Background Since HIV-1 Tat and Vpr genes are involved in promoter transactivation, apoptosis, etc, we carried out studies to find out nature and extent of natural variation in the two genes from seropositive patients from Northern India and determined their functional implications. Methods HIV-1 tat exon 1 and vpr were amplified from the genomic DNA isolated from the blood of HIV-1 infected individuals using specific primers by Polymerase Chain reaction (PCR) and subjected to extensive genetic analysis (CLUSTAL W, Simplot etc). Their expression was monitored by generating myc fusion clones. Tat exon 1 and Vpr variants were co-transfected with the reporter gene construct (LTR-luc) and their transactivation potential was monitored by measuring luciferase activity. Apoptosis and cell cycle analysis was done by Propidium Iodide (PI) staining followed by FACS. Results Exon 1 of tat was amplified from 21 samples and vpr was amplified from 16 samples. One of the Tat exon 1 variants showed phylogenetic relatedness to subtype B & C and turned out to be a unique recombinant. Two of the Vpr variants were B/C/D recombinants. These natural variations were found to have no impact on the stability of Tat and Vpr. These variants differed in their ability to transactivate B LTR and C LTR promoters. B/C recombinant Tat showed better co-operative interaction with Vpr. B/C/D recombination in Vpr was found to have no effect on its co-operativity with Tat. Recombinant Tat (B/C) induced more apoptosis than wild type B and C Tat. The B/C/D recombination in Vpr did not affect its G2 arrest induction potential but reduced its apoptosis induction ability. Conclusions Extensive sequence and region-specific variations were observed in Tat and Vpr genes from HIV-1 infected individuals from Northern India. These variations have functional implications & therefore important for the pathogenicity of virus.
References
[1]
Siddappa NB, Dash PK, Mahadevan A, Desai A, Jayasuryan N, et al. (2005) Identification of unique B/C recombinant strains of HIV-1 in the southern state of Karnataka, India. AIDS 19 (13) 1426–1429.
[2]
Neogi U, Sood V, Chowdhury A, Das S, Ramachandran VG, et al. (2009) Genetic analysis of HIV-1 Circulating Recombinant Form 02_AG, B and C subtype-specific envelope sequences from Northern India and their predicted co-receptor usage. AIDS Res Ther 6: 28.
[3]
Ouyang Y, Shao Y, Ma L (2012) HIV-1 CRF_BC Recombinants Infection in China: Molecular Epidemic and Characterizations. Current HIV Research 10: 151–161.
[4]
Robertson DL, Anderson JP, Bradac JA, Carr JK, Foley B, et al.. (1999) HIV-1 nomenclature proposal: a reference guide to HIV-1 classification. In: Kuiken CL, Foley B, Hahn BH, Korber B, McCutchan F and Marx PA, editors. Human Retroviruses and AIDS: A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences. NM: Theoretical Biology and Biophysics Group, Los Alamos National Laboratory. pp. 492–505.
[5]
Lole KS, Bollinger RC, Paranjape RS, Gadkari D, Kulkarni SS, et al. (1999) Full-length human immunodeficiency virus type 1 genomes from subtype C-infected seroconverters in India, with evidence of intersubtype recombination. J Virol 73 (1) 152–160.
[6]
Tripathy SP, Kulkarni SS, Jadhav SD, Agnihotri KD, Jere AJ, et al. (2005) Subtype B and subtype C HIV type 1 recombinants in the northeastern state of Manipur, India. AIDS Res Hum Retroviruses 21 (2) 152–157.
[7]
Robertson DL, Hahn BH, Sharp PM (1995a) Recombination in AIDS viruses. J Mol Evol 40: 249–259.
Kuiken C, Foley B, Hahn B, Marx P, McCutchan F, et al.. (2000) HIV Sequence Compendium 2000. Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, NM, USA.
[10]
Robertson DL, Anderson JP, Bradac JA, Carr JK, Foley B, et al. (2000) HIV-1 nomenclature proposal. Science 288: 55–56.
[11]
Plantier JC, Leoz M, Dickerson JE, De Oliveira F, Cordonnier F, et al. (2009) A new human immunodeficiency virus derived from gorillas. Nature Medicine 15 (8) 871–872.
[12]
Sodroski JG, Rosen CA, Wong-Staal F, Salahuddin SZ, Popovic M, et al. (1985) Trans-acting transcriptional regulation of human T-cell leukemia virus type III long terminal repeat. Science 227: 171–173.
