Purpose of review

Recent findings

Summary

The cellular mRNA decay protein AUF1 acts as a restriction factor during infection by picornaviruses including poliovirus, coxsackievirus, and human rhinovirus. AUF1 relocalizes from the nucleus to the cytoplasm during infection by these viruses due to the disruption of nucleocytoplasmic trafficking by viral proteinases. Previous studies have demonstrated that AUF1 binds to poliovirus and coxsackievirus B3 (CVB3) RNA during infection, with binding shown to occur within the internal ribosome entry site (IRES) of the 5’ non-coding region (NCR) or the 3’ NCR, respectively. Binding to different sites within the viral RNA suggests that AUF1 may negatively regulate infection by these viruses using different mechanisms. The work presented in this dissertation addresses the mechanism of AUF1 inhibition of the replication of poliovirus and CVB3. It is demonstrated that AUF1 knockdown in human cells results in increased viral translation, RNA synthesis, and virus production and that negative regulation of some picornaviruses may occur in a cell type- or species-specific manner. AUF1 is shown to negatively regulate translation of poliovirus and CVB3 IRES reporter RNAs during infection, but not in uninfected cells. This inhibitory activity is not mediated through destabilization of viral genomic RNA; however, it does require virus-induced relocalization of AUF1 from the nucleus to the cytoplasm during the early phases of infection. All four isoforms of AUF1 are shown to inhibit poliovirus replication when expressed individually; however, endogenous AUF1 may associate with the poliovirus 5’ NCR in an isoform-specific manner during infection. These findings suggest that AUF1 restriction of poliovirus and CVB3 replication uses a common mechanism through the viral IRES, which is distinct from the canonical role that AUF1 plays in regulated mRNA decay in uninfected host cells.

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.

Internal ribosome entry sites (IRESs) are regions of mRNA that facilitate direct binding of components of the initiation complex for protein synthesis independent of the 5’-terminus. While first discovered in viruses, there are many cellular mRNAs that harbor these elements. The current study is evaluating the requirements (both canonical and non-canonical) for translation initiation on the voltage-gated potassium-channel (Kv1.4) IRES. Kv1.4 is a shaker related family member which contributes to the repolarizing phase of the cardiac action potential. Kv1.4 has a highly tissue-specific expression, despite its relatively non-specific promoter, and is regulated at the post-transcriptional level. Using both a genetic (yeast 3-hybrid screen) and a biochemical approach (biotinylated-RNA capture), we probed for novel interacting proteins that might modulate the activity of these IRES elements. Parallel screens reveal a diversity of interacting proteins, which may represent trans-acting factors for translation initiation. Gene set enrichment analysis as well as manual cross-reference identify a high abundance of RNA and translation associated proteins with and without ascribed functions in IRES-mediated translation. Characterizations of a select group of these interactions indicate roles in translation initiation and allude to tissue specific mechanisms of post-transcriptional regulation.