Rotavirus Genomic RNA Complex Forms via Specific RNA-RNA Interactions: Disruption of RNA Complex Inhibits Virus Infectivity.

Teodoro Fajardo Jr,Cristina Celma,Po-Yu Sung,Polly Roy

doi:10.3390/v9070167

Teodoro Fajardo Jr, Cristina Celma + Show 2 more

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https://doi.org/10.3390/v9070167

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Abstract

Rotavirus (RV), a member of the Reoviridae family, causes infection in children and infants, with high morbidity and mortality. To be viable, the virus particle must package a set of eleven RNA segments. In order to understand the packaging mechanism, here, we co-synthesized sets of RNA segments in vitro in different combinations and detected by two alternate methods: the electrophoretic mobility shift assay (EMSA) and the RNA-bead pull-down assay. We showed that viral positive-sense RNA segments interact with each other in a specific manner, forming RNA complexes, and that the RNA–RNA interactions followed a sequential order initiated by small RV segments. Further, we demonstrated that RNA complexes were perturbed by targeted specific antisense oligoribonucleotides (ORNs) complementary to short RNA sequences, indicating that the RNA–RNA interactions between different segments were sequence-specific. The same inhibitory ORNs also had the capability to inhibit virus replication. The combined in vitro and in vivo data inferred that RNA–RNA interactions and specific complex formation are essential for sorting different segments, possibly prior to, or during, genome packaging. As genome assembly is a universal requirement in the Reoviridae family, this work offers an approach towards a further understanding of the sorting and packaging mechanisms of RV and related dsRNA (double-stranded RNA) viruses.

Highlights

Rotavirus (RV) gastroenteritis is a highly contagious, acute viral disease that remains a major cause of childhood diarrhoeal disease worldwide, notably in developing countries
Rhesus rotavirus (RRV) strain was kindly provided by Harry Greenberg (Stanford, CA, USA), and human rotavirus (HRV) strain ST-3 genotype G4P [6] was kindly provided by David Allen (Public Health England, UK)
To investigate if specific RNA–RNA interactions occurred among RV RNA segments, first, we synthesized exact copies of the small RNA segments (S8–S11) from the RV strain RRV by generating complementary DNA (cDNA) copies of genomic dsRNA segments from purified RV

Summary

Introduction

Rotavirus (RV) gastroenteritis is a highly contagious, acute viral disease that remains a major cause of childhood diarrhoeal disease worldwide, notably in developing countries. RV is a non-enveloped virus (a member of the Reoviridae family) with three layers of capsid proteins enclosing the viral polymerase complex and a double-stranded (ds)RNA genome of 11 segments (S1–S11) with different sequences and lengths (small: S7–S11; medium: S4–S6; and large: S1–S3) [1,2,3]. Following entry into the host cell, like all other members of the family, the triple-layered RV particle (TLP) uncoats to generate a cytoplasmic double-layered particle (DLP) of two major structural proteins, VP6 (outer layer) and VP2. The DLP remains intact, but becomes transcriptionally active, synthesizing and extruding multiple copies of capped, positive-sense, single-stranded (ss)RNA transcripts of the 11 dsRNA segments [4,5].

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