Partial purification and characterization of foxtail mosaic potexvirus RNA-dependent RNA polymerase.
The RNA-dependent RNA polymerase (RdRp) of foxtail mosaic virus (FMV) was partially purified from infected leaves of Chenopodium quinoa. The membrane fraction of crude plant extracts contained most of the FMV RdRp activity. Additional purification was obtained by solubilization of the RdRp using KCl and dodecyl-sucrose and by centrifugation through a glycerol gradient. The RNA template endogenous to RdRp preparations could be removed using micrococcal nuclease but the resulting fraction was unable to copy added template purified from FMV virions. However, supplementation of fractions containing RdRp activity with FMV RNA resulted in a significant decrease in the level of RNA synthesis. This effect was specific to potexviral RNAs since a similar interference was also observed with clover yellow mosaic virus RNA but not with brome mosaic virus RNA or yeast RNA. RNA transcripts corresponding to various regions of the FMV genome were tested for their ability to inhibit RNA synthesis on endogenous template. The simultaneous presence of both 5' and 3' terminal regions of the viral genome was necessary to interfere with RNA synthesis suggesting that this inhibition resulted from competition for the binding of component(s) of the RdRp complex.
Rouleau
M
M
Department of Biochemistry, University of Western Ontario, London, Canada.
Bancroft
J B
JB
Mackie
G A
GA
D001483:Base Sequence; D007887:Fabaceae / Q000382:microbiology; D016679:Genome, Viral; D001467:Hordeum / Q000382:microbiology; D008836:Micrococcal Nuclease / Q000378:metabolism; D008969:Molecular Sequence Data; D010944:Plants / Q000382:microbiology; D010946:Plants, Medicinal; D010957:Plasmids; D017863:Potexvirus / Q000201:enzymology*; D012333:RNA, Messenger / Q000096:biosynthesis; D012367:RNA, Viral / Q000096:biosynthesis; D012324:RNA-Dependent RNA Polymerase / Q000302:isolation & purification* / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000378:metabolism; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism
D012333:RNA, Messenger; D012367:RNA, Viral; D012324:RNA-Dependent RNA Polymerase; D012259:Ribonuclease, Pancreatic; D015719:Single-Strand Specific DNA and RNA Endonucleases; D008836:Micrococcal Nuclease