Vif Proteins from Diverse Human Immunodeficiency Virus/Simian Immunodeficiency Virus Lineages Have Distinct Binding Sites in A3C

Abstract

Lentiviruses have evolved the Vif protein to counteract APOBEC3 (A3) restriction factors by targeting them for proteasomal degradation. Previous studies have identified important residues in the interface of human immunodeficiency virus type 1 (HIV-1) Vif and human APOBEC3C (hA3C) or human APOBEC3F (hA3F). However, the interaction between primate A3C proteins and HIV-1 Vif or natural HIV-1 Vif variants is still poorly understood. Here, we report that HIV-1 Vif is inactive against A3Cs of rhesus macaques (rhA3C), sooty mangabey monkeys (smmA3C), and African green monkeys (agmA3C), while HIV-2, African green monkey simian immunodeficiency virus (SIVagm), and SIVmac Vif proteins efficiently mediate the depletion of all tested A3Cs. We identified that residues N/H130 and Q133 in rhA3C and smmA3C are determinants for this HIV-1 Vif-triggered counteraction. We also found that the HIV-1 Vif interaction sites in helix 4 of hA3C and hA3F differ. Vif alleles from diverse HIV-1 subtypes were tested for degradation activities related to hA3C. The subtype F-1 Vif was identified to be inactive for degradation of hA3C and hA3F. The residues that determined F-1 Vif inactivity in the degradation of A3C/A3F were located in the C-terminal region (K167 and D182). Structural analysis of F-1 Vif revealed that impairing the internal salt bridge of E171-K167 restored reduction capacities to A3C/A3F. Furthermore, we found that D101 could also form an internal interaction with K167. Replacing D101 with glycine and R167 with lysine in NL4-3 Vif impaired its counteractivity to A3F and A3C. This finding indicates that internal interactions outside the A3 binding region in HIV-1 Vif influence the capacity to induce degradation of A3C/A3F. IMPORTANCEThe APOBEC3 restriction factors can serve as potential barriers to lentiviral cross-species transmissions. Vif proteins from lentiviruses counteract APOBEC3 by proteasomal degradation. In this study, we found that monkey-derived A3C, rhA3C and smmA3C, were resistant to HIV-1 Vif. This was determined by A3C residues N/H130 and Q133. However, HIV-2, SIVagm, and SIVmac Vif proteins were found to be able to mediate the depletion of all tested primate A3C proteins. In addition, we identified a natural HIV-1 Vif (F-1 Vif) that was inactive in the degradation of hA3C/hA3F. Here, we provide for the first time a model that explains how an internal salt bridge of E171-K167-D101 influences Vif-mediated degradation of hA3C/hA3F. This finding provides a novel way to develop HIV-1 inhibitors by targeting the internal interactions of the Vif protein. S imian immunodeficiency virus (SIV) naturally infects manyOld World primate species in Africa. The pandemic of human immunodeficiency virus (HIV) originated from cross-species transmission events of SIVs to humans. HIV-1 was introduced into the human population by multiple transmissions of a chimpanzee (cpz) virus, which is known as SIVcpz. The less virulent human lentivirus, HIV-2, was derived from SIVsmm, which was obtained from sooty mangabey monkeys (sm...

Loop 1 of APOBEC3C Regulates its Antiviral Activity against HIV-1

“…The study revealed several key residues in the hydrophobic V-shaped groove formed by the 2 and 3 helices of A3C that facilitate Vif binding resulting in proteasome-mediated degradation of A3C [63]. We extended this finding and identified additional Vif interaction sites in 4 helix of A3C [64]. Other than a previous study that predicted putative DNA substrate binding pockets [52], biochemical and structural aspects of A3C enzymatic activity and their relevance for antiviral activity are not well investigated to date [3,4].…”

Stably expressed APOBEC3H forms a barrier for cross-species transmission of simian immunodeficiency virus of chimpanzee to humans

“…Chimpanzee APOBEC3 (A3) expression plasmids (A3D, A3F, A3G and A3H) were provided by Michael Emerman [ 40 ], chimpanzee A3C plasmid was described recently [ 61 ]. Human A3s (A3A-A3H) were expressed by PTR600 vector with a carboxyl-terminal triple hemaggutinin (HA) tag [ 33 ].…”

“…To generate the SIV Vif expression plasmids, Vif fragments from the following molecular clones: SIVcpz Ptt EK505 (DQ373065), Gab1 (X52154), MB897 (EF535994) and SIVcpz Pts TAN1 (AF447763), TAN2 (DQ374657) and SIVgor CP2139 (FJ424866) were amplified and inserted into pCRV1 by EcoRI and NotI . Vif expression plasmids of HIV-1 LAI, F-1, N-116 and O-127 were provided by Viviana Simon [ 33 , 61 ]. All SIVcpz Ptt MB897 Vif mutants were generated by overlapping PCR and cloned into pCRV1 without any tag, verified by sequencing.…”

Feline APOBEC3s, Barriers to Cross-Species Transmission of FIV?

“… Schematic representation of A3-Vif interaction sites. ( A ) HIV-1 Vif binding sites in human A3C, A3F, A3G, and A3H [ 60 , 94 , 95 , 96 , 97 ]; ( B ) FIV, HIV-2, SIVmac, and SIVsmm Vifs binding sites in feline A3Z2, A3Z3, and A3Z2Z3, respectively [ 79 ]; ( C ) The human A3F, A3G, A3H, CUL5, CBF-β, and ElonginB/C binding sites in HIV-1 Vif (see review [ 98 ]); ( D ) The feline A3Z2, A3Z3, and CUL5 binding sites in FIV Vif [ 46 ]. FIV Vif domains that interact with additional cofactors and sites for oligomerization are not identified.…”