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

Abstract

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.

Autographa californica Multiple Nucleopolyhedrovirus 38K Is a Novel Nucleocapsid Protein That Interacts with VP1054, VP39, VP80, and Itself

“…The ORFs were amplified by PCR using primer pairs as follows: ac5452 and ac5433 for vp1054, ac8952 and ac8932 for vp39, ac98529 and ac98326 for 38K, and ac10452 and ac10432 for vp80. The PCR products were first cloned into the pMD 18-T vector for sequencing before being subcloned into pHSFLAGHisViϩ (kindly provided by A. Lorena Passarelli, Kansas State University) (8) to generate the FLAG-tagged constructs pFLAG-VP1054, pFLAG-VP39, pFLAG-38K, and pFLAG-VP80, respectively. To generate the HA-tagged 38K expression plasmid, the 38K PCR product was subcloned into pHSEpiHisVIϩ (kindly provided by A. L. Passarelli) (8) to give pHA-38K.…”

“…To increase the transfection efficiency, a second cotransfection was performed 12 h after the first cotransfection. The cells were then incubated in fresh medium for 24 h at 27°C, heat shocked for 30 min at 42°C, and then treated for 30 min at 27°C with 50 g/ml Z-Leu-Leu-Leu-Leu-Al (MG 132; Sigma-Aldrich Co.), a proteasome inhibitor (8). After 4 to 6 h, cells were lysed with 500 l of lysis/immunoprecipitation buffer [as described in reference 51, except that (NH 4 ) 2 SO 4 was changed from 150 to 100 mM].…”

Interactions Between Nucleopolyhedroviruses and Polydnaviruses in Larval Lepidoptera

“…The pif genes include p74-pif, and pif genes 1-5, Ac119, Ac22, Ac115, Ac96, and Ac148, respectively. Deletion of any of the genes from a viral genome significantly decreases but does not eliminate per os infectivity (d'Alencon et al, 2004;Crouch et al, 2007). The PIFs, with the exception of PIF3, are thought to be involved in binding or interacting with the midgut cells that leads to infection (Ohkawa, et al, 2005;Li et al, 2007;Peng et al, 2010;Horton & Burand, 1993).…”

Quality Control of Baculoviral Bioinsecticide Production

“…Other two genes (p49 and 38 K) encode proteins associated to packaging, assembly, and release of virions (Wu et al, 2008;Lin et al, 2010). Meanwhile, lef9 gene encodes a polypeptide involved in virus transcription machinery (Crouch et al, 2007). Using multiple alignments derived from sequences corresponding to P74, lef9 and 38k genes from all alpha and betabaculovirus members were selected the two better regions of homology to design a set of primers (Figure 4).…”