Department of Molecular & Medical Pharmacology, UCLA AIDS Institute, Jonsson Comprehensive Cancer Center, and the Molecular Biology Institute, University of California at Los Angeles, Los Angeles, CA 90095, USA1
Author for correspondence: Ren Sun (at Department of Molecular & Medical Pharmacology). Fax +1 310 825 6267. e-mail rsun{at}mednet.ucla.edu
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() |
---|
![]() |
Main text |
---|
![]() ![]() ![]() ![]() |
---|
A number of cell lines derived from PEL carry HHV-8 predominantly in a latent state (Arvanitakis et al., 1996 ; Boshoff et al., 1998
; Cesarman et al., 1995b
; Renne et al., 1996
; Said et al., 1996
). However, treatment of these cells with chemicals such as 12-O-tetradecanoylphorbol 13-acetate (TPA) or sodium butyrate induces HHV-8 to initiate lytic replication in a subset of the cell population (Miller et al., 1997
; Nicholas et al., 1997
; Renne et al., 1996
; Sarid et al., 1998
). Using these cell lines, several immediate-early genes have been identified (Lukac et al., 1999
; Sun et al., 1998
; Zhu et al., 1999
). However, a biological function has only been demonstrated for the product of one immediate-early gene, rta. Expression of Rta alone in latently infected cells disrupted latency and activated the expression of viral lytic genes; induction of a viral late gene, ORF65, by Rta indicated that Rta drives the lytic cycle to completion (Lukac et al., 1998
; Sun et al., 1998
). In addition, introduction of a dominant-negative mutant of Rta into latently infected cells abolished viral reactivation (Lukac et al., 1999
). Therefore, HHV-8 Rta is both necessary and sufficient to mediate the switch from latency to lytic replication.
Rta is highly conserved among gammaherpesviruses (Nicholas et al., 1991 ; Sun et al., 1998
; Telford et al., 1995
; van Santen, 1993
; Wu et al., 2000
). Recently, it was shown that Rta of murine gammaherpesvirus 68 is also capable of disrupting latency and driving the lytic cycle to completion (Wu et al., 2000
). In EpsteinBarr virus, Rta (also called BRLF1 or R) and ZEBRA (also termed BZLF1, Zta, Z or EB1) are the earliest viral gene products synthesized during virus reactivation. They both exhibit auto-activation and cross-activation in certain cell lines. These two proteins in turn act in a cooperative manner to synergistically activate a cascade of lytic gene expression (Chevallier-Greco et al., 1986
; Cox et al., 1990
; Flemington & Speck, 1990
; Hardwick et al., 1988
; Holley-Guthrie et al., 1990
; Manet et al., 1989
; Ragoczy et al., 1998
; Zalani et al., 1996
). However, unlike Rta, a homologue of the ZEBRA protein has only been identified in HHV-8 among gamma2-herpesviruses (Gruffat et al., 1999
; Lin et al., 1999
; Sun et al., 1998
). Ectopic expression of this protein, K-bZip, did not disrupt latency (Sun et al., 1998
). Moreover, K-bZip was shown to be an early protein whose expression was activated by Rta, placing K-bZip kinetically downstream of Rta (Sun et al., 1999
). These studies highlight the conservation and critical role of Rta in controlling the balance between latency and lytic replication.
Functional identification of the rta gene of HHV-8 has laid the foundation for us to dissect the mechanism of HHV-8 reactivation. Two groups of experiments have been undertaken, one to identify viral genes that are activated by Rta, and the other to study the regulation of rta gene expression. The convergence of these two groups of studies led to our discovery that Rta positively regulates its own expression.
