Sequences Intervening between the Core Packaging D
http://www.100md.com
病菌学杂志 2005年第21期
Departments of Biochemistry
Medical Microbiology, Faculty of Medicine and Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates
ABSTRACT
The packaging determinants of feline immunodeficiency virus (FIV) consist of two discontinuous core regions, extending from R to 150 bp of the 5' untranslated region and the first 100 bp of gag. However, the role of sequences intervening between the core regions in packaging has not been clear. A mutational analysis was conducted to determine whether the intervening sequences played a role in FIV RNA packaging, using an in vivo packaging assay complemented with semiquantitative reverse transcriptase PCR. Our analyses reveal that the intervening sequences are dispensable not only for vector RNA packaging but also for propagation, confirming the discontinuous nature of the FIV packaging signal.
TEXT
A series of recent studies have suggested that the packaging determinants of feline immunodeficiency virus (FIV) are complex and multipartite, consisting of at least two discontinuous core regions, one located upstream of the major splice donor sequence from R/U5 at the 5' end to the first 150 bp of the 5' untranslated region (UTR), while the other is within the first 100 bp of gag (2, 3, 6, 7). To determine whether the region intervening between the core determinants was required for maintaining the stability of the FIV packaging signal, we modified our previously described vector, MB15 (2), and constructed a series of transfer vectors, AG002 to AG004, that maintained the R/U5 and 100 bp of gag but kept only the first 90, 120, or 150 bp of the 5' UTR, generating incremental deletions between the core packaging determinants (Fig. 1). In a second series, AG013 to AG015, the deletions were replaced with heterologous sequences of the same lengths to further determine whether the deleted/substituted region had a role at the structural level or acted only as a spacer (Fig. 1).
The two series of mutant vector RNAs were tested for their ability to be packaged by FIV proteins, using our well-established in vivo packaging assay along with TR394, which contains the entire 270-bp UTR with a 333-bp gag sequence (1-3). The amount of transfer vector RNA packaged into viral particles was analyzed by reverse transcriptase PCR (RT-PCR) (10) for variable cycle numbers, and the resulting products were Southern blotted and hybridized using an R/U5 probe. All mutant vector RNAs, either with deletions or deletion/substitution, were packaged into the virus particles but with different efficiencies, while the control vectors, MB15 and TR394, were packaged at similar levels (Fig. 2A). This was despite the fact that all cultures produced similar levels of viral particles and the transfection efficiencies were within twofold of each other (Fig. 2B and C). Since the PCRs were conducted across the deleted or deleted/substituted region, the size of the PCR fragment varied with each construct, confirming that correct packaging constructs were being expressed in each transfection.
To determine the packaging efficiencies of the various constructs accurately, RT-PCRs were repeated using primers within the R/U5 region that would result in same-sized fragments. Optical densities observed in the various bands were normalized to the amount of luciferase expression observed from the cotransfected luciferase expression vector, and the packaging efficiencies obtained were compared to that for MB15, which was assigned a value of 1. The transfer vector RNAs with only 90 bp of UTR, AG002 and AG013, were least efficiently packaged (0.2 and 0.1, respectively) compared to MB15 (Fig. 2D). The encapsidation efficiency increased substantially with the increase of 30 bp at the 5' UTR with AG003 and AG014, which contain 120 bp of UTR (0.6 and 0.4, respectively), and increased to nearly the same as that of MB15 by 150 bp of UTR with AG004 and AG015 (0.8 and 0.9, respectively) (Fig. 2D).
The reduced effect on packaging observed for AG013 over and above the lack of sufficient 5' UTR sequences is probably due to the 180-bp heterologous sequences that were inserted to maintain the spacing between the two core regions. Other than for AG013, the packaging efficiencies between the vectors with deletions were similar to those between the vectors with deletions/substitutions, suggesting that sequences in between the core packaging determinants were not needed as spacers and the core elements could fold into functional packaging determinants independently of the intervening region.
Finally, propagation of transfer vector RNAs by transduction of the hygromycin marker gene to target cells was determined by infecting HeLa cells with virions produced by the 293T cells. Colonies observed upon hygromycin selection of the infected cells were adjusted to the luciferase expression observed to normalize for various transfection efficiencies and amounts of virions produced in the supernatant. Overall, the transfer vector RNAs were propagated with 1- to 6-fold less efficiency than MB15 (Table 1). (Similar observations were made when Cos cells were used as virus-producing cells; data not shown.) Transfer vector RNAs with the UTR deletions, AG002 and AG004, were propagated with 2- to 4-fold-reduced efficiency, while those with the deletion/insertions were propagated with a 1- to 6-fold reduction compared to MB15 (Table 1). Consistent with the packaging data, vectors with only 90 bp of UTR (AG002 and AG013) exhibited the greatest reduction in titers (4- and 6-fold), reflecting their poor packaging efficiency, while vectors with 120 (AG003 and AG014) or 150 (AG004 and AG015) bp of UTR resulted in near-wild-type titers (within 1- to 2.5-fold) either with or without insertions, reflecting the improved packaging efficiencies observed with these vectors (Table 1).
