Paramyxovirus-Induced Shutoff of Host and Viral Protein Synthesis: Role of the P and V Proteins in Limiting PKR Activation

Abstract

The paramyxovirus simian virus 5 (SV5) establishes highly productive persistent infections of epithelial cells without inducing a global inhibition of translation. Here we show that an SV5 mutant (the P/V-CPI ؊ mutant) with substitutions in the P subunit of the viral polymerase and the accessory V protein also establishes highly productive infections like wild-type (WT) SV5 but that cells infected with the P/V-CPI ؊ mutant show an overall shutdown of both host and viral translation at late times postinfection. Reduced host and viral protein synthesis with the P/V-CPI ؊ virus was not due to lower levels of mRNA or caspase-dependent apoptosis and correlated with phosphorylation of the translation initiation factor eIF-2␣. WT SV5 was a poor activator of the eIF-2␣ kinase protein kinase R (PKR). By contrast, the P/V-CPI ؊ mutant induced PKR phosphorylation, which correlated with the time course of translation inhibition but was independent of interferon signaling. In HeLa cells that expressed the PKR inhibitor influenza A virus NS1 or reovirus sigma3, the rate of host protein synthesis at late times after infection with the P/V-CPI ؊ mutant was restored to ϳ50% that of control HeLa cells. By contrast, the rates of P/V-CPI ؊ viral protein synthesis in HeLa cells expressing NS1 or sigma3 were dramatically enhanced, between 5-and 20-fold, while levels of viral mRNA were increased only slightly (NS1-expressing cells) or remained constant (sigma3-expressing cells). Similar results were found using HeLa cells where PKR levels were reduced due to knockdown by small interfering RNA. Expression of either the WT P or the WT V protein from the genome of the P/V-CPI ؊ mutant resulted in lower levels of PKR activation and rates of host and viral protein synthesis that closely matched those seen with WT SV5. Despite higher rates of translation, cells infected with the V-or P-complemented virus accumulated viral mRNAs to lower levels than that seen with the parental P/V-CPI ؊ mutant. We present a model in which the paramyxovirus P/V gene products limit induction of PKR by limiting the synthesis of aberrant viral mRNAs and double-stranded RNA and thus prevent the shutdown of translation by a mechanism that differs from that of other PKR inhibitors such as NS1 and sigma3.

Role for the Paramyxovirus Genomic Promoter in Limiting Host Cell Antiviral Responses and Cell Killing

“…2D). As we have reported previously (13), the enhanced gene expression and WT growth properties for the Le-(U5C, A14G) mutant are not consistent with defective viral particles, and the PFUto-HA ratios were very similar between WT (10,766 Ϯ 1,335) and mutant (8,723 Ϯ 1,897) viruses.…”

“…The Le-(U5C, A14G) mutant expresses a functional WT V protein with kinetics that match that of the parental WT SV5 virus, making it reasonable to assume that mda-5 is not involved in responses to the leader mutant. Le-(U5C, A14G) infection does not significantly activate PKR or induce a shutdown of translation (data not shown), two properties of a previously described SV5 P/V mutant (13). This differential effect of Le-(U5C, A14G) on activation of RIG-I pathways but not PKR could reflect the relative sensitivities of these two cytoplasmic receptors to levels or lengths of dsRNA, with RIG-I being more easily activated than PKR.…”

“…For knockdown of RIG-I, A549 cells in 24-well plates were transfected using Transit siQuest (Mirus, Inc.) with siRNA specific for RIG-I (Dharmacon M-0125-01; 100 nM final concentration) or nontarget control RNA (Dharmacon D-001206-13; 100 nM final concentration) according to the manufacturer's instructions and as previously described (13). Forty-eight hours posttransfection, cells were infected as described above.…”

“…Phase microscopy and fluorescent microscopy were carried out as described previously (50). Analysis of IRF-3 nuclear translocation was performed as detailed elsewhere (13). Images were captured using a QImaging digital camera and processed using Q-capture software.…”

“…Virus stock was generated in Vero cells by low-multiplicity of infection (MOI) infection, as described previously (13), in order to prevent generation of defective particles. To assay virus growth, ϳ10 6 A549 cells in six-well dishes were infected at an MOI of 0.05 (multistep growth) or 10 (single-step growth) for 1 h and replaced with DMEM containing 2% fetal bovine serum.…”