The major coding region for the Rta protein is located in ORF50 of the HHV-8 genome. A splicing event that removes ORF49 introduces a new methionine initiation codon at nucleotide 71596 [all positions cited are according to the published sequence (Russo et al., 1996 )], adding 6 amino acids in exon 1 plus an additional 54 amino acids in exon 2 to ORF50 (Lukac et al., 1999
; Sun et al., 1998
; Zhu et al., 1999
). A 3 kb sequence upstream of the Rta translation initiation codon was amplified from total cellular DNA prepared from BC-1 cells, using primers NheRta (5' acgctagcGCTCCTTCAATTGGAAGCA 3') and BglRta2 (5' acaagatctTTGTGGCTGCCTGGACAGTATTC 3'). The PCR fragment was cloned into the pGL2-basic vector (Promega), with NheI and BglII as cloning sites, to derive pRpluc. pGL2-basic has only the coding sequence for firefly luciferase. pRpluc was responsive to TPA or sodium butyrate, which are inducers of HHV-8 lytic replication (H. Deng & R. Sun, unpublished results). Therefore, pRpluc can be used to study the transcriptional regulation of the rta promoter.
The reporter plasmid pRpluc (7·5 µg) was electroporated into 107 HHV-8 latently infected KS-1 or BCBL-1 cells, with either an Rta expression plasmid, pcDNA3/Rta (Sun et al., 1998 ), or the pcDNA3 vector (2·5 µg). Alternatively, 200 ng each of pRpluc and expression plasmid were co-transfected into an HHV-8-negative cell line, 293T, in 24-well plates, using LipofectAmine PLUS (Gibco). A control plasmid, pRL-CMV, which constitutively expresses Renilla luciferase, was included in each transfection. Cells were harvested at 48 h post-transfection, and Dual-Luciferase Reporter Assays (Promega) were performed. The rta promoter was activated 144·4-fold by the Rta protein in BCBL-1 cells, and 38·7-fold in KS-1 cells. In 293T, the rta promoter was activated 40·3-fold (Fig. 1A
). Therefore, the Rta protein activated the rta promoter independently of a B cell-specific or other virus-specific factor. Seaman et al. (1999)
previously reported that in BCBL-1 cells an rta promoter construct, p.50p-CAT, was activated 11·6-fold by Rta. However, activation of p.50p-CAT by pcDNA3 was 4·27-fold. Thus, the -fold activation by Rta was 2·7, in contrast to the 144-fold activation of pRpluc by Rta that we demonstrated using the same cell line. One difference between these two studies is that p.50p-CAT only contains a 655 bp fragment upstream of the Rta coding region.
|
Results from the reporter system demonstrated that Rta activated the rta promoter from a plasmid that lacks chromatin structure. We next examined whether Rta can activate the expression of the rta gene from endogenous viral genomes. To distinguish between the rta transcripts from the endogenous viral genomes and the transcripts from transfected pcDNA3/Rta (Fig. 2A), we cloned the 5' untranslated region (nucleotides 7142971594) of rta into pBluescript KS(-) (Stratagene), to generate riboprobes for ribonuclease protection assays (RPAs).
|
Previous studies have identified the transcription initiation site of the rta gene at nucleotide 71513 in BC-1 cells using 5' RACE (Sun et al., 1998 ; Zhu et al., 1999
), or at nucleotide 71560 in BCBL-1 cells using 5' RACE and primer extension (Lukac et al., 1999
). In our RPAs, sequencing ladders were run in parallel to determine the sizes of the protected fragments. With correction for the slower mobility of RNA compared to DNA of the same length in a denaturing polyacrylamide gel, the shortest protected fragments (the doublet bands in Fig. 2 B
, arrowhead) represented transcripts initiated at nucleotides 71489 and 71490 in both KS-1 and BCBL-1 cells. This result was reproducible in three independent experiments. No shorter specific bands were observed. The discrepancy between previous results and our RPA result most likely stems from differences in the experimental methods. In 5' RACE and primer extension experiments, reverse transcription reactions are usually performed at 37 or 42 °C, temperatures at which RNA secondary structures may not be completely disrupted. Moreover, consistent with our result, there is a putative TATA box (TAAATA) at nucleotide 71452, 3738 nucleotides upstream of our mapped transcription initiation sites.
