Hepatitis B Virus pX Interacts with HBXAP, a PHD Finger Protein to Coactivate Transcription
2002; Elsevier BV; Volume: 277; Issue: 12 Linguagem: Inglês
10.1074/jbc.m111354200
ISSN1083-351X
AutoresMeir Shamay, Orr Barak, Gilad Doitsh, Israel Ben‐Dor, Yosef Shaul,
Tópico(s)Ubiquitin and proteasome pathways
ResumoHepatitis B virus (HBV) gene expression is mainly regulated at the transcription initiation level. The viral X protein (pX) is a transcription coactivator/mediator targeting TFIIB for the recruitment of RNA polymerase II. Here we report a novel pX nuclear target designated HBXAP (hepatitis B virus X-associated protein). HBXAP is a novel cellular nuclear protein containing a PHD (planthomology domain) finger, a domain shared by many proteins that play roles in chromatin remodeling, transcription coactivation, and oncogenesis. pX physically interacts with HBXAPin vitro and in vivo via the HBXAP region containing the PHD finger. At the functional level HBXAP increases HBV transcription in a pX-dependent manner suggesting a role for this interaction in the virus life cycle. Interestingly, HBXAP collaborates with pX in coactivating the transcriptional activator NF-κB. Coactivation of NF-κB was also observed in tumor necrosis factor α-treated cells suggesting that pX-HBXAP functional collaboration localized downstream to the NF-κB nuclear import. Collectively our data suggest that pX recruits and potentiates a novel putative transcription coactivator to regulate NF-κB. The implication of pX-HBXAP interaction in the development of hepatocellular carcinoma is discussed. Hepatitis B virus (HBV) gene expression is mainly regulated at the transcription initiation level. The viral X protein (pX) is a transcription coactivator/mediator targeting TFIIB for the recruitment of RNA polymerase II. Here we report a novel pX nuclear target designated HBXAP (hepatitis B virus X-associated protein). HBXAP is a novel cellular nuclear protein containing a PHD (planthomology domain) finger, a domain shared by many proteins that play roles in chromatin remodeling, transcription coactivation, and oncogenesis. pX physically interacts with HBXAPin vitro and in vivo via the HBXAP region containing the PHD finger. At the functional level HBXAP increases HBV transcription in a pX-dependent manner suggesting a role for this interaction in the virus life cycle. Interestingly, HBXAP collaborates with pX in coactivating the transcriptional activator NF-κB. Coactivation of NF-κB was also observed in tumor necrosis factor α-treated cells suggesting that pX-HBXAP functional collaboration localized downstream to the NF-κB nuclear import. Collectively our data suggest that pX recruits and potentiates a novel putative transcription coactivator to regulate NF-κB. The implication of pX-HBXAP interaction in the development of hepatocellular carcinoma is discussed. hepatitis B virus TBP-associated factor Sos recruitment system plant homology domain glutathioneS-transferase tetramethyl rhodamine isothiocyanate hepatitis B virus X-associated protein histone deacetylase hemagglutinin tumor necrosis factor-α Study of the interactions of the virus with the host cell is a promising approach to understand, at the molecular level, how the virus evades the defense strategies of the cells they infect. Hepatitis B virus (HBV)1 is a hepatotropic virus containing a partially double-stranded circular DNA genome that causes both acute and chronic hepatic injuries. Persistent HBV infection is strongly associated with the development of hepatocellular carcinoma (1Ganem D. Varmus H.E. Annu. Rev. Biochem. 1987; 56: 651-693Crossref PubMed Scopus (813) Google Scholar). The compact 3.2-kbp genome contains enhancers and multiple promoters that are regulated by the cellular transcription machinery. HBV encodes a single known regulatory polypeptide, called pX (or HBx). pX is likely to be an important regulatory protein since its sequence is conserved among the mammalian hepadnaviridae members. A debate exists regarding the role of this protein in the HBV life-cycle, however, it is evident that at least, in woodchucks, pX plays an essential role in woodchuck HBV infection (2Chen H.S. Kaneko S. Girones R. Anderson R.W. Hornbuckle W.E. Tennant B.C. Cote P.J. Gerin J.L. Purcell R.H. Miller R.H. J. Virol. 