Expression of L protein of vesicular stomatitis virus Indiana serotype from recombinant baculovirus in insect cells: requirement of a host factor(s) for its biological activity in vitro.
The 241-kDa large (L) protein of vesicular stomatitis virus (VSV) Indiana serotype, a multifunctional catalytic subunit of the viral RNA polymerase, has been expressed in Spodoptera frugiperda cells infected with recombinant baculovirus BacPAK6-L containing the L gene under the control of a polyhedrin promoter. The recombinant L protein was biologically active and supported viral mRNA synthesis in vitro. When the expressed L protein was purified by phosphocellulose column chromatography, it eluted in two peaks, one at 0.4 M NaCl (peak I) and the second at 0.75 M NaCl (peak II). The L protein in peak I showed significant transcriptional activity in an in vitro transcription reconstitution experiment, whereas the L protein in peak II was inactive. Interestingly, the addition of cytoplasmic extract from uninfected Sf21 cells to peak II completely restored transcription in vitro, indicating the requirement of a host factor(s) for the activity of the L protein. This factor is relatively heat stable and is dissociable from the recombinant L protein. It is also present in BHK, COS, and HeLa cells in detectable levels. The role of the putative host protein(s) in the activation of the L protein is discussed.
Mathur
M
M
Department of Molecular Biology, Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
Das
T
T
Banerjee
A K
AK
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001685:Biological Factors / Q000502:physiology*; D002460:Cell Line; D003001:Cloning, Molecular; D006367:HeLa Cells; D006801:Humans; D012324:RNA-Dependent RNA Polymerase; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D018411:Spodoptera; D014158:Transcription, Genetic; D014721:Vesicular stomatitis Indiana virus / Q000235:genetics*; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification
D001685:Biological Factors; D011994:Recombinant Proteins; D014764:Viral Proteins; C040530:L protein, vesicular stomatitis virus; D012324:RNA-Dependent RNA Polymerase