1 Tumor Virology Program, Children's Cancer Research Institute, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA
2 Departments of Pediatrics, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA
3 Microbiology, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA
4 Medicine, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA
5 San Antonio Cancer Institute, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA
Correspondence
Shou-Jiang Gao (at Tumor Virology Program, Children's Cancer Research Institute)
gaos{at}uthscsa.edu
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Similar to other tumour viruses, KSHV has evolved specific mechanisms to evade host antiviral defences. KSHV encodes a unique set of non-structural genes that target specific cellular signal pathways, contributing to the pathogenesis of KSHV-related diseases (Russo et al., 1996). One of these non-structural genes is the open reading frame (ORF) K9, which encodes the viral interferon regulatory factor (vIRF), a homologue of cellular IRFs (Gao et al., 1997
). IRFs are a family of transcriptional factors that regulate interferon signal transduction through binding to interferon-stimulated response elements (ISREs) in the promoters of interferon-responsive genes (Fujita et al., 1989
; Harada et al., 1989
; Imam et al., 1990
; Nguyen et al., 1995
; Pine et al., 1990
; Taniguchi, 1995
; Taniguchi et al., 1998
). Similar to IRF2, overexpression of vIRF transforms NIH3T3 and Rat-1 cells (Gao et al., 1997
; Zimring et al., 1998
). Early reports have demonstrated that vIRF represses interferon signal transduction through direct binding to IRFs, and p300 and CREB-binding protein (CBP) transcriptional coadaptors (Burysek et al., 1999
; Seo et al., 2000
). Through interaction with p300, vIRF displaces p300/CBP-associated factor from the transcriptional complexes, inhibits the histone acetyltransferase activity of p300 and blocks IRF3 recruitment of p300/CBP (Li et al., 2000
; Lin et al., 2001
). vIRF is also a transcriptional activator and regulates the expression of other KSHV genes. More recently, vIRF has been shown to interact with p53 tumour suppressor protein and inhibits p53-mediated transcriptional regulation and apoptosis (Nakamura et al., 2001
; Seo et al., 2001
).
Given the likely important function of vIRF in KSHV-related pathogenesis, elucidation of the molecular mechanisms controlling vIRF gene expression could lead to an understanding of its precise role in regulating the expression of cellular and KSHV genes. In previous studies, we have mapped the vIRF core promoter region and a transcriptional silencer, Tis, and defined a 12-O-tetradecanoylphorbol 13-acetate (TPA)-responsive region in the upstream regulatory sequence of the vIRF gene (Wang et al., 2001, 2002
). Here, we have shown that vIRF also auto-activates its own promoter through two unidentified cis elements that are unresponsive to interferons.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Construction of plasmids.
vIRF expression plasmid pCMV-Tag2/vIRF was generated by inserting the PCR-amplified ORF K9 gene DNA fragment into the EcoRI/XhoI site of pCMV-Tag2B (Stratagene).
vIRF promoter reporter plasmids for mapping the region responsive to vIRF auto-activation have been described previously (Wang et al., 2001). Briefly, a 1·052 kbp DNA fragment spanning the region -991 to +62 relative to the vIRF gene transcriptional start site (+1) was inserted into the HindIII/XbaI sites of a promoterless and enhancerless chloramphenicol acetyltransferase (CAT) vector, pCAT-Basic, to generate the reporter construct pCAT-991. The 5' end sequence of pCAT-991 was then sequentially deleted to generate constructs pCAT-499 (-499 to +62), pCAT-337 (-337 to +62), pCAT-125 (-125 to +62) and pCAT-56 (-56 to +62) (see Fig. 3A
).
|
Treatment of cells with interferons.
To determine the effect of interferons on vIRF promoter activity, cells were either transfected alone with CAT reporter constructs pCAT-125, pCAT-337, pCAT-499 and pCAT-991 or cotransfected with pCMV-Tag2/vIRF and were then treated for 24 h with either 1000 U interferon- or -
ml-1 at 24 h post-transfection. The cells were then harvested for CAT assay.
Western blot analysis.
Ectopic expression of pCMV-Tag2/vIRF plasmids transfected in 293, HeLa and COS7 cells was detected by Western blot analysis. Briefly, cells (1x107) were harvested, washed twice with PBS, pelleted and resuspended in 200 µl SDS sample buffer. The samples were then subjected to SDS-PAGE before proteins were transferred to nitrocellulose membrane. The membrane was probed with vIRF-specific monoclonal antibody 2H5, followed by probing with a 1 : 5000 dilution of the rabbit anti-mouse immunoglobulin alkaline phosphatase conjugate (Sigma) and developed using NBT/BCIP as substrates.
RNA isolation and Northern blot analysis.
