Mechanism of Enhanced HIV Restriction by Virion Coencapsidated Cytidine Deaminases APOBEC3F and APOBEC3G

Abstract

The APOBEC3 (A3) enzymes, A3G and A3F, are coordinately expressed in CD4 ϩ T cells and can become coencapsidated into HIV-1 virions, primarily in the absence of the viral infectivity factor (Vif). A3F and A3G are deoxycytidine deaminases that inhibit HIV-1 replication by inducing guanine-to-adenine hypermutation through deamination of cytosine to form uracil in minus-strand DNA. The effect of the simultaneous presence of both A3G and A3F on HIV-1 restriction ability is not clear. Here, we used a single-cycle infectivity assay and biochemical analyses to determine if coencapsidated A3G and A3F differ in their restriction capacity from A3G or A3F alone. Proviral DNA sequencing demonstrated that compared to each A3 enzyme alone, A3G and A3F, when combined, had a coordinate effect on hypermutation. Using size exclusion chromatography, rotational anisotropy, and in vitro deamination assays, we demonstrate that A3F promotes A3G deamination activity by forming an A3F/G hetero-oligomer in the absence of RNA which is more efficient at deaminating cytosines. Further, A3F caused the accumulation of shorter reverse transcripts due to decreasing reverse transcriptase efficiency, which would leave singlestranded minus-strand DNA exposed for longer periods of time, enabling more deamination events to occur. Although A3G and A3F are known to function alongside each other, these data provide evidence for an A3F/G hetero-oligomeric A3 with unique properties compared to each individual counterpart.IMPORTANCE The APOBEC3 enzymes APOBEC3F and APOBEC3G act as a barrier to HIV-1 replication in the absence of the HIV-1 Vif protein. After APOBEC3 enzymes are encapsidated into virions, they deaminate cytosines in minus-strand DNA, which forms promutagenic uracils that induce transition mutations or proviral DNA degradation. Even in the presence of Vif, footprints of APOBEC3-catalyzed deaminations are found, demonstrating that APOBEC3s still have discernible activity against HIV-1 in infected individuals. We undertook a study to better understand the activity of coexpressed APOBEC3F and APOBEC3G. The data demonstrate that an APOBEC3F/ APOBEC3G hetero-oligomer can form that has unique properties compared to each APOBEC3 alone. This hetero-oligomer has increased efficiency of virus hypermutation, raising the idea that we still may not fully realize the antiviral mechanisms of endogenous APOBEC3 enzymes. Hetero-oligomerization may be a mechanism to increase their antiviral activity in the presence of Vif.

Biochemical Basis of APOBEC3 Deoxycytidine Deaminase Activity on Diverse DNA Substrates

“…Reprinted with permission from Adolph et al, 2018 [1]. [74,108,117,118]. Similar data has been found using in vitro substrates for A3A that also deaminate at 5'TTC [119].…”

“…The C→U deaminations catalyzed on the (-) DNA strand become G→A mutations when reverse transcriptase is forced to use uracil as a template during (+) DNA strand synthesis (Figure 1.2B) [67][68][69][70]. Throughout this process, the A3 enzymes also directly disrupt proviral DNA synthesis by oligomerizing on the template DNA resulting in a "roadblock" for the reverse transcriptase [71][72][73][74]. In addition, A3G can interact with reverse transcriptase directly and inhibit polymerase activity [71,75].…”

“…The ability to induce stop codons arises Depending on the surrounding sequence context, A3F may also be able to induce a stop codon (Figure 1.8A). However, in proviral DNA sequences, the A3G-induced tryptophan mutation to a stop codon is recovered more often than the A3F-induced tryptophan mutation to a stop codon [74,108] (Figure 1.8B). A3F is more likely to induce a variety of missense (amino acid changing) mutations than A3G, which primarily causes mutations at glycine codons ( Figure 1.7B) [74,108].…”

“…However, in proviral DNA sequences, the A3G-induced tryptophan mutation to a stop codon is recovered more often than the A3F-induced tryptophan mutation to a stop codon [74,108] (Figure 1.8B). A3F is more likely to induce a variety of missense (amino acid changing) mutations than A3G, which primarily causes mutations at glycine codons ( Figure 1.7B) [74,108]. The A3G-induced mutations at glycine codons tend to be nonconservative and inactivating mutations whereas the A3F-induced missense mutations have less inactivation ability Deaminations by A3G at 5′CC motifs in the (-) DNA lead to codon changes in the (+) DNA primarily at glycine.…”

“…The A3F deaminations at 5′TC motifs in the (-) DNA result in much more variable amino acid changes in the (+) DNA. Sequences of the protease gene of ΔVif HIV-1 exposed to A3G or A3F were analyzed from Ara et al [74,108]. Reprinted with permission from Adolph et al, 2018 [1].…”