The upper bands in RPAs represent protection of the complete viral sequence in the riboprobe (reproducible in three independent experiments). The appearance of the multiple bands was likely caused by end-breathing of the RNARNA hybrids and partial protection at the end of the RNA duplexes. Since gel-purified, full-length riboprobes were utilized, the doublet bands and the upper bands presumably reflected heterogeneity of the rta transcripts. Several open reading frames immediately upstream of nucleotide position 71489 (ORFs 4548) are all transcribed in the opposite orientation to ORF50, and would not be protected by the antisense rta probe. Furthermore, all the protected transcripts from chemically induced cells were resistant to cycloheximide, a protein synthesis inhibitor (H. Deng & R. Sun, unpublished results), indicating that there are multiple transcription initiation sites for the rta gene. Our results are also consistent with previous reports of a complex transcription pattern across the rta locus of HHV-8 (Sun et al., 1998 ; Zhu et al., 1999
). A precedent for this phenomenon is the herpesvirus saimiri rta homologue, which is transcribed from two distinct promoters (Whitehouse et al., 1997
).
The rta gene was classified as an immediate-early gene, based on the resistance of its expression to cycloheximide (Sun et al., 1998 , 1999
; Zhu et al., 1999
). However, it was noted that although treatment with cycloheximide did not abolish expression of the rta gene, it did cause a reduction in the level of rta gene expression in BC-1 cells. Rta auto-activation provides one explanation for this observation.
In summary, we have demonstrated Rta auto-activation of HHV-8, using two independent approaches. Thus, Rta establishes a positive feedback loop in the cascade of viral lytic gene expression. Auto-activation of rta, the master switch gene, may be an important strategy for the virus to amplify environmental stimuli, thereby allowing the virus to be efficiently reactivated from latency. Further analysis of the regulation of HHV-8 rta gene expression will help elucidate the mechanism controlling the dynamic balance between latency and lytic replication.
![]() |
Acknowledgments |
---|
We thank members of the Sun laboratory and Wendy Aft for helpful discussions and critical reading of the manuscript. This work was supported by the UCLA AIDS Institute and the Stop Cancer Award. H.D. is supported by a Tumor Immunology Training Grant (T32 CA0912024).
![]() |
References |
---|
![]() ![]() ![]() ![]() |
---|
Boshoff, C., Gao, S. J., Healy, L. E., Matthews, S., Thomas, A. J., Coignet, L., Warnke, R. A., Strauchen, J. A., Matutes, E., Kamel, O. W., Moore, P. S., Weiss, R. A. & Chang, Y.(1998). Establishing a KSHV+ cell line (BCP-1) from peripheral blood and characterizing its growth in Nod/SCID mice.Blood91, 1671-1679.
Cesarman, E., Chang, Y., Moore, P. S., Said, J. W. & Knowles, D. M.(1995a). Kaposis sarcoma-associated herpesvirus-like DNA sequences in AIDS-related body-cavity-based lymphomas.New England Journal of Medicine332, 1186-1191.
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 Kaposis sarcoma-associated herpesvirus-like (KSHV) DNA sequences.Blood86, 2708-2714.
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 Kaposis sarcoma.Science266, 1865-1869.[Medline]
Chevallier-Greco, A., Manet, E., Chavrier, P., Mosnier, C., Daillie, J. & Sergeant, A.(1986). Both EpsteinBarr virus (EBV)-encoded trans-acting factors, EB1 and EB2, are required to activate transcription from an EBV early promoter.EMBO Journal5, 3243-3249.[Abstract]
Cox, M. A., Leahy, J. & Hardwick, J. M.(1990). An enhancer within the divergent promoter of EpsteinBarr virus responds synergistically to the R and Z transactivators.Journal of Virology64, 313-321.[Medline]
Flemington, E. & Speck, S. H.(1990). Autoregulation of EpsteinBarr virus putative lytic switch gene BZLF1. Journal of Virology64, 1227-1232.[Medline]
Gruffat, H., Portes-Sentis, S., Sergeant, A. & Manet, E.(1999). Kaposis sarcoma-associated herpesvirus (human herpesvirus-8) encodes a homologue of the EpsteinBarr virus bZip protein EB1.Journal of General Virology80, 557-561.[Abstract]
Hardwick, J. M., Lieberman, P. M. & Hayward, S. D.(1988). A new EpsteinBarr virus transactivator, R, induces expression of a cytoplasmic early antigen.Journal of Virology62, 2274-2284.[Medline]
Holley-Guthrie, E. A., Quinlivan, E. B., Mar, E. C. & Kenney, S.(1990). The EpsteinBarr virus (EBV) BMRF1 promoter for early antigen (EA-D) is regulated by the EBV transactivators, BRLF1 and BZLF1, in a cell- specific manner. Journal of Virology64, 3753-3759.[Medline]
Lin, S. F., Robinson, D. R., Miller, G. & Kung, H. J.(1999). Kaposis sarcoma-associated herpesvirus encodes a bZIP protein with homology to BZLF1 of EpsteinBarr virus.Journal of Virology73, 1909-1917.
