The HIV-1 primary transcript undergoes a complex splicing process by which more than 40 different spliced RNAs are generated. One of the factors contributing to HIV-1 splicing complexity is the multiplicity of 3′ splice sites (3'ss) used for generation of rev RNAs, with two 3'ss, A4a and A4b, being most commonly used, a third site, A4c, used less frequently, and two additional sites, A4d and A4e, reported in only two and one isolates, respectively. HIV-1 splicing has been analyzed mostly in subtype B isolates, and data on other group M clades are lacking. Here we examine splice site usage in three primary isolates of subtype C, the most prevalent clade in the HIV-1 pandemic, by using an in vitro infection assay of peripheral blood mononuclear cells. Viral spliced RNAs were identified by RT-PCR amplification using a fluorescently-labeled primer and software analyses and by cloning and sequencing the amplified products. The results revealed that splice site usage for generation of rev transcripts in subtype C differs from that reported for subtype B, with most rev RNAs using two previously unreported 3'ss, one located 7 nucleotides upstream of 3'ss A4a, designated A4f, preferentially used by two isolates, and another located 14 nucleotides upstream of 3'ss A4c, designated A4g, preferentially used by the third isolate. A new 5′ splice site, designated D2a, was also identified in one virus. Usage of the newly identified splice sites is consistent with sequence features commonly found in subtype C viruses. These results show that splice site usage may differ between HIV-1 subtypes.
References
[1]
Schwartz SB, Felber K, Benko DM, Feny？ EM, Pavlakis GN (1990) Cloning and functional analysis of multiply spliced mRNA species of human immunodeficiency virus type 1. J Virol 64: 2519–2529.
[2]
Purcell DF, Martin M (1993) Alternative splicing of human immunodeficiency virus type 1 mRNA modulates viral protein expression, replication, and infectivity. J Virol 67: 6365–6378.
[3]
Benko DM, Schwartz S, Pavlakis GN, Felber BK (1990) A novel human immunodeficiency virus type 1 protein, tev, shares sequences with tat, env, and rev proteins. J Virol 64: 2505–2518.
[4]
Salfeld JH, G？ttlinger G, Sia RA, Park RE, Sodroski JG, et al. (1990) A tripartite HIV-1 tat-env-rev fusion protein. EMBO J 9: 965–970.
[5]
Furtado MR, Balachandran R, Gupta P, Wolinski SM (1991) Analysis of alternatively spliced human immunodeficiency virus type-1 mRNA species, one of which encodes a novel Tat-Env fusion protein. Virology 185: 258–270.
[6]
Smith J, Azad J, Deacon N (1992) Identification of two novel human immunodeficiency splice acceptor sites in infected T cell lines. J Gen Virol 73: 1825–1828.
[7]
Berkhout B, van Wamel JLB (1996) Identification of a novel splice acceptor in the HIV-1 genome: independent expression of the cytoplasmic tail of the envelope protein. Arch Virol 141: 839–855.
[8]
Bilodeau PS, Domsic JK, Stoltzfus CM (1999) Splicing regulatory elements within tat exon 2 of human immunodeficiency virus type 1 (HIV-1) are characteristic of group M but not group O HIV-1 strains. J Virol 73: 9764–9772.
[9]
Lützelberger ML, Reinert S, Das AT, Berkhout B, Kjems J (2006) A novel splice donor site in the gag-pol gene is required for HIV-1 RNA stability. J Biol Chem 281: 18644–18651.
[10]
Carrera C, Pinilla M, Pérez-álvarez L, Thomson MM (2010) Identification of unusual and novel HIV type 1 spliced transcripts generated in vivo. AIDS Res Hum Retroviruses 26: 815–820.
[11]
Staffa A, Cochrane A (1994) The tat/rev intron of human immunodeficiency virus type 1 is inefficiently spliced because of suboptimal signals in the 3′ splice site. J Virol 68: 3071–3079.
[12]
O'Reilly MM, McNally T, Beemon KL (1995) Two strong 5′ splice sites and competing, suboptimal 3′ splice sites involved in alternative splicing of human immunodeficiency virus type 1 RNA. Virology 213: 373–385.
[13]
Si ZB, Amendt A, Stoltzfus CM (1997) Splicing efficiency of human immunodeficiency virus type 1 tat RNA is determined by both a suboptimal 3′ splice site and a 10 nucleotide exon splicing silencer element located within tat exon 2. Nucleic Acids Res 25: 861–867.
