Redundant Roles for Nucleocapsid and Matrix RNA-Bi
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病菌学杂志 2005年第22期
Basic Research Program, SAIC-Frederick, Inc., National Cancer Institute at Frederick, Frederick, Maryland 21702-1201
ABSTRACT
RNA appears to be required for the assembly of retroviruses. This is likely due to binding of RNA by multiple Gags, which in turn organizes and stabilizes the Gag-Gag interactions that form the virion. While the nucleocapsid (NC) domain is the most conspicuous RNA-binding region of the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein, we have previously shown that NC is not strictly required for efficient particle production. To determine if an RNA requirement for HIV-1 assembly exists, we analyzed virions produced by an NC deletion mutant for the presence of RNA. The results revealed that virions without NC still contained significant amounts of RNA. Since these packaged RNAs are probably incorporated by other RNA-binding sequences in Gag, an RNA-binding site in the matrix protein (MA) of Gag was mutated. While this mutation did not interfere with HIV-1 replication, a construct with both MA and NC mutations (MX/NX) failed to produce particles. The MX/NX mutant was rescued in trans by coassembly with several forms of Gag: wild-type Gag, either of the single-mutant Gags, or Gag truncations that contain MA or NC sequences. Addition of basic sequences to the MX/NX mutant partially restored particle production, consistent with a requirement for Gag-RNA binding in addition to Gag-Gag interactions. Together, these results support an RNA-binding requirement for Gag assembly, which relies on binding of RNA by MA or NC sequences to condense, organize, and stabilize the HIV-1 Gag-Gag interactions that form the virion.
INTRODUCTION
Retrovirus assembly is a concerted process by which the virion components coalesce to produce a particle that is released from the cell. Only three proteins are strictly required toproduce an infectious retrovirus (reviewed in references 15and 18): Gag, the polyprotein that is the primary structural protein of the virion; Gag-Pol, the polyprotein created by a –1 frameshift in Gag that provides the protease, reverse transcriptase, and integrase enzymes required for replication; and Env, the protein complex on the viral surface that binds cellular receptors and allows the virus to enter cells.
Of these three proteins, Gag is the only viral protein strictly required for particle formation. However, cellular components also play critical roles in particle formation. The Gag polyprotein consists of the mature internal structural proteins, minimally matrix (MA), capsid (CA), and nucleocapsid (NC), that are produced from this precursor by viral protease processing (60). (The organization of human immunodeficiency virus type 1 [HIV-1] Gag and its mature cleavage products are presented in the NL4-3 cartoon in Fig. 1.) For HIV-1, Gag processing begins during assembly and concludes some time after the virion is released from the cell (30, 31). Premature cleavage of Gag can abolish assembly (4, 7, 33, 39). Thus, the Gag polyprotein, not the individual mature proteins themselves, is required for assembly and budding.
One of the key questions in assembly is what provides the organizing force for Gag in the formation of particles. While CA-CA interactions between Gag molecules are a vital force in Gag assembly (27, 61), other multimerization forces also are required for assembly (1, 28, 63). In vitro, purified Gag is unable to form spherical particles without RNA (9-11, 19), demonstrating that Gag-RNA binding is required for assembly in cell-free systems. Also, RNA has been found to be a structural component of murine leukemia virus (MuLV) particles, supporting a role for RNA in assembly (45). Based on these results and those from other groups, RNA has been proposed to be required for retrovirus assembly. RNA bound by Gag could function either as a structural part of the nascent immature virion, by providing a scaffold upon which multiple Gag proteins assemble, or as a bridge between two Gag molecules that allows them to form dimers which then form the particle (8, 9, 11, 12, 14, 24, 29, 40, 41, 45, 46, 57). In either case, the interactions between RNA and Gag promote and stabilize the Gag-to-Gag protein interactions necessary for particle formation. While genomic RNA is not required for assembly (5), other cellular species, including nonviral mRNAs and small RNAs (e.g., tRNA), may also interact with RNA-binding sequences in Gag and provide the RNA assembly and structural function (16, 36, 37, 43, 45, 59).
NC is the most prominent and extensively studied RNA-binding domain within Gag (reviewed in references 5, 55, and60). While the zinc fingers of NC are necessary for specific incorporation of genomic RNA (21, 22), the 15 basic amino acids present throughout the NC region nonspecifically bind RNA (52), which can result in the incorporation of cellular RNAs into the virion (3). Thus, the NC domain is the leading candidate in Gag for providing the RNA-mediated multimerization interactions required for assembly (14, 34, 40, 41, 44). Additionally, NC-RNA interactions may also be required for HIV-1 particle stability (62). Despite the importance of RNA in assembly, the HIV-1 NC domain can be replaced by protein domains that multimerize (1, 28, 63). Furthermore, the NC regions of HIV-1 Gag can be intermolecularly cross-linked, while those in CA cannot, suggesting that the NC regions inthe assembling Gag proteins interact with each other in theimmature particle (42). Thus, dimerization mediated by NC-NC protein interactions has been proposed to be an initiating force in Gag assembly (28, 40, 41, 63).
Recently, we showed that HIV-1 could form particles efficiently when all but seven amino acids of NC were deleted from Gag in the absence of protease activity (48). This construct packaged essentially no viral RNA, experimentally a 104-fold decrease in the RNA level compared to that in the wild type. While these data imply that the NC domain within Gag and the RNA (both viral and cellular) that it incorporates do not necessarily contribute to assembly, other, nonviral RNA molecules could bind another region of Gag to provide the RNA-mediated function needed to produce the NC-deficient virions. To examine the requirement for RNA in assembly, we assayed the RNA present in a protease-deficient NC deletion mutant. We found that RNA was incorporated into these NC-deficient particles. Additional mutagenesis revealed that the presence of either NC or an RNA-binding site in MA is required for assembly, consistent with a requisite role for RNA binding by HIV-1 Gag in assembly.
MATERIALS AND METHODS
DNA mutagenesis. The pNL4-3 infectious molecular clone of HIV-1 (2) (GenBank accession no. AF324493) was used for these studies and altered by site-directed mutagenesis using PCR-based methods, either by direct amplification with a mutagenic primer or by two rounds of amplification using the overlap extension procedure (26). NL4-3 mutants constructed and used (diagramed in Fig. 1) were as follows: MX, the pNL4-3 clone with nucleotides (nt) 865 to 866 and 868 to 869 changed from AA to GC, resulting in lysine-to-alanine substitutions at MA positions 26 and 27, and nt 877 changed from A to G, resulting in a change of position 30 from lysine to glutamic acid; NX, formerly named DelNC (48), the pNL4-3 clone with all but seven amino acids of NC (IQKG-QAN) deleted; NX/PX, formerly named DelNC/PRR57G (48), the NX construct with a protease-inactivating R57G mutation (49); MX/NX, the NX construct combined with the MX construct (48); MX/NX/PX, the MX/NX construct with the R57G mutation; LNX, the pNL4-3 construct with the Gag initiator methionine altered by a nonsense mutation (51); MA-CA, the NX construct with a tryptophan-to-nonsense mutation in SP2 at codon 6 (nt 2102 G to A), which produces a truncated Gag ending in IQKGQANFLGKI after SP1; MA-p6, the pNL4-3 clone with the N terminus of MA (amino acids 1 to 36) fused to the C-terminal 4 amino acids of NC with a serine at the junction (IVW-S-RQAN), which produces a truncated MA-SP1-p6Gag product; MKK, the MX/NX/PX construct with a KKKYK insertion at the NC deletion (IQKG-KKKYK-QAN); MRR, the MX/NX/PX construct with an arginine-rich sequence insertion at the NC deletion (IQKG-AARNRRRRPRSGSR-QAN); Mp10, the MX/NX construct with an insertion of MuLV NC (58) in the NC deletion region (IQKG-MuLV NC-QAN); MZip, the MX construct with a complete replacement of NC by a modified GCN4 sequence (25) that forms tetramers (TPPGSSRLKQIEDKL LEILSKLYHEKELARIKKSSGER); the PX protease mutant, formerly named PRR57G, described previously (49); and the Px protease mutant, formerly named PRD25A, described previously (50). After construction, the regions of DNA that were PCR amplified were sequenced to confirm the mutation and to rule out the possibility of any additional changes being introduced during the mutagenesis process. The pLP-VSVg plasmid, which produces the G surface glycoprotein from vesicular stomatitis virus, was obtained from Invitrogen Corporation. (Carlsbad, CA)
Cell culture. The 293T human embryonic kidney and HeLa-CD4-LTR-lacZ (HCLZ) cell lines were cultured in Dulbecco's modified Eagle's medium supplemented with 10% (vol/vol) fetal bovine serum, 2 mM L-glutamine, 100 U per mlpenicillin, and 100 μg per ml streptomycin. All cell culture products were obtained from Invitrogen. Transient transfections of 293T cells were carried out using calcium phosphate transfection (23), except for the RNA analysis samples described below. For the 1-to-1 cotransfection experiments, single DNA construct controls were combined with an equal amount of sheared salmon sperm DNA (sssDNA) to maintain equal DNA input with the matching two construct cotransfections. The HIV-1 infection assays using HCLZ cells as a lacZ-Tat transcomplementation reporter were carried out as previously described (22). The cells were infected with dilutions of virus, and the assay was developed for ?-galactosidase activity by staining with 5-bromo-4-chloro-3-indolyl-?-galactoside 48 h postinfection. Infected cells (those staining blue) were observed by light microscopy and counted to score infection events.