[13]
Jeang KT (1996) A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences. In: Los Alamos National Laboratory, ed. Human Retroviruses and AIDS. pp. III-3–III-18.
[14]
Seigel LJ, Ratner L, Josephs SF, Derse D, Feinberg MB, et al. (1986) Transactivation induced by human T-lymphotropic virus type III (HTLV-III) maps to a viral sequence encoding 58 amino acids and lacks tissue specificity. Virology 148: 226–231.
[15]
Cullen BR (1986) Transactivation of human immunodeficiency virus occurs via a bimodal mechanism. Cell 46: 973–982.
[16]
Jones KA, Peterlin BM (1994) Control of RNA initiation and elongation at the HIV-1 promoter. Annu Rev Biochem 63: 717.
[17]
Kuppuswamy M, Subramanian T, Srinivasan A, Chinnadurai G (1989) Multiple functional domains of Tat, the trans-activator of HIV-1 defined by mutational analysis. Nucleic Acids Res 17: 3551–3561.
[18]
Rappaport J, Lee SJ, Khalili K, Wong-Staal F (1989) The acidic amino-terminal region of the HIV-1 Tat protein constitutes an essential activating domain. New Biol 1: 101–110.
[19]
Frankel AD, Bredt D, Pabo C (1988) Tat protein from immunodeficiency virus forms a metal-linked dimer. Science 240: 70–73.
[20]
Hauber J, Malim M, Cullen B (1989) Mutational analysis of the conserved basic domain of human immunodeficiency virus Tat protein. J Virol 63: 1181–1187.
[21]
Ruben S, Perkins A, Purcell R, Joung K, Sia R, et al. (1989) Structural and functional characterization of human immunodeficiency virus Tat protein. J Virol 63: 1–8.
[22]
Roy S, Delling U, Chen C-H, Rosen CA, Sonenberg N (1990) A bulge structure in HIV-1 TAR RNA is required for Tat binding and Tat-mediated trans-activation. Genes Dev 4: 1365–1373.
[23]
Bayer P, Kraft M, Ejchart A, Westendrop M, Frank R, et al. (1995) Structural studies of HIV-1 Tat protein. J Mol Biol 247: 529–535.
[24]
Klostermeier D, Bayer P, Kraft M, Frank RW, R？sch P (1997) Spectroscopiuc investigations of HIV-1 trans-activator and related peptides in aqueous solutions. Biophys Chem 63: 87–96.
[25]
Metzger AU, Bayer P, Willbold D, Hoffmann S, Frank RW, et al. (1997) The interaction of HIV-1 Tat (32–72) with its target RNA: a fluorescence and nuclear magnetic resonance study. Biochem Biophys Res Comm 241: 31–36.
[26]
Rana TM, Jeang KT (1999) Biochemical and functional interactions between HIV-1 Tat protein and TAR RNA. Arch Biochem Biophys 365: 175–185.
[27]
Chiu YL, Ho CK, Saha N, Schwer B, Shuman S, et al. (2002) Tat stimulates co-transcriptional capping of HIV mRNA. Mol Cell 10: 585–597.
[28]
McCloskey TW, Ott M, Tribble E, Khan SA, Teichberg S, et al. (1997) Dual Role of HIV Tat in Regulation of Apoptosis in T Cells. J Immunol 158: 1014–1019.
[29]
Ott M, Lovett JL, Mueller L, Verdin E (1998) Superinduction of IL-8 in T Cells by HIV-1 Tat Protein Is Mediated Through NF-kB Factors. J Immunol 160: 2872–2880.
[30]
de la Fuente C, Santiago F, Dend L, Eadie C, Zilberman I, et al. (2002) Gene Expression Profile of HIV-1 Tat expressing cells: a close interplay between proliferative and differentiative signals. BMC Biochemistry 3: 14.
[31]
Morellet N, Bouaziz S, Petitjean P, Roques BP (2003) NMR structure of the HIV-1 regulatory protein VPR. J Mol Biol 327: 215–227.
[32]
Cohen EA, Dehni G, Sodroski JG, Haseltine WA (1990) Human immunodeficiency virus vpr product is a virion-associated regulatory protein. J Virol 64: 3097–3099.
[33]
Heinzinger NK, Bukrinsky MI, Haggerty SA, Ragland AM, Kewalramani V, et al. (1994) The Vpr protein of human immunodeficiency virus type 1 influences nuclear localization of viral nucleic acids in nondividing host cells. Proc Natl Acad Sci USA 91: 7311–7315.
