| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan
2 Department of Life Sciences, National Chung Hsing University, Taichung 402, Taiwan
Correspondence
Yu Chuan Chao
mbycchao{at}gate.sinica.edu.tw
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Supplementary tables are available with the online version of this paper.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the most characterized member of the family Baculoviridae, is a model virus routinely used for foreign protein expression in insect cells. In recent years, there has been increasing interest in its use as a vehicle for delivering foreign genes into mammalian cells for gene expression or gene therapy (Boyce & Bucher, 1996; Hofmann et al., 1995; Hu et al., 2003; Huser & Hofmann, 2003; Huser et al., 2001; Shoji et al., 1997), or as an antigen-presenting, immune-stimulating particle (Abe et al., 2005, 2003; Chang et al., 2004). These wide-ranging potential applications of baculovirus in mammalian systems provide an incentive for an in-depth study of its gene regulation pathways in mammalian cells.
The 133 kb double-stranded, circular DNA genome of AcMNPV has been completely sequenced (Ayres et al., 1994) and is predicted to contain 155 open reading frames (ORFs). In insect cells, these genes are expressed in three temporally ordered phases: early, late and very late. The early genes are further divided into two subclasses: the immediate-early (ie) type and the delayed-early type genes. The ie genes require no viral factor for activation and are believed to govern the instigation of the baculovirus life cycle within host cells. Two of these genes are ie1 and ie2, the major trans-activators of baculovirus. Both of them are involved in the activation of late expression factor genes, which are necessary for virus propagation (Lu & Miller, 1995).
Extensive studies of genes ie1 and ie2 and their protein products (IE1 and IE2) have been carried out in insect cells. Mutational and deletion studies of ie1 and ie2 genes have previously been used to study their domain functions, and their effects on viral viability and infectivity (Kovacs et al., 1992; Prikhod'ko et al., 1999; Yoo & Guarino, 1994). The IE1 protein trans-activates several viral promoters, including promoters of the genes 39K (Guarino & Summers, 1986), p35 (Nissen & Friesen, 1989), gp64 (Blissard & Rohrmann, 1991), p143 (Lu & Carstens, 1993) and he65 (Murges et al., 1997). Some of these promoters are greatly enhanced by being cis-linked to palindromic sequences from AcMNPV homologous repeat (hr) regions (Leisy et al., 1995), which contain IE1-binding sites (Choi & Guarino, 1995). The IE2 protein has been shown to trans-activate viral promoters of ie1, ie0 and ie2 genes (Carson et al., 1991; Prikhod'ko et al., 1999). It also augments IE1 activation of the promoter of 39K (Carson et al., 1988).
In transient expression studies, IE1 protein was shown to activate baculoviral early promoter of he65 in BHK-21 cells (Murges et al., 1997). In another study, the acidic activation domain of AcMNPV IE1 protein was shown to be active in both NIH-3T3 and CHO cells (Dai et al., 2004). When whole baculoviruses were transduced into mammalian cells, ie1 gene transcript was detected in AcMNPV-transduced HeLa14 cells on an AcMNPV DNA microarray (Fujita et al., 2006), and in Bombyx mori NPV-transduced BEK293 cells and rat primary Schwann cells by RT-PCR (Kenoutis et al., 2006). The presence and functionality of IE1 in these mammalian cells convinced us to explore transcriptional pathways of baculovirus genes in mammalian cells.
In this study, we designed a novel approach to examine the activation of the AcMNPV transcriptome by IE1 and IE2 in a mammalian system. Firstly, we discovered that Vero E6 cells are ideal for this type of study because of their high sensitivity to baculovirus transduction and extremely low levels of endogenous baculoviral gene expression. Secondly, proper ie gene expression was achieved in Vero E6 cells by using the cytomegalovirus immediate-early (CMVie) promoter to drive the baculoviral genes, which were delivered into the cells by recombinant baculoviruses. Lastly, the IE1- or IE2-activated AcMNPV transcriptome within the transduced Vero E6 cells was analysed using an AcMNPV DNA microarray. Using this method, we have identified novel sets of baculoviral genes activated by IE1 or IE2 in a mammalian system. More importantly, we also discovered that these two ie trans-activators had a synergistic effect on AcMNPV genome activation. This is the first study to draw a clear correlation between baculoviral trans-activators and AcMNPV genome activation in a mammalian system.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Lo et al., 2002) (Fig. 1a).
