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RNAi screening reveals requirement for host cell secretory pathway in infection by diverse families of negative-strand RNA viruses.

ABSTRACT: Negative-strand (NS) RNA viruses comprise many pathogens that cause serious diseases in humans and animals. Despite their clinical importance, little is known about the host factors required for their infection. Using vesicular stomatitis virus (VSV), a prototypic NS RNA virus in the family Rhabdoviridae, we conducted a human genome-wide siRNA screen and identified 72 host genes required for viral infection. Many of these identified genes were also required for infection by two other NS RNA viruses, the lymphocytic choriomeningitis virus of the Arenaviridae family and human parainfluenza virus type 3 of the Paramyxoviridae family. Genes affecting different stages of VSV infection, such as entry/uncoating, gene expression, and assembly/release, were identified. Depletion of the proteins of the coatomer complex I or its upstream effectors ARF1 or GBF1 led to detection of reduced levels of VSV RNA. Coatomer complex I was also required for infection of lymphocytic choriomeningitis virus and human parainfluenza virus type 3. These results highlight the evolutionarily conserved requirements for gene expression of diverse families of NS RNA viruses and demonstrate the involvement of host cell secretory pathway in the process.

SUBMITTER: Panda D

PROVIDER: S-EPMC3223448 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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RNAi screening reveals requirement for host cell secretory pathway in infection by diverse families of negative-strand RNA viruses.

Panda Debasis D Das Anshuman A Das Anshuman A Dinh Phat X PX Subramaniam Sakthivel S Nayak Debasis D Barrows Nicholas J NJ Pearson James L JL Thompson Jesse J Kelly David L DL Ladunga Istvan I Pattnaik Asit K AK

Proceedings of the National Academy of Sciences of the United States of America 20111107 47

Negative-strand (NS) RNA viruses comprise many pathogens that cause serious diseases in humans and animals. Despite their clinical importance, little is known about the host factors required for their infection. Using vesicular stomatitis virus (VSV), a prototypic NS RNA virus in the family Rhabdoviridae, we conducted a human genome-wide siRNA screen and identified 72 host genes required for viral infection. Many of these identified genes were also required for infection by two other NS RNA viru ...[more]

PMID: 22065774

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Project description:The nucleocapsid of a negative-strand RNA virus is assembled with a single nucleocapsid protein and the viral genomic RNA. The nucleocapsid protein polymerizes along the length of the single-strand genomic RNA (viral RNA) or its cRNA. This process of encapsidation occurs concomitantly with genomic replication. Structural comparisons of several nucleocapsid-like particles show that the mechanism of RNA encapsidation in negative-strand RNA viruses has many common features. Fundamentally, there is a unifying mechanism to keep the capsid protein protomer monomeric prior to encapsidation of viral RNA. In the nucleocapsid, there is a cavity between two globular domains of the nucleocapsid protein where the viral RNA is sequestered. The viral RNA must be transiently released from the nucleocapsid in order to reveal the template RNA sequence for transcription/replication. There are cross-molecular interactions among the protein subunits linearly along the nucleocapsid to stabilize its structure. Empty capsids can form in the absence of RNA. The common characteristics of RNA encapsidation not only delineate the evolutionary relationship of negative-strand RNA viruses but also provide insights into their mechanism of replication.What separates negative-strand RNA viruses (NSVs) from the rest of the virosphere is that the nucleocapsid of NSVs serves as the template for viral RNA synthesis. Their viral RNA-dependent RNA polymerase can induce local conformational changes in the nucleocapsid to temporarily release the RNA genome so that the viral RNA-dependent RNA polymerase can use it as the template for RNA synthesis during both transcription and replication. After RNA synthesis at the local region is completed, the viral RNA-dependent RNA polymerase processes downstream, and the RNA genome is restored in the nucleocapsid. We found that the nucleocapsid assembly of all NSVs shares three essential elements: a monomeric capsid protein protomer, parallel orientation of subunits in the linear nucleocapsid, and a (5H + 3H) motif that forms a proper cavity for sequestration of the RNA. This observation also suggests that all NSVs evolved from a common ancestor that has this unique nucleocapsid.

Project description:The spectrum of viruses in insects is important for subjects as diverse as public health, veterinary medicine, food production, and biodiversity conservation. The traditional interest in vector-borne diseases of humans and livestock has drawn the attention of virus studies to hematophagous insect species. However, these represent only a tiny fraction of the broad diversity of Hexapoda, the most speciose group of animals. Here, we systematically probed the diversity of negative strand RNA viruses in the largest and most representative collection of insect transcriptomes from samples representing all 34 extant orders of Hexapoda and 3 orders of Entognatha, as well as outgroups, altogether representing 1243 species. Based on profile hidden Markov models we detected 488 viral RNA-directed RNA polymerase (RdRp) sequences with similarity to negative strand RNA viruses. These were identified in members of 324 arthropod species. Selection for length, quality, and uniqueness left 234 sequences for analyses, showing similarity to genomes of viruses classified in Bunyavirales (n = 86), Articulavirales (n = 54), and several orders within Haploviricotina (n = 94). Coding-complete genomes or nearly-complete subgenomic assemblies were obtained in 61 cases. Based on phylogenetic topology and the availability of coding-complete genomes we estimate that at least 20 novel viral genera in seven families need to be defined, only two of them monospecific. Seven additional viral clades emerge when adding sequences from the present study to formerly monospecific lineages, potentially requiring up to seven additional genera. One long sequence may indicate a novel family. For segmented viruses, cophylogenies between genome segments were generally improved by the inclusion of viruses from the present study, suggesting that in silico misassembly of segmented genomes is rare or absent. Contrary to previous assessments, significant virus-host codivergence was identified in major phylogenetic lineages based on two different approaches of codivergence analysis in a hypotheses testing framework. In spite of these additions to the known spectrum of viruses in insects, we caution that basing taxonomic decisions on genome information alone is challenging due to technical uncertainties, such as the inability to prove integrity of complete genome assemblies of segmented viruses.

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RNAi screening reveals requirement for host cell secretory pathway in infection by diverse families of negative-strand RNA viruses.

Publications

RNAi screening reveals requirement for host cell secretory pathway in infection by diverse families of negative-strand RNA viruses.

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