1993; 67: 1218-1226Crossref PubMed Google Scholar,3Zoulim F. Saputelli J. Seeger C. J. Virol. 1994; 68: 2026-2030Crossref PubMed Google Scholar).At the cellular level pX supports transcription and signaling (for review, see Ref. 4Murakami S. J. Gastroenterol. 2001; 36: 651-660Crossref PubMed Scopus (287) Google Scholar). pX increases HBV transcription by trans-activating the viral enhancer-I via the sequence named the E-element (5Faktor O. Shaul Y. Oncogene. 1990; 5: 867-872PubMed Google Scholar). This element binds a number of bZip cellular transcription activators including cAMP-response element-binding protein (CREB) and activating transcription factor (ATF) whose binding is potentiated by the presence of pX (6Faktor O. Budlovsky S. Ben-Levy R. Shaul Y. J. Virol. 1990; 64: 1861-1863Crossref PubMed Google Scholar, 7Maguire H.F. Hoeffler J.P. Siddiqui A. Science. 1991; 252: 842-844Crossref PubMed Scopus (378) Google Scholar, 8Williams J.S. Andrisani O.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3819-3823Crossref PubMed Scopus (146) Google Scholar). In addition, pX activates transcription through NF-κB (5Faktor O. Shaul Y. Oncogene. 1990; 5: 867-872PubMed Google Scholar, 9Twu J.S. Chu K. Robinson W.S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5168-5172Crossref PubMed Scopus (92) Google Scholar, 10Siddiqui A. Gaynor R. Srinivasan A. Mapoles J. Farr R.W. Virology. 1989; 169: 479-484Crossref PubMed Scopus (107) Google Scholar, 11Lucito R. Schneider R.J. J. Virol. 1992; 66: 983-991Crossref PubMed Google Scholar). pX also interacts with the general transcription factors (12Haviv I. Vaizel D. Shaul Y. EMBO J. 1996; 15: 3413-3420Crossref PubMed Scopus (87) Google Scholar), consistent with its transcription coactivation function (13Haviv I. Vaizel D. Shaul Y. Mol. Cell. Biol. 1995; 15: 1079-1085Crossref PubMed Google Scholar). Interaction of pX with TFIIH was reported by a number of groups (12Haviv I. Vaizel D. Shaul Y. EMBO J. 1996; 15: 3413-3420Crossref PubMed Scopus (87) Google Scholar, 14Wang X.W. Forrester K. Yeh H. Feitelson M.A. Gu J.R. Harris C.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2230-2234Crossref PubMed Scopus (632) Google Scholar, 15Qadri I. Conaway J.W. Conaway R.C. Schaack J. Siddiqui A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10578-10583Crossref PubMed Scopus (129) Google Scholar). This interaction may be relevant not only to the role of pX in transcription but also in DNA repair. Significantly, pX interacts simultaneously with TFIIB and RNA-polymerase II possibly to facilitate polymerase recruitment to promoters (16Lin Y. Nomura T. Cheong J. Dorjsuren D. Iida K. Murakami S. J. Biol. Chem. 1997; 272: 7132-7139Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 17Haviv I. Shamay M. Doitsh G. Shaul Y. Mol. Cell. Biol. 1998; 18: 1562-1569Crossref PubMed Scopus (127) Google Scholar).In addition to the transcription activators and general transcription factors a third group of proteins is needed to support transcription, collectively called coactivators or mediators. TBP-associated factors (TAFs) belong to this group of proteins. Interestingly, pX supports transcription in the absence of TAFs (12Haviv I. Vaizel D. Shaul Y. EMBO J. 1996; 15: 3413-3420Crossref PubMed Scopus (87) Google Scholar). In vitro studies on naked DNA templates revealed that pX functions in a TAF independent manner. In vivo, pX rescues the temperature-sensitive phenotype of the ts13 cell line which exhibits growth arrest at restrictive temperature due to a mutation in TAFII250. In addition, TBP mutants lacking TAF binding that poorly respond to activators exhibit wild type activity in the presence of pX, in vivo (18Haviv I. Matza Y. Shaul Y. Genes Dev. 1998; 12: 1217-1226Crossref PubMed Scopus (30) Google Scholar). Thus, pX can support transcription on chromatinized template in the absence of some of the transcription coactivators/mediators. While pX may be capable of alone coactivating transcription, it also may act by recruiting specific cellular coactivators. The latter possibility was addressed in this study by identifying pX-associated cellular proteins.The two-hybrid screen is a promising experimental approach to identifying additional pX interacting proteins. The conventional screen, although very powerful, is based on a transcriptional readout, and may provide spurious data when attempting to identify proteins that interact with general transcription factors, as pX does. Recently, a two-hybrid screen which relies on a cytoplasmic signaling event was described called the Sos Recruitment System (SRS). We utilized this system and isolated two clones that specifically and repeatedly interacted with pX in our screen. One of the isolated clones is the Tat-binding protein 1 that is described elsewhere (19Barak O. Aronheim A. Shaul Y. Virology. 