Feline Calicivirus Infection Disrupts Assembly of Cytoplasmic Stress Granules and Induces G3BP1 Cleavage.

UNLABELLED: In response to stress such as virus infection, cells can stall translation by storing mRNAs away in cellular compartments called stress granules (SGs). This defense mechanism favors cell survival by limiting the use of energy and nutrients until the stress is resolved. In some cases it may also block viral propagation as viruses are dependent on the host cell resources to produce viral proteins. Human norovirus is a member of the Caliciviridae family responsible for gastroenteritis outbreaks worldwide. Previous studies on caliciviruses have identified mechanisms by which they can usurp the host translational machinery, using the viral protein genome-linked VPg, or regulate host protein synthesis through the mitogen-activated protein kinase (MAPK) pathway. Here, we examined the effect of feline calicivirus (FCV) infection on SG accumulation. We show that FCV infection impairs the assembly of SGs despite an increased phosphorylation of eukaryotic initiation factor eIF2α, a hallmark of stress pathway activation. Furthermore, SGs did not accumulate in FCV-infected cells that were stressed with arsenite or hydrogen peroxide. FCV infection resulted in the cleavage of the SG-nucleating protein Ras-GTPase activating SH3 domain-binding protein (G3BP1), which is mediated by the viral 3C-like proteinase NS6(Pro) Using mutational analysis, we identified the FCV-induced cleavage site within G3BP1, which differs from the poliovirus 3C proteinase cleavage site previously identified. Finally, we showed that NS6(Pro)-mediated G3BP1 cleavage impairs SG assembly. In contrast, murine norovirus (MNV) infection did not impact arsenite-induced SG assembly or G3BP1 integrity, suggesting that related caliciviruses have distinct effects on the stress response pathway. IMPORTANCE: Human noroviruses are a major cause of viral gastroenteritis, and it is important to understand how they interact with the infected host cell. Feline calicivirus (FCV) and murine norovirus (MNV) are used as models to understand norovirus biology. Recent studies have suggested that the assembly of stress granules is central in orchestrating stress and antiviral responses to restrict viral replication. Overall, our study provides the first insight on how caliciviruses impair stress granule assembly by targeting the nucleating factor G3BP1 via the viral proteinase NS6(Pro) This work provides new insights into host-pathogen interactions that regulate stress pathways during FCV infection.

Translational Control during Calicivirus Infection.

In this review, we provide an overview of the strategies developed by caliciviruses to subvert or regulate the host protein synthesis machinery to their advantage. As intracellular obligate parasites, viruses strictly depend on the host cell resources to produce viral proteins. Thus, many viruses have developed strategies that regulate the function of the host protein synthesis machinery, often leading to preferential translation of viral mRNAs. Caliciviruses lack a 5' cap structure but instead have a virus-encoded VPg protein covalently linked to the 5' end of their mRNAs. Furthermore, they encode 2-4 open reading frames within their genomic and subgenomic RNAs. Therefore, they use alternative mechanisms for translation whereby VPg interacts with eukaryotic initiation factors (eIFs) to act as a proteinaceous cap-substitute, and some structural proteins are produced by reinitiation of translation events. This review discusses our understanding of these key mechanisms during caliciviruses infection as well as recent insights into the global regulation of eIF4E activity.

Murine norovirus 1 (MNV1) replication induces translational control of the host by regulating eIF4E activity during infection.

Protein synthesis is a tightly controlled process responding to several stimuli, including viral infection. As obligate intracellular parasites, viruses depend on the translation machinery of the host and can manipulate it by affecting the availability and function of specific eukaryotic initiation factors (eIFs). Human norovirus is a member of the Caliciviridae family and is responsible for gastroenteritis outbreaks. Previous studies on feline calicivirus and murine norovirus 1 (MNV1) demonstrated that the viral protein, genome-linked (VPg), acts to direct translation by hijacking the host protein synthesis machinery. Here we report that MNV1 infection modulates the MAPK pathway to activate eIF4E phosphorylation. Our results show that the activation of p38 and Mnk during MNV1 infection is important for MNV1 replication. Furthermore, phosphorylated eIF4E relocates to the polysomes, and this contributes to changes in the translational state of specific host mRNAs. We propose that global translational control of the host by eIF4E phosphorylation is a key component of the host-pathogen interaction.

