The Interaction of Bluetongue Virus VP6 and Genomic RNA Is Essential for Genome Packaging.

Po-Yu Sung,Polly Roy,Guanghui Yi,Robert Vaughan,C Cheng Kao,Adeline Kerviel,Shah Kamranur Rahman,Tom Gallagher

doi:10.1128/jvi.02023-18

Po-Yu Sung, Polly Roy + Show 6 more

Open Access

https://doi.org/10.1128/jvi.02023-18

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Abstract

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.

Highlights

The genomes of the Reoviridae, including the animal pathogen bluetongue virus (BTV), are multisegmented double-stranded RNA
During BTV replication, newly synthesized sense RNAs (ssRNAs) segments are first packaged into the assembling viral cores, which, in turn, serve as templates for the synthesis of genomic double-stranded RNA (dsRNA) segments [5]
To confirm the correlation between a functional VP6 and RNA packaging, an available BTV strain with a truncated VP6 protein [16] was Journal of Virology jvi.asm.org 2

Summary

Introduction

The genomes of the Reoviridae, including the animal pathogen bluetongue virus (BTV), are multisegmented double-stranded RNA (dsRNA). 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. 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. Core particles become transcriptionally active, producing and extruding single-stranded positive-sense RNAs (ssRNAs) through the local channels at the 5-fold axis, without further disassembly These ssRNAs act both as mRNAs for viral protein synthesis and as templates for nascent genomic RNA synthesis. Genomic dsRNA molecules are subsequently synthesized within this assembled particle (known as the “subcore”) prior to encapsidation by the VP7 layer, leading to robust core particle formation [5]

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