Unique capping activity of the recombinant RNA polymerase (L) of vesicular stomatitis virus: association of cellular capping enzyme with the L protein.
Vesicular stomatitis virus (VSV), a prototype of non-segmented negative strand RNA viruses, packages an RNA-dependent RNA polymerase (L) which, together with an associated phosphoprotein (P), transcribes the genome RNA, in vitro and in vivo, into mRNAs that are capped at the 5'-ends. However, unlike cellular guanlylyltransferase (GT), the RNA polymerase incorporates GDP in the capped structure, as Gp(alpha)p(beta)-p(alpha)A. In an effort to characterize the capping activity of the RNA polymerase, we have purified recombinant L (rL) protein expressed in insect cells. The rL, like the virion L polymerase, also caps transcribed mRNAs with identical unique cap structure. Interestingly, the purified rL is found to be tightly bound to the GT of the insect cell during all stages of purification. VSV grown in baby hamster kidney cells also packages cellular GT of the murine cell, suggesting that VSV L protein or its associated proteins may have a strong affinity for the cellular GT. The GT bound to rL, however, formed E-GMP complex, whereas no such complex was detected with the rL protein. It appears that the L protein may contain the putative active site for the unique capping reaction or the tightly bound cellular GT may by some unknown mechanism participate in the unique capping reaction.
Gupta
Ashim K
AK
Department of Virology, Lerner Research Institute at NN-10, The Cleveland Clinic Foundation, Cleveland, OH 44195, USA.
Mathur
Manjula
M
Banerjee
Amiya K
AK
D000818:Animals; D002460:Cell Line; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D006157:Guanosine Monophosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D009713:Nucleotidyltransferases / Q000378:metabolism; D012315:RNA Caps / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000378:metabolism; D014158:Transcription, Genetic / Q000502:physiology*; D014721:Vesicular stomatitis Indiana virus / Q000201:enzymology* / Q000235:genetics
D012315:RNA Caps; D011994:Recombinant Proteins; D006157:Guanosine Monophosphate; D006160:Guanosine Triphosphate; D009713:Nucleotidyltransferases; C020842:guanylyltransferase; D012321:DNA-Directed RNA Polymerases