Identification of 81LGxGxxIxW89 and 171EDRW174 Domains from Human Immunodeficiency Virus Type 1 Vif That Regulate APOBEC3G and APOBEC3F Neutralizing Activity

The human cytidine deaminases APOBEC3G (A3G) and APOBEC3F (A3F) potently restrict human immunodeficiency virus type 1 (HIV-1) replication, but they are neutralized by the viral protein Vif. Vif bridges A3G and A3F with a Cullin 5 (Cul5)-based E3 ubiquitin ligase and mediates their proteasomal degradation. This mechanism has been extensively studied, and several Vif domains have been identified that are critical for A3G and A3F neutralization. Here, we identified two additional domains. Via sequence analysis of more than 2,000 different HIV-1 Vif proteins, we identified two highly conserved amino acid sequences, ⁸¹LGxGxSIEW⁸⁹ and ¹⁷¹EDRWN¹⁷⁵. Within the ⁸¹LGxGxSIEW⁸⁹ sequence, residues L81, G82, G84, and, to a lesser extent, I87 and W89 play very critical roles in A3G/A3F neutralization. In particular, residues L81 and G82 determine Vif binding to A3F, residue G84 determines Vif binding to both A3G and A3F, and residues ⁸⁶SIEW⁸⁹ affect Vif binding to A3F, A3G, and Cul5. Accordingly, this ⁸¹LGxGxSIEW⁸⁹ sequence was designated the ⁸¹LGxGxxIxW⁸⁹ domain. Within the ¹⁷¹EDRWN¹⁷⁵ sequence, all residues except N175 are almost equally important for regulation of A3F neutralization, and consistently, they determine Vif binding only to A3F. Accordingly, this domain was designated ¹⁷¹EDRW¹⁷⁴. The LGxGxxIxW domain is also partially conserved in simian immunodeficiency virus Vif from rhesus macaques (SIVmac239) and has a similar activity. Thus, ⁸¹LGxGxxIxW⁸⁹ and ¹⁷¹EDRW¹⁷⁴ are two novel functional domains that are very critical for Vif function. They could become new targets for inhibition of Vif activity during HIV replication.The function of the lentiviral protein Vif is to neutralize the major host antiretroviral cytidine deaminases that belong to the APOBEC (apolipoprotein B mRNA-editing catalytic polypeptide) family, as recently reviewed by several investigators (18, 29, 31). This family consists of APOBEC1; activation-induced deaminase (AID); APOBEC2; a subgroup of APOBEC3 (A3) proteins, including A3A, A3B, A3C, A3DE, A3F, A3G, and A3H; and APOBEC4 in humans. They have one or two copies of a cytidine deaminase (CDA) domain with a signature motif (HxEx_23-28PCx_2-4C), only one of which normally has deaminase activity.All seven A3 genes have been shown to inhibit replication of various types of retrovirus via cytidine deamination-dependent or -independent mechanisms. In particular, human A3B, A3DE, A3F, A3G, and A3H inhibit human immunodeficiency virus type 1 (HIV-1) replication, whereas A3A and A3C do not (1, 3, 5, 26, 33, 37). Among these proteins, the expression of human A3G and A3F in vivo has been demonstrated, and in vitro studies indicate that they have the most potent anti-HIV-1 activity. A3G and A3F share ∼50% sequence similarity but have different biochemical properties (32) and different target sequence preferences while catalyzing cytidine deamination of viral cDNAs (13). Expression of human A3B has not been detected (7), and a 29.5-kb deletion spanning from the 3′ end of the A3A gene to the 8th exon of the A3B gene, leading to the complete removal of the A3B gene, has been detected in certain human populations (12). Human A3H is also poorly expressed in vivo (20). It was reported that human A3H has four haplotypes (Hap I, II, III, and IV), and only Hap II, which is maintained primarily in African populations, could be stably expressed in vitro (19). However, expression of this protein has not been detected in any human populations. Thus, the primary function of HIV-1 Vif is to neutralize A3G, A3F, and, to a lesser extent, A3DE.Vif hijacks cellular proteasomal machinery to destroy these host cytidine deaminases by protein degradation (15, 27, 30). Vif acts as an adaptor protein that bridges A3 proteins with a Cullin 5-based E3 ubiquitin ligase complex, which includes Cul5, Elongin B (EloB), and Elongin C (EloC) (35). Vif has a BC-box motif (¹⁴⁴SLQYLALA¹⁴⁹) that binds to EloC (16, 36) and an HCCH motif (¹⁰⁸Hx₅Cx_17-18Cx_3-5H¹³⁹) that binds to Cul5 (14, 17, 34). On the other hand, Vif also interacts with A3G and A3F. As a consequence of these interactions, A3G and A3F are polyubiquitylated and directed to 26S proteasomes for degradation. In addition, Vif may also inhibit A3 activity independently of proteasomal degradation (10, 11, 24).Interactions between Vif and A3G/A3F are a key step for their proteasomal degradation, and this mechanism has been extensively studied. First, unique surfaces in A3G and A3F important for Vif interaction were identified, and interestingly, they are located in different regions of the two proteins (9, 23). Second, several discontinuous surfaces on Vif have been found to regulate A3G and/or A3F degradation. The ⁴⁰YRHHY⁴⁴ domain specifically binds to A3G and determines Vif specificity for A3G (22); the ¹¹WxxDRMR¹⁷ and ⁷⁴TGERxW⁷⁹ domains specifically bind to A3F and determine Vif specificity for A3F (8, 22); and the ²¹WxSLVK²⁶, ⁵⁵VxIPLx₄L⁶⁴, and ⁶⁹YxxL⁷²domains determine Vif specificity for both A3G and A3F (2, 6, 8, 21). These results indicate that the mechanism that regulates Vif recognition of A3G and A3F is quite complicated, and understanding this mechanism is critical for pharmaceutical protection of A3G and A3F from Vif-mediated proteasomal degradation.Based on our current knowledge of these functional domains, it has been thought that Vif interacts with A3G and A3F mainly via its N-terminal region and with Cul5 E3 ubiquitin ligase machinery via its C-terminal region. However, here we identify a new A3G and A3F regulatory domain from the central region and a new A3F regulatory domain from the C-terminal region of HIV-1 Vif. Our results indicate that A3G and A3F interaction surfaces on HIV-1 Vif are structurally complex, and more efforts are required for a complete understanding of this host-pathogen interactive mechanism.