RNA analysis. Virions were prepared from culture supernatants of 293T cells (3-T150 flasks) transfected with either pHLA-II? (an HLA class II beta chain expression plasmid used as a negative control), PX, or NX/PX by use of TransIT 293T reagent (Mirus Corporation, Madison, WI). Virions were isolated by centrifugation through a 20% (wt/vol) sucrose pad in an SW28 Ti rotor (Beckman Coulter, Fullerton, CA) at 112,000 x g and 4°C for 1 h. The preparations (300μl each in phosphate-buffered saline without Mg2+ and Ca2+) were then digested with 20 U RQ1 RNase-free DNase (Promega, Madison, WI) and RNase cocktail, consisting of 7.5 U RNase A and 1,333 U RNase T1 (Ambion, Austin, TX), for 2 h at 37°C in the RQ1 buffer. Next, the preparations were digested with 1 mg/ml (wt/vol) subtilisin (Roche Applied Science [formerly Boehringer-Mannheim], Indianapolis, IN) in 40 mM Tris HCl (pH 7)-2 mM CaCl2 for 3 h at 37°C. Then the virions were reisolated by centrifugation in an SW60 Ti rotor at 308,000x g for 1 h at 4°C. Virion pellets were resuspended in 50 mM Tris HCl (pH 7.4), 100 mM NaCl, 10 mM disodium EDTA, 10% (wt/vol) sodium dodecyl sulfate (SDS), 2 μg per μl linear acrylamide (Ambion), and 100 μg per ml proteinase K (Roche Applied Science) and incubated at 37°C for 2 h. The virion lysates were then extracted twice with phenol-chloroform-isoamyl alcohol (Invitrogen) and ethanol precipitated.
Total virion RNA was quantitated with the RiboGreen RNA kit (Molecular Probes, Eugene, OR) according to the manufacturer's instructions, using a FluoroMax-2 fluorimeter (Instruments SA, Edison, NJ) with settings of 482-nm excitation, 526-nm emission, and 5 trials. Measurements of total RNA were made and repeated after an additional RQ1 DNA digestion. The two sets of data were essentially equivalent. RNA quantitation for the virions was expressed as the signal minus that from the negative control (an equal volume of the negative control, the HLA-II ? expression plasmid). Samples of virions analyzed for RNA were normalized for equal amounts of material based on immunoblotting as described below.
Protein analysis. Virions were isolated as described in the preceding section. Immunoblot analysis was performed as previously described (47). Primary goat antisera against p24CA (number 81) and p17MA (number 83) were obtained from the AIDS Vaccine Program, NCI-Frederick, Frederick, MD. Proteins were detected by developing blots with a horseradish peroxidase (HRP)-conjugated anti-goat secondary antibody (Biochain Institute, Hayward, CA) and the Immun-star HRP substrate kit (Bio-Rad, Hercules, CA) on LumiFilm (Roche Applied Science, Indianapolis, IN).
Metabolic labeling of transfected cells with [35S]Met and [35S]Cys was carried out as previously described (56). Briefly, cells were first placed in Met– Cys– medium for 1 h and then transferred to a medium containing a mixture of 150 μCiof [35S]Met and 60 μCi of [35S]Cys (Promix; Amersham Biosciences, Piscataway, NJ). Virions were isolated by sucrose density centrifugation as described above. Gag was immunoprecipitated from viral and cellular lysates with goat antibodies to p24CA and EZview red protein G beads (Sigma). Precipitates were separated on 10% Tris-glycine SDS-polyacrylamide gel electrophoresis (PAGE) gels (Invitrogen) and analyzed with a PMI phosphorimager using Quantity One software (both from Bio-Rad Laboratories). Signals measured were adjusted for the relative amounts of Met and Cys amino acids in the observed Gag products.
RESULTS
The DelNC/PRR57G proviral construct (renamed NX/PX for this report) contains both an NC deletion and a protease mutation yet produces virions efficiently (48). To determine if these virions contain RNA, we isolated mutant NX/PX virions and those with only the protease mutation, PRR57G (NL4-3 with a protease-inactivating mutation; renamed PX for this report), for RNA quantitation. Virions were isolated from transfected 293T cell supernatants by density centrifugation. Since any carryover DNA and cellular RNA binding to the surface of the virion would contaminate a virion RNA preparation, the virus preparations were first treated with DNase and RNase. Still, microvesicles which contain RNA and DNA could also contaminate these virion preparations (6, 20). Therefore, the samples were then treated with subtilisin and reisolated by density centrifugation (50). RNA was extracted from the preparations and quantitated by Ribogreen assay, a high-sensitivity, fluorescence-based RNA detection method. The results showed that the amount of RNA in the NX/PX virions was approximately 50% of the amounts detected in the PX preparations (Fig. 2). These data show that HIV-1 virions can incorporate significant amounts of RNA even without NC, the most conspicuous nucleic acid-binding domain in Gag.
An MA RNA-binding site mutant assembles normally. Since NX/PX contains RNA, we considered other sequences in Gag that could bind RNA and potentially provide an RNA-mediated function in Gag assembly. Previously, we found that Pol proteins were not required for efficient DelNC assembly and budding (48). Also, Gag-Pol incorporation does not appear to be mediated by RNA (32). Therefore, the Pol proteins are not likely candidates for RNA incorporation and assembly. In addition to the highly basic NC sequence, a basic region of MAconsisting of lysines at positions 26, 27, and 30 in the N terminus of NL4-3 MA can also bind RNA (8, 13, 38, 53). Since the RNA present in NX/PX virions could be incorporated by interactions with this RNA-binding region, we mutated the basic stretch of amino acids in MA, K26K27QYK30, to AAQYE to produce the MX construct. This region is important for RNA-mediated incorporation of the tRNA-binding protein EF1- into virions; apparently, EF1- binds indirectly to Gag by means of an RNA bridge (13). p17MA immunoblotting of virion preparations isolated from equal amounts of 293T transfection supernatants demonstrated that the MX mutant generally produced near-wild-type levels of virions (Fig.3). The immunoblot data also revealed that the processing of this mutant Gag was normal, comparable to that of the wild type (Fig.3). To confirm the immunoblot data, the production of virions was examined by metabolically labeling Gag in transfected cell cultures with [35S]Met-Cys for 6 h, followed by immunoprecipitation of Gag from lysates of virion and cytoplasmic preparations with an anti-p24CA serum. Like the immunoblot results, these data showed that the MX mutant produced virions in amounts similar to those of the wild-type construct (Fig.4A). To normalize for potential differences in protein expression, we used phosphorimager analysis to quantify the total amounts of labeled Gag precipitated from the virion preparations and the cell lysates in order to calculate the virus release factor, i.e., the amount of virions released in 6 h (Gag pelletable by density centrifugation) divided by total Gag (pelletable Gag and Gag in cell lysates). Quantitation of phosphorimages revealed that the release factor for MX was 0.29, compared to 0.33 for the wild type (Fig. 4B). Thus, the MX mutant produces particles efficiently. Infectivity tests showed that MX particles were approximately 300-fold less infectious than wild-type particles, with typical titers of 2 x 103 versus 6 x 105, respectively. However, this defect was rescued by coexpression with the Gsurface glycoprotein from vesicular stomatitis virus, producing titers of approximately 5 x 106. Thus, the lower infectivity of the MX mutant is due to a block at entry and not to an overt defect in genomic packaging. Other MA basic-region mutants produced in the HXB2 proviral clone have yielded similar results (13, 35).