Untreated BC-1, BCBL-1 and BJAB cells and cells either treated with TPA (Sigma) or transfected with vIRF expression plasmids (pCMV-Tag2/vIRF) were collected for total RNA isolation using TRI AGENT according to the instructions of the manufacturer (Sigma). Northern blot analysis was performed as previously reported (Wang et al., 2001). Briefly, the isolated total RNA was treated with DNase I (Promega) at 37 °C for 30 min, fractionated on agarose gel containing 1 % formaldehyde and transferred to Hybond-N+ nylon membrane (Amersham Pharmacia Biotech). The vIRF probe was prepared by labelling the vIRF DNA with [
-32P]dCTP (NEN Life Science Products) using the Rediprime II kit (Amersham Pharmacia Biotech). Hybridization of the probe to the nylon membrane was carried out at 68 °C for 3 h with the PerfectHyb Plus Hybridization Buffer (Sigma).
-Actin probe was used to calibrate RNA loading quantity. The specific hybridization signals on the membrane were captured and analysed with a Molecular Imager.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
vIRF activation of its own promoter is dose-dependent
To determine further the specificity of vIRF activation of its own promoter, we assayed the dose-responsiveness of Vac1 and Vac2 to ectopic expression of vIRF protein. vIRF promoter reporter constructs were cotransfected with different doses (0·1, 0·5, 0·75, 1·0, 1·5, 2·0, 2·5, 3·0 and 3·5 µg) of vIRF expression plasmid pCMV-Tag2/vIRF into 293 cells. As shown in Fig. 4(A) and (B), the expression levels of vIRF protein detected by monoclonal antibody 2H5 increased in a step-wise fashion following the increasing amount of vIRF plasmid DNA. As expected, the CAT activities of cells transfected with pCAT-56 and pCAT-125 containing Vac1 and pCAT-991 containing Vac1 and Vac2 also increased in a dose-dependent fashion, ranging from 2·3- to 11-fold (Fig. 4C
). Again, the increase in vIRF protein expression had minimal effect on the reporter constructs pCAT-337 and pCAT-499.
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
vIRF activates its own promoter through two cis elements, Vac1 and Vac2. Vac1 is located between -56 and +1, a region that contains the core promoter of the vIRF gene (Wang et al., 2001). Vac2 is located between -499 and -991, a region that contains the TRR (Wang et al., 2001
). vIRF auto-activation through Vac1 is suppressed by Tis (-241 to -219) (Wang et al., 2002
); however, vIRF auto-activation of its own promoter through Vac2 overcomes the suppression effect of Tis. It is likely that these two vIRF-responsive cis elements cooperate with each other to produce a synergistic effect to ensure efficient vIRF expression during KSHV lytic replication. The fact that Vac2 is located within a region containing the TRR suggests that this region is most likely a common target site of viral or cellular transactivators. Further delineation of this region could shed light on the transcriptional regulation of other KSHV lytic genes during viral lytic replication.
All the cellular IRFs bind to ISREs to regulate the expression of interferon-stimulated genes (Darnell et al., 1994). Although vIRF does not directly bind to the ISRE sequence, it interacts with IRF1, IRF3 and IRF7 to inhibit interferon signalling and regulate the expression of interferon-stimulated genes (Burysek et al., 1999
; Gao et al., 1997
; Seo et al., 2000
). Unlike other vIRF-responsive genes identified so far, the expression of the vIRF gene itself is not responsive to treatments with interferon-
and -
. Indeed, both Vac1 and Vac2, regardless of activation by vIRF, are not responsive to treatments with interferon-
and -
, indicating that vIRF also targets genes whose promoters do not contain ISRE-like sequences. Further mapping of Vac1 and Vac2 will most likely provide insight into the new function of vIRF and transcriptional regulation of viral and cellular genes by vIRF.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Boshoff, C., Whitby, D., Hatziionnou, T., Fisher, C., van der Walt, J., Hatzakis, A., Weiss, R. & Schulz, T. (1995). Kaposi's sarcoma-associated herpesvirus in HIV-negative Kaposi's sarcoma. Lancet 345, 10431044.[Medline]
Burysek, L., Yeow, W. S., Lubyova, B., Kellum, M., Schafer, S. L., Huang, Y. Q. & Pitha, P. M. (1999). Functional analysis of human herpesvirus 8-encoded viral interferon regulatory factor 1 and its association with cellular interferon regulatory factors and p300. J Virol 73, 73347342.
Carbone, A., Gloghini, A., Vaccher, E. & 8 other authors (1996). Kaposi's sarcoma-associated herpesvirus DNA sequences in AIDS-related and AIDS-unrelated lymphomatous effusions. Br J Haematol 94, 533543.[CrossRef][Medline]
Cesarman, E., Chang, Y., Moore, P. S., Said, J. W. & Knowles, D. M. (1995a). Kaposi's sarcoma-associated herpesvirus-like DNA sequences in AIDS-related body-cavity-based lymphomas. N Engl J Med 332, 11861191.
