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

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.

In Vitro Reassortment between Endemic Bluetongue Viruses Features Global Shifts in Segment Frequencies and Preferred Segment Combinations

“…Segment 5 encodes the NS1 protein, which is associated with microtubules and has been shown to enhance BTV translation; segment 6 encodes VP5, one of the two major outer capsid proteins; and segment 9 encodes VP6 and NS4 [ 49 , 50 ]. VP6 is the RNA helicase, while NS4 is translated from another open reading frame and acts as an interferon antagonist [ 51 , 52 ]. Specific interactions between these three segments have not been described, nor do these segments share the highest nucleotide or amino acid identity of the ten segments between the BTV-2 and BTV-10 used in our study.…”

RNA Origami: Packaging a Segmented Genome in Orbivirus Assembly and Replication

“…The same site was also found to be critical for RNA binding by caVP6. Further in vitro and in vivo studies confirmed that a motif within this shared binding region preferentially binds BTV ssRNAs over non-BTV ssRNAs shown by an RNA-binding completion assay and is essential for RNA packaging ( Figure 9 ) [ 20 ]. While these data suggest that VP6 acts as an RNA chaperone protein, the precise RNA sequence(s) that VP6 binds to for packaging and whether VP6 is the only active participant in RNA recruitment by the core still remain unknown.…”

“…The outer capsid is removed shortly after cell entry to release the inner capsid, the ‘core’, into the cytoplasm [ 11 , 12 , 13 , 14 ]. The core is composed of two major proteins, VP7 and VP3, and three minor proteins that make up the transcription complex (TC), including VP1 polymerase, VP4 capping enzyme, and an RNA binding/putative helicase and RNA packaging protein VP6 [ 15 , 16 , 17 , 18 , 19 , 20 ]. Within the core, the TC repeatedly transcribes the ten genomic dsRNA segments and the ssRNA transcripts extrude from the core to serve as messengers for viral protein synthesis and nascent dsRNA segment synthesis, thereby initiating virus replication.…”

Vaccination as a Strategy to Prevent Bluetongue Virus Vertical Transmission

“…Core-like particle assembly experiments have also indicated that the RNA capping enzyme and methyl transferase VP4 is associated with the VP3-VP1 complex [23]. The spatial distribution within the core of the RNA helicase VP6 is less well characterized, but its presence is important for the correct packaging of the dsRNA genome [24]. The inner core also contains the segmented RNA genome.…”