|
) promoters was inserted into the XhoI site of the modified TriEx plasmid to make pAscmR (Fig. 2a). Baculoviruses containing this reporter cassette were able to express DsR2 protein in both mammalian (SV40) and insect cells (CMVm); thus, this simplified the process of single clone purification in insect cells and it doubled as a visual marker for transduction in mammalian cells. Both ie1 and ie2 gene fragments were amplified as above and placed under the CMVie promoter of pAscmR, to make pAcIE1scmR and pAcIE2scmR (Fig. 2a). All plasmid constructions were confirmed by DNA sequencing. PCR primers for generating the reporter cassette are also listed in Supplementary Table S1 (available in JGV Online).
|
. Briefly, the expression vectors shown in Fig. 2(a)
were co-transfected with vAcRP23.Laz (PharMingen), a linearized viral DNA of AcMNPV, into Sf21 cells using Cellfectin (Life Technologies). The resulting baculoviruses were termed vAscmR, vAcIE1scmR and vAcIE2scmR. Homologous recombination occurs at the site of the polyhedrin gene, and produces a baculovirus lacking this gene. Recombinant baculoviruses were isolated through end-point dilutions using DsR2 as the marker, and their titres were determined by quantitative PCR (Lo & Chao, 2004).
Mammalian cell culture and viral transduction conditions.
African green monkey kidney epithelial Vero E6 cells were cultured in the presence of 10 % FBS, 100 U penicillin ml–1 and 100 µg streptomycin ml–1 in minimum essential medium (MEM; Gibco) at 37 °C and 5 % CO2. Baculoviruses were transduced into Vero E6 cells at an m.o.i. equal to 10. Transduced cells were harvested at various time points by trypsin digestion, and total RNA was extracted using the Qiagen RNeasy mini kit. The quantity and quality of the RNA were checked by a NanoDrop ND-1000 Spectrophotometer (J & H Technology).
Construction of AcMNPV cDNA microarray.
All of the 154 ORFs of AcMNPV were amplified by PCR, purified using the QIAquick PCR purification kit (Qiagen) and spotted in duplicate onto GAPS-2 coated slides (Corning). Most of the PCR amplified products were 300–500 bp in length. A set of 10 artificial cDNA fragments from the Stratagene SpotReport Alien cDNA Validation System were also spotted onto glass slides as controls (Stratagene), together with the corresponding exogenous RNA spiked into the labelling reaction; these control spots were used to normalize both dye incorporation efficiencies and signal intensities between different arrays. After spotting, the glass slides were UV-cross-linked at 300 mJ cm–2 and blocked for 20 min in 1.55 % succinic anhydride and 84.5 mM sodium borate in N-methyl-2-pyrrolidone.
Hybridization of AcMNPV transcriptome.
Recombinant baculoviruses vAscmR (reporter-only control), vAcIE1scmR and vAcIE2scmR were transduced into Vero E6 cells at an m.o.i. of 10. After 48 h, total RNAs from these transduced cells were examined by the AcMNPV cDNA microarray. Total RNA (20 µg) from these cells was reverse-transcribed using SuperScript II (Invitrogen). Probe synthesis, purification and microarray washing were conducted as described previously (Zheng et al., 2004). We used two-colour microarrays, where the total cDNA from vAscmR and vAcIE1scmR (or vAcIE2scmR) transductions were labelled with cy3 and cy5 dyes, respectively. The cy5- and cy3-labelled cDNAs were mixed and applied onto a single AcMNPV microarray block. The microarray was hybridized with a MAUI hybridization system (BioMicro Systems), and then examined using an Axon 4000B scanner (AronInstrument). The levels of total AcMNPV transcripts from both cy5- and cy3-labelled samples were analysed by Axon GenePix Pro 6.0 software. The numerical data obtained were normalized by comparison to the signal intensities of the spike cDNA controls (Stratagene).
Construction of luciferase plasmids.
