The viral serpin SPI-1 directly inhibits the host cell serine protease FAM111A

The host-range mutant of rabbitpox virus (RPXV) with a deletion in the gene encoding the serpin serine protease inhibitor 1 (SPI-1) fails to replicate efficiently in restrictive host cells. Depletion of the host cell serine protease FAM111A restores viral replication in these cells, suggesting that SPI-1 targets FAM111A to facilitate infection. However, direct evidence of SPI-1 inhibiting FAM111A has been lacking. Here, we demonstrate that SPI-1 directly inhibits FAM111A's protease activity in vitro through covalent complex formation, a hallmark of the serpin inhibition mechanism. SPI-1 also exhibits specificity for FAM111A compared to other serine proteases in vitro. Through mutagenesis studies, we identified residues and regions within SPI-1's reactive center loop (RCL) that are critical for FAM111A inhibition and covalent complex formation in vitro, with varying degrees of impact. Notably, these RCL mutations showed a spectrum of effects on SPI-1's ability to support RPXV replication in non-permissive cells, which strongly correlated with their impact on SPI-1's capacity to inhibit FAM111A activity in vitro. Altogether, our study provides direct evidence that SPI-1 inhibits FAM111A protease activity, highlighting FAM111A's antiviral role and its significance as a target of SPI-1 during orthopoxvirus infection.