Human herpesvirus 6 major immediate early promoter has strong activity in T cells and is useful for heterologous gene expression
© Matsuura et al; licensee BioMed Central Ltd. 2011
Received: 18 August 2010
Accepted: 11 January 2011
Published: 11 January 2011
Human herpesvirus-6 (HHV-6) is a beta-herpesvirus. HHV-6 infects and replicates in T cells. The HHV-6-encoded major immediate early gene (MIE) is expressed at the immediate-early infection phase. Human cytomegalovirus major immediate early promoter (CMV MIEp) is commercially available for the expression of various heterologous genes. Here we identified the HHV-6 MIE promoter (MIEp) and compared its activity with that of CMV MIEp in various cell lines.
The HHV-6 MIEp and some HHV-6 MIEp variants were amplified by PCR from HHV-6B strain HST. These fragments and CMV MIEp were subcloned into the pGL-3 luciferase reporter plasmid and subjected to luciferase reporter assay. In addition, to investigate whether the HHV-6 MIEp could be used as the promoter for expression of foreign genes in a recombinant varicella-zoster virus, we inserted HHV-6 MIEp-DsRed expression casette into the varicella-zoster virus genome.
HHV-6 MIEp showed strong activity in T cells compared with CMV MIEp, and the presence of intron 1 of the MIE gene increased its activity. The NF-κB-binding site, which lies within the R3 repeat, was critical for this activity. Moreover, the HHV-6 MIEp drove heterologous gene expression in recombinant varicella-zoster virus-infected cells.
These data suggest that HHV-6 MIEp functions more strongly than CMV MIEp in various T-cell lines.
Human herpesvirus 6 (HHV-6) was first isolated in 1986 from the peripheral blood of patients with lymphoproliferative disorders and AIDS [1, 2]. The virus was subsequently shown to be ubiquitous in healthy adults . HHV-6 has been isolated from infants with exanthema subitum, a common childhood disease . Later, HHV-6 isolates were classified into two variants, A and B (HHV-6A and HHV-6B), based on molecular and biological criteria [5–8]. HHV-6B causes exanthema subitum , while the pathogenesis of HHV-6A is still unknown. HHV-6 has the unique feature of being able to replicate and produce progeny in T cells [9, 10]. The HHV-6 genome is a double-stranded DNA of approximately 160 kbp, consisting of a unique long region of 140 kbp flanked by 10-kbp direct repeats, and there is 90% identity between the two variants .
HHV-6 belongs to the beta-herpesvirus subfamily, which includes human cytomegalovirus (HCMV) and human herpesvirus 7 (HHV-7) . The betaherpesviruses have extensive domains of similar genomic organization, with conserved herpesvirus gene blocks in the unique region of their genome . HCMV's major immediate early (MIE) enhancer-containing promoter has been developed [14, 15]; it is currently commercially available and is used to drive the expression of various genes. The MIE promoter controls the expression of two IE transcripts, designated IE1 (UL123) and IE2 (UL122) . HHV-6 has positional homologs of UL123 and UL122; they are U89 and U86, which are designated IE1 and IE2, respectively [11, 13, 17, 18]. The HHV-6 IE1 and IE2 transcripts are formed by alternative splicing [19, 20]. Recently Takemoto et al. reported that the R3 region in the right end of HHV-6 is a strong enhancer of another HHV-6 immediate early gene, U95 . R3 is positioned between U95 and U89; therefore, the region containing R3 is predicted to also contain promoter activity for the IE1 and IE2 genes. In other words, this location is predicted to be a positional homolog of the HCMV MIE promoter.
In this study, we identified the promoter region that regulates the HHV-6 MIE gene, and analyzed its activity. As expected, part of the R3 region was critical for the promoter activity. We also found that the first intron encoded by the IE1 gene enhanced HHV-6 MIE promoter (HHV-6 MIEp) activity, and that HHV-6 MIEp with the first intron had significantly stronger activity than the HCMV MIE promoter, especially in T-cell lines. The HHV-6 MIEp was able to express heterologous genes in a recombinant varicella-zoster virus, indicating that it could be useful for expressing various genes in a similar manner as the CMV MIE promoter.
The HHV-6 major immediate-early promoter had stronger activity than the CMV promoter in T-cell lines
The HHV-6 MIE promoter could drive the expression of foreign gene in a recombinant varicella virus
We recently constructed a recombinant varicella vaccine Oka strain (vOka) expressing the MuV (mumps virus) HN (hemaglutinin-neuraminidase) gene, as a possible candidate for a polyvalent vaccine for both varicella zoster virus (VZV) and MuV infections . In that study, the CMV promoter was used to control the HN gene. Since the HHV-6 MIE promoter and CMV promoter showed similar activity in MRC-5 cells and MeWo cells, which are susceptible to VZV infection, we next examined whether the HHV-6 MIE promoter could control the expression of foreign genes in VZV.
