HIV-1 Mutant Assembly, Processing and Infectivity Expresses Pol Independent of Gag
Abstract
1. Introduction
2. Materials and Methods
2.1. Plasmid Construction
2.2. Cell Culture, Transfection, and Infection
2.3. Sucrose Density Gradient Fractionation
2.4. Western Immunoblot Analysis
3. Results
3.1. HIV-1 Gag and Pol Expression in a Single Plasmid
3.2. G2AP Exhibits Wild-Type HIV-1 Particle Density and Possesses Infectivity
3.3. Reduced Pol Expression Significantly Increases Virus Yields
3.4. PR Activity Attenuation Increases Virus Titers
3.5. PR-RT Cleavage Blocking Enhances G2AP Virus Yields
3.6. G2AP Is Defective in Pol Incorporation
3.7. 2A exerts no Major Effects on Post-Assembly Post-Processing Stages of Virus Infectivity
4. Discussion
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
- Petropoulos, C. Retroviral Taxonomy, Protein Structures, Sequences, and Genetic Maps. In Retroviruses; Coffin, J.M., Hughes, S.H., Varmus, H.E., Eds.; Cold Spring Harbor Laboratory Press: New York, NY, USA, 1997. [Google Scholar]
- Jacks, T.; Power, M.D.; Masiarz, F.R.; Luciw, P.A.; Barr, P.J.; Varmus, H.E. Characterization of ribosomal frameshifting in HIV-1 gag-pol expression. Nature 1988, 331, 280–283. [Google Scholar] [CrossRef] [PubMed]
- Freed, E.O. HIV-1 assembly, release and maturation. Nat. Rev. Microbiol. 2015, 13, 484–496. [Google Scholar] [CrossRef] [PubMed]
- Lee, S.K.; Potempa, M.; Swanstrom, R. The choreography of HIV-1 proteolytic processing and virion assembly. J. Biol. Chem. 2012, 287, 40867–40874. [Google Scholar] [CrossRef] [PubMed]
- Swanstrom, R.; Wills, J.W. Synthesis, Assembly, and Processing of Viral Proteins. In Retroviruses; Coffin, J.M., Hughes, S.H., Varmus, H.E., Eds.; Cold Spring Harbor Laboratory Press: New York, NY, USA, 1997. [Google Scholar]
- Arrigo, S.J.; Huffman, K. Potent inhibition of human immunodeficiency virus type 1 (HIV-1) replication by inducible expression of HIV-1 PR multimers. J. Virol. 1995, 69, 5988–5994. [Google Scholar] [CrossRef]
- Hill, M.K.; Hooker, C.W.; Harrich, D.; Crowe, S.M.; Mak, J. Gag-Pol supplied in trans is efficiently packaged and supports viral function in human immunodeficiency virus type 1. J. Virol. 2001, 75, 6835–6840. [Google Scholar] [CrossRef]
- Krausslich, H.G. Human immunodeficiency virus proteinase dimer as component of the viral polyprotein prevents particle assembly and viral infectivity. Proc. Natl. Acad. Sci. USA 1991, 88, 3213–3217. [Google Scholar] [CrossRef]
- Park, J.; Morrow, C.D. Overexpression of the gag-pol precursor from human immunodeficiency virus type 1 proviral genomes results in efficient proteolytic processing in the absence of virion production. J. Virol. 1991, 65, 5111–5117. [Google Scholar] [CrossRef]
- Rose, J.R.; Babe, L.M.; Craik, C.S. Defining the level of human immunodeficiency virus type 1 (HIV-1) protease activity required for HIV-1 particle maturation and infectivity. J. Virol. 1995, 69, 2751–2758. [Google Scholar] [CrossRef]
