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Ultrastructural characterization of arterivirus replication structures: reshaping the endoplasmic reticulum to accommodate viral RNA synthesis.

ABSTRACT: Virus-induced membrane structures support the assembly and function of positive-strand RNA virus replication complexes. The replicase proteins of arteriviruses are associated with double-membrane vesicles (DMVs), which were previously proposed to derive from the endoplasmic reticulum (ER). Using electron tomography, we performed an in-depth ultrastructural analysis of cells infected with the prototypic arterivirus equine arteritis virus (EAV). We established that the outer membranes of EAV-induced DMVs are interconnected with each other and with the ER, thus forming a reticulovesicular network (RVN) resembling that previously described for the distantly related severe acute respiratory syndrome (SARS) coronavirus. Despite significant morphological differences, a striking parallel between the two virus groups, and possibly all members of the order Nidovirales, is the accumulation in the DMV interior of double-stranded RNA, the presumed intermediate of viral RNA synthesis. In our electron tomograms, connections between the DMV interior and cytosol could not be unambiguously identified, suggesting that the double-stranded RNA is compartmentalized by the DMV membranes. As a novel approach to visualize and quantify the RNA content of viral replication structures, we explored electron spectroscopic imaging of DMVs, which revealed the presence of phosphorus in amounts equaling on average a few dozen copies of the EAV RNA genome. Finally, our electron tomograms revealed a network of nucleocapsid protein-containing protein tubules that appears to be intertwined with the RVN. This potential intermediate in nucleocapsid formation, which was not observed in coronavirus-infected cells, suggests that arterivirus RNA synthesis and assembly are coordinated in intracellular space.

SUBMITTER: Knoops K

PROVIDER: S-EPMC3302280 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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Ultrastructural characterization of arterivirus replication structures: reshaping the endoplasmic reticulum to accommodate viral RNA synthesis.

Knoops Kèvin K Bárcena Montserrat M Limpens Ronald W A L RW Koster Abraham J AJ Mommaas A Mieke AM Snijder Eric J EJ

Journal of virology 20111221 5

Virus-induced membrane structures support the assembly and function of positive-strand RNA virus replication complexes. The replicase proteins of arteriviruses are associated with double-membrane vesicles (DMVs), which were previously proposed to derive from the endoplasmic reticulum (ER). Using electron tomography, we performed an in-depth ultrastructural analysis of cells infected with the prototypic arterivirus equine arteritis virus (EAV). We established that the outer membranes of EAV-induc ...[more]

PMID: 22190716

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Project description:Hepatitis E virus (HEV) causes acute hepatitis in many parts of the world including Asia, Africa and Latin America. Though self-limiting in normal individuals, it results in ~30% mortality in infected pregnant women. It has also been reported to cause acute and chronic hepatitis in organ transplant patients. Of the seven viral genotypes, genotype-1 virus infects humans and is a major public health concern in South Asian countries. Sporadic cases of genotype-3 and 4 infection in human and animals such as pigs, deer, mongeese have been reported primarily from industrialized countries. Genotype-5, 6 and 7 viruses are known to infect animals such as wild boar and camel, respectively. Genotype-3 and 4 viruses have been successfully propagated in the laboratory in mammalian cell culture. However, genotype-1 virus replicates poorly in mammalian cell culture and no other efficient model exists to study its life cycle. Here, we report that endoplasmic reticulum (ER) stress promotes genotype-1 HEV replication by inducing cap-independent, internal initiation mediated translation of a novel viral protein (named ORF4). Importantly, ORF4 expression and stimulatory effect of ER stress inducers on viral replication is specific to genotype-1. ORF4 protein sequence is mostly conserved among genotype-1 HEV isolates and ORF4 specific antibodies were detected in genotype-1 HEV patient serum. ORF4 interacted with multiple viral and host proteins and assembled a protein complex consisting of viral helicase, RNA dependent RNA polymerase (RdRp), X, host eEF1α1 (eukaryotic elongation factor 1 isoform-1) and tubulinβ. In association with eEF1α1, ORF4 stimulated viral RdRp activity. Furthermore, human hepatoma cells that stably express ORF4 or engineered proteasome resistant ORF4 mutant genome permitted enhanced viral replication. These findings reveal a positive role of ER stress in promoting genotype-1 HEV replication and pave the way towards development of an efficient model of the virus.

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Ultrastructural characterization of arterivirus replication structures: reshaping the endoplasmic reticulum to accommodate viral RNA synthesis.

Publications

Ultrastructural characterization of arterivirus replication structures: reshaping the endoplasmic reticulum to accommodate viral RNA synthesis.

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