Portal de Periódicos da CAPES

Src kinases involved in hepatitis B virus replication

1999; Springer Nature; Volume: 18; Issue: 18 Linguagem: Inglês

10.1093/emboj/18.18.5019

1460-2075

Nicola P. Klein,

NF-κB Signaling Pathways

Article15 September 1999free access Src kinases involved in hepatitis B virus replication Nicola P. Klein Nicola P. Klein Present address: Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, 94305 USA Search for more papers by this author Michael J. Bouchard Michael J. Bouchard Department of Microbiology, New York University School of Medicine, 550 First Avenue, New York, NY, 10016 USA Search for more papers by this author Li-Hua Wang Li-Hua Wang Department of Microbiology, New York University School of Medicine, 550 First Avenue, New York, NY, 10016 USA Search for more papers by this author Claudia Kobarg Claudia Kobarg Present address: Centro de Biologia Molecular, Universidade de Campinas, Campinas, Brazil Search for more papers by this author Robert J. Schneider Corresponding Author Robert J. Schneider Department of Biochemistry, New York University School of Medicine, 550 First Avenue, New York, NY, 10016 USA Search for more papers by this author Nicola P. Klein Nicola P. Klein Present address: Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, 94305 USA Search for more papers by this author Michael J. Bouchard Michael J. Bouchard Department of Microbiology, New York University School of Medicine, 550 First Avenue, New York, NY, 10016 USA Search for more papers by this author Li-Hua Wang Li-Hua Wang Department of Microbiology, New York University School of Medicine, 550 First Avenue, New York, NY, 10016 USA Search for more papers by this author Claudia Kobarg Claudia Kobarg Present address: Centro de Biologia Molecular, Universidade de Campinas, Campinas, Brazil Search for more papers by this author Robert J. Schneider Corresponding Author Robert J. Schneider Department of Biochemistry, New York University School of Medicine, 550 First Avenue, New York, NY, 10016 USA Search for more papers by this author Author Information Nicola P. Klein2, Michael J. Bouchard1, Li-Hua Wang1, Claudia Kobarg3 and Robert J. Schneider 4 1Department of Microbiology, New York University School of Medicine, 550 First Avenue, New York, NY, 10016 USA 2Present address: Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, 94305 USA 3Present address: Centro de Biologia Molecular, Universidade de Campinas, Campinas, Brazil 4Department of Biochemistry, New York University School of Medicine, 550 First Avenue, New York, NY, 10016 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (1999)18:5019-5027https://doi.org/10.1093/emboj/18.18.5019 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Chronic infection by hepatitis B virus is a leading cause of human liver cancer and liver disease. The hepatitis B virus HBx protein is a regulatory factor that is essential for virus infection in mammals and is implicated in development of liver cancer and liver disease. Among the reported activities of HBx is the ability to stimulate Src tyrosine kinases, Ras-GTPases and transcriptional activation. We now demonstrate that HBx activation of Src tyrosine kinases, but not Ras, promotes a high level of viral replication in cell culture. HBx is shown to stimulate reverse transcription of the viral pregenomic mRNA into genomic DNA through a Src-mediated pathway in tissue culture cells. Targeted inhibition of Src tyrosine kinase activity, mutational inactivation of the HBx gene or retargeting of HBx to the nucleus to abolish cytoplasmic signal transduction activity, are shown to impair viral reverse transcription strongly. These studies implicate HBx stimulation of the Src family of tyrosine kinases in stimulation of viral polymerase activity. Introduction Hepatitis B viruses (HBVs) are hepadnaviruses that preferentially infect the liver of their mammalian and avian hosts, often establishing chronic infection. Chronic infection is strongly associated with development of liver disease and liver cancer in mammals (hepatocellular carcinoma, HCC), and is a leading cause of human cancer worldwide (Wands and Blum, 1991). Prototypic members of the HBV family include human hepatitis B