APOBEC3-driven host mechanism promotes poxvirus diversification to overcome host immune restriction

Project Summary/Abstract The recent global spread of monkeypox (Mpox) virus and the subsequent declaration of a public health emergency by the World Health Organization highlighted the continuous threat of re-emerging infectious agents. With the possible establishment of new animal reservoirs in non-endemic countries, selection of variants for transmissibility, and potential continued spread in humans, there is an urgent need to be able to predict Mpox evolution. Analysis of viral genomes, by our group and others, from the recent Mpox outbreak showed recent acquisition of C>T mutations at TC hotspots, which is consistent with host APOBEC3-driven mutagenesis. This highlighted the gap in knowledge surrounding the mechanisms by which poxviruses undergo genome remodeling, gene mutation, and acquisition of new genes. AID/APOBEC enzymes, specifically the APOBEC3 branch cause mutations in the viral genome with 2 seemingly opposing consequences: viral restriction and enhanced evasion of host immunity. APOBECs mutate C to U in single-stranded DNA (ssDNA), preferentially in di- or tri-nucleotide "hotspot" sequence motifs that are unique to each APOBEC family member. Human APOBEC3A, for example, preferentially mutates at TC hot spots in ssDNA, whereas APOBEC3G prefers CCC in addition to TC. The role of APOBECs as viral restriction factors has been established through many studies on retroviruses, notably HIV. APOBECs are also implicated as a major source of mutagenesis across many non-retroviruses. Viral genes targeted by APOBEC3s have been shown to have an "APOBEC co-evolutionary footprint" either by having fewer APOBEC3 hotspots, or by being enriched for hotspots in positions where APOBEC3-directed mutagenesis favors the generation of immune- or drug-escape variants. Thus, although APOBEC3s are classically viewed as viral restriction factors, they also have pro-viral effects (e.g., mutating viral cytotoxic T-cell epitopes to drive immune escape). Recent observation of Mpox evolution suggests that the Mpox genome will undergo further APOBEC3-mediated diversification as the virus spreads in the human population. Thus, our long-term goal is to be able to predict the potential variation of Mpox. Toward this goal, we will use an experimental evolution model system in human cells to investigate how and which APOBEC3s affect poxviruses. We propose to conduct out study in 2 stages: (1) examine in vitro biochemical activities of APOBEC3s toward Mpox targets and (2) understand the impact of APOBEC3s on C>T mutagenesis and non-homologous recombination in orthopoxviruses such as vaccinia virus as a surrogate to Mpox using an experimental evolution system. Through this comprehensive investigation of the direct impact of APOBEC3s on poxvirus evolution, we will contribute knowledge that can be used for prediction of virus evolution trajectories and therefore reforming policies to protect public health.