Molecular mechanisms of human innate immune restriction factors APOBEC3G and APOBEC3H for inhibiting the retrovirus HIV-1 and retrotransposon LINE1

The APOBEC3 restriction factors belong to a family of cytidine deaminases that are able to suppress the replication of viruses with a single-stranded DNA intermediate by inducing mutagenesis and functional inactivation of the virus. Of the seven common human APOBEC3 enzymes, only APOBEC3-D, -F, -G and -H appear to be relevant to HIV-1 restriction in CD4+ T cells. The restriction of HIV-1 by APOBEC3 enzymes occurs most potently in the absence of HIV-1 Vif, which counteracts APOBEC3s by inducing polyubiquitination and subsequent degradation of APOBEC3 enzymes. Virion-encapsidated APOBEC3s can deaminate cytosines to uracils in viral (-)DNA. Upon replication of (-)DNA to (+)DNA, the HIV-1 reverse transcriptase incorporates adenines opposite to the uracils hereby inducing C/G to T/A transition mutations. Among all APOBEC3 enzymes that are relevant to HIV-1 restriction, APOBEC3G is the most studied APOBEC3 enzyme. APOBEC3G has been shown to processively catalyze deamination reactions on single-stranded (-)DNA using a mechanism called facilitated diffusion, which involves sliding and jumping movements in search of target cytosine-containing motifs. This single-stranded DNA scanning mechanism allows APOBEC3G to efficiently deaminate multiple cytosines within one enzyme-DNA encounter and it is important for the mutational inactivation of HIV-1 in vivo. Vif attempts to neutralize APOBEC3G’s function not only by inducing proteasomal degradation, but also by several degradation-independent mechanisms, such as inhibiting APOBEC3G virion encapsidation, mRNA translation, and for those APOBEC3G molecules that still become virion encapsidated, Vif has been shown to inhibit APOBEC3G’s deamination activity. My Ph.D. thesis work investigated the molecular mechanism of degradation-independent Vif-mediated inhibition of APOBEC3G and APOBEC3H deamination activity. This research led to the development of the hypothesis that Vif has developed a unique interaction with each APOBEC3 enzyme due to the different selection pressures they impose on HIV-1. Thus, we investigated how the interaction of Vif differs between APOBEC3G and APOBEC3H and characterized the activity of APOBEC3H as a restriction factor. This research allowed us to have a better understanding of the molecular determinants that govern an efficient APOBEC3-degradatation by HIV-1 Vif and provide insights for APOBEC3-based HIV-1 therapeutics. Two Vif variants obtained from HIV-1 laboratory isolates, VifHXB2 and VifIIIB, were used to determine the degradation-independent effects of Vif on APOBEC3G. Biochemical assays using a model HIV-1 replication assay and synthetic single-stranded or partially double-stranded DNA substrates demonstrated that APOBEC3G has an altered processive mechanism in the presence of Vif, and this caused APOBEC3G to be less effective at inducing mutagenesis in a model HIV-1 replication assay. APOBEC3H is unique in that it is the only single-domain common APOBEC3 enzyme that restricts HIV-1. APOBEC3H exists in humans as seven haplotypes (I-VII) with different cellular stabilities. Amongst three stable APOBEC3H haplotypes, haplotype II and V occur most frequently in the population. I characterized the single-stranded DNA scanning mechanisms that haplotype II and V use to search their single-stranded substrate for cytosine-containing deamination motif. APOBEC3H haplotype II was able to processively deaminate its substrate using Brownian motion-driven movements termed sliding, jumping and intersegmental transfer, whereas haplotype V showed compromised sliding and intersegmental transfer abilities but was able to jump along single-stranded DNA. Since an Asp or Glu at amino acid 178 differentiates these two haplotypes, these data suggest this amino acid on predicted helix 6 contributes to processivity. I found the optimal processivity on ssDNA also required dimerization of APOBEC3H through the β2 strands. The diminished processivity of APOBEC3H haplotype V did not result in a reduced efficiency to restrict HIV-1 replication in single-cycle infectivity assay. This suggests a redundancy in the contribution of jumping and intersegmental transfer to mutagenic efficiency. VifHXB2, but not VifIIIB, can cause degradation of APOBEC3H even though APOBEC3H interacts with both Vif variants. APOBEC3G is degraded after interaction with both of these Vif variants. To define the parameters for efficient Vif-induced degradation of an APOBEC3 enzyme, I used an in vitro quantitative method to determine the binding strength of APOBEC3G and APOBEC3H with Vif variant heterotetramers (Vif/CBFβ/EloB/EloC), the most stable form of Vif. Our biochemical analysis, along with cellular experiments to determine Vif-induced degradation efficiency and APOBEC3-Vif interactions in cells, support a model in which the degradation efficiency of Vifs correlates with both the APOBEC3-Vif binding strength and APOBEC3-Vif interface. I also investigated how APOBEC3 enzymes restrict the replication of retrotransposon LINE-1. Retrotransposons are DNA sequences that replicate using a “copy-and-paste” mechanism through an RNA intermediate. The degradation of deaminated L1 cDNA rendered it difficult to detect any APOBEC3-induced G-to-A mutations while the addition of uracil DNA glycosylase inhibitor allowed for the recovery of the APOBEC3-mediated deamination events. I found that two stable A3H haplotypes (haplotype II and haplotype V) use a deamination-independent mechanism to restrict L1 mobilization and compared the ability of APOBEC3H’s to inhibit LINE1 with that of APOBEC3A and APOBEC3G, two APOBEC3s whose LINE1 restriction ability have been previously characterized. Taken together, these studies of the molecular mechanisms that APOBEC3G and APOBEC3H use to inhibit HIV-1 and LINE1 have allowed us to better understand their biological properties as cytidine deaminases and the determinants in APOBEC3s that made them efficient host innate immune restriction factors.

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