Measles Virus C Protein Impairs Production of Defective Copyback Double-Stranded Viral RNA and Activation of Protein Kinase R

Abstract

Measles virus (MV) lacking expression of C protein (C KO ) is a potent activator of the double-stranded RNA (dsRNA)-dependent protein kinase (PKR), whereas the isogenic parental virus expressing C protein is not. Here, we demonstrate that significant amounts of dsRNA accumulate during C KO mutant infection but not following parental virus infection. dsRNA accumulated during late stages of infection and localized with virus replication sites containing N and P proteins. PKR autophosphorylation and stress granule formation correlated with the timing of dsRNA appearance. Phospho-PKR localized to dsRNA-containing structures as revealed by immunofluorescence. Production of dsRNA was sensitive to cycloheximide but resistant to actinomycin D, suggesting that dsRNA is a viral product. Quantitative PCR (qPCR) analyses revealed reduced viral RNA synthesis and a steepened transcription gradient in C KO virus-infected cells compared to those in parental virus-infected cells. The observed alterations were further reflected in lower viral protein expression levels and reduced C KO virus infectious yield. RNA deep sequencing confirmed the viral RNA expression profile differences seen by qPCR between C KO mutant and parental viruses. After one subsequent passage of the C KO virus, defective interfering RNA (DI-RNA) with a duplex structure was obtained that was not seen with the parental virus. We conclude that in the absence of C protein, the amount of PKR activator RNA, including DI-RNA, is increased, thereby triggering innate immune responses leading to impaired MV growth.

Editing of Cellular Self-RNAs by Adenosine Deaminase ADAR1 Suppresses Innate Immune Stress Responses

“…We earlier found that ADAR1 suppressed multiple dsRNA-dependent innate activities, including the activation of PKR, the IPS-dependent induction of IFN, and the formation of SG in cells infected with WT and V mutant measles virus (7,25,27). However, suppression of these dsRNA-dependent responses by ADAR1 remarkably was not observed with the measles C mutant (7,25,27), a mutant now understood to produce much higher amounts of viral dsRNA than either WT or the V mutant virus (23,27,64). Our results described herein, together with those of others (65)(66)(67), reveal that in the absence of ADAR1 at least some of the cytoplasmic sensors of dsRNA are also triggered by cellular (self) dsRNAs.…”

“…A simple explanation is that the suppression results from the destabilization of duplex RNAs, either cellular dsRNA regions as shown herein (Figs. 4 and 5) and also by Walkley and co-workers (65) or viral dsRNA structures as seen with the measles virus (23,64).…”

“…In the case of several viruses, PKR displays antiviral and proapoptotic activities (12,21,22). Exemplified by measles virus, PKR sufficiency and kinase activation correlate with reduced virus growth (23,24), enhanced induction of IFN␤ (25,26), increased SG responses (7), and increased cytotoxicity and apoptosis (27,28). The central importance of PKR furthermore is illustrated both by the large number of viruses that encode gene products that antagonize PKR activation and function, and by the effects of genetic disruption of the Pkr gene (12,13,29).…”

Measles Virus Defective Interfering RNAs Are Generated Frequently and Early in the Absence of C Protein and Can Be Destabilized by Adenosine Deaminase Acting on RNA-1-Like Hypermutations

“…Protein extracts of infected cells (MOI ϭ 0.1, 30 to 48 h postinfection) were generated, and protein concentrations were determined as described previously (14). Protein (25 g) was fractionated using 12% polyacrylamide gels and transferred to Immobilon-P membranes (EMD Millipore, Billerica, MA) using a semidry protocol.…”

“…Infection of HeLa cells and immunostaining for dsRNA and MV N protein were performed as described previously (14). Slides were analyzed using an LSM 780 inverted laser scanning confocal microscope and ZEN 2012 (Black Edition, v8.0) software (Carl Zeiss Microscopy, Jena, Germany).…”