Isolation of p10 gene fromBombyx mori nuclear polyhedrosis virus and study of its promoter activity in recombinant baculovirus vector system

A homologue ofAutographa californica NPV (AcNPV) p10 gene was identified and cloned fromBombyx mori NPV (BmNPV). BmNPV p10 gene encodes truncated protein of 70 amino acid residues that lacks carboxyl terminus comparing with the p10 protein encoded by AcNPV. The putative TATA box sequence and the ATAAG motif which is the consensus sequence of baculovirus very late promoter were conserved. A transfer vector, pBNT1, which includes the p10 promoter region of BmNPV for foreign gene expression was constructed. By using pBNT1, a recombinant BmNPV, Bmp10-Luc, in which the p10 gene was replaced by the firefly luciferase gene, was obtained. We also obtained another recombinant virus, BmPH-Luc, in which the polyhedrin gene was replaced by the luciferase gene. The luciferase activity detected in BoMo-15AIIc insect cells infected with Bmp10-Luc was approximately 50% of that infected with BmPH-Luc, suggesting that although both the p10 and polyhedrin promoters of BrnNPV are effective in high-level expression of foreign gene, the p10 promoter is not so strong as the polyhedrin promoter.     

Novel baculovirus DNA elements strongly stimulate activities of exogenous and endogenous promoters.

A DNA sequence upstream from the polyhedrin gene of baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) was found to activate strongly the expression of full or minimal promoters derived from AcMNPV and other sources. Promoters tested included the minimal CMV (CMVm) promoter from human cytomegalovirus, the full heat shock 70 promoter from Drosophila, and the minimal p35 promoter from baculovirus. Deletion and mutagenesis analyses showed that this functional polyhedrin upstream (pu) activator sequence contains three open reading frames (ORFs), ORF4, ORF5, and lef2. In plasmid transfection assays, the pu sequence was able to confer high level luciferase expression driven by all of these full or minimal promoters in insect Sf21 cells. A known baculovirus enhancer, the homologous region (hr) of AcMNPV, further enhanced the expression of these promoters. Experiments showed that although multiple hr sequences function in an additive manner, pu and hr together function synergistically, resulting in as much as 18,000-fold promoter activation. Furthermore, a modified CMVm promoter containing pu and/or hr was inserted into the baculovirus genome to drive the luciferase coding region. The CMVm promoter expressed luciferase much earlier, and although it expressed a bit less than did the p10 promoter, the CMVm promoter gave rise to greater luciferase activity. Therefore, we have uncovered a cryptic viral sequence capable of activating a diverse group of promoters. Finally, these experiments demonstrate that synthetic sequences containing pu, hr, and different full or minimal promoters can generate a set of essentially unlimited novel promoters for weak to very strong expression of foreign proteins using baculovirus.     

Factors regulating baculovirus late and very late gene expression in transient-expression assays.

Eighteen genes of Autographa californica nuclear polyhedrosis virus are necessary and sufficient to transactivate expression from the late vp39 promoter in transient-expression assays in SF-21 cells. These 18 genes, known as late expression factor genes (lefs), are also required to transactivate the very late promoter of the polyhedrin gene, polh, but expression from this promoter is relatively weak compared with expression from the vp39 promoter. To further define the factors required for late and very late promoter expression, we first determined that the eighteen lefs were also required for expression from two other major baculovirus promoters: the late basic 6.9-kDa protein gene, p6.9, and the very late 10-kDa protein gene, p10. We next examined the effect of the very late expression factor 1 gene (vlf-1), a gene previously identified by analysis of a temperature-sensitive mutant, in the transient expression assay and found that vlf-1 specifically transactivated the two very late promoters but not the two late promoters. We then surveyed the Autographa californica nuclear polyhedrosis virus genome for additional genes which might specifically regulate very late gene expression; no additional vlf genes were detected, suggesting that VLF-1 is the primary regulator of very late gene expression. Finally, we found that the relative contribution of the antiapoptosis gene p35, which behaves as a lef in these transient-expression assays, depended on the nature of the other viral genes provided in the cotransfection mixtures, suggesting that other viral genes also contribute to the ability of the virus to block apoptosis.     

