Human immunodeficiency virus type 1 envelope proteins traffic toward virion assembly sites via a TBC1D20/Rab1-regulated pathway
© Nachmias et al; licensee BioMed Central Ltd. 2012
Received: 18 October 2011
Accepted: 19 January 2012
Published: 19 January 2012
The cellular activity of many factors and pathways is required to execute the complex replication cycle of the human immunodeficiency virus type 1 (HIV-1). To reveal these cellular components, several extensive RNAi screens have been performed, listing numerous 'HIV-dependency factors'. However, only a small overlap between these lists exists, calling for further evaluation of the relevance of specific factors to HIV-1 replication and for the identification of additional cellular candidates. TBC1D20, the GTPase-activating protein (GAP) of Rab1, regulates endoplasmic reticulum (ER) to Golgi trafficking, was not identified in any of these screens, and its involvement in HIV-1 replication cycle is tested here.
Excessive TBC1D20 activity perturbs the early trafficking of HIV-1 envelope protein through the secretory pathway. Overexpression of TBC1D20 hampered envelope processing and reduced its association with detergent-resistant membranes, entailing a reduction in infectivity of HIV-1 virion like particles (VLPs).
These findings add TBC1D20 to the network of host factors regulating HIV replication cycle.
KeywordsHIV-1 envelope assembly TBCID20 Rab1 secretory pathway
Numerous host factors regulate, directly and indirectly, different steps of HIV-1 infection. To reveal these factors, several extensive RNAi screens have been performed, listing over a thousand proteins [1–5]. Surprisingly, there is only a small overlap between these lists, calling for further evaluation of the relevance of specific factors to HIV-1 replication [6, 7]. The small GTPase Rab1 has been marked in one of these screens as a putative HIV-dependency factor . Here we identified TBC1D20, a specific GAP of this Rab [8, 9], as a new host factor that regulates HIV replication.
Rab1, which cycles between active GTP-bound and inactive GDP-bound forms , and is present as Rab1a/b isoforms, regulates the early secretory pathway by controlling ER to Golgi traffic ; yet, unconventional Rab1-independent secretion pathway(s) have also been described [12–14]. TBC1D20, a Rab1-GAP, inactivates Rab1 through the stimulation of GTP hydrolysis; accordingly, TBC1D20 overproduction blocks Rab1-mediated ER-to-Golgi transport [8, 9]. The utilization of the Rab1-dependent secretory pathway by HIV-1 and the influence of TBC1D20/Rab1 axis on its infectivity remain unexplored.
Because of the profound effect of TBC1D20 overexpression on gp41 migration (JRCSF strain), we expanded this analysis to the JRFL (R5 strain) and LAI (X4 strain) envelope proteins [22, 24, 25] and to the VSV-G. TBC1D20 overexpression resulted in abnormal migration of all these proteins (Figure 2B). This result is in line with TBC1D20 effect on ER-to-Golgi trafficking of VSV-G [8, 9] and with the reduced infectivity of HIV-1 VLPs pseudotyped with VSV-G (Figure 1A). Overall, these results indicate the generality of TBC1D20 activity on the cellular processing of the envelope glycoproteins of these viruses. In accord with this conclusion, TBC1D20 overexpression also resulted in abnormal migration of the transforming growth factor beta receptor II (TGFβ receptor II) - a glycosylated cellular protein that traffics through the secretory pathway to the plasma membrane (data not shown).
The above effect of TBC1D20 on the migration of envelope glycoproteins is expected to reflect a lack of Rab1 activity - the target of TBC1D20. We next examined if direct modulations of Rab1 activity will similarly affect gp41 migration. We transfected cells with the JRCSF Env and Gag-Pol proteins (as in Figure 2A) and either TBC1D20, R105A, Rab1a, Rab1b, constitutively-active Rab1b (Q67L)  or the dominant negative (DN) forms of Rab1a (N124I) or Rab1b (N121I) . All of these proteins were expressed as GFP fusions , accordingly a GFP only negative control was used. All treatments, except for the negative controls (R105A or GFP), induced the aberrant migration of gp41 (Figure 2C). The DN activity of N124I and N121I, similar to TBC1D20 overexpression, reduces Rab1 function and thus the aberrant migration of gp41 is expected. Surprisingly, overexpression of Rab1a, Rab1b or Q67L, which enhances Rab1 activity, also resulted in abnormal gp41 migration. An explanation for this might be the observation that excesses of Rab1 activity increase ER-to-Golgi transport , and this too may impair proper glycosylation. Thus, a precise regulation of the Rab1/TBC1D20 axis may be necessary for proper gp41 processing.
