Divergent requirement of a DNA repair enzyme during picornavirus infections
Viruses of the Picornaviridae family, including poliovirus, coxsackievirus B3 (CVB3), human rhinovirus, and encephalomyocarditis virus (EMCV), commandeer the functions of host cell proteins to aid in the replication of their small viral RNA genomes during infection. One of these host proteins is a cellular DNA repair enzyme known as 5’ tyrosyl-DNA phosphodiesterase 2 (TDP2). TDP2 was previously demonstrated to mediate the cleavage of a unique covalent linkage between a viral protein (VPg) and the 5’ end of picornavirus RNAs. Although VPg is absent from actively translating poliovirus mRNAs, the removal of VPg is not required for the in vitro translation and replication of the RNA. However, TDP2 appears to be excluded from replication and encapsidation sites during peak times of poliovirus infection of HeLa cells, suggesting a role for TDP2 during the viral replication cycle. Using a mouse embryonic fibroblast cell line lacking TDP2, we found that TDP2 is differentially required among picornaviruses. Our single-cycle viral growth analysis shows that CVB3 replication has a greater dependency on TDP2 compared to poliovirus, human rhinovirus, or EMCV replication in murine cells. During infection CVB3 protein accumulation is undetectable by Western blot analysis in the absence of TDP2, while poliovirus and EMCV protein accumulation is still detectable at reduced levels. Following transfection of an infectious CVB3 RNA with a reporter, CVB3 RNA replication could still be detected in the absence of TDP2, albeit at reduced levels. EMCV showed the least dependence on TDP2 of the picornaviruses we studied and a possible alternative mechanism for modulating TDP2 activity during infection. TDP2 was shown to relocalize from the nucleus to the cytoplasm during EMCV infection. Unlike during poliovirus infection, TDP2 was not excluded from putative replication sites at peak times of EMCV infection. TDP2 was also cleaved during peak times of EMCV infection, but not during other picornavirus infections, suggesting that EMCV regulates TDP2 activity differently than enteroviruses. Despite these differences, these findings collectively show that TDP2 potentiates viral replication during picornavirus infections of cultured mouse cells, making TDP2 a putative target for antiviral therapeutic development for picornavirus infections.