Signal Research Division of Celgene, 5555 Oberlin Drive, San Diego, CA 92121, USA1
Author for correspondence: Jun Wu. Fax +1 858 623 0870. e-mail jwu{at}signalpharm.com
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Abstract |
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Introduction |
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HCMV replicates in various terminally differentiated cell types, including smooth muscle, endothelial, epithelial, neuronal, and microglial cells, fibroblasts and differentiated cells of the monocyte/macrophage lineage (Drew et al., 1979 ; Fish et al., 1995
, 1998
; Gonczol et al., 1984
; Ibanez et al., 1991
; Kondo et al., 1994
; Sinzger et al., 1995
). Productive infection is species- and cell-specific (McDonough & Spector, 1983
) and requires the tightly coordinated expression of viral genes. This sequential, cascade-like, viral gene expression is divided into three kinetic classes: immediate early (IE) or
, early (E) or
and late (L) or
(Spector, 1996
; Stenberg, 1996
; Chambers et al., 1999
). The major IE gene products, regulated by a complex enhancer promoter, are synthesized immediately after viral infection and rely primarily on host factors for their expression. Transcriptional regulation of immediate early genes has been investigated in detail (Cherrington & Mocarski, 1989
; Jahn et al., 1984
; Lang & Stamminger, 1993
; Pizzorno & Hayward, 1990
; Wu et al., 1993
). The three major IE proteins, IE1 (IE72) and IE2 (IE86 and IE55), play a critical role in the HCMV gene regulatory cascade (Hermiston et al., 1987
; Baracchini et al., 1992
; Hagemeier et al., 1992
). Early genes are transcribed prior to viral DNA replication and their expression requires one or more viral IE gene products (Malone et al., 1990
; Spaete & Mocarski, 1985
; Schwartz et al., 1994
; Sommer et al., 1994
). The late genes, which constitute a majority of the viral genome, are transcribed in abundance only after viral DNA replication (Geballe et al., 1986
; Stenberg et al., 1989
).
Although studies on the mechanism of regulation of herpesvirus IE and E gene expression have been reported, the mechanism regulating L gene expression has not been well characterized. It has been shown that two early-late genes (1), encoding ICP36 (UL44) and pp65 (UL83), are transcriptionally active at early and late times in infection, suggesting that these early-late genes are controlled by post-transcriptional events (Geballe et al., 1986
; Goins & Stinski, 1986
; Depto & Stenberg, 1989
). It has been reported that the pp28 (UL99) protein is expressed as a true late gene product (Depto & Stenberg, 1992
). These workers further suggested that the pp28 upstream (pp28US) promoter contains two regulatory components: one that is dependent on the onset of viral DNA synthesis and a second that is replication independent and responds to viral trans-acting factors. Using a recombinant virus, Kohler et al. (1994)
further showed that the sequence from -40 to +106 in the pp28US promoter is sufficient to confer true late kinetics.
Traditionally, drug discovery has relied on the systematic screening of natural products and synthetic chemicals in biological and pharmacological assays. The rapid pace at which discoveries have been made in the field of transcriptional regulation over the past decades has opened the possibility for rational targeting of transcription factors that are involved in human disease. Cell-based viral assays hold promise for the development of therapeutic agents. Virus-encoded regulatory proteins present attractive targets for antiviral therapy since interruption of the viral gene expression cascade should prevent virus propagation without affecting the host-cell machinery (Peterson & Baichwal, 1993 ).
We were interested in determining if the temporal regulation of the HCMV true late gene pp28 (UL99) occurs only within the context of the viral genome and what role the immediate early proteins play in the regulation of its promoter. Toward that goal we tested the ability of a construct containing the pp28US promoter fused to the luciferase reporter gene to respond to viral infection when present extrachromosomally, or integrated into the host-cell chromosome. In addition, we studied the effect of the immediate early proteins, IE72, IE86 and IE55, alone or together on the pp28US promoter. Our results demonstrate that the pp28US promoter has undetectable basal activity and requires viral infection for its activation. Expression of the luciferase reporter gene, regulated by the pp28US promoter, was synchronous with that of the endogenous viral pp28 gene, independently of whether the reporter was episomal or integrated. In addition, we found that IE86 was able to transactivate the pp28US promoter but IE72 and IE55 were not. Finally, we found that activation of the pp28US promoter was specific to HCMV, as herpes simplex virus (HSV) infection did not significantly induce luciferase expression.
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Methods |
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Cells and viruses.
