Norovirus RNA synthesis is modulated by an interaction between the viral RNA-dependent RNA polymerase and the major capsid protein, VP1.
Using a cell-based assay for RNA synthesis by the RNA-dependent RNA polymerase (RdRp) of noroviruses, we previously observed that VP1, the major structural protein of the human GII.4 norovirus, enhanced the GII.4 RdRp activity but not that of the related murine norovirus (MNV) or other unrelated RNA viruses (C. V. Subba-Reddy, I. Goodfellow, and C. C. Kao, J. Virol. 85:13027-13037, 2011). Here, we examine the mechanism of VP1 enhancement of RdRp activity and the mechanism of mouse norovirus replication. We determined that the GII.4 and MNV VP1 proteins can enhance cognate RdRp activities in a concentration-dependent manner. The VP1 proteins coimmunoprecipitated with their cognate RdRps. Coexpression of individual domains of VP1 with the viral RdRps showed that the VP1 shell domain (SD) was sufficient to enhance polymerase activity. Using SD chimeras from GII.4 and MNV, three loops connecting the central β-barrel structure were found to be responsible for the species-specific enhancement of RdRp activity. A differential scanning fluorimetry assay showed that recombinant SDs can bind to the purified RdRps in vitro. An MNV replicon with a frameshift mutation in open reading frame 2 (ORF2) that disrupts VP1 expression was defective for RNA replication, as quantified by luciferase reporter assay and real-time quantitative reverse transcription-PCR (qRT-PCR). Trans-complementation of VP1 or its SD significantly recovered the VP1 knockout MNV replicon replication, and the presence or absence of VP1 affected the kinetics of viral RNA synthesis. The results document a regulatory role for VP1 in the norovirus replication cycle, further highlighting the paradigm of viral structural proteins playing additional functional roles in the virus life cycle.
Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana, USA.
Chennareddy V
CV
Subba-Reddy
Muhammad Amir
MA
Yunus
Ian G
IG
Goodfellow
C Cheng
CC
Kao
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D036022:Capsid Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D055786:Gene Knockout Techniques; D005814:Genes, Viral; D005816:Genetic Complementation Test; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D029322:Norovirus / Q000145:classification / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D054730:Protein Interaction Domains and Motifs; D012367:RNA, Viral / Q000096:biosynthesis* / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012093:Replicon; D017386:Sequence Homology, Amino Acid; D014779:Virus Replication / Q000235:genetics / Q000502:physiology
D036022:Capsid Proteins; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D012324:RNA-Dependent RNA Polymerase