virus (HBV), woodchuck hepatitis B virus (WHV) and duck hepatitis B virus (DHBV). HBVs are distantly related to retroviruses, since they undergo reverse transcription of the viral mRNA pregenome into genomic DNA, which is packaged as a partially double-stranded DNA (dsDNA) circular genome within virus particles (Ganem and Varmus, 1987; Nassal and Schaller, 1993). Upon infection, the HBV dsDNA genome is converted to a covalently closed circular (ccc) DNA episome that remains within the nucleus of the infected hepatocyte, where it serves as the template for transcription of viral mRNAs (Newbold et al., 1995). Transcripts consist of the pregenomic mRNA which encodes the viral reverse transcriptase (Pol protein), and the core protein (HBcAg), a component of the virus particle that also participates in reverse transcription and replication of the viral genome (Hatton et al., 1992; Nassal, 1992). Other mRNAs are synthesized that encode precore protein, the three envelope proteins (surface antigens, HBsAgs) and the regulatory protein HBx (Nassal and Schaller, 1993). Reverse transcription of the pregenomic mRNA and synthesis of the viral dsDNA genome occurs within core protein particles in the cytoplasm (Summers and Mason, 1982), which are subsequently enveloped by HBsAgs and exported from the cell. Mammalian HBVs are all highly related and are the only HBVs that encode HBx protein. Studies in the woodchuck model system have shown that expression of WHV HBx (WHx) is essential to establish infection, in that direct injection of WHV genomes into the livers of animals establishes infection only if WHx is expressed (Chen et al., 1993; Zoulim et al., 1994). WHx expression is also strongly correlated with active centers of viral replication in the liver during chronic WHV infection (Dandri et al., 1996), and therefore it is thought to be essential for human HBV replication as well. HBx and WHx are highly related and appear to possess similar functional properties. Although HBx and WHx do not bind DNA (Twu and Schloemer, 1987; Spandau and Lee, 1988; Zahm et al., 1988; Yen, 1996), they stimulate a variety of cellular and viral transcriptional elements (Yen, 1996), including RNA polymerase II- and III-dependent promoters (Aufiero and Schneider, 1990; Kwee et al., 1992; Wang et al., 1995). HBx and WHx are predominantly cytoplasmic proteins, but may also be found in small amounts in the nucleus (Doria et al., 1995; Dandri et al., 1998). In vitro, HBx binds several transcription factors (Maguire et al., 1991; Cheong et al., 1995; Haviv et al., 1995; Williams and Andrisani, 1995; Haviv et al., 1996; Qadri et al., 1996; Lin et al., 1997), and can stimulate transcription if tethered to a DNA binding domain (Haviv et al., 1995) or experimentally directed to the nucleus (Doria et al., 1995). HBx also binds the DNA repair protein UV-DDB and possibly impairs cellular DNA repair (Lee et al., 1995b; Sitterlin et al., 1997; Becker et al., 1998). A number of studies have shown that HBx and WHx are in vivo cytoplasmic activators of signal transduction pathways in transfected cells (Cross et al., 1993; Kekule et al., 1993; Lucito and Schneider, 1993; Benn and Schneider, 1994, 1995; Natoli et al., 1994; Doria et al., 1995; Wang et al., 1995; Benn et al., 1996; Chirillo et al., 1996; Su and Schneider, 1996, 1997; Cong et al., 1997; Klein and Schneider, 1997; Wang et al., 1997, 1998). The ability to influence cellular signal transduction pathways might be important for HBV and WHV infection and replication, which is the subject of this report. HBx and WHx activate the Ras-Raf-MEK-mitogen activated protein kinase (MAPK) signaling pathway, which is essential for many HBx transcriptional activities, including activation of transcription factor AP1/Fos-Jun and NF-κB (Cross et al., 1993; Benn and Schneider, 1994; Natoli et al., 1994; Doria et al., 1995; Benn et al., 1996; Chirillo et al., 1996; Su and Schneider, 1996; Klein and Schneider, 1997), and RNA polymerase III-directed transcription (Wang et al., 1997). HBx also modestly stimulates deregulation of early cell cycle checkpoint controls (Koike et al., 1994; Benn and Schneider, 1995) in a manner that involves stimulation of signal transduction pathways (Benn