Experiments were done with the human glioblastoma cell line U373 MG. Conditions for their growth and infection have been described (Baracchini et al., 1992 ). The Towne strain of HCMV, Vero cells and HSV-1 were purchased from the ATCC. For all infection experiments, cells were treated at an m.o.i. of 510 p.f.u. per cell. Phosphonoacetic acid (PAA, Sigma 99·7% purity) treatment was done as previously described (Depto & Stenberg, 1989
). For UV treatment, the virus was exposed to UV light at 254nm for 20 min.
Transfection and infection assays.
U373 MG cells were first transfected with the reporter construct by the calcium phosphate method using the Profection mammalian transfection system (Promega); 1416 h post-transfection, cells were infected at the indicated m.o.i. Cells were harvested at specific times and assayed for luciferase activity as prescribed by the assay system manufacturer (Analytical Luminescence Laboratory).
Establishment of pp28-luc stable cell line.
The pp28-luciferase reporter and pSV2Neo selection plasmid were cotransfected into U373 MG cells by the calcium phosphate method. Transfectants were selected in medium containing 0·6 mg/ml G418 on the third day after transfection. G418-resistant clones were expanded and 3x104 cells seeded in triplicate in a 96-well plate. Cells were infected with HCMV at 510 p.f.u. per cell; 48 h post-infection, cells were harvested at the indicated times and assayed for luciferase activity. Clones showing high luciferase activity were further analysed by PCR to ascertain the integrity of the reporter transcriptional unit integrated into the genomic DNA.
mRNA isolation and Northern blot analysis.
Transfected and infected U373 MG cells were processed for messenger RNA as indicated by the manufacturer (Stratagene) of the Northern blot kit. Equal aliquots of mRNA were subjected to Northern blot analysis. Probes were labelled with [-32P]dCTP (3000Ci/mmol, Amersham) using Prime-It RmT random primer labelling kit (Stratagene). Blots were hybridized to the radiolabelled probes for each gene using QuikHyb hybridization solution following the manufacturers protocol (Stratagene).
IE2 antisense oligonucleotide treatment.
The sequence of oligonucleotides complementary to RNA of the IE2 region (GCGTTTGCTCTTCTTCTTGCG) and nonspecific RNA (TGGAAAGTGTACACAGGCGAA) has been described previously (Azad et al., 1993 ). The pp28-luc stable cell lines were seeded in duplicate in a 96-well plate and treated the following day with different concentrations of IE2 or nonspecific antisense oligonucleotide/Lipofectin mixture for 4 h (Azad et al., 1993
). The medium was changed and cells were infected with HCMV at 5 p.f.u. per cell; 48 h after infection, cell were harvested, and assayed for luciferase activity.
Cytotoxicity assays.
Cytotoxicity of PAA under viral DNA replication inhibition conditions was evaluated with a non-radioactive cell proliferation assay (Promega). Briefly, U373 MG cells seeded in 96-well plates were treated with 200 or 400 µg/ml PAA for 72 h, and then assayed according to the assay manufacturers protocol (Promega).
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Results |
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IE86 is required for transactivation of the pp28US promoter
It has been reported that some viral late genes are regulated by IE gene products (Depto & Stenberg, 1989 ; Stasiak & Mocarski, 1992
). Therefore, we were interested in determining if any of the three HCMV major IE proteins, IE86, IE72 and IE55, transactivated the pp28US promoter. Toward this goal the pp28US-luc construct was cotransfected with individual RSV expression vectors for IE86, IE72 and IE55 into U373MG cells. We found that only IE86 activated luciferase activity (18-fold), while neither IE72 nor IE55 induced luciferase activity above background (Fig. 5A
). However, if pp28US-luc was cotransfected with a plasmid (pSVH) which expresses IE1 and IE2 under control of its own promoter (major immediate early promoter, MIEP), a 35-fold activation of pp28US promoter activity was seen. Western blot analysis indicated that the difference in pp28US promoter activation by different expression vectors is most likely a reflection of the higher expression level of IE86 protein from the pSVH expression vector (data not shown).