- Shehu-Xhilaga, M.; Crowe, S.M.; Mak, J. Maintenance of the Gag/Gag-Pol ratio is important for human immunodeficiency virus type 1 RNA dimerization and viral infectivity. J. Virol. 2001, 75, 1834–1841. [Google Scholar] [CrossRef]
- Wang, C.T.; Chou, Y.C.; Chiang, C.C. Assembly and processing of human immunodeficiency virus Gag mutants containing a partial replacement of the matrix domain by the viral protease domain. J. Virol. 2000, 74, 3418–3422. [Google Scholar] [CrossRef]
- Huang, M.; Martin, M.A. Incorporation of Pr160(gag-pol) into virus particles requires the presence of both the major homology region and adjacent C-terminal capsid sequences within the Gag-Pol polyprotein. J. Virol. 1997, 71, 4472–4478. [Google Scholar] [CrossRef] [PubMed]
- Smith, A.J.; Srinivasakumar, N.; Hammarskjöld, M.L.; Rekosh, D. Requirements for incorporation of Pr160gag-pol from human immunodeficiency virus type 1 into virus-like particles. J. Virol. 1993, 67, 2266–2275. [Google Scholar] [CrossRef] [PubMed]
- Srinivasakumar, N.; Hammarskjöld, M.L.; Rekosh, D. Characterization of deletion mutations in the capsid region of human immunodeficiency virus type 1 that affect particle formation and Gag-Pol precursor incorporation. J. Virol. 1995, 69, 6106–6114. [Google Scholar] [CrossRef] [PubMed]
- Park, J.; Morrow, C.D. The nonmyristylated Pr160gag-pol polyprotein of human immunodeficiency virus type 1 interacts with Pr55gag and is incorporated into viruslike particles. J. Virol. 1992, 66, 6304–6313. [Google Scholar] [CrossRef] [PubMed]
- Han, G.-Z.; Worobey, M. An Endogenous Foamy-like Viral Element in the Coelacanth Genome. PLoS Pathog. 2012, 8, e1002790. [Google Scholar] [CrossRef]
- Löchelt, M.; Flügel, R.M. The human foamy virus pol gene is expressed as a Pro-Pol polyprotein and not as a Gag-Pol fusion protein. J. Virol. 1996, 70, 1033–1040. [Google Scholar] [CrossRef]
- Yu, S.F.; Baldwin, D.N.; Gwynn, S.R.; Yendapalli, S.; Linial, M.L. Human Foamy Virus Replication: A Pathway Distinct from That of Retroviruses and Hepadnaviruses. Science 1996, 271, 1579–1582. [Google Scholar] [CrossRef]
- Cen, S.; Niu, M.; Saadatmand, J.; Guo, F.; Huang, Y.; Nabel, G.J.; Kleiman, L. Incorporation of Pol into Human Immunodeficiency Virus Type 1 Gag Virus-Like Particles Occurs Independently of the Upstream Gag Domain in Gag-Pol. J. Virol. 2004, 78, 1042–1049. [Google Scholar] [CrossRef]
- Chiu, H.-C.; Yao, S.-Y.; Wang, C.-T. Coding Sequences Upstream of the Human Immunodeficiency Virus Type 1 Reverse Transcriptase Domain in Gag-Pol Are Not Essential for Incorporation of the Pr160gag-pol into Virus Particles. J. Virol. 2002, 76, 3221–3231. [Google Scholar] [CrossRef]
- Buchschacher, G.L.; Yu, L.; Murai, F.; Friedmann, T.; Miyanohara, A. Association of Murine Leukemia Virus Pol with Virions, Independent of Gag-Pol Expression. J. Virol. 1999, 73, 9632–9637. [Google Scholar] [CrossRef]