To determine the effect of mutations on any putative secondary RNA structure that this region may assume, the region between R and the first 333 bp of FIV gag was folded using the RNAstructure program (8). Similar to the case with human immunodeficiency virus, simian immunodeficiency virus, and Mason-Pfizer monkey virus (4, 5, 9, 11), this region folded into several stem-loops, of which four were stable, named stem-loops 1 to 4 (SL1 to SL4). Folding of the mutant vector RNAs revealed that most of the conformation of the wild-type UTR was destroyed in the vectors with only 90 and 120 bp of the UTR, except for SL1, which was conserved in all transfer vector RNAs (Fig. 3C to F). However, since it was present both in RNAs that were packaged efficiently and in those that were packaged poorly, SL1 does not seem to play a significant role as a packaging determinant (Fig. 3C to H).
SL2, an unusually large stem-loop found within the first 180 bp of the UTR (the region directly tested in our deletion analysis), was the most consistently formed stem-loop structure found to be associated with enhanced packaging (Fig. 2 and 3). It was completely absent in vectors with 90 bp of UTR (AG002 and AG013) (Fig. 3C and D). However, in the presence of 120 bp of UTR in AG003 and AG014, the first few loops of SL2 started to emerge, irrespective of the insertion (Fig. 3E and F), while in the presence of 150 bp of UTR in AG004 and AG015, two-thirds of SL2 had reformed (Fig. 3G and H). Even restoration of the first two bulges of SL2 formed within 120 bp of UTR improved packaging significantly (0.62 and 0.40 for AG003 and AG014, respectively), while emergence of two-thirds of SL2 restored packaging nearly to that of MB15 (0.80 and 0.92 for AG004 and AG015, respectively) (Fig. 3G and H). The reformation of partial SL2 in vectors with 120- and 150-bp UTR sequences (irrespective of the insertions) suggests that FIV may not need the entire SL2 for packaging, but only a part of this structure that is probably sufficiently formed by the presence of sequences within 150 bp of the 5' UTR for efficient packaging.
Kemler et al. (6) mutated part of the region encompassing the fourth stem/bulge region of SL2 but observed no effect of the mutations on packaging (6). Our structural analysis indicates that their mutations did not really perturb the structure of SL2 enough to affect packaging due to its size and stability (G = –75.5) (data not shown), and perhaps more drastic mutations (large deletions) will be needed to affect RNA packaging significantly, such as those in AG002.
Together, this study reveals that the sequences intervening between the core packaging determinants are dispensable not only for efficient FIV RNA packaging but for vector RNA propagation as well. These data should enhance our understanding of how complex retroviruses package their genomic RNAs and help streamline the design of FIV-based transfer vectors for human gene therapy.
ACKNOWLEDGMENTS
This work was accomplished with a grant from the Faculty of Medicine and Health Sciences (New Project Grant 2002-NP/02/30) and in part with funds from the Terry Fox Foundation for Cancer Research (2001/02 and 2001/03) and Sheikh Hamdan Award for Medical Sciences (MRG26/2001-2002). A.G. was supported by a scholarship from the School of Graduate Studies, UAE University, United Arab Emirates.
REFERENCES
Browning, M. T., R. D. Schmidt, K. A. Lew, and T. A. Rizvi. 2001. Primate and feline lentiviral vector RNA packaging and propagation by heterologous lentiviral virions. J. Virol. 75:5129-5140.
Browning, M. T., F. Mustafa, R. D. Schmidt, K. A. Lew, and T. A. Rizvi. 2003. Sequences within the gag gene of feline immunodeficiency virus (FIV) are important for efficient RNA encapsidation. Virus Res. 93:199-209.
Browning, M. T., F. Mustafa, R. D. Schmidt, K. A. Lew, and T. A. Rizvi. 2003. Delineation of sequences important for efficient FIV RNA packaging. J. Gen. Virol. 84:621-627.
Clever, J. L., D. Miranda, Jr., and T. G. Parslow. 2002. RNA structure and packaging signals in the 5' leader region of the human immunodeficiency virus type 1 genome. J. Virol. 76:12381-12387.
