., P. Cotton, A. Lustig, W. Yonemoto, L. Lipsich, P. Coussens, J. N. Barrett, D. Nonner, and R. W. Keane. 1987. Characterization of the altered form of the c-src gene product in neuronal cells. Genes Dev. 1:287-296.
Cai, W., T. L. Astor, L. M. Liptak, C. Cho, D. M. Coen, and P. A. Schaffer. 1993. The herpes simplex virus type 1 regulatory protein ICP0 enhances virus replication during acute infection and reactivation from latency. J. Virol. 67:7501-7512.
Cartwright, C. A., R. Simantov, P. L. Kaplan, T. Hunter, and W. Eckhart. 1987. Alterations in pp60c-src accompany differentiation of neurons from rat embryo striatum. Mol. Cell. Biol. 7:1830-1840.
Chee, A. V., P. Lopez, P. P. Pandolfi, and B. Roizman. 2003. Promyelocytic leukemia protein mediates interferon-based anti-herpes simplex virus 1 effects. J. Virol. 77:7101-7105.
Collette, Y., and D. Olive. 1997. Non-receptor protein tyrosine kinases as immune targets of viruses. Immunol. Today 18:393-400.
Ejercito, P., E. D. Kieff, and B. Roizman. 1968. Characterization of herpes simplex virus strains differing in their effects on social behavior of infected cells. J. Gen. Virol. 2:357-364.
Gu, H., Y. Liang, G. Mandel, and B. Roizman. 2005. Components of the REST/CoREST/histone deacetylase repressor complex are disrupted, modified, and translocated in HSV-1-infected cells. Proc. Natl. Acad. Sci. USA 102:7571-7576.
Hagglund, R., and B. Roizman. 2004. Role of ICP0 in the strategy of conquest of the host cell by herpes simplex virus 1. J. Virol. 78:2169-2178.
Ichaso, N., and S. M. Dilworth. 2001. Cell transformation by the middle T-antigen of polyoma virus. Oncogene 20:7908-7916.
Imamoto, A., and P. Soriano. 1993. Disruption of the csk gene, encoding a negative regulator of Src family tyrosine kinases, leads to neural tube defects and embryonic lethality in mice. Cell 73:1117-1124.
Kawaguchi, Y., C. Van Sant, and B. Roizman. 1997. Herpes simplex virus 1 alpha regulatory protein ICP0 interacts with and stabilizes the cell cycle regulator cyclin D3. J. Virol. 71:7328-7336.
Klages, S., D. Adam, E. Eiseman, J. Fargnoli, S. M. Dymecki, S. V. Desiderio, and J. B. Bolen. 1993. Molecular cloning and analysis of cDNA encoding the murine c-yes tyrosine protein kinase. Oncogene 8:713-719.
Klein, N. P., and R. J. Schneider. 1997. Activation of Src family kinases by hepatitis B virus HBx protein and coupled signaling to Ras. Mol. Cell. Biol. 17:6427-6436.
Klein, N. P., M. J. Bouchard, L. H. Wang, C. Kobarg, and R. J. Schneider. 1999. Src kinases involved in hepatitis B virus replication. EMBO J. 18:5019-5027.
Klinghoffer, R. A., C. Sachsenmaier, J. A. Cooper, and P. Soriano. 1999. Src family kinases are required for integrin but not PDGFR signal transduction. EMBO J. 18:2459-2471.
Kowanetz, K., I. Szymkiewicz, K. Haglund, M. Kowanetz, K. Husnjak, J. D. Taylor, P. Soubeyran, U. Engstrom, J. E. Ladbury, and I. Dikic. 2003. Identification of a novel proline-arginine motif involved in CIN85-dependent clustering of Cbl and down-regulation of epidermal growth factor receptors. J. Biol. Chem. 278:39735-39746.
Kurakin, A. V., S. Wu, and D. E. Bredesen. 2003. Atypical recognition consensus of CIN85/SETA/Ruk SH3 domains revealed by target-assisted iterative screening. J. Biol. Chem. 278:34102-34109.
Levy, J. B., T. Dorai, L. H. Wang, and J. S. Brugge. 1987. The structurally distinct form of pp60c-src detected in neuronal cells is encoded by a unique c-src mRNA. Mol. Cell. Biol. 7:4142-4145.
Liang, Y., A. Kurakin, and B. Roizman. 2005. Herpes simplex virus 1 infected cell protein 0 forms a complex with CIN85 and Cbl and mediates the degradation of EGF receptor from cell surfaces. Proc. Natl. Acad. Sci. USA 102:5838-5843.
Lowell, C. A., and P. Soriano. 1996. Knockouts of src-family kinases: stiff bones, wimpy T-cells and bad memories. Genes Dev. 10:1845-1857.
Macdonald, A., K. Crowder, A. Street, C. McCormick, and M. Harris. 2004. The hepatitis C virus NS5A protein binds to members of the Src family of tyrosine kinases and regulates kinase activity. J. Gen. Virol. 85:721-729.
Martinez, R., B. Mathey-Prevot, A. Bernards, and D. Baltimore. 1987. Neuronal pp60c-src contains a six-amino acid insertion relative to its non-neuronal counterpart. Science 237:411-415.
Mayer, B. J. 2001. SH3 domains: complexity in moderation. J. Cell Sci. 114:1253-1263.
Melroe, G. T., N. A. DeLuca, and D. M. Knipe. 2004. Herpes simplex virus 1 has multiple mechanisms for blocking virus-induced interferon production. J. Virol. 78:8411-8420.
Miller, C. L., A. L. Burkhardt, J. H. Lee, B. Stealey, R. Longnecker, J. B. Bolen, and E. Kieff. 1995. Integral membrane-protein 2 of Epstein-Barr virus regulates reactivation from latency through dominant-negative effects on protein-tyrosine kinases. Immunity 2:155-166.
Poon, A. P., and B. Roizman. 1997. Differentiation of the shutoff of protein synthesis by virion host shutoff and mutant gamma (1)34.5 genes of herpes simplex virus 1. Virology 229:98-105.
Post, L. E., and B. Roizman. 1981. A generalized technique for deletion of specific genes in large genomes: gene 22 of herpes simplex virus is not essential for growth. Cell 25:227-232.
Purves, F. C., R. M. Longnecker, D. P. Leader, and B. Roizman. 1987. Herpes simplex virus 1 protein kinase is encoded by open reading frame US3, which is not essential for growth in cell culture. J. Virol. 61:2896-2901.
Purves, F. C., W. O. Ogle, and B. Roizman. 1993. Processing of the herpes simplex virus regulatory protein 22 mediated by the UL13 protein kinase determines the accumulation of a subset of mRNAs and proteins in infected cells. Proc. Natl. Acad. Sci. USA 90:6701-6705.
Roskoski, R., Jr. 2004. Src protein-tyrosine kinase structure and regulation. Biochem. Biophys. Res. Commun. 324:1155-1164.
Saksela, K., G. Cheng, and D. Baltimore. 1995. Proline-rich (PxxP) motifs in HIV-1 Nef bind to SH3 domains of a subset of Src kinases and are required for the enhanced growth of Nef+ viruses but not for down-regulation of CD4. EMBO J. 14:484-491.
Sicheri, F., and J. Kuriyan. 1997. Structures of Src-family tyrosine kinases. Curr. Opin. Struct. Biol. 7:777-778.
Thomas, S. M., and J. S. Brugge. 1997. Cellular functions regulated by src family kinases. Annu. Rev. Cell Dev. Biol. 13:513-609.(Yu Liang and Bernard Roiz)