Overall, TBC1D20 may affect virus replication cycle either directly, as in the case of the hepatitis C virus where interaction of TBC1D20 with the viral protein NS5A is required for viral RNA replication [9, 40]; or indirectly, as described here for HIV-1 and recently for herpes simplex virus , where TBC1D20 affects the ER-trafficking of the viral envelope glycoproteins.
To summarize, here, we showed that enhancement of TBC1D20 activity, a negative regulator of Rab1 function, perturbs HIV-1 infectivity. This adds the TBC1D20/Rab1 axis to other identified factors of the secretory pathway that influence HIV replication cycle, such as Rab5, 6a, 7, 9 and 11a, [1, 42–45]; and places TBC1D20 in the network of host regulators of the late stages of HIV infection.
We thank Drs. Andrea Cimarelli, Jonathan Gershoni, Mia Horowitz, and Yechiel Shai for providing various reagents. The following reagents were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: Chessie 8 hybridoma from Dr. George Lewis and the HIV-1 p24 Hybridoma (183-H12-5C) from Dr. Bruce Chesebro. This work was supported in part by the Israel Science Foundation (grant 169/09); the Ela Kodesz Institute for Research on Cancer Development and Prevention; the Jakov, Marianna and Jorge Saia Scholarship Fund for HIV and Parkinson Diseases Research; and the Tel-Aviv University Recanati research fund.
- Brass AL, Dykxhoorn DM, Benita Y, Yan N, Engelman A, Xavier RJ, Lieberman J, Elledge SJ: Identification of host proteins required for HIV infection through a functional genomic screen. Science. 2008, 319: 921-926. 10.1126/science.1152725.View ArticlePubMedGoogle Scholar
- Konig R, Zhou Y, Elleder D, Diamond TL, Bonamy GM, Irelan JT, Chiang CY, Tu BP, De Jesus PD, Lilley CE, et al: Global analysis of host-pathogen interactions that regulate early-stage HIV-1 replication. Cell. 2008, 135: 49-60. 10.1016/j.cell.2008.07.032.PubMed CentralView ArticlePubMedGoogle Scholar
- Rato S, Maia S, Brito PM, Resende L, Pereira CF, Moita C, Freitas RP, Moniz-Pereira J, Hacohen N, Moita LF, Goncalves J: Novel HIV-1 knockdown targets identified by an enriched kinases/phosphatases shRNA library using a long-term iterative screen in Jurkat T-cells. PLoS One. 5: e9276-Google Scholar
- Yeung ML, Houzet L, Yedavalli VS, Jeang KT: A genome-wide short hairpin RNA screening of jurkat T-cells for human proteins contributing to productive HIV-1 replication. J Biol Chem. 2009, 284: 19463-19473. 10.1074/jbc.M109.010033.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhou H, Xu M, Huang Q, Gates AT, Zhang XD, Castle JC, Stec E, Ferrer M, Strulovici B, Hazuda DJ, Espeseth AS: Genome-scale RNAi screen for host factors required for HIV replication. Cell Host Microbe. 2008, 4: 495-504. 10.1016/j.chom.2008.10.004.View ArticlePubMedGoogle Scholar
- Bushman FD, Malani N, Fernandes J, D'Orso I, Cagney G, Diamond TL, Zhou H, Hazuda DJ, Espeseth AS, Konig R, et al: Host cell factors in HIV replication: meta-analysis of genome-wide studies. PLoS Pathog. 2009, 5: e1000437-10.1371/journal.ppat.1000437.PubMed CentralView ArticlePubMedGoogle Scholar
- Goff SP: Knockdown screens to knockout HIV-1. Cell. 2008, 135: 417-420. 10.1016/j.cell.2008.10.007.PubMed CentralView ArticlePubMedGoogle Scholar
