Generic placeholder image

Current HIV Research


ISSN (Print): 1570-162X
ISSN (Online): 1873-4251

Research Article

Molecular and Genetic Characterization of Natural Variants of HIV-1 Nef Gene from North India and its Functional Implication in Down-Regulation of MHC-I and CD-4

Author(s): J. Singh*, L. Ronsard, M. Pandey, R. Kapoor, V. G. Ramachandran and A. C. Banerjea

Volume 19 , Issue 2 , 2021

Published on: 25 September, 2020

Page: [172 - 187] Pages: 16

DOI: 10.2174/1570162X18666200925160755

Price: $65


Background: HIV-1 Nef is an important accessory protein with multiple effector functions. Genetic studies of the HIV-1 Nef gene show extensive genetic diversity and the functional studies have been carried out mostly with Nef derived from regions dominated by subtype B (North America & Europe).

Objective: This study was carried out to characterize genetic variations of the Nef gene from HIV-1 infected individuals from North India and to find out their functional implications.

Methods: The unique representative variants were sub-cloned in a eukaryotic expression vector and further characterized with respect to their ability to downregulate cell surface expression of CD4 and MHC-1 molecules.

Results: The phylogenetic analysis of Nef variants revealed sequence similarity with either consensus subtype B or B/C recombinants. Boot scan analysis of some of our variants showed homology to B/C recombinant and some to wild type Nef B. Extensive variations were observed in most of the variants. The dN/dS ratio revealed 80% purifying selection and 20% diversifying selection implying the importance of mutations in Nef variants. Intracellular stability of Nef variants differed greatly when compared with wild type Nef B and C. There were some variants that possessed mutations in the functional domains of Nef and responsible for its differential CD4 and MHC-1 downregulation activity.

Conclusion: We observed enhanced biological activities in some of the variants, perhaps arising from amino acid substitutions in their functional domains. The CD4 and MHC-1 down-regulation activity of Nef is likely to confer immense survival advantage allowing the most rare genotype in a population to become the most abundant after a single selection event.

Keywords: Cluster of Differentiation-4 (CD4), functional domain, genetic variations, human immunodeficiency virus (HIV), major histocompatibility complex-1 (MHC-1), Nef.