Lukac, D. M., Renne, R., Kirshner, J. R. & Ganem, D.(1998). Reactivation of Kaposis sarcoma-associated herpesvirus infection from latency by expression of the ORF 50 transactivator, a homolog of the EBV R protein. Virology252, 304-312.[Medline]
Lukac, D. M., Kirshner, J. R. & Ganem, D.(1999). Transcriptional activation by the product of open reading frame 50 of Kaposis sarcoma-associated herpesvirus is required for lytic viral reactivation in B cells.Journal of Virology73, 9348-9361.
Manet, E., Gruffat, H., Trescol, B. M. C., Moreno, N., Chambard, P., Giot, J. F. & Sergeant, A.(1989). EpsteinBarr virus bicistronic mRNAs generated by facultative splicing code for two transcriptional trans-activators.EMBO Journal8, 1819-1826.[Abstract]
Miller, G., Heston, L., Grogan, E., Gradoville, L., Rigsby, M., Sun, R., Shedd, D., Kushnaryov, V. M., Grossberg, S. & Chang, Y.(1997). Selective switch between latency and lytic replication of Kaposis sarcoma herpesvirus and EpsteinBarr virus in dually infected body cavity lymphoma cells. Journal of Virology71, 314-324.[Abstract]
Neipel, F. & Fleckenstein, B.(1999). The role of HHV-8 in Kaposis sarcoma.Seminars in Cancer Biology9, 151-164.[Medline]
Nicholas, J., Coles, L. S., Newman, C. & Honess, R. W.(1991). Regulation of the herpesvirus saimiri (HVS) delayed-early 110- kilodalton promoter by HVS immediate-early gene products and a homolog of the EpsteinBarr virus R trans activator.Journal of Virology65, 2457-2466.[Medline]
Nicholas, J., Ruvolo, V., Zong, J., Ciufo, D., Guo, H. G., Reitz, M. S. & Hayward, G. S.(1997). A single 13-kilobase divergent locus in the Kaposi sarcoma-associated herpesvirus (human herpesvirus 8) genome contains nine open reading frames that are homologous to or related to cellular proteins.Journal of Virology71, 1963-1974.[Abstract]
Ragoczy, T., Heston, L. & Miller, G.(1998). The EpsteinBarr virus Rta protein activates lytic cycle genes and can disrupt latency in B lymphocytes.Journal of Virology72, 7978-7984.
Renne, R., Zhong, W., Herndier, B., McGrath, M., Abbey, N., Kedes, D. & Ganem, D.(1996). Lytic growth of Kaposis sarcoma-associated herpesvirus (human herpesvirus 8) in culture.Nature Medicine2, 342-346.[Medline]
Russo, J. J., Bohenzky, R. A., Chien, M. C., Chen, J., Yan, M., Maddalena, D., Parry, J. P., Peruzzi, D., Edelman, I. S., Chang, Y. & Moore, P. S.(1996). Nucleotide sequence of the Kaposi sarcoma-associated herpesvirus (HHV8).Proceedings of the National Academy of Sciences, USA93, 14862-14867.
