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ISSN 0582-9879                                    ACTA BIOCHIMICA et BIOPHYSICA SINICA 2003, 35(1): 18-26                                    CN 31-1300/Q

Promoter Activities in the Baculovirus Envelope Glycoprotein gp64 Gene

ZHOU Ya-Jing1,2￥, YI Yong-Zhu1, ZHANG Zhi-Fang1*, HE Jia-Lu1, ZHANG Yuan-Xing2, WU Xiang-Fu3

( 1 Key Laboratory of Silkworm Biotechnology, Ministry of Agriculture, Sericultural Research Institute,

Chinese Academy of Agricultural Sciences, Zhenjiang 212018, China;

2 State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China;

3 Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, the Chinese Academy of Sciences, Shanghai 200031, China )

 

Abstract      Baculovirus GP64 envelope glycoprotein is a specific major component of the envelope of the budded virus and is involved in virus entry into the host cells by endocytosis. For promoter activity analysis in the baculovirus gp64 gene, two DNA fragments containing 437 and 439 bp upstream of 5' ends of the BmNPV and AcMNPV gp64 ORF were amplified by polymerase chain reaction and cloned, respectively. The sequence analysis indicated that two gp64 genes have both early (CAGT) and late (A/GTAAG) transcriptional start sites. By use of the plasmids with a reporter luciferase gene (Luc) driven by gp64 promoter to transfect insect cells, transient expression assay showed that pBmgp64Luc had high expression levels in permissive Bm-N cells and very low levels in non-permissive Sf-21 cells, while pAcgp64Luc had relatively high expression levels both in permissive Sf-21 cells and in non-permissive Bm-N cells. Furthermore, the transcription of both gp64 promoters appeared to be transactivated by 2.4 – 4 times in corresponding permissive cells by corresponding viral factors, separately. By inserting BmNPV homologous region-3 (hr3) into the downstream of luciferase reporter gene driven by gp64 promoter, it enhanced transcription from both gp64 promoters by 13 – 22 times in Bm-N cells and over 7000-14 000 times in Sf-21 cells, respectively. In the presence of BmNPV hr3, correspondingly, the viral factors transactivated the transcriptional activity from two promoters by about 73 – 78 times in corresponding permissive cells. It suggested that BmNPV hr3 plays an important role in co-activation with viral factors onto the gp64 promoter besides the functions of viral DNA origin and enhancer.

 

Key words     baculovirus; gp64 gene promoter; homologous region-3; viral factors; transfection

 

The baculoviruses are a large and diverse family of occluded viruses with double-stranded DNA genomes of 100 to 180 kb depending on the virus strain. They are pathogenic for insects particularly members of the lepidopteran, dipteran, and hymenopteran[1]. Two baculovirus genera, the nucleopolyhedroviruses (NPVs) that have large occlusion bodies (OBs) containing numerous virions[2] and the granuloviruses (GVs) that normally have single virions occluded within small granular OBs[3], have been described. During baculovirus replication in the host insects and cell culture, two virion phenotypes are produced[4]. The virions of occluded virus (OV) phenotype acquire an envelope in the nucleus and are subsequently occluded in large polyhedron-shaped occlusion bodies. This virus phenotype is necessary for the transmission of infection between insects[5,6]. The virions of the budded virus (BV) phenotype, while, are not occluded and acquire an envelope by budding through the virus-modified plasma membrane at the cell surface. The BV phenotype serves to spread the infection from cell to cell within an infected individual[7,8]. A major difference between these two types of virions is their envelope proteins. The envelope of OV may be composed of multiple proteins and the mechanism of its facilitating the initiation of infection of insect midgut cells is unclear[9]. While, the BV envelope contains an envelope fusion protein that causes the merging of the virion envelope and the membrane of cellular endocytic vesicles when exposed to low pH. Current evidence suggests that lepidopteran baculoviruses may be divided into two phylogenetic groups based on their envelope fusion proteins[10]. One group, including Autographa californica multinucleocapsid NPV (AcMNPV), Orgyia pseudotsugata MNPV (OpMNPV) and other relatively closely related viruses, utilizes GP64, a low pH-dependent envelope fusion protein[11,12], whereas the other employs a protein family, for example, the LD130 in the Lymantria dispar NPV, unrelated to GP64, but that is also low pH-dependent[10]. Similar results have been reported for the LD130 homologs in SeMNPV[13], Plutella xylostella GV (PxGV)[14], and Xestia c-nigrum GV (XcGV)[15]. Zhang et al.[16,17] found that SL136 protein in SpltMNPV might also be an envelope fusion protein.

