Investigation into genome-scale ordered RNA structure (GORS) in murine norovirus and other positive-stranded RNA viruses
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Date
2010Author
Blundell, Richard James
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Abstract
Genome-scale ordered RNA structure (GORS) was first identified in 2004. It
refers to the presence of secondary structure throughout the length of the RNA
genomes of certain genera of RNA virus families, as predicted by bioinformatic
analysis. It was also observed that the viruses containing GORS were able to
establish persistent infections in their natural hosts, raising the possibility that
the presence of GORS could play a role in viral avoidance of the innate immune
system.
This thesis describes the first study of GORS and its possible role in persistence.
Two GORS viruses have been studied, equine rhinitis A virus (ERAV) and
murine norovirus (MNV). A 55% seroprevalence of ERAV has been
determined in a cohort of Scottish horses indicating a wide exposure to the
virus. Equine faecal samples were screened for ERAV by PCR with the
intention of identifying a virus, possibly from a persistently infected animal,
which would not have undergone any cell culture adaptations as laboratory
strains have. Newly identified viruses would then be sequenced, their secondary
structures predicted and further studies carried out. Unfortunately, none of the
50 faecal samples screened were positive and clinical isolates of ERAV
provided by the Animal Health Trust were sequenced but were identical to
laboratory strains, so the study then focussed on MNV. Prevalence of MNV in
laboratory mice was determined by PCR of faecal samples to be 67%. MNV
was also discovered in the faeces of a pet shop mouse and a wild wood mouse
(Apodemus sylvaticus). The complete genomes of 4 laboratory mouse MNVs,
the pet shop mouse and wood mouse MNVs were sequenced. Phylogenetic
analysis showed the wood mouse MNV had a p distance of 23% from other
MNVs, although the laboratory mice and pet shop mouse were closely related to
other MNVs. Structural analysis of the genomes of 6 sequenced MNVs,
including the wood mouse virus, showed all were GORS viruses. A laboratory
strain of MNV, MNV-3, was serially passaged in RAW 264.7 cells to test the
hypothesis that in an animal with an intact immune system, there is a pressure
for GORS viruses to maintain their genomic RNA structure as a means of
immune avoidance, and that cell culture adaptation would attenuate the degree
of secondary structure. The complete genome of passage 33 was sequenced, which revealed 7 base mutations, a mutation rate of 0.1 %, which was not
considered significant enough to have affected the degree of secondary
structure.
In order to assess if structured and unstructured RNA behaved differently in
cells, replication deficient RNA transcripts were made from the infectious
clones of a panel of GORS and non-GORS viruses. These transcripts were
electroporated into cells and their rate of decay measured, but there was no
difference between the GORS and non-GORS transcripts. The full length and 4
kilobase transcripts were transfected into NIH3T3 cells and the degree of
interferon-β induction measured by quantitative PCR and a luciferase reporter
assay. The IFN-β response differed across the panel of viruses, and although
none of the GORS viruses induced strongly, the non-GORS viruses were
variable in their ability to induce an IFN-β response, some inducing strongly,
other not at all. This result indicates that during exposure of viral genomes in
the cytoplasm during infection, GORS-virus RNAs are unlikely to induce an
interferon response, possibly contributing to their ability to persist. It is unclear
why some non-GORS-viruses failed to induce IFN and there are likely to be
other contributory factors.