Reporter plasmids (pxL where x is a baculoviral promoter) were constructed using the promoter-less pGL3-basic luciferase vector (Promega) as the backbone. Promoter region of genes: ie1, ie2, pe38, gp64, 39K, he65, orf16, orf17, orf25 and pcna (up to 500 bp) were amplified by PCR and cloned into the pGL3 vector. Primer sequences used to amplify each promoter are given in Supplementary Table S1 (available in JGV Online).
Transfection of Vero E6 cells.
Cells (1x104) were seeded in each well of a 96-well plate, and the plate was incubated with 5 % CO2 at 37 °C 24 h prior to the experiment. Transfection was performed using Lipofectamin 2000 as the transfection reagent, following the protocol from the manufacturer (Invitrogen). For each well, 30 ng reporter plasmid (pxL) DNA was mixed with 70 ng effector plasmids (pAcscmR, pAcIE1scmR or pAcIE2scmR) in 10 µl serum-free MEM. A volume of 0.3 µl Lipofectamin 2000 was diluted in 20 µl serum-free MEM and then incubated at room temperature for 5 min, before being combined with the diluted DNA and incubated at room temperature for 20 min. A volume of 30 µl serum-free MEM was then added to each sample before the samples were added to the Vero E6 cells. Cells were incubated at 37 °C with 5 % CO2 for 4 h before changing the medium to 10 % FBS/2 % antibiotic MEM. Cells were incubated further at 37 °C with 5 % CO2 for another 48 h before their luciferase activity was measured.
Luciferase assay.
After the serum-containing medium was removed, transfected cells in a 96-well plate were washed with Dulbecco's PBS (Invitrogen), then lysed with 100 µl culture cell lysis reagent [100 mM potassium phosphate (pH 7.8), 1 mM EDTA, 10 % glycerol, 1 % Triton X-100 and 2 mM 
-mercaptoethanol]. The plate was placed on a shaker at 200 r.p.m. for 20 min at 4 °C to complete cell lysis. Cell lysates were transferred into a 96-well conical-based Nunc MicroWell plate and centrifuged at 1885 g for 30 min at 4 °C. After centrifugation, 20 µl supernatant from each well was combined with 180 µl luciferase activity reagent [25 mM Tricine (pH 7.8), 15 mM potassium phosphate (pH 7.8), 15 mM MgSO4, 4 mM EGTA, 1 mM ATP and 0.1 mM dithiothreitol] in a luciferase assay plate. Luciferase activity was measured on a luminometer (Lumat LB 9501; Berthold) by injecting 50 µl 0.2 mM luciferin (Promega) into each well. The protein concentration of each well was measured using a Coomassie protein assay kit (Pierce).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Unlike the full-length CMVie promoter with enhancer element, the CMV minimal (CMVm) promoter, containing the sequence between –53 and +75 region of CMVie, is not active in mammalian cells (Gossen & Bujard, 1992) and requires the hr viral fragment for effective activation in Sf21 insect cells (Lo et al., 2002). We hypothesized that this activation is IE1-dependent, as hr contains IE1-binding motifs (Leisy et al., 1995). The pAcE-positive control expressed enhanced green fluorescent protein (EGFP) with or without IE1 protein. The pApE-negative control showed that the promoter of polyhedrin was not active in mammalian cells as reported previously (Fujita et al., 2006; Kenoutis et al., 2006). As predicted, the hr-CMVm promoter was clearly dependent on IE1 for trans-activation (Fig. 1b). This result was further confirmed when the two recombinant baculoviruses vAhcmE (made from plasmid pAhcmE) and vAcIE1scmR were co-transduced into Vero E6 cells (Fig. 2b). The former virus expresses EGFP by the hr-CMVm promoter; and the latter virus expresses both IE1 and DsR2 proteins by CMVie and SV40 promoters, respectively. Using an m.o.i. of 10 for each virus, a large proportion of cells were transduced by both viruses. Fig. 2(b) clearly shows that the green fluorescence from the egfp gene of vAhcmE was only present in cells also emitting red fluorescence (from expression of the dsR2 gene from vAcIE1scmR). This not only reconfirmed that hr-CMVm promoter requires IE1 for activation, but also established that baculovirus vAcIE1scmR can effectively express functional IE1 protein in Vero E6 cells. As the transduced virus contains a complete set of AcMNPV genes (apart from the polyhedrin gene), we hypothesized that upon the expression of IE1 there will be transcripts of IE1-responsive genes from AcMNPV genome within the transduced mammalian cells.
