SummaryPicornavirus infection alters the endoplasmic reticulum (ER) membrane but it is unclear whether this induces ER stress. Infection of rhabdomyosarcoma cells with enterovirus 71 (EV71), a picornavirus, caused overexpression of the ER-resident chaperone proteins, BiP and calreticulin, and phosphorylation of eIF2a, but infection with UV-inactivated virus did not, indicating that ER stress was induced by viral replication and not by viral attachment or entry. Silencing (si)RNA knockdown demonstrated that phosphorylation of eIF2a was dependent on PKR: eIF2a phosphorylation was reduced by siPKR but not by siPERK. We provided evidence showing that PERK is upstream of PKR and is thus able to negatively regulate the PKR-eIF2a pathway. Pulse-chase experiments revealed that EV71 infection inhibited translation and activation of ATF6. Expression of BiP at the protein level was activated by a virus-dependent, ATF6-independent mechanism. EV71 upregulated XBP1 mRNA level, but neither IRE1-mediated XBP1 splicing nor its active spliced protein was detected, and its downstream gene, EDEM, was not activated. Epigenetic BiP overexpression alleviated EV71-induced ER stress and reduced viral protein expression and replication. Our results suggest that EV71 infection induces ER stress but modifies the outcome to assist viral replication.

The aim of this study was to identify the antiviral mechanism of a novel compound, BPR3P0128. From a large-scale screening of a library of small compounds, BPR3P compounds were found to be potent inhibitors of influenza viral replication in MadinDarby canine kidney (MDCK) cells. BPR3P0128 exhibited inhibitory activity against both influenza A and B viruses. The 50% inhibitory concentrations were in the range of 51 to 190 nM in MDCK cells, as measured by inhibition-of-cytopathic-effect assays. BPR3P0128 appeared to target the viral replication cycle but had no effect on viral adsorption. The inhibition of capdependent mRNA transcription by BPR3P0128 was more prominent with a concurrent increase in cap-independent cRNA replication in a primer extension assay, suggesting a role of BPR3P0128 in switching transcription to replication. This reduction in mRNA expression resulted from the BPR3P-mediated inhibition of the cap-dependent endoribonuclease (cap-snatching) activities of nuclear extracts containing the influenza virus polymerase complex. No inhibition of binding of 5= viral RNA to the viral polymerase complex by this compound was detected. BPR3P0128 also effectively inhibited other RNA viruses, such as enterovirus 71 and human rhinovirus, but not DNA viruses, suggesting that BPR3P0128 targets a cellular factor(s) associated with viral PB2 cap-snatching activity. The identification of this factor(s) could help redefine the regulation of viral transcription and replication and thereby provide a potential target for antiviral chemotherapeutics.

Enterovirus 71 (EV-A71) causes hand, foot and mouth disease in young children and infants, but can also cause severe neurological complications or even death. The double-stranded RNA (dsRNA)-dependent protein kinase R (PKR), an interferon-induced antiviral protein, phosphorylates the regulatory α-subunit of the eukaryotic translation initiation factor 2 in response to viral infection, thereby blocking the translation of cellular and viral mRNA and promoting apoptosis. The cleavage of PKR after infection with poliovirus, a prototype enterovirus, has been reported by others, but the underlying mechanism of this cleavage and its role in viral replication remain unclear. In the present study, we show that viral 3C protease cleaves PKR at a site, Q188, which differs from the site cleaved during apoptosis, D251. In contrast to the conventional phosphorylation of PKR by dsRNA, EV-A71 3C physically interacts with PKR to mediate the phosphorylation of PKR; this effect is dependent on 3C protease activity. Overexpression of a catalytically inactive PKR mutant (K296H) accelerates viral protein accumulation and increases virus titer, whereas a K64E substitution in the dsRNA binding site abolishes this advantage. We also demonstrate that PKR cleavage mediated by EV-A71 3C protease produces a short N-terminal PKR fragment that can enhance EV-A71 replication, in terms of viral RNA, viral protein, and viral titers. We conclude that PKR is co-opted by EV-A71 via viral protease 3C-mediated proteolytic activation to facilitate viral replication.

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