Novel Baculovirus DNA Elements Strongly Stimulate Activities of Exogenous and Endogenous Promoters

Abstract

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.

RING and Coiled-Coil Domains of Baculovirus IE2 Are Critical in Strong Activation of the Cytomegalovirus Major Immediate-Early Promoter in Mammalian Cells

“…We have previously found that IE1 and IE2 can activate some early baculovirus promoters within Vero E6 cells (24). To test whether they also activate the mammalian CMV promoter, we cotransfected IE1-or IE2-expressing plasmids with the reporter plasmid pAcL (where CMV drives the luciferase gene) or pA h cL (where CMV drives luciferase with an additional hr1 baculovirus enhancer) (26). We found that IE2 dramatically improved the CMV promoter-driven luciferase activity, which increased 122-fold in the presence of the hr1 sequence and over 25-fold without the hr1 sequence (Fig.…”

Identification of a High-Efficiency Baculovirus DNA Replication Origin That Functions in Insect and Mammalian Cells

“…(B) DpnI digestion showed that subfragments p143-2 and p143-3 can function as replication origins in Sf-21 cells in the presence of baculovirus infection. Sf-21 cells were transfected with plasmids containing the indicated baculovirus genomic regions and pABpLhGFP (11) and pABhcmEpL, which contain egfp and hr1 elements, respectively, and which served as the necessary positive and negative controls, respectively, followed by AcMNPV infection at an MOI of 1. ity is located in the overlapping region between p143-2 and p143-3. It has been reported previously that a plasmid containing the p143 gene sequence can replicate in the presence of baculovirus infection, indicating that an element in the sequence of this gene can function as a replication origin (14).…”

“…The transfected/infected cells were harvested at 48 h postinfection for luciferase activity assays. The detailed procedures were described previously (11). The luciferase activity data (average Ϯ standard deviation) were collected from triplicate assays of three independent transfections and are presented as the number of relative light units (RLUs) per microgram protein.…”

“…The hrs contain 2 to 8 imperfect, 30-bp palindromes with an EcoRI site at the center of each palindrome (9). hrs are bifunctional and can function as both transcriptional enhancers and oris, and these two functions are independent of one another (10)(11)(12). The enhancer function of hr was also reported in mammalian Vero E6 cells in a position-and orientation-independent manner; however, hr did not support ori activity in the tested mammalian system (13).…”

Homologous Region 3 fromBombyx moriNucleopolyhedrovirus Enhancing the Transcriptional Activity of Heat Shock Cognate 70-4 Promoter fromBombyx moriandBombyx mandarinain Vitroandin Vivo

“…1,3,4) We cloned the hr3 fragment from the BmNPV ZJ8 genome (Genbank accession No. U51328) and found that it can function as the origin of viral DNA replication and increase the transcriptional activity of viral gene promoters such as helicase and gp64 and of nonviral gene promoters such as hsp70 from Drosophila.…”