Cleavage of specific host proteins by viral proteases is one mechanism to evade host defenses and modify the cell to favour replication. Murine norovirus (MNV) protease (Pro) cleaves the non-structural viral polyprotein during replication. Additionally, Pro can cleave poly-A binding protein to modulate host cell translation, raising the question of whether Pro can cleave other host proteins.
Recombinant MNV Pro was incubated with murine RAW264.7 cell lysate. The resulting cleavage products were N-terminally labelled and then further processed for identification via mass spectrometry. Sequences of labelled peptides were investigated for known protease recognition motifs. One cleaved protein identified was heterologous nuclear ribonuclear protein K (hnRNPK), a nucleic acid binding protein with multiple cellular functions.
Western blotting confirmed that MNV Pro cleaves hnRNPK in a cell lysate to produce a fragment corresponding to cleavage at the predicted site. This processing removes a KH nucleic acid binding domain from hnRNPK. Furthermore, we confirmed that total hnRNPK levels declined during an MNV infection.
MNV infection during siRNA-mediated knockdown of hnRNPK showed a 3-fold increase in viral NS1-2 protein and a 2-fold increase in viral RNA at 18 hpi in hnRNPK depleted cells compared to the control.
To test if cleavage of hnRNPK is conserved by norovirus genogroups, recombinant Pro from a human norovirus (HuNv) was incubated with cell lysate from a HEK293T cell. Western blotting confirmed that HuNv Pro could cleave hnRNPK to produce a fragment of 55 kDa.
In conclusion, we have identified a new cellular target of the protease from MNV and shown that depletion of hnRNPK increases virus replication. Additionally, we have shown that a HuNv protease can also cleave hnRNPK in a cell lysate, implying that viral protease targeting of hnRNPK is conserved across norovirus genogroups.