Physicochemical analysis of rotavirus segment 11 supports a ‘modified panhandle’ structure and not the predicted alternative tRNA-like structure (TRLS)

Subhajit Biswas,Wilson Li,Laura E Easton,Emily Manktelow,Jonathan Lever,Ulrich Desselberger,Andrew M Lever,Peter J Lukavsky

doi:10.1007/s00705-013-1802-8

Abstract

Rotaviruses are a major cause of acute gastroenteritis, which is often fatal in infants. The viral genome consists of 11 double-stranded RNA segments, but little is known about their cis-acting sequences and structural elements. Covariation studies and phylogenetic analysis exploring the potential structure of RNA11 of rotaviruses suggested that, besides the previously predicted “modified panhandle” structure, the 5’ and 3’ termini of one of the isoforms of the bovine rotavirus UKtc strain may interact to form a tRNA-like structure (TRLS). Such TRLSs have been identified in RNAs of plant viruses, where they are important for enhancing replication and packaging. However, using tRNA mimicry assays (in vitro aminoacylation and 3’- adenylation), we found no biochemical evidence for tRNA-like functions of RNA11. Capping, synthetic 3’ adenylation and manipulation of divalent cation concentrations did not change this finding. NMR studies on a 5’- and 3’-deletion construct of RNA11 containing the putative intra-strand complementary sequences supported a predominant panhandle structure and did not conform to a cloverleaf fold despite the strong evidence for a predicted structure in this conserved region of the viral RNA. Additional viral or cellular factors may be needed to stabilise it into a form with tRNA-like properties.

Highlights

Rotaviruses (RVs) are a major cause of acute gastroenteritis in infants and young children worldwide and are responsible for considerable mortality, mainly in developing countries
Rotaviruses are a major cause of acute gastroenteritis, which is often fatal in infants
Covariation studies and phylogenetic analysis exploring the potential structure of RNA11 of rotaviruses suggested that, besides the previously predicted ‘‘modified panhandle’’ structure, the 5’ and 3’ termini of one of the isoforms of the bovine rotavirus UKtc strain may interact to form a tRNA-like structure (TRLS)

Summary

Introduction

Rotaviruses (RVs) are a major cause of acute gastroenteritis in infants and young children worldwide and are responsible for considerable mortality, mainly in developing countries. Rotaviruses form a genus of the family Reoviridae and are icosahedral triple-layered particles (TLPs). They contain a genome of 11 segments of doublestranded RNA encoding six structural viral proteins (VP1VP4, VP6 and VP7) and six non-structural viral proteins (NSP1-NSP6). Enzymatic digestion of the outer particle layer in the cytoplasm produces double-layered particles (DLPs) that are transcriptionally active and extrude 11 ssRNA segments of positive polarity. These RNA molecules are either translated into viral proteins or function as templates for RNA replication, producing the double-stranded genome in progeny virions. Viral morphogenesis and RNA replication occur in cytoplasmic inclusion bodies called viroplasms [1]

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