2001; 283: 110-120Crossref PubMed Scopus (29) Google Scholar). Tat-binding protein-1 and its homologues, such as Sug1, are components of the proteasome 19S regulatory cap particle. These proteins have also been identified as transcription mediators. The second clone is a novel gene which we designated HBXAP (hepatitis B virus X-associated protein). HBXAP is a nucleoprotein of 240 kDa in size that contains a PHD/leukemia-associated protein finger, a motif shared by a variety of chromatin-associated proteins (20Aasland R. Gibson T.J. Stewart A.F. Trends Biochem. Sci. 1995; 20: 56-59Abstract Full Text PDF PubMed Scopus (747) Google Scholar). We show that pX interacts with HBXAP both in vitro and in vivo and that this interaction potentiates the ability of HBXAP to coactivate NF-κB.DISCUSSIONBoth viral and cellular regulatory proteins participate in multiple protein-protein interactions. In contrast to other viruses, such as HIV, which encode many regulatory proteins, only a few proteins control the HBV life cycle. Yet, it has an outstanding capacity to infect and propagate with an efficiency that by far exceeds that of HIV as calculated by particle number per ml of serum. Although many factors might be responsible for this efficacy, we may assume that it is in part due to pX, the HBV regulatory protein, which has an extraordinary capacity to perform multiple functions. In this report we provide evidence for a physical and functional interaction between pX and HBXAP, a novel cellular nuclear protein with the attributes of a transcription coactivator. pX, like many other viral regulators, interacts with cellular proteins to recruit the machinery needed to support viral propagation and to counteract the cellular defense systems. A favorable approach to investigate the molecular mechanisms of HBV-host cell interaction is the characterization of pX target cellular proteins. Previous biochemical experiments have revealed a number of pX-interacting cellular proteins that are components of the transcription machinery. However, none of the pX-interacting proteins isolated by the two-hybrid screen (26Cong Y.S. Yao Y.L. Yang W.M. Kuzhandaivelu N. Seto E. J. Biol. Chem. 1997; 272: 16482-16489Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 27Huang J. Kwong J. Sun E.C. Liang T.J. J. Virol. 1996; 70: 5582-5591Crossref PubMed Google Scholar, 28Lee T.H. Elledge S.J. Butel J.S. J. Virol. 1995; 69: 1107-1114Crossref PubMed Google Scholar, 29Kuzhandaivelu N. Cong Y.S. Inouye C. Yang W.M. Seto E. Nucleic Acids Res. 1996; 24: 4741-4750Crossref PubMed Scopus (94) Google Scholar, 30Melegari M. Scaglioni P.P. Wands J.R. J. Virol. 1998; 72: 1737-1743Crossref PubMed Google Scholar), are known components of this machinery. We hypothesized that the conventional two-hybrid screen that is based on a transcriptional readout may not be suitable for identifying transcriptional activators and effectors because the system may be compromised by pX activity. Given this rationale we utilized the SRS two-hybrid screen (31Aronheim A. Zandi E. Hennemann H. Elledge S.J. Karin M. Mol. Cell. Biol. 1997; 17: 3094-3102Crossref PubMed Scopus (389) Google Scholar) that acts outside the nucleus at the plasma membrane. Two proteins were isolated, both are nuclear and have the characteristics of transcription coactivators, one is the Tat-binding protein 1 (TBP-1) that is described elsewhere (19Barak O. Aronheim A. Shaul Y. Virology. 2001; 283: 110-120Crossref PubMed Scopus (29) Google Scholar) and the other HBXAP. Notably, recently we have identified three HBXAP isoforms designated α, β, and γ. In this study we used the HBXAPγ isoform that in comparison to α is N-terminal truncated (32Shamay M. Barak O. Shaul Y. GENOMICS. 