Influence of genome-scale RNA structure disruption on the replication of murine norovirus—similar replication kinetics in cell culture but attenuation of viral fitness in vivo

Abstract

Mechanisms by which certain RNA viruses, such as hepatitis C virus, establish persistent infections and cause chronic disease are of fundamental importance in viral pathogenesis. Mammalian positive-stranded RNA viruses establishing persistence typically possess genome-scale ordered RNA secondary structure (GORS) in their genomes. Murine norovirus (MNV) persists in immunocompetent mice and provides an experimental model to functionally characterize GORS. Substitution mutants were constructed with coding sequences in NS3/4- and NS6/7-coding regions replaced with sequences with identical coding and (di-)nucleotide composition but disrupted RNA secondary structure (F1, F2, F1/F2 mutants). Mutants replicated with similar kinetics to wild-type (WT) MNV3 in RAW264.7 cells and primary macrophages, exhibited similar (highly restricted) induction and susceptibility to interferon-coupled cellular responses and equal replication fitness by serial passaging of co-cultures. In vivo, both WT and F1/F2 mutant viruses persistently infected mice, although F1, F2 and F1/F2 mutant viruses were rapidly eliminated 1–7 days post-inoculation in competition experiments with WT. F1/F2 mutants recovered from tissues at 9 months showed higher synonymous substitution rates than WT and nucleotide substitutions that potentially restored of RNA secondary structure. GORS plays no role in basic replication of MNV but potentially contributes to viral fitness and persistence in vivo.

Subtype-specific structural constraints in the evolution of influenza A virus hemagglutinin genes

“…Strong conservation of these hairpins in some HA subtypes and their disappearance in others is consistent with frequently observed non-random reassortment of compatible segments26303132. In positive-sense mRNA, locally stable structures may be involved in modulation of protein folding, with their folding free energies slowing down the ribosome movement and thus facilitating correct cotranslational formation of native domain structures55, or in interference with host innate immunity5657.…”

“…Plaque assays of mutant viruses with disrupted or destabilized structures predicted in the HA cleavage site region also did not reveal any replication defects (Supplementary Figs S6 and S7). Similar inconsistency between evolutionary conservation of virus structures and their influence on virus replication assays was previously noted for HIV-1 and murine norovirus5657. It has been suggested that RNA folding could have a function that contributes to virus fitness and persistence in nature but is not essential for basic replication under the experimental conditions.…”

RNA structural constraints in the evolution of the influenza A virus genome NP segment

“…One of the possible functions of structured regions in RNA genomes is to interfere with the host innate systems recognizing a pathogen RNA. 2,[57][58][59] Future experimental studies will elucidate the structurefunction relationships in the influenza virus RNA genome. Apparently, RNA folding is an important factor contributing to the virus evolution.…”

Advances in Norovirus Biology

“…A polypyrimidine (pY) tract within a conserved stem-loop in the 3′ untranslated region of the MuNoV-1 genome, which binds cellular poly(rC) binding protein and polypyrimidine tract binding protein, is not required for virus replication in vitro , but its removal results in a partially attenuated virus in STAT1 -/- mice (Bailey et al, 2010). Similarly, secondary structure elements in NS3/4 and NS6/7 of MuNoV-3 enhance viral fitness, although they are not required for replication in vitro or persistence establishment in immunocompetent mice (McFadden et al, 2013). It will be important in future investigations and as the necessary research tools become available to validate these factors as virulence determinants of HuNoVs.…”