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

Molecular, Cellular, and Developmental Biology Program, Division of Biology, 116 Ackert Hall, Kansas State University, Manhattan, KS 66506-4901, USA.
Virology (Impact Factor: 3.32). 11/2007; 367(2):265-74. DOI: 10.1016/j.virol.2007.05.026
Source: PubMed


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.

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    • "The consequence of transcriptional shutdown is that cellular protein levels deteriorate in baculovirus infections between 10–12 hours postinfection and cell lysis usually occurs sometime after 76 hours postinfection. AcMNPV overcomes this transcriptional downregulation by expressing its own DNAdependent RNA polymerase complex, which is responsible for the transcription of late and very late genes [26] [27] [28] [29] [30] [31] [32] [33]. Coupled to a high gene copy number as a result of the replication of the virus genome, this polymerase potentiates high level synthesis of recombinant protein. "
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