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Tombusvirus recruitment of host translational machinery via the 3' UTR.

ABSTRACT: RNA viruses recruit the host translational machinery by different mechanisms that depend partly on the structure of their genomes. In this regard, the plus-strand RNA genomes of several different pathogenic plant viruses do not contain traditional translation-stimulating elements, i.e., a 5'-cap structure and a 3'-poly(A) tail, and instead rely on a 3'-cap-independent translational enhancer (3'CITE) located in their 3' untranslated regions (UTRs) for efficient synthesis of viral proteins. We investigated the structure and function of the I-shaped class of 3'CITE in tombusviruses--also present in aureusviruses and carmoviruses--using biochemical and molecular approaches and we determined that it adopts a complex higher-order RNA structure that facilitates translation by binding simultaneously to both eukaryotic initiation factor (eIF) 4F and the 5' UTR of the viral genome. The specificity of 3'CITE binding to eIF4F is mediated, at least in part, through a direct interaction with its eIF4E subunit, whereas its association with the viral 5' UTR relies on complementary RNA-RNA base-pairing. We show for the first time that this tripartite 5' UTR/3'CITE/eIF4F complex forms in vitro in a translationally relevant environment and is required for recruitment of ribosomes to the 5' end of the viral RNA genome by a mechanism that shares some fundamental features with cap-dependent translation. Notably, our results demonstrate that the 3'CITE facilitates the initiation step of translation and validate a molecular model that has been proposed to explain how several different classes of 3'CITE function. Moreover, the virus-host interplay defined in this study provides insights into natural host resistance mechanisms that have been linked to 3'CITE activity.

SUBMITTER: Nicholson BL

PROVIDER: S-EPMC2885689 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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Tombusvirus recruitment of host translational machinery via the 3' UTR.

Nicholson Beth L BL Wu Baodong B Chevtchenko Irina I White K Andrew KA

RNA (New York, N.Y.) 20100527 7

RNA viruses recruit the host translational machinery by different mechanisms that depend partly on the structure of their genomes. In this regard, the plus-strand RNA genomes of several different pathogenic plant viruses do not contain traditional translation-stimulating elements, i.e., a 5'-cap structure and a 3'-poly(A) tail, and instead rely on a 3'-cap-independent translational enhancer (3'CITE) located in their 3' untranslated regions (UTRs) for efficient synthesis of viral proteins. We inv ...[more]

PMID: 20507975

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Project description:UnlabelledViruses require the host translational apparatus to synthesize viral proteins. Host stress response mechanisms that suppress translation, therefore, represent a significant obstacle that viruses must overcome. Here, we report a strategy whereby the mammalian orthoreoviruses compartmentalize the translational machinery within virus-induced inclusions known as viral factories (VF). VF are the sites of reovirus replication and assembly but were thought not to contain ribosomes. It was assumed viral mRNAs exited the VF to undergo translation by the cellular machinery, and proteins reentered the factory to participate in assembly. Here, we used ribopuromycylation to visualize active translation in infected cells. These studies revealed that active translation occurs within VF and that ribosomal subunits and proteins required for translation initiation, elongation, termination, and recycling localize to the factory. Interestingly, we observed components of the 43S preinitiation complex (PIC) concentrating primarily at factory margins, suggesting a spatial and/or dynamic organization of translation within the VF. Similarly, the viral single-stranded RNA binding protein ?NS localized to the factory margins and had a tubulovesicular staining pattern that extended a short distance from the margins of the factories and colocalized with endoplasmic reticulum (ER) markers. Consistent with these colocalization studies, ?NS was found to associate with both eukaryotic translation initiation factor 3 subunit A (eIF3A) and the ribosomal subunit pS6R. Together, these findings indicate that ?NS functions to recruit 43S PIC machinery to the primary site of viral translation within the viral factory. Pathogen-mediated compartmentalization of the translational apparatus provides a novel mechanism by which viruses might avoid host translational suppression.ImportanceViruses lack biosynthetic capabilities and depend upon the host for protein synthesis. This dependence requires viruses to evolve mechanisms to coerce the host translational machinery into synthesizing viral proteins in the face of ongoing cellular stress responses that suppress global protein synthesis. Reoviruses replicate and assemble within cytoplasmic inclusions called viral factories. However, synthesis of viral proteins was thought to occur in the cytosol. To identify the site(s) of viral translation, we undertook a microscopy-based approach using ribopuromycylation to detect active translation. Here, we report that active translation occurs within viral factories and that translational factors are compartmentalized within factories. Furthermore, we find that the reovirus nonstructural protein ?NS associates with 43S preinitiation complexes at the factory margins, suggesting a role for ?NS in translation. Together, virus-induced compartmentalization of the host translational machinery represents a strategy for viruses to spatiotemporally couple viral protein synthesis with viral replication and assembly.

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Tombusvirus recruitment of host translational machinery via the 3' UTR.

Publications

Tombusvirus recruitment of host translational machinery via the 3' UTR.

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