The MX/NX mutant fails to produce virions. To determine if both the NC and MA RNA-binding regions are important for assembly, we combined the MX and NX mutations to produce the MX/NX proviral construct (Fig. 1). Since we previously found that the budding defect from the NX mutation was rescued by protease inactivation (48), we produced a protease-deficient version of this construct, MX/NX/PX. Both the MX/NX and MX/NX/PX constructs failed to produce detectable levels of virions as assayed by immunoblot analysis of virion preparations produced from 293T transfections (Fig. 3). [35S]Met-Cys radioimmunoprecipitation of Gag from MX/NX or MX/NX/PX virion preparations and transfected 293T cell lysates with a p24CA antiserum detected only a faint signal in the virion preparations yet a readily detectable signal in the cell lysates (Fig. 4A). p24CA immunoblot analysis of a 48-h harvest (the supernatant from the cells just before radiolabeling) produced results similar to those obtained from the 6-h radiolabeling (Fig. 4B). Phosphorimager quantitation of the radioimmunoprecipitates revealed that the MX/NX mutant produced only 5% of the wild-type levels of p24CA and Gag in the virion fractions. The release factor for MX/NX was 0.02, compared to 0.33 for the wild type, i.e., MX/NX virion production was 4% of the wild-type level when normalized for precursor expression in the cells. The MX/NX/PX construct gave equivalent results (Fig. 4B). Electron microscopy of MX/NX-transfected cells failed to visualize any virus-like assembly structures (data not shown), consistent with a failure of Gag to begin even the earliest observable assembly steps. Together, these results confirm that the MX/NX mutant fails to efficiently assemble and release from cells.
Complementation of MX/NX with Gag. If the defect in assembly is simply the loss of a function in MX/NX Gag (e.g., a reflection of an RNA assembly requirement) and not a dominant-negative Gag-Gag interaction, then this mutant should be rescued by coexpression with Gag. To examine this possibility, 293T cells were cotransfected with either the MX/NX or the MX/NX/PX mutant and a protease active-site mutant, PRD25A (renamed Px for this report), at a 1-to-1 ratio. Although this protease mutant is different from the K57R PX construct, these two protease mutants have similar properties. p17MA immunoblotting of virion preparations (with equal volumes of material) revealed the presence of both Pr55Gag from the protease mutant and Pr48Gag from the MX/NX/ PX construct (Fig. 5). In contrast, the MX/NX sample contained littledetectable Gag protein. The MX/NX-plus-Px cotransfection sample contained processed p17MA as well as both Pr55Gag and Pr48Gag. Since only the MX/NX provirus expresses an active protease, the processing of Gag in these particles is the result of incorporation of MX/NX Gag-Pol into the particles. These results demonstrate that the MX/NX defect is readily complemented by wild-type Gag. Thus, the defect in MX/NX is a loss-of-function mutation, consistent with a loss of RNA binding that can be restored in trans.
MX or NX coexpression can rescue MX/NX Gag. Since Gag that contains both RNA-binding regions can rescue MX/NX budding, the abilities of the single mutants, MX and NX, to complement the MX/NX double mutant were tested in the 1-to-1 cotransfection assay. To avoid any potential processing-related assembly defects previously observed with NX (48), we used protease-deficient versions of all the mutants. Immunoblot analysis of virion preparations from MX/NX/PX cotransfected with MX/PX showed that the MX/NX/PX mutant was rescued by this MX mutant: virion preparations from the cotransfection supernatants contained both Pr55Gag from MX/PX and Pr48Gag from MX/NX/PX (Fig. 6A). Samples of the preparations from the MX/NX/PX-and-NX/PX cotransfection produced a more intense Pr48Gag signal than those from the NX/PX-plus-sssDNA cotransfection (Fig. 6B). Since both the MX/NX/PX and NX/PX constructs produce Pr48Gag, we designed an experiment in such a way that the ability of NX/PX to complement MX/NX was evidenced by the processing ofvirions. The MX/NX-plus-NX/PX cotransfection produced virions that were processed (Fig. 6C). Since active protease can come only from the MX/NX mutant, processing confirms that the NC deletion mutant NX can complement the MX/NX mutation to a similar extent as wild-type Gag in this system (Fig. 6C, lane MX/NX + PX). Together, these results demonstrate that either the basic MA sequence from amino acid 27 to 30 or NC sequences within Gag can provide the assembly function required for HIV-1 virion formation.
Complementation requires Gag interaction. The abilities of two truncated Gag proteins (LNX and MA-CA) to complement MX/NX assembly were also tested. While these shortened Gag proteins cannot produce virions themselves, they can be rescued by wild-type Gag (51) (unpublished results). Thus, the formation of any particles by cotransfection with MX/NX is due to the complementation of the MX/NX defect. One mutant, the LNX proviral clone, contains a mutation that eliminates the normal Gag translation initiator methionine and produces a truncated Gag, which initiates at Met10 in CA to express a Pr40Gag polyprotein, CA-SP1-NC-SP2-p6Gag (51). Since LNX Gag does not contain MA, p17MA immunoblotting will detect only MX/NX in the samples from LNX complementation experiments. Cotransfection (at a 1-to-1 ratio) of LNX and MX/NX revealed that this truncated Gag could rescue the MX/NX assembly defect: a p17MA immunoblot assay detected both unprocessed and processed particles in cotransfected samples (Fig. 7A, lane 3). In comparison, samples from cotransfection of MX/NX and PX produced more p48Gag signal than the LNX-plus-MX/NX samples (Fig. 7A; compare lane 4 with lane 3). Thus, the rescue of the double-mutant Gag by LNX Gag was less efficient than that by full-length Gag. Since LNX Gag typically is incorporated into wild-type particles at about 25% the level of full-length Gag (51), the decreased ability of this truncated Gag to rescue MX/NX Gag could be due to the limited copackaging of LNX. Nevertheless, LNX complemented MX/NX assembly.
To confirm that MA sequences can complement the MX/NX defect, another Gag truncation construct, MA-CA, was tested. This construct produces an MA-CA-SP1 Gag product with seven amino acids from DelNC and five from SP2 (IQKGQANFLGKI) at its C terminus. Cotransfection of MA-CA with MX/NX/PX produced particles containing both Gag proteins, Pr40Gag and Pr48Gag (Fig. 7A, lane 7), although the levels were reduced from those of the MX/NX/PX-plus-PX cotransfection (Fig. 7A, lane 8). Thus, like LNX Gag, MA-CA does not rescue MX/NX with the same efficiency as wild-type Gag. Since both these truncated Gag proteins cannot produce particles themselves, their assembly defect is also complemented by MX/NX Gag, which provides a potential explanation for the low rescue efficiency. Together, these data confirm that the presence of either the MA or the NC region of Gag can provide an assembly function, likely RNA binding, to MX/NX in trans.
If the binding of Gag to RNA organizes and stabilizes Gag-Gag interactions, then Gag truncations that fail to coassemble with Gag should not rescue MX/NX. The experiments described above rescued MX/NX budding with portions of Gag that contain CA, a primary determinant in close-contact Gag-Gag interactions (27). The MA-p6 construct, a proviral clone that expresses only the first 36 amino acids of MA (which include the RNA-binding region) linked to SP2-p6Gag (i.e., deletion of two-thirds of MA and of the entire CA and NC regions), produces a short protein that failed to form particles or coassemble with full-length Gag (data not shown). Immunoblotting of samples produced from cotransfection of MX/NX and MA-p6 (at a 1-to-1 ratio) revealed that this highly truncated form of Gag was unable to rescue the MX/NX mutant (Fig.7A, lane 5). Thus, the ability to maintain Gag-Gag interaction is critical for rescue, as expected.