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Induction of the integrated pp28 promoter is virus specific
The cascade-like pattern of gene expression in different herpesviruses is similar (Honess & Roizman, 1974 , 1975
). In addition, the immediate early viral proteins appear to have a regulatory role in early and late gene expression. Therefore, we compared the response of the pp28US-luc reporter to two herpesviruses, HCMV and HSV. The pp28US-luc reporter cell line was infected with HCMV or HSV-1, and luciferase activity quantified at different times post-infection. As shown in Fig. 6
, HCMV infection resulted in significant expression of luciferase while HSV infection had no effect on expression of the reporter gene. Western blot analysis revealed that HSV-1 gC, a late viral gene product, was efficiently expressed in HSV-1-infected U373 MG cells (data not shown). Although HSV and HCMV replicate at vastly different rates, we did not observe luciferase induction at 4 to 8 h post-infection as would be expected for HSV-1-induced gene expression. Therefore, while HSV-1 infects U373 MG cells and is able to express its late genes, it does not efficiently induce the HCMV pp28US promoter, suggesting that activation of the pp28US promoter is virus specific.
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Discussion |
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This study demonstrates that the pp28US (UL99) promoter requires viral DNA replication for its maximal expression even when present on a plasmid. This is consistent with studies on the true late US11 promoter of HSV-1 (Johnson & Everett, 1986 ; Johnson et al., 1986
). In that study, the authors found that the US11 promoter present on a plasmid was expressed with similar kinetics to the viral US11 promoter, and required viral DNA replication for abundant expression. However, the temporal regulation of the HCMV pp28US promoter is different from that reported for the HSV late gene, gC (Arsenakis et al., 1986
). In that study, the authors found that the gC transcriptional unit integrated into the host cell genome was expressed as an early gene. In addition, they found that in the presence of PAA the integrated gC was expressed at higher levels, as expected of a natural viral early gene. In contrast, we did not observe an increase in luciferase activity, protein or mRNA levels in the presence of PAA at any concentration. It has been reported that the pp28US promoter is activated early in a cotransfection assay (Depto & Stenberg, 1992
). In this study, we also detected luciferase activity at 24 h post-infection in a transfection/infection assay (see Fig. 2A
). However, when the promoter is integrated into the host chromosome, this effect was greatly decreased (see Fig. 6
). This difference between the response of transiently transfected and integrated reporters may reflect the expected tighter regulation of the chromatin structure of the integrated promoter. Our results demonstrate that the temporal regulation of the integrated pp28US promoter mimics that of its endogenous counterpart. Therefore, we conclude that the pp28US promoter behaves as a late promoter when removed from the context of the viral genome and integrated into the host cell genome.
The cascade model of gene regulation predicts that late gene expression is dependent on the production of viral transactivators early in infection. It has been demonstrated that IE1 and IE2 are sufficient for activation of a late gene encoding pp65 (Depto & Stenberg, 1989 ) and that the activation of the ICP36 promoter requires viral immediate early proteins, IE86, IE72 and IE55 (Stasiak & Mocarski, 1992
). In cotransfection assays, Depto & Stenberg (1992)
showed that the IE proteins are insufficient for activation of the pp28US promoter in human fibroblast cells. In our study, we found that IE proteins were able to transactivate the pp28US promoter in the permissive human glioblastoma cell line U373 MG. This difference may be a reflection of the different cell types used. In addition, use of different reporters and possible distinct transfection efficiencies could exacerbate stoichiometric differences in viral and cellular factors necessary for transactivation. Nevertheless, our results show that the kinetics of activation of the endogenous viral pp28US promoter is identical to that of the integrated copy in HCMV-susceptible U373 MG cells. Furthermore, our data suggest that IE86 is a viral transcription factor required for pp28US promoter activation. However, the higher activation observed upon HCMV infection may reflect accessory activation by other viral factors.
Comparative analysis of luciferase activity in the stable cell line infected with either HCMV or HSV-1 showed that robust induction was seen only with the homologous virus, HCMV. As expected from our results with PAA, inhibition of HCMV virus replication blocks expression of the luciferase gene, thus making this assay also sensitive to inhibition of viral DNA replication. This virus replication-dependent assay is a facile, microtitre plate-formatted assay amenable for high throughput screening. In addition, the assay system can be completed in 2448 h and thus is more expedient than the 34 weeks required for an HCMV plaque assay. Therefore, this system provides a quick, sensitive and quantitative HCMV detection assay.
In summary, we developed a reporter cell line with two important biological applications: first, it is specific for HCMV and thus can be used for quick and sensitive detection of HCMV; second, it provides a method for identifying inhibitors of HCMV virus replication. Potentially, two classes of inhibitors may be detected. Those that directly inhibit virus replication; and those that indirectly inhibit virus replication by decreasing expression of a viral gene required for genomic replication.
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Acknowledgments |
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Footnotes |
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References |
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Received 22 August 2000;
accepted 15 January 2001.
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