Preferential Translation of Reovirus mRNA by a σ3-Dependent Mechanism

Progeny virions of mammalian reoviruses are assembled in the cytoplasm of infected cells at discrete sites termed viral inclusions. Studies of temperature-sensitive (ts) mutant viruses indicate that nonstructural protein NS and core protein 2 are required for synthesis of double-stranded (ds) RNA, a process that occurs at sites of viral assembly. We used confocal immunofluorescence microscopy and ts mutant reoviruses to define the roles of NS and 2 in viral inclusion formation. In cells infected with wild-type (wt) reovirus, NS and 2 colocalize to large, perinuclear structures that correspond to viral inclusions. In cells infected at a nonpermissive temperature with NS-mutant virus tsE320, NS is distributed diffusely in the cytoplasm and 2 is contained in small, punctate foci that do not resemble viral inclusions. In cells infected at a nonpermissive temperature with 2-mutant virus tsH11.2, 2 is distributed diffusely in the cytoplasm and the nucleus. However, NS localizes to discrete structures in the cytoplasm that contain other viral proteins and are morphologically indistinguishable from viral inclusions seen in cells infected with wt reovirus. Examination of cells infected with wt reovirus over a time course demonstrates that NS precedes 2 in localization to viral inclusions. These findings suggest that viral RNA-protein complexes containing NS nucleate sites of viral replication to which other viral proteins, including 2, are recruited to commence dsRNA synthesis.

Section: Resultsmentioning

confidence: 98%

Reovirus ςNS Protein Is Required for Nucleation of Viral Assembly Complexes and Formation of Viral Inclusions

Becker

Goral

Hazelton

et al. 2001

“…1B). We chose an MOI of 80 PFU/cell for the rest of our protein labeling experiments because this MOI was historically used for examining cellular translation in reovirus-infected cells (26,40,41) and maximized strain-specific phenotypes.…”

Section: Resultsmentioning

confidence: 99%

“…However, the extent of reovirus-induced host shutoff varies in a strain-specific manner; infection with strain Dearing has a minimal effect on cellular translation, whereas infection with other strains, such as Jones, clone 8 (c8), clone 87 (c87), and clone 93 (c93), leads to dramatic host shutoff (40,41). In most situations, even when cellular translation is inhibited, reovirus proteins are efficiently synthesized.…”

mentioning

confidence: 99%

Involvement of the Interferon-Regulated Antiviral Proteins PKR and RNase L in Reovirus-Induced Shutoff of Cellular Translation

Smith

Schmechel

Williams

et al. 2005

Cellular translation is inhibited following infection with most strains of reovirus, but the mechanisms responsible for this phenomenon remain to be elucidated. The extent of host shutoff varies in a straindependent manner; infection with the majority of strains leads to strong host shutoff, while infection with strain Dearing results in minimal inhibition of cellular translation. A genetic study with reassortant viruses and subsequent biochemical analyses led to the hypothesis that the interferon-induced, double-stranded RNA-activated protein kinase, PKR, is responsible for reovirus-induced host shutoff. To directly determine whether PKR is responsible for reovirus-induced host shutoff, we used a panel of reovirus strains and mouse embryo fibroblasts derived from knockout mice. This approach revealed that PKR contributes to but is not wholly responsible for reovirus-induced host shutoff. Studies with cells lacking RNase L, the endoribonuclease component of the interferon-regulated 2,5-oligoadenylate synthetase-RNase L system, demonstrated that RNase L also down-regulates cellular protein synthesis in reovirus-infected cells. In many viral systems, PKR and RNase L have well-characterized antiviral functions. An analysis of reovirus replication in cells lacking these molecules indicated that, while they contributed to host shutoff, neither PKR nor RNase L exerted an antiviral effect on reovirus growth. In fact, some strains of reovirus replicated more efficiently in the presence of PKR and RNase L than in their absence. Data presented in this report illustrate that the inhibition of cellular translation following reovirus infection is complex and involves multiple interferon-regulated gene products. In addition, our results suggest that reovirus has evolved effective mechanisms to avoid the actions of the interferon-stimulated antiviral pathways that include PKR and RNase L and may even benefit from their expression.Cellular translation is inhibited following infection with most strains of mammalian reovirus, a phenomenon known as host shutoff (54). However, the extent of reovirus-induced host shutoff varies in a strain-specific manner; infection with strain Dearing has a minimal effect on cellular translation, whereas infection with other strains, such as Jones, clone 8 (c8), clone 87 (c87), and clone 93 (c93), leads to dramatic host shutoff (40,41). In most situations, even when cellular translation is inhibited, reovirus proteins are efficiently synthesized. Although the mechanisms responsible for reovirus-induced host shutoff remain to be elucidated, they have been defined for other viruses, including poliovirus and rotavirus (14). In poliovirusinfected cells, virus-encoded protease 2A pro leads to cleavage of eukaryotic translation initiation factors eIF4GI and eIF4GII, thus preventing translation of the vast majority of capped cellular mRNAs (10, 17). Uncapped and internal ribosome entry site-containing poliovirus mRNAs, in contrast, require only the C-terminal cleaved portion of eIF4G for translation initi...

“…By contrast, some RNA viruses replicate very efficiently without inducing an overall inhibition of host cell gene expression. Examples of this include dengue virus (15), arenaviruses (4), some paramyxoviruses (e.g., human respiratory syncytial virus [11]), and some strains of reovirus (43).…”

mentioning

confidence: 99%

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

Gainey

Dillon

Clark

et al. 2008

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.