Structural constraints in the packaging of bluetongue virus genomic segments.

Christiane Burkhardt,Polly Roy,Cristina C Celma,Po-Yu Sung

doi:10.1099/vir.0.066647-0

Christiane Burkhardt, Polly Roy + Show 2 more

Open Access

https://doi.org/10.1099/vir.0.066647-0

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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.

Highlights

Bluetongue virus (BTV) is the prototype species of the genus Orbivirus within the Reoviridae, a family consisting of non-enveloped viruses with segmented dsRNA genomes
As the length of the 39 untranslated region (UTR) differs between the 10 segments of BTV, but is highly conserved among the same segments of different serotypes, it was hypothesized that the packaging signals could be located in the 39 UTR
We demonstrated that the hairpin loop structure was essential for packaging of Segment 4 (S4) of BTV-9, whilst it was unclear whether this structure was specific for this segment of BTV-9 or the function of this structure could apply to other segments of other serotypes

Summary

Introduction

Bluetongue virus (BTV) is the prototype species of the genus Orbivirus within the Reoviridae, a family consisting of non-enveloped viruses with segmented dsRNA genomes. An outer capsid composed of two proteins, VP2 and VP5, encapsidates the inner core In addition to these seven structural proteins, BTV synthesizes four nonstructural proteins (NS1–NS4) in infected cells, each of which is involved in various stages of virus replication and assembly. The released cores become transcriptionally active (Huismans et al, 1987), releasing 10 positive-sense ssRNAs through the pores located at the 12 vertices of the icosahedral core (Diprose et al, 2001). These ssRNA molecules act as templates (mRNAs) for synthesis of viral proteins as well as for genomic dsRNA segments for progeny virions. Of nascent BTV cores takes place within VIBs in the cytoplasm (Eaton et al, 1990)

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