Rescue of MX/NX by RNA-binding sequences. The one common attribute of the regions of MA and NC that were mutated in this study is the ability to bind RNA. To determine if RNA binding could restore particle production in the Gag double mutant, we inserted potential RNA-binding sequences into the NC deletion region of the MX/NX/PX mutant. In one construct, MKK, a KKKYK sequence was inserted. Previously, addition of this sequence to Rous sarcoma virus (RSV) Gag allowed it to bind EF1- in vitro (13), presumably due to binding of RNA by this basic region. In another construct, MRR, a series of basic amino acids (AARNRRRRPRSGSR) loosely based on the RNA-binding, arginine-rich motif from bromoviruses (54) was inserted into the NC deletion region of MX/NX/PX. Immunoblotting of virion preparations revealed that both the MKK and MRR constructs produced particles (Fig. 7B), though at an apparently lower level than the wild type. Acomparison of release factors calculated from the results ofp24CA radioimmunoprecipitation found that, while both MKK and MRR produced particles more efficiently than theMX/NX/PX mutant (two- to fourfold higher, respectively [Fig. 7C]), their particle production was three- to sixfold lower than that of the PX construct expressing wild- type Gag.
To determine if a defined RNA-binding sequence could rescue the MX/NX mutant, we inserted the NC protein of MuLV (p10NC) into the NC deletion of the MX/NX mutant to produce the Mp10 construct. As expected, the MuLV NC protein was able to complement the loss of both the MA basic region and the HIV-1 NC domain in this background (Fig. 7B). While this chimera expresses protease, processing was inefficient; immunoblotting revealed mostly polyprotein and processing intermediates, with very little processed protein. The release factor for this construct was similar to that for the MKK construct, higher than that for the MX/NX/PX mutant but certainly lower than that for wild-type Gag (Fig. 7C). Together, these sequence replacement data show that RNA-binding sequences can partially complement the MX/NX mutations in cis. Thus, addition of RNA-binding sequences to this mutant Gag restores some budding, consistent with the idea that RNA binding is an important factor in assembly.
Rescue of MX/NX by protein multimerizing sequences. Previous studies have shown that the assembly function of NC in Gag can be replaced by sequences that dimerize or multimerize (1, 28, 63). To determine if MX/NX can be rescued by protein-protein interactions, we constructed MZip, in which a modified GCN4 leucine zipper sequence was inserted into the NC deletion in MX/NX. This zipper sequence tetramerizes to form four-stranded coils (25). Virion preparations of MZip showed that this sequence was able to rescue MX/NX budding (Fig. 7B). However, as with the RNA sequences discussed above, the rescue was not as efficient as wild-type virus budding (Fig. 7B and C), though it was better than that with the RNA sequence insertion mutants (Fig. 7C). As with Mp10, the processing of Gag with the MZip mutant was very inefficient. The ability of this multimerizing sequence to rescue the MX/NX mutant confirms that the loss of particle formation is due to a deficiency in Gag-Gag interaction, entirely consistent with a role for RNA-Gag interactions in virus assembly.
DISCUSSION
Our results show that either one of two RNA-binding domains in Gag is required for assembly of HIV-1 Gag. These regions appear to provide a redundant function, since either mutation of the RNA-binding region of MA or deletion of NC alone does not dramatically reduce virion production per se. Gag assembly defects caused by the removal of these RNA-binding sites can be rescued in trans by coassembly with Gag proteins containing either both MA and NC sites (wild-type Gag) or only one of the single sites alone (i.e., either truncated Gag proteins or a single-site mutant). Rescue of viral budding in trans depended on the ability of the two Gag proteins to interact, which is consistent with a binding of Gag to RNA that promotes Gag-Gag interactions. This requirement for an RNA-binding sequence in Gag assembly could be supplied by RNA-binding sequences in cis, though less efficiently than when the GCN4 tetramerizing mutant sequences are used. The ability of the protein multimerizing sequences to promote Gag assembly in the absence of NC, seen here and in previous studies (1, 28, 63), confirms that particle formation in the absence of NC can be driven by strong protein-protein binding. However, in nature, this required interaction likely occurs by Gag-RNA-Gag binding. Taken together, our data support a requirement for RNA in HIV-1 assembly, offering solid genetic evidence for RNA-Gag binding as an integral step in virion formation.
Similar results were obtained in vitro by Burniston et al. In their study, the RNA-binding region of MA was necessary for Gag-Gag interaction when two-thirds of NC was deleted from Gag (8). Additionally, MA contributed to Gag-Gag interaction in a yeast two-hybrid screen for the minimal interacting sequences in Gag. In cells, Gag without the MA basic region and positively charged amino acids (e.g., arginines and lysines) in NC had a severe assembly defect (13). Combined with our data presented here, these results are entirely consistent with HIV-1 Gag containing two RNA-binding regions that perform a redundant role in RNA-mediated Gag assembly.
While there is essentially no HIV-1 genomic RNA inside the NX/PX particles (48), we show that there still remains a significant level of other RNAs, presumably incorporated by the basic region of MA. These RNAs could be nonviral mRNAs or small RNAs, possibly tRNAs, that are incorporated into retrovirus particles (13, 16, 36). Further characterization of the RNA species is required to determine what RNAs are incorporated in these particles. Even though small RNAs might be limited in their ability to bind multiple Gag molecules, dA15 to dA20 oligonucleotides are long enough to support HIV-1 Gag assembly in vitro (9). Also, HIV-1 NC appears to require only 8 nucleotides for nucleic acid binding, so several Gag molecules could readily bind small RNAs such as tRNA (17). Similarly, RSV Gag can use a 16-nt DNA for efficient particle formation in vitro (40, 41). Therefore, even small RNAs such as tRNA bound to Gag could provide the functional RNA requirement for HIV-1 Gag assembly (8).
In contrast to our results, MuLV and RSV Gag proteins strictly require NC for assembly (34, 44). For MuLV, this difference is likely due to the absence of the basic region in MA, analogous to the HIV-1 MA KKYQK construct that could bind RNA. Furthermore, MuLV and RSV do not incorporate EF1-, a tRNA-binding protein that appears to be incorporated by its association with RNA, which, in turn, is bound by the basic region in HIV-1 MA. Thus, it appears that these viruses do not have an RNA-binding site equivalent to the KKYQK sequence in HIV-1. Interestingly, RSV Gag can be engineered to bind EF1-, and presumably RNA, by inserting the HIV-1 HXB2 KKKYKL MA sequence (the same sequence that partially complemented the MX/NX mutation incis) into RSV MA (13). Because Pr55Gag possesses two RNA-binding regions, it does not display the same strict dependence on NC for particle production as MuLV or RSV Gag. However, since the MX mutant has lost the RNA-binding site in MA, it behaves similarly to MuLV and RSV, which are strictly dependent on the NC protein for assembly. Interestingly, only 8 positively charged residues in RSV NC are required for particle production, further supporting the idea that nonspecific RNA interactions are important for assembly in Gag (34).
This difference in assembly requirements for various Gag proteins highlights the fact that, even though they perform essentially the same function, significant differences may exist in the precise way in which each type of Gag assembles. As discussed above, some of the assembly requirements for MuLV and RSV Gag appear to be significantly different from those for HIV-1 Gag. Considering this point, findings pertaining to one particular Gag do not necessarily apply to all retroviruses. Thus, while a plethora of studies implicate NC as strictly required for retroviral particle formation in several contexts (34), it is the ability of Gag to bind RNA and provide for Gag multimerization that appears to be required for assembly, rather than the NC protein itself.
The role of NC as a driving force in virus assembly has been debated for some time. One model posits that NC-NC protein dimerization interactions assist in Gag-Gag interactions (28,40, 41, 63). The ability to delete NC and maintain particle formation suggests that direct NC interactions are not strictly required for this part of HIV-1 assembly (48). While these data also initially appeared to be at odds with a role for RNA in assembly, the results presented here reconcile our previous findings with the RNA-mediated assembly model, in that an RNA-binding region in either MA or NC is required for virion formation. Thus, Gag-RNA interactions play an essential role in Gag assembly, in addition to those contributed by Gag-Gag interactions.
ACKNOWLEDGMENTS
We thank Kunio Nagashima for electron microscopy, David Morcock for assistance with the fluorimeter measurements, and Alan Rein and Robert Gorelick for critical reading of the manuscript and helpful suggestions.