and Schneider, 1995). We have recently demonstrated that HBx and WHx activate Ras signaling by stimulating the Src family of protein tyrosine kinases (Klein and Schneider, 1997). Here we investigated whether activation of Src or Ras by HBx and WHx proteins is required for viral replication in cultured cells. We show that activation of the Src family of tyrosine kinases by both HBx and WHx promotes strong viral replication by enhancing reverse transcription of packaged viral pregenomic mRNAs. Inhibition of HBx or WHx activation of Src signaling did not strongly impair the formation of viral core particles that contain pregenomic mRNA, and only modestly reduced the level of viral transcription in the short time frame of these studies. Instead, HBx activation of Src signaling was found to stimulate reverse transcription of viral pregenomic mRNA and second-strand genomic DNA synthesis. These results imply a specific requirement for Src-type kinases in mammalian hepadnavirus replication. Results HBx and WHx induce activation of Src–Ras signaling during viral replication Cultured cells cannot be efficiently infected by WHV or HBV, which is thought to result from lack of expression of the viral receptor in cultured cell lines. However, HBV replication in cultured cells can be established by transfection of plasmid cDNA copies of viral RNA genomes (Seeger and Maragos, 1989; Zoulim et al., 1994), or a head-to-tail dimer of viral genomic DNA (Blum et al., 1983). Both templates initiate synthesis of viral mRNAs and polypeptides that reverse transcribe, replicate and package viral genomic DNAs within cytoplasmic viral particles. HBV and WHV replicate to levels 2- to 10-fold less in the absence of HBx or WHx during transient studies in many cell lines (Yaginuma et al., 1987; Colgrove et al., 1989; Nakatake et al., 1993; Zoulim et al., 1994; Lee et al., 1995a; Melegari et al., 1998). Here we show that HBx and WHx activate Src and Ras signaling during the course of viral replication in cultured cells. Chang cells or HepG2 cells were transfected with plasmids encoding wild-type (wt) or HBx(−) forms of HBV or WHV genomes. Initial transfection efficiencies were similar for the different constructs (∼30% in Chang cells, 5% in HepG2 cells) based on cotransfection of a green fluorescent protein (GFP) reporter (data not shown). Cells were maintained for up to 3 weeks, passaged when confluent and placed in low serum (0.5%) for 1 day before assay of Ras-Raf-MAPK signaling. There was no evidence for cell killing following transfection (data not shown). Activation of immunoprecipitated MAPK Erk2 was assayed to measure HBx or WHx stimulation of the Ras-Raf-MAPK pathway, using in vitro phosphorylation of the MAPK substrate myelin basic protein (MBP) with [γ-32P]ATP (Klein and Schneider, 1997). As shown for the 2-week samples, Erk2 activity was stimulated ∼3- to 4-fold only in Chang cells expressing wtWHV or wtHBV viral genomes, and not in cells containing the HBx(−) virus genomes, CWHV or X21-HBV, nor in control cells (Figure 1A). Examination of Erk2 activity at 1 and 3 weeks (the last time point tested) provided identical results (data not shown). Similar but slightly less pronounced results were observed for activation of Erk2 in HepG2 cells containing wtWHV (Figure 1B). Stimulation of Ras by HBx and WHx was previously shown to result from Src family tyrosine kinase activation (Klein and Schneider, 1997). It was therefore determined whether WHx stimulates Src kinases in the context of WHV gene expression. Chang cells and HepG2 cells were transfected with plasmids expressing wt or WHx(−) CWHV genomes, with and without co-expression of the Csk tyrosine kinase. Csk specifically phosphorylates the C-terminus of the family of Src kinases and blocks their activity (Sabe et al., 1992; Nada et al., 1993). Cells were also transfected with the dominant-inhibiting N17 form of p21 Ras (Feig and Cooper, 1988) to block Ras activation. MAPK Erk2 activity was elevated by 3- to 4-fold only in cells containing replicating wtWHV(WHx+), and was inhibited by overexpression of Csk, which blocks activation of Src kinases, or by inhibition of Ras with the N17 inhibitor (Figure 2A). These results implicate HBx stimulation