Said, W., Chien, K., Takeuchi, S., Tasaka, T., Asou, H., Cho, S. K., de Vos, S., Cesarman, E., Knowles, D. M. & Koeffler, H. P.(1996). Kaposis sarcoma-associated herpesvirus (KSHV or HHV8) in primary effusion lymphoma: ultrastructural demonstration of herpesvirus in lymphoma cells.Blood87, 4937-4943.
Sarid, R., Flore, O., Bohenzky, R. A., Chang, Y. & Moore, P. S.(1998). Transcription mapping of the Kaposis sarcoma-associated herpesvirus (human herpesvirus 8) genome in a body cavity-based lymphoma cell line (BC-1). Journal of Virology72, 1005-1012.
Schulz, T. F. & Moore, P. S.(1999). Kaposis sarcoma-associated herpesvirus: a new human tumor virus, but how? Trends in Microbiology7, 196-200.[Medline]
Seaman, W. T., Ye, D., Wang, R. X., Hale, E. E., Weisse, M. & Quinlivan, E. B.(1999). Gene expression from the ORF50/K8 region of Kaposis sarcoma-associated herpesvirus.Virology263, 436-449.[Medline]
Soulier, J., Grollet, L., Oksenhendler, E., Cacoub, P., Cazals-Hatem, D., Babinet, P., dAgay, M. F., Clauvel, J. P., Raphael, M., Degos, L. and others (1995). Kaposis sarcoma-associated herpesvirus-like DNA sequences in multicentric Castlemans disease. Blood 86, 12761280.
Sun, R., Lin, S. F., Gradoville, L., Yuan, Y., Zhu, F. & Miller, G.(1998). A viral gene that activates lytic cycle expression of Kaposis sarcoma-associated herpesvirus.Proceedings of the National Academy of Sciences, USA95, 10866-10871.
Sun, R., Lin, S. F., Staskus, K., Gradoville, L., Grogan, E., Haase, A. & Miller, G.(1999). Kinetics of Kaposis sarcoma-associated herpesvirus gene expression.Journal of Virology73, 2232-2242.
Telford, E. A., Watson, M. S., Aird, H. C., Perry, J. & Davison, A. J.(1995). The DNA sequence of equine herpesvirus 2.Journal of Molecular Biology249, 520-528.[Medline]
van Santen, V. L.(1993). Characterization of a bovine herpesvirus 4 immediate-early RNA encoding a homolog of the EpsteinBarr virus R transactivator.Journal of Virology67, 773-784.[Abstract]
Vieira, J., Huang, M. L., Koelle, D. M. & Corey, L.(1997). Transmissible Kaposis sarcoma-associated herpesvirus (human herpesvirus 8) in saliva of men with a history of Kaposis sarcoma.Journal of Virology71, 7083-7087.[Abstract]
Whitehouse, A., Carr, I. M., Griffiths, J. C. & Meredith, D. M.(1997). The herpesvirus saimiri ORF50 gene, encoding a transcriptional activator homologous to the EpsteinBarr virus R protein, is transcribed from two distinct promoters of different temporal phases.Journal of Virology71, 2550-2554.[Abstract]
Wu, T. T., Usherwood, E. J., Stewart, J. P., Nash, A. A. & Sun, R.(2000). Rta of murine gammaherpesvirus 68 reactivates the complete lytic cycle from latency.Journal of Virology74, 3659-3667.
Zalani, S., Holley-Guthrie, E. & Kenney, S.(1996). EpsteinBarr viral latency is disrupted by the immediate-early BRLF1 protein through a cell-specific mechanism.Proceedings of the National Academy of Sciences, USA93, 9194-9199.
Zhong, W., Wang, H., Herndier, B. & Ganem, D.(1996). Restricted expression of Kaposi sarcoma-associated herpesvirus (human herpesvirus 8) genes in Kaposi sarcoma.Proceedings of the National Academy of Sciences, USA93, 6641-6646.
Zhu, F. X., Cusano, T. & Yuan, Y.(1999). Identification of the immediate-early transcripts of Kaposis sarcoma-associated herpesvirus. Journal of Virology73, 5556-5567.
Received 24 May 2000;
accepted 24 August 2000.