This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract NO1-CO-12400.
The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.
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ABSTRACT
RNA appears to be required for the assembly of retroviruses. This is likely due to binding of RNA by multiple Gags, which in turn organizes and stabilizes the Gag-Gag interactions that form the virion. While the nucleocapsid (NC) domain is the most conspicuous RNA-binding region of the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein, we have previously shown that NC is not strictly required for efficient particle production. To determine if an RNA requirement for HIV-1 assembly exists, we analyzed virions produced by an NC deletion mutant for the presence of RNA. The results revealed that virions without NC still contained significant amounts of RNA. Since these packaged RNAs are probably incorporated by other RNA-binding sequences in Gag, an RNA-binding site in the matrix protein (MA) of Gag was mutated. While this mutation did not interfere with HIV-1 replication, a construct with both MA and NC mutations (MX/NX) failed to produce particles. The MX/NX mutant was rescued in trans by coassembly with several forms of Gag: wild-type Gag, either of the single-mutant Gags, or Gag truncations that contain MA or NC sequences. Addition of basic sequences to the MX/NX mutant partially restored particle production, consistent with a requirement for Gag-RNA binding in addition to Gag-Gag interactions. Together, these results support an RNA-binding requirement for Gag assembly, which relies on binding of RNA by MA or NC sequences to condense, organize, and stabilize the HIV-1 Gag-Gag interactions that form the virion.
INTRODUCTION
Retrovirus assembly is a concerted process by which the virion components coalesce to produce a particle that is released from the cell. Only three proteins are strictly required toproduce an infectious retrovirus (reviewed in references 15and 18): Gag, the polyprotein that is the primary structural protein of the virion; Gag-Pol, the polyprotein created by a –1 frameshift in Gag that provides the protease, reverse transcriptase, and integrase enzymes required for replication; and Env, the protein complex on the viral surface that binds cellular receptors and allows the virus to enter cells.
Of these three proteins, Gag is the only viral protein strictly required for particle formation. However, cellular components also play critical roles in particle formation. The Gag polyprotein consists of the mature internal structural proteins, minimally matrix (MA), capsid (CA), and nucleocapsid (NC), that are produced from this precursor by viral protease processing (60). (The organization of human immunodeficiency virus type 1 [HIV-1] Gag and its mature cleavage products are presented in the NL4-3 cartoon in Fig. 1.) For HIV-1, Gag processing begins during assembly and concludes some time after the virion is released from the cell (30, 31). Premature cleavage of Gag can abolish assembly (4, 7, 33, 39). Thus, the Gag polyprotein, not the individual mature proteins themselves, is required for assembly and budding.
One of the key questions in assembly is what provides the organizing force for Gag in the formation of particles. While CA-CA interactions between Gag molecules are a vital force in Gag assembly (27, 61), other multimerization forces also are required for assembly (1, 28, 63). In vitro, purified Gag is unable to form spherical particles without RNA (9-11, 19), demonstrating that Gag-RNA binding is required for assembly in cell-free systems. Also, RNA has been found to be a structural component of murine leukemia virus (MuLV) particles, supporting a role for RNA in assembly (45). Based on these results and those from other groups, RNA has been proposed to be required for retrovirus assembly. RNA bound by Gag could function either as a structural part of the nascent immature virion, by providing a scaffold upon which multiple Gag proteins assemble, or as a bridge between two Gag molecules that allows them to form dimers which then form the particle (8, 9, 11, 12, 14, 24, 29, 40, 41, 45, 46, 57). In either case, the interactions between RNA and Gag promote and stabilize the Gag-to-Gag protein interactions necessary for particle formation. While genomic RNA is not required for assembly (5), other cellular species, including nonviral mRNAs and small RNAs (e.g., tRNA), may also interact with RNA-binding sequences in Gag and provide the RNA assembly and structural function (16, 36, 37, 43, 45, 59).
NC is the most prominent and extensively studied RNA-binding domain within Gag (reviewed in references 5, 55, and60). While the zinc fingers of NC are necessary for specific incorporation of genomic RNA (21, 22), the 15 basic amino acids present throughout the NC region nonspecifically bind RNA (52), which can result in the incorporation of cellular RNAs into the virion (3). Thus, the NC domain is the leading candidate in Gag for providing the RNA-mediated multimerization interactions required for assembly (14, 34, 40, 41, 44). Additionally, NC-RNA interactions may also be required for HIV-1 particle stability (62). Despite the importance of RNA in assembly, the HIV-1 NC domain can be replaced by protein domains that multimerize (1, 28, 63). Furthermore, the NC regions of HIV-1 Gag can be intermolecularly cross-linked, while those in CA cannot, suggesting that the NC regions inthe assembling Gag proteins interact with each other in theimmature particle (42). Thus, dimerization mediated by NC-NC protein interactions has been proposed to be an initiating force in Gag assembly (28, 40, 41, 63).
Recently, we showed that HIV-1 could form particles efficiently when all but seven amino acids of NC were deleted from Gag in the absence of protease activity (48). This construct packaged essentially no viral RNA, experimentally a 104-fold decrease in the RNA level compared to that in the wild type. While these data imply that the NC domain within Gag and the RNA (both viral and cellular) that it incorporates do not necessarily contribute to assembly, other, nonviral RNA molecules could bind another region of Gag to provide the RNA-mediated function needed to produce the NC-deficient virions. To examine the requirement for RNA in assembly, we assayed the RNA present in a protease-deficient NC deletion mutant. We found that RNA was incorporated into these NC-deficient particles. Additional mutagenesis revealed that the presence of either NC or an RNA-binding site in MA is required for assembly, consistent with a requisite role for RNA binding by HIV-1 Gag in assembly.
MATERIALS AND METHODS
DNA mutagenesis. The pNL4-3 infectious molecular clone of HIV-1 (2) (GenBank accession no. AF324493) was used for these studies and altered by site-directed mutagenesis using PCR-based methods, either by direct amplification with a mutagenic primer or by two rounds of amplification using the overlap extension procedure (26). NL4-3 mutants constructed and used (diagramed in Fig. 1) were as follows: MX, the pNL4-3 clone with nucleotides (nt) 865 to 866 and 868 to 869 changed from AA to GC, resulting in lysine-to-alanine substitutions at MA positions 26 and 27, and nt 877 changed from A to G, resulting in a change of position 30 from lysine to glutamic acid; NX, formerly named DelNC (48), the pNL4-3 clone with all but seven amino acids of NC (IQKG-QAN) deleted; NX/PX, formerly named DelNC/PRR57G (48), the NX construct with a protease-inactivating R57G mutation (49); MX/NX, the NX construct combined with the MX construct (48); MX/NX/PX, the MX/NX construct with the R57G mutation; LNX, the pNL4-3 construct with the Gag initiator methionine altered by a nonsense mutation (51); MA-CA, the NX construct with a tryptophan-to-nonsense mutation in SP2 at codon 6 (nt 2102 G to A), which produces a truncated Gag ending in IQKGQANFLGKI after SP1; MA-p6, the pNL4-3 clone with the N terminus of MA (amino acids 1 to 36) fused to the C-terminal 4 amino acids of NC with a serine at the junction (IVW-S-RQAN), which produces a truncated MA-SP1-p6Gag product; MKK, the MX/NX/PX construct with a KKKYK insertion at the NC deletion (IQKG-KKKYK-QAN); MRR, the MX/NX/PX construct with an arginine-rich sequence insertion at the NC deletion (IQKG-AARNRRRRPRSGSR-QAN); Mp10, the MX/NX construct with an insertion of MuLV NC (58) in the NC deletion region (IQKG-MuLV NC-QAN); MZip, the MX construct with a complete replacement of NC by a modified GCN4 sequence (25) that forms tetramers (TPPGSSRLKQIEDKL LEILSKLYHEKELARIKKSSGER); the PX protease mutant, formerly named PRR57G, described previously (49); and the Px protease mutant, formerly named PRD25A, described previously (50). After construction, the regions of DNA that were PCR amplified were sequenced to confirm the mutation and to rule out the possibility of any additional changes being introduced during the mutagenesis process. The pLP-VSVg plasmid, which produces the G surface glycoprotein from vesicular stomatitis virus, was obtained from Invitrogen Corporation. (Carlsbad, CA)
Cell culture. The 293T human embryonic kidney and HeLa-CD4-LTR-lacZ (HCLZ) cell lines were cultured in Dulbecco's modified Eagle's medium supplemented with 10% (vol/vol) fetal bovine serum, 2 mM L-glutamine, 100 U per mlpenicillin, and 100 μg per ml streptomycin. All cell culture products were obtained from Invitrogen. Transient transfections of 293T cells were carried out using calcium phosphate transfection (23), except for the RNA analysis samples described below. For the 1-to-1 cotransfection experiments, single DNA construct controls were combined with an equal amount of sheared salmon sperm DNA (sssDNA) to maintain equal DNA input with the matching two construct cotransfections. The HIV-1 infection assays using HCLZ cells as a lacZ-Tat transcomplementation reporter were carried out as previously described (22). The cells were infected with dilutions of virus, and the assay was developed for ?-galactosidase activity by staining with 5-bromo-4-chloro-3-indolyl-?-galactoside 48 h postinfection. Infected cells (those staining blue) were observed by light microscopy and counted to score infection events.