- Kim, J.H.; Lee, S.-R.; Li, L.-H.; Park, H.-J.; Park, J.-H.; Lee, K.Y.; Kim, M.-K.; Shin, B.A.; Choi, S.-Y. High Cleavage Efficiency of a 2A Peptide Derived from Porcine Teschovirus-1 in Human Cell Lines, Zebrafish and Mice. PLoS ONE 2011, 6, e18556. [Google Scholar] [CrossRef]
- Guo, T.-W.; Yu, F.-H.; Huang, K.-J.; Wang, C.-T. P6gag domain confers cis HIV-1 Gag-Pol assembly and release capability. J. Gen. Virol. 2016, 97, 209–219. [Google Scholar] [CrossRef] [PubMed]
- Doyon, L.; Payant, C.; Brakier-Gingras, L.; Lamarre, D. Novel Gag-Pol Frameshift Site in Human Immunodeficiency Virus Type 1 Variants Resistant to Protease Inhibitors. J. Virol. 1998, 72, 6146–6150. [Google Scholar] [CrossRef] [PubMed]
- Yu, F.-H.; Huang, K.-J.; Wang, C.-T. C-Terminal HIV-1 Transframe p6* Tetrapeptide Blocks Enhanced Gag Cleavage Incurred by Leucine Zipper Replacement of a Deleted p6* Domain. J. Virol. 2017, 91, e00103–e00117. [Google Scholar] [CrossRef] [PubMed]
- Yu, F.-H.; Chou, T.-A.; Liao, W.-H.; Huang, K.-J.; Wang, C.-T. Gag-Pol Transframe Domain p6* Is Essential for HIV-1 Protease-Mediated Virus Maturation. PLoS ONE 2015, 10, e0127974. [Google Scholar] [CrossRef] [PubMed]
- Burns, J.C.; Friedmann, T.; Driever, W.; Burrascano, M.; Yee, J. Vesicular Stomatitis Virus G Glycoprotein Pseudotyped Retroviral Vectors: Concentration to Very High Titer and Efficient Gene Transfer into Mammalian and Nonmammalian Cells. Proc. Natl. Acad. Sci. USA 1993, 90, 8033–8037. [Google Scholar] [CrossRef] [PubMed]
- Ferris, A.L.; Hizi, A.; Showalter, S.D.; Pichuantes, S.; Babe, L.; Craik, C.S.; Hughes, S.H. Immunologic and proteolytic analysis of HIV-1 reverse transcriptase structure. Virology 1990, 175, 456–464. [Google Scholar] [CrossRef]
- Hizi, A.; McGill, C.; Hughes, S.H. Expression of Soluble, Enzymatically Active, Human Immunodeficiency Virus Reverse Transcriptase in Escherichia coli and Analysis of Mutants. Proc. Natl. Acad. Sci. USA 1988, 85, 1218–1222. [Google Scholar] [CrossRef]
- Donnelly, M.L.L.; Luke, G.; Mehrotra, A.; Li, X.; Hughes, L.E.; Gani, D.; Ryan, M.D. Analysis of the aphthovirus 2A/2B polyprotein ‘cleavage’ mechanism indicates not a proteolytic reaction, but a novel translational effect: A putative ribosomal ‘skip’. J. Gen. Virol. 2001, 82, 1013–1025. [Google Scholar] [CrossRef]
- Donnelly, M.L.L.; Hughes, L.E.; Luke, G.; Mendoza, H.; ten Dam, E.; Gani, D.; Ryan, M.D. The ‘cleavage’ activities of foot-and-mouth disease virus 2A site-directed mutants and naturally occurring ‘2A-like’ sequences. J. Gen. Virol. 2001, 82, 1027–1041. [Google Scholar] [CrossRef]
- Cherry, E.; Liang, C.; Rong, L.; Quan, Y.; Inouye, P.; Li, X.; Morin, N.; Kotler, M.; Wainberg, M.A. Characterization of human immunodeficiency virus type-1 (HIV-1) particles that express protease-reverse transcriptase fusion proteins11Edited by J. Karn. J. Mol. Biol. 1998, 284, 43–56. [Google Scholar] [CrossRef] [PubMed]
