Natural Polymorphisms in Human APOBEC3H and HIV-1 Vif Combine in Primary T Lymphocytes to Affect Viral G-to-A Mutation Levels and Infectivity

Abstract

The Vif protein of HIV-1 allows virus replication by degrading several members of the host-encoded APOBEC3 family of DNA cytosine deaminases. Polymorphisms in both host APOBEC3 genes and the viral vif gene have the potential to impact the extent of virus replication among individuals. The most genetically diverse of the seven human APOBEC3 genes is APOBEC3H with seven known haplotypes. Overexpression studies have shown that a subset of these variants express stable and active proteins, whereas the others encode proteins with a short half-life and little, if any, antiviral activity. We demonstrate that these stable/unstable phenotypes are an intrinsic property of endogenous APOBEC3H proteins in primary CD4+ T lymphocytes and confer differential resistance to HIV-1 infection in a manner that depends on natural variation in the Vif protein of the infecting virus. HIV-1 with a Vif protein hypo-functional for APOBEC3H degradation, yet fully able to counteract APOBEC3D, APOBEC3F, and APOBEC3G, was susceptible to restriction and hypermutation in stable APOBEC3H expressing lymphocytes, but not in unstable APOBEC3H expressing lymphocytes. In contrast, HIV-1 with hyper-functional Vif counteracted stable APOBEC3H proteins as well as all other endogenous APOBEC3s and replicated to high levels. We also found that APOBEC3H protein levels are induced over 10-fold by infection. Finally, we found that the global distribution of stable/unstable APOBEC3H haplotypes correlates with the distribution a critical hyper/hypo-functional Vif amino acid residue. These data combine to strongly suggest that stable APOBEC3H haplotypes present as in vivo barriers to HIV-1 replication, that Vif is capable of adapting to these restrictive pressures, and that an evolutionary equilibrium has yet to be reached.

The Structural Interface between HIV-1 Vif and Human APOBEC3H

“…Human A3H is polymorphic, and not every human individual carries the stable A3H haplotypes which exert anti-HIV activity (9,10,12,13). As a result, not every HIV-1 strain is able to counteract A3H.…”

“…Of note, because C-terminal tagging of A3H may interfere with Vif binding, we decided to leave A3H untagged (2,20) and detected A3H expression using an A3H-specific antibody ( Fig. 3A) (13). Infection of these T cell lines with HIV-WT and HIV-ΔVif showed that HIV-ΔVif only replicated in the absence of A3H, indicating that all of the A3H variants selected restricted HIV efficiently ( Fig.…”

“…We generated a panel of 13 Vif mutants consisting of positively charged arginine and negatively charged glutamate at and around positions deemed important for A3H recognition based on previous work (Fig. 5A) (12)(13)(14)(15)(16) and tested their activity against selected Vif-resistant A3H mutants in single-cycle infectivity assays. We found that Vif-WT, Vif-G37R, and Vif-D61R efficiently counteracted A3H-WT, Vif mutants G37E, G60E, A62E, A62R, R63E, and R90E showed reduced anti-A3H activity, and Vif mutants F39E, F39R, E54R, H56E, and R93E completely lost their activity against A3H-WT (Fig.…”

“…A3H haplotypes II, V, and VII potently restrict HIV-1, whereas haplotypes I, III, IV, and VI express unstable proteins and lack anti-HIV activity. We have previously shown that endogenously expressed stable A3H haplotype II restricts HIV-1 variants that are unable to counteract it (12,13). Moreover, HIV-1 Vif alleles derived from HIV-infected patients encoding unstable A3H haplotypes are more likely to lack anti-A3H activity, indicating that HIV-1 Vif adapts to the stable A3H (12).…”

“…To date, only a single amino acid at position 121 in A3H has been implicated in the interaction with HIV Vif: mutating aspartic acid (D) at position 121 to a lysine (K) renders A3H resistant to Vif (14,15). On the Vif side, amino acid positions 39, 48, and 60 to 63 are important for the counteraction of A3H, indicating they are part of the A3H binding site (12,13,15,16).…”