GP64, peculiar to BV, is a major virion envelope glycoprotein of the baculovirus. It is encoded by virus with reported molecular weight ranging from 64 000 to 70 000[18] and is required for endocytosis of the virus into host insect cells[19,20]. It is presented on the surface of infected cells and on virions as a homotrimer, forming typical peplomer structures[21]. During the infection cycle, GP64 is abundantly expressed and transported to the cell surface to be incorporated into budding virions. When a foreign protein is fused at a suitable site to a copy of the entire coat protein GP64 or, alternatively, to just the membrane anchor of GP64, it is packaged into the viral coat and is present on the surface of baculovirus particles and infected insect cells. Thus, fusion to an endogenous viral glycoprotein provides the mechanism for the display of proteins on the virus and infected cell surface[22]. Therefore, GP64 is considered suitable as the basis for the display of fusion proteins and benefits the choice of baculovirus as a candidate for development as an eukaryotic display vector, which will be widely applied for problems in molecular biology, diagnostics and medicine in animals and humans.

The structure and function of many genes of baculovirus have been identified and sequenced[23–28]. The gp64 envelope glycoprotein genes of AcMNPV and OpMNPV, encoding 511 and 509 amino acid, respectively, with 78% homologues, have been mapped, cloned, and sequenced, too[11,12]. Although most baculovirus structural proteins are expressed as late genes, gp64 gene expression is regulated by a bi-phasic promoter that contains both immediate early and late promoter functions[12]. From the early promoter, GP64 is shed from infected cells early in infection before any progeny BV could be detected[20]. From the late promoter during infection, the expressed GP64 is transported to the cell surface continually to compensate its pickup by the assembling virus during the process of budding[22]. Its synthesis peaks at 8 and 24 hours post infection[11].

For investigation of promoter activities in baculovirus gp64 gene, in this work, two gp64 promoters of including 437 bp, from BmNPV, and 439 bp, of AcMNPV, of upstream of second ATG in gp64 long open reading frame (ORF), were cloned and sequenced, respectively. Furthermore, the second ATG was eliminated by site-directed mutagenesis to construct a non-fused reporter plasmid by using luciferase gene as the reporter gene that was under the control of gp64 promoter to investigate gp64 promoter transcriptional regulation in insect cells and/or silkworm larvae through transient expression assay system. In addition, the effects of transactivation of baculoviral factors and enhancement of BmNPV homologous region-3 (hr3) enhancer on the gp64 promoter transient transcriptional activity were examined.

1    Materials and Methods

1.1   Materials

The Bombyx mori cell line (Bm-N), Spodoptera frugiperda cell line (Sf-21), and hyperexpression variety of silkworm (JY1) were maintained in the Key Laboratory of Silkworm Biotechnology, Ministry of Agriculture, China. The wild-type BmNPV-ZJ8 and AcMNPV, plasmid pUL220[29] containing an entire luciferase gene with 3' polyadenylic acid (polyA), and plasmid pSK-hr3[30] containing BmNPV homologous region-3 enhancer were kindly provided by professor WU Xiang-Fu. The enzymes, reagents, and chemicals used throughout this work were obtained from Life Technologies (USA) and/or Sigma Chemical (USA), unless otherwise stated.

1.2   Cell culture

The cells were cultured with TC-100 medium supplemented with 10% fetal bovine serum and were incubated at 27 ℃ and subcultured every 3 – 5 days using a split ratio of 1:2 – 3. The details of cell culture were referred to Summers et al.[31].

1.3   Cloning and sequencing of upstream region of the second ATG in gp64 ORF

The polymerase chain reaction (PCR) was used to amplify the fragments of upstream region of the second ATG in gp64 ORF that had either early and late transcriptional initiation sites. Each genomic DNA was isolated from BmNPV-ZJ8 or AcMNPV as described by Summers et al.[31] and about 20 ng was used as template for standard PCR. Based on the BmNPV[32] and AcMNPV[33] gp64 ORF, two pairs of PCR primers were devised as:

Bmgp64, 5'-TTTCTAGATATTTAAATAAACCAAACACATG-3' (forward) and

                                           XbaI

5'-GCGGATCCAATCTCGCTTGTGTGTTTCTTA-3' (reverse);

BamHI

Acgp64, 5'-TTTCTAGATATTTAAATAAACCAAACACATG-3' (forward) and

                                        XbaI

5'-GCGGATCCAATCTTGCTTGTGTGTTCCTTA-3' (reverse).

