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Structural constraints in the packaging of bluetongue virus genomic segments.

ABSTRACT: The mechanism used by bluetongue virus (BTV) to ensure the sorting and packaging of its 10 genomic segments is still poorly understood. In this study, we investigated the packaging constraints for two BTV genomic segments from two different serotypes. Segment 4 (S4) of BTV serotype 9 was mutated sequentially and packaging of mutant ssRNAs was investigated by two newly developed RNA packaging assay systems, one in vivo and the other in vitro. Modelling of the mutated ssRNA followed by biochemical data analysis suggested that a conformational motif formed by interaction of the 5' and 3' ends of the molecule was necessary and sufficient for packaging. A similar structural signal was also identified in S8 of BTV serotype 1. Furthermore, the same conformational analysis of secondary structures for positive-sense ssRNAs was used to generate a chimeric segment that maintained the putative packaging motif but contained unrelated internal sequences. This chimeric segment was packaged successfully, confirming that the motif identified directs the correct packaging of the segment.

SUBMITTER: Burkhardt C

PROVIDER: S-EPMC4165931 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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Structural constraints in the packaging of bluetongue virus genomic segments.

Burkhardt Christiane C Sung Po-Yu PY Celma Cristina C CC Roy Polly P

The Journal of general virology 20140630 Pt 10

The mechanism used by bluetongue virus (BTV) to ensure the sorting and packaging of its 10 genomic segments is still poorly understood. In this study, we investigated the packaging constraints for two BTV genomic segments from two different serotypes. Segment 4 (S4) of BTV serotype 9 was mutated sequentially and packaging of mutant ssRNAs was investigated by two newly developed RNA packaging assay systems, one in vivo and the other in vitro. Modelling of the mutated ssRNA followed by biochemical ...[more]

PMID: 24980574

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Project description:The genomes of the Reoviridae, including the animal pathogen bluetongue virus (BTV), are multisegmented double-stranded RNA (dsRNA). During replication, single-stranded (ss) positive-sense RNA segments are packaged into the assembling virus capsid, triggering genomic dsRNA synthesis. However, exactly how this packaging event occurs is not clear. A minor capsid protein, VP6, unique for the orbiviruses, has been proposed to be involved in the RNA-packaging process. In this study, we sought to characterize the RNA binding activity of VP6 and its functional relevance. A novel proteomic approach was utilized to map the ssRNA/dsRNA binding sites of a purified recombinant protein and the genomic dsRNA binding sites of the capsid-associated VP6. The data revealed that each VP6 protein has multiple distinct RNA-binding regions and that only one region is shared between recombinant and capsid-associated VP6. A combination of targeted mutagenesis and reverse genetics identified the RNA-binding region that is essential for virus replication. Using an in vitro RNA-binding competition assay, a unique cell-free assembly assay, and an in vivo single-cycle replication assay, it was possible to identify a motif within the shared binding region that binds BTV ssRNA preferentially in a manner consistent with specific RNA recruitment during capsid assembly. These data highlight the critical roles that this unique protein plays in orbivirus genome packaging and replication.IMPORTANCE Genome packaging is a critical stage during virus replication. For viruses with segmented genomes, the genome segments need to be correctly packaged into a newly formed capsid. However, the detailed mechanism of this packaging is unclear. Here we focus on VP6, a minor viral protein of bluetongue virus, which is critical for genome packaging. We used multiple approaches, including a robust RNA-protein fingerprinting assay, to map the ssRNA binding sites of recombinant VP6 and the genomic dsRNA binding sites of capsid-associated VP6. By these means, together with virological and biochemical methods, we identify the viral RNA-packaging motif of a segmented dsRNA virus for the first time.

Dataset Information

Structural constraints in the packaging of bluetongue virus genomic segments.

Publications

Structural constraints in the packaging of bluetongue virus genomic segments.

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