Norovirus cleavage of hnRNP-K enhances virus replication

Vernon Ward, University of Otago

G. M. McKenzie-Goldsmith1., T. Kleffmann2., M. J. Edwards1, A, M, McSweeney1., Ward, V. K1 1 Department of Microbiology and Immunology, 2 Department of Biochemistry, School of Biomedical Sciences, University of Otago, Dunedin, NZ,

Entry and Replication

Cleavage of host proteins by viral proteases is an important mechanism for modifying the cell to favour replication. To identify cellular targets of norovirus proteases we used iTRAQ-TAILS mass spectrometry to label and analyse murine RAW264.7 cell lysate incubated with recombinant MNV-1 protease (NS6) and the precursor ProPol (NS6-7). Cleavage sites detected were filtered for known viral protease P1/P1ʹ scissile bonds to identify MNV-1 protease cleavage of SUG1 (proteasome), actin and heterogeneous nuclear ribonucleoprotein K (hnRNP-K), a multifunctional RNA-binding protein that influences numerous cellular processes in both the nucleus and cytoplasm of cells. Western blotting confirmed cleavage of hnRNP-K with generation of a product consistent with removal of the c-terminal KH3 nucleic acid binding domain from hnRNP-K as predicted from the identified cleavage site. In addition, total hnRNP-K levels were shown to decline during an MNV infection. Knockdown of hnRNP-K during infection increased viral NS1-2 from 12 hours post-infection (hpi) and improved virus yield approximately 3-fold, as determined by RT-qPCR of cellular supernatant from hnRNP-K depleted cells at 18 hpi. Furthermore, HEK293T cell lysate treated with recombinant Pro/NS6 from GI.2 (Southampton) and GII.4 (Sydney 2012) human noroviruses and analysed by SDS-PAGE and western blotting confirmed that both GI and GII viral proteases also cleave hnRNP-K. In conclusion, we have identified cellular proteins cleaved by MNV-1 protease and shown that hnRNP-K is a conserved cellular target of noroviruses that antagonises viral replication.