Effects of Staurosporine on Transcription by Vesicular Stomatitis Virus

Abstract

The protein kinase inhibitor staurosporine has been found to inhibit vesicular stomatitis virus transcription in infected BHK cells. Both primary and secondary transcription were virtually abolished by 10 μM staurosporine. In contrast, transcription by purified virions, or by nucleocapsids isolated from them, was unaffected by staurosporine. Staurosporine inhibition did not occur at or prior to the uncoating step, since: (i) the drug was equally effective if added at t = 0 or at t = 1 hr after infection; (ii) immunofluorescence microscopy of infected cells in the presence of staurosporine showed that M protein was diffusely present in the cytoplasm, indicative of normal uncoating. Staurosporine caused a modest (<25%) reduction in virus internalization from the cell surfaces. This was not sufficient to account for the transcription inhibition, however. Inhibition of transcription by staurosporine was not observed in extracts from infected cells, nor could it be induced by addition of uninfected cell cytosol to purified nucleocapsids. This suggests that inhibition of transcription in infected cells is secondary to substrate depletion or other cytotoxic effects of the drug. The addition of staurosporine to purified nucleocapsids, while not affecting cell-free transcription, inhibited phosphorylation of L protein completely and of P (NS) protein partially, providing addition evidence that at least two different kinases are present in nucleocapsids, and that the staurosporine inhibited ones are unnecessary for cell-free viral transcription.