Abstract
Human noroviruses are highly infectious viruses that cause the majority of acute, non-bacterial epidemic gastroenteritis cases worldwide. Noroviruses belong to the family Caliciviridae and have a positive-sense RNA genome of around 7.5 kb. Murine norovirus is a useful model for the uncultivable human strains, and has a 7.4 kb genome with four open reading frames (ORFs). Two of these, ORF2 and ORF3, encode structural proteins and ORF4 encodes a virulence factor. The first open reading frame (ORF1) encodes a polyprotein that is cleaved by the viral protease into six nonstructural proteins. There is limited functional and biophysical information available for two of these nonstructural proteins, NS1-2 and NS4. The aims of this research were to characterise these two proteins.
The NS1-2 protein lacks any significant sequence similarity to other viral or cellular proteins. Bioinformatic analyses identified an inherently disordered region (residues 1 – 142) in the highly divergent N-terminal region of the NS1-2 protein. Expression and purification of the NS1-2 protein of murine norovirus confirmed these predictions by identifying features typical of an inherently disordered protein. These were a biased amino acid composition with enrichment in the disorder promoting residues serine and proline, a lack of predicted secondary structure, a hydrophilic nature, an aberrant electrophoretic migration, an increased Stokes radius similar to that predicted for a protein from the pre-molten globule family, a high sensitivity to thermolysin proteolysis and a circular dichroism spectrum typical of an inherently disordered protein. The purification of the NS1-2 protein identified the presence of an NS1-2 dimer when expressed in Escherichia coli, which was also identified in transfected HEK293T cells and MNV-infected RAW264.7 cells.
The NS4 protein is often referred to as the 3A-like protein due to a similar position in the genome as the 3A protein of poliovirus. However, NS4 shares only limited sequence similarity to polio 3A. The NS4 protein of murine norovirus was expressed in Escherichia coli and purified for the generation of a polyclonal antibody.
Whole transcriptome RNA-Seq analysis was conducted on transfected RAW264.7 cells to provide important leads on functional roles, effects and host cell responses to in vitro transcripts encoding the complete MNV genome, the ORF1 nonstructural polyprotein, the full-length NS1-2 protein and the disordered region of NS1-2. Each transfected cell sample showed an upregulation in anti-apoptotic genes and a downregulation of pro-apoptotic genes, suggesting that MNV, and in particular the disordered region of NS1-2, manipulates the induction of apoptosis. A downregulation of sterol lipid synthesis genes and an upregulation of Th1-type chemokines were observed in MNV- and ORF1-transfected cells. NS1-2-transfected cells showed an increased expression for genes encoding intercellular junctions and regulatory proteins, indicating that NS1-2 is likely to have a multi-functional role affecting several metabolic pathways within an infected cell.