Differential Anti-APOBEC3G Activity of HIV-1 Vif Proteins Derived from Different Subtypes

Abstract

Antiretroviral cytidine deaminase APOBEC3G, which is abundantly expressed in peripheral blood lymphocytes and macrophages, strongly protects these cells against HIV-1 infection. The HIV-1 Vif protein overcomes this antiviral effect by enhancing proteasome-mediated APOBEC3G degradation and is key for maintaining viral infectivity. The 579-bp-long vif gene displays high genetic diversity among HIV-1 subtypes. Therefore, it is intriguing to address whether Vif proteins derived from different subtypes differ in their viral defense activity against APOBEC3G. Expression plasmids encoding Vif proteins derived from subtypes A, B, C, CRF01_AE, and CRF02_AG isolates were created, and their anti-APOBEC3G activities were compared. Viruses produced from cells expressing APOBEC3G and Vif proteins from different subtypes showed relatively different viral infectivities. Notably, subtype C-derived Vif proteins tested had the highest activity against APOBEC3G that was ascribed to its increased binding activity, for which the N-terminal domain of the Vif protein sequences was responsible. These results suggest that the biological differences of Vif proteins belonging to different subtypes might affect viral fitness and quasispecies in vivo.Among the seven human APOBEC3 3 cytidine deaminase proteins (from A to H) that act as intrinsic restriction factors against endogenous and exogenous retroviruses (1-9), APOBEC3G provides the most potent retroviral restriction in vitro and in vivo (10 -12). This host protein is abundantly expressed in peripheral blood mononuclear cells (PBMCs) and macrophages. APOBEC3G deaminates deoxycytidine to deoxyuridine in nascent viral minus-strand cDNA, thereby inducing G-to-A hypermutations during reverse transcription (13-16). It also partially restricts viral replication in a deamination-independent fashion, mainly by blocking DNA synthesis (17)(18)(19)(20). Human immunodeficiency virus type 1 (HIV-1) is armed with Vif 3 protein, which induces proteasome-mediated APOBEC3G degradation (21-24) via a mechanism involving the Cullin5 (Cul5)-containing E3 ubiquitin ligase (25-28). As a result, Vif protein reduces virion incorporation of APOBEC3G in virus-producer cells (29 -32), leading to efficient reverse transcription in the target cells. By the same mechanism, Vif protein can inactivate APOBEC3F and APOBEC3DE, which are expressed in PBMCs and suppress Vif-deficient HIV-1 infection to a lesser extent than does APOBEC3G (33-35).Several in vivo studies have demonstrated that APOBEC3G-induced G-to-A hypermutation is frequently observed in patient-derived proviral DNA (36 -42) even in the presence of full-length but polymorphic vif genes (43). The vif genes also have high in vivo genetic variability (11, 44 -46) and subtypedependent amino acid substitutions (47). These findings imply that the sequence diversity of vif genes (possibly in a subtypedependent manner) might harbor differential levels of anti-APOBEC3G activity. Among the strains tested in the present study, Vif protein derived from subtype C strain...

Determinants of Efficient Degradation of APOBEC3 Restriction Factors by HIV-1 Vif

“…HXB2 . A3G degradation is induced by all HIV Vif variants (referred to as Vif) tested in the literature (22,41,47,57), including the Vif IIIB and Vif HXB2 used in our study ( Fig. 2A).…”

Anti-APOBEC3G Activity of HIV-1 Vif Protein Is Attenuated in Elite Controllers

“…At the least, we found that Vifinduced intracellular degradation of APOBEC3G could be one of the possible mechanisms of attenuated Vif function in EC. Although we did not evaluate Vif's interactions with other APOB EC3G proteins, the intrinsic potencies of other APOBEC3 proteins (such as APOBEC3DE/3F/3H) appear to be lower than those of APOBEC3G (46,(72)(73)(74)(75). Moreover, we did not observe interpatient differences in Vif-mediated infectivity in control experiments where the APOBEC3G expression plasmid was not delivered into 293 T cells (data not shown).…”

“…In addition, second-round vif amplicons were digested with KpnI and MscI and cloned into pCAGGS-FLAG-RRE (a FLAG-tagged Vif expression plasmid containing a Rev-responsive element), as previously described (46). A single representative clone per patient, exhibiting an amino acid sequence identical to that of the original bulk sequence, was selected for subsequent analysis.…”

“…Briefly, to prepare VSV-G-pseudotyped HIV-1 luciferase reporter viruses, 1.75 ϫ 10 5 293T cells were cotransfected with 12.5 ng of pCA-hA3G-HA, 50 ng of VSV-G expression plasmid pHIT/G, 4 ng of the patient-derived Vif expression plasmid, and 0.43 g of an empty vector (to adjust the total DNA amount to reach 1 g) together with 500 ng of pNL-Luc-vif(-)env(-) using FuGENE 6. The amount of APOBEC3G expression plasmid was optimized to physiologically relevant levels (46). After medium exchange and DNase treatment, pseudoviruses were harvested 48 h following transfection.…”

“…The viral infectivity assay for comparing the levels of anti-APOB EC3G activity of Vif has been previously described (46). Briefly, to prepare VSV-G-pseudotyped HIV-1 luciferase reporter viruses, 1.75 ϫ 10 5 293T cells were cotransfected with 12.5 ng of pCA-hA3G-HA, 50 ng of VSV-G expression plasmid pHIT/G, 4 ng of the patient-derived Vif expression plasmid, and 0.43 g of an empty vector (to adjust the total DNA amount to reach 1 g) together with 500 ng of pNL-Luc-vif(-)env(-) using FuGENE 6.…”