TRIM56 restricts Coxsackievirus B infection by mediating the ubiquitination of viral RNA-dependent RNA polymerase 3D

Coxsackievirus B (CVB) is the major causative pathogen for severe diseases such as viral myocarditis, meningitis, and pancreatitis. There is no effective antiviral therapy currently available for CVB infection primarily due to that the pathogenesis of CVB has not been completely understood. Viruses are obligate intracellular pathogens which subvert cellular processes to ensure viral replication. Dysregulation of ubiquitination has been implicated in CVB infection. However, how ubiquitination is involved in CVB infection remains unclear. Here we found that the 3D protein of CVB3, the RNA-dependent RNA polymerase, was modified at by K48-linked polyubiquitination which promoted its degradation through proteasome. Proteomic analysis showed that the E3 ligase TRIM56 was upregulated in CVB3-infected cells, while the majority of TRIMs remained unchanged. Pull-down and immunoprecipitation analyses showed that TRIM56 interacted with CVB3 3D. Immunofluorescence observation showed that viral 3D protein was colocalized with TRIM56. TRIM56 overexpression resulted in enhanced ubiquitination of CVB3 3D and decreased virus yield. Moreover, TRIM56 was cleaved by viral 3C protease in CVB3-infected cells. Taken together, this study demonstrated that TRIM56 mediates the ubiquitination and proteasomal degradation of the CVB3 3D protein. These findings demonstrate that TRIM56 is an intrinsic cellular restriction factor against CVB infection, and enhancing viral protein degradation could be a potential strategy to control CVB infection. Coxsackievirus B (CVB) is the major causative agent for a range of human diseases. As small infectious agent with a simple genome, CVB relies on the subversion of host processes to replicate. On the other hand, host cells contain antiviral strategies to restrict CVB replication. The present study aims to address the role played by a cellular protein termed TRIM56 in the infection of CVB3, a representative serotype of CVB, which is the causative agent of inflammatory heart disease. Our study found that viral RNA replicase 3D harbors a type of covalent modification termed polyubiquitination, resulting in the accelerated degradation of this viral replicase. We show that TRIM56 is the cellular enzyme which promotes the polyubiquitination of viral 3D protein. We demonstrated that when TRIM56 was overexpressed in the cell, the production of viral particles was significantly reduced. Moreover, we found that the protease 3C of CVB3 counteracts the antiviral effect of TRIM56 through its protease activity. Together, these data demonstrate that TRIM56 is an intrinsic cellular restriction factor against CVB infection. Enhancing viral protein degradation could be a potential strategy to control CVB infection.