Harrison, G. P., E. Hunter, and A. M. L. Lever. 1995. Secondary structure model of the Mason-Pfizer monkey virus 5' leader sequence: identification of a structural motif common to a variety of retroviruses. J. Virol. 69:2175-2186.
Kemler, I., R. Barraza, and E. M. Poeschla. 2002. Mapping the encapsidation determinants of feline immunodeficiency virus. J. Virol. 76:11889-11903.
Kemler, I., I. Azmi, and E. M. Poeschla. 2004. The critical role of proximal gag sequences in feline immunodeficiency virus genome encapsidation. Virology 327:111-120.
Mathews, D. H., J. Sabina, M. Zuker, and D. H. Turner. 1999. Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. J. Mol. Biol. 288:911-940.
McBride, M. S., and A. T. Panganiban. 1996. The human immunodeficiency virus type 1 encapsidation site is a multipartite RNA element composed of functional hairpin structures. J. Virol. 70:2963-2973.
Mustafa, F., P. Jayanth, P. S. Phillip, A. Ghazawi, R. D. Schmidt, K. A. Lew, and T. A. Rizvi. 2005. Relative activity of the feline immunodeficiency virus promoter in feline and primate cell lines. Microbes Infect. 7:233-239.
Strappe, P. M., J. Greatorex, J. Thomas, P. Biswas, E. McCann, and A. M. Lever. 2003. The packaging signal of simian immunodeficiency virus is upstream of the major splice donor at a distance from the RNA cap site similar to that of human immunodeficiency virus types 1 and 2. J. Gen. Virol. 84:2423-2430.
Talbott, R. T., E. E. Sparger, K. M. Lovelace, W. M. Fitch, N. C. Pedersen, P. A. Luciw, and J. H. Elder. 1989. Nucleotide sequence and genomic organization of feline immunodeficiency virus. Proc. Natl. Acad. Sci. USA 86:5743-5747.
Tan, W., B. K. Felber, A. S. Zolotukhin, G. N. Pavlakis, and S. Schwartz. 1995. Efficient expression of the human papillomavirus type 16 L1 protein in epithelial cells by using Rev and the Rev-responsive element of human immunodeficiency virus or the cis-acting transactivation element of simian retrovirus type 1. J. Virol. 69:5607-5620.(Farah Mustafa, Akela Ghaz)
Medical Microbiology, Faculty of Medicine and Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates
ABSTRACT
The packaging determinants of feline immunodeficiency virus (FIV) consist of two discontinuous core regions, extending from R to 150 bp of the 5' untranslated region and the first 100 bp of gag. However, the role of sequences intervening between the core regions in packaging has not been clear. A mutational analysis was conducted to determine whether the intervening sequences played a role in FIV RNA packaging, using an in vivo packaging assay complemented with semiquantitative reverse transcriptase PCR. Our analyses reveal that the intervening sequences are dispensable not only for vector RNA packaging but also for propagation, confirming the discontinuous nature of the FIV packaging signal.
TEXT
A series of recent studies have suggested that the packaging determinants of feline immunodeficiency virus (FIV) are complex and multipartite, consisting of at least two discontinuous core regions, one located upstream of the major splice donor sequence from R/U5 at the 5' end to the first 150 bp of the 5' untranslated region (UTR), while the other is within the first 100 bp of gag (2, 3, 6, 7). To determine whether the region intervening between the core determinants was required for maintaining the stability of the FIV packaging signal, we modified our previously described vector, MB15 (2), and constructed a series of transfer vectors, AG002 to AG004, that maintained the R/U5 and 100 bp of gag but kept only the first 90, 120, or 150 bp of the 5' UTR, generating incremental deletions between the core packaging determinants (Fig. 1). In a second series, AG013 to AG015, the deletions were replaced with heterologous sequences of the same lengths to further determine whether the deleted/substituted region had a role at the structural level or acted only as a spacer (Fig. 1).
The two series of mutant vector RNAs were tested for their ability to be packaged by FIV proteins, using our well-established in vivo packaging assay along with TR394, which contains the entire 270-bp UTR with a 333-bp gag sequence (1-3). The amount of transfer vector RNA packaged into viral particles was analyzed by reverse transcriptase PCR (RT-PCR) (10) for variable cycle numbers, and the resulting products were Southern blotted and hybridized using an R/U5 probe. All mutant vector RNAs, either with deletions or deletion/substitution, were packaged into the virus particles but with different efficiencies, while the control vectors, MB15 and TR394, were packaged at similar levels (Fig. 2A). This was despite the fact that all cultures produced similar levels of viral particles and the transfection efficiencies were within twofold of each other (Fig. 2B and C). Since the PCRs were conducted across the deleted or deleted/substituted region, the size of the PCR fragment varied with each construct, confirming that correct packaging constructs were being expressed in each transfection.
