Abstract
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.
Highlights
Enterovirus A71 (EV-A71), which belongs to the Enterovirus genus of the Picornaviridae family, is the major pathogen of human hand, foot, and mouth disease (HFMD)
We have shown previously that EV-A71 infection induces the phosphorylation of eIF2α by protein kinase R (PKR), resulting in a gradual shutoff of translation during infection (Jheng et al, 2010)
It is worth noting that PKR is phosphorylated and degraded by EV-A71, consistent with observations during PV infection (Black et al, 1989, 1993; Figure 1A)
Summary
Enterovirus A71 (EV-A71), which belongs to the Enterovirus genus of the Picornaviridae family, is the major pathogen of human hand, foot, and mouth disease (HFMD). IFN-induced double-stranded RNA (dsRNA)-activated protein kinase R (PKR) is an IFN-stimulated gene (Gale and Katze, 1998; Peters et al, 2001; Pindel and Sadler, 2011) and acts as a pathogen recognition receptor (Gilfoy and Mason, 2007) by recognizing dsRNA, a typical by-product of viral infection, for IFN induction. Activated PKR catalyzes the phosphorylation of the regulatory α-subunit of the eukaryotic translation initiation factor 2 (eIF2α; Meurs et al, 1992; Clemens and Elia, 1997), blocking the initiation of mRNA translation, which results in the global arrest of both cellular and viral protein synthesis and can lead to apoptosis in response to virus infection (Balachandran et al, 1998). Our results indicate that the 3C protease-generated fragments of PKR facilitated viral replication
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