Hybridization of baculovirus transcripts onto an AcMNPV microarray
To observe the activation of AcMNPV transcriptome by IE1 and another early trans-activator, IE2, within Vero E6 cells, we hybridized total cellular cDNAs from vAcIE1scmR- and vAcIE2scmR (cDNAs labelled with cy5)-transduced Vero E6 cells onto an in-house AcMNPV DNA microarray, together with a wild-type baculovirus control, vAscmR (cDNAs labelled with cy3). No significant level of green cy3-labelled AcMNPV transcript was seen; the only spots were from labelled positive control DNA (Fig. 3a). In contrast, within vAcIE1scmR- or vAcIE2scmR-transduced Vero E6 cells, several viral genes were clearly activated because of IE1 or IE2 trans-activation (Fig. 3b, c). The activation effects of IE1 and IE2 were synergistically enhanced when both vAcIE1scmR and vAcIE2scmR viruses were co-transduced into the same pool of Vero E6 cells (Fig. 3d). This result indicates that some AcMNPV genes require the cooperative effects of both IE1 and IE2 trans-activators for transcription to commence.
|
). On the AcMNPV microarray, the highest value for negative control spots was 127±16 fluorescence units (F.U.); therefore, we chose a cy3 or cy5 with an F.U. of 180 as a lowered cut-off point for positive results. At 48 h post-transduction, we found that AcMNPV genes gp64, pe38, ie2, he65, orf16 and orf17 were significantly transcribed in the vAcIE1scmR-transduced sample, but no transcription of these genes was observed in the vAscmR control (Fig. 3a). One surprising finding was that the level of early and late viral surface glycoprotein gp64 gene expression was extremely high, surpassing the expression level of the ie1 gene, which was driven by mammalian CMVie promoter.
To obtain the onset time and expression profile of these genes that were activated by IE1, we performed time-course studies of IE1-activated AcMNPV transcriptomes in Vero E6 cells. The total RNA from vAcIE1scmR-transduced cells was harvested at 4, 12, 24, 48, 72 and 96 h post-transduction. These samples were analysed with AcMNPV microarrays, and profiles of genes activated by IE1, including pe38, ie2, he65, orf16, orf17, orf25 and pcna, are shown in Fig. 4(a). The surprisingly strong strength of the promoter of gp64 in Vero E6 cells was compared with the CMVie-driven ie1 gene as shown in Fig. 4(b). The levels of gp64 transcript were higher than that of ie1 at 48, 72 and 96 h post-transduction. This result was double checked by Western blot analysis of total protein from transduced Vero E6 cells, probed with anti-gp64 antibody. In both the 48 and 72 h samples, a clear gp64 protein band was seen in vAcIE1scmR-transduced Vero E6 cells (Fig. 4c). At the earlier time points, either the gp64 transcripts had not been translated, or the amount of gp64 protein was below the level of detection. At the 96 h time point, the transduced cells were inactive due to either virus burden and/or confluence; the cellular translation mechanism was most likely not functional.
|
), their promoters activated by IE2 protein.
Surprisingly, when both vAcIE1scmR and vAcIE2scmR were used to transduce Vero E6 cells, the combined effects of the two ie genes were far greater than that of each individual gene. Around 38 % of the AcMNPV genome was clearly detectable on the IE1+IE2 microarray (>180 F.U.) (Figs 3d, 5); the most intense gene spots (>1000 F.U.) were gp64, orf25, orf17, he65, pe38, lef3, 39K, pcna and orf16. The values of all 59 activated genes are listed in Supplementary Table S2 (available in JGV Online). All the high level transcripts were from early genes or early and late genes, classified by their promoter sequences (Ayres et al., 1994). Some of these genes, he65, 39K and gp64, had been identified as IE1-activated genes by transient assays in insect cells (Blissard & Rohrmann, 1991; Guarino & Summers, 1986; Murges et al., 1997). Interestingly, it is known that the 39K gene requires both IE1 and IE2 activators for efficient activation in insect cells (Carson et al., 1988). In Vero E6 cells, the level of 39K transcript was found to be barely detectable on the IE1 microarray (63 F.U., 48 h post-transduction); however, it became highly abundant on the IE1+IE2 array (1485 F.U., 48 h post-transduction). The activation ratios of the combined IE1+IE2 trans-activators compared with IE1-only for he65, orf17, gp64, 39k, lef3, orf25 and pcna genes are shown in Fig. 6.
