Stress granules (SGs) are granular aggregates in the cytoplasm that are formed under a variety of stress situations including viral infection. Previous studies indicate that poliovirus, a member of Picornaviridae, can induce SG formation. However, the exact mechanism by which the picornaviruses induce SG formation is unknown. The localization of SG markers in cells infected with coxsackievirus B3 (CVB3) or enterovirus 71 (EV71) and in cells expressing each viral protein was determined via immunofluorescence assays or plasmid transfection. Eight plasmids expressing mutants of the 2A protease (2Apro) of CVB3 were generated using a site-directed mutagenesis strategy. The cleavage efficiencies of eIF4G by CVB3 2Apro and its mutants were determined via western blotting assays. In this study, we found that CVB3 infection induced SG formation, as evidenced by the co-localization of some accepted SG markers in viral infection-induced granules. Furthermore, we identified that 2Apro of CVB3 was the key viral component that triggered SG formation. A 2Apro mutant with the G122E mutation, which exhibited very low cleavage efficiency toward eIF4G, significantly attenuated its capacity for SG induction, indicating that the protease activity was required for 2Apro to initiate SG formation. Finally, we observed that SGs also formed in EV71-infected cells. Expression of EV71 2Apro alone was also sufficient to cause SG formation. Both CVB3 and EV71 infections can induce SG formation, and 2Apro plays a crucial role in the induction of SG formation during these infections. This finding may help us to better understand how picornaviruses initiate the SG response.

Coxsackievirus B (CVB) belongs to Enterovirus genus within the Picornaviridae family, and it is one of the most common causative pathogens of viral myocarditis in young adults. The pathogenesis of myocarditis caused by CVB has not been completely elucidated. In CVB infection, autophagy is manipulated to facilitate viral replication. Here we report that protein 2B, one of the non-structural proteins of CVB3, possesses autophagy-inducing capability. The autophagy-inducing motif of protein 2B was identified by the generation of truncated 2B and site-directed mutagenesis. The expression of 2B alone was sufficient to induce the formation of autophagosomes in HeLa cells, while truncated 2B containing the two hydrophobic regions of the protein also induced autophagy. In addition, we demonstrated that a single amino acid substitution (56V→A) in the stem loop in between the two hydrophobic regions of protein 2B abolished the formation of autophagosomes. Moreover, we found that 2B and truncated 2B with autophagy-inducting capability were co-localized with LC3-II. This study indicates that protein 2B relies on its transmembrane hydrophobic regions to induce the formation of autophagosomes, while 56 valine residue in the stem loop of protein 2B might exert critical structural influence on its two hydrophobic regions. These results may provide new insight for understanding the molecular mechanism of autophagy triggered by CVB infection.

Group B Coxsackieviruses (CVB) are one of the causative pathogens of myocarditis, which may progress to cardiomyopathy. The pathogenesis of CVB is not fully understood, and effective antiviral therapy is not available. N-acetylcysteine (NAC), the classic antioxidant, has been used in clinical practice for several decades to treat various medical conditions. In this study, the anti-CVB effect of NAC was investigated. We show that NAC dramatically suppressed viral replication and alleviated cardiac injury induced by CVB3. To further study the antiviral mechanism of NAC, RNA-sequencing was performed for CVB3-infected cells with NAC treatment. We found that eukaryotic elongation factor 1 alpha 1 (EEF1A1) is one of the most upregulated genes in CVB3-infected cells. However, EEF1A2, the highly homologous isoform of EEF1A1, remains unchanged. EEF1A1 expression was significantly suppressed by NAC treatment in CVB3-infected cells, while EEF1A2 was not affected. eEF1A1 knockdown significantly inhibited CVB3 replication, implicating that eEF1A1 facilitates viral replication. Importantly, we show that eEF1A1, which was not expressed in the myocardia of newborn mice, was significantly upregulated by CVB3 infection. NAC markedly downregulated the expression of eEF1A1 but not eEF1A2 in the myocardia of CVB3-infected mice. Furthermore, NAC accelerated eEF1A1 degradation by promoting autophagy in CVB3-infected cells. We show that p62, one of the critical adaptors of autophagic targets, interacts with eEF1A1 and was downregulated in CVB3-infected cells upon NAC treatment. Taken together, this study demonstrated that NAC shows a potent anti-CVB effect through the downregulation of eEF1A1.

Human papillomavirus (HPV) is a dsDNA virus and its high-risk subtypes increase cancer risks. Yet, the mechanism of HPV infection and pathogenesis still remain unclear. Therefore, understanding the molecular mechanisms, and the pathogenesis of HPV are crucial in the prevention of HPV related cancers. In this study, we analyzed cervix squamous cell carcinoma (CESC) and head and neck carcinoma (HNSC) combined data to investigate various HPV induced cancer common feature. We showed that epidermal growth factor receptor (EGFR) was downregulated in HPV positive (HPV+) cancer, and that HPV+ cancer patients exhibited better prognosis than HPV negative (HPV−) cancer patients. Our study also showed that TP53 mutation rate is lower in HPV+ cancer than in HPV− cancer and that TP53 can be modulated by HPV E7 protein. However, there was no significant difference in the expression of wildtype TP53 in both groups. Subsequently, we constructed HPV-human interaction network and found that EGFR is a critical factor. From the network, we also noticed that EGFR is regulated by HPV E7 protein and hsa-miR-944. Moreover, while phosphorylated EGFR is associated with a worse prognosis, EGFR total express level is not significantly correlated with prognosis. This indicates that EGFR activation will induce a worse outcome in HPV+ cancer patients. Further enrichment analysis showed that EGFR downstream pathway and cancer relative pathway are diversely activated in HPV+ cancer and HPV− cancer. In summary, HPV E7 protein downregulates EGFR that downregulates phosphorylated EGFR and inhibit EGFR related pathways which in turn and consequently induce better prognosis. Importance: Although HPV infection has been studied in various cancer types, there are only limited studies that have focused on the common effect of HPV related cancer. Consequently, this study focused on CESC and HNSC, two cancer types with high HPV infection proportion in cohort, thereby, intending to dig out the common effects and mechanisms of HPV+ cancers. Unlike some virus-human interaction prediction studies, the P-HIPSter database provides virus-human protein interaction based on protein structure prediction. Through this data, our interaction network was able to uncover previously unnoticed protein interactions. Our finding revealed that HPV infection caused various gene expression differences, and a great amount of which interact with EGFR, a cancer related gene. Therefore, since EGFR is associated with HPV+ cancer patients’ survival, some FDA proved EGFR inhibitors would be potential anti-HPV drugs.