Human FAM111A inhibits vaccinia virus replication by degrading viral uncoating factor I3 and is antagonized by poxvirus host range factor SPI-1

Poxviruses are large double-stranded DNA viruses that infect a wide range of animals including humans. Smallpox virus, the most notorious poxvirus, was eradicated globally in the 1970s. Since then, other members of the poxvirus family, such as monkeypox virus (MPXV) are still posing a great threat to public health. Vaccinia virus (VACV) is a prototypic poxvirus and was used as the vaccine strain for smallpox eradication. VACV encodes a serine protease inhibitor 1 (SPI-1) conserved in all orthopoxviruses, which has been recognized as a host range factor for modified vaccinia virus Ankara (MVA), an approved smallpox vaccine and a promising vaccine vector. FAM111A, a nuclear protein that regulates host DNA replication, was previously identified as a putative target of VACV-ΔSPI-1, which was unable to replicate in human cells unless FAM111A was repressed. Nevertheless, the detailed antiviral mechanisms of FAM111A were unresolved. Here, we show that FAM111A is a potent antiviral factor for VACV-ΔSPI-1 and MVA. Deletion of FAM111A was able to rescue the replication of MVA and VACV-ΔSPI-1. Overexpression of FAM111A significantly reduced viral DNA replication and virus titers but did not affect viral early gene expression. The antiviral effect of FAM111A required its functional serine protease domain and DNA binding domain but not the PCNA-interacting motif. We further discovered that FAM111A translocated into the cytoplasm upon VACV infection and this process was activated through the cGAS-STING signaling pathway. Infection-triggered FAM111A degraded the nuclear pore complex via its serine protease activity, translocated to the cytoplasm, and interacted with and promoted the degradation of virus uncoating factor I3 in a DNA-dependent manner. Interestingly, the serine protease activity of FAM111A was only needed for nuclear export but not I3 degradation. Further analysis suggested I3 was degraded through autophagy. Moreover, VACV SPI-1 was found primarily in the nucleus of infected cells and antagonized FAM111A by prohibiting its nuclear export. Mutations in the SPI-1 reactive-site loop (RSL) abolished its anti-FAM111A activity. SPI-1 from MPXV and lumpy skin disease virus could also inhibit human FAM111A. Our findings reveal the detailed mechanism by which FAM111A functions to restrict a cytoplasmic DNA virus and provide explanations for the immune evasive function of VACV SPI-1. ### Competing Interest Statement The authors have declared no competing interest.