Baculovirus immediately early 1, a mediator for homologous regions enhancer function in trans
© Lin et al; licensee BioMed Central Ltd. 2010
Received: 25 November 2009
Accepted: 10 February 2010
Published: 10 February 2010
Enhancers are DNA sequences that serve as binding sites for regulatory proteins, and stimulate transcriptional activity independent of their positions and orientations with respect to the transcriptional initiation site. Previous studies considered that baculovirus homologous regions (hrs) function as enhancers in cis. In our study, a plasmid containing homologous region 3 (hr3) enhancer from Bombyx mori nucleopolyhedrovirus (BmNPV) failed to enhance transcription of promoter in other plasmid in co-transfection assays, but strong stimulation occurred when cells were infected by BmNPV.
The cotransfection results of each BmNPV genomic library plasmid, hr3 plasmid and reporter plasmid showed that there were eight library plasmids stimulated the luciferase gene expression remarkably. Sequencing these plasmids revealed that each of them contained the ie-1 gene. Transfected plasmids, containing ie-1, hr3 and various origin promoter drove reporter gene showed the function was even retained. Cotransfection of hr3 functional dissected fragment and ie-1 revealed that the 30-bp imperfect palindrome destroyed fragment can't enhance reporter gene expression even though transfected with ie-1.
IE-1 was the only early factor of BmNPV that could act as a mediator for hr enhancer function in trans and the trans-function was achieved with a broad-spectrum of promoters. The 30-bp imperfect palindrome was the elementary molecular structure by which IE-1 participated in the enhancer function in trans.
The genome of baculovirus contains interspersed homologous regions (hrs) that function as transcriptional enhancers linking in cis to viral or heterologous promoters in either insect or mammalian cells . The immediately early gene 1, ie-1, is one of six essential genes required for DNA replication in transient replication assays, and the 67-kDa encoded product of ie-1 is the principal transcriptional regulator of baculovirus . As assayed by plasmid transfection, IE-1 transactivates the expression of various baculovirus early genes and some housekeeping genes . When the affected promoter links in cis to the hr enhancer, IE-1 protein also markedly stimulates promoter activity through binding to the 28-mer palindrome units [4–6].
Transcriptional enhancers for eukaryotic genes are binding sites for regulatory proteins; they lie at a distance upstream or downstream of the transcriptional start sites, and the regulatory proteins that bind to them activate (or sometimes inhibit) transcription [7, 8]. A previous report showed that the hr enhancer stimulated transcription only in the cis-linked conformation . In contrast, another study found that when plasmid p39CAT was co-transfected with Bgl II-digested viral DNA and a Pst I DNA library of Autographa californica Nucleopolyhedrovirus (AcNPV), the CAT activity increased remarkably .
In our study, hr3 from BmNPV failed to enhance the expression of the luciferase gene (luc) in trans in co-transfection assays, but strong enhancement occurred when the two independent plasmids were co-transfected into silkworm cells along with BmNPV. Therefore, we assumed that certain viral factor(s) participate in the trans-activation effect. A random BmNPV genomic library was constructed and used to screen viral factor(s) mediating hr3 enhancer function in trans through co-transfection with DNAs from reporter plasmid and hr3 enhancer-containing plasmid. According to the structural characteristics of the hr3 enhancer, dissection analyses with different amounts of palindromes were conducted to uncover the basic requirement for hr3 enhancer function in trans.
T4 DNA ligase, platinum pfx DNA polymerase and the lipofectin kit were purchased from Invitrogen (USA). Taq DNA polymerase, restriction endonucleases, pGEM-T easy vector, DNA purification kit, luciferase assay kit and pRL-CMV vector for internal control transfections were purchased from Promega Corp (USA). E. coli strain DH10B was maintained in our lab. The reporter plasmids pKS-hel510-luc, pKS-Bmgp64-luc and pGEM3Z-lsp-luc, containing helicase, gp64 and the silkworm larvae serum protein (lsp) gene promoter respectively, were from our previous work [11–13]. The enhancer vectors, pKS-hr114, pKS-hr198 and pKS-hr3 containing 0, 1 or 3 30-bp incomplete palindromes respectively, were constructed and maintained in our lab .
Virus, cell lines and random library
The BmNPV-ZJ8 strain was maintained in our lab. Bm-N cells were propagated at 27°C in TC-100 insect medium supplemented with 10% heat-inactivated (56°C, 30 min) fetal bovine serum (FBS) (Invitrogen). The details for cell culture were from Summers and Smith's manual . A random genomic library of BmNPV was constructed according to the "partial filling-in" method that contained a 3 kb to 5 kb fragment in the pUC19 vector [16, 17]. Plasmid DNAs of 238 positive colonies were extracted for further transient assays .
Transfection in insect cells
Bm-N cells were seeded in 24-well plates and allowed to attach at 27°C overnight. Transfection assays were conducted using lipofectin following the manufacturer's instructions. The co-transfection solution contained 0.3 μg reporter plasmid DNA, 0.1 μg internal control plasmid DNA in some cases, 0.3 μg of each plasmid DNA from the random library, and hr enhancer when necessary, along with 2 μl lipofectin in a total volume of 50 μl. pBlueScript DNA was introduced in some reactions to maintain a constant quantity of DNA. If virus infection was required, the virus was added to the serum-free medium and left for 1 h before the supernatant was replaced with complete medium. Each transfection contained at least three separate experiments.
