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PLOS ONE  2012 

The HIV-1 Rev Protein Enhances Encapsidation of Unspliced and Spliced, RRE-Containing Lentiviral Vector RNA

DOI: 10.1371/journal.pone.0048688

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Abstract:

Background During the RNA encapsidation process of human immunodeficiency virus (HIV) viral genomic, unspliced RNA (gRNA) is preferentially incorporated into assembling virions. However, a certain amount of spliced viral transcripts can also be detected in viral particles. Recently, we observed that nuclear export of HIV and lentiviral vector gRNA by Rev is required for efficient encapsidation. Since singly-spliced HIV transcripts also contain the Rev-response element (RRE), we investigated if the encapsidation efficiency of RRE-containing spliced HIV-vector transcripts is also increased by the viral Rev protein. Findings Starting with a lentiviral vector imitating the splicing pattern of HIV, we constructed vectors that express an unspliced transcript either identical in sequence to the singly-spliced or the fully-spliced RNA of the parental construct. After transfection of the different lentiviral vectors cytoplasmic and virion-associated RNA levels and vector titers were determined in the presence and absence of Rev. Rev enhanced the infectious titer of vectors containing an RRE 6 to 37-fold. Furthermore, Rev strongly increased encapsidation efficiencies of all RRE-containing transcripts up to 200-fold. However, a good correlation between encapsidation efficiency and lentiviral vector titer could only be observed for the gRNA. The infectious titer of the vector encoding the fully-spliced RNA without RRE as well as the encapsidation efficiency of all transcripts lacking the RRE was not influenced by Rev. Interestingly, the splicing process itself did not seem to interfere with packaging, since the encapsidation efficiencies of the same RNA expressed either by splicing or as an unspliced transcript did not differ significantly. Conclusions Rev-mediated nuclear export enhances the encapsidation efficiency of RRE-containing lentiviral vector RNAs independently of whether they have been spliced or not.

