Molecular analysis of the p48 gene of Choristoneura fumiferana multicapsidnucleopolyhedroviruses CfMNPV and CfDEFNPV

Attempts were made to linearize the DNA of Choristoneura fumiferana (Cf) mu lticapsid nucleopolyhedrovirus (MNPV), in order to improve the efficiency o f generation of recombinant viruses after transfection. A unique site for t he restriction enzyme Sse83871 was found in ORF p48. The requirement for th is ORF during virus replication was investigated by molecular analyses incl uding sequencing, transcriptional analysis and inactivation by insertion of marker genes. Sequence analysis showed that ORF p48 consists of 1233 nucle otides encoding a potential protein of 47.88 kDa. The proteins encoded by O RF p48 from CfMNPV and Orgyia pseudotsugata MNPV contain 411 amino acids wh ile that from CfDEFNPV (a virus that is defective for infection by the per os route) is slightly smaller, at 408 amino acids, Transcriptional and prim er extension analyses showed that the mRNA is initiated from a typical bacu lovirus late gene ATAAG motif, The mRNA was detected at 24 h post-infection (p.i.), reached maximum levels at 48 h p.i. and declined by 96 h p.i., whi ch confirmed the late property of the gene. Inactivation of the gene was at tempted by inserting a cassette containing either the gene encoding beta-ga lactosidase or that encoding green fluorescent protein. Blue or fluorescent green plaques of infected cells were observed after transfection. Attempts to generate a plaque-purified virus were not successful. Restriction enzym e analysis showed that the marker genes were inserted randomly at positions other than the p48 locus. This indicated that the gene may be needed for v irus replication. The gene is relatively well conserved among baculoviruses but its function remains unclear.