Following infection with most reovirus strains, viral protein synthesis is robust, even when cellular translation is inhibited. To gain further insight into pathways that regulate translation in reovirus-infected cells, we performed a comparative microarray analysis of cellular gene expression following infection with two strains of reovirus that inhibit host translation (clone 8 and clone 87) and one strain that does not (Dearing). Infection with clone 8 and clone 87 significantly increased the expression of cellular genes characteristic of stress responses, including the integrated stress response. Infection with these same strains decreased transcript and protein levels of P58 IPK , the cellular inhibitor of the eukaryotic initiation factor 2␣ (eIF2␣) kinases PKR and PERK. Since infection with host shutoff-inducing strains of reovirus impacted cellular pathways that control eIF2␣ phosphorylation and unphosphorylated eIF2␣ is required for translation initiation, we examined reovirus replication in a variety of cell lines with mutations that impact eIF2␣ phosphorylation. Our results revealed that reovirus replication is more efficient in the presence of eIF2␣ kinases and phosphorylatable eIF2␣. When eIF2␣ is phosphorylated, it promotes the synthesis of ATF4, a transcription factor that controls cellular recovery from stress. We found that the presence of this transcription factor increased reovirus yields 10-to 100-fold. eIF2␣ phosphorylation also led to the formation of stress granules in reovirus-infected cells. Based on these results, we hypothesize that eIF2␣ phosphorylation facilitates reovirus replication in two ways-first, by inducing ATF4 synthesis, and second, by creating an environment that places abundant reovirus transcripts at a competitive advantage for limited translational components.As representative members of the Reoviridae family, mammalian orthoreoviruses (reoviruses) have genomes composed of 10 segments of double-stranded RNA (dsRNA) that are surrounded by two concentric protein capsids (reviewed in reference 54). In natural infections, reoviruses replicate in cells of the respiratory or enteric tract; however, in experimental infections of neonatal mice, their tropism is much broader. Pathogenesis studies with the mouse model have demonstrated that reoviruses replicate in cells of the brain, heart, liver, muscle, and pancreas (reviewed in reference 73). At the cellular level, the consequences of reovirus infection have been extensively analyzed and include the inhibition of DNA synthesis, cell cycle arrest at the G 2 /M stage, apoptosis induction, translational inhibition, and type

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By atomic absorption analysis, we determined that the reovirus outer capsid protein cr3, which binds double-stranded RNA (dsRNA), is a zinc metalloprotein. Using Northwestern blots and a novel zinc blotting technique, we localized the zinc-and dsRNA-binding activities of cr3 to distinct V8 protease-generated fragments. Zinc-binding activity was contained within an amino-terminal fragment that contained a transcription factor IIIA-like zinc-binding sequence, and dsRNA-binding activity was associated with a carboxyterminal fragment. By these techniques, new zinc-and dsRNA-binding activities were also detected in reovirus core proteins. A sequence similarity was observed between the catalytic site of the picornavirus proteases and the transcription factor lIlA-Like zinc-binding site within a3. We suggest that the zinc-and dsRNA-binding activities of cr3 may be important for its proposed regulatory effects on viral and host cell transcription and translation.The mammalian reoviruses represent the prototype of a group of nonenveloped plant and animal viruses whose segmented, double-stranded RNA (dsRNA) genomes are surrounded by an inner capsid core and an outer capsid shell. Three serotypes of mammalian reoviruses have been identified, and the different strains of these viruses studied to date have been found to vary with respect to a number of biochemical markers and biological properties. As a result of these polymorphisms, reoviruses offer an excellent model system for the study of virion morphogenesis and virus-host cell interactions because the genetic analysis of reassortant viruses has provided a means to assign biological phenotypes and biochemical properties to particular viral genome segments. Results of studies with reassortant viruses have suggested that, in addition to playing structural roles, the reovirus outer capsid proteins influence the virus-host cell interaction at a variety of stages in the viral replicative cycle. S4, the smallest of the 10 mammalian reovirus genes, encodes the a3 protein (38, 40), a major component of the virion outer capsid (53). The or3 protein is the only reovirus protein that has been reported to have affinity for dsRNA (27). This is an unusual property for an outer capsid protein which is removed from infecting parental virus by protease digestion early in infection (52, 55), and suggests that this property may be one of newly synthesized a3 that operates in the cell cytoplasm. Results of biochemical and genetic studies have implied a role for the S4 gene product in the regulation of viral transcription (4) and translation (33, 34), the inhibition of host cell RNA and protein synthesis (50), and the establishment of persistent reovirus infection (1). To begin to determine whether the ability of cr3 to bind to dsRNA is mechanistically important for virion morphogenesis or any of the biological properties which have been mapped to the S4 gene segment, we initiated a study to investigate the structural basis for the dsRNA-binding activity of cr3. * Corresponding author. A re...

Entry of reovirus virions has been well studied in several tissue culture systems. After attachment to junctional adhesion molecule A (JAM-A), virions undergo clathrin-mediated endocytosis followed by proteolytic disassembly of the capsid and penetration to the cytoplasm. However, during in vivo infection of the intestinal tract, and likely in the tumor microenvironment, capsid proteolysis (uncoating) is initiated extracellularly. We used multiple approaches to determine if uncoated reovirus particles, called intermediate subviral particles (ISVPs), enter cells by directly penetrating the limiting membrane or if they take advantage of endocytic pathways to establish productive infection. We found that entry and infection by reovirus ISVPs was inhibited by dynasore, an inhibitor of dynamin-dependent endocytosis, as well as by genistein and dominant-negative caveolin-1, which block caveolar endocytosis. Inhibition of caveolar endocytosis also reduced infection by reovirus virions. Extraction of membrane cholesterol with methyl-␤-cyclodextrin inhibited infection by virions but had no effect when infection was initiated with ISVPs. We found this pathway to be independent of both clathrin and caveolin. Together, these data suggest that reovirus virions can use both dynamin-dependent and dynamin-independent endocytic pathways during cell entry, and they reveal that reovirus ISVPs can take advantage of caveolar endocytosis to establish productive infection.

We previously hypothesized that efficient translation of influenza virus mRNA requires the recruitment of P58 IPK , the cellular inhibitor of PKR, an interferon-induced kinase that targets the eukaryotic translation initiation factor eIF2␣. P58 IPK also inhibits PERK, an eIF2␣ kinase that is localized in the endoplasmic reticulum (ER) and induced during ER stress. The ability of P58 IPK to interact with and inhibit multiple eIF2␣ kinases suggests it is a critical regulator of both cellular and viral mRNA translation. In this study, we sought to definitively define the role of P58IPK during viral infection of mammalian cells. Using mouse embryo fibroblasts from P58 IPK؊/؊ mice, we demonstrated that the absence of P58 IPK led to an increase in eIF2␣ phosphorylation and decreased influenza virus mRNA translation. The absence of P58 IPK also resulted in decreased vesicular stomatitis virus replication but enhanced reovirus yields. In cells lacking the P58 IPK target, PKR, the trends were reversed-eIF2␣ phosphorylation was decreased, and influenza virus mRNA translation was increased. Although P58 IPK also inhibits PERK, the presence or absence of this kinase had little effect on influenza virus mRNA translation, despite reduced levels of eIF2␣ phosphorylation in cells lacking PERK. Finally, we showed that influenza virus protein synthesis and viral mRNA levels decrease in cells that express a constitutively active, nonphosphorylatable eIF2␣. Taken together, our results support a model in which P58 IPK regulates influenza virus mRNA translation and infection through a PKR-mediated mechanism which is independent of PERK.