                                     BamHI

After 30 PCR cycles with viral DNA and primers, the 437 bp and 439 bp reaction products, from BmNPV and AcMNPV, were purified on a 1% low gelling temperature Seaplaque agarose gel, recovered, and inserted into the XbaI/BamHI sites of pSK, respectively. The end products, plasmid pBmgp64 and pAcgp64, were then sequenced by T3 primer.

1.4   Plasmids construction

To investigate the transient transcriptional activity of baculovirus gp64 promoter, the reporter plasmids were generated. Briefly, the plasmid pUL220 containing an entire luciferase gene (Luc) (1.8 kb) with 3' polyadenylic acid (polyA) was digested with BamHI. The excised luciferase gene fragment was then subcloned into same sites of pBmgp64 and pAcgp64, respectively, under the control of the gp64 promoter in the right orientation. The generated reporter plasmids were named as pBmgp64Luc and pAcgp64Luc. To examine the effect of BmNPV hr3 on the transcription of gp64 promoter, furthermore, the plasmids, pBmgp64Luc-hr3 and pAcgp64Luc-hr3, were constructed by inserting hr3, excised from plasmid pSK-hr3, into the pBmgp64Luc and pAcgp64Luc under the downstream of luciferase gene, respectively. The plasmids construction, by using the methods described in Sambrook et al.[34], was devised as shown in Fig.1. For normalization of luciferase activity from each transfection, a control plasmid pHSP70LacZ was generated by which a fragment of about 3.7 kb containing the E.coli LacZ gene with simian virus 40 (SV40) polyadenylation signals under the control of HSP70 promoter was excised from pAcDZ1[35] at the XbaI and BamHI sites and then cloned into XbaI/BamHI-digested pSK.

1.5   Transfection in insect cells and/or fifth-instar silkworm larvae

For transfections, Bm-N or Sf-21 cells were seeded in each 15 cm2 flask at a density of about 5×105 cells/ml and cells were allowed to attach at 27 ℃ overnight. TC-100 medium was then replaced with 1 ml of serum-free medium, and 100 μl of transfection solution containing 6 μl of lipofectin and 1 μg of reporter plasmid DNA and 1 μg of control plasmid was added to each flask. Cells were incubated at 27 ℃ for 4 – 6 h before the supernatant was decanted and replaced with 3 ml of conditioned medium. Each treatment consisted of at least three separate transfections. Similarly, each fifth instar larva at 48 h post-molting was injected with 20 μl transfectional solution into the larval hemolymph containing 6 μl lipofectin and 1 μg reporter plasmid DNA and 1 μg control plasmid. Each treatment consisted of at least 3 separated groups (selecting 5 larvae with about the same weight as one group) and was repeated three times. At 48 h post transfection (hpt), the transfected Bm-N, Sf-21, and hemolymph cells in larvae were collected by centrifugation at 10 000 r/min for 5 min at 4 ℃ and ready for enzymatic activity assay.

1.6   Transient expression assay

The cell extracts were prepared with a luciferase assay kit (Promega). The harvested cells were washed twice by resuspended in phosphate buffered saline (PBS), then centrifuged at 5000 r/min for 4 min at 4 ℃. After washing, the cells were lysed by a single freeze-thawing cycle with the kit. The lysate was centrifuged at 4 ℃ to remove cell debris and supernatant on ice was ready for measurement. Measurements of luciferase activity[36] on three separate transfections were taken in triplicate using a liquid scintillation spectrometer (Beckman). The specific activity of E. coli β-galactosidase was assayed by the method described as Sambrook et al.[34] The LacZ reporter data from each extract were used to normalize the luciferase activity. The amount of protein in the lysate was measured using the Bradford method as described[37].

2    Results

2.1   Cloning and sequence analysis of baculovirus gp64 gene promoter

There are two in-frame ATGs in the 5' region of the BmNPV or AcMNPV gp64 gene at positions +1 and +55. Using in vitro transcription-translation assay, Jarvis et al.[20] found that the downstream ATG at position +55 serves as the translational initiation codon in the AcMNPV gp64 long ORF, as predicted by Kozak’s rules, and that downstream sequences encode a functional signal peptide. Based on the reported complete nucleotide sequence of BmNPV T3 strain[32,38] and AcMNPV C6 strain[33], we devised two pairs of primers in which each second ATG was changed to ATT by site-directed mutagenesis for the construction of non-fused reporter plasmids. Making BmNPV-ZJ8 or AcMNPV as template, two fragments of upstream region of the second ATG in gp64 ORF, with 437 bp from BmNPV-ZJ8 and 439 bp from AcMNPV, were amplified by PCR. These products were purified, recovered, and inserted into the XbaI/BamHI sites of pSK, and named as pBmgp64 and pAcgp64, respectively.

The sequence analysis showed that the produced gp64 promoter region from BmNPV-ZJ8 or AcMNPV has the same promoter sequence in individual corresponding region compared with the reported genomes of BmNPV T3 and AcMNPV C6 strain. In the AcMNPV gp64 promoter region, there exist two ATGs at +1 and +55 site. The early transcription start site, CAGT at +16 site, is located downstream of two late transcription start sites T/ATAAG. The TATA box is located at –16 site. A small minicistron at –8 site with an ATG in keeping with the Kozak’s rules is located on late transcripts, but not on early transcripts. Different from AcMNPV gp64 promoter partially, the late transcription start site ATAAG at –73 site is changed to ATAGA with a difference of two nucleotides in BmNPV gp64 promoter region. Two nucleotides of A at –35 site and T at –90 site are vacated.

2.2   Transient expression of reporter plasmids in uninfected insect cells 

To determine whether these reporter plasmids, pBmgp64Luc and pAcgp64Luc, generated by inserting luciferase gene fragment with 3' polyA excised from pUL220[29] at BamHI into the same sites of pBmgp64 and pAcgp64, respectively, under the control of gp64 gene promoter, are functional in uninfected insect cells, two plasmids were transfected into Bm-N or Sf-21 cell lines mediated by lipofectin separately. At 48 h post transfection (hpt), the transfected cells were collected and the transient expression activity of luciferase was measured, and the data were shown in Table 1.

 

Table 1   Transient expression activity of luciferase in uninfected insect cells

Plasmids

Luciferase activity (cpm)

(in Bm-N cells)

Luciferase activity (cpm)

(in Sf-21 cells)

pBmgp64Luc

45 877.0 ± 5 168.9

316.0 ± 43.3

pAcgp64Luc

12 274.0 ± 2 872.6

1 206.0 ± 85.2

Luciferase activity is indicated as counts per minute (cpm) in 15 s. pBmgp64Luc or pAcgp64Luc was transfected into Bm-N and Sf-21 cells mediated by lipofectin separately. The β-gal normalizing system was introduced into each transfection. Each reaction contained 20 μg of protein extracted from the uninfected cells. The results represented averages from three separate transfections at 48 h post transfection.

 

From Table 1, the luciferase activity was 45 877.0 cpm in permissive Bm-N cells transfected by pBmgp64Luc. However, in the non-permissive Sf-21 cells transfected with the same plasmid, a very lower level of luciferase activity, 316.0 cpm only, was detected. It indicated that transcription from BmNPV gp64 early promoter is recognized by host RNA polymerase II and requires no other viral gene products and is limited to the host in which the BmNPV virus normally replicates. Interestingly, a reverse trend appeared by using pAcgp64Luc for transfection in two cell lines. In non-permissive Bm-N cells transfected with pAcgp64Luc, the enzymatic activity appeared in a higher level of 12 274.0 cpm while in a relative lower level of 1206.0 cpm in permissive Sf-21 cells.

2.3   Transcriptional transactivation of baculovirus gp64 promoter by viral factors

Because some baculovirus transcriptional transactivators, such as immediate early gene products like IE-1, a key factor in baculovirus cascade regulation, influence the regulation of some baculovirus early genes and are required for replication of viral DNA[39–43], we examined the effect of the baculovirus factors on the transcription from both gp64 promoters in insect cells. For this study, cells were transfected with each plasmid in individuals for 4 h then treated with BmNPV-ZJ8 in Bm-N cells or AcMNPV in Sf-21 cells at a MOI of 0.5 for another 1 h. At 48 hpt, the transfected cells were gathered and the enzymatic activity of luciferase was measured. The data from LacZ were normalized to luciferase activity.

As shown in Table 2(a), in permissive Bm-N cells, transfection with pBmgp64Luc alone gave a 51 618.4 cpm of luciferase activity. By treatment with BmNPV-ZJ8 after transfection with this plasmid, the enzymatic activity increased to 121 468.0 cpm with 2.35-fold enhancement. Similarly, treatment with AcMNPV gave about 4-fold increase in enzymatic activity over the pAcgp64Luc transfection alone in permissive Sf-21 cells as shown in Table 2(b). It suggested that the viral factors transactivated the transcription of baculovirus gp64 promoter in transfected permissive cells.

 

Table 2   Augmented luciferase activity by viral factors

(a)  Augmented luciferase activity from BmNPV gp64 promoter by BmNPV-ZJ8 factors in permissive Bm-N cells

Luciferase activity (cpm)

Stimulating folds

pBmgp64Luc alone

51 618.4 ± 13 596.2

1.00 ± 0.26

pBmgp64Luc+BmNPV

121 468.0 ± 29 559.4

2.35 ± 0.57

(b)  Augmented luciferase activity from AcMNPV gp64 promoterby AcMPNV factors in permissive Sf-21 cells

Luciferase activity (cpm)

Stimulating folds

pAcgp64Luc alone

1 453.0 ± 242.1

1.00 ± 0.17

pAcgp64Luc+AcMNPV

5 771.69 ± 1 247.6

3.97 ± 0.86

Luciferase activity is indicated as cpm in 15 s. Transactivating ability of viruses is presented as stimulating folds over each corresponding plasmid transfection alone that is arbitrarily set as 1.00. Each reaction contained 20 μg of protein extracted from the transfected cells. The β-gal normalizing system was introduced into each transfection. The results represented averages from three separate transfections at 48 h post transfection. (a) pBmgp64Luc was transfected into permissive Bm-N cells and then treated with BmNPV-ZJ8. (b) pAcgp64Luc was transfected into permissive Sf-21 cells and then treated with AcMNPV.

 

2.4   Enhancement of transcriptional activity of baculovirus gp64 promoter by BmNPV hr3

Baculovirus homologous region (hr) functions as both viral DNA origin and an enhancer[44]. To examine the effect of BmNPV hr3 on the transcriptional activity of gp64 promoter, it was subcloned into the downstream site of luciferase gene under the control of BmNPV or AcMNPV gp64 promoter, respectively. Diagrammatic representation of construction, named as pBmgp64Luc-hr3 and pAcgp64Luc-hr3, respectively, was devised as shown in Fig.1.

The cells were co-transfected with 1 μg control plasmid pHSP70LacZ and 1 μg plasmid with or without hr3, respectively, mediated by lipofectin. At 48 hpt, the transfected cells were collected and the enzymatic activity was assayed. The data from LacZ were used to normalize each luciferase activity.

From Table 3(a), the luciferase activity was 41 897.4 cpm in transfected Bm-N cells with pBmgp64Luc. When the Bm-N cells were transfected with pBmgp64Luc-hr3 that contained BmNPV hr3, the enzymatic activity increased to 925 031.0 cpm with about 22-fold enhancement. Surprisingly, in non-permissive Sf-21 cells transfected with the same plasmids, the enzymatic activity from pBmgp64Luc-hr3 was as high as 5 062 105 cpm with 14 292.46-fold enhancement over that from pBmgp64Luc in which the enzymatic activity was only 354.2 cpm as shown in Table 3(b). Similar trends were showed when Bm-N or Sf-21 cells were tranfected with pAcgp64Luc and pAcgp64Luc-hr3, respectively. In transfected Bm-N cells, the luciferase activity from pAcgp64Luc-hr3 was enhanced by 12.7 times over that from pAcgp64Luc as shown in Table 3(a). In transfected Sf-21 cells, the enzymatic activity from pAcgp64Luc-hr3 was enhanced by 7367.2 times compared with that from pAcgp64Luc as shown in Table 3(b).

 

Fig.1       Diagrammatic representation of the pBmgp64Luc-hr3/pAcgp64Luc-hr3 constructs used in transient expression assay

 

Table 3   Enhancement of transcriptional activity of baculovirus gp64 promoter by BmNPV hr3

(a)   Augmented luciferase activity from BmNPV gp64 promoter by BmNPV hr3 in transfected Bm-N cells

Plasmids

Luciferase activity (cpm)

Stimulating folds

pBmgp64Luc

41 897.4 ± 10 103.0

1.00 ± 0.24

pBmgp64Luc-hr3

925 031.0 ± 95 367.2

22.08 ± 2.28

pAcgp64Luc

9 766.7 ± 1 719.2

1.00 ± 0.17

pAcgp64Luc-hr3

124 185.3 ± 18 752.2

12.72 ± 1.92

(b)  Augmented luciferase activity from AcMNPV gp64 promoter by BmNPV hr3 in transfected Sf-21 cells

Plasmids

Luciferase activity (cpm)

Stimulating folds

pBmgp64Luc

354.2 ± 37.6

1.00 ± 0.11

pBmgp64Luc-hr3

(506 210.5 ± 72 372.0) ´10

14 292.46 ± 2 043.37

pAcgp64Luc

1 148.0 ± 90.5

1.00 ± 0.08

pAcgp64Luc-hr3

(845 753.2 ± 59 008.1) ´10

7 367.2 ± 514.0

Luciferase activity is presented as cpm in 15 seconds. Enhancing ability of BmNPV hr3 inserted in each plasmid is presented as stimulating folds over the corresponding plasmid with no BmNPV hr3 that is arbitrarily set as 1.00. The β-gal normalizing system was introduced into each transfection. The results represented averages from three separate transfections at 48 h post transfection. (a) Transfection of each plasmid containing hr3 compared with that of the corresponding plasmid with no hr3 in transfected Bm-N cells. Each reaction contained 20 μg of protein extracted from transfected Bm-N cells. (b) Transfection of each plasmid containing hr3 compared with that of the corresponding plasmid with no hr3 in transfected Sf-21 cells. Each reaction contained 20 μg of protein, for plasmids with no hr3, or 2 μg of protein for plasmids with hr3, extracted from transfected Sf-21 cells.

 

BmNPV hr3 significantly enhanced the transcriptional activity from either BmNPV or AcMNPV gp64 promoter in two cell lines, especially in transfected Sf-21 cells.

2.5   Transcriptional co-activation of baculovirus gp64 promoter by BmNPV hr3 and viral factors

The baculovirus factors appeared to transactivate the transcriptional activity of gp64 promoters. To investigate whether they could co-activate the transcription of gp64 promoters together with BmNPV hr3, the Bm-N/Sf-21 cells were transfected with plasmids pBmgp64Luc-hr3/pAcgp64Luc-hr3 containing BmNPV hr3 for 4 h then treated with BmNPV/AcMNPV (MOI=0.5) for 1 h, parallelly. At 48 hpt, the transfected cells were collected and enzymatic activity was measured.

As shown in Fig.2, the BmNPV treatment in permissive Bm-N cells increased expression level of luciferase from pBmgp64Luc-hr3 by 72.7 times. By parallel operation, the AcMNPV treatment in permissive Sf-21 cells increased enzymatic activity from pAcgp64Luc-hr3 by 78.22 times. It suggested that baculovirus factors transactivated transcriptional activity of gp64 promoter significantly in the presence of BmNPV hr3.

 

Fig.2       Augmented luciferase activity from baculovirus gp64 promoter containing BmNPV hr3 by viral factors in insect cells

1, pBmgp64Luc-hr3 alone； 2, pBmgp64Luc-hr3+BmNPV； 3, pAcgp64Luc-hr3 alone； 4, pAcgp64Luc-hr3 + AcMNPV. The increase of luciferase activity is indicated on the Y axis as stimulating folds over the transfection in the absence of virus which was arbitrarily set at 1.00. Parallel operation is presented on X axis in Bm-N cell line transfected with pBmgp64Luc-hr3 and treated with BmNPV and in Sf-21 cell line transfected with pAcgp64Luc-hr3 and treated with AcMNPV. The β-gal normalizing system was introduced into each transfection. Each reaction contained 2 μg of protein extracted from transfected cells. The results represented averages from three separate transfections at 48 h post transfection.

 

Because the in vivo transfection conditions greatly differ from that in transfected cells and the plasmids containing hr3 are functional in transfected silkworm larvae[45], we made the fifth-instar silkworm larvae at 48 h post-molting to be transfected with pBmgp64Luc-hr3 by injection with 20 μl transfection solution containing plasmids and lipofectin. Four hours later, the BmNPV-ZJ8 was injected into the larval hemolymph to observe the in vivo transactivation effect of viral factors on the transcription of gp64 promoter in the presence of BmNPV hr3. At 48 hpt, the hemolymph cells were collected by centrifugation and the enzymatic activity was assayed. The data from LacZ were used to normalize each luciferase activity.

From Table 4, the trend of in vivo co-activational effect of viral factors with hr3 was similar to that of in vitro. The pBmgp64Luc-hr3 transfection alone gave a basal expression level of 6932 cpm. In the presence of BmNPV, the enzymatic activity increased to 443 541.3 cpm. The viral factors in vivo co-activated the transcriptional activity of gp64 promoter by 64 times with BmNPV hr3.

 

Table 4   Augmented luciferase activity from BmNPV gp64 promoter containing BmNPV hr3 by BmNPV-ZJ8 in permissive silkworm larvae

 

Luciferase activity (cpm)

Stimulating folds

pBmgp64Luc-hr3 alone

6932.0±1134.3

1.00±0.16

pBmgp64Luc-hr3+BmNPV

443 541.3±132 249.6

64.00±19.08

Each fifth-instar larva at 48 h post-molting was injected with pBmgp64Luc-hr3 and control plasmid mediated by lipofectin for transfection. Four hours later, 5 μl solution containing about 1.0×105 pfu viruses diluted in serum-free medium was injected for 1 h. The β-gal normalizing system was introduced into each transfection. Transactivating ability of virus is presented as stimulating folds over pBmgp64Luc-hr3 transfection alone that is arbitrarily set as 1.00. Each reaction contained 20 μg of protein extracted from larval hemolymph cells. The results represented averages from three separate treatments. Each treatment consisted of three separate groups (selecting 5 larvae with about the same weight as one group).

 

3    Discussion

GP64, a baculovirus major envelope glycoprotein, plays an important role in viral infection, mediating penetration of BV into host cells by endocytic pathway. Previous work[20] has shown that GP64 was synthesized in a biphasic fashion, with peaks at 8 and 24 h postinfection (hpi) in both the intracellular and extracellular fractions, and its expression was regulated by transcription from both early and late promoters. Early transcription of AcMNPV gp64 gene initiated around the CAGT motif and peaked at 4 hpi. After the onset of DNA replication, the late transcription initiated mainly around the distal ATAAG motif (position –73) and peaked after 12 hpi. However, there are few reports on the BmNPV gp64 promoter activity. In this study, the constructed pBmgp64Luc from BmNPV gp64 promoter had high expression levels in permissive Bm-N cell line and low levels in non-permissive Sf-21 cell line in the absence of viral factors, indicating that transcription from BmNPV gp64 promoter was recognized by permissive host RNA polymerase and regulated by early promoter. While the plasmid pAcgp64Luc from AcMNPV gp64 promoter had relatively high expression levels in both permissive Sf-21 cells and non-permissive Bm-N cells, indicating that the transcription from AcMNPV gp64 promoter was recognized by either Sf-21 or Bm-N cell RNA polymerase and regulated by early promoter and required no viral factors. It is unclear why the transcriptional activity of AcMNPV gp64 promoter in non-permissive Bm-N cell line was higher than that in permissive Sf-21 cells (Table 1). By treatment with virus after transfection, the luciferase activity accumulated in cells and reached the maximum at 48 – 60 hpt (data not shown), indicating that gp64 transcription was also regulated by late promoter. Compared with the late core start motif ATAAG at -73 site of AcMNPV gp64 promoter region, the one of BmNPV gp64 promoter region at this position is changed as ATAGA with two nucleotides difference. Therefore, it is presumed that the late transcript initiates farther upstream, mainly around the late core motif TTAAG at –86 site.

Baculoviruses replicate and are transcribed in the nuclei of infected cells. During the infection, viral genes are expressed in a coordinately regulated cascade fashion and divided into immediate early, delayed-early, late, and very late phases. The regulation of these phases appears to be determined primarily at the level of transcription. Early genes are transcribed by host RNA polymerase II prior to viral DNA replication. Late genes are transcribed by a virus-induced RNA polymerase activity that appears to be encoded mostly by viral genes[40,46,47]. The transcription from some early promoters, such as, derived from baculovirus 39K, p26, gp64, and DNA polymerase genes, was found to be transactivated by 3-10 times by the nuclear extracts prepared from the AcMNPV-infected Spodoptera frugiperda cells[48]. Xiao et al.[49] analyzed BmNPV helicase gene promoter functions and found that BmNPV factors could transactivate the transcription of helicase gene promoter by 12 – 20 times in transfected Bm-5 or Bm-N cells[45]. In this work, the transcription from BmNPV gp64 promoter was found to be transactivated by the BmNPV factors by about 2.4 times and the one from AcMNPV gp64 promoter was transactivated by 4 times by AcMNPV (Table 2).

It has been confirmed that homologous region 3 (hr3) from BmNPV-ZJ8 has the same functions as AcMNPV hr3 both as viral DNA origin and as an enhancer[30,44]. We placed the BmNPV hr3 downstream of luciferase gene driven by gp64 promoter and found that in permissive Bm-N cells, the hr3 enhanced transcription of BmNPV gp64 promoter by about 22 times. Surprisingly, in non-permissive Sf-21 cells transfected with the same plasmids, hr3 enhanced transcription from this promoter by over 14 000 times because of a very low transcriptional level of gp64 promoter containing no hr3 (Table 3). BmNPV hr3 seemed to be recognized by non-permissive Sf-21 RNA polymerase and gave a significant enhancement to BmNPV gp64 promoter in non-permissive Sf-21 cells. A reverse trend appeared for enhancement of transcription from AcMNPV gp64 promoter by BmNPV hr3. In non-permissive Bm-N cells, BmNPV hr3 enhanced transcriptional activity of AcMNPV gp64 promoter by about 13 times, while in permissive Sf-21 cells, it enhanced transcription from this promoter by over 7000 times (Table 3). The reason of which needs addressing further.

From absolute cpm value, the transient transcriptional activities of gp64 promoter from either BmNPV or AcMNPV in plasmid-transfected Bm-N cell line, with no BmNPV hr3 inserting, were higher than that in transfected Sf-21 cell line with the same plasmids, respectively (Table 1). A reverse trend appeared when these two plasmids, inserted with hr3, were transfected into these two cell lines. The enzymatic activities in transfected Sf-21 cell line were significantly higher than that in Bm-N cell line, respectively (Table 3). In transfected Sf-21 cell line, the BmNPV hr3 enhanced transcriptional activities of gp64 promoter over 7000-14 000 times, which were greatly higher than the enhancing ability of 12.7-22 times in transfected Bm-N cell line. It suggested that the enhancing behavior of BmNPV hr3 might be related by different cell line factors.

At the same time, the transcription from gp64 promoter with BmNPV hr3 was found to be transactivated by viral factors to a great extend with enhancement of about 73 times in permissive Bm-N cells with pBmgp64Luc-hr3 by BmNPV or about 78 times in permissive Sf-21 cells with pAcgp64Luc-hr3 by AcMNPV (Fig. 2). It implies that BmNPV hr3 plays an important role in co-activation with viral factors in addition to the functions of viral DNA origin and enhancer. This was supported by the transactivation (64 times) of BmNPV to transcription from BmNPV gp64 promoter containing hr3 in the fifth-instar silkworm larvae transfected with pBmgp64Luc-hr3 (Table 4).

This work may, thus, provide some information to understand baculovirus gp64 expression and regulation deep. It will be useful for gp64 promoter based-constructs, for example, the improvement of baculovirus surface display system for problems in molecular biology, diagnostics and medicine, which is widely used in gene delivery, antigen presentation, functional genomics, generation of cDNA libraries, etc.

 

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Received: July 11, 2002      Accepted: August 27, 2002
This work was supported by grants from the National Natural Science Foundation of China (No.39970571) and the National High Technology Research and Development Program of China (863 Program) (No.102-11-02-06)
￥He is studying for his doctoral degree in East China University of Science and Technology
*Corresponding author: Tel, 86-511-5616659; Fax, 86-511-5615044; e-mail, [email protected]