The expression of the DsRed was confirmed by Western blotting analysis (Figure 7B). Recombinant vOka-infected MRC-5 cell lysates were separated by SDS-PAGE and analyzed by Western blotting with an anti-DsRed mAb or anti-VZV gB Ab. The expression of gB, which is a late gene , was examined as a positive control of VZV infection. As shown in Figure 7B, the expression of gB was found in lysates from cells infected with either the control rvOka-BAC or HHV-6MIEpin1-DsRed-rvOka-BAC, while the anti-DsRed mAb specifically reacted with a 29-kDa band only in the HHV-6MIEpin1-DsRed-rVoka-BAC-infected cell lysates. These data indicated that the HHV-6 MIE promoter can be used to drive the expression of foreign genes in VZV-infected cells.
The HCMV major immediate early promoter (HCMV MIEp) has been established and used as a tool to drive gene expression by researchers worldwide. HHV-6 also belongs to the beta-herpesviruses, and has a positional homolog of the HCMV MIE gene. As described in the Introduction, HHV-6 replicates and produces progeny in T cells very well; we therefore speculated that the MIE promoter would have stronger promoter activity in T cells than in other cells. Here we identified the region of the HHV-6 major immediate early promoter (HHV-6 MIEp), described in Figure 1. The promoter activity of HHV-6 MIEp was stronger than that of HCMV MIEp in T- cell lines, but not in other adherent cell lines. This feature of HHV-6 MIEp activity is consistent with the fact that HHV-6 is T-cell tropic.
HHV-6 MIEp is predicted to have an NF-κB-binding site. The activity of a mutant HHV-6 MIEp, with the NF-κB-binding site deleted, was dramatically decreased, indicating that the NF-κB-binding site is critical for the promoter activity of HHV-6 MIEp. However, the HCMV MIEp activity was weak compared to that of HHV-6 MIEp in T-cell lines in our study, even though HCMV MIEp also has an NF-κB-binding site that plays a major role in its promoter activity [27, 28]. Therefore, another binding site in addition to the NF-κB-binding site might contribute to the T-cell-specific promoter activity of HHV-6 MIEp, or another binding site in HCMV MIEp might have a repressive effect in T cells.
Although the AP-2 and PEA3 binding sites were not found in HHV-6 MIE promoter region by TFSEARCH, R3 region has these binding sites[17, 29]. However, in the study of U95 promoter, it has been reported that PEA3 binding sites in R3 region did not bind any proteins. Therefore, PEA3 binding site might have no or low effect on the MIEp activity. The deletion promoter, HHV-6 MIEp-d1, lost two complete AP-2 binding sites and one AP-2 binding site with one nucleotide mutation, compared to full length promoter. Nevertheless, the activity of HHV-6 MIEp-d1 was similar to that of HHV-6 MIEp. Therefore, the AP-2 binding sites might have low effect on the MIEp activitiy.
Adding the first intron (intron 1) of IE1 to HHV-6 MIEp enhanced the promoter activity significantly. When intron 1 was added, the activity of HHV-6 MIEp became markedly greater than that of HCMV in T cells. In adherent cell lines such as MRC-5 and MeWo cells, the activity of HHV-6 MIEp with intron 1 became similar to that of HCMV MIEp. Intron1 of the IE1 region is predicted to have two CCAAT enhancer binding protein (C/EBP) binding sites and an OCT-1-binding site (Figure 3). The transcriptional regulators that bind to these sites might enhance the promoter activity of HHV-6 MIEp. Interestingly, the promoter construct that contained introns 1 and 2 was less active than the promoter containing only intron 1. Further investigation is needed to elucidate the mechanisms involving the intron regions.
We recently developed a recombinant VZV vaccine strain containing the mumps virus HN gene. In this study, we examined whether the HHV-6 MIEp containing intron 1 functioned as a heterologous expression promoter in the VZV vaccine strain. Indeed, in the recombinant VZV, HHV-6 MIEp functioned to drive the expression of the DsRed gene, which is a heterologous gene. These findings indicate that, like the commercially available HCMVp, HHV-6 MIEp is useful for expressing heterologous genes in a VZV vaccine strain.
Our results show that HHV-6 MIE promoter functions more strongly than CMV MIEp in various T-cell lines. Moreover, the first intron of HHV-6 IE1 gene enhances the promoter activity of HHV-6 MIEp. In addition, the HHV-6 MIEp could drive heterologous gene expression in recombinant varicella-zoster virus-infected cells. These results suggest that HHV-6 MIEp can be used for driving gene expression.