- Haas AK, Yoshimura S, Stephens DJ, Preisinger C, Fuchs E, Barr FA: Analysis of GTPase-activating proteins: Rab1 and Rab43 are key Rabs required to maintain a functional Golgi complex in human cells. J Cell Sci. 2007, 120: 2997-3010. 10.1242/jcs.014225.View ArticlePubMedGoogle Scholar
- Sklan EH, Serrano RL, Einav S, Pfeffer SR, Lambright DG, Glenn JS: TBC1D20 is a Rab1 GTPase-activating protein that mediates hepatitis C virus replication. J Biol Chem. 2007, 282: 36354-36361. 10.1074/jbc.M705221200.View ArticlePubMedGoogle Scholar
- Pfeffer S, Aivazian D: Targeting Rab GTPases to distinct membrane compartments. Nat Rev Mol Cell Biol. 2004, 5: 886-896.View ArticlePubMedGoogle Scholar
- Tisdale EJ, Bourne JR, Khosravi-Far R, Der CJ, Balch WE: GTP-binding mutants of rab1 and rab2 are potent inhibitors of vesicular transport from the endoplasmic reticulum to the Golgi complex. J Cell Biol. 1992, 119: 749-761. 10.1083/jcb.119.4.749.View ArticlePubMedGoogle Scholar
- Gee HY, Noh SH, Tang BL, Kim KH, Lee MG: Rescue of DeltaF508-CFTR Trafficking via a GRASP-Dependent Unconventional Secretion Pathway. Cell. 146: 746-760.Google Scholar
- Wu G, Zhao G, He Y: Distinct pathways for the trafficking of angiotensin II and adrenergic receptors from the endoplasmic reticulum to the cell surface: Rab1-independent transport of a G protein-coupled receptor. J Biol Chem. 2003, 278: 47062-47069. 10.1074/jbc.M305707200.View ArticlePubMedGoogle Scholar
- Yoo JS, Moyer BD, Bannykh S, Yoo HM, Riordan JR, Balch WE: Non-conventional trafficking of the cystic fibrosis transmembrane conductance regulator through the early secretory pathway. J Biol Chem. 2002, 277: 11401-11409. 10.1074/jbc.M110263200.View ArticlePubMedGoogle Scholar
- Swanstrom R, Wills JW: Synthesis, assembly, and processing of viral proteins. Retroviruses. Edited by: Coffin JM, Hughes SH, Varmus HE. 1997, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, 263-334.Google Scholar
- Ono A, Freed EO: Plasma membrane rafts play a critical role in HIV-1 assembly and release. Proc Natl Acad Sci USA. 2001, 98: 13925-13930. 10.1073/pnas.241320298.PubMed CentralView ArticlePubMedGoogle Scholar
- Yang P, Ai LS, Huang SC, Li HF, Chan WE, Chang CW, Ko CY, Chen SS: The cytoplasmic domain of human immunodeficiency virus type 1 transmembrane protein gp41 harbors lipid raft association determinants. J Virol. 84: 59-75.Google Scholar
- Naldini L, Blomer U, Gallay P, Ory D, Mulligan R, Gage FH, Verma IM, Trono D: In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science. 1996, 272: 263-267. 10.1126/science.272.5259.263.View ArticlePubMedGoogle Scholar
- Miyoshi H, Takahashi M, Gage FH, Verma IM: Stable and efficient gene transfer into the retina using an HIV-based lentiviral vector. Proc Natl Acad Sci USA. 1997, 94: 10319-10323. 10.1073/pnas.94.19.10319.PubMed CentralView ArticlePubMedGoogle Scholar
- Telesnitsky A, Blain S, Goff SP: Assays for retroviral reverse transcriptase. Methods Enzymol. 1995, 262: 347-362.View ArticlePubMedGoogle Scholar
- Platt EJ, Wehrly K, Kuhmann SE, Chesebro B, Kabat D: Effects of CCR5 and CD4 cell surface concentrations on infections by macrophagetropic isolates of human immunodeficiency virus type 1. J Virol. 1998, 72: 2855-2864.PubMed CentralPubMedGoogle Scholar