[34]
Fouchier RA, Meyer BE, Simon JH, Fisher U, Albright AV, et al. (1998) Interaction of the human immunodeficiency virus type 1 Vpr protein with the nuclear pore complex. J Virol 72: 6004–6013.
[35]
Nie Z, Bergeron D, Subbramanian R, Yao X-J, Checroune F, et al. (1998) The putative alpha helix 2 of human immunodeficiency virus type 1 Vpr contains a determinant which is responsible for the nuclear translocation of the proviral DNA in growth-arrested cells. J Virol 72: 4104–4115.
[36]
Popov S, Rexach M, Zybarth G, Reiling N, Lee M-A, et al. (1998) Viral protein R regulates nuclear import of HIV-1 pre-integration complex. EMBO J 17: 909–917.
[37]
Jowett JB, Planelles V, Poon B, Shah NP, Chen ML, et al. (1995) The human immunodeficiency virus type 1 vpr gene arrests infected T cells in the G2+M phase of the cell cycle. J Virol 69: 6304–6313.
[38]
He J, Choe S, Walker R, Di Marzio P, Morgan DO, et al. (1995) Human immunodeficiency virus type 1 viral protein R (Vpr) arrests cells in the G2 phase of the cell cycle by inhibiting p34cdc2 activity. J Virol 69: 6705–6711.
[39]
Goh WC, Rogel ME, Kinsey CM, Michael SF, Fultz PN, et al. (1998) HIV-1 Vpr increases viral expression by manipulation of the cell cycle: a mechanism for selection of Vpr in vivo. Nat Med 4: 65–71.
[40]
Subbramanian RA, Kessous-Elbaz A, Lodge R, Forget J, Yao X-J, et al. (1998) Human immunodeficiency virus type 1 Vpr is a positive regulator of viral transcription and infectivity in primary human macrophages. J Exp Med 187: 1103–1111.
[41]
Stewart SA, Poon B, Jowett JBM, Chen IS (1997) Human immunodeficiency virus type 1 Vpr induces apoptosis following cell cycle arrest. J Virol 71: 5579–5592.
[42]
Pauza CD, Trivedi P, Wallace M, Ruckwardt TJ, Buanec HL, et al. (2000) Vaccination with Tat toxoid attenuates disease in simian HIV-challenged macaques. PNAS 97 (7) 3515–3519.
[43]
Allen TM, Mortara L, Mothé BR, Liebl M, Jing P, et al. (2002) Tat-Vaccinated Macaques Do Not Control Simian Immunodeficiency Virus SIVmac239 Replication. J Virol 76 (8) 4108–4112.
[44]
Nkolola JP, Wee EG, Im EJ, Jewell CP, Chen N, et al. (2004) Engineering RENTA, a DNA prime-MVA boost HIV vaccine tailored for Eastern and Central Africa Gene Ther. 11 (13) 1068–1080.
[45]
Dougherty JP, Temin HM (1988) Determination of the rate of base-pair substitution and insertion mutations in retrovirus replication. J Virol 62: 2817–2822.
[46]
Preston BD, Poiesz BJ, Loeb LA (1988) Fidelity of HIV-1 reverse transcriptase. Science 242: 1168–1171.
[47]
Roberts JD, Bebenek K, Kunkel TA (1988) The accuracy of reverse transcriptase from HIV-1. Science 242: 1171–1173.
[48]
Ho DD, Neumann AU, Perelson AS, Chen W, Leonard JM, et al. (1995) Rapid turnover of plasma virions and CD4 lymphocytes in HIV-1 infection. Nature 373: 123–126.
[49]
Mullick R, Sengupta S, Sarkar K, Chakrabarti S (2010) Molecular characterization of tat gene and long terminal repeat region of Human Immunodeficiency virus type-1 detected among the injecting drug users (IDUs) of Manipur, India: Identification of BC recombinants. Virus Res 147: 195–201.
[50]
Neogi U, Gupta S, Sahoo PN, Shet A, Rao SD, et al. (2012) Genetic Characterization of HIV Type 1 Tat Exon 1 from a Southern Indian Clinical Cohort: Identification of Unique Epidemiological Signature Residues. Aids Res Hum Retroviruses 28 (9) 1152–1156.
[51]
Sawaya BE, Khalili K, Gordon J, Taube R, Amini S (2000) Cooperative Interaction between HIV-1 Regulatory Proteins Tat and Vpr Modulates Transcription of the Viral Genome. J Biol Chem 275 (45) 35209–35214.