Structural features of the Seneca Valley virus internal ribosome entry site (IRES) element: a picornavirus with a pestivirus-like IRES.

The RNA genome of Seneca Valley virus (SVV), a recently identified picornavirus, contains an internal ribosome entry site (IRES) element which has structural and functional similarity to that from classical swine fever virus (CSFV) and hepatitis C virus, members of the Flaviviridae. The SVV IRES has an absolute requirement for the presence of a short region of virus-coding sequence to allow it to function either in cells or in rabbit reticulocyte lysate. The IRES activity does not require the translation initiation factor eIF4A or intact eIF4G. The predicted secondary structure indicates that the SVV IRES is more closely related to the CSFV IRES, including the presence of a bipartite IIId domain. Mutagenesis of the SVV IRES, coupled to functional assays, support the core elements of the IRES structure model, but surprisingly, deletion of the conserved IIId(2) domain had no effect on IRES activity, including 40S and eIF3 binding. This is the first example of a picornavirus IRES that is most closely related to the CSFV IRES and suggests the possibility of multiple, independent recombination events between the genomes of the Picornaviridae and Flaviviridae to give rise to similar IRES elements.

Infection of bovine dendritic cells by rinderpest or measles viruses induces different changes in host transcription.

The morbilliviruses are a closely related genus which are very similar in their sequences and share a common receptor, but nevertheless show significant restriction in the host species in which they cause disease. One contribution to this restriction might be the nature of the hosts' responses to infection. We have used microarrays to study the changes in the transcriptome of bovine dendritic cells after infection with wild-type (pathogenic) and vaccine (apathogenic) strains of rinderpest virus (RPV), a bovine pathogen, and a wild-type isolate of measles virus (MV), a morbillivirus that causes disease only in humans and some other primates. We found that, as previously observed in human cells, MV induces a rapid interferon response, while that induced by RPV was delayed and much reduced in magnitude. Pathogenic and apathogenic RPV also showed significant differences, with the latter inducing a slightly higher interferon response as well as significant effects on transcription of genes involved in cell cycle regulation.

Monocistronic mRNAs containing defective hepatitis C virus-like picornavirus internal ribosome entry site elements in their 5' untranslated regions are efficiently translated in cells by a cap-dependent mechanism.

The initiation of protein synthesis on mRNAs within eukaryotic cells is achieved either by a 5' cap-dependent mechanism or through internal initiation directed by an internal ribosome entry site (IRES). Picornavirus IRES elements, located in the 5' untranslated region (5'UTR), contain extensive secondary structure and multiple upstream AUG codons. These features can be expected to inhibit cap-dependent initiation of translation. However, we have now shown that certain mutant hepatitis C virus-like picornavirus IRES elements (from porcine teschovirus-1 and avian encephalomyelitis virus), which are unable to direct internal initiation, are not significant barriers to efficient translation of capped monocistronic mRNAs that contain these defective elements within their 5'UTRs. Moreover, the translation of these mRNAs is highly sensitive to the expression of an enterovirus 2A protease (which induces cleavage of eIF4G) and is also inhibited by hippuristanol, a specific inhibitor of eIF4A function, in contrast to their parental wild-type IRES elements. These results provide a possible basis for the evolution of viral IRES elements within the context of functional mRNAs that are translated by a cap-dependent mechanism.

The picornavirus avian encephalomyelitis virus possesses a hepatitis C virus-like internal ribosome entry site element.

Avian encephalomyelitis virus (AEV) is a picornavirus that causes disease in poultry worldwide, and flocks must be vaccinated for protection. AEV is currently classified within the hepatovirus genus, since its proteins are most closely related to those of hepatitis A virus (HAV). We now provide evidence that the 494-nucleotide-long 5' untranslated region of the AEV genome contains an internal ribosome entry site (IRES) element that functions efficiently in vitro and in mammalian cells. Unlike the HAV IRES, the AEV IRES is relatively short and functions in the presence of cleaved eIF4G and it is also resistant to an inhibitor of eIF4A. These properties are reminiscent of the recently discovered class of IRES elements within certain other picornaviruses, such as porcine teschovirus 1 (PTV-1). Like the PTV-1 IRES, the AEV IRES shows significant similarity to the hepatitis C virus (HCV) IRES in sequence, function, and predicted secondary structure. Furthermore, mutational analysis of the predicted pseudoknot structure at the 3' end of the AEV IRES lends support to the secondary structure we present. AEV is therefore another example of a picornavirus harboring an HCV-like IRES element within its genome, and thus, its classification within the hepatovirus genus may need to be reassessed in light of these findings.

