Inter-subunit interactions of the Autographa californica M nucleopolyhedrovirus RNA polymerase

Autographa californica M nucleopolyhedrovirus transcribes genes using two DNA-directed RNA polymerases; early genes are transcribed by the host RNA polymerase II, and late and very late genes are transcribed by a viral-encoded multisubunit RNA polymerase. The viral RNA polymerase is composed of four proteins: Late Expression Factor-4 (LEF-4), LEF-8, LEF-9, and P47. The predicted amino acid sequences of lef-9 and lef-8 contain motifs that are similar to those that participate at the catalytic center of known RNA polymerases. The requirement for the motif present in LEF-8 in late gene expression has been previously demonstrated. We have assessed the requirement of specific residues within the motif in LEF-9 for late gene expression. The conserved aspartic acid residues within the LEF-9 motif, corresponding to those essential for activity of the Escherichia coli RNA polymerase largest subunit, were required for late gene expression. Furthermore, we found that LEF-8 and LEF-9 interacted in coimmunoprecipitation experiments. We determined possible interactions of all the RNA polymerase subunits in pairwise combinations and found associations between LEF-9 and P47, LEF-4 and P47, and LEF-8 and P47. In contrast, LEF-4 and LEF-8 did not coimmunoprecipitate but coimmunoprecipitated in the presence of P47, suggesting that they do not associate directly. A weak association was observed between LEF-4 and LEF-9. Further analysis also suggested that LEF-8, LEF-9, and P47 have the ability to self-associate. Studies on protein-protein interactions may provide insight into the structural design of the complex and mechanistic aspects affecting late and very late gene expression.