Purification and characterization of ORF69 of Autographa californica nuclear polyhedrosis virus

Abstract

Autographa californica nuclear polyhedrosis virus (AcNPV) late genes are transcribed by a viral-encoded RNA polymerase that is composed of four subunits. The LEF-4 subunit has guanylyltransferase and RNA triphosphatase activities which are needed to form the 5' cap structure. But whether AcNPV encodes its own capping methyltransferases (MTase) is yet unknown. The ORF69 protein was identified as a candidate for capping MTases based on its sequence similarity to FtsJ. ORF69 was overexpressed in E.coli and purified to homogeneity. Purified ORF69 had AdoMet binding activity. The pattern of protein expression in vivo and its dependency on DNA replication suggest that ORF69 is a late protein. Primer extension analysis identified the presence of a late promoter motif at the transcription start site, thus further confirming this hypothesis. A mutant virus with the E.coli lacZ gene inserted into the middle of orf69 was constructed and orf69 was shown to be non-essential for virus replication. Single-step growth curves of mutant and wild type viruses showed that the rate of virus replication was not affected by the absence of ORF69. As an approach to the identification of the viral mRNA (guanine-7-) methyltransferase (cap MTase), the enzymatic activity was partially purified from the infected and uninfected cells. Cap MTase exhibited identical purification patterns from both sources on three different columns. Together, these data showed that the AcNPV orf69 gene encodes a late, non-essential protein with the potential to be a MTase. Partial purification of cap MTase from infected cells suggests that AcNPV uses host cap MTase to cap its late transcripts.