of Src signaling in stimulation of the Ras-Raf-MAPK pathway in the context of virus gene expression. Activation of c-Src kinase was shown directly by the ability of immunoprecipitated c-Src to phosphorylate the substrate enolase in vitro with [γ-32P]ATP in Chang cells (Figure 2B) and HepG2 cells (Figure 2C). WHx stimulated Src kinase activity 2.5- to 4-fold, which was blocked by co-expression with the Csk inhibitor of Src. Coexpression of WHx and the N17 Ras inhibitor had no effect, demonstrating that HBx stimulation of Src kinases lies upstream of Ras stimulation. Thus, HBx and WHx proteins moderately stimulate Src kinases and Ras-Raf-MAPK signaling in the context of virus replication in cultured cells (as shown next). Figure 1.Prolonged expression of HBx or WHx activates Ras and Src signaling. (A) Chang cells were transfected with head-to-tail dimer genomic forms of HBV encoding wtHBV or a mutant that fails to synthesize HBx, X-21HBV; or pregenomic WHV DNA corresponding to wtWHV or the mutant, CWHV(WHx−). (B) HepG2 cells were transfected with pregenomic forms. Cells were passaged when confluent, then serum-starved for 1 day before analysis of MAPK activity, at 2 weeks post-transfection. Equal amounts of cell lysate were immunoprecipitated with anti-Erk2 antibody, and MAPK activity was measured by in vitro kinase assay using the substrate MBP and [γ-32P]ATP. Labeled MBP was resolved by 15% SDS–PAGE and visualized by autoradiography. Control vector refers to transfection with plasmid pGEM3 devoid of viral sequences. Results are typical of at least three independent experiments and were quantified by digital densitometry. In the control lane cells were transfected with vector alone. Download figure Download PowerPoint Figure 2.Prolonged expression of WHx stimulates Src kinases. Chang and HepG2 cells were transfected with cDNA forms of pregenomic wtWHV or CWHV(WHx−) plasmid, and a plasmid encoding Csk or dominant inhibitor RasN17. Four days post-transfection, cells were serum-starved and Erk2 or c-Src were immunoprecipitated from equal amounts of cell lysate and their activity measured by in vitro phosphorylation of MBP (A) or enolase (B and C), respectively, using [γ-32P]ATP. Immunoblots are shown for immunoprecipitated Erk2 or c-Src protein levels. In the control lane cells were transfected with vector alone. Control vector as in Figure 1 legend. Results are typical of at least three independent experiments and were quantified by digital densitometry. Download figure Download PowerPoint HBx or WHx activation of Src family kinases stimulates viral replication It was determined whether activation of Src kinases or Ras constitutes a relevant activity of HBx or WHx that might be involved in mammalian hepadnavirus replication. Chang and HepG2 cells were transfected with plasmids encoding wtWHV(WHx+), or the WHx(−) mutant CWHV. The effect of inhibition of Ras or Src kinases on WHV replication in Chang cells was examined by cotransfecting plasmids encoding the RasN17 inhibitor or the Src family inhibitory kinase, Csk. Viral core protein particles (core particles) were isolated from equal numbers of cells at 4 days post-transfection. Core particles contain either viral pregenomic mRNA, or reverse-transcribed viral genomic DNA. Viral DNA genomes range from single-stranded (ss) DNAs to mature circular double-stranded (ds) forms, and hence the population migrates electrophoretically as a smear. The amount of replicated viral dsDNA within core particles was examined by gel electrophoresis and Southern blot analysis. Replication of WHV DNA was reduced 10- to 15-fold in Chang cells by the absence of WHx expression (Figure 3A, compare wt and WHx(−) genomes, lanes 2 and 5). Surprisingly, cotransfection of the Ras N17 dominant inhibitor had little negative effect on WHV replication (Figure 3A, compare lanes 2 and 3). The Ras N17 inhibitor was shown to ablate Ras activity under these conditions (Figure 2). In contrast, transfection of Chang cells with a Csk expression vector strongly impaired WHV replication, reducing it 10- to 15-fold to the low level of replication observed for the CWHV(WHx−) virus (Figure 3A). The expression of Csk was shown to inhibit Src family tyrosine kinase