Development of baculovirus triple and quadruple expression vectors: co-expression of three or four bluetongue virus proteins and the synthesis of bluetongue virus-like particles in insect cells.

Baculovirus multiple gene transfer vectors pAcAB3 and pAcAB4 have been developed to facilitate the insertion of three or four foreign genes respectively into the Autographa californica nuclear polyhedrosis virus (AcNPV) genome by a single co-transfection experiment. The pAcAB3 vector contains a polyhedrin promoter and two p10 promoters on either side of the polyhedrin promoter but in opposite orientations. The pAcAB4 vector has an additional polyhedrin promoter in opposite orientation to the first copy that is in juxtaposition to the first p10 promoter. Each of these derived vectors (pAcAB3, pAcAB4) have been used for the simultaneous expression of three or four bluetongue virus (BTV) genes respectively. When Spodoptera frugiperda cells were infected with the recombinant virus (AcBT-3/2/7/5) expressing the four major structural genes of BTV, double-capsid, virus-like particles consisting of VP2, VP3, VP5 and VP7 of BTV were assembled.     

The promoter of the late p10 gene in the insect nuclear polyhedrosis virus Autographa californica: activation by viral gene products and sensitivity to DNA methylation.

In lepidopteran insect cells infected with the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV), two major late viral gene products are expressed: the polyhedrin, a 28 000 mol. wt. protein which makes up the mass of the nuclear inclusion bodies, and a 10 000 mol. wt. protein (p10) whose function is unknown. The nucleotide sequences of these strong promoters conform to those of other eukaryotic promoters and are rich in AT base pairs. We used the pSVO-CAT construct containing the prokaryotic gene chloramphenicol acetyl transferase (CAT) to study the function of the p10 gene promoter in insect and mammalian cells. Upon transfection of the pAcp10-CAT construct, which contained 402 bp of the p10 gene of AcNPV DNA in the HindIII site of pSVO-CAT, CAT activity was determined. The p10 gene promoter was inactive in human HeLa cells and in uninfected Spodoptera frugiperda insect cells. The same promoter was active, however, in AcNPV-infected S. frugiperda cells and exhibited optimal activity when cells were transfected 18 h after infection with the insect virus. This finding demonstrated directly that the p10 gene promoter required other viral gene products for its activity in insect cells. The nature of these products was unknown. The p10 gene promoter sequence contained one 5'-CCGG-3' site 40 bp upstream from the cap site of the gene and two such sites 178 and 192 bp downstream from the ATG initiation codon of the gene. Since Drosophila DNA or S. frugiperda DNA contained no 5-methylcytosine or extremely small amounts of it, we were interested in determining the effect of site-specific methylations on the p10 gene insect virus promoter. Methylation at the 5'-CCGG-3' sites led to a block of this promoter.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Influence of SV40 polyA on Gene Expression of Baculovirus Expression Vector Systems

The simian virus 40 polyadenylation signal (SV40 polyA) has been routinely inserted downstream of the polyhedrin promoter in many baculovirus expression vector systems (BEVS). In the baculovirus prototype Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the polyhedrin promoter (very late promoter) transcribes its gene by a viral RNA polymerase therefore there is no supporting evidence that SV40 polyA is required for the proper gene expression under the polyhedrin promoter. Moreover, the effect of the SV40 polyA sequence on the polyhedrin promoter activity has not been tested either at its natural polyhedrin locus or in other loci in the viral genome. In order to test the significance of adding the SV40 polyA sequence on gene expression, the expression of the enhanced green fluorescent protein (egfp) was evaluated with and without the presence of SV40 polyA under the control of the polyhedrin promoter at different genomic loci (polyherin, ecdysteroid UDP-glucosyltransferase (egt), and gp37). In this study, spectrofluorometry and western blot showed reduction of EGFP protein for all recombinant viruses with SV40 polyA, whereas qPCR showed an increase in the egfp mRNA levels. Therefore, we conclude that SV40 polyA increases mRNA levels but decreases protein production in the BEVS when the polyhedrin promoter is used at different loci. This work suggests that SV40 polyA in BEVSs should be replaced by an AcMNPV late gene polyA for optimal protein production or left untouched for optimal RNA production (RNA interference applications).     