Cesarman, E., Moore, P. S., Rao, P. H., Inghirami, G., Knowles, D. M. & Chang, Y. (1995b). In vitro establishment and characterization of two acquired immunodeficiency syndrome-related lymphoma cell lines (BC-1 and BC-2) containing Kaposi's sarcoma-associated herpesvirus-like (KSHV) DNA sequences. Blood 86, 27082714.
Chang, Y., Cesarman, E., Pessin, M. S., Lee, F., Culpepper, J., Knowles, D. M. & Moore, P. S. (1994). Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi's sarcoma. Science 266, 18651869.[Medline]
Chang, Y., Ziegler, J., Wabinga, H. & 8 other authors (1996). Kaposi's sarcoma-associated herpesvirus and Kaposi's sarcoma in Africa. Uganda Kaposi's Sarcoma Study Group. Arch Intern Med 156, 202204.[Abstract]
Darnell, J. E., Jr, Kerr, I. M. & Stark, G. R. (1994). Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 264, 14151421.[Medline]
Deng, H., Young, A. & Sun, R. (2000). Auto-activation of the rta gene of human herpesvirus-8/Kaposi's sarcoma-associated herpesvirus. J Gen Virol 81, 30433048.
Dupin, N., Grandadam, M., Calvez, V. & 8 other authors (1995). Herpesvirus-like DNA in patients with Mediterranean Kaposi's sarcoma. Lancet 345, 761762.[CrossRef][Medline]
Flemington, E. & Speck, S. H. (1990). Autoregulation of EpsteinBarr virus putative lytic switch gene BZLF1. J Virol 64, 12271232.[Medline]
Fujita, T., Kimura, Y., Miyamoto, M., Barsoumian, E. L. & Taniguchi, T. (1989). Induction of endogenous IFN-alpha and IFN-beta genes by a regulatory transcription factor, IRF-1. Nature 337, 270272.[CrossRef][Medline]
Gao, S. J., Boshoff, C., Jayachandra, S., Weiss, R. A., Chang, Y. & Moore, P. S. (1997). KSHV ORF K9 (vIRF) is an oncogene which inhibits the interferon signaling pathway. Oncogene 15, 19791985.[CrossRef][Medline]
Gessain, A., Sudaka, A., Briere, J. & 8 other authors (1996). Kaposi sarcoma-associated herpes-like virus (human herpesvirus type 8) DNA sequences in multicentric Castleman's disease: is there any relevant association in non-human immunodeficiency virus-infected patients? Blood 87, 414416.
Harada, H., Fujita, T., Miyamoto, M., Kimura, Y., Maruyama, M., Furia, A., Miyata, T. & Taniguchi, T. (1989). Structurally similar but functionally distinct factors, IRF-1 and IRF-2, bind to the same regulatory elements of IFN and IFN-inducible genes. Cell 58, 729739.[Medline]
Imam, A. M., Ackrill, A. M., Dale, T. C., Kerr, I. M. & Stark, G. R. (1990). Transcription factors induced by interferons alpha and gamma. Nucleic Acids Res 18, 65736580.[Abstract]
Jayachandra, S., Low, K. G., Thlick, A. E., Yu, J., Ling, P. D., Chang, Y. & Moore, P. S. (1999). Three unrelated viral transforming proteins (vIRF, EBNA2 and E1A) induce the MYC oncogene through the interferon-responsive PRF element by using different transcription coadaptors. Proc Natl Acad Sci U S A 96, 1156611571.
Karcher, D. S. & Alkan, S. (1995). Herpes-like DNA sequences, AIDS-related tumors, and Castleman's disease. N Engl J Med 333, 797798.
Li, M., Lee, H., Guo, J., Neipel, F., Fleckenstein, B., Ozato, K. & Jung, J. U. (1998). Kaposi's sarcoma-associated herpesvirus viral interferon regulatory factor. J Virol 72, 54335440.
Li, M., Damania, B., Alvarez, X., Ogryzko, V., Ozato, K. & Jung, J. U. (2000). Inhibition of p300 histone acetyltransferase by viral interferon regulatory factor. Mol Cell Biol 20, 82548263.
Lin, R., Genin, P., Mamane, Y., Sgarbanti, M., Battistini, A., Harrington, W. J., Jr, Barber, G. N. & Hiscott, J. (2001). HHV-8 encoded vIRF-1 represses the interferon antiviral response by blocking IRF-3 recruitment of the CBP/p300 coactivators. Oncogene 20, 800811.[CrossRef][Medline]
Moore, P. S. & Chang, Y. (1995). Detection of herpesvirus-like DNA sequences in Kaposi's sarcoma lesions from persons with and without HIV infection. N Engl J Med 332, 11811185.