[14]
Swanson AK, Stoltzfus CM (1998) Overlapping cis sites used for splicing of HIV-1 env/nef and rev mRNAs. J Biol Chem 273: 34551–34557.
[15]
Madsen JM, Stoltzfus CM (2006) A suboptimal 5′ splice site downstream of HIV-1 splice site A1 is required for unspliced viral mRNA accumulation and efficient virus replication. Retrovirology 3: 10.
[16]
Stoltzfus CM (2009) Regulation of HIV-1 alternative splicing and its role in virus replication. Adv Virus Res 74: 1–40.
[17]
Madsen JM, Stoltzfus CM (2005) An exonic splicing silencer downstream of the 3′ splice site A2 is required for efficient human immunodeficiency virus type 1 replication. J Virol 79: 10478–10486.
[18]
Hemelaar J, Gouws E, Ghys PD, Osmanov S, WHO-UNAIDS Network for HIV Isolation and Characterisation (2011) Global trends in molecular epidemiology of HIV-1 during 2000–2007. AIDS 25: 679–689.
[19]
Cuevas MT, Fernández-García A, Pinilla M, García-Alvarez V, Thomson M, et al. (2009) Biological and genetic characterization of HIV type 1 subtype B and nonsubtype B transmitted viruses: usefulness for vaccine candidate assessment. AIDS Res Hum Retroviruses 26: 1019–1025.
[20]
Fernández-García A, Cuevas MT, Mu？oz-Nieto M, Ocampo A, Pinilla M, et al. (2009) Development of a panel of well-characterized human immunodeficiency virus type 1 isolates from newly diagnosed patients including acute and recent infections. AIDS Res Hum Retroviruses 25: 93–102.
[21]
Gao K, Masuda A, Matsuura T, Ohno K (2008) Human branch point consensus sequence is yUnAy. Nucleic Acids Res 36: 2257–2267.
[22]
Corvelo A, Halleger M, Smith CWJ, Eyras E (2010) Genome-wide association between branch point properties and alternative splicing. PLoS Comput Biol 6: e1001016.
[23]
HIV Sequence Database Los Alamos National Laboratory. Available: http://www.hiv.lanl.gov/content/sequence？/HIV/mainpage.html. Accessed 2011 Mar 10.
[24]
Wu J, Manley JL (1989) Mammalian pre-mRNA branch site selection by U2 snRNP involves base pairing. Genes Dev 3: 1553–1561.
[25]
Nelson KK, Green MR (1989) Mammalian U2 snRNP has a sequence-specific RNA-binding activity. Genes Dev 3: 1562–1571.
[26]
Reed R (1989) The organization of 3′ splice-site sequences in mammalian introns. Genes Dev 3: 2113–2123.
[27]
Roscigno RF, Weiner M, Garcia-Blanco M (1993) A mutational analysis of the polypyrimidine tract of introns. J Biol Chem 15: 11222–1229.
[28]
Norton PA (1994) Polypyrimidine tract sequences direct selection of alternative branch sites and influence protein binding. Nucleic Acids Res 22: 3854–3860.
[29]
Coolidge CJ, Seely RJ, Patton JG (1997) Functional analysis of the polypyrimidine tract in pre-mRNA splicing. Nucleic Acids Res 25: 888–896.
[30]
Buvoli M, Mayer SA, Patton JG (1997) Functional crosstalk between exon enhancers, polypyrimidine tracts and branchpoint sequences. EMBO J 16: 7174–7183.
[31]
Lear AL, Eperon LP, Wheatley IM, Eperon IC (1990) Hierarchy for 5′ splice site preference determined in vivo. J Mol Biol 211: 103–115.
[32]
Roca X, Sachidanandam R, Krainer AR (2005) Determinants of the inherent strength of human 5′ splice sites. RNA 11: 683–698.
[33]
Smith CW, Chu TT, Nadal-Ginard B (1993) Scanning and competition between AGs are involved in 3′ splice site selection in mammalian introns. Mol Cell Biol 13: 4939–4952.
[34]
Kol G, Levy-Maor G, Ast G (2005) Human-mouse comparative analysis reveals that branch-site plasticity contributes to splicing regulation. Hum Mol Genet 14: 1559–1568.
[35]
Zhuang Y, Goldstein AM, Weiner AM (1989) UACUAAC is the preferred branch site for mammalian mRNA splicing. Proc Natl Acad Sci USA 86: 2752–2756.