|
|
). This element was found to exist both within the AcMNPV hr region, as well as the promoter regions of ie0, ie2 and pe38. Of the more highly activated IE1 and IE2 genes (>1000 F.U.), the element ACTTGTAA with one allowed mismatch was found in the 5' UTR of pe38, he65, orf16, ie2 and gp64 genes (Table 1). It was also found in the promoter region of 39K and ie1, albeit on the complementary strand. Further testing is needed to confirm the significant role of these IE1-binding motifs to the promoter regulation of these genes.
|
). Ten baculoviral promoters were tested for their ability to be activated by IE1 and IE2. These genes were discovered through microarray analysis to have a high to moderate level of transcription induced by IE1- or IE2-expressing recombinant baculoviruses. The results showed that IE1 protein can directly activate ie1, ie2, pe38, 39K, he65, pcna, orf16 and orf25, but not gp64 or orf17. IE2 protein can weakly activate he65 and orf16. Surprisingly, when in combination, IE1 and IE2 proteins in transient assay do not have a synergistic effect on gene activation. This result most likely indicated that the synergistic characteristic of IE1 plus IE2 activation involves other viral or cellular components, and this effect can only be seen in vivo in conjunction with the whole AcMNPV genome.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
In Vero E6 cells, we optimized the baculovirus transduction dosage to an m.o.i. of 10. At this minimal inoculation ratio, we observed almost no endogenous viral gene transcription. A recently published AcMNPV DNA microarray study by Fujita et al. (2006) has shown low but detectable levels of ie1 and ie2 gene transcripts in BHK-21 cells and ie1 transcript in HeLa14 cells when inoculated with AcMNPV at an m.o.i. of 30. The normally low endogenous expression level of baculoviral genes in mammalian cells, however, made microarray study of baculovirus gene regulation in mammalian systems difficult, as some transcripts may not be detectable above the background signal. In this study, a different approach was used where we selectively overexpressed specific baculoviral trans-activators and saw a significant induction of many viral genes in response to that.
A set of IE1-responsive genes were identified from the AcMNPV microarray hybridized with cDNAs from vAcIE1scmR-transduced Vero E6 cells, including gp64, pe38, ie2, he65, orf16, orf17 and orf25. From previous transient transfection assays in insect systems, it had been shown that gp64 and he65 genes are positively regulated by IE1 (Blissard & Rohrmann, 1991; Murges et al., 1997), while promoters of ie2 and pe38 contain an IE1-binding element (ACBYGTAA) and are negatively regulated (Leisy et al., 1997). In mammalian cells, ie2 and pe38 gene expression was activated by IE1, and this discrepancy was probably because of host factor differences between the two systems. By analysing the promoters of IE1-responsive genes in Vero E6 cells, we identified several other IE1-responsive genes that also contain a sequence almost identical to this IE1-binding element (Table 1). It is highly probable that IE1 activated these genes by binding to this sequence.
Several other genes previously unlinked to IE1 were found to be activated by the vAcIE1scmR virus in the microarray experiment, including pcna, orf16, orf17 and orf25. When all the vAcIE1scmR-virus-activated genes were tested by transient expression study in Vero E6 cells, two groups were found. One set of genes, pcna, orf16 and orf25, was directly switched on by the IE1 protein, while the other set, gp64 and orf17, was not. Because of the proximities of orf16 and orf17, the possibility that the two gene signals on the microarray were from a single overlapping transcript could not be overlooked. However, a closer look at the gene expression pattern of orf16 and orf17 (Fig. 4a) in a time-course study showed that the two adjacent genes on the same strand have different expression profiles. Therefore, it is unlikely that a single transcript was solely responsible for the signals on their corresponding spots on the microarray. The AcMNPV microarray contains unique gene fragments from each gene; care was taken to use a region of gene not overlapped with other genes on either strand. Also, gel-electrophoresis showed most cy3- or cy5-labelled cDNA samples were 0.5–1 kb in length, therefore only a small percentage of the signals should be from run-through transcripts. Nonetheless, the signals from adjacent genes oriented from the same strand should be viewed with caution; in this case, both time-course studies and transient expression assays were performed to support our microarray data.
