Protein-primed and de novo initiation of RNA synthesis by norovirus 3Dpol.
Noroviruses (Caliciviridae) are RNA viruses with a single-stranded, positive-oriented polyadenylated genome. To date, little is known about the replication strategy of norovirus, a so-far noncultivable virus. We have examined the initiation of replication of the norovirus genome in vitro, using the active norovirus RNA-dependent RNA polymerase (3D(pol)), homopolymeric templates, and synthetic subgenomic or antisubgenomic RNA. Initiation of RNA synthesis on homopolymeric templates as well as replication of subgenomic polyadenylated RNA was strictly primer dependent. In this context and as observed for other enteric RNA viruses, i.e., poliovirus, a protein-primed initiation of RNA synthesis after elongation of the VPg by norovirus 3D(pol) was postulated. To address this question, norovirus VPg was expressed in Escherichia coli and purified. Incubation of VPg with norovirus 3D(pol) generated VPg-poly(U), which primed the replication of subgenomic polyadenylated RNA. In contrast, replication of antisubgenomic RNA was not primer dependent, nor did it depend on a leader sequence, as evidenced by deletion analysis of the 3' termini of subgenomic and antisubgenomic RNA. On nonpolyadenylated RNA, i.e., antisubgenomic RNA, norovirus 3D(pol) initiated RNA synthesis de novo and terminated RNA synthesis by a poly(C) stretch. Interestingly, on poly(C) RNA templates, norovirus 3D(pol) initiated RNA synthesis de novo in the presence of high concentrations of GTP. We propose a novel model for initiation of replication of the norovirus genome by 3D(pol), with a VPg-protein-primed initiation of replication of polyadenylated genomic RNA and a de novo initiation of replication of antigenomic RNA.
Rohayem
Jacques
J
Institut für Virologie, The Calicilab, Fiedlerstrasse 42, D-01307 Dresden, Germany. Jacques.Rohayem@mailbox.tu-dresden.de.
Robel
Ivonne
I
Jäger
Katrin
K
Scheffler
Ulrike
U
Rudolph
Wolfram
W
D002474:Cell-Free System; D004926:Escherichia coli / Q000235:genetics; D016679:Genome, Viral / Q000502:physiology*; D006160:Guanosine Triphosphate / Q000378:metabolism; D029322:Norovirus / Q000502:physiology*; D009843:Oligoribonucleotides / Q000235:genetics / Q000378:metabolism; D011066:Poly C / Q000235:genetics / Q000378:metabolism; D016372:RNA, Antisense / Q000096:biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics; D012367:RNA, Viral / Q000096:biosynthesis* / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication / Q000502:physiology*
D009843:Oligoribonucleotides; D016372:RNA, Antisense; D012333:RNA, Messenger; D012367:RNA, Viral; D011994:Recombinant Proteins; D014764:Viral Proteins; D011066:Poly C; D006160:Guanosine Triphosphate; D012324:RNA-Dependent RNA Polymerase