RNA analysis. Virions were prepared from culture supernatants of 293T cells (3-T150 flasks) transfected with either pHLA-II? (an HLA class II beta chain expression plasmid used as a negative control), PX, or NX/PX by use of TransIT 293T reagent (Mirus Corporation, Madison, WI). Virions were isolated by centrifugation through a 20% (wt/vol) sucrose pad in an SW28 Ti rotor (Beckman Coulter, Fullerton, CA) at 112,000 x g and 4°C for 1 h. The preparations (300μl each in phosphate-buffered saline without Mg2+ and Ca2+) were then digested with 20 U RQ1 RNase-free DNase (Promega, Madison, WI) and RNase cocktail, consisting of 7.5 U RNase A and 1,333 U RNase T1 (Ambion, Austin, TX), for 2 h at 37°C in the RQ1 buffer. Next, the preparations were digested with 1 mg/ml (wt/vol) subtilisin (Roche Applied Science [formerly Boehringer-Mannheim], Indianapolis, IN) in 40 mM Tris HCl (pH 7)-2 mM CaCl2 for 3 h at 37°C. Then the virions were reisolated by centrifugation in an SW60 Ti rotor at 308,000x g for 1 h at 4°C. Virion pellets were resuspended in 50 mM Tris HCl (pH 7.4), 100 mM NaCl, 10 mM disodium EDTA, 10% (wt/vol) sodium dodecyl sulfate (SDS), 2 μg per μl linear acrylamide (Ambion), and 100 μg per ml proteinase K (Roche Applied Science) and incubated at 37°C for 2 h. The virion lysates were then extracted twice with phenol-chloroform-isoamyl alcohol (Invitrogen) and ethanol precipitated.
Total virion RNA was quantitated with the RiboGreen RNA kit (Molecular Probes, Eugene, OR) according to the manufacturer's instructions, using a FluoroMax-2 fluorimeter (Instruments SA, Edison, NJ) with settings of 482-nm excitation, 526-nm emission, and 5 trials. Measurements of total RNA were made and repeated after an additional RQ1 DNA digestion. The two sets of data were essentially equivalent. RNA quantitation for the virions was expressed as the signal minus that from the negative control (an equal volume of the negative control, the HLA-II ? expression plasmid). Samples of virions analyzed for RNA were normalized for equal amounts of material based on immunoblotting as described below.
Protein analysis. Virions were isolated as described in the preceding section. Immunoblot analysis was performed as previously described (47). Primary goat antisera against p24CA (number 81) and p17MA (number 83) were obtained from the AIDS Vaccine Program, NCI-Frederick, Frederick, MD. Proteins were detected by developing blots with a horseradish peroxidase (HRP)-conjugated anti-goat secondary antibody (Biochain Institute, Hayward, CA) and the Immun-star HRP substrate kit (Bio-Rad, Hercules, CA) on LumiFilm (Roche Applied Science, Indianapolis, IN).
Metabolic labeling of transfected cells with [35S]Met and [35S]Cys was carried out as previously described (56). Briefly, cells were first placed in Met– Cys– medium for 1 h and then transferred to a medium containing a mixture of 150 μCiof [35S]Met and 60 μCi of [35S]Cys (Promix; Amersham Biosciences, Piscataway, NJ). Virions were isolated by sucrose density centrifugation as described above. Gag was immunoprecipitated from viral and cellular lysates with goat antibodies to p24CA and EZview red protein G beads (Sigma). Precipitates were separated on 10% Tris-glycine SDS-polyacrylamide gel electrophoresis (PAGE) gels (Invitrogen) and analyzed with a PMI phosphorimager using Quantity One software (both from Bio-Rad Laboratories). Signals measured were adjusted for the relative amounts of Met and Cys amino acids in the observed Gag products.
RESULTS
The DelNC/PRR57G proviral construct (renamed NX/PX for this report) contains both an NC deletion and a protease mutation yet produces virions efficiently (48). To determine if these virions contain RNA, we isolated mutant NX/PX virions and those with only the protease mutation, PRR57G (NL4-3 with a protease-inactivating mutation; renamed PX for this report), for RNA quantitation. Virions were isolated from transfected 293T cell supernatants by density centrifugation. Since any carryover DNA and cellular RNA binding to the surface of the virion would contaminate a virion RNA preparation, the virus preparations were first treated with DNase and RNase. Still, microvesicles which contain RNA and DNA could also contaminate these virion preparations (6, 20). Therefore, the samples were then treated with subtilisin and reisolated by density centrifugation (50). RNA was extracted from the preparations and quantitated by Ribogreen assay, a high-sensitivity, fluorescence-based RNA detection method. The results showed that the amount of RNA in the NX/PX virions was approximately 50% of the amounts detected in the PX preparations (Fig. 2). These data show that HIV-1 virions can incorporate significant amounts of RNA even without NC, the most conspicuous nucleic acid-binding domain in Gag.
An MA RNA-binding site mutant assembles normally. Since NX/PX contains RNA, we considered other sequences in Gag that could bind RNA and potentially provide an RNA-mediated function in Gag assembly. Previously, we found that Pol proteins were not required for efficient DelNC assembly and budding (48). Also, Gag-Pol incorporation does not appear to be mediated by RNA (32). Therefore, the Pol proteins are not likely candidates for RNA incorporation and assembly. In addition to the highly basic NC sequence, a basic region of MAconsisting of lysines at positions 26, 27, and 30 in the N terminus of NL4-3 MA can also bind RNA (8, 13, 38, 53). Since the RNA present in NX/PX virions could be incorporated by interactions with this RNA-binding region, we mutated the basic stretch of amino acids in MA, K26K27QYK30, to AAQYE to produce the MX construct. This region is important for RNA-mediated incorporation of the tRNA-binding protein EF1- into virions; apparently, EF1- binds indirectly to Gag by means of an RNA bridge (13). p17MA immunoblotting of virion preparations isolated from equal amounts of 293T transfection supernatants demonstrated that the MX mutant generally produced near-wild-type levels of virions (Fig.3). The immunoblot data also revealed that the processing of this mutant Gag was normal, comparable to that of the wild type (Fig.3). To confirm the immunoblot data, the production of virions was examined by metabolically labeling Gag in transfected cell cultures with [35S]Met-Cys for 6 h, followed by immunoprecipitation of Gag from lysates of virion and cytoplasmic preparations with an anti-p24CA serum. Like the immunoblot results, these data showed that the MX mutant produced virions in amounts similar to those of the wild-type construct (Fig.4A). To normalize for potential differences in protein expression, we used phosphorimager analysis to quantify the total amounts of labeled Gag precipitated from the virion preparations and the cell lysates in order to calculate the virus release factor, i.e., the amount of virions released in 6 h (Gag pelletable by density centrifugation) divided by total Gag (pelletable Gag and Gag in cell lysates). Quantitation of phosphorimages revealed that the release factor for MX was 0.29, compared to 0.33 for the wild type (Fig. 4B). Thus, the MX mutant produces particles efficiently. Infectivity tests showed that MX particles were approximately 300-fold less infectious than wild-type particles, with typical titers of 2 x 103 versus 6 x 105, respectively. However, this defect was rescued by coexpression with the Gsurface glycoprotein from vesicular stomatitis virus, producing titers of approximately 5 x 106. Thus, the lower infectivity of the MX mutant is due to a block at entry and not to an overt defect in genomic packaging. Other MA basic-region mutants produced in the HXB2 proviral clone have yielded similar results (13, 35).