- Pfrepper, K.-I.; Rackwitz, H.-R.; Schnölzer, M.; Heid, H.; Löchelt, M.; Flügel, R.M. Molecular Characterization of Proteolytic Processing of the Pol Proteins of Human Foamy Virus Reveals Novel Features of the Viral Protease. J. Virol. 1998, 72, 7648–7652. [Google Scholar] [CrossRef] [PubMed]
- Figueiredo, A.; Moore, K.L.; Mak, J.; Sluis-Cremer, N.; de Bethune, M.P.; Tachedjian, G. Potent nonnucleoside reverse transcriptase inhibitors target HIV-1 Gag-Pol. PLoS Pathog. 2006, 2, e119. [Google Scholar] [CrossRef] [PubMed]
- Tachedjian, G.; Orlova, M.; Sarafianos, S.G.; Arnold, E.; Goff, S.P. Nonnucleoside reverse transcriptase inhibitors are chemical enhancers of dimerization of the HIV type 1 reverse transcriptase. Proc. Natl. Acad. Sci. USA 2001, 98, 7188–7193. [Google Scholar] [CrossRef] [PubMed]
- Freed, E.O. HIV-1 Gag Proteins: Diverse Functions in the Virus Life Cycle. Virology 1998, 251, 1–15. [Google Scholar] [CrossRef] [PubMed]
- Donnelly, M.L.; Gani, D.; Flint, M.; Monaghan, S.; Ryan, M.D. The cleavage activities of aphthovirus and cardiovirus 2A proteins. J. Gen. Virol. 1997, 78, 13–21. [Google Scholar] [CrossRef]
- Groot Bramel-Verheije, M.H.; Rottier, P.J.M.; Meulenberg, J.J.M. Expression of a Foreign Epitope by Porcine Reproductive and Respiratory Syndrome Virus. Virology 2000, 278, 380–389. [Google Scholar] [CrossRef]
- Liao, W.H.; Wang, C.T. Characterization of human immunodeficiency virus type 1 Pr160 gag-pol mutants with truncations downstream of the protease domain. Virology 2004, 329, 180–188. [Google Scholar] [CrossRef]
- Abbink, T.E.M.; Ooms, M.; Haasnoot, P.C.J.; Berkhout, B. The HIV-1 Leader RNA Conformational Switch Regulates RNA Dimerization but Does Not Regulate mRNA Translation. Biochemistry 2005, 44, 9058–9066. [Google Scholar] [CrossRef]
- Clever, J.L.; Miranda, J.D.; Parslow, T.G. RNA Structure and Packaging Signals in the 5′ Leader Region of the Human Immunodeficiency Virus Type 1 Genome. J. Virol. 2002, 76, 12381–12387. [Google Scholar] [CrossRef]
- Clever, J.L.; Taplitz, R.A.; Lochrie, M.A.; Polisky, B.; Parslow, T.G. A Heterologous, High-Affinity RNA Ligand for Human Immunodeficiency Virus Gag Protein Has RNA Packaging Activity. J. Virol. 2000, 74, 541–546. [Google Scholar] [CrossRef] [PubMed][Green Version]
- Lu, K.; Heng, X.; Summers, M.F. Structural Determinants and Mechanism of HIV-1 Genome Packaging. J. Mol. Biol. 2011, 410, 609–633. [Google Scholar] [CrossRef] [PubMed]
- Lever, A.; Gottlinger, H.; Haseltine, W.; Sodroski, J. Identification of a sequence required for efficient packaging of human immunodeficiency virus type 1 RNA into virions. J. Virol. 1989, 63, 4085–4087. [Google Scholar] [CrossRef] [PubMed]
- Luban, J.; Goff, S.P. Mutational analysis of cis-acting packaging signals in human immunodeficiency virus type 1 RNA. J. Virol. 1994, 68, 3784–3793. [Google Scholar] [CrossRef] [PubMed]