[52]
Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucleic Acids Res 22: 4673–4680.
[53]
Korber B (2000) HIV Signature and Sequence Variation Analysis. In: Rodrigo AG, Learn GH, editors. Computational Analysis of HIV Molecular Sequences. Dordrecht, Netherlands: Kluwer Academic Publishers. pp. 55–72.
[54]
Sorgel S, Fraedrich K, Votteler J, Thomas M, Stamminger T, et al. (2012) Perinuclear localization of the HIV-1 regulatory protein Vpr is important for induction of G2-arrest. Virology 432: 444–451.
[55]
Saitou N, Nei M (1987) The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol Biol Evol 4: 406–425.
[56]
Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24: 1596–1599.
[57]
Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16: 111–120.
[58]
Bano AS, Sood V, Neogi U, Goel N, Kuttiat VS, et al. (2009) Genetic and functional characterization of human immunodeficiency virus type 1 VprC variants from north India: presence of unique recombinants with mosaic genomes from B, C and D subtypes within the open reading frame of Vpr. J Gen Virol 90: 2768–2776.
[59]
Ammosova T, Berro R, Jerebtsova M, Jackson A, Charles S, et al. (2006) Phosphorylation of HIV-1 Tat by CDK2 in HIV-1 transcription. Retrovirology 3: 78.
[60]
Fang Z, Xing H, Meng Z, Hong K, Liao L, et al. (2010) Genetic Characterization Analysis of the Tat Exon-1 Region of HIV Type 1 CRF07_BC Strains in China. Aids Res Hum Retroviruses 26 (3) 359–363.
[61]
Garber ME, Wei P, Kewal Ramani VN, Mayall TP, Herrmann CH, et al. (1998) The interaction between HIV-1 Tat and human cyclin T1 requires zinc and a critical cysteine residue that is not conserved in the murine CycT1 protein. Genes Dev 12: 3512–3527.
[62]
Wei P, Garber ME, Fang SM, Fischer WH, Jones KA (1998) A novel CDK9-associated C-type cyclin interacts directly with HIV-1 Tat and mediates its high-affinity, loop-specific binding to TAR RNA. Cell 92: 451–462.
[63]
Verhoef K, Koper M, Berkhout B (1997) Determination of the minimal amount of Tat activity required for human immunodeficiency virus type 1 replication. Virology 237: 228–236.
[64]
Sadaie MR, Rappaport J, Benter T, Josephs SF, Willis R, et al. (1988) Missense mutations in an infectious human immunodeficiency viral genome: functional mapping of tat and identification of the rev splice acceptor. Proc Natl Acad Sci USA 85 (23) 9224–9228.
[65]
Ranga U, Shankarappa R, Siddappa NB, Ramakrishna L, Nagendran R, et al. (2004) Tat protein of human immunodeficiency virus type 1 subtype C strains is a defective chemokine. J Virol 78 (5) 2586–2590.
[66]
Dey SS, Xue Y, Joachimiak MP, Friedland GD, Burnett JC, et al. (2012) Mutual Information Analysis Reveals Coevolving Residues in Tat that Compensate for Two Distinct Functions in HIV-1 Gene Expression. J Biol Chem 287 (11) 7945–7955.
[67]
Verhoef K, Berkhout B (1999) A Second-Site Mutation That Restores Replication of a Tat Defective Human Immunodeficiency Virus. J Virol 73 (4) 2781–2789.
[68]
Neogi U, Sood V, Goel N, Wanchu A, Banerjea AC (2008) Novel HIV-1 long terminal repeat (LTR) sequences of subtype B and mosaic intersubtype B/C recombinants in North India. Arch Virol 153 (10) 1961–66.
[69]
Tumas KM, Poszgay JM, Avidan N, Ksiazek SJ, Overmoyer B, et al. (1993) Loss of antigenic epitopes as the result of env gene recombination in retrovirus-induced leukemia in immunocompetent mice. Virology 192 (2) 587–595.
[70]
Golovkina T, Jaffe A, Ross S (1994) Coexpression of exogenous and endogenous mouse mammary tumor viruses RNA in vivo results in viral recombination and broadens the virus host range. J Virol 68: 5019–5026.
[71]
Moutouh L, Corbeil J, Richman D (1996) Recombination leads to the rapid emergence of HIV-1 dually resistant mutants under selective drug pressure. Proc Natl Acad Sci USA 93: 6106–6111.