Luciferase activity assay
The cells were harvested at 48 h post transfection (hpt) and cell extracts were prepared following the instructions with the luciferase assay kit (Promega). The amount of protein in the lysate was measured using the Bradford method . Measurements of dual-luciferase activity were performed with a liquid scintillation spectrometer (Beckman LS6000 Series, USA) . Luciferase activity was indicated as counts per minute (CPM) in 15 s.
Cloning of Orf121, Orf122 and ie-1 genes
Primers used to amplify the orf121, orf122 and ie-1 genes, as well as 5' UTR
Function of the hr enhancer in trans via virus infection
Transactivation effects of hr3 enhancer on target promoters via BmNPV infection in Bm-N cells
22.3 ± 4.5
17 ± 3.8
80 ± 13.3
1303384 ± 74692
8 ± 2.7
11.6 ± 4.1
68 ± 21.2
227636 ± 37514
Genome-wide screening for viral factors mediating the hr enhancer function in trans
Plasmids involved in hr3 enhancer function in trans
Corresponding site in T3 strain
Intact coding regions contained
odv-e18, odv-ec27, orf-121, orf-122, ie-1
orf-121, orf-122, ie-1
orf-121, orf-122, ie-1
orf-121, orf-122, ie-1
odv-ec27, orf-121, orf-122, ie-1, odv-e56, orf-125
orf-121, orf-122, ie-1
odv-ec27, orf-121, orf-122, ie-1, odv-e56, orf-125
orf-121, orf-122, ie-1
IE-1 protein affected hr enhancer function in trans alone
Transactivation effects of hr3 enhancer on target promoters via IE-1 protein bridge.
21.4 ± 5.6
16093 ± 1432
735880.1 ± 119032
74.9 ± 15.4
40080 ± 2947
5738241 ± 609222
14.4 ± 3.9
208 ± 17.3
26876 ± 4811
78 ± 11.8
404 ± 53.5
76400 ± 9312
Functional dissection of hr enhancer structure essential for function in trans
Baculovirus homologous regions are repeated sequences that are interspersed in the genomes of baculoviruses. It is known that these regions contain the origins of DNA replication, and augment the expression of a number of genes in an orientation-independent manner .
In the random genomic library of BmNPV, the average size of fragments were 3 to 5 kb, while the largest open reading frame of BmNPV, helicase, was 3669 bp, so we ensured that the largest coding region in the genome was included in the random library. According to the average size of cloned fragments, the representation of the random library was more than 99% . Therefore, the completeness and representation of the constructed library was adequate for genome-wide screening for regulatory products or elements.
In some cases, enhancers can function in trans on a separate DNA molecule via a protein bridge to the promoter by covalent linkage of molecules or a cellular factor binding with the two elements [21, 22]. The simian virus 40 (SV40) enhancer functions in trans to the β-globin promoter when they are linked by a protein bridge . A recent report even suggests that certain enhancers can interact with several target promoters in trans on different chromosomes . Genome-wide screening revealed eight plasmids, all containing orf-121, orf-122 and ie-1 coding regions could stimulate the expression of luciferase. Subsequent experiments showed that IE-1 was the only factor of BmNPV that acted as a mediator for the hr enhancer functioning in trans. This may be because the sizes of the two hypothetical proteins ORF121 and ORF122 are too small to afford the binding domain and activating domain required for a transcription factor. Previous reports using gel shift assays and mutational analyses confirm that IE-1 binds directly to the hr enhancers [24, 25]. The direct interaction between IE-1 and hr enhancer may be consistent with the theory that some eukaryotic enhancers reach their targets (as yet undefined) by means of DNA loops, IE-1 bring the hr enhancer to the proximal promoter and the complex is easily recruited by the transcriptional apparatus [26, 27]. In this study, we assumed that IE-1 plays two distinct roles, one as a transactivator to stimulate the transcription of the basal promoter, and the other as a mediator for hr enhancer functioning in trans by binding to the hr enhancer and then reaching the target promoter to give another 40 to 100-fold activation. So the enormous enhancement of luciferase activity was obtained by the cumulative effects of these mechanisms. When hr114 was co-transfected with pGEM-T-ie1 and reporter plasmids, the CPM values were lower than control assays with co-transfected pGEM-T-ie1 and reporter plasmids. Since IE-1 can bind with a half palindrome of hr enhancer, the competitive binding of IE-1 by the two half palindromes might have resulted in the decrease of IE-1 transactivator. It was reported that the imperfect palindrome, especially the naturally occurring Eco RI site, is essential for its enhancing function in cis. The present study demonstrated that the intact palindrome is also the elementary structural requirement for hr enhancer functioning in trans.
AcMNPV hr5 functions in trans in an IE-1-dependent 39K promoter and the p35 promoter, and the stimulating effects of hr5 in trans are about 2 and 7-fold [28, 29]. In this report, the IE-1 induced much higher activation of all the differently derived promoters to the trans-presented hr3 enhancer from BmNPV, and this suggests that IE-1 is a generic mediator for hr enhancer functioning in trans. Screening of the genomic library confirmed that IE-1 is the only viral factor that mediates hr enhancer functioning in trans. Furthermore, this result implies that in some transfection experiments, particularly for co-transfection molecules and internal controls, the trans-effects should not be ignored .
We thank Iain C. Bruce and Gen Wang for critical reading of the manuscript. This work was supported by grants from the National Natural Sciences Foundation of China (30670082) and the "863" Project (2006AA10A119).
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