Characterization of the double-stranded RNA implicated in the inhibition of protein synthesis in cells infected with a mutant adenovirus defective for VA RNA

ABSTRACT Sendai virus (SeV) C protein is a multifunctional protein that plays important roles in regulating viral genome replication and transcription, antagonizing the host interferon system, suppressing virus-induced apoptosis, and facilitating virus assembly and budding. We here report a novel role of SeV C protein, the limitation of double-stranded RNA (dsRNA) generation for maintaining the rate of protein synthesis in infected cells. It was found that the intracellular protein synthesis rate was maintained even after wild-type (wt) SeV infection, but markedly suppressed following C-knockout SeV infection. This indicates the requirement of C protein for maintaining protein synthesis after infection. In contrast to wt SeV infection, C-knockout SeV infection caused phosphorylation of both the translation initiation factor eIF2α and dsRNA-dependent protein kinase (PKR). Phosphorylation of eIF2α occurred mainly due to the action of PKR, since knockdown of PKR by small interfering RNA limited eIF2α phosphorylation. C protein, however, could inhibit neither poly(I):poly(C)-activated nor Newcastle disease virus-induced phosphorylation of PKR and eIF2α, suggesting that C protein does not target common pathways leading to PKR activation. Immunofluorescent staining experiments with a monoclonal antibody specifically recognizing dsRNA revealed generation of a large amount of dsRNA in cells infected with C-knockout SeV but not wt SeV. The dsRNA generation as well as phosphorylation of PKR and eIF2α induced by C-knockout SeV was markedly suppressed in cells constitutively expressing C protein. Taken together, these results demonstrate that the SeV C protein limits generation of dsRNA, thereby keeping PKR inactive to maintain intracellular protein synthesis.

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Activation of p38 Mitogen-Activated Protein Kinase and c-Jun NH2-Terminal Kinase by Double-Stranded RNA and Encephalomyocarditis Virus: Involvement of RNase L, Protein Kinase R, and Alternative Pathways

Molecular and Cellular Biology ◽

10.1128/mcb.20.2.617-627.2000 ◽

2000 ◽

Vol 20 (2) ◽

pp. 617-627 ◽

Cited By ~ 156

Author(s):

Mihail S. Iordanov ◽

Jayashree M. Paranjape ◽

Aimin Zhou ◽

John Wong ◽

Bryan R. G. Williams ◽

...

Keyword(s):

Protein Synthesis ◽

Protein Kinase ◽

P38 Mapk ◽

Encephalomyocarditis Virus ◽

Mitogen Activated Protein Kinase ◽

Rnase L ◽

Double Stranded Rna ◽

Mitogen Activated Protein ◽

Stimulation Of ◽

In Cells

ABSTRACT Double-stranded RNA (dsRNA) accumulates in virus-infected mammalian cells and signals the activation of host defense pathways of the interferon system. We describe here a novel form of dsRNA-triggered signaling that leads to the stimulation of the p38 mitogen-activated protein kinase (p38 MAPK) and the c-Jun NH2-terminal kinase (JNK) and of their respective activators MKK3/6 and SEK1/MKK4. The dsRNA-dependent signaling to p38 MAPK was largely intact in cells lacking both RNase L and the dsRNA-activated protein kinase (PKR), i.e., the two best-characterized mediators of dsRNA-triggered antiviral responses. In contrast, activation of both MKK4 and JNK by dsRNA was greatly reduced in cells lacking RNase L (or lacking both RNase L and PKR) but was restored in these cells when introduction of dsRNA was followed by inhibition of ongoing protein synthesis or transcription. These results are consistent with the notion that the role of RNase L and PKR in the activation of MKK4 and JNK is the elimination, via inhibition of protein synthesis, of a labile negative regulator(s) of the signaling to JNK acting upstream of SEK1/MKK4. In the course of these studies, we identified a long-sought site of RNase L-mediated cleavage in the 28S rRNA, which could cause inhibition of translation, thus allowing the activation of JNK by dsRNA. We propose that p38 MAPK is a general participant in dsRNA-triggered cellular responses, whereas the activation of JNK might be restricted to cells with reduced rates of protein synthesis. Our studies demonstrate the existence of alternative (RNase L- and PKR-independent) dsRNA-triggered signaling pathways that lead to the stimulation of stress-activated MAPKs. Activation of p38 MAPK (but not of JNK) was demonstrated in mouse fibroblasts in response to infection with encephalomyocarditis virus (ECMV), a picornavirus that replicates through a dsRNA intermediate. Fibroblasts infected with EMCV (or treated with dsRNA) produced interleukin-6, an inflammatory and pyrogenic cytokine, in a p38 MAPK-dependent fashion. These findings suggest that stress-activated MAPKs participate in mediating inflammatory and febrile responses to viral infections.

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Phosphorylation and the control of protein synthesis

Philosophical Transactions of the Royal Society of London Series B Biological Sciences ◽

10.1098/rstb.1983.0045 ◽

1983 ◽

Vol 302 (1108) ◽

pp. 127-134 ◽

Cited By ~ 7

Keyword(s):

Protein Synthesis ◽

Protein Kinases ◽

Molecular Mechanism ◽

The Other ◽

Initiation Factor ◽

Reversible Phosphorylation ◽

Double Stranded Rna ◽

Inhibition Of Protein Synthesis ◽

The One ◽

Rabbit Reticulocytes

This paper reviews the evidence that protein synthesis in rabbit reticulocytes is regulated by the reversible phosphorylation of the initiation factor eIF-2 by protein kinases under the control of the cytoplasmic haemin concentration on the one hand, and double-stranded RNA on the other. A molecular mechanism is proposed to account for the observation that inhibition of protein synthesis occurs when considerably less than half the eIF-2 present has been phosphorylated. The question of whether phosphorylation regulates protein synthesis in other types of cell is discussed.

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