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.

The APOBEC3 family of cytosine deaminase enzymes are able to restrict replication of retroelements, such as LINE-1. However, each of the seven APOBEC3 enzymes have been reported to act differentially to prevent LINE-1 retrotransposition and the mechanisms of APOBEC3-mediated LINE-1 inhibition has not been well understood. The prevailing view for many years was that APOBEC3-mediated LINE-1 inhibition was deamination-independent and relied on APOBEC3s blocking the LINE-1 reverse transcriptase DNA polymerization or transport of the LINE-1 RNA into the nucleus. However, recently it was shown that APOBEC3A can deaminate cytosine, to form uracil, on transiently exposed single-stranded LINE-1 cDNA and this leads to LINE-1 cDNA degradation. In this study, we confirmed that APOBEC3A is a potent deamination-dependent inhibitor of LINE-1 retrotransposition, but show that in contrast, A3H haplotype II and haplotype V restrict LINE-1 activity using a deamination-independent mechanism. Our study supports the model that different APOBEC3 proteins have evolved to inhibit LINE-1 retrotransposition through distinct mechanisms.

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SIVsmm infecting sooty mangabeys has been transmitted to humans on at least nine occasions, giving rise to HIV-2 groups A to I. SIVsmm isolates replicate in human T cells and seem capable of overcoming major human restriction factors without adaptation. However, only groups A and B are responsible for the HIV-2 epidemic in Sub-Saharan Africa and it is largely unclear whether adaptive changes were associated with spread in humans. To address this, we examined the sensitivity of infectious molecular clones (IMCs) of five HIV-2 strains and representatives of five different SIVsmm lineages to various APOBEC3 proteins. We confirmed that SIVsmm strains replicate in human T cells, albeit with more variable and frequently lower efficiency than HIV-2 IMCs. Efficient viral propagation was generally dependent on intact vif genes, highlighting the need for counteraction of APOBEC3 proteins. On average, SIVsmm was more susceptible to inhibition by human APOBEC3D, F, G and H than HIV-2. For example, human APOBEC3F reduced infectious virus yield of SIVsmm by ∼80% but achieved only ∼40% in the case of HIV-2. Functional and mutational analyses of human and monkey derived alleles revealed that an R128T polymorphism in APOBEC3F contributes to species-specific counteraction by HIV-2 and SIVsmm Vif proteins. In addition, a T84S substitution in SIVsmm Vif increased its ability to counteract human APOBEC3F. Altogether, our results confirm that SIVsmm Vifs show intrinsic activity against human ABOBEC3 proteins but also demonstrate that epidemic HIV-2 strains evolved an increased ability to counteract this class of restriction factors during human adaptation. IMPORTANCE Viral zoonoses pose a significant threat to human health and it is important to understand determining factors. SIVs infecting great apes gave rise to HIV-1. In contrast, SIVs infecting African monkey species have not been detected in humans, with one notable exception. SIVsmm from sooty mangabeys crossed the species barrier to humans on at least nine independent occasions and seems capable of overcoming many innate defense mechanisms without adaptation. Here, we confirmed that SIVsmm Vif proteins show significant activity against human APOBEC3 proteins. Our analyses also revealed, however, that different lineages of SIVsmm are significantly more susceptible to inhibition by various human APOBEC3 proteins than HIV-2 strains. Mutational analyses suggest that a R128T substitution in APOBEC3F and a T84S change in Vif contribute to species-specific counteraction by HIV-2 and SIVsmm. Altogether, our results support that epidemic HIV-2 strains acquired increased activity against human APOBEC3 proteins to clear this restrictive barrier.

SIVsmm infecting sooty mangabeys has been transmitted to humans on at least nine independent occasions, giving rise to HIV-2 groups A to I. SIVsmm isolates replicate in human T cells and seem capable of overcoming major human restriction factors without adaptation. However, only groups A and B are responsible for the HIV-2 epidemic in Sub-Saharan Africa and it is largely unclear whether adaptive changes were associated with significant spread in humans. To address this, we examined the sensitivity of infectious molecular clones (IMCs) of five HIV-2 strains (4 group A and one AB recombinant) and representatives of five different SIVsmm lineages to inhibition by type I interferon (IFN) and various APOBEC3 proteins. We confirmed that SIVsmm strains replicate in primary human CD4+ T cells. However, SIVsmm replication was highly variable, typically lower relative to HIV-2 isolates and almost entirely prevented by type I IFN treatment. Viral propagation was generally dependent on intact vif genes, highlighting the need for efficient counteraction of APOBEC3 proteins. On average, SIVsmm strains were significantly more susceptible to inhibition by human APOBEC3D, F, G and H than HIV-2 IMCs. For example, human APOBEC3F reduced infectious virus yield of SIVsmm by ∼80% but achieved only ∼40% in the case of HIV-2. Functional and mutational analyses of human, sooty mangabey and rhesus macaque derived alleles revealed that an R128T polymorphism in APOBEC3F is important for species-specific counteraction by HIV-2 and SIVsmm Vif proteins. In addition, we found that changes of Y45H and T84S in SIVsmm Vif increase its ability to antagonize human APOBEC3F. Altogether, our results show that SIVsmm Vifs show some intrinsic activity against human ABOBEC3 proteins, but HIV-2 Vifs acquired adaptive changes to efficiently clear this barrier in the human host.AUTHOR SUMMARYSIVs infecting African monkey species do not infect humans, with one notable exception. SIVsmm from sooty mangabeys managed to cross the species barrier to humans on at least nine independent occasions. This is because SIVsmm strains seem capable of overcoming many innate defense mechanisms without adaptation and that their Vif proteins are active against human APOBEC3 proteins. Here, we show that replication of SIVsmm is highly variable in human CD4 T cells and more sensitive to interferon inhibition compared to HIV-2. While different lineages of SIVsmm were capable of counteracting human APOBEC3 proteins in a Vif-dependent manner, they were significantly more susceptible to inhibition by APOBEC3D/F/G/H compared to HIV-2. Mutational analyses revealed an R128T substitution in APOBEC3F and a T84S change in Vif are relevant for species-specific counteraction by HIV-2 and SIVsmm. Altogether, our results support that HIV-2 group A adapted to humans prior to or during epidemic spread.