Graphical Abstract
Sharp PM, Hahn BH. The evolution of HIV-1 and the origin of AIDS. Philos Trans R Soc Lond B Biol Sci 2010; 365(1552): 2487-94.
[] [PMID: 20643738]
Vidya Vijayan KK, Karthigeyan KP, Tripathi SP, Hanna LE. Pathophysiology of CD4+ T-Cell Depletion in HIV-1 and HIV-2 Infections. Front Immunol 2017; 8: 580.
[] [PMID: 28588579]
Hu WS, Hughes SH. HIV-1 reverse transcription. Cold Spring Harb Perspect Med 2012; 2(10): a006882.
[] [PMID: 23028129]
Emerman M, Malim MH. HIV-1 regulatory/accessory genes: keys to unraveling viral and host cell biology. Science 1998; 280(5371): 1880-4.
[] [PMID: 9632380]
Coffin JM, Hughes SH, Varmus HE. Retroviruses. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press The Place of Retroviruses in Biology 1997.
Coffin JM, Hughes SH, Varmus HE. Retroviruses. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press Accessory Proteins and Assembly 1997.
Sagar M, Kirkegaard E, Long EM, et al. Human immunodeficiency virus type 1 (HIV-1) diversity at time of infection is not restricted to certain risk groups or specific HIV-1 subtypes. J Virol 2004; 78(13): 7279-83.
[] [PMID: 15194805]
Taylor BS, Sobieszczyk ME, McCutchan FE, Hammer SM. The challenge of HIV-1 subtype diversity. N Engl J Med 2008; 358(15): 1590-602.
[] [PMID: 18403767]
Hemelaar J. Implications of HIV diversity for the HIV-1 pandemic. J Infect 2013; 66(5): 391-400.
[] [PMID: 23103289]
Hemelaar J, Gouws E, Ghys PD, Osmanov S. Global and regional distribution of HIV-1 genetic subtypes and recombinants in 2004. AIDS 2006; 20(16): W13-23.
[] [PMID: 17053344]
Neogi U, Sood V, Chowdhury A, et al. 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 2009; 6: 28.
[] [PMID: 19954551]
Bobkov AF, Kazennova EV, Sukhanova AL, et al. An HIV type 1 subtype A outbreak among injecting drug users in Kazakhstan. AIDS Res Hum Retroviruses 2004; 20(10): 1134-6.
[] [PMID: 15585106]
Gupta N, Sood V, Bano AS, Banerjea AC. X Protein of hepatitis B virus potently activates HIV-1 subtype C long terminal repeat promoter: implications for faster spread of HIV-1 subtype C. AIDS 2007; 21(11): 1491-2.
[] [PMID: 17589200]
Sood V, Ranjan R, Banerjea AC. Functional analysis of HIV-1 subtypes B and C HIV-1 Tat exons and RGD/QGD motifs with respect to Tat-mediated transactivation and apoptosis. AIDS 2008; 22(13): 1683-5.
[] [PMID: 18670233]
Bano AS, Gupta N, Sood V, Banerjea AC. Vpr from HIV-1 subtypes B and C exhibit significant differences in their ability to transactivate LTR-mediated gene expression and also in their ability to promote apoptotic DNA ladder formation. AIDS 2007; 21(13): 1832-4.
[] [PMID: 17690591]
Neogi U, Sharma Y, Sood V, Wanchu A, Banerjea AC. Diversity of HIV type 1 long terminal repeat (LTR) sequences following mother-to-child transmission in North India. AIDS Res Hum Retroviruses 2010; 26(12): 1299-305.
[] [PMID: 20929348]
Verma S, Ronsard L, Kapoor R, Banerjea AC. Genetic characterization of natural variants of Vpu from HIV-1 infected individuals from Northern India and their impact on virus release and cell death. PLoS One 2013; 8(3): e59283.
[] [PMID: 23555649]
Kirchhoff F, Münch J, Carl S, et al. The human immunodeficiency virus type 1 nef gene can to a large extent replace simian immunodeficiency virus nef in vivo. J Virol 1999; 73(10): 8371-83.
[] [PMID: 10482588]
Hahn T, Ramakrishnan R, Ahmad N. Evaluation of genetic diversity of human immunodeficiency virus type 1 NEF gene associated with vertical transmission. J Biomed Sci 2003; 10(4): 436-50.
[] [PMID: 12824703]
Basmaciogullari S, Pizzato M. The activity of Nef on HIV-1 infectivity. Front Microbiol 2014; 5: 232.
[] [PMID: 24904546]