activity under these conditions (Figure 2). The absence of WHx gene expression was also found to impair WHV replication in HepG2 cells. The levels of core particle-associated viral genomic DNA was reduced by ∼4- to 5-fold in the absence of WHx (Figure 3B). It is not known why the effect of WHx gene expression on viral DNA replication was somewhat less pronounced in HepG2 cells than in Chang cells. Nevertheless, the ability of WHx to stimulate viral replication, as observed in Chang cells, was demonstrated in HepG2 cells. As the response to WHx was better in Chang cells they were used for the remainder of this study. Figure 3.Activation of Src family kinases is involved in replication of WHV. (A) Chang cells or (B) HepG2 cells were transfected with pregenomic forms of the WHx(−) mutant CWHV. Chang cells were also cotransfected with wtWHV and Csk or the dominant inhibitor RasN17. Four days post-transfection viral core particles were purified from equal numbers of cells, encapsidated viral DNA was extracted and analyzed by Southern blot hybridization. Control refers to cells transfected with vector alone. Download figure Download PowerPoint Control studies next demonstrated that the poor replication of the CWHV(WHx−) genomes resulted from the lack of WHx gene expression and stimulation of cytoplasmic Src family signal transduction, and not from a mutation of the WHV genome unrelated to WHx activity. Chang cells were transfected with a wild-type or CWHV(WHx−) mutant genome and either wtHBx or an HBx containing a potent nuclear localization signal (HBx-NLS). Previous studies showed that HBx-NLS is relocalized exclusively to the nucleus, which abolishes cytoplasmic activation of signal transduction but not certain nuclear transcriptional activities (Doria et al., 1995; Klein and Schneider, 1997). Viral core particles were isolated 4 days post-transfection, genomic WHV DNA was purified and resolved by Southern analysis. Human wtHBx complemented CWHV replication in trans whereas the HBx-NLS mutant did not (Figure 4). Thus, the poor replication of the CWHV(WHx−) genome resulted specifically from the lack of WHx expression, and stimulation of cytoplasmic (Src family kinase) signal transduction. Figure 4.Expression of HBx in trans complements lack of WHx in WHV replication. Chang cells were transfected with plasmids encoding wtWHV pregenomic viral DNA, the pregenomic CWHV (WHx−) form or CWHV and 10 μg of pCMV-HBx, which expresses the human HBV HBx protein, or pCMV-HBx-NLS, which expresses a variant containing the SV40 T-antigen nuclear localization signal. Four days post-transfection, intracellular viral core particles were isolated, viral DNA was purified and analyzed by Southern blot hybridization. No transf. refers to cells transfected with pCMV vector alone. Download figure Download PowerPoint WHx stimulates reverse transcription of pregenomic mRNA and second-strand DNA synthesis by Pol Studies were carried out to determine the step at which viral replication was impaired by inhibition or absence of WHx. The dependence of viral transcription on WHx was determined by Northern blot analysis. Equal amounts of whole cytoplasmic RNA were prepared from Chang cells at 4 days post-transfection with wtWHV or the CWHV(WHx−) mutant, with or without cotransfection of plasmids encoding the Ras N17 dominant inhibitor, or the Src kinase inhibitor, Csk (Figure 5A). Steady-state levels of viral pregenomic and preS/S antigen mRNAs were enhanced only 2- to 4-fold in cells containing wtWHV compared with the CWHV(WHx−) genome. These results are consistent with previous reports demonstrating only moderate reduction in viral mRNA levels for HBx mutants of WHV, GSHV and HBV (Yaginuma et al., 1987; Colgrove et al., 1989; Nakatake et al., 1993; Zoulim et al., 1994; H.Lee et al., 1995; Melegari et al., 1998). Inhibition of Ras signaling had little or no effect on viral mRNA levels, while inhibition of the Src family of kinases decreased the abundance of WHV mRNAs between 2- and 4-fold, to the same extent as in WHx(−) samples. Control studies showed that inhibition of Src kinases by Csk only reduced the activity of the CMV promoter, which drives pregenomic mRNA transcription, by 50% in Chang cells (Klein