The IE2-expressing recombinant baculovirus only weakly activated two genes on the microarray, pe38 and orf17. Strikingly, in the presence of both vAcIE1scmR and vAcIE2scmR, both the number and intensity of the downstream genes increased significantly. Two likely reasons may be (i) the activated genes require the presence of both ie factors for activation, and/or (ii) IE1 and IE2 initiate a cascade of gene activation through subsequent transcriptional activators. The transient assay showed that a combination of IE1 and IE2 proteins has no obvious activation effect on the 10 baculoviral promoters. The mechanism of IE1+IE2 activation of AcMNPV genome is still unclear, but it obviously requires other viral factors/elements not present in a transient assay. For example, a distant cis-element could be missing in the reporter plasmid construct, thus rendering the trans-activators ineffective.
Protein expression experiments using baculovirus are currently predominantly performed in insect cells, due to a high yield. However, the differences in post-translational modifications between insect and mammalian cells are evident, and thus using mammalian cells as an alternative for protein expression experiments using baculovirus has been discussed in several studies (Boyce & Bucher, 1996; Hofmann et al., 1995; Hu et al., 2003; Shoji et al., 1997). In this study, we found very late promoters were not directly activated by IE1 and IE2. However, an interesting finding was the strong activation of the AcMNPV gp64 gene by the IE1-expressing recombinant baculovirus. The amount of gp64 transcripts even surpassed ie1 at 48 h post-transduction, even though ie1 transcription was driven by the strong mammalian CMVie promoter.
The analysis of the AcMNPV transcriptome in Vero E6 cells in this study provided valuable information on baculovirus early gene regulation in the mammalian system. The IE1-activated genes in Vero E6 cells match well with ones found in insect cells, including 39K, he65 and gp64. In addition, the observation that 39K gene activation required both IE1 and IE2 proteins agreed well with a previous study by Carson et al.(1988), where IE2 augmented IE1-induced 39K gene activation in insect cells. The two systems, insect and mammalian, while evolutionarily distant, appear to have similar cellular machineries. Perhaps, given the right combination of baculoviral trans-activators, we could eventually utilize the extremely strong promoters of p10 or polyhedrin for protein production in mammalian cells.
A promoter analysis of IE1/IE2-responsive genes showed all of them contained the baculovirus early transcription initiation sites C (CAGT) or E [CGTGC; A(A/T)CGT(G/T)] (Table 1). This concurred with our hypothesis that IE1/IE2 turns on other baculovirus genes in gene cascades similar to ones observed in insect cells. Most of these genes also contain a TATA motif (Table 1), which together with the partial eukaryotic transcriptional initiation site CAGT forms a promoter structure typical for eukaryotic RNA polymerase II. The involvement of IE1 or IE2 must be the last key step needed for these promoters to become active in mammalian cells.
Except for the ie2 gene, all the major IE1- and IE2-responsive genes in Table 1 conform to Kozak rules [sequence: ANNATG(A/G) or GNNATGG; N=any nucleotide] (Kozak, 1987), while only 59 % of the genes in the AcMNPV genome do (Ayres et al., 1994). Although a previous report indicated that the Kozak sequence does not affect yield of foreign protein expression in Sf21 cells when driven by the very late polyhedrin promoter (Hills & Crane-Robinson, 1995), our result suggests that a Kozak consensus sequence might be important for translation initiation of early baculoviral genes in mammalian cells.
In this study, we have attempted to map the interaction of some baculoviral genes, in detail, in a mammalian system. So far, by overexpressing IE1 and IE2, we have successfully turned on over a third of the baculoviral genome in cells that normally do not support baculoviral gene expression. Future experiments, involving expressing key late expression factor genes in mammalian cells, will hopefully enable us to activate late and very late promoters of AcMNPV. This approach may lead to a strong baculovirus expression system for mammalian cells, as well as give some insights into baculoviral gene regulation in its original host system.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Abe, T., Hemmi, H., Miyamoto, H., Moriishi, K., Tamura, S., Takaku, H., Akira, S. & Matsuura, Y. (2005). Involvement of the Toll-like receptor 9 signaling pathway in the induction of innate immunity by baculovirus. J Virol 79, 2847–2858.