2001; (in press)Google Scholar). Although the number of interactions in which pX is engaged is unexpectedly large, these multiple interactions appear to be a hallmark of viral regulatory proteins.Experiments were conducted to show that HBXAP is a genuine pX target. HBXAP physical interaction with pX was demonstrated in vitroand in vivo. We found that recombinant HBXAP is preferentially retained on the GST-pX column in a series of pull down experiments, suggesting that these proteins directly interact. The observed interaction must be specific and with relatively high affinity as HBXAP that is expressed in transfected cells is selectively retained by a GST-pX column. Furthermore, these proteins, when expressed in cells by co-transfection experiments are coimmunoprecipitated. HA-tagged HBXAP that is immunoprecipitated by anti-HA is associated with GFP-pX. Collectively, these data suggest that pX and HBXAP are in physical and direct contact.We provide evidence in support of the possibility that HBXAP and pX physical interaction has a functional significance. We show that HBXAP collaborates with HBV genome-encoded pX, to support HBV transcription. Co-transfection of HBV DNA with HBXAP expression vector resulted in an elevation in the level of the HBV transcripts. As this effect was not observed in the context of an HBV mutant that does not express pX, we assumed that pX is required for this process. This possibility was further supported by the fact that HBXAP activity was recapitulated by a co-transfected pX expressor plasmid. To gain mechanistic insight on the functional interaction between these proteins we employed reporter assays. Interestingly, functional collaboration between HBXAP and pX was observed in the context of HBV and NFκB-derived enhancers.To date several mechanisms were reported to explain the pX role in activating NF-κB (33Weil R. Sirma H. Giannini C. Kremsdorf D. Bessia C. Dargemont C. Brechot C. Israel A. Mol. Cell. Biol. 1999; 19: 6345-6354Crossref PubMed Google Scholar, 34Purcell N.H. Yu C. He D. Xiang J. Paran N. DiDonato J.A. Yamaoka S. Shaul Y. Lin A. Am. J. Physiol. Gastrointest. Liver Physiol. 2001; 280: G669-G677Crossref PubMed Google Scholar, 35Waris G. Huh K.W. Siddiqui A. Mol. Cell. Biol. 2001; 21: 7721-7730Crossref PubMed Scopus (280) Google Scholar). Here we provide evidence for the nuclear pX role in NF-κB coactivation. Previously it has been reported that at least a fraction of pX is localized in the nucleus (17Haviv I. Shamay M. Doitsh G. Shaul Y. Mol. Cell. Biol. 1998; 18: 1562-1569Crossref PubMed Scopus (127) Google Scholar) and that pX directly interacts with IκBα, which is able to transport it to the nucleus by a piggyback mechanism (33Weil R. Sirma H. Giannini C. Kremsdorf D. Bessia C. Dargemont C. Brechot C. Israel A. Mol. Cell. Biol. 1999; 19: 6345-6354Crossref PubMed Google Scholar). We took this event a step further to show that in the nucleus coactivation of NF-κB by pX is modulated by a novel transcription coactivator, HBXAP. We have performed a set of experiments to localize pX function along the NF-κB activation pathway. Given the fact that pX-HBXAP stimulate NF-κB activity in the presence of TNF-α, where the majority of NF-κB is nuclear, it is reasonable to localize the effect of this complex downstream to nuclear translocation of NF-κB. Thus, pX-HBXAP effect is restricted to the DNA-bound NF-κB, and therefore displays the attributes of transcription coactivators.The finding that HBXAP blocks TNF-α-induced NF-κB activation is rather interesting given the fact that some of the well described members of the PHD family, including Trithorax, ALL-1, TIF1, CBP, and p300, are transcription coactivators. However, a number of studies indicate that the PHD finger might be involved in transcription repression. The PHD and bromo-domain of KAP-1 form a cooperative unit with silencing activity. In that case KAP-1, mediated silencing requires association with NuRD and HDAC activity (36Schultz D.C. Friedman J.R. Rauscher F.J. Genes Dev. 2001; 15: 428-443Crossref PubMed Scopus (393) Google Scholar). Furthermore, the PHD finger in context with the bromo-domain of KAP-1 are sufficient to represses transcription when artificially recruited to DNA via GAL4-DBD (36Schultz D.C. Friedman J.R. Rauscher F.J. Genes Dev. 2001; 15: 428-443Crossref PubMed Scopus (393) Google Scholar). Mi-2β, a PHD finger protein was also reported to interact