and Schneider, 1997). Inhibition of Src family kinases therefore does not strongly block synthesis of pregenomic mRNA and cannot account for the stronger inhibition of virus replication in these short-term replication assays. Sustained inhibition of viral DNA replication would ultimately be expected to reduce viral mRNA and protein levels severely as a result of depletion of genome templates for transcription. Immunoblot analysis of cell extracts showed a 2- to 3-fold stimulation of core protein levels resulting from WHx expression during WHV replication (Figure 5B). These results are consistent with core and pregenomic mRNA levels. Thus, the modest reduction in viral mRNA levels, or the absence of WHx, cannot account for the much greater decrease in viral DNA replication observed during the short (3–4 day) period of Src kinase inhibition. Figure 5.Role of WHx in WHV replication in cell culture. Chang cells were transfected as described for Figure 3. (A) Total cellular RNA was isolated 4 days post-transfection, equal amounts (based on levels of 18S and 28S rRNA, not shown) were resolved by formaldehyde–agarose gel electrophoresis, transferred to nylon membrane and identified by Northern blot hybridization to a 32P-labeled full-length WHV probe. mRNAs were identified by electrophoretic mobilities relative to standards. Control refers to cells transfected with vector alone. (B) Equal amounts of total cellular protein were resolved by SDS–PAGE and Western immunoblotted with antisera to WHV core protein. Download figure Download PowerPoint These results indicate that WHx either (i) increases the abundance of core protein particles that package viral genomes, (ii) enhances the packaging of viral pregenomic mRNA into viral core particles, or (iii) stimulates the reverse transcription of viral pregenomic mRNA into genomic DNA. To discriminate between these possibilities, we examined the level of viral core particles and encapsidated pregenomic mRNA. Chang cells were transfected with wtWHV or CWHV pregenomic cDNA for 4 days. WHV core protein particles were purified from equal numbers of cells by sedimentation, resolved by SDS–PAGE and immunoblotted for core protein (Figure 6A). WHx stimulated formation of core protein particles 2-fold compared with Csk inhibition of its ability to stimulate Src kinases. The increase in core particles is consistent with the abundance of core protein (Figure 5B). The significant effect of WHx on WHV replication does not result from a significantly increased abundance of core protein or core protein particles. The level of encapsidated pregenomic WHV mRNA was therefore determined. Core particles were purified at 4 days post-transfection from equal numbers of cells expressing wtWHV or CWHV(WHx−) genomes, or from cells cotransfected with wtWHV and the Csk expression vector to inhibit Src kinases. DNA was exhaustively degraded with DNase I, pregenomic mRNA was extracted, and hybridized to a radiolabeled riboprobe complementary to the 5′ end of pregenomic mRNA, and the products resistant to RNase A were resolved by denaturing urea–gel electrophoresis (Figure 6B). Approximately 2-fold higher levels of WHV pregenomic mRNA were evident in core particles obtained from cells expressing wtWHV, compared with CWHV(WHx−) or wtWHV with Csk. RNase A treatment of samples prior to hybridization eliminated protected fragments (no RNA sample), demonstrating that the pregenomic mRNA 5′ end was specifically detected. As an independent measure of pregenomic mRNA packaging, the amount of pregenomic mRNA within core particles was determined by semi-quantitative RT–PCR (Figure 6C). Reverse transcription of the 5′ end of pregenomic mRNA and PCR amplification showed similar levels of viral pregenomic mRNA in core particles obtained from cells expressing wtWHV, wtWHV with Csk or CWHV(WHx−). Extracts were exhaustively digested with DNase I prior to RT–PCR analysis, to exclude contamination by viral genomic DNA, as demonstrated in the PCR-only samples. RT–PCR was performed semi-quantitatively, as shown by three independent but consistent levels of amplified ribosomal S15 reporter mRNA that was included. The range of experimental error for RT–PCR is inclusive of the 2- to 