West Nile Virus Infections Suppress Early Viral RNA Synthesis and Avoid Inducing the Cell Stress Granule Response

Japanese encephalitis virus (JEV) is an enveloped flavivirus with a single-stranded, positive-sense RNA genome encoding three structural and seven nonstructural proteins. To date, the role of JEV nonstructural protein 2A (NS2A) in the viral life cycle is largely unknown. The interferon (IFN)-induced double-stranded RNA (dsRNA)-activated protein kinase (PKR) phosphorylates the eukaryotic translation initiation factor 2α subunit (eIF2α) after sensing viral RNA and results in global translation arrest as an important host antiviral defense response. In this study, we found that JEV NS2A could antagonize PKR-mediated growth inhibition in a galactose-inducible PKR-expressing yeast system. In human cells, PKR activation, eIF2α phosphorylation, and the subsequent translational inhibition and cell death triggered by dsRNA and IFN-α were also repressed by JEV NS2A. Moreover, among the four eIF2α kinases, NS2A specifically blocked the eIF2α phosphorylation mediated by PKR and attenuated the PKR-promoted cell death induced by the chemotherapeutic drug doxorubicin. A single point mutation of NS2A residue 33 from Thr to Ile (T33I) abolished the anti-PKR potential of JEV NS2A. The recombinant JEV mutant carrying the NS2A-T33I mutation showed reduced in vitro growth and in vivo virulence phenotypes. Thus, JEV NS2A has a novel function in blocking the host antiviral response of PKR during JEV infection.

Section: Discussionmentioning

confidence: 84%

Blocking Double-Stranded RNA-Activated Protein Kinase PKR by Japanese Encephalitis Virus Nonstructural Protein 2A

Liang

et al. 2012

“…As shown in Fig. 2D, both heat shock and TBEV infection induced eIF2␣ phosphorylation in U2OS cells, most likely through protein kinase R (PKR) (19,33). To determine if TBEV prevents SG formation in response to heat shock, U2OS cells were electroporated with TNd/⌬ME_24ϫMS2.…”

Section: Resultsmentioning

confidence: 99%

“…They concluded that these viruses inhibit SG formation by targeting TIA-1/TIAR either through the nonstructural protein NS3, which could be immunoprecipitated by both TIA-1 and TIAR, or through the viral 3=(Ϫ)SL RNA, which was earlier shown to bind TIAR and, to a lesser extent, TIA-1 (17). However, more recently, they reinterpreted their own data, this time looking at G3BP1 as a marker for SG, showing that natural WNV genotypes, such as Eg101, induced SG less efficiently than the lineage 2/1 chimeric WNV infectious clone W956IC, which produced high levels of early viral RNA (19). They proposed that induction of SG by WNV is closely linked to the availability of dsRNA to PKR signaling and that fast-replicating genotypes, such as W956IC, release more dsRNA from virus-induced vesicles, thus triggering the Ϫ/Ϫ and their control MEF WT were electroporated with TNd/⌬ME_C17_fluc_GAA.…”

Section: Discussionmentioning

confidence: 99%

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The Stress Granule Component TIA-1 Binds Tick-Borne Encephalitis Virus RNA and Is Recruited to Perinuclear Sites of Viral Replication To Inhibit Viral Translation

Albornoz

Carletti

Corazza

et al. 2014

Flaviviruses are a major cause of disease in humans and animals worldwide. Tick-borne encephalitis virus (TBEV) is the most important arthropod-borne flavivirus endemic in Europe and is the etiological agent of tick-borne encephalitis, a potentially fatal infection of the central nervous system. However, the contributions of host proteins during TBEV infection are poorly understood. In this work, we investigate the cellular protein TIA-1 and its cognate factor TIAR, which are stress-induced RNA

“…Formation of SG is PKR dependent in a number of virus-infected cell systems (49), including MV (19), hepatitis C virus (50), respiratory syncytial virus (51), and West Nile virus (52). Subcellular localization, kinetics of accumulation, and sensitivity to cycloheximide, a potent inhibitor of MV replication (40), suggest that the RNA responsible for the dsRNA signal observed in C KO mutant virus-infected cells is of viral and not of cellular origin.…”

Section: Discussionmentioning

confidence: 99%

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

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.