- Short report
- Open Access
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.
- 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.
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