- Koyanagi Y, Miles S, Mitsuyasu RT, Merrill JE, Vinters HV, Chen IS: Dual infection of the central nervous system by AIDS viruses with distinct cellular tropisms. Science. 1987, 236: 819-822. 10.1126/science.3646751.View ArticlePubMedGoogle Scholar
- Yang YS, Strittmatter SM: The reticulons: a family of proteins with diverse functions. Genome Biol. 2007, 8: 234-10.1186/gb-2007-8-12-234.PubMed CentralView ArticlePubMedGoogle Scholar
- Munk C, Wei G, Yang OO, Waring AJ, Wang W, Hong T, Lehrer RI, Landau NR, Cole AM: The theta-defensin, retrocyclin, inhibits HIV-1 entry. AIDS Res Hum Retroviruses. 2003, 19: 875-881. 10.1089/088922203322493049.View ArticlePubMedGoogle Scholar
- Peden K, Emerman M, Montagnier L: Changes in growth properties on passage in tissue culture of viruses derived from infectious molecular clones of HIV-1LAI, HIV-1MAL, and HIV-1ELI. Virology. 1991, 185: 661-672. 10.1016/0042-6822(91)90537-L.View ArticlePubMedGoogle Scholar
- Wilson AL, Sheridan KM, Erdman RA, Maltese WA: Prenylation of a Rab1B mutant with altered GTPase activity is impaired in cell-free systems but not in intact mammalian cells. Biochem J. 1996, 318 (Pt 3): 1007-1014.PubMed CentralView ArticlePubMedGoogle Scholar
- Cooper AA, Gitler AD, Cashikar A, Haynes CM, Hill KJ, Bhullar B, Liu K, Xu K, Strathearn KE, Liu F, et al: Alpha-synuclein blocks ER-Golgi traffic and Rab1 rescues neuron loss in Parkinson's models. Science. 2006, 313: 324-328. 10.1126/science.1129462.PubMed CentralView ArticlePubMedGoogle Scholar
- Helenius A, Aebi M: Intracellular functions of N-linked glycans. Science. 2001, 291: 2364-2369. 10.1126/science.291.5512.2364.View ArticlePubMedGoogle Scholar
- Mizuochi T, Matthews TJ, Kato M, Hamako J, Titani K, Solomon J, Feizi T: Diversity of oligosaccharide structures on the envelope glycoprotein gp 120 of human immunodeficiency virus 1 from the lymphoblastoid cell line H9. Presence of complex-type oligosaccharides with bisecting N-acetylglucosamine residues. J Biol Chem. 1990, 265: 8519-8524.PubMedGoogle Scholar
- Ron I, Horowitz M: ER retention and degradation as the molecular basis underlying Gaucher disease heterogeneity. Hum Mol Genet. 2005, 14: 2387-2398. 10.1093/hmg/ddi240.View ArticlePubMedGoogle Scholar
- Trimble RB, Maley F: Optimizing hydrolysis of N-linked high-mannose oligosaccharides by endo-beta-N-acetylglucosaminidase H. Anal Biochem. 1984, 141: 515-522. 10.1016/0003-2697(84)90080-0.View ArticlePubMedGoogle Scholar
- Plummer TH, Elder JH, Alexander S, Phelan AW, Tarentino AL: Demonstration of peptide:N-glycosidase F activity in endo-beta-N-acetylglucosaminidase F preparations. J Biol Chem. 1984, 259: 10700-10704.PubMedGoogle Scholar
- Molloy SS, Thomas L, VanSlyke JK, Stenberg PE, Thomas G: Intracellular trafficking and activation of the furin proprotein convertase: localization to the TGN and recycling from the cell surface. EMBO J. 1994, 13: 18-33.PubMed CentralPubMedGoogle Scholar
- Earl PL, Moss B, Doms RW: Folding, interaction with GRP78-BiP, assembly, and transport of the human immunodeficiency virus type 1 envelope protein. J Virol. 1991, 65: 2047-2055.PubMed CentralPubMedGoogle Scholar
