Summary auto-generated
This study analyzed the p48 gene of Choristoneura fumiferana multicapsid nucleopolyhedroviruses (CfMNPV and CfDEFNPV), baculoviruses infecting spruce budworm. Researchers identified a unique restriction enzyme site (Sse8387I) in the p48 open reading frame, which encodes a potential 47–88 kDa protein of 411 amino acids in CfMNPV and OpMNPV, and 408 amino acids in CfDEFNPV. Transcriptional analysis using primer extension and Northern blotting revealed that p48 is a late baculovirus gene, with mRNA initiation beginning at a conserved ATAAG motif. Expression peaked at 48 hours post-infection. The gene is well conserved across multiple baculovirus species, sharing 50% amino acid identity. When researchers attempted to inactivate p48 by inserting reporter genes (lacZ or gfp), all insertions occurred at random genomic locations rather than the intended p48 locus. This inability to generate functional recombinant viruses with inactivated p48 suggests the gene is essential for viral replication. However, immunoblot analysis failed to detect p48 protein in infected cells, indicating very low or no protein expression, suggesting p48 may function at the RNA level rather than through protein synthesis.
Key findings
- The p48 gene of CfMNPV contains 1233 nucleotides encoding a 47–88 kDa protein and is transcribed as a late baculovirus gene, peaking at 48 hours post-infection
- p48 is conserved among baculoviruses with ≥50% amino acid identity across species, indicating evolutionary significance
- All attempts to inactivate p48 by inserting reporter gene cassettes failed, with insertions occurring randomly elsewhere in the genome, suggesting p48 is essential for viral replication
- Immunoblot analysis could not detect p48 protein in infected cells despite successful transcription, suggesting the gene may function at the RNA level rather than as a translated protein
- The p48 gene and surrounding gene organization (p40–p12–p48–p82) are highly conserved among baculoviruses including CfMNPV, CfDEFNPV, OpMNPV, and AcMNPV
This summary was generated automatically from the article PDF and is not part of the original publication. Refer to the PDF for the authoritative text.
Abstract
Attempts were made to linearize the DNA of Choristoneura fumiferana (Cf) multicapsid nucleopolyhedrovirus (MNPV), in order to improve the efficiency of generation of recombinant viruses after transfection. A unique site for the restriction enzyme Sse8387I was found in ORF p48. The requirement for this ORF during virus replication was investigated by molecular analyses including sequencing, transcriptional analysis and inactivation by insertion of marker genes. Sequence analysis showed that ORF p48 consists of 1233 nucleotides encoding a potential protein of 47.88 kDa. The proteins encoded by ORF p48 from CfMNPV and Orgyia pseudotsugata MNPV contain 411 amino acids while that from CfDEFNPV (a virus that is defective for infection by the per os route) is slightly smaller, at 408 amino acids. Transcriptional and primer extension analyses showed that the mRNA is initiated from a typical baculovirus 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., which confirmed the late property of the gene. Inactivation of the gene was attempted by inserting a cassette containing either the gene encoding beta-galactosidase 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 enzyme 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 virus replication. The gene is relatively well conserved among baculoviruses but its function remains unclear.