Deciphering the mechanism of TDP2/VPg unlinkase activity during picornavirus infections
- Author(s): Holmes, Autumn
- Advisor(s): Semler, Bert L
- et al.
Members of the Picornaviridae family are responsible for many highly prevalent human illnesses. These include: the common cold, hepatitis, myocarditis, and paralytic poliomyelitis, which are caused by human rhinoviruses, hepatitis A virus, coxsackieviruses, and poliovirus, respectively. Given their limited coding capacity, picornaviruses must repurpose host factors to carry out their replication cycles. One such host protein is the DNA repair enzyme, tyrosyl-DNA phosphodiesterase 2 (TDP2), also known as VPg unlinkase. Picornaviruses use TDP2 to cleave the bond between viral genomic RNA and VPg, the primer for RNA synthesis that is covalently linked to the 5’ end of virion RNA. Notably, viral RNA destined for RNA packaging must be VPg-linked, while VPg is absent from polysome-associated viral mRNAs and positive-strand RNA templates used to prime negative-strand RNA synthesis. Previous studies in cultured murine cells have revealed a significant dependence on TDP2 during infection by poliovirus, coxsackievirus, or human rhinovirus. However, the precise biological consequences of VPg removal during picornavirus infections of human cells are unknown.
To determine the impact of TDP2 removal of VPg during picornavirus infections of human cells, we first established a human retinal pigment epithelial (RPE) cell model for the study of viral replication in the absence of TDP2. We then determined if premature encapsidation occurs in the absence of TDP2 VPg unlinkase activity. Finally, we assessed virus translation in wild type (WT) and knock-out (KO) TDP2 RPE cells by polysome analysis. We found that titers of either poliovirus or coxsackievirus B3 are reduced by 1-2 log10 units in the absence of TDP2 in human RPE cells and that this effect is multiplicity of infection-dependent. We also demonstrated that premature encapsidation is not responsible for the virus growth defect observed in the absence of TDP2. Finally, we determined that viral RNA is actively translated in both WT and KO TDP2 RPE cells at early times of infection. Additionally, we found that TDP2 is relocalized from the nucleus to the cytoplasm in a panel of cell lines and colocalizes with poliovirus structural protein, VP1 in the nucleus of MCF7 cells. Finally, we found that virion RNA that has been treated with proteinase K (which degrades VPg), acts as a more efficient template for both positive and negative strand RNA synthesis. From these data, we conclude that TDP2 is an important host cell factor for picornavirus infections of human cells and that TDP2 removal of VPg from viral RNA at early times during infection is involved in step(s) other than translation or encapsidation, a likely candidate being RNA synthesis.