Antiviral activity of Tripartite-Motif 2 protein against New World Arenaviruses

Abstract

Background: New World Arenaviruses (NWA) are single-stranded bisegmented RNA viruses that cause viral hemorrhagic fevers in humans. NWAs are endemic in New World rodents and are transmitted to humans by aerosol. Host factors that alter entry and infection by Junín virus (JUNV), a NWA found in Argentina, were identified through a high throughput RNAi screen. Among them was an antiviral restriction factor, TRIM2, a member of the tripartite-motif (TRIM) family, many of which have effector roles responses to viral infections. TRIM proteins share a RING finger-B box-Coiled coil (RBCC) motif at the N-terminus but have diverse C-terminal domains; TRIM2 contains FIL/NHL domain. TRIMs are E3 ubiquitin ligases whose activity is conferred by the RING domain. Our data suggests that TRIM2 acts at the viral entry step, which would be unique for TRIM proteins. Methods & Materials: Using CRISPR/Cas9 technology we generated different TRIM2 mutant mice with different deletions. Bone Marrow-derived macrophages (BMDMs) were isolated from the mutant mice, and infected with the JUNV vaccine strain Candid 1 for 24 hours. RNA was extracted from the cells and subjected to RT-qPCR to assess the levels of infection. JUNV was also detected by immunoblotting using an anti NP monoclonal antibody. Eight old weeks mice were infected intracranially with 1 × 104 PFU of Candid 1 for 5 days and the RNA was assayed as described above. Constructs containing different domains of TRIM2 were cloned into expression vectors and transfected into 293T cells followed by JUNV infection to assess the domain involved in the antiviral activity. TRIM2-interacting proteins were identified by co-immunoprecipitation from BMDMs and JUNV-infected brains using an anti-TRIM2 monoclonal antibody. Results: We observed higher levels of JUNV infection in the TRIM2 mutant cells and mice in comparison to wild-type. We found that the FIL domain, but not the RING domain, was involved in the antiviral activity and that TRIM2 interacted with signal regulatory protein alpha (SIRPA), which had anti-NWA activity. Conclusion: TRIM2 is an anti-arenaviral host factor and might work through SIRPA by a mechanism not fully elucidated yet. This type of restriction has not been previously described for any TRIM protein.