- Leonard CK, Spellman MW, Riddle L, Harris RJ, Thomas JN, Gregory TJ: Assignment of intrachain disulfide bonds and characterization of potential glycosylation sites of the type 1 recombinant human immunodeficiency virus envelope glycoprotein (gp120) expressed in Chinese hamster ovary cells. J Biol Chem. 1990, 265: 10373-10382.PubMedGoogle Scholar
- Fenouillet E, Gluckman JC, Bahraoui E: Role of N-linked glycans of envelope glycoproteins in infectivity of human immunodeficiency virus type 1. J Virol. 1990, 64: 2841-2848.PubMed CentralPubMedGoogle Scholar
- Abacioglu YH, Fouts TR, Laman JD, Claassen E, Pincus SH, Moore JP, Roby CA, Kamin-Lewis R, Lewis GK: Epitope mapping and topology of baculovirus-expressed HIV-1 gp160 determined with a panel of murine monoclonal antibodies. AIDS Res Hum Retroviruses. 1994, 10: 371-381. 10.1089/aid.1994.10.371.View ArticlePubMedGoogle Scholar
- Chan WE, Lin HH, Chen SS: Wild-type-like viral replication potential of human immunodeficiency virus type 1 envelope mutants lacking palmitoylation signals. J Virol. 2005, 79: 8374-8387. 10.1128/JVI.79.13.8374-8387.2005.PubMed CentralView ArticlePubMedGoogle Scholar
- Hauri HP, Schweizer A: The endoplasmic reticulum-Golgi intermediate compartment. Curr Opin Cell Biol. 1992, 4: 600-608. 10.1016/0955-0674(92)90078-Q.View ArticlePubMedGoogle Scholar
- Sklan EH, Staschke K, Oakes TM, Elazar M, Winters M, Aroeti B, Danieli T, Glenn JS: A Rab-GAP TBC domain protein binds hepatitis C virus NS5A and mediates viral replication. J Virol. 2007, 81: 11096-11105. 10.1128/JVI.01249-07.PubMed CentralView ArticlePubMedGoogle Scholar
- Zenner HL, Yoshimura S, Barr FA, Crump CM: Analysis of Rab GTPase-Activating Proteins Indicates that Rab1a/b and Rab43 Are Important for Herpes Simplex Virus 1 Secondary Envelopment. J Virol. 85: 8012-8021.Google Scholar
- Chu H, Wang JJ, Spearman P: HIV Interactions with Host Cell Proteins. Current Topics in Microbiology and Immunology. Edited by: Spearman P. 2009, Freed EO: Springer, 339: 78-80.Google Scholar
- Murray JL, Mavrakis M, McDonald NJ, Yilla M, Sheng J, Bellini WJ, Zhao L, Le Doux JM, Shaw MW, Luo CC, et al: Rab9 GTPase is required for replication of human immunodeficiency virus type 1, filoviruses, and measles virus. J Virol. 2005, 79: 11742-11751. 10.1128/JVI.79.18.11742-11751.2005.PubMed CentralView ArticlePubMedGoogle Scholar
- Nydegger S, Foti M, Derdowski A, Spearman P, Thali M: HIV-1 egress is gated through late endosomal membranes. Traffic. 2003, 4: 902-910. 10.1046/j.1600-0854.2003.00145.x.View ArticlePubMedGoogle Scholar
- Vidricaire G, Tremblay MJ: Rab5 and Rab7, but not ARF6, govern the early events of HIV-1 infection in polarized human placental cells. J Immunol. 2005, 175: 6517-6530.View ArticlePubMedGoogle Scholar
- Chesebro B, Wehrly K, Nishio J, Perryman S: Macrophage-tropic human immunodeficiency virus isolates from different patients exhibit unusual V3 envelope sequence homogeneity in comparison with T-cell-tropic isolates: definition of critical amino acids involved in cell tropism. J Virol. 1992, 66: 6547-6554.PubMed CentralPubMedGoogle Scholar
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