Ayres, M. D., Howard, S. C., Kuzio, J., Lopez-Ferber, M. & Possee, R. D. (1994). The complete DNA sequence of Autographa californica nuclear polyhedrosis virus. Virology 202, 586–605.[CrossRef][Medline]
Blissard, G. W. & Rohrmann, G. F. (1991). Baculovirus gp64 gene expression: analysis of sequences modulating early transcription and transactivation by IE1. J Virol 65, 5820–5827.
Boyce, F. M. & Bucher, N. L. (1996). Baculovirus-mediated gene transfer into mammalian cells. Proc Natl Acad Sci U S A 93, 2348–2352.
Carson, D. D., Guarino, L. A. & Summers, M. D. (1988). Functional mapping of an AcNPV immediately early gene which augments expression of the IE1 trans-activated 39K gene. Virology 162, 444–451.[CrossRef][Medline]
Carson, D. D., Summers, M. D. & Guarino, L. A. (1991). Molecular analysis of a baculovirus regulatory gene. Virology 182, 279–286.[CrossRef][Medline]
Chang, Y. J., Liu, C. Y., Chiang, B. L., Chao, Y. C. & Chen, C. C. (2004). Induction of IL-8 release in lung cells via activator protein-1 by recombinant baculovirus displaying severe acute respiratory syndrome-coronavirus spike proteins: identification of two functional regions. J Immunol 173, 7602–7614.
Choi, J. & Guarino, L. A. (1995). The baculovirus transactivator IE1 binds to viral enhancer elements in the absence of insect cell factors. J Virol 69, 4548–4551.[Abstract]
Dai, X., Willis, L. G., Huijskens, I., Palli, S. R. & Theilmann, D. A. (2004). The acidic activation domains of the baculovirus transactivators IE1 and IE0 are functional for transcriptional activation in both insect and mammalian cells. J Gen Virol 85, 573–582.
Fujita, R., Matsuyama, T., Yamagishi, J., Sahara, K., Asano, S. & Bando, H. (2006). Expression of Autographa californica multiple nucleopolyhedrovirus genes in mammalian cells and upregulation of the host -actin gene. J Virol 80, 2390–2395.
Gossen, M. & Bujard, H. (1992). Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci U S A 89, 5547–5551.
Guarino, L. A. & Summers, M. D. (1986). Functional mapping of a trans-activating gene required for expression of a baculovirus delayed-early gene. J Virol 57, 563–571.
Hills, D. & Crane-Robinson, C. (1995). Baculovirus expression of human basic fibroblast growth factor from a synthetic gene: role of the Kozak consensus and comparison with bacterial expression. Biochim Biophys Acta 1260, 14–20.[Medline]
Hofmann, C., Sandig, V., Jennings, G., Rudolph, M., Schlag, P. & Strauss, M. (1995). Efficient gene transfer into human hepatocytes by baculovirus vectors. Proc Natl Acad Sci U S A 92, 10099–10103.
Hu, Y. C., Tsai, C. T., Chang, Y. J. & Huang, J. H. (2003). Enhancement and prolongation of baculovirus-mediated expression in mammalian cells: focuses on strategic infection and feeding. Biotechnol Prog 19, 373–379.[CrossRef][Medline]
Huser, A. & Hofmann, C. (2003). Baculovirus vectors: novel mammalian cell gene-delivery vehicles and their applications. Am J Pharmacogenomics 3, 53–63.[CrossRef][Medline]
Huser, A., Rudolph, M. & Hofmann, C. (2001). Incorporation of decay-accelerating factor into the baculovirus envelope generates complement-resistant gene transfer vectors. Nat Biotechnol 19, 451–455.[CrossRef][Medline]
Kenoutis, C., Efrose, R. C., Swevers, L., Lavdas, A. A., Gaitanou, M., Matsas, R. & Iatrou, K. (2006). Baculovirus-mediated gene delivery into mammalian cells does not alter their transcriptional and differentiating potential but is accompanied by early viral gene expression. J Virol 80, 4135–4146.
