The role of template-primer interactions in cleavage and initiation by the influenza virus polymerase.
An in vitro cleavage/initiation assay was used to analyse cleavage site choice and transcription initiation by the influenza virus polymerase. A synthetic mRNA which is cleaved by the polymerase to produce a single 11 base primer fragment was altered around this cleavage site. Depending upon the mutations made, alternative cleavage sites were used. This system was then used in extracts from recombinant vaccinia virus infected cells which express the polymerase. These extracts require the addition of a synthetic vRNA in order to induce cleavage and initiation activity. The data show that the choice of cleavage site is wholely controlled by the mRNA and does not depend upon interactions with the vRNA template. However, the site of initiation of the cleaved primer on the template is influenced by template-primer interactions.
Hagen
M
M
Department of Virology, Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, CT 06492.
Tiley
L
L
Chung
T D
TD
Krystal
M
M
D001483:Base Sequence; D004722:Endoribonucleases / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D009980:Influenza A virus / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D017354:Point Mutation / Q000502:physiology; D012313:RNA / Q000235:genetics / Q000378:metabolism*; D012315:RNA Caps; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D013698:Templates, Genetic; D014158:Transcription, Genetic; D014616:Vaccinia virus / Q000235:genetics
D012315:RNA Caps; C034477:RNA primers; D012333:RNA, Messenger; D012367:RNA, Viral; D011994:Recombinant Proteins; D012313:RNA; D012324:RNA-Dependent RNA Polymerase; D004722:Endoribonucleases