with HDAC1, and its two PHD fingers are essential but not sufficient for the interaction (23Zhang Y. LeRoy G. Seelig H.P. Lane W.S. Reinberg D. Cell. 1998; 95: 279-289Abstract Full Text Full Text PDF PubMed Scopus (680) Google Scholar). In addition, it was reported that the PHD-like motif in the DNA methyltransferase Dnmt3a, represses transcription via the recruitment of HDAC-1 (37Fuks F. Burgers W.A. Godin N. Kasai M. Kouzarides T. EMBO J. 2001; 20: 2536-2544Crossref PubMed Scopus (463) Google Scholar). Collectively, it appears that a number of PHD proteins are active in transcription silencing and that the PHD finger via interaction with HDAC is at least partially responsible for this function. Likewise, when we recruited HBXAP to DNA via GAL4-DBD it represses transcription. Furthermore, the PHD finger of HBXAP as an independent structural unit has very strong repression activity (32Shamay M. Barak O. Shaul Y. GENOMICS. 2001; (in press)Google Scholar). The fact that pX interacts with HBXAP in a region containing this domain might be significant. pX by binding the PHD finger may displace the associated HDAC corepressor to permit transcription activation, a switching that we have observed in the context of TNF-α-induced NF-κB activation.PHD containing proteins are often associated with cancer. Their chromosomal locations are amplified and rearranged in many tumors, including MOZ in leukemia (38Aguiar R.C. Chase A. Coulthard S. Macdonald D.H. Carapeti M. Reiter A. Sohal J. Lennard A. Goldman J.M. Cross N.C. Blood. 1997; 90: 3130-3135Crossref PubMed Google Scholar, 39Carapeti M. Aguiar R.C. Goldman J.M. Cross N.C. Blood. 1998; 91: 3127-3133Crossref PubMed Google Scholar), CBP in acute myologenous leukemia (40Giles R.H. Dauwerse J.G. Higgins C. Petrij F. Wessels J.W. Beverstock G.C. Dohner H. Jotterand-Bellomo M. Falkenburg J.H. Slater R.M. van Ommen G.J. Hagemeijer A. van der Reijden B.A. Breuning M.H. Leukemia. 1997; 11: 2087-2096Crossref PubMed Scopus (62) Google Scholar), and ALL-1 in acute lymphocytic leukemia (41Gu Y. Nakamura T. Alder H. Prasad R. Canaani O. Cimino G. Croce C.M. Canaani E. Cell. 1992; 71: 701-708Abstract Full Text PDF PubMed Scopus (789) Google Scholar). At the moment it is too early to conclude whether HBXAP is associated with cancer, but there is evidence in support of this possibility. The region 11q13.4–14.1, where the hbxap gene is located was reported to be amplified in 7–10% of breast cancer (42Szepetowski P. Ollendorff V. Grosgeorge J. Courseaux A. Birnbaum D. Theillet C. Gaudray P. Oncogene. 1992; 7: 2513-2517PubMed Google Scholar), amplified, and rearranged in multiple endocrine neoplasia type I syndrome (MEN1) (43Larsson C. Skogseid B. Oberg K. Nakamura Y. Nordenskjold M. Nature. 1988; 332: 85-87Crossref PubMed Scopus (896) Google Scholar), and B-cells malignancies. We have found the amplification of thehbxap gene in a few cases of breast cancers that we have analyzed (data not shown). However, since pX is the putative HBV oncogene, the behavior of this genomic locus in hepatocellular carcinomas is of particular interest. Therefore, we found it rather remarkable that 11q13 is amplified in 15% of HBV-positive tumors, but only a rare event in HCV-positive cases (44Kusano N. Shiraishi K. Kubo K. Oga A. Okita K. Sasaki K. Hepatology. 1999; 29: 1858-1862Crossref PubMed Scopus (196) Google Scholar). It has been suggested that this amplification is related to the progression of HBV-infected HCCs. Interestingly, we have identified in one of the HCC cell lines an HBV integrant containing a functional enhancer at this chromosomal locus (45Shamay M. Agami R. Shaul Y. Oncogene. 2001; 20: 6811-6819Crossref PubMed Scopus (37) Google Scholar). The possibility that the hbxap gene is indeed amplified in these HCCs or activated by the integrated HBV sequences is of highest interest and may shed new light on the mechanism of oncogenic function of HBV. Study of the interactions of the virus with the host cell is a promising approach to understand, at the molecular level, how the virus evades the defense strategies of the cells they infect. Hepatitis B virus (HBV)1 is a hepatotropic virus containing a partially double-stranded circular DNA genome that causes both acute and chronic hepatic injuries. Persistent HBV infection is strongly associated with the development of hepatocellular carcinoma (1Ganem D. Varmus H.E. Annu. Rev. Biochem. 