3-fold change derived from RNase protection, which probably explains the failure to detect differences in packaged pregenomic mRNA by this approach. These results are consistent with WHx promotion of reverse transcription of viral pregenomic mRNA into viral genomic DNA. Figure 6.Core-associated pregenomic mRNA levels and reverse transcription. (A–C) Chang cells were transfected for 4 days with wtWHV or CWHV pregenomic cDNA expression vectors and plasmid pCsk, and core protein particles were purified from equal numbers of cells by sedimentation. (A) Proteins were resolved by SDS–PAGE and immunoblotted with WHV core-specifc antisera. Mock refers to cells transfected with vector alone. (B and C) Purified core particles were disrupted, core-associated genomic DNA was degraded with RNase-free DNase I, and encapsidated pregenomic mRNA was extracted. Amounts of encapsidated viral pregenomic mRNA were determined by (B) specific RNase A protection and resolved by urea–gel electrophoresis, or (C) semi-quantitative RT–PCR in comparison with internal controls, as shown. No RNA refers to RNase A digestion of samples prior to RNase A protection or RT–PCR assay. (D) Chang cells were transfected with pWHV or pCWHV, or pCWHV and pSrcF527, which expresses a constitutively active form of human c-Src (Cooper and MacAuley, 1988). Core particles were purified from equal numbers of cells and used for analysis of endogenous viral polymerase activity by incubation with [α-32P]dATP and unlabeled dTTP, dCTP and dGTP. Products of reverse transcription and second-strand DNA synthesis were resolved by electrophoresis in 1% agarose gels containing SDS and visualized by autoradiography. Download figure Download PowerPoint To investigate directly whether HBx stimulates Pol activity for reverse transcription and (+) strand DNA synthesis, cytoplasmic core particles were purified from cells transfected for 3 days with either wtWHV, CWHV or CWHV and a plasmid expressing a constitutively activated c-Src kinase (SrcF527) (Cooper and MacAuley, 1988). Endogenous viral polymerase activity within core particles was examined by incubating particles with [α-32P]dNTPs, thereby measuring the activity of endogenous viral polymerase for reverse transcription and/or second-strand DNA synthesis (Nassal, 1992). Products were resolved by gel electrophoresis and visualized by autoradiography (Figure 6D). Wild-type WHV generated predominantly products of ∼3 kb in length descending to ∼2 kb, consistent with relaxed circular DNA and linear duplex molecules, respectively (Nassal, 1992). In the absence of WHx protein (CWHV), only very low levels of endogenous polymerase activity were detected, consistent with the reduced levels of core-associated viral genomic DNA. Importantly, a constitutively active c-Src kinase (SrcF527) partially (∼30%) but not fully stimulated endogenous polymerase activity in CWHV samples. Transfection of increased amounts of SrcF527 did not further increase polymerase activity (data not shown). Studies have also excluded the possibility that Src kinase activity decreased the rate of virus particle secretion, thereby increasing virus export in the WHx-deficient samples. Although the level of WHV secreted into medium is very low, much higher levels were detected in the presence of WHx expression (data not shown). These results therefore indicate that the stimulation of Src family kinases by HBx or WHx is an important but not sufficient activity for viral replication. Discussion This study demonstrated that activation of Src signal transduction by HBx or WHx protein stimulates WHV replication in cultured cells. These results are consistent with those from infection of woodchucks with a WHV deleted of WHx (Chen et al., 1993; Zoulim et al., 1994). In animal studies, infection of hepatocytes in young woodchucks could not be established by the direct injection into the liver of WHx(−) CWHV genomes, whereas injection of wtWHV genomes led to successful infection. The augmentation of WHV replication by WHx or HBx in cultured cells is therefore consistent with animal infection studies in which WHx was essential, but which were not open to the molecular analysis carried out here. A key result of these studies was the d