表达乙型肝炎病毒表面抗原基因又形成多角体的杆状病毒

用形成包含体(OCC~+)并能利用人工合成启动序列和多角体XIV启动子表达外源基因的转移载体质粒pSXIVVI~+X3,将乙型肝炎病毒表面抗原(HBsAg)基因和多角体基因同时插入无包含体的粉纹夜蛾核型多角体病毒TnNPV-SVI-G基因组中,得到表达HBsAg基因又形成包含体(多角体)的重组毒侏TnNPV-HBs85-OCC~+。与利用野生型多角体启动子表达HBsAg基因的无包含体毒株TnNPV-HBsD4不同,TnNPV-HBs85-OCC~+由于具包含体,能以口服方式大规模感染粉纹夜蛾(Trichoplusia ni)幼虫,且HBsAg基因在草地夜蛾(Spodoptera frugiperda)离体细胞中的表达量要比前者高约37％,在虫体中的表达则更高。     

Expression of EGFP driven by BmNPV orf4 promoter, a novel immediate early promoter

Bombyx mori nucleopolyhedrovirus (BmNPV) orf4 has been shown to be expressed at very early stage of Bm-NPV infection cycle. In this study, using transient expression experiment, we demonstrated for the first time that orf4 promoter is an immediate early promoter, indicating that orf4 may play a role in the immediate-early stage of BmNPV infection. Moreover, with the recently developed Bac-to-Bac/BmNPV baculovirus expression system and a modified pFast-Bac1 whose polyhedrin promoter was replaced with orf4 promoter, a recombinant bacmid baculovirus expressing enhanced green fluorescent protein (EGFP) under the control of orf4 promoter in Bombyx mori (Bm) cells was successfully constructed. The result not only showed that the polyhedrin promoter can be replaced easily with other promoters to direct the expression of foreign genes by using this novel system but also laid the foundation for the rescue experiment of orf4 deletion mutant.     

Use of Scorpion Neurotoxins to Improve the Insecticidal Activity of Rachiplusia ou Multicapsid Nucleopolyhedrovirus

The nucleopolyhedrovirus of the mint looper, Rachiplusia ou (RoMNPV, also known as Anagrapha falcifera multicapsid nucleopolyhedrovirus), has previously been shown to kill the European corn borer, Ostrinia nubilalis, and to protect corn plants against this pest more effectively than the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). To further improve the performance of RoMNPV against O. nubilalis, we engineered it to express the insect-selective scorpion neurotoxins AaIT and LqhIT2 using the promoters of the AcMNPV p10 and p6.9 genes. RoMNPV recombinant virus expressing AaIT from the p10 promoter produced a reduced quantity of viral occlusions and polyhedrin protein and failed to occlude virions efficiently. Western blot analysis revealed that the amount of AaIT in hemolymph of recombinant virus-infected O. nubilalis was very low compared to that in the hemolymph of infected Heliothis virescens. In all but one case, insertion of the toxins did not alter the dose of virus required to kill three different hosts. Viruses expressing toxins from the p6.9 promoter killed larvae of O. nubilalis significantly faster than wild-type virus, but no significant difference in speed of kill was observed between AaIT- and LqhIT2-expressing viruses in this host. Expression of LqhIT2 reduced survival times of Helicoverpa zea and H. virescens to a significantly greater extent than expression of AaIT. An AcMNPV recombinant expressing LqhIT2 from the p6.9 promoter killed larvae faster than a recombinant utilizing the p10 promoter for expression, but recombinants using these promoters to drive AaIT expression did not exhibit any significant differences in speed of kill.