Kost, T. A., Condreay, J. P. & Jarvis, D. L. (2005). Baculovirus as versatile vectors for protein expression in insect and mammalian cells. Nat Biotechnol 23, 567–575.[CrossRef][Medline]
Kovacs, G. R., Choi, J., Guarino, L. A. & Summers, M. D. (1992). Functional dissection of the Autographa californica nuclear polyhedrosis virus immediate-early 1 transcriptional regulatory protein. J Virol 66, 7429–7437.
Kozak, M. (1987). An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res 15, 8125–8148.
Leisy, D. J., Rasmussen, C., Kim, H. T. & Rohrmann, G. F. (1995). The Autographa californica nuclear polyhedrosis virus homologous region 1a: identical sequences are essential for DNA replication activity and transcriptional enhancer function. Virology 208, 742–752.[CrossRef][Medline]
Leisy, D. J., Rasmussen, C., Owusu, E. O. & Rohrmann, G. F. (1997). A mechanism for negative gene regulation in Autographa californica multinucleocapsid nuclear polyhedrosis virus. J Virol 71, 5088–5094.[Abstract]
Lo, H. R. & Chao, Y. C. (2004). Rapid titer determination of baculovirus by quantitative real-time polymerase chain reaction. Biotechnol Prog 20, 354–360.[CrossRef][Medline]
Lo, H. R., Chou, C. C., Wu, T. Y., Yuen, J. P. & Chao, Y. C. (2002). Novel baculovirus DNA elements strongly stimulate activities of exogenous and endogenous promoters. J Biol Chem 277, 5256–5264.
Lu, A. & Carstens, E. B. (1993). Immediate-early baculovirus genes transactivate the p143 gene promoter of Autographa californica nuclear polyhedrosis virus. Virology 195, 710–718.[CrossRef][Medline]
Lu, A. & Miller, L. K. (1995). The roles of eighteen baculovirus late expression factor genes in transcription and DNA replication. J Virol 69, 975–982.[Abstract]
Murges, D., Kremer, A. & Knebel-Morsdorf, D. (1997). Baculovirus transactivator IE1 is functional in mammalian cells. J Gen Virol 78, 1507–1510.[Abstract]
Nissen, M. S. & Friesen, P. D. (1989). Molecular analysis of the transcriptional regulatory region of an early baculovirus gene. J Virol 63, 493–503.
O'Reilly, D. R., Miller, L. K. & Luckow, V. A. (1994). Baculovirus Expression Vectors: a Laboratory Manual. New York: Oxford University Press.
Prikhod'ko, E. A., Lu, A., Wilson, J. A. & Miller, L. K. (1999). In vivo and in vitro analysis of baculovirus ie-2 mutants. J Virol 73, 2460–2468.
Shoji, I., Aizaki, H., Tani, H., Ishii, K., Chiba, T., Saito, I., Miyamura, T. & Matsuura, Y. (1997). Efficient gene transfer into various mammalian cells, including non-hepatic cells, by baculovirus vectors. J Gen Virol 78, 2657–2664.[Abstract]
Yoo, S. & Guarino, L. A. (1994). Functional dissection of the ie2 gene product of the baculovirus Autographa californica nuclear polyhedrosis virus. Virology 202, 164–172.[CrossRef][Medline]
Zheng, D., Constantinidou, C., Hobman, J. L. & Minchin, S. D. (2004). Identification of the CRP regulon using in vitro and in vivo transcriptional profiling. Nucleic Acids Res 32, 5874–5893.
Received 24 October 2006;
accepted 24 April 2007.
This article has been cited by other articles:
|
|
 |
|
  C. Y.-Y. Liu, C.-H. Wang, W.-K. Hsiao, H.-R. Lo, C. P. Wu, and Y. C. Chao RING and Coiled-Coil Domains of Baculovirus IE2 Are Critical in Strong Activation of the Cytomegalovirus Major Immediate-Early Promoter in Mammalian Cells J. Virol., April 15, 2009; 83(8): 3604 - 3616. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
, gp64; 
, IE1; 
, IE2; 
, actin; 
, dsR2; the spike-in controls are underlined.
