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Analysis of Group IV Viral SSHHPS Using In Vitro and In Silico Methods
Published on: December 21, 2019
The viral serpin SPI-1 directly inhibits the host cell serine protease FAM111A
Allison L Welter1, Sowmiya Palani2, Yuka Machida3
1Developmental Therapeutics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, USA; Mayo Clinic Graduate School of Biomedical Sciences, Mayo Clinic, Rochester, Minnesota, USA.
Abstract:
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.
Insights
Rabbitpox virus (RPXV) serpin SPI-1 directly inhibits host serine protease FAM111A. This interaction is crucial for viral replication in restrictive cells, revealing FAM111A
Area of Science:
- Virology
- Biochemistry
- Molecular Biology
Background:
- Rabbitpox virus (RPXV) host-range mutants lacking the serpin serine protease inhibitor 1 (SPI-1) exhibit impaired replication in restrictive host cells.
- Host cell serine protease FAM111A depletion restores RPXV replication, suggesting SPI-1 targets FAM111A to promote viral infection.
- Direct experimental evidence for SPI-1 inhibition of FAM111A has been previously lacking.
Purpose of the Study:
- To provide direct evidence of SPI-1 inhibiting FAM111A protease activity.
- To investigate the mechanism and specificity of SPI-1 inhibition of FAM111A.
- To elucidate the role of SPI-1-FAM111A interaction in orthopoxvirus infection.
Main Methods:
- In vitro assays to assess direct inhibition of FAM111A protease activity by SPI-1.
- Covalent complex formation analysis to confirm serpin inhibition mechanism.
- Mutagenesis studies of SPI-1's reactive center loop (RCL) and correlation with inhibition and viral replication.
Main Results:
- SPI-1 directly inhibits FAM111A protease activity in vitro via covalent complex formation.
- SPI-1 demonstrates specificity for FAM111A over other tested serine proteases.
- Mutations in SPI-1's RCL critically affected FAM111A inhibition and RPXV replication, showing a strong correlation.
Conclusions:
- This study provides the first direct evidence that SPI-1 inhibits FAM111A protease activity.
- FAM111A plays a significant antiviral role by restricting orthopoxvirus replication.
- SPI-1's inhibition of FAM111A is essential for efficient RPXV infection in non-permissive cells.
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