The MX/NX mutant fails to produce virions. To determine if both the NC and MA RNA-binding regions are important for assembly, we combined the MX and NX mutations to produce the MX/NX proviral construct (Fig. 1). Since we previously found that the budding defect from the NX mutation was rescued by protease inactivation (48), we produced a protease-deficient version of this construct, MX/NX/PX. Both the MX/NX and MX/NX/PX constructs failed to produce detectable levels of virions as assayed by immunoblot analysis of virion preparations produced from 293T transfections (Fig. 3). [35S]Met-Cys radioimmunoprecipitation of Gag from MX/NX or MX/NX/PX virion preparations and transfected 293T cell lysates with a p24CA antiserum detected only a faint signal in the virion preparations yet a readily detectable signal in the cell lysates (Fig. 4A). p24CA immunoblot analysis of a 48-h harvest (the supernatant from the cells just before radiolabeling) produced results similar to those obtained from the 6-h radiolabeling (Fig. 4B). Phosphorimager quantitation of the radioimmunoprecipitates revealed that the MX/NX mutant produced only 5% of the wild-type levels of p24CA and Gag in the virion fractions. The release factor for MX/NX was 0.02, compared to 0.33 for the wild type, i.e., MX/NX virion production was 4% of the wild-type level when normalized for precursor expression in the cells. The MX/NX/PX construct gave equivalent results (Fig. 4B). Electron microscopy of MX/NX-transfected cells failed to visualize any virus-like assembly structures (data not shown), consistent with a failure of Gag to begin even the earliest observable assembly steps. Together, these results confirm that the MX/NX mutant fails to efficiently assemble and release from cells.
Complementation of MX/NX with Gag. If the defect in assembly is simply the loss of a function in MX/NX Gag (e.g., a reflection of an RNA assembly requirement) and not a dominant-negative Gag-Gag interaction, then this mutant should be rescued by coexpression with Gag. To examine this possibility, 293T cells were cotransfected with either the MX/NX or the MX/NX/PX mutant and a protease active-site mutant, PRD25A (renamed Px for this report), at a 1-to-1 ratio. Although this protease mutant is different from the K57R PX construct, these two protease mutants have similar properties. p17MA immunoblotting of virion preparations (with equal volumes of material) revealed the presence of both Pr55Gag from the protease mutant and Pr48Gag from the MX/NX/ PX construct (Fig. 5). In contrast, the MX/NX sample contained littledetectable Gag protein. The MX/NX-plus-Px cotransfection sample contained processed p17MA as well as both Pr55Gag and Pr48Gag. Since only the MX/NX provirus expresses an active protease, the processing of Gag in these particles is the result of incorporation of MX/NX Gag-Pol into the particles. These results demonstrate that the MX/NX defect is readily complemented by wild-type Gag. Thus, the defect in MX/NX is a loss-of-function mutation, consistent with a loss of RNA binding that can be restored in trans.
MX or NX coexpression can rescue MX/NX Gag. Since Gag that contains both RNA-binding regions can rescue MX/NX budding, the abilities of the single mutants, MX and NX, to complement the MX/NX double mutant were tested in the 1-to-1 cotransfection assay. To avoid any potential processing-related assembly defects previously observed with NX (48), we used protease-deficient versions of all the mutants. Immunoblot analysis of virion preparations from MX/NX/PX cotransfected with MX/PX showed that the MX/NX/PX mutant was rescued by this MX mutant: virion preparations from the cotransfection supernatants contained both Pr55Gag from MX/PX and Pr48Gag from MX/NX/PX (Fig. 6A). Samples of the preparations from the MX/NX/PX-and-NX/PX cotransfection produced a more intense Pr48Gag signal than those from the NX/PX-plus-sssDNA cotransfection (Fig. 6B). Since both the MX/NX/PX and NX/PX constructs produce Pr48Gag, we designed an experiment in such a way that the ability of NX/PX to complement MX/NX was evidenced by the processing ofvirions. The MX/NX-plus-NX/PX cotransfection produced virions that were processed (Fig. 6C). Since active protease can come only from the MX/NX mutant, processing confirms that the NC deletion mutant NX can complement the MX/NX mutation to a similar extent as wild-type Gag in this system (Fig. 6C, lane MX/NX + PX). Together, these results demonstrate that either the basic MA sequence from amino acid 27 to 30 or NC sequences within Gag can provide the assembly function required for HIV-1 virion formation.
Complementation requires Gag interaction. The abilities of two truncated Gag proteins (LNX and MA-CA) to complement MX/NX assembly were also tested. While these shortened Gag proteins cannot produce virions themselves, they can be rescued by wild-type Gag (51) (unpublished results). Thus, the formation of any particles by cotransfection with MX/NX is due to the complementation of the MX/NX defect. One mutant, the LNX proviral clone, contains a mutation that eliminates the normal Gag translation initiator methionine and produces a truncated Gag, which initiates at Met10 in CA to express a Pr40Gag polyprotein, CA-SP1-NC-SP2-p6Gag (51). Since LNX Gag does not contain MA, p17MA immunoblotting will detect only MX/NX in the samples from LNX complementation experiments. Cotransfection (at a 1-to-1 ratio) of LNX and MX/NX revealed that this truncated Gag could rescue the MX/NX assembly defect: a p17MA immunoblot assay detected both unprocessed and processed particles in cotransfected samples (Fig. 7A, lane 3). In comparison, samples from cotransfection of MX/NX and PX produced more p48Gag signal than the LNX-plus-MX/NX samples (Fig. 7A; compare lane 4 with lane 3). Thus, the rescue of the double-mutant Gag by LNX Gag was less efficient than that by full-length Gag. Since LNX Gag typically is incorporated into wild-type particles at about 25% the level of full-length Gag (51), the decreased ability of this truncated Gag to rescue MX/NX Gag could be due to the limited copackaging of LNX. Nevertheless, LNX complemented MX/NX assembly.
To confirm that MA sequences can complement the MX/NX defect, another Gag truncation construct, MA-CA, was tested. This construct produces an MA-CA-SP1 Gag product with seven amino acids from DelNC and five from SP2 (IQKGQANFLGKI) at its C terminus. Cotransfection of MA-CA with MX/NX/PX produced particles containing both Gag proteins, Pr40Gag and Pr48Gag (Fig. 7A, lane 7), although the levels were reduced from those of the MX/NX/PX-plus-PX cotransfection (Fig. 7A, lane 8). Thus, like LNX Gag, MA-CA does not rescue MX/NX with the same efficiency as wild-type Gag. Since both these truncated Gag proteins cannot produce particles themselves, their assembly defect is also complemented by MX/NX Gag, which provides a potential explanation for the low rescue efficiency. Together, these data confirm that the presence of either the MA or the NC region of Gag can provide an assembly function, likely RNA binding, to MX/NX in trans.
If the binding of Gag to RNA organizes and stabilizes Gag-Gag interactions, then Gag truncations that fail to coassemble with Gag should not rescue MX/NX. The experiments described above rescued MX/NX budding with portions of Gag that contain CA, a primary determinant in close-contact Gag-Gag interactions (27). The MA-p6 construct, a proviral clone that expresses only the first 36 amino acids of MA (which include the RNA-binding region) linked to SP2-p6Gag (i.e., deletion of two-thirds of MA and of the entire CA and NC regions), produces a short protein that failed to form particles or coassemble with full-length Gag (data not shown). Immunoblotting of samples produced from cotransfection of MX/NX and MA-p6 (at a 1-to-1 ratio) revealed that this highly truncated form of Gag was unable to rescue the MX/NX mutant (Fig.7A, lane 5). Thus, the ability to maintain Gag-Gag interaction is critical for rescue, as expected.
