Distinct Antiretroviral Mechanisms Elicited by a Viral Mutagen.

5-aza-cytidine (5-aza-C) has been shown to be a potent human immunodeficiency virus type 1 (HIV-1) mutagen that induces G-to-C hypermutagenesis by incorporation of the reduced form (i.e., 5-aza-dC, 5-aza-dCTP). Evidence to date suggests that this lethal mutagenesis is the primary antiretroviral mechanism for 5-aza-C. To investigate the breadth of application of 5-aza-C as an antiretroviral mutagen, we have conducted a comparative, parallel analysis of the antiviral mechanism of 5-aza-C between HIV-1 and gammaretroviruses - i.e., murine leukemia virus (MuLV) and feline leukemia virus (FeLV). Intriguingly, in contrast to the hallmark G-to-C hypermutagenesis observed with HIV-1, MuLV and FeLV did not reveal the presence of a significant increase in mutational burden, particularly that of G-to-C transversion mutations. The effect of 5-aza-dCTP on DNA synthesis revealed that while HIV-1 RT was not inhibited by 5-aza-dCTP even at 100 µM, 5-aza-dCTP was incorporated and significantly inhibited MuLV RT, generating pause sites and reducing the fully extended product. 5-aza-dCTP was found to be incorporated into DNA by MuLV RT or HIV-1 RT, but only acted as a non-obligate chain terminator for MuLV RT. This biochemical data provides an independent line of experimental evidence in support of the conclusion that HIV-1 and MuLV have distinct primary mechanisms of antiretroviral action with 5-aza-C. Taken together, our data provides striking evidence that an antiretroviral mutagen can have strong potency via distinct mechanisms of action among closely related viruses, unlinking antiviral activity from antiviral mechanism of action.

Deamination hotspots among APOBEC3 family members are defined by both target site sequence context and ssDNA secondary structure.

The human apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3 (APOBEC3, A3) family member proteins can deaminate cytosines in single-strand (ss) DNA, which restricts human immunodeficiency virus type 1 (HIV-1), retrotransposons, and other viruses such as hepatitis B virus, but can cause a mutator phenotype in many cancers. While structural information exists for several A3 proteins, the precise details regarding deamination target selection are not fully understood. Here, we report the first parallel, comparative analysis of site selection of A3 deamination using six of the seven purified A3 member enzymes, oligonucleotides having 5'TC3' or 5'CT3' dinucleotide target sites, and different flanking bases within diverse DNA secondary structures. A3A, A3F and A3H were observed to have strong preferences toward the TC target flanked by A or T, while all examined A3 proteins did not show a preference for a TC target flanked by a G. We observed that the TC target was strongly preferred in ssDNA regions rather than dsDNA, loop or bulge regions, with flanking bases influencing the degree of preference. CT was also shown to be a potential deamination target. Taken together, our observations provide new insights into A3 enzyme target site selection and how A3 mutagenesis impacts mutation rates.

Distinct dual antiviral mechanism that enhances hepatitis B virus mutagenesis and reduces viral DNA synthesis.

Reverse transcriptase (RT) is an essential enzyme for the replication of retroviruses and hepadnaviruses. Current therapies do not eliminate the intracellular viral replication intermediate termed covalently closed circular (ccc) DNA, which has enhanced interest in hepatitis B virus (HBV) reverse transcription and cccDNA formation. The HBV cccDNA is generated as a plasmid-like episome in the host cell nucleus from the protein-linked relaxed circular (rc) DNA genome in incoming virions during HBV replication. The creation of the cccDNA via conversion from rcDNA remains not fully understood. Here, we sought to investigate whether viral mutagens can effect HBV replication. In particular, we investigated whether nucleoside analogs that act as viral mutagens with retroviruses could impact hepadnaviral DNA synthesis. We observed that a viral mutagen (e.g., 5-aza-2'-deoxycytidine, 5-aza-dC or 5-azacytidine, 5-aza-C) severely diminished the ability of a HBV vector to express a reporter gene following virus transfer and infection of target cells. As predicted, the treatment of 5-aza-dC or 5-aza-C elevated the HBV rcDNA mutation frequency, primarily by increasing the frequency of G-to-C transversion mutations. A reduction in rcDNA synthesis was also observed. Intriguingly, the cccDNA nick/gap region transcription was diminished by 5-aza-dC, but did not enhance viral mutagenesis. Taken together, our results demonstrate that viral mutagens can impact HBV reverse transcription, and propose a model in which viral mutagens can induce mutagenesis during rcDNA formation and diminish viral DNA synthesis during both rcDNA formation and the conversion of rcDNA to cccDNA.