Effects of TDP2/VPg Unlinkase Activity on Picornavirus Infections Downstream of Virus Translation

Abstract

In this study, we characterized the role of host cell protein tyrosyl-DNA phosphodiesterase 2 (TDP2) activity, also known as VPg unlinkase, in picornavirus infections in a human cell model of infection. TDP2/VPg unlinkase is used by picornaviruses to remove the small polypeptide, VPg (Virus Protein genome-linked, the primer for viral RNA synthesis), from virus genomic RNA. We utilized a CRISPR/Cas-9-generated TDP2 knock out (KO) human retinal pigment epithelial-1 (hRPE-1) cell line, in addition to the wild type (WT) counterpart for our studies. We determined that in the absence of TDP2, virus growth kinetics for two enteroviruses (poliovirus and coxsackievirus B3) were delayed by about 2 h. Virus titers were reduced by ~2 log10 units for poliovirus and 0.5 log10 units for coxsackievirus at 4 hours post-infection (hpi), and by ~1 log10 unit at 6 hpi for poliovirus. However, virus titers were nearly indistinguishable from those of control cells by the end of the infectious cycle. We determined that this was not the result of an alternative source of VPg unlinkase activity being activated in the absence of TPD2 at late times of infection. Viral protein production in TDP2 KO cells was also substantially reduced at 4 hpi for poliovirus infection, consistent with the observed growth kinetics delay, but reached normal levels by 6 hpi. Interestingly, this result differs somewhat from what has been reported previously for the TDP2 KO mouse cell model, suggesting that either cell type or species-specific differences might be playing a role in the observed phenotype. We also determined that catalytically inactive TDP2 does not rescue the growth defect, confirming that TDP2 5′ phosphodiesterase activity is required for efficient virus replication. Importantly, we show for the first time that polysomes can assemble efficiently on VPg-linked RNA after the initial round of translation in a cell culture model, but both positive and negative strand RNA production is impaired in the absence of TDP2 at mid-times of infection, indicating that the presence of VPg on the viral RNA affects a step in the replication cycle downstream of translation (e.g., RNA synthesis). In agreement with this conclusion, we found that double-stranded RNA production (a marker of viral RNA synthesis) is delayed in TDP2 KO RPE-1 cells. Moreover, we show that premature encapsidation of nascent, VPg-linked RNA is not responsible for the observed virus growth defect. Our studies provide the first lines of evidence to suggest that either negative- or positive-strand RNA synthesis (or both) is a likely candidate for the step that requires the removal of VPg from the RNA for an enterovirus infection to proceed efficiently.

A Proximity biotinylation assay with a host protein bait reveals multiple factors modulating enterovirus replication

“…Over the years, several other host nuclear proteins have been identified that interact with specific structural elements of the RNAs of enteroviruses and other picornaviruses, however, the full extent of the effects exerted by the complex mixture of the nuclear RNA-binding factors that are translocated to the cytoplasm upon infection is still poorly understood [ 15 , 27 ]. Interestingly, the accumulating evidence demonstrates that none of these RNA binding and/or processing proteins is absolutely required for enterovirus infection, suggesting a significant redundancy of the host protein functions in supporting viral RNA translation/replication cycle [ 22 , 23 , 28 , 29 ]. Rather, the cumulative effect of multiple host proteins on viral RNA stability, translation, and replication efficiency is likely cell type-dependent, and may contribute to the determination of viral tropism in the host [ 30 , 31 ].…”

TRIM52 is a primate-specific player in the DNA repair process under tight proteolytic control by a triad of giant E3 ligases

“…1j) [34][35][36] . These activities are critical for cell fitness, as their inhibition results in p53 activation, and cell cycle arrest even in the absence of exogenous DNA damage 28,29,31,37 , as seen upon loss of TRIM52 8 . Therefore, we hypothesized that TRIM52 plays a role in the repair of TOP2-associated DSBs, playing an important function in the TDP2-dependent HDR pathway (Fig.…”

Protein Nucleotidylylation in +ssRNA Viruses

“…Picornaviruses have been shown to hijack a host protein, TDP2, to cleave the phosphodiester bond linking VPg to the genomic RNA, with this RNA becoming the template for translation [ 48 , 49 , 50 ]. In addition to a polymerase, viruses in the picornavirus family require cis-acting RNA elements for VPg nucleotidylylation and replication of the viral genome.…”