Murine Norovirus Manipulation of the Cell Cycle
Davies, Colin Thomas Rainsford
Human norovirus (HuNoV) causes the majority of acute, non-bacterial endemic gastroenteritis worldwide. Noroviruses belong to the Caliciviridae family and have a positive-sense, single single-stranded, RNA genome of around 7.5 kb. The historic inability to cultivate HuNoV in cell culture and animals has led to the use of closely related models to advance understanding of norovirus replication and pathogenicity. Murine norovirus (MNV) is a routinely researched model for HuNoV and allows for studies of viral replication within cell culture. The MNV genome consists of four open reading frames (ORF), with the non-structural proteins expressed from the polyprotein of ORF1 and a single protein from ORF4. The structural proteins are expressed from ORF2 and ORF3. In order to develop successful antiviral agents against norovirus, an increased knowledge of viral replication and viral-host interactions is needed. Previous microarray results from MNV-1-infected cells identified changes in the levels of transcripts of genes that regulate host cell division. This research aimed to characterise this interaction between MNV-1 and the host cell cycle. Western blot analysis of MNV-1 infected murine macrophages (RAW-Blue cells) confirmed at a protein level, the down-regulation of cyclins expressed in late phases of the cell cycle. The decrease in cyclin A and cyclin B2 expression is consistent with the effects observed on cell division, with MNV-1 infection causing a decrease in progression through the G1/S checkpoint, leading to an accumulation of cells in the G0/G1 phase. Furthermore, the G1 phase arrest was revealed to be beneficial to viral replication, as cells progressing through the G1 phase supported a two-fold or more increase in viral progeny and VP1 expression over cells synchronised into an alternate cell cycle phase or an unsynchronised population. These findings suggest that MNV-1 infection manipulates the host cell cycle, arresting cell division in a phase favourable to viral replication. This manipulation of the host cell is proposed as a strategy used by MNV-1 to enhance viral replication. The mechanism by which MNV-1 induces its cell cycle effects was explored. The induction of cell cycle effects was shown to be independent of interferon (IFN) type 1 production as cells non-responsive to IFN, still arrested at the G1/S restriction point during MNV-1 infection. Expression of individual viral proteins identified virus protein genome-linked (VPg or NS5) as the causative agent of the cell cycle arrest. Expression of NS5 from an in vitro transcript induced an arrest at the G1/S checkpoint and increased the G0/G1 population of cells in an analogous manner to MNV-1 infection. Expression of cyclin A was also inhibited, consistent with the observed decrease of this cyclin during MNV-1 infection. Expression of truncated NS5 linked the activity to the first 62 amino acids, indicating a novel mechanism of cell cycle manipulation, independent of the host elongation initiation factor (eIF) binding motif at the C-terminus of NS5. Furthermore, the first 10 amino acids at the N-terminus of NS5 were revealed to be essential in inducing the cell cycle arrest. Bioinformatic analysis of the N-terminus of NS5 suggests this region is involved in nucleotide interactions and proposes a possible mechanism of cell cycle control, due to interactions with host mRNA. This research is the first documentation of a calicivirus interacting with the host cell cycle, in order to favour viral replication. Moreover, this is the first identification of a VPg protein manipulating the host cell cycle. Due to similarities in NS5 sequences especially at the N-terminal between norovirus genogroups, we propose that the cell cycle manipulation reported here may be conserved in other noroviruses.
Advisor: Ward, Vernon K.; Mercer, Andrew A.
Degree Name: Doctor of Philosophy
Degree Discipline: Microbiology and Immunology
Publisher: University of Otago
Keywords: Murine Norovirus; Cell Cycle; VPg; NS5
Research Type: Thesis