Wildum S, Schindler M, Münch J, Kirchhoff F. Contribution of Vpu, Env, and Nef to CD4 down-modulation and resistance of human immunodeficiency virus type 1-infected T cells to superinfection. J Virol 2006; 80(16): 8047-59.
[] [PMID: 16873261]
Lubben NB, Sahlender DA, Motley AM, Lehner PJ, Benaroch P, Robinson MS. HIV-1 Nef-induced down-regulation of MHC class I requires AP-1 and clathrin but not PACS-1 and is impeded by AP-2. Mol Biol Cell 2007; 18(9): 3351-65.
[] [PMID: 17581864]
Saxena SK, Shrivastava G, Tiwari S, Nair MP. HIV-1 Nef: hacker of the host cell. Future Virol 2012; 7(2): 117-20.
[] [PMID: 23847689]
Foster JL, Garcia JV. HIV-1 Nef: at the crossroads. Retrovirology 2008; 5: 84.
[] [PMID: 18808677]
Grzesiek S, Stahl SJ, Wingfield PT, Bax A. The CD4 determinant for downregulation by HIV-1 Nef directly binds to Nef. Mapping of the Nef binding surface by NMR. Biochemistry 1996; 35(32): 10256-61.
[] [PMID: 8756680]
Preusser A, Briese L, Baur AS, Willbold D. Direct in vitro binding of full-length human immunodeficiency virus type 1 Nef protein to CD4 cytoplasmic domain. J Virol 2001; 75(8): 3960-4.
[] [PMID: 11264384]
Lindwasser OW, Smith WJ, Chaudhuri R, Yang P, Hurley JH, Bonifacino JS. A diacidic motif in human immunodeficiency virus type 1 Nef is a novel determinant of binding to AP-2. J Virol 2008; 82(3): 1166-74.
[] [PMID: 18032517]
Chaudhuri R, Mattera R, Lindwasser OW, Robinson MS, Bonifacino JS. A basic patch on alpha-adaptin is required for binding of human immunodeficiency virus type 1 Nef and cooperative assembly of a CD4-Nef-AP-2 complex. J Virol 2009; 83(6): 2518-30.
[] [PMID: 19129443]
Piguet V, Wan L, Borel C, et al. HIV-1 Nef protein binds to the cellular protein PACS-1 to downregulate class I major histocompatibility complexes. Nat Cell Biol 2000; 2(3): 163-7.
[] [PMID: 10707087]
Blagoveshchenskaya AD, Thomas L, Feliciangeli SF, Hung CH, Thomas G. HIV-1 Nef downregulates MHC-I by a PACS-1- and PI3K-regulated ARF6 endocytic pathway. Cell 2002; 111(6): 853-66.
[] [PMID: 12526811]
Greenberg ME, Iafrate AJ, Skowronski J. The SH3 domain-binding surface and an acidic motif in HIV-1 Nef regulate trafficking of class I MHC complexes. EMBO J 1998; 17(10): 2777-89.
[] [PMID: 9582271]
Mangasarian A, Piguet V, Wang JK, Chen YL, Trono D. Nef-induced CD4 and major histocompatibility complex class I (MHC-I) down-regulation are governed by distinct determinants: N-terminal alpha helix and proline repeat of Nef selectively regulate MHC-I trafficking. J Virol 1999; 73(3): 1964-73.
[] [PMID: 9971776]
Agopian K, Wei BL, Garcia JV, Gabuzda D. A hydrophobic binding surface on the human immunodeficiency virus type 1 Nef core is critical for association with p21-activated kinase 2. J Virol 2006; 80(6): 3050-61.
[] [PMID: 16501114]
Noviello CM, Benichou S, Guatelli JC. Cooperative binding of the class I major histocompatibility complex cytoplasmic domain and human immunodeficiency virus type 1 Nef to the endosomal AP-1 complex via its mu subunit. J Virol 2008; 82(3): 1249-58.
[] [PMID: 18057255]
Wonderlich ER, Williams M, Collins KL. The tyrosine binding pocket in the adaptor protein 1 (AP-1) mu1 subunit is necessary for Nef to recruit AP-1 to the major histocompatibility complex class I cytoplasmic tail. J Biol Chem 2008; 283(6): 3011-22.
[] [PMID: 18073204]
Singh RK, Lau D, Noviello CM, Ghosh P, Guatelli JC. An MHC-I cytoplasmic domain/HIV-1 Nef fusion protein binds directly to the mu subunit of the AP-1 endosomal coat complex. PLoS One 2009; 4(12): e8364.
[] [PMID: 20020046]
Roeth JF, Williams M, Kasper MR, Filzen TM, Collins KL. HIV-1 Nef disrupts MHC-I trafficking by recruiting AP-1 to the MHC-I cytoplasmic tail. J Cell Biol 2004; 167(5): 903-13.
