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

Abstract

Poxviruses are large double-stranded DNA viruses that infect a wide range of animals including humans. Since the eradication of smallpox, 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 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 shown to restrict the replication of VACV-{Delta}SPI-1 in human cells. Nevertheless, the detailed antiviral mechanisms of FAM111A were unresolved. Here, we show that FAM111A is a potent restriction factor for VACV-{Delta}SPI-1 and MVA. Deletion of FAM111A rescued the replication of MVA and VACV-{Delta}SPI-1 and overexpression of FAM111A significantly reduced viral DNA replication and virus titers but did not affect viral early gene expression. The antiviral effect of FAM111A necessitated its trypsin-like 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 mediated by the cGAS-STING signaling pathway. Infection-triggered FAM111A degraded the nuclear pore complex via its protease activity, translocated to the cytoplasm, and interacted with and promoted the degradation of virus DNA binding protein I3 in a DNA-dependent manner. Interestingly, the protease activity of FAM111A was only needed for nuclear export but not I3 degradation as further analysis showed 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. MPXV and lumpy skin disease virus SPI-1s also inhibited 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.