1987; 56: 651-693Crossref PubMed Scopus (813) Google Scholar). The compact 3.2-kbp genome contains enhancers and multiple promoters that are regulated by the cellular transcription machinery. HBV encodes a single known regulatory polypeptide, called pX (or HBx). pX is likely to be an important regulatory protein since its sequence is conserved among the mammalian hepadnaviridae members. A debate exists regarding the role of this protein in the HBV life-cycle, however, it is evident that at least, in woodchucks, pX plays an essential role in woodchuck HBV infection (2Chen H.S. Kaneko S. Girones R. Anderson R.W. Hornbuckle W.E. Tennant B.C. Cote P.J. Gerin J.L. Purcell R.H. Miller R.H. J. Virol. 1993; 67: 1218-1226Crossref PubMed Google Scholar,3Zoulim F. Saputelli J. Seeger C. J. Virol. 1994; 68: 2026-2030Crossref PubMed Google Scholar). At the cellular level pX supports transcription and signaling (for review, see Ref. 4Murakami S. J. Gastroenterol. 2001; 36: 651-660Crossref PubMed Scopus (287) Google Scholar). pX increases HBV transcription by trans-activating the viral enhancer-I via the sequence named the E-element (5Faktor O. Shaul Y. Oncogene. 1990; 5: 867-872PubMed Google Scholar). This element binds a number of bZip cellular transcription activators including cAMP-response element-binding protein (CREB) and activating transcription factor (ATF) whose binding is potentiated by the presence of pX (6Faktor O. Budlovsky S. Ben-Levy R. Shaul Y. J. Virol. 1990; 64: 1861-1863Crossref PubMed Google Scholar, 7Maguire H.F. Hoeffler J.P. Siddiqui A. Science. 1991; 252: 842-844Crossref PubMed Scopus (378) Google Scholar, 8Williams J.S. Andrisani O.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3819-3823Crossref PubMed Scopus (146) Google Scholar). In addition, pX activates transcription through NF-κB (5Faktor O. Shaul Y. Oncogene. 1990; 5: 867-872PubMed Google Scholar, 9Twu J.S. Chu K. Robinson W.S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5168-5172Crossref PubMed Scopus (92) Google Scholar, 10Siddiqui A. Gaynor R. Srinivasan A. Mapoles J. Farr R.W. Virology. 1989; 169: 479-484Crossref PubMed Scopus (107) Google Scholar, 11Lucito R. Schneider R.J. J. Virol. 1992; 66: 983-991Crossref PubMed Google Scholar). pX also interacts with the general transcription factors (12Haviv I. Vaizel D. Shaul Y. EMBO J. 1996; 15: 3413-3420Crossref PubMed Scopus (87) Google Scholar), consistent with its transcription coactivation function (13Haviv I. Vaizel D. Shaul Y. Mol. Cell. Biol. 1995; 15: 1079-1085Crossref PubMed Google Scholar). Interaction of pX with TFIIH was reported by a number of groups (12Haviv I. Vaizel D. Shaul Y. EMBO J. 1996; 15: 3413-3420Crossref PubMed Scopus (87) Google Scholar, 14Wang X.W. Forrester K. Yeh H. Feitelson M.A. Gu J.R. Harris C.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2230-2234Crossref PubMed Scopus (632) Google Scholar, 15Qadri I. Conaway J.W. Conaway R.C. Schaack J. Siddiqui A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10578-10583Crossref PubMed Scopus (129) Google Scholar). This interaction may be relevant not only to the role of pX in transcription but also in DNA repair. Significantly, pX interacts simultaneously with TFIIB and RNA-polymerase II possibly to facilitate polymerase recruitment to promoters (16Lin Y. Nomura T. Cheong J. Dorjsuren D. Iida K. Murakami S. J. Biol. Chem. 1997; 272: 7132-7139Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 17Haviv I. Shamay M. Doitsh G. Shaul Y. Mol. Cell. Biol. 1998; 18: 1562-1569Crossref PubMed Scopus (127) Google Scholar). In addition to the transcription activators and general transcription factors a third group of proteins is needed to support transcription, collectively called coactivators or mediators. TBP-associated factors (TAFs) belong to this group of proteins. Interestingly, pX supports transcription in the absence of TAFs (12Haviv I. Vaizel D. Shaul Y. EMBO J. 1996; 15: 3413-3420Crossref PubMed Scopus (87) Google Scholar). In vitro studies on naked DNA templates revealed that pX functions in a TAF independent manner. In vivo, pX rescues the temperature-sensitive phenotype of the ts13 cell line which exhibits growth arrest at restrictive temperature due to a mutation