MRC-5 cells, human lung fibroblasts, were cultured in modified minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS). MeWo cells, a human melanoma cell line, and U373 cells, a human astrocytoma cell line, were cultured in Dulbecco's modified Eagle's medium supplemented with 8% FBS. Molt-3 cells, SupT1 cells, and Jurkat cells, which are lymphoblastic T-cell lines, were cultured in RPMI1640 medium supplemented with 8% FBS.
Plasmids for the luciferase reporter assay
5'-tct ctc gag agt taa aga tca gcg ggt ac-3'
5'-agt cgg tac c gg cga atg aga act cta aaa gct c-3'
5'-agt cgg tac c ta ctg tgg ttg ggg tct ttc cta c-3'
5'-acc ggt acc tac cca ggc taa cga gaa cc-3'
5'-agt cgg tac c ac att cct gtt tca tga tgt gta gc-3'
5'-agt cgg tac c tc ctg ttt ttg agt aag ata tga c-3'
5'-agt cgg tac c ag cta att tcc att cca tat ttg tc-3'
5'-agt cgg tac c ta cag cga ttg gct cct tca tcc tc-3'
5'-agt cct cga g ca ctg aac tgg ctg taa ctt ctg c-3'
5'-tct aag ctt cag caa tcc aat aat tga tg-3'
5'-cat aag ctt gca tac gtt cct cat tgg at-3'
5'-cat aag ctt cca aag ttt tga att ctt ca-3'
5'-cat aag ctt ttt gga tgc aag tgc caa cg-3'
5'-acc aag ctt tac cgg tcg cca cca tgg cct-3'
5'-acc aag ctt tta tct aga tcc ggt gga tcc-3'
5'-tat ctc gag agg tac cgg tga ctt cag ag-3'
5'-cga gga tcc aat caa cca atc aga cct-3'
5'-gag gat cc g tac cca caa tat caa gtg gt-3'
5'-gac tcg ag c cta ttc gtg tca tct aga tgg-3'
The CMV MIE promoter sequence was excised with Nru I and Bam HI from pcDNA3.1(+) (Invitrogen), and inserted into pGL3-basic (Promega) at the Sma I and Bgl II sites.
The pRL-TK plasmid (Promega), which contains the Renilla luciferase reporter gene under the HSV TK promoter, was used to normalize the transfection efficiency.
Luciferase reporter assay
Adherent cells (MRC-5, MeWo, and U373) were plated on 24-well plates at a density of 1 × 105 cells per well on the day before transfection, and were transfected with 1 μg of reporter plasmid and 0.25 μg of pRL-TK plasmid (Promega), using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Samples containing 4 × 105 suspended cells (Molt-3, Jurkat, or SupT1) were transfected with 1 μg of reporter plasmid and 0.25 μg of pRL-TK using Lipofectamine2000.
Firefly and Renilla luciferase activities were measured with the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's protocol, using a luminometer (Berthold, TriStar LB941). Cells were lysed in 1 × lysis buffer (50 μL/well) for 15 min at room temperature, and each cell lysate was added to a luminometer tube containing 100 μL of assay reagent. The mixture was blended quickly by flicking, and placed in the luminometer for a 1-sec measurement. The transfection efficiency was normalized to the Renilla luciferase activity. The data (mean + SD) were collected from three independent transfections.
Generation of a recombinant vOka-BAC genome containing HHV-6 MIE promoter
To generate the HHV-6MIEpin1-pFastBac plasmid, the gentamicin-resistance gene and the polyhedrin (PH)-promoter region of the pFastBac1 plasmid (Invitrogen) were replaced with 6MIEp including the intron 1 (HHV-6MIEpin1) sequence.
The DsRed fragment was amplified by PCR using the primer pair DsRed2-HindF and DsRed2-HindR, and Hin dIII sites were introduced at both the 5' and 3' ends. The pDsRed2-C1 plasmid (Clontech), in which the Hin dIII site had been eliminated, was used as the PCR template. Following amplification, the PCR products were inserted into the HHV-6MIEpin1-pFastBac plasmid at the Hin dIII site, generating the HHV-6MIEpin1-DsRed-pFastBac plasmid (Figure 5C). The BGH poly (A) signal sequence was derived from pFastBac plasmid.