[] [PMID: 15569716]
Foster JL, Denial SJ, Temple BR, Garcia JV. Mechanisms of HIV-1 Nef function and intracellular signaling. J Neuroimmune Pharmacol 2011; 6(2): 230-46.
[] [PMID: 21336563]
Kienzle N, Bachmann M, Müller WE, Müller-Lantzsch N. Expression and cellular localization of the Nef protein from human immunodeficiency virus-1 in stably transfected B-cells. Arch Virol 1992; 124(1-2): 123-32.
[] [PMID: 1571013]
Liang SH, Hong D, Clarke MF. Cooperation of a single lysine mutation and a C-terminal domain in the cytoplasmic sequestration of the p53 protein. J Biol Chem 1998; 273(31): 19817-21.
[] [PMID: 9677415]
Murti KG, Brown PS, Ratner L, Garcia JV. Highly localized tracks of human immunodeficiency virus type 1 Nef in the nucleus of cells of a human CD4+ T-cell line. Proc Natl Acad Sci USA 1993; 90(24): 11895-9.
[] [PMID: 8265644]
Duan L, Ozaki I, Oakes JW, Taylor JP, Khalili K, Pomerantz RJ. The tumor suppressor protein p53 strongly alters human immunodeficiency virus type 1 replication. J Virol 1994; 68(7): 4302-13.
[] [PMID: 8207805]
Sawaya BE, Khalili K, Mercer WE, Denisova L, Amini S. Cooperative actions of HIV-1 Vpr and p53 modulate viral gene transcription. J Biol Chem 1998; 273(32): 20052-7.
[] [PMID: 9685344]
Greenway AL, McPhee DA, Allen K, et al. Human immunodeficiency virus type 1 Nef binds to tumor suppressor p53 and protects cells against p53-mediated apoptosis. J Virol 2002; 76(6): 2692-702.
[] [PMID: 11861836]
Hansen S, Lane DP, Midgley CA. The N terminus of the murine p53 tumour suppressor is an independent regulatory domain affecting activation and thermostability. J Mol Biol 1998; 275(4): 575-88.
[] [PMID: 9466932]
Xiong Y, Zhang H, Beach D. Subunit rearrangement of the cyclin-dependent kinases is associated with cellular transformation. Genes Dev 1993; 7(8): 1572-83.
[] [PMID: 8101826]
Xiong Y, Hannon GJ, Zhang H, Casso D, Kobayashi R, Beach D. p21 is a universal inhibitor of cyclin kinases. Nature 1993; 366(6456): 701-4.
[] [PMID: 8259214]
el-Deiry WS, Harper JW, O’Connor PM, et al. WAF1/CIP1 is induced in p53-mediated G1 arrest and apoptosis. Cancer Res 1994; 54(5): 1169-74.
[PMID: 8118801]
el-Deiry WS, Tokino T, Velculescu VE, et al. WAF1, a potential mediator of p53 tumor suppression. Cell 1993; 75(4): 817-25.
[] [PMID: 8242752]
Di Leonardo A, Linke SP, Clarkin K, Wahl GM. DNA damage triggers a prolonged p53-dependent G1 arrest and long-term induction of Cip1 in normal human fibroblasts. Genes Dev 1994; 8(21): 2540-51.
[] [PMID: 7958916]
Pietenpol JA, Tokino T, Thiagalingam S, el-Deiry WS, Kinzler KW, Vogelstein B. Sequence-specific transcriptional activation is essential for growth suppression by p53. Proc Natl Acad Sci USA 1994; 91(6): 1998-2002.
[] [PMID: 8134338]
Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 1994; 22(22): 4673-80.
[] [PMID: 7984417]
Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 1987; 4(4): 406-25.
[PMID: 3447015]
Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 2007; 24(8): 1596-9.
[] [PMID: 17488738]
Resh MD. Fatty acylation of proteins: new insights into membrane targeting of myristoylated and palmitoylated proteins. Biochim Biophys Acta 1999; 1451(1): 1-16.
[] [PMID: 10446384]
Geyer M, Fackler OT, Peterlin BM. Structure- function relationships in HIV-1 Nef. EMBO Rep 2001; 2(7): 580-5.
[] [PMID: 11463741]
Peitzsch RM, McLaughlin S. Binding of acylated peptides and fatty acids to phospholipid vesicles: pertinence to myristoylated proteins. Biochemistry 1993; 32(39): 10436-43.
[] [PMID: 8399188]
Fackler OT, Luo W, Geyer M, Alberts AS, Peterlin BM. Activation of Vav by Nef induces cytoskeletal rearrangements and downstream effector functions. Mol Cell 1999; 3(6): 729-39.
