Sendai Virus C Protein Plays a Role in Restricting PKR Activation by Limiting the Generation of Intracellular Double-Stranded RNA

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.

Section: Discussionsupporting

confidence: 62%

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

Pfaller

Radeke

Cattaneo

et al. 2014

“…1 and 5), together with the observed activation of PKR phosphorylation observed for C ko virus-infected cells (Fig. 2), is consistent with an enhanced generation of activator RNA with significant double-stranded character by this mutant virus (32,35).…”

Section: Irf-3 Phosphorylation Is Enhanced In C Ko -Infected Comparedsupporting

confidence: 58%

Mechanisms of Protein Kinase PKR-Mediated Amplification of Beta Interferon Induction by C Protein-Deficient Measles Virus

McAllister

Toth

Zhang

et al. 2010

106

The measles virus P gene products V and C antagonize the host interferon (IFN) response, blocking both IFN signaling and production. Using Moraten vaccine strain-derived measles virus and isogenic mutants deficient for either V or C protein production (V ko and C ko , respectively), we observed that the C Measles virus (MV), a member of the genus Morbillivirus of the Paramyxoviridae, causes an acute febrile illness. Despite an effective vaccine, measles continues to cause extreme morbidity and mortality worldwide (10), and recently, there has been a resurgence of measles in industrialized countries, where a lack of adherence to vaccine recommendations is an increasing problem (5, 12). The need for improved MV vaccines (11), together with the potential for use of engineered MV vaccine strains with defined mechanisms of attenuation as oncolytic viruses for cancer therapy (4), further justify ongoing efforts to gain an enhanced understanding of the host response to MV infection at the molecular level.The 15.9-kb negative-stranded RNA genome of MV consists of six genes (N, P/V/C, M, F, H, and L). The P gene is polycistronic, encoding the V and C nonstructural proteins in addition to P, a structural phosphoprotein and essential cofactor for the viral polymerase (2, 3, 10). The V protein shares its N-terminal 231 amino acids with P, but the C-terminal 68 amino acids are unique because of the pseudotemplated G insertion that causes a frameshift in V mRNA, whereas the C protein is synthesized by an alternative translation initiation AUG codon positioned 22 nucleotides downstream of the P/V translation start codon (3, 11). V and C are accessory proteins that serve a variety of functions including the modulation of the host innate immune response to MV infection (8, 9, 27). Isogenic MV mutants that are defective for the expression of either V or C have been generated. Strong adaptive immune responses, but dysregulated innate responses, are seen with these mutants (4,8,35).An important component of the host innate antiviral response is the interferon (IFN) response. IFNs are proinflammatory cytokines that possess antiviral activity (27,31). IFN action involves IFN binding to cognate receptors and subsequent prototypical JAK-STAT signal transduction that leads to the expression of IFN-stimulated genes, whose products inhibit virus growth. IFN production involves the recognition of pathogen-associated molecular patterns including viral RNAs by retinoic acid-inducible protein (RIG-I)-like cytosolic receptors (RLRs) and membrane-associated Toll-like receptors (TLRs). The RLR and TLR3 sensors signal through cognate adapter proteins, including IPS-1 and TRIF, respectively, to transcriptionally activate IFN expression (36,40). In the case of the IFN-␤ gene, IFN regulatory factor 3 (IRF-3) and nuclear factor B (NF-B) are activated by RLR or TLR signaling, enter the nucleus, and function together with activating transcription factor 2 (ATF-2)/c-jun to constitute the IFN-␤ enhanceosome that drives IFN-␤ transcription (22).The antago...

“…Moreover, we observed about 10 times more DI-RNA in C KO virus infection than in parental virus infection, implying a genetic link between the C KO phenotype and DI-RNA generation. Notably, C KO mutants of SeV and human parainfluenza virus type 1 have also been reported to generate double-stranded RNA and activate PKR, but these studies did not address the origin or the structure of these RNAs (43,44). Although C KO mutants of several paramyxoviruses can be rescued and are only moderately attenuated in cultured cells (16), they are strongly attenuated in vivo (45,46).…”

Section: Discussionmentioning

confidence: 99%

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

Pfaller

Mastorakos

Matchett

et al. 2015

Defective interfering RNAs (DI-RNAs) of the viral genome can form during infections of negative-strand RNA viruses and outgrow full-length viral genomes, thereby modulating the severity and duration of infection. Here we document the frequent de novo generation of copy-back DI-RNAs from independent rescue events both for a vaccine measles virus (vac2) and for a wildtype measles virus (IC323) as early as passage 1 after virus rescue. Moreover, vaccine and wild-type C-protein-deficient (C-protein-knockout [C KO ]) measles viruses generated about 10 times more DI-RNAs than parental virus, suggesting that C enhances the processivity of the viral polymerase. We obtained the nucleotide sequences of 65 individual DI-RNAs, identified breakpoints and reinitiation sites, and predicted their structural features. Several DI-RNAs possessed clusters of A-to-G or U-to-C transitions. Sequences flanking these mutation sites were characteristic of those favored by adenosine deaminase acting on RNA-1 (ADAR1), which catalyzes in double-stranded RNA the C-6 deamination of adenosine to produce inosine, which is recognized as guanosine, a process known as A-to-I RNA editing. In individual DI-RNAs the transitions were of the same type and occurred on both sides of the breakpoint. These patterns of mutations suggest that ADAR1 edits unencapsidated DI-RNAs that form doublestrand RNA structures. Encapsidated DI-RNAs were incorporated into virus particles, which reduced the infectivity of virus stocks. The C KO phenotype was dominant: DI-RNAs derived from vac2 with a C KO suppressed the replication of vac2, as shown by coinfections of interferon-incompetent lymphatic cells with viruses expressing different fluorescent reporter proteins. In contrast, coinfection with a C-protein-expressing virus did not counteract the suppressive phenotype of DI-RNAs. IMPORTANCE Recombinant measles viruses (MVs) are in clinical trials as cancer therapeutics and as vectored vaccines for HIV-AIDS and other infectious diseases. The efficacy of MV-based vectors depends on their replication proficiency and immune activation capacity.Here we document that copy-back defective interfering RNAs (DI-RNAs) are generated by recombinant vaccine and wild-type MVs immediately after rescue. The MV C protein interferes with DI-RNA generation and may enhance the processivity of the viral polymerase. We frequently detected clusters of A-to-G or U-to-C transitions and noted that sequences flanking individual mutations contain motifs favoring recognition by the adenosine deaminase acting on RNA-1 (ADAR1). The consistent type of transitions on the DI-RNAs indicates that these are direct substrates for editing by ADAR1. The ADAR1-mediated biased hypermutation events are consistent with the protein kinase R (PKR)-ADAR1 balancing model of innate immunity activation. We show by coinfection that the C-defective phenotype is dominant.