The vOka-BAC was obtained using pHA-2 cloning vector (a kind gift from Dr. Ulrich Koszinowski), as described previously. The LacZα-mini-att Tn7 cassette was inserted into vOka-BAC (Figure 5A) to produce vOka-BAC-Tn (Figure 5B) using RecA-mediated recombination, essentially as described previously . In brief, E. coli DH10B electrocompetent cells harboring circular vOka-BAC DNA were co-transformed with 1 μg of the targeting vector, pKO5M-Tn (pKO5M is a kind gift from Dr. Kawaguchi), which contain the LacZα-mini-attTn7 region[33, 34], and 3 μg of pDF25(Tet)-- (a kind gift from Dr. J. Heath ) by electroporation, using a Gene Pulser II (Bio-Rad, Hercules, CA). The surviving co-integrant colonies, selected by their resistance to chloramphenicol and zeocin, and by a Lac + phenotype on an LB plate containing X-Gal and IPTG, were made electrocompetent and transformed with 1 μg of pDF25(Tet). The E. coli DH10B colonies containing the correct survival recombination were then selected by the following criteria: resistance to chloramphenicol, sensitivity to zeocin, and a Lac + phenotype on LB containing X-Gal and IPTG. The insertion of the LacZα-mini-attTn7 sequence into the BAC genome was confirmed by PCR and Southern blotting (Data not shown).
The HHV-6MIEpin1-DsRed cassette was inserted into the vOka-BAC-Tn genome using Tn7-mediated site-specific transposition, essentially as described previously . In brief, E. coli DH10B harboring the vOka-BAC-Tn genome was transformed with HHV-6MIEpin1-DsRed-pFastBac and pMON7124 (Invitrogen), a helper plasmid for transposition. The pMON7124 plasmid DNA was isolated from DH10Bac cells (Invitrogen). The transformed E. coli was cultured on LB containing X-gal and IPTG for blue/white selection. The white colonies were analyzed by PCR to verify the insertion of the DsRed expression cassette (data not shown). This completed the construction of the HHV-6MIEpin1-DsRed-vOka-BAC genome (Figure 5D).
Southern blot analysis
The HHV-6MIEpin1-DsRed-vOka-BAC DNA was extracted using a NucleoBond BAC 100 kit (Macherey-Nagel) following the manufacturer's instructions.
The BAC DNA was then digested with Bam HI, loaded onto a 0.5% agarose gel, and separated by electrophoresis at 20 V for 72 hrs. The DNA fragments were visualized with a UV transilluminator and then transferred onto a nylon membrane (Hybond-N+) (GE Healthcare Bio-sciences). The blots were hybridized with ORF12, ORF13, DsRed, or HHV-6MIEp probes labeled with horseradish peroxidase. These probes were amplified by PCR using the following primer pairs: ORF12TnFw/ORF12TnRv, ORF13TnFw/ORF13TnRv, DsRed-HindF/DsRed-HindR, and 6MIEpF-552/6MIEpex2R, respectively (the primer sequences are shown in Table 1). Bands were detected by the Enhanced Chemiluminescence (ECL) Direct Nucleic Acid Labeling and Detection System (GE Healthcare Bio-sciences) following the manufacturer's instructions.
Reconstitution of infectious virus from vOka-BAC DNA
Reconstitution of the recombinant virus, named HHV-6MIEpin1-DsRed-rvOka, was performed as described previously [32, 36]. Briefly, MRC-5 cells were transfected with 1 μg of HHV-6MIEpin1-DsRed-vOka-BAC DNA by electroporation, using a Nucleofection unit (Amaxa Biosystems). The transfected cells were then cultured in MEM supplemented with 3% FBS for 3-5 days, and were observed under a microscope until a typical cytopathic effect with green and red fluorescence appeared.
Western blot analysis
The HHV-6MIEp-DsRed-vOka-BAC-infected MRC-5 cells were lysed in sample buffer [32 mM Tris-HCl (pH 6.8), 1.5% SDS, 5% glycerol, 2.5% 2-mercaptoethanol], separated by SDS-polyacrylamide gel electrophoresis (PAGE), and electrotransferred onto PVDF membranes (Bio-Rad Laboratories). A monoclonal antibody (mAb) against DsRed (Clontech) was purchased, and an anti-VZV gB monospecific antibody (Ab) was produced in our laboratory . Blots were blocked with blocking buffer (PBS, 5% skim milk, 0.1% Tween-20) and reacted with the anti-DsRed mAb or anti-gB Ab in blocking buffer. The protein bands were developed with horseradish peroxidase-conjugated secondary antibodies (GE Healthcare) and ECL detection reagents (GE Healthcare Bio-Sciences), following the manufacturer's instructions.
We thank Dr. Ulrich Koszinowski (Max von Pettenkofer Institut fur Virologie, Ludwig-Maximilians-Universitat Munchen, Germany) for providing the pHA-2 plasmid, Dr. John Heath (School of Biosciences, University of Birmingham, Birmingham, UK) for providing the pDF25(Tet) plasmid, Dr. Yasushi Kawaguchi (The Institute of Medical Science, The University of Tokyo, Japan) for providing the pKO5M plasmid.
This study was supported in part by a grant in aid of Cluster, Ministry of Education, Culture, Sports, Science and Technology of Japan.
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