The Structural Interface between HIV-1 Vif and Human APOBEC3H

Abstract

Human APOBEC3H (A3H) is a cytidine deaminase that inhibits HIV-1 replication. To evade this restriction, the HIV-1 Vif protein binds A3H and mediates its proteasomal degradation. To date, little information on the Vif-A3H interface has been available. To decipher how both proteins interact, we first mapped the Vifbinding site on A3H by functionally testing a large set of A3H mutants in singlecycle infectivity and replication assays. Our data show that the two A3H ␣-helixes ␣3 and ␣4 represent the Vif-binding site of A3H. We next used viral adaptation and a set of Vif mutants to identify novel, reciprocal Vif variants that rescued viral infectivity in the presence of two Vif-resistant A3H mutants. These A3H-Vif interaction points were used to generate the first A3H-Vif structure model, which revealed that the A3H helixes ␣3 and ␣4 interact with the Vif ␤-sheet (␤2-␤5). This model is in good agreement with previously reported Vif and A3H amino acids important for interaction. Based on the predicted A3H-Vif interface, we tested additional points of contact, which validated our model. Moreover, these experiments showed that the A3H and A3G binding sites on HIV-1 Vif are largely distinct, with both host proteins interacting with Vif ␤-strand 2. Taken together, this virus-host interface model explains previously reported data and will help to identify novel drug targets to combat HIV-1 infection.IMPORTANCE HIV-1 needs to overcome several intracellular restriction factors in order to replicate efficiently. The human APOBEC3 locus encodes seven proteins, of which A3D, A3F, A3G, and A3H restrict HIV-1. HIV encodes the Vif protein, which binds to the APOBEC3 proteins and leads to their proteasomal degradation. No HIV-1 Vif-APOBEC3 costructure exists to date despite extensive research. We and others previously generated HIV-1 Vif costructure models with A3G and A3F by mapping specific contact points between both proteins. Here, we applied a similar approach to HIV-1 Vif and A3H and successfully generated a Vif-A3H interaction model. Importantly, we find that the HIV-1 Vif-A3H interface is distinct from the Vif-A3G and Vif-A3F interfaces, with a small Vif region being important for recognition of both A3G and A3H. Our Vif-A3H structure model informs on how both proteins interact and could guide toward approaches to block the Vif-A3H interface to target HIV replication.KEYWORDS HIV-1, APOBEC3H, Vif, restriction factor, APOBEC3, A3H, HIV T he human APOBEC3 family consists of seven distinct proteins (A3A to A3H), several of which have potent activity against HIV-1 (1, 2). During infection of the new cell, virion-incorporated APOBEC3 proteins target the process of reverse transcription through directly deaminating the viral single-stranded DNA as well as by deaminaseindependent mechanisms (3-6). In turn, HIV-1 counteracts APOBEC3 restriction by

Recurrent Loss of APOBEC3H Activity during Primate Evolution

“…Haplotypes with a CGRELP motif compose one clade on the tree, while the other has an SRQKRQ motif. Residues 127 and 128 are located on the α4 helix, which has been implicated in A3H-Vif binding (35, 36). Furthermore, the regions of A3H with the greatest number of nonsynonymous mutations are in the predicted loops 1 and 7, and the α6 helix (Table 1), which were recently shown by structural studies to be involved in an interaction between A3H and a co-crystalized RNA (32–34).…”

Biochemical Basis of APOBEC3 Deoxycytidine Deaminase Activity on Diverse DNA Substrates

“…A3H was previously thought to have a unique interface to A3G however these residues identified overlap with the residues known for A3G. A3H can also interact with Vif on unique residues at position 63 and 90 [147].…”

“…Studies have identified that the 14DRMR17 motif that interacts with A3F is also the interface for the interaction with A3C and A3D [144,146]. Most recently, studies have shown that A3H interacts with Vif through another unique interface composed of residues on the β-sheet of Vif, (residues 40-44) [147]. A3H was previously thought to have a unique interface to A3G however these residues identified overlap with the residues known for A3G.…”

“…Vif interacts with A3s through three interfaces on the A3s that are grouped as an A3G, A3F/C/D or A3H interface (Figure 1.9B) [143,[146][147][148]. Although the A3F/C/D interaction site is identified as one, there are small differences in the amino acid interactions for each A3, but overall the interface is largely the same [146,149].…”

Studying Evolutionary Adaptation of MERS-CoV

“…While the majority of these mutations will have neutral or negative effects on viral fitness, a small subset of these mutations may prove beneficial and enhance the ability for certain variants to replicate despite selective pressures of interest such as the host immune response or an antiviral drug. Forced adaptation experiments have been used to determine viral mutations that facilitate escape from drugs [4][5][6], monoclonal antibodies [7,8], host restriction factors [9][10][11], and species variation in host receptors [12][13][14] and to elucidate various viral mechanisms of infection and replication [15][16][17].…”