Hepatitis B virus X protein enhances Myc stability by inhibiting SCF(Skp2) ubiquitin E3 ligase-mediated Myc ubiquitination and contributes to oncogenesis.

The stability of Myc oncoprotein is regulated by Skp/cullin/F-box (SCF) ubiquitin E3 ligase, in particular, via either SCF(Skp2) or SCF(Fbw7) ubiquitin E3 ligases. An earlier study has shown that hepatitis B virus X protein (HBx) augments Myc stability via a mechanism involving the inhibition of Myc ubiquitination. However, the underlying mechanism by which HBx inhibits Myc ubiquitination remained to be elucidated. Moreover, to what extent HBx-mediated Myc stabilization contributes to viral oncogenesis is unknown. First, we corroborated the physiological significance of HBx-mediated Myc stabilization in HBV-replicating cells by demonstrating that (1) the elevation of Myc level in a HBV-replicating HepG2.2.15 cell compared to parental cells; (2) HBx-mediated Myc stabilization in a HBV replicon transfected cells; and (3) the inhibition of Myc ubiquitination by HBx in a HBV replicon transfected cells. Then, the molecular interaction between HBx and Myc protein was revealed via coimmunoprecipitation and immunofluorescence experiments. Subsequent analysis indicated that HBx stabilizes Myc oncoprotein by blocking Skp2, as opposed to Fbw7, -mediