https://www.abbs.info e-mail:[email protected] 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-Jing^1,2￥, 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

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

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

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).