Rescue of MX/NX by RNA-binding sequences. The one common attribute of the regions of MA and NC that were mutated in this study is the ability to bind RNA. To determine if RNA binding could restore particle production in the Gag double mutant, we inserted potential RNA-binding sequences into the NC deletion region of the MX/NX/PX mutant. In one construct, MKK, a KKKYK sequence was inserted. Previously, addition of this sequence to Rous sarcoma virus (RSV) Gag allowed it to bind EF1- in vitro (13), presumably due to binding of RNA by this basic region. In another construct, MRR, a series of basic amino acids (AARNRRRRPRSGSR) loosely based on the RNA-binding, arginine-rich motif from bromoviruses (54) was inserted into the NC deletion region of MX/NX/PX. Immunoblotting of virion preparations revealed that both the MKK and MRR constructs produced particles (Fig. 7B), though at an apparently lower level than the wild type. Acomparison of release factors calculated from the results ofp24CA radioimmunoprecipitation found that, while both MKK and MRR produced particles more efficiently than theMX/NX/PX mutant (two- to fourfold higher, respectively [Fig. 7C]), their particle production was three- to sixfold lower than that of the PX construct expressing wild- type Gag.
To determine if a defined RNA-binding sequence could rescue the MX/NX mutant, we inserted the NC protein of MuLV (p10NC) into the NC deletion of the MX/NX mutant to produce the Mp10 construct. As expected, the MuLV NC protein was able to complement the loss of both the MA basic region and the HIV-1 NC domain in this background (Fig. 7B). While this chimera expresses protease, processing was inefficient; immunoblotting revealed mostly polyprotein and processing intermediates, with very little processed protein. The release factor for this construct was similar to that for the MKK construct, higher than that for the MX/NX/PX mutant but certainly lower than that for wild-type Gag (Fig. 7C). Together, these sequence replacement data show that RNA-binding sequences can partially complement the MX/NX mutations in cis. Thus, addition of RNA-binding sequences to this mutant Gag restores some budding, consistent with the idea that RNA binding is an important factor in assembly.
Rescue of MX/NX by protein multimerizing sequences. Previous studies have shown that the assembly function of NC in Gag can be replaced by sequences that dimerize or multimerize (1, 28, 63). To determine if MX/NX can be rescued by protein-protein interactions, we constructed MZip, in which a modified GCN4 leucine zipper sequence was inserted into the NC deletion in MX/NX. This zipper sequence tetramerizes to form four-stranded coils (25). Virion preparations of MZip showed that this sequence was able to rescue MX/NX budding (Fig. 7B). However, as with the RNA sequences discussed above, the rescue was not as efficient as wild-type virus budding (Fig. 7B and C), though it was better than that with the RNA sequence insertion mutants (Fig. 7C). As with Mp10, the processing of Gag with the MZip mutant was very inefficient. The ability of this multimerizing sequence to rescue the MX/NX mutant confirms that the loss of particle formation is due to a deficiency in Gag-Gag interaction, entirely consistent with a role for RNA-Gag interactions in virus assembly.
DISCUSSION
Our results show that either one of two RNA-binding domains in Gag is required for assembly of HIV-1 Gag. These regions appear to provide a redundant function, since either mutation of the RNA-binding region of MA or deletion of NC alone does not dramatically reduce virion production per se. Gag assembly defects caused by the removal of these RNA-binding sites can be rescued in trans by coassembly with Gag proteins containing either both MA and NC sites (wild-type Gag) or only one of the single sites alone (i.e., either truncated Gag proteins or a single-site mutant). Rescue of viral budding in trans depended on the ability of the two Gag proteins to interact, which is consistent with a binding of Gag to RNA that promotes Gag-Gag interactions. This requirement for an RNA-binding sequence in Gag assembly could be supplied by RNA-binding sequences in cis, though less efficiently than when the GCN4 tetramerizing mutant sequences are used. The ability of the protein multimerizing sequences to promote Gag assembly in the absence of NC, seen here and in previous studies (1, 28, 63), confirms that particle formation in the absence of NC can be driven by strong protein-protein binding. However, in nature, this required interaction likely occurs by Gag-RNA-Gag binding. Taken together, our data support a requirement for RNA in HIV-1 assembly, offering solid genetic evidence for RNA-Gag binding as an integral step in virion formation.
Similar results were obtained in vitro by Burniston et al. In their study, the RNA-binding region of MA was necessary for Gag-Gag interaction when two-thirds of NC was deleted from Gag (8). Additionally, MA contributed to Gag-Gag interaction in a yeast two-hybrid screen for the minimal interacting sequences in Gag. In cells, Gag without the MA basic region and positively charged amino acids (e.g., arginines and lysines) in NC had a severe assembly defect (13). Combined with our data presented here, these results are entirely consistent with HIV-1 Gag containing two RNA-binding regions that perform a redundant role in RNA-mediated Gag assembly.
While there is essentially no HIV-1 genomic RNA inside the NX/PX particles (48), we show that there still remains a significant level of other RNAs, presumably incorporated by the basic region of MA. These RNAs could be nonviral mRNAs or small RNAs, possibly tRNAs, that are incorporated into retrovirus particles (13, 16, 36). Further characterization of the RNA species is required to determine what RNAs are incorporated in these particles. Even though small RNAs might be limited in their ability to bind multiple Gag molecules, dA15 to dA20 oligonucleotides are long enough to support HIV-1 Gag assembly in vitro (9). Also, HIV-1 NC appears to require only 8 nucleotides for nucleic acid binding, so several Gag molecules could readily bind small RNAs such as tRNA (17). Similarly, RSV Gag can use a 16-nt DNA for efficient particle formation in vitro (40, 41). Therefore, even small RNAs such as tRNA bound to Gag could provide the functional RNA requirement for HIV-1 Gag assembly (8).
In contrast to our results, MuLV and RSV Gag proteins strictly require NC for assembly (34, 44). For MuLV, this difference is likely due to the absence of the basic region in MA, analogous to the HIV-1 MA KKYQK construct that could bind RNA. Furthermore, MuLV and RSV do not incorporate EF1-, a tRNA-binding protein that appears to be incorporated by its association with RNA, which, in turn, is bound by the basic region in HIV-1 MA. Thus, it appears that these viruses do not have an RNA-binding site equivalent to the KKYQK sequence in HIV-1. Interestingly, RSV Gag can be engineered to bind EF1-, and presumably RNA, by inserting the HIV-1 HXB2 KKKYKL MA sequence (the same sequence that partially complemented the MX/NX mutation incis) into RSV MA (13). Because Pr55Gag possesses two RNA-binding regions, it does not display the same strict dependence on NC for particle production as MuLV or RSV Gag. However, since the MX mutant has lost the RNA-binding site in MA, it behaves similarly to MuLV and RSV, which are strictly dependent on the NC protein for assembly. Interestingly, only 8 positively charged residues in RSV NC are required for particle production, further supporting the idea that nonspecific RNA interactions are important for assembly in Gag (34).
This difference in assembly requirements for various Gag proteins highlights the fact that, even though they perform essentially the same function, significant differences may exist in the precise way in which each type of Gag assembles. As discussed above, some of the assembly requirements for MuLV and RSV Gag appear to be significantly different from those for HIV-1 Gag. Considering this point, findings pertaining to one particular Gag do not necessarily apply to all retroviruses. Thus, while a plethora of studies implicate NC as strictly required for retroviral particle formation in several contexts (34), it is the ability of Gag to bind RNA and provide for Gag multimerization that appears to be required for assembly, rather than the NC protein itself.
The role of NC as a driving force in virus assembly has been debated for some time. One model posits that NC-NC protein dimerization interactions assist in Gag-Gag interactions (28,40, 41, 63). The ability to delete NC and maintain particle formation suggests that direct NC interactions are not strictly required for this part of HIV-1 assembly (48). While these data also initially appeared to be at odds with a role for RNA in assembly, the results presented here reconcile our previous findings with the RNA-mediated assembly model, in that an RNA-binding region in either MA or NC is required for virion formation. Thus, Gag-RNA interactions play an essential role in Gag assembly, in addition to those contributed by Gag-Gag interactions.
ACKNOWLEDGMENTS
We thank Kunio Nagashima for electron microscopy, David Morcock for assistance with the fluorimeter measurements, and Alan Rein and Robert Gorelick for critical reading of the manuscript and helpful suggestions.
This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract NO1-CO-12400.
The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.
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