Summary auto-generated
This study examined the expression and cellular localization of LEF-11, a viral protein encoded by the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). LEF-11 is a late transcription factor previously identified as necessary for optimal late gene expression. The researchers used 5' and 3' RACE analysis and Northern blotting to map lef-11 transcripts, finding a 1.376 kb mRNA with its transcription start site located 196 nucleotides upstream of the lef-11 translation initiation codon, within the upstream orf38 gene. The lef-11 transcript shares a common 3' end with the downstream pp31 gene mRNA. Western blot analysis detected LEF-11 protein from 4 to 72 hours post-infection, with maximum levels between 8 and 24 hours. Immunofluorescence microscopy revealed that LEF-11 localizes to dense regions within infected cell nuclei, consistent with its presumed role as a transcription factor. The relatively low abundance of lef-11 mRNA compared to overlapping pp31 transcripts required affinity-purified antibodies for reliable protein detection.
Key findings
- LEF-11 mRNA (1.376 kb) initiates 196 nucleotides upstream of the translation start codon within the upstream orf38 gene and shares a 3' end with pp31 transcripts
- LEF-11 protein is detected from 4 to 72 hours post-infection with maximal expression between 8-24 hours, while mRNA levels decline after 36 hours
- LEF-11 localizes to dense regions within the nucleus, consistent with a role as a late transcription factor
- The lef-11 transcript contains a small upstream open reading frame of 58 amino acids that overlaps the lef-11 ORF by 35 nucleotides
- LEF-11 mRNA abundance is substantially lower than overlapping pp31 transcripts, limiting detection without affinity-purified antibodies
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Abstract
The Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) lef-11 gene was found previously to be necessary to support optimal levels of transient expression from an AcMNPV late promoter. The lef-11 gene is unusual in that it overlaps both upstream (orf38) and downstream (pp31) genes. In this study, the expression and cellular localization of LEF-11 were examined. The lef-11 transcripts were detected from 4 to 36 h post-infection (p.i.). The 1·5 kb lef-11 mRNA initiates 196 nt upstream of the lef-11 translation initiation codon, within the upstream orf38 gene. This relatively long 5' upstream region encodes a potential small upstream open reading frame (ORF) of 58 amino acids that overlaps the lef-11 ORF. The 3' end of the lef-11 mRNA was mapped as co-terminal with mRNAs from the downstream pp31 gene. Using affinity purified anti-LEF-11 antibodies, levels of LEF-11 expression were found to be maximal between approximately 8 and 24 h p.i., although LEF-11 could be detected as late as 72 h p.i. Using immunofluorescence microscopy, it was determined that LEF-11 localized to dense regions of infected cell nuclei, consistent with its role as a possible late transcription factor.