- McBride, M.S.; Panganiban, A.T. The human immunodeficiency virus type 1 encapsidation site is a multipartite RNA element composed of functional hairpin structures. J. Virol. 1996, 70, 2963–2973. [Google Scholar] [CrossRef] [PubMed]
- Chamanian, M.; Purzycka, K.J.; Wille, P.T.; Ha, J.S.; McDonald, D.; Gao, Y.; Le Grice, S.F.J.; Arts, E.J. A cis-Acting Element in Retroviral Genomic RNA Links Gag-Pol Ribosomal Frameshifting to Selective Viral RNA Encapsidation. Cell Host Microbe 2013, 13, 181–192. [Google Scholar] [CrossRef]
- Nikolaitchik, O.A.; Hu, W.-S. Deciphering the Role of the Gag-Pol Ribosomal Frameshift Signal in HIV-1 RNA Genome Packaging. J. Virol. 2014, 88, 4040–4046. [Google Scholar] [CrossRef]
- Comas-Garcia, M.; Datta, S.A.K.; Baker, L.; Varma, R.; Gudla, P.R.; Rein, A. Dissection of specific binding of HIV-1 Gag to the ‘packaging signal’ in viral RNA. elife 2017, 6, e27055. [Google Scholar] [CrossRef]
- Lee, E.-G.; Sinicrope, A.; Jackson, D.L.; Yu, S.F.; Linial, M.L. Foamy Virus Pol Protein Expressed as a Gag-Pol Fusion Retains Enzymatic Activities, Allowing for Infectious Virus Production. J. Virol. 2012, 86, 5992–6001. [Google Scholar] [CrossRef]
- Swiersy, A.; Wiek, C.; Reh, J.; Zentgraf, H.; Lindemann, D. Orthoretroviral-like prototype foamy virus gag-pol expression is compatible with viral replication. Retrovirology 2011, 8, 66. [Google Scholar] [CrossRef]
- Chiu, H.-C.; Wang, F.-D.; Chen, Y.-M.A.; Wang, C.-T. Effects of human immunodeficiency virus type 1 transframe protein p6* mutations on viral protease-mediated Gag processing. J. Gen. Virol. 2006, 87, 2041–2046. [Google Scholar] [CrossRef] [PubMed]
Construct a | Titer (c.f.u./mL) | Relative Titer | Mean Relative Titer | |
---|---|---|---|---|
Mutant | Dp6*PR b | (%) | (%) ± SD c | |
D2APol | 9 | 5500 | 0.164 | |
11 | 5000 | 0.220 | ||
8 | 3750 | 0.213 | ||
11 | 5500 | 0.200 | 0.199 ± 0.025 | |
D2A/P22APol | 11,000 | 5500 | 200.0 | |
11,000 | 5000 | 220.0 | ||
9000 | 3750 | 240.0 | ||
8750 | 5500 | 159.1 | 204.8 ± 34.56 | |
D2A/D15EPol | 8250 | 5500 | 150.0 | |
10,500 | 5000 | 210.0 | ||
6250 | 3750 | 166.7 | ||
8250 | 5500 | 150.0 | 169.2 ± 28.33 |
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Yu, F.-H.; Huang, K.-J.; Wang, C.-T. HIV-1 Mutant Assembly, Processing and Infectivity Expresses Pol Independent of Gag. Viruses 2020, 12, 54. https://doi.org/10.3390/v12010054
Yu F-H, Huang K-J, Wang C-T. HIV-1 Mutant Assembly, Processing and Infectivity Expresses Pol Independent of Gag. Viruses. 2020; 12(1):54. https://doi.org/10.3390/v12010054
Chicago/Turabian StyleYu, Fu-Hsien, Kuo-Jung Huang, and Chin-Tien Wang. 2020. "HIV-1 Mutant Assembly, Processing and Infectivity Expresses Pol Independent of Gag" Viruses 12, no. 1: 54. https://doi.org/10.3390/v12010054
APA StyleYu, F.-H., Huang, K.-J., & Wang, C.-T. (2020). HIV-1 Mutant Assembly, Processing and Infectivity Expresses Pol Independent of Gag. Viruses, 12(1), 54. https://doi.org/10.3390/v12010054