Bombyx mori nucleopolyhedrosis virus: expression from very late promoters

Abstract

The baculovirus expression system has been widely used to synthesise soluble, posttranslationally processed recombinant proteins in biologically active form. For highlevel expression of cloned foreign genes, the promoters of the hypertranscribed very late genes, polyhedrin (polh) and p10, are mainly utilised. Autographa californica nucleopolyhedrosis virus (AcMNPV) has been the most extensively exploited, followed by Bombyx mori nucleopolyhedrosis virus (BmNPV), for expression in permissive insect cell lines. Although these viruses share about 90% DNAlevel homology, they differ in host range. More than 600 proteins have been overexpressed in insect cell lines using the AcMNPV system. The BmNPV system offers an economically viable alternative for highlevel expression through larval caterpillars, which are readily reared in the laboratory. Highlevel expression of biologically active proteins and simple methods for protein isolation from recombinantBmNPVinfected silkworm larvae have been documented. Although significant work has been done to develop and optimise baculovirus expression systems, little is known about the molecular mechanisms governing the high levels of transcription from very late promoters. Transcription from late and very late promoters is mediated by a virally encoded, amanitinresistant RNA polymerase. So far, 18 virally encoded late gene expression factors (Lefs) have been identified as necessary and sufficient for late gene expression from AcMNPV, but the molecular basis of their action is not well established. The present studies were undertaken with two major objectives:

To improve the BmNPVbased expression system for highlevel expression of recombinant genes in silkworm larvae, and To analyse the role of the late gene expression factor Lef2 in very late gene expression.

1. Expression from AcMNPV verylate promoters in Bombyx cells and generation of recombinant BmNPVs via homeologous recombination Although many proteins have been highly expressed in silkworm larvae, the BmNPV system has limitations: • limited transfer vectors for optimised expression, • absence of simple recombinant selection procedures, • low transfection efficiencies in BmN cells. We previously established a highefficiency lipofection method enabling reliable generation of recombinant viruses. To address the vector limitation, we examined whether AcMNPVbased vectors function in the BmNPV system. A luciferase (luc) reporter gene was cloned under AcMNPV polh and p10 promoters and analysed by transient transfection in BmN cells infected with BmNPV. The heterologous promoters functioned efficiently in BmN cells. Gene location relative to the promoter influenced expression levels (+35 > 3 > 8 nt relative to the start codon). Given the high homology between AcMNPV and BmNPV at the polh and p10 loci, homeologous recombination was attempted, successfully generating recombinant BmNPVs containing luc under these promoters. Luciferasebased screening simplified selection, and Southern blotting confirmed recombination. High expression levels were observed in BmN cells and B. mori larvae infected with these recombinants. These recombinant viruses can also serve as tools for studying viral infection pathology.

2. Cloning and characterization of lef2 from BmNPV A late gene expression factor, lef2, encoding a 209aa ORF, was identified based on homology to AcMNPV and cloned by PCR. Detailed characterization included:

Temporal transcription: A 1.2 kb transcript appeared during delayedearly and late phases. Transcription start sites:

A late, aphidicolinsensitive transcript from a TAAG motif at 352 nt. An early, aphidicolininsensitive transcript from a TTGT motif located 35 nt downstream of a TATA box.

Termination and polyadenylation sites were mapped by RNase protection. Protein localization: Lef2 (~24 kDa) is present at 48 h postinfection and is found in both nucleus and cytoplasm, specifically associating with the virogenic stroma.

3. Functional role of Lef2 Lef2 transactivated verylate gene expression from both polh and p10 promoters. Deletion of the Cysrich Cterminal domain abolished activation. To distinguish between effects on transcription versus DNA replication, antisense lef2 constructs were used under early (ie1) or very late (polh) promoters. Key findings:

Antisense expressed early 45-50% reduction in viral titre and ~55-60% inhibition of viral DNA synthesis. Antisense expressed late Strong inhibition of verylate gene transcription but minimal effect on DNA replication.

Lef2 also stimulated plasmidbased viral DNA replication mediated by hr sequences. Thus, Lef2 functions in both viral DNA replication and verylate gene transcription.

4. Lef2 as a transcriptional coactivator Sequence searches revealed no DNAbinding motifs. GSTLef2 did not bind heparin or polh promoter DNA. Lef2 did not activate transcription from the yeast Gal1 promoter, suggesting it is not a general activator. A Cterminal Cysrich region showed homology to the CH3 domain of mammalian p300, a transcriptional coactivator. Coimmunoprecipitation identified three interacting proteins; one (39 kDa) resembled the basal transcription factor TBP. Direct interaction between Lef2 and TBP was confirmed by TBPagarose pulldowns. Two LXXLLlike motifs (LYMLL and VMHLL), known for mediating protein-protein interactions, were identified in Lef2. Mutation of these motifs abolished Lef2’s ability to activate verylate transcription in a lef2-mutant virus background, while WT Lef2 restored promoter activity. Thus, Lef2 plays a direct role in verylate gene expression as a transcriptional coactivator.