Identification and characterisation of an overlapping open reading frame (ORF4) within the murine norovirus genome

Caliciviruses are single-stranded positive-sense RNA viruses, most commonly associated with outbreaks of gastroenteritis in humans. In addition to the three open reading frames (ORFs) typical of caliciviruses a highly conserved fourth overlapping ORF within the murine norovirus (MNV) genome was identified and characterised during this project. ORF4 overlaps and is contained within the capsid encoding ORF2 in a +1 frame. Once a suitable antibody had been generated, immunoblotting was used to show that the ORF4 protein is produced during MNV infection. Although ORF4 mutant viruses were viable and replicated to wild-type MNV levels in tissue culture, pressure to restore ORF4 expression upon serial passage under specific conditions was demonstrated. Importantly, the ORF4 knockout virus was significantly attenuated in STAT1-/- mice. Proteomic analysis and a commercial yeast two-hybrid screen were used to identify host cell factors which interact with the ORF4 protein and confocal imaging was employed to examine cellular localisation. These approaches indicated that the ORF4 protein localises to the mitochondria and in vitro assays were subsequently used to demonstrate an involvement of the ORF4 protein in MNV induced apoptosis. As cells infected with the knockout virus showed an earlier and higher degree of apoptosis induction compared to wild-type infected cells, it is possible that the ORF4 protein may function to delay the onset of apoptosis during MNV infection. Whether or not the ORF4 protein has antiapoptotic activity or whether the difference in apoptotic phenotype is an indirect consequence of ORF4 protein function remains to be investigated. These data indicate that the ORF4 protein represents a novel, previously uncharacterised virulence determinant in MNV

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

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

Functional analysis of RNA structures present at the 3' extremity of the murine norovirus genome: the variable polypyrimidine tract plays a role in viral virulence.

Interactions of host cell factors with RNA sequences and structures in the genomes of positive-strand RNA viruses play various roles in the life cycles of these viruses. Our understanding of the functional RNA elements present in norovirus genomes to date has been limited largely to in vitro analysis. However, we recently used reverse genetics to identify evolutionarily conserved RNA structures and sequences required for norovirus replication. We have now undertaken a more detailed analysis of RNA structures present at the 3′ extremity of the murine norovirus (MNV) genome. Biochemical data indicate the presence of three stable stem-loops, including two in the untranslated region, and a single-stranded polypyrimidine tract [p(Y)] of variable length between MNV isolates, within the terminal stem-loop structure. The well-characterized host cell pyrimidine binding proteins PTB and PCBP bound the 3′-untranslated region via an interaction with this variable sequence. Viruses lacking the p(Y) tract were viable both in cell culture and upon mouse infection, demonstrating that this interaction was not essential for virus replication. However, competition analysis with wild-type MNV in cell culture indicated that the loss of the p(Y) tract was associated with a fitness cost. Furthermore, a p(Y)-deleted mutant showed a reduction in virulence in the STAT1−/− mouse model, highlighting the role of RNA structures in norovirus pathogenesis. This work highlights how, like with other positive-strand RNA viruses, RNA structures present at the termini of the norovirus genome play important roles in virus replication and virulence

The Student Movement Volume 107 Issue 18: Ain\u27t I A Woman? : AU Students Reflect on the Feminist Movement

HUMANS Interview with the WEAAU President, Interviewed by: Grace No What is Feminism to You?, Interviewed by: Caryn Cruz What\u27s Your Major?, Interviewed by: Nora Martin ARTS & ENTERTAINMENT Currently: The Idol, Solana Campbell Faculty Art Exhibition, Ysabelle Fernando Music Seniors: A Day in the Life, Aiko J. Ayala Rios Through the Paintings, Kaela McFadden NEWS Empowering our Neighbors: Interview with H.E.L.P. Program Director Carlisle Suttton, Isabella Koh John Wesley Taylor V Elected to be the Next Andrews University President Students Open the Month with Cultural Showcase, Andrew Francis The Agora at AU: A Conversation on Police Brutality, Solana Campbell What\u27s Going on East Palestine, Ohio?, Julia Randall IDEAS The Biblical Bechdel: How Much are Women Represented in the Bible?, Bella Hamann The Murder of Abby Choi: A Spotlight on Relationship Violence Against Women, Abby Shim PULSE Nilah Mataafa: An End to Period Poverty, Interviewed by: Chris Ngugi Remember the Ladies! : The Female Perspective on Women\u27s History Month, Amelia Stefanescu The Aerial Workout You\u27ve Never Heard Of, Melissa Moore LAST WORD Perfect Imperfections, Shania Wattshttps://digitalcommons.andrews.edu/sm-107/1017/thumbnail.jp

The influence of viral RNA secondary structure on interactions with innate host cell defences

RNA viruses infecting vertebrates differ fundamentally in their ability to establish persistent infections with markedly different patterns of transmission, disease mechanisms and evolutionary relationships with their hosts. Although interactions with host innate and adaptive responses are complex and persistence mechanisms likely multi-factorial, we previously observed associations between bioinformatically predicted RNA secondary formation in genomes of positive-stranded RNA viruses with their in vivo fitness and persistence. To analyse this interactions functionally, we transfected fibroblasts with non-replicating, non-translated RNA transcripts from RNA viral genomes with differing degrees of genome-scale ordered RNA structure (GORS). Single-stranded RNA transcripts induced interferon-β mediated though RIG-I and PKR activation, the latter associated with rapid induction of antiviral stress granules. A striking inverse correlation was observed between induction of both cellular responses with transcript RNA structure formation that was independent of both nucleotide composition and sequence length. The consistent inability of cells to recognize RNA transcripts possessing GORS extended to downstream differences from unstructured transcripts in expression of TNF-α, other interferon-stimulated genes and induction of apoptosis. This functional association provides novel insights into interactions between virus and host early after infection and provides evidence for a novel mechanism for evading intrinsic and innate immune responses

Norovirus Regulation of the Innate Immune Response and Apoptosis Occurs via the Product of the Alternative Open Reading Frame 4

Small RNA viruses have evolved many mechanisms to increase the capacity of their short genomes. Here we describe the identification and characterization of a novel open reading frame (ORF4) encoded by the murine norovirus (MNV) subgenomic RNA, in an alternative reading frame overlapping the VP1 coding region. ORF4 is translated during virus infection and the resultant protein localizes predominantly to the mitochondria. Using reverse genetics we demonstrated that expression of ORF4 is not required for virus replication in tissue culture but its loss results in a fitness cost since viruses lacking the ability to express ORF4 restore expression upon repeated passage in tissue culture. Functional analysis indicated that the protein produced from ORF4 antagonizes the innate immune response to infection by delaying the upregulation of a number of cellular genes activated by the innate pathway, including IFN-Beta. Apoptosis in the RAW264.7 macrophage cell line was also increased during virus infection in the absence of ORF4 expression. In vivo analysis of the WT and mutant virus lacking the ability to express ORF4 demonstrated an important role for ORF4 expression in infection and virulence. STAT1-/- mice infected with a virus lacking the ability to express ORF4 showed a delay in the onset of clinical signs when compared to mice infected with WT virus. Quantitative PCR and histopathological analysis of samples from these infected mice demonstrated that infection with a virus not expressing ORF4 results in a delayed infection in this system. In light of these findings we propose the name virulence factor 1, VF1 for this protein. The identification of VF1 represents the first characterization of an alternative open reading frame protein for the calicivirus family. The immune regulatory function of the MNV VF1 protein provide important perspectives for future research into norovirus biology and pathogenesis