in TAFII250. In addition, TBP mutants lacking TAF binding that poorly respond to activators exhibit wild type activity in the presence of pX, in vivo (18Haviv I. Matza Y. Shaul Y. Genes Dev. 1998; 12: 1217-1226Crossref PubMed Scopus (30) Google Scholar). Thus, pX can support transcription on chromatinized template in the absence of some of the transcription coactivators/mediators. While pX may be capable of alone coactivating transcription, it also may act by recruiting specific cellular coactivators. The latter possibility was addressed in this study by identifying pX-associated cellular proteins. The two-hybrid screen is a promising experimental approach to identifying additional pX interacting proteins. The conventional screen, although very powerful, is based on a transcriptional readout, and may provide spurious data when attempting to identify proteins that interact with general transcription factors, as pX does. Recently, a two-hybrid screen which relies on a cytoplasmic signaling event was described called the Sos Recruitment System (SRS). We utilized this system and isolated two clones that specifically and repeatedly interacted with pX in our screen. One of the isolated clones is the Tat-binding protein 1 that is described elsewhere (19Barak O. Aronheim A. Shaul Y. Virology. 2001; 283: 110-120Crossref PubMed Scopus (29) Google Scholar). Tat-binding protein-1 and its homologues, such as Sug1, are components of the proteasome 19S regulatory cap particle. These proteins have also been identified as transcription mediators. The second clone is a novel gene which we designated HBXAP (hepatitis B virus X-associated protein). HBXAP is a nucleoprotein of 240 kDa in size that contains a PHD/leukemia-associated protein finger, a motif shared by a variety of chromatin-associated proteins (20Aasland R. Gibson T.J. Stewart A.F. Trends Biochem. Sci. 1995; 20: 56-59Abstract Full Text PDF PubMed Scopus (747) Google Scholar). We show that pX interacts with HBXAP both in vitro and in vivo and that this interaction potentiates the ability of HBXAP to coactivate NF-κB. DISCUSSIONBoth viral and cellular regulatory proteins participate in multiple protein-protein interactions. In contrast to other viruses, such as HIV, which encode many regulatory proteins, only a few proteins control the HBV life cycle. Yet, it has an outstanding capacity to infect and propagate with an efficiency that by far exceeds that of HIV as calculated by particle number per ml of serum. Although many factors might be responsible for this efficacy, we may assume that it is in part due to pX, the HBV regulatory protein, which has an extraordinary capacity to perform multiple functions. In this report we provide evidence for a physical and functional interaction between pX and HBXAP, a novel cellular nuclear protein with the attributes of a transcription coactivator. pX, like many other viral regulators, interacts with cellular proteins to recruit the machinery needed to support viral propagation and to counteract the cellular defense systems. A favorable approach to investigate the molecular mechanisms of HBV-host cell interaction is the characterization of pX target cellular proteins. Previous biochemical experiments have revealed a number of pX-interacting cellular proteins that are components of the transcription machinery. However, none of the pX-interacting proteins isolated by the two-hybrid screen (26Cong Y.S. Yao Y.L. Yang W.M. Kuzhandaivelu N. Seto E. J. Biol. Chem. 1997; 272: 16482-16489Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 27Huang J. Kwong J. Sun E.C. Liang T.J. J. Virol. 1996; 70: 5582-5591Crossref PubMed Google Scholar, 28Lee T.H. Elledge S.J. Butel J.S. J. Virol. 1995; 69: 1107-1114Crossref PubMed Google Scholar, 29Kuzhandaivelu N. Cong Y.S. Inouye C. Yang W.M. Seto E. Nucleic Acids Res. 1996; 24: 4741-4750Crossref PubMed Scopus (94) Google Scholar, 30Melegari M. Scaglioni P.P. Wands J.R. J. Virol. 1998; 72: 1737-1743Crossref PubMed Google Scholar), are known components of this machinery. We hypothesized that the conventional two-hybrid screen that is based on a transcriptional readout may not be suitable for identifying transcriptional activators and effectors because the system may be compromised by pX activit
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