Transcriptomic analysis of human norovirus NS1-2 protein highlights a multifunctional role in murine monocytes.

BACKGROUND: The GII.4 Sydney 2012 strain of human norovirus (HuNoV) is a pandemic strain that is responsible for the majority of norovirus outbreaks in healthcare settings. The function of the non-structural (NS)1-2 protein from HuNoV is unknown. RESULTS: In silico analysis of human norovirus NS1-2 protein showed that it shares features with the murine NS1-2 protein, including a disordered region, a transmembrane domain and H-box and NC sequence motifs. The proteins also contain caspase cleavage and phosphorylation sites, indicating that processing and phosphorylation may be a conserved feature of norovirus NS1-2 proteins. In this study, RNA transcripts of human and murine norovirus full-length and the disordered region of NS1-2 were transfected into monocytes, and next generation sequencing was used to analyse the transcriptomic profile of cells expressing virus proteins. The profiles were then compared to the transcriptomic profile of MNV-infected cells. CONCLUSIONS: RNAseq analysis showed that NS1-2 proteins from human and murine noroviruses affect multiple immune systems (chemokine, cytokine, and Toll-like receptor signaling) and intracellular pathways (NFκB, MAPK, PI3K-Akt signaling) in murine monocytes. Comparison to the transcriptomic profile of MNV-infected cells indicated the pathways that NS1-2 may affect during norovirus infection.

Inherent structural disorder and dimerisation of murine norovirus NS1-2 protein.

Human noroviruses are highly infectious viruses that cause the majority of acute, non-bacterial epidemic gastroenteritis cases worldwide. The first open reading frame of the norovirus RNA genome encodes for a polyprotein that is cleaved by the viral protease into six non-structural proteins. The first non-structural protein, NS1-2, lacks any significant sequence similarity to other viral or cellular proteins and limited information is available about the function and biophysical characteristics of this protein. Bioinformatic analyses identified an inherently disordered region (residues 1-142) in the highly divergent N-terminal region of the norovirus NS1-2 protein. Expression and purification of the NS1-2 protein of Murine norovirus confirmed these predictions by identifying several 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 also identified the presence of an NS1-2 dimer in Escherichia coli and transfected HEK293T cells. Inherent disorder provides significant advantages including structural flexibility and the ability to bind to numerous targets allowing a single protein to have multiple functions. These advantages combined with the potential functional advantages of multimerisation suggest a multi-functional role for the NS1-2 protein.