Norovirus proteinase-polymerase and polymerase are both active forms of RNA-dependent RNA polymerase.
In vitro mapping studies of the MD145 norovirus (Caliciviridae) ORF1 polyprotein identified two stable cleavage products containing the viral RNA-dependent RNA polymerase (RdRp) domains: ProPol (a precursor comprised of both the proteinase and polymerase) and Pol (the mature polymerase). The goal of this study was to identify the active form (or forms) of the norovirus polymerase. The recombinant ProPol (expressed as Pro(-)Pol with an inactivated proteinase domain to prevent autocleavage) and recombinant Pol were purified after synthesis in bacteria and shown to be active RdRp enzymes. In addition, the mutant His-E1189A-ProPol protein (with active proteinase but with the natural ProPol cleavage site blocked) was active as an RdRp, confirming that the norovirus ProPol precursor could possess two enzymatic activities simultaneously. The effects of several UTP analogs on the RdRp activity of the norovirus and feline calicivirus Pro(-)Pol enzymes were compared and found to be similar. Our data suggest that the norovirus ProPol is a bifunctional enzyme during virus replication. The availability of this recombinant ProPol enzyme might prove useful in the development of antiviral drugs for control of the noroviruses associated with acute gastroenteritis.
Belliot
Gaël
G
National Institutes of Health/DHHS, NIAID/LID, Building 50, Room 6316, 9000 Rockville Pike, Bethesda, MD 20892-8007, USA. gbelliot@niaid.nih.gov
Sosnovtsev
Stanislav V
SV
Chang
Kyeong-Ok
KO
Babu
Vijay
V
Uche
Uzo
U
Arnold
Jamie J
JJ
Cameron
Craig E
CE
Green
Kim Y
KY
D002139:Caliciviridae / Q000201:enzymology*; D002460:Cell Line; D010450:Endopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004792:Enzyme Precursors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism*; D012367:RNA, Viral / Q000096:biosynthesis; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism*
D004792:Enzyme Precursors; D012367:RNA, Viral; D012324:RNA-Dependent RNA Polymerase; D010450:Endopeptidases; D010447:Peptide Hydrolases