[] [PMID: 10394361]
Saksela K, Cheng G, Baltimore D. Proline-rich (PxxP) motifs in HIV-1 Nef bind to SH3 domains of a subset of Src kinases and are required for the enhanced growth of Nef+ viruses but not for down-regulation of CD4. EMBO J 1995; 14(3): 484-91.
[] [PMID: 7859737]
Fackler OT, Lu X, Frost JA, et al. p21-activated kinase 1 plays a critical role in cellular activation by Nef. Mol Cell Biol 2000; 20(7): 2619-27.
[] [PMID: 10713183]
Renkema GH, Manninen A, Mann DA, Harris M, Saksela K. Identification of the Nef-associated kinase as p21-activated kinase 2. Curr Biol 1999; 9(23): 1407-10.
[] [PMID: 10607567]
Kumar M, Jain SK, Pasha ST, Chattopadhaya D, Lal S, Rai A. Genomic diversity in the regulatory nef gene sequences in Indian isolates of HIV type 1: emergence of a distinct subclade and predicted implications. AIDS Res Hum Retroviruses 2006; 22(12): 1206-19.
[] [PMID: 17209762]
Bresnahan PA, Yonemoto W, Ferrell S, Williams-Herman D, Geleziunas R, Greene WC. A dileucine motif in HIV-1 Nef acts as an internalization signal for CD4 downregulation and binds the AP-1 clathrin adaptor. Curr Biol 1998; 8(22): 1235-8.
[] [PMID: 9811606]
Craig HM, Pandori MW, Guatelli JC. Interaction of HIV-1 Nef with the cellular dileucine-based sorting pathway is required for CD4 down-regulation and optimal viral infectivity. Proc Natl Acad Sci USA 1998; 95(19): 11229-34.
[] [PMID: 9736718]
Greenberg M, DeTulleo L, Rapoport I, Skowronski J, Kirchhausen T. A dileucine motif in HIV-1 Nef is essential for sorting into clathrin-coated pits and for downregulation of CD4. Curr Biol 1998; 8(22): 1239-42.
[] [PMID: 9811611]
Hodge DR, Dunn KJ, Pei GK, et al. Binding of c-Raf1 kinase to a conserved acidic sequence within the carboxyl-terminal region of the HIV-1 Nef protein. J Biol Chem 1998; 273(25): 15727-33.
[] [PMID: 9624170]
Korber B, Gaschen B, Yusim K, Thakallapally R, Kesmir C, Detours V. Evolutionary and immunological implications of contemporary HIV-1 variation. Br Med Bull 2001; 58: 19-42.
[] [PMID: 11714622]
Hemelaar J, Gouws E, Ghys PD, Osmanov S. WHO-UNAIDS Network for HIV Isolation and Characterisation. Global trends in molecular epidemiology of HIV-1 during 2000-2007. AIDS 2011; 25(5): 679-89.
[] [PMID: 21297424]
Gupta N, Banerjea AC. C-terminal half of HIV-1 Vif C possesses major determinant for APOBEC3G degradation. AIDS 2009; 23(1): 141-3.
[] [PMID: 19050398]
Ronsard L, Lata S, Singh J, Ramachandran VG, Das S, Banerjea AC. Molecular and genetic characterization of natural HIV-1 Tat Exon-1 variants from North India and their functional implications. PLoS One 2014; 9(1): e85452.
[] [PMID: 24465566]
Ronsard L, Raja R, Panwar V, et al. Genetic and functional characterization of HIV-1 Vif on APOBEC3G degradation: First report of emergence of B/C recombinants from North India. Sci Rep 2015; 5: 15438.
[] [PMID: 26494109]
Wei BL, Arora VK, Foster JL, Sodora DL, Garcia JV. In vivo analysis of Nef function. Curr HIV Res 2003; 1(1): 41-50.
[] [PMID: 15043211]
Garcia JV, Miller AD. Serine phosphorylation-independent downregulation of cell-surface CD4 by nef. Nature 1991; 350(6318): 508-11.
[] [PMID: 2014052]
Schwartz O, Maréchal V, Le Gall S, Lemonnier F, Heard JM. Endocytosis of major histocompatibility complex class I molecules is induced by the HIV-1 Nef protein. Nat Med 1996; 2(3): 338-42.
[] [PMID: 8612235]
Lamers SL, Fogel GB, Singer EJ, et al. HIV-1 Nef in macrophage-mediated disease pathogenesis. Int Rev Immunol 2012; 31(6): 432-50.
[] [PMID: 23215766]

Rights & Permissions Print Export Cite as
© 2022 Bentham Science Publishers | Privacy Policy