[36]
Caputi M, Freund M, Kammler S, Asang C, Schaal H (2004) A bidirectional SF2/ASF- and SRp40-dependent splicing enhancer regulates HIV-1 rev, env, vpu, and nef gene expression. J Virol 78: 6517–6526.
[37]
Borensztajn KM, Sobrier L, Duquesnoy P, Fischer AM, Tapon-Bretaudière J, et al. (2006) Oriented scanning is the leading mechanism underlying 5′ splice site selection in mammals. PLoS Genet 2: e138.
[38]
Korber B, Gaschen B, Yusim K, Thakallapally R, Kesmir C, et al. (2001) Evolutionary and immunological implications of contemporary HIV-1 variation. Br Med Bull 58: 19–42.
[39]
Verhoef K, Klein A, Berkhout B (1996) Paracrine activation of the HIV-1 LTR promoter by the viral Tat protein is mechanistically similar to trans-activation within a cell. Virology 225: 316–327.
[40]
Montano MA, Nixon CP, Essex M (1998) Dysregulation through the NF-κB enhancer and TATA box of the human immunodeficiency virus type 1 subtype E promoter. J Virol 72: 8446–8452.
[41]
Montano MA, Nixon CP, Ndung'u T, Bussmann H, Novitsky VA, et al. (2000) Elevated tumor necrosis factor-alpha activation of human immunodeficiency virus type 1 subtype C in Southern Africa is associated with an NF-κB enhancer gain-of-function. J Infect Dis 181: 76–81.
[42]
Quivy V, Adam E, Collette Y, Demonte D, Chariot A, et al. (2002) Synergistic activation of human immunodeficiency virus type 1 promoter activity by NF-κB and inhibitors of deacetylases: potential perspectives for the development of therapeutic strategies. J Virol 76: 11091–11103.
[43]
Lemieux AM, Pare ME, Audet B, Legault E, Lefort S, et al. (2004) T-cell activation leads to poor activation of the HIV-1 clade E long terminal repeat and weak association of nuclear factor-κB and NFAT with its enhancer region. J Biol Chem 279: 52949–52960.
[44]
Jeeninga RE, Hoogenkamp M, Armand-Ugon M, de Baar M, Verhoef K (2000) Functional differences between the long terminal repeat transcriptional promoters of human immunodeficiency virus type 1 subtypes A through G. J Virol 74: 3740–3751.
[45]
Ball SC, Abraha A, Collins KR, Marozsan AJ, Baird H, et al. (2003) Comparing the ex vivo fitness of CCR5-tropic human immunodeficiency virus type 1 isolates of subtypes B and C. J Virol 77: 1021–1038.
[46]
Arien KK, Abraha A, Qui？ones-Mateu ME, Kestens L, Vanham G, et al. (2005) The replicative fitness of primary human immunodeficiency virus type 1 (HIV-1) group M, HIV-1 group O, and HIV-2 isolates. J Virol 79: 8979–8990.
[47]
Tscherning C, Alaeus A, Fredriksson R, Bjorndal A, Deng H, et al. (1998) Differences in chemokine coreceptor usage between genetic subtypes of HIV-1. Virology 241: 181–188.
[48]
Abebe A, Demissie D, Goudsmit J, Brouwer M, Kuiken CL, et al. (1999) HIV-1 subtype C syncytium- and non-syncytium-inducing phenotypes and coreceptor usage among Ethiopian patients with AIDS. AIDS 13: 1305–1311.
[49]
Bjorndal A, Sonnerborg A, Tscherning C, Albert J, Fenyo EM (1999) Phenotypic characteristics of human immunodeficiency virus type 1 subtype C isolates of Ethiopian AIDS patients. AIDS Res Hum Retroviruses 15: 647–653.
[50]
Kaleebu P, Nankya IL, Yirrell DL, Shafer LA, Kyosiimire-Lugemwa J, et al. (2007) Relation between chemokine receptor use, disease stage, and HIV-1 subtypes A and D: results from a rural Ugandan cohort. J Acquir Immune Defic Syndr 45: 28–33.
[51]
Huang W, Eshleman SH, Toma J, Fransen S, Stawiski E, et al. (2007) Coreceptor tropism in human immunodeficiency virus type 1 subtype D: high prevalence of CXCR4 tropism and heterogeneous composition of viral populations. J Virol 81: 7885–7893.
[52]
Iordanskiy , Waltke M, Feng Y, Wood C (2010) Subtype-associated differences in HIV-1 reverse transcription affect the viral replication. Retrovirology 7: 85.