References

[1]  D'Souza V, Summers MF (2005) How retroviruses select their genomes. Nat Rev Microbiol 3: 643–655.
[2]  Purcell DF, Martin MA (1993) Alternative splicing of human immunodeficiency virus type 1 mRNA modulates viral protein expression, replication, and infectivity. J Virol 67: 6365–6378.
[3]  Stoltzfus CM, Madsen JM (2006) Role of viral splicing elements and cellular RNA binding proteins in regulation of HIV-1 alternative RNA splicing. Curr HIV Res 4: 43–55.
[4]  Bohne J, Wodrich H, Krausslich HG (2005) Splicing of human immunodeficiency virus RNA is position-dependent suggesting sequential removal of introns from the 5′ end. Nucleic Acids Res 33: 825–837.
[5]  Lever AML (1999) Lentiviral Vectors. In: Meager A, editor. Gene Therapy Technologies, Applications and Regulations. Chichester, West Sussex, England: John Wiley & Sons Ltd. 61–86.
[6]  Saurya S, Lichtenstein Z, Karpas A (2005) Defective rev response element (RRE) and rev gene in HAART treated AIDS patients with discordance between viral load and CD4+ T-cell counts. J Clin Virol 33: 324–327.
[7]  Geigenmuller U, Linial ML (1996) Specific binding of human immunodeficiency virus type 1 (HIV-1) Gag-derived proteins to a 5′ HIV-1 genomic RNA sequence. J Virol 70: 667–671.
[8]  Didierlaurent L, Racine PJ, Houzet L, Chamontin C, Berkhout B, et al. (2011) Role of HIV-1 RNA and protein determinants for the selective packaging of spliced and unspliced viral RNA and host U6 and 7SL RNA in virus particles. Nucleic Acids Res 39: 8915–8927.
[9]  Houzet L, Paillart JC, Smagulova F, Maurel S, Morichaud Z, et al. (2007) HIV controls the selective packaging of genomic, spliced viral and cellular RNAs into virions through different mechanisms. Nucleic Acids Res 35: 2695–2704.
[10]  Houzet L, Morichaud Z, Mougel M (2007) Fully-spliced HIV-1 RNAs are reverse transcribed with similar efficiencies as the genomic RNA in virions and cells, but more efficiently in AZT-treated cells. Retrovirology 4: 30.
[11]  Liang C, Hu J, Russell RS, Kameoka M, Wainberg MA (2004) Spliced human immunodeficiency virus type 1 RNA is reverse transcribed into cDNA within infected cells. AIDS Res Hum Retroviruses 20: 203–211.
[12]  Blissenbach M, Grewe B, Hoffmann B, Brandt S, Uberla K (2010) Nuclear RNA export and packaging functions of HIV-1 Rev revisited. J Virol 84: 6598–6604.
[13]  Brandt S, Blissenbach M, Grewe B, Konietzny R, Grunwald T, et al. (2007) Rev proteins of human and simian immunodeficiency virus enhance RNA encapsidation. PLoS Pathog 3: e54.
[14]  Grewe B, überla K (2010) Rev Revisited: Additional Functions of the HIV-1 Rev Protein. In: Lever AML, Jeang K-T, Berkhout B, editors. Recent Adcances in Human Retroviruses: Principles of Replication and Pathogenesis. Singapore: World Scientific Publishing Co. Pte. Ltd. 439–470.
[15]  Cockrell AS, van Praag H, Santistevan N, Ma H, Kafri T (2011) The HIV-1 Rev/RRE system is required for HIV-1 5′ UTR cis elements to augment encapsidation of heterologous RNA into HIV-1 viral particles. Retrovirology 8: 51.
[16]  Lucke S, Grunwald T, Uberla K (2005) Reduced mobilization of Rev-responsive element-deficient lentiviral vectors. J Virol 79: 9359–9362.
[17]  Miyoshi H, Blomer U, Takahashi M, Gage FH, Verma IM (1998) Development of a self-inactivating lentivirus vector. J Virol 72: 8150–8157.
[18]  Grewe B, Hoffmann B, Ohs I, Blissenbach M, Brandt S, et al. (2012) Cytoplasmic utilization of human immunodeficiency virus type 1 genomic RNA is not dependent on a nuclear interaction with Gag. J Virol 86: 2990–3002.
[19]  Galla M, Will E, Kraunus J, Chen L, Baum C (2004) Retroviral pseudotransduction for targeted cell manipulation. Mol Cell 16: 309–315.
[20]  Haas DL, Case SS, Crooks GM, Kohn DB (2000) Critical factors influencing stable transduction of human CD34(+) cells with HIV-1-derived lentiviral vectors. Mol Ther 2: 71–80.
[21]  Kim SS, Kothari N, You XJ, Robinson WE Jr, Schnell T, et al. (2001) Generation of replication-defective helper-free vectors based on simian immunodeficiency virus. Virology 282: 154–167.
[22]  Anson DS, Fuller M (2003) Rational development of a HIV-1 gene therapy vector. J Gene Med 5: 829–838.
[23]  Cui Y, Iwakuma T, Chang LJ (1999) Contributions of viral splice sites and cis-regulatory elements to lentivirus vector function. J Virol 73: 6171–6176.
[24]  Kotsopoulou E, Kim VN, Kingsman AJ, Kingsman SM, Mitrophanous KA (2000) A Rev-independent human immunodeficiency virus type 1 (HIV-1)-based vector that exploits a codon-optimized HIV-1 gag-pol gene. J Virol 74: 4839–4852.
[25]  Mautino MR, Ramsey WJ, Reiser J, Morgan RA (2000) Modified human immunodeficiency virus-based lentiviral vectors display decreased sensitivity to trans-dominant Rev. Hum Gene Ther 11: 895–908.
[26]  Graf M, Bojak A, Deml L, Bieler K, Wolf H, et al. (2000) Concerted action of multiple cis-acting sequences is required for Rev dependence of late human immunodeficiency virus type 1 gene expression. J Virol 74: 10822–10826.
[27]  Seguin B, Staffa A, Cochrane A (1998) Control of human immunodeficiency virus type 1 RNA metabolism: role of splice sites and intron sequences in unspliced viral RNA subcellular distribution. J Virol 72: 9503–9513.
[28]  Greatorex JS, Palmer EA, Pomerantz RJ, Dangerfield JA, Lever AM (2006) Mutation of the Rev-binding loop in the human immunodeficiency virus 1 leader causes a replication defect characterized by altered RNA trafficking and packaging. J Gen Virol 87: 3039–3044.
[29]  Houzet L, Morichaud Z, Didierlaurent L, Muriaux D, Darlix JL, et al. (2008) Nucleocapsid mutations turn HIV-1 into a DNA-containing virus. Nucleic Acids Res 36: 2311–2319.
[30]  Luban J, Goff SP (1994) Mutational analysis of cis-acting packaging signals in human immunodeficiency virus type 1 RNA. J Virol 68: 3784–3793.
[31]  Bagnarelli P, Valenza A, Menzo S, Sampaolesi R, Varaldo PE, et al. (1996) Dynamics and modulation of human immunodeficiency virus type 1 transcripts in vitro and in vivo. J Virol 70: 7603–7613.
[32]  Butera ST, Roberts BD, Lam L, Hodge T, Folks TM (1994) Human immunodeficiency virus type 1 RNA expression by four chronically infected cell lines indicates multiple mechanisms of latency. J Virol 68: 2726–2730.
[33]  Peng H, Reinhart TA, Retzel EF, Staskus KA, Zupancic M, et al. (1995) Single cell transcript analysis of human immunodeficiency virus gene expression in the transition from latent to productive infection. Virology 206: 16–27.
[34]  Pomerantz RJ, Seshamma T, Trono D (1992) Efficient replication of human immunodeficiency virus type 1 requires a threshold level of Rev: potential implications for latency. J Virol 66: 1809–1813.
[35]  Tange TO, Nott A, Moore MJ (2004) The ever-increasing complexities of the exon junction complex. Curr Opin Cell Biol 16: 279–284.
[36]  Malim MH, Hauber J, Fenrick R, Cullen BR (1988) Immunodeficiency virus rev trans-activator modulates the expression of the viral regulatory genes. Nature 335: 181–183.
[37]  Fouchier RA, Meyer BE, Simon JH, Fischer U, Malim MH (1997) HIV-1 infection of non-dividing cells: evidence that the amino-terminal basic region of the viral matrix protein is important for Gag processing but not for post-entry nuclear import. EMBO J 16: 4531–4539.
[38]  Wagner R, Graf M, Bieler K, Wolf H, Grunwald T, et al. (2000) Rev-independent expression of synthetic gag-pol genes of human immunodeficiency virus type 1 and simian immunodeficiency virus: implications for the safety of lentiviral vectors. Hum Gene Ther 11: 2403–2413.
[39]  Schnell T, Foley P, Wirth M, Munch J, Uberla K (2000) Development of a self-inactivating, minimal lentivirus vector based on simian immunodeficiency virus. Hum Gene Ther 11: 439–447.

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