To determine the packaging efficiencies of the various constructs accurately, RT-PCRs were repeated using primers within the R/U5 region that would result in same-sized fragments. Optical densities observed in the various bands were normalized to the amount of luciferase expression observed from the cotransfected luciferase expression vector, and the packaging efficiencies obtained were compared to that for MB15, which was assigned a value of 1. The transfer vector RNAs with only 90 bp of UTR, AG002 and AG013, were least efficiently packaged (0.2 and 0.1, respectively) compared to MB15 (Fig. 2D). The encapsidation efficiency increased substantially with the increase of 30 bp at the 5' UTR with AG003 and AG014, which contain 120 bp of UTR (0.6 and 0.4, respectively), and increased to nearly the same as that of MB15 by 150 bp of UTR with AG004 and AG015 (0.8 and 0.9, respectively) (Fig. 2D).
The reduced effect on packaging observed for AG013 over and above the lack of sufficient 5' UTR sequences is probably due to the 180-bp heterologous sequences that were inserted to maintain the spacing between the two core regions. Other than for AG013, the packaging efficiencies between the vectors with deletions were similar to those between the vectors with deletions/substitutions, suggesting that sequences in between the core packaging determinants were not needed as spacers and the core elements could fold into functional packaging determinants independently of the intervening region.
Finally, propagation of transfer vector RNAs by transduction of the hygromycin marker gene to target cells was determined by infecting HeLa cells with virions produced by the 293T cells. Colonies observed upon hygromycin selection of the infected cells were adjusted to the luciferase expression observed to normalize for various transfection efficiencies and amounts of virions produced in the supernatant. Overall, the transfer vector RNAs were propagated with 1- to 6-fold less efficiency than MB15 (Table 1). (Similar observations were made when Cos cells were used as virus-producing cells; data not shown.) Transfer vector RNAs with the UTR deletions, AG002 and AG004, were propagated with 2- to 4-fold-reduced efficiency, while those with the deletion/insertions were propagated with a 1- to 6-fold reduction compared to MB15 (Table 1). Consistent with the packaging data, vectors with only 90 bp of UTR (AG002 and AG013) exhibited the greatest reduction in titers (4- and 6-fold), reflecting their poor packaging efficiency, while vectors with 120 (AG003 and AG014) or 150 (AG004 and AG015) bp of UTR resulted in near-wild-type titers (within 1- to 2.5-fold) either with or without insertions, reflecting the improved packaging efficiencies observed with these vectors (Table 1).
To determine the effect of mutations on any putative secondary RNA structure that this region may assume, the region between R and the first 333 bp of FIV gag was folded using the RNAstructure program (8). Similar to the case with human immunodeficiency virus, simian immunodeficiency virus, and Mason-Pfizer monkey virus (4, 5, 9, 11), this region folded into several stem-loops, of which four were stable, named stem-loops 1 to 4 (SL1 to SL4). Folding of the mutant vector RNAs revealed that most of the conformation of the wild-type UTR was destroyed in the vectors with only 90 and 120 bp of the UTR, except for SL1, which was conserved in all transfer vector RNAs (Fig. 3C to F). However, since it was present both in RNAs that were packaged efficiently and in those that were packaged poorly, SL1 does not seem to play a significant role as a packaging determinant (Fig. 3C to H).
SL2, an unusually large stem-loop found within the first 180 bp of the UTR (the region directly tested in our deletion analysis), was the most consistently formed stem-loop structure found to be associated with enhanced packaging (Fig. 2 and 3). It was completely absent in vectors with 90 bp of UTR (AG002 and AG013) (Fig. 3C and D). However, in the presence of 120 bp of UTR in AG003 and AG014, the first few loops of SL2 started to emerge, irrespective of the insertion (Fig. 3E and F), while in the presence of 150 bp of UTR in AG004 and AG015, two-thirds of SL2 had reformed (Fig. 3G and H). Even restoration of the first two bulges of SL2 formed within 120 bp of UTR improved packaging significantly (0.62 and 0.40 for AG003 and AG014, respectively), while emergence of two-thirds of SL2 restored packaging nearly to that of MB15 (0.80 and 0.92 for AG004 and AG015, respectively) (Fig. 3G and H). The reformation of partial SL2 in vectors with 120- and 150-bp UTR sequences (irrespective of the insertions) suggests that FIV may not need the entire SL2 for packaging, but only a part of this structure that is probably sufficiently formed by the presence of sequences within 150 bp of the 5' UTR for efficient packaging.