Nador, R. G., Cesarman, E., Knowles, D. M. & Said, J. W. (1995). Herpes-like DNA sequences in a body-cavity-based lymphoma in an HIV-negative patient. N Engl J Med 333, 943.
Nakamura, H., Li, M., Zarycki, J. & Jung, J. U. (2001). Inhibition of p53 tumor suppressor by viral interferon regulatory factor. J Virol 75, 75727582.
Nguyen, H., Mustafa, A., Hiscott, J. & Lin, R. (1995). Transcription factor IRF-2 exerts its oncogenic phenotype through the DNA binding/transcription repression domain. Oncogene 11, 537544.[Medline]
Pastore, C., Gloghini, A., Volpe, G., Nomdedeu, J., Leonardo, E., Mazza, U., Saglio, G., Carbone, A. & Gaidano, G. (1995). Distribution of Kaposi's sarcoma herpesvirus sequences among lymphoid malignancies in Italy and Spain. Br J Haematol 91, 918920.[Medline]
Pine, R., Decker, T., Kessler, D. S., Levy, D. E. & Darnell, J. J. (1990). Purification and cloning of interferon-stimulated gene factor 2 (ISGF2): ISGF2 (IRF-1) can bind to the promoters of both beta interferon- and interferon-stimulated genes but is not a primary transcriptional activator of either. Mol Cell Biol 10, 24482457.[Medline]
Ragoczy, T. & Miller, G. (2001). Autostimulation of the EpsteinBarr virus BRLF1 promoter is mediated through consensus Sp1 and Sp3 binding sites. J Virol 75, 52405251.
Renne, R., Zhong, W., Herndier, B., McGrath, M., Abbey, N., Kedes, D. & Ganem, D. (1996). Lytic growth of Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) in culture. Nature Med 2, 342346.[Medline]
Roan, F., Zimring, J. C., Goodbourn, S. & Offermann, M. K. (1999). Transcriptional activation by the human herpesvirus-8-encoded interferon regulatory factor. J Gen Virol 80, 22052209.
Russo, J. J., Bohenzky, R. A., Chien, M. C. & 8 other authors (1996). Nucleotide sequence of the Kaposi sarcoma-associated herpesvirus (HHV8). Proc Natl Acad Sci U S A 93, 1486214867.
Schalling, M., Ekman, M., Kaaya, E. E., Linde, A. & Biberfeld, P. (1995). A role for a new herpesvirus (KSHV) in different forms of Kaposi's sarcoma. Nature Med 1, 707708.[Medline]
Seo, T., Lee, D., Lee, B., Chung, J. H. & Choe, J. (2000). Viral interferon regulatory factor 1 of Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) binds to, and inhibits transactivation of, CREB-binding protein. Biochem Biophys Res Commun 270, 2327.[CrossRef][Medline]
Seo, T., Park, J., Lee, D., Hwang, S. G. & Choe, J. (2001). Viral interferon regulatory factor 1 of Kaposi's sarcoma-associated herpesvirus binds to p53 and represses p53-dependent transcription and apoptosis. J Virol 75, 61936198.
Soulier, J., Grollet, L., Oksenhendler, E. & 7 other authors (1995). Kaposi's sarcoma-associated herpesvirus-like DNA sequences in multicentric Castleman's disease. Blood 86, 12761280.
Taniguchi, T. (1995). IRF-1 and IRF-2 as regulators of the interferon system and cell growth. Indian J Biochem Biophys 32, 235239.[Medline]
Taniguchi, T., Tanaka, N. & Taki, S. (1998). Regulation of the interferon system, immune response and oncogenesis by the transcription factor interferon regulatory factor-1. Eur Cytokine Netw 9, 4348.[Medline]
Wang, X., Zhang, Y., Deng, J., Pan, H., Zhou, F., Montalvo, E. A. & Gao, S. J. (2001). Characterization of the promoter region of viral interferon regulatory factor encoded by Kaposi's sarcoma-associated herpesvirus. Oncogene 20, 523530.[CrossRef][Medline]
Wang, X. P., Zhang, Y. J., Deng, J. H., Pan, H. Y., Zhou, F. C. & Gao, S. J. (2002). Transcriptional regulation of Kaposi's sarcoma-associated herpesvirus-encoded oncogene viral interferon regulatory factor by a novel transcriptional silencer, Tis. J Biol Chem 277, 1202312031.
Zimring, J. C., Goodbourn, S. & Offermann, M. K. (1998). Human herpesvirus 8 encodes an interferon regulatory factor (IRF) homolog that represses IRF-1-mediated transcription. J Virol 72, 701707.
Received 12 June 2002;
accepted 4 September 2002.