A cross-kingdom internal ribosome entry site reveals a simplified mode of internal ribosome entry.

Rhopalosiphum padi virus (RhPV) is an insect virus of the Dicistroviridae family. Recently, the 579-nucleotide-long 5' untranslated region (UTR) of RhPV has been shown to contain an internal ribosome entry site (IRES) that functions efficiently in mammalian, plant, and insect in vitro translation systems. Here, the mechanism of action of the RhPV IRES has been characterized by reconstitution of mammalian 48S initiation complexes on the IRES from purified components combined with the toeprint assay. There is an absolute requirement for the initiation factors eIF2 and eIF3 and the scanning factor eIF1 to form 48S complexes on the IRES. In addition, eIF1A, eIF4F (or the C-terminal fragment of eIF4G), and eIF4A strongly stimulated the assembly of this complex, whereas eIF4B had no effect. Although the eIF4-dependent pathway is dominant in the RhPV IRES-directed cell-free translation, omission of either eIF4G or eIF4A or both still allowed the assembly of 48S complexes from purified components with approximately 23% of maximum efficiency. Deletions of up to 100 nucleotides throughout the 5'-UTR sequence produced at most a marginal effect on the IRES activity, suggesting the absence of specific binding sites for initiation factors. Only deletion of the U-rich unstructured 380-nucleotide region proximal to the initiation codon resulted in a complete loss of the IRES activity. We suggest that the single-stranded nature of the RhPV IRES accounts for its strong but less selective potential to bind key mRNA recruiting components of the translation initiation apparatus from diverse origins.

A bunyamwera virus minireplicon system in mosquito cells.

Artificial minigenomes are powerful tools for studying the replication and transcription of negative-strand RNA viruses. Bunyamwera virus (BUN; genus Orthobunyavirus, family Bunyaviridae) is an arbovirus that shows fundamental biological differences when replicating in mammalian versus mosquito cells. To study BUN RNA synthesis in mosquito cells, we developed a bacteriophage T7 RNA polymerase-based minireplicon system similar to that described previously for mammalian cells. An Aedes albopictus C6/36-derived mosquito cell line stably expressing T7 RNA polymerase was established. Viral proteins and artificial minigenomes (containing Renilla luciferase as a reporter) were transcribed and expressed in these cells from transfected T7 promoter-containing plasmids. Transcription of the minigenome required two viral proteins, the nucleocapsid protein N and the RNA-dependent RNA polymerase L, a situation similar to that in mammalian cells. However, unlike the situation in mammalian cells, the viral polymerase was not inhibited by the viral nonstructural protein NSs. We also report that promoter strength is different for vertebrate versus invertebrate cells. The development of this system opens the way for a detailed comparison of bunyavirus replication in cells of disparate phylogeny.

The Rhopalosiphum padi virus 5' internal ribosome entry site is functional in Spodoptera frugiperda 21 cells and in their cell-free lysates: implications for the baculovirus expression system.

Cap-independent internal initiation of translation occurs on a number of viral and cellular mRNAs and is directed by internal ribosome entry site (IRES) elements. Rhopalosiphum padi virus (RhPV) is a member of the Dicistroviridae. These viruses have single-stranded, positive-sense RNA genomes that contain two open reading frames, both preceded by IRES elements. Previously, the activity of the RhPV 5' UTR IRES has been demonstrated in mammalian, Drosophila and wheat germ in vitro translation systems. It is now shown that this IRES also functions within Spodoptera frugiperda (Sf21) cells which are widely used in the baculovirus expression system, and in a novel Sf21 cell-based lysate system. Inclusion of the RhPV IRES in a dicistronic reporter mRNA transcript increased translation of the second cistron 23-fold within Sf21 cells. In contrast, the encephalomyocarditis virus IRES was inactive in both systems. The RhPV IRES therefore has the potential to be utilized in insect cell expression systems.