Kemler et al. (6) mutated part of the region encompassing the fourth stem/bulge region of SL2 but observed no effect of the mutations on packaging (6). Our structural analysis indicates that their mutations did not really perturb the structure of SL2 enough to affect packaging due to its size and stability (G = –75.5) (data not shown), and perhaps more drastic mutations (large deletions) will be needed to affect RNA packaging significantly, such as those in AG002.
Together, this study reveals that the sequences intervening between the core packaging determinants are dispensable not only for efficient FIV RNA packaging but for vector RNA propagation as well. These data should enhance our understanding of how complex retroviruses package their genomic RNAs and help streamline the design of FIV-based transfer vectors for human gene therapy.
ACKNOWLEDGMENTS
This work was accomplished with a grant from the Faculty of Medicine and Health Sciences (New Project Grant 2002-NP/02/30) and in part with funds from the Terry Fox Foundation for Cancer Research (2001/02 and 2001/03) and Sheikh Hamdan Award for Medical Sciences (MRG26/2001-2002). A.G. was supported by a scholarship from the School of Graduate Studies, UAE University, United Arab Emirates.
REFERENCES
Browning, M. T., R. D. Schmidt, K. A. Lew, and T. A. Rizvi. 2001. Primate and feline lentiviral vector RNA packaging and propagation by heterologous lentiviral virions. J. Virol. 75:5129-5140.
Browning, M. T., F. Mustafa, R. D. Schmidt, K. A. Lew, and T. A. Rizvi. 2003. Sequences within the gag gene of feline immunodeficiency virus (FIV) are important for efficient RNA encapsidation. Virus Res. 93:199-209.
Browning, M. T., F. Mustafa, R. D. Schmidt, K. A. Lew, and T. A. Rizvi. 2003. Delineation of sequences important for efficient FIV RNA packaging. J. Gen. Virol. 84:621-627.
Clever, J. L., D. Miranda, Jr., and T. G. Parslow. 2002. RNA structure and packaging signals in the 5' leader region of the human immunodeficiency virus type 1 genome. J. Virol. 76:12381-12387.
Harrison, G. P., E. Hunter, and A. M. L. Lever. 1995. Secondary structure model of the Mason-Pfizer monkey virus 5' leader sequence: identification of a structural motif common to a variety of retroviruses. J. Virol. 69:2175-2186.
Kemler, I., R. Barraza, and E. M. Poeschla. 2002. Mapping the encapsidation determinants of feline immunodeficiency virus. J. Virol. 76:11889-11903.
Kemler, I., I. Azmi, and E. M. Poeschla. 2004. The critical role of proximal gag sequences in feline immunodeficiency virus genome encapsidation. Virology 327:111-120.
Mathews, D. H., J. Sabina, M. Zuker, and D. H. Turner. 1999. Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. J. Mol. Biol. 288:911-940.
McBride, M. S., and A. T. Panganiban. 1996. The human immunodeficiency virus type 1 encapsidation site is a multipartite RNA element composed of functional hairpin structures. J. Virol. 70:2963-2973.
Mustafa, F., P. Jayanth, P. S. Phillip, A. Ghazawi, R. D. Schmidt, K. A. Lew, and T. A. Rizvi. 2005. Relative activity of the feline immunodeficiency virus promoter in feline and primate cell lines. Microbes Infect. 7:233-239.
Strappe, P. M., J. Greatorex, J. Thomas, P. Biswas, E. McCann, and A. M. Lever. 2003. The packaging signal of simian immunodeficiency virus is upstream of the major splice donor at a distance from the RNA cap site similar to that of human immunodeficiency virus types 1 and 2. J. Gen. Virol. 84:2423-2430.
Talbott, R. T., E. E. Sparger, K. M. Lovelace, W. M. Fitch, N. C. Pedersen, P. A. Luciw, and J. H. Elder. 1989. Nucleotide sequence and genomic organization of feline immunodeficiency virus. Proc. Natl. Acad. Sci. USA 86:5743-5747.
Tan, W., B. K. Felber, A. S. Zolotukhin, G. N. Pavlakis, and S. Schwartz. 1995. Efficient expression of the human papillomavirus type 16 L1 protein in epithelial cells by using Rev and the Rev-responsive element of human immunodeficiency virus or the cis-acting transactivation element of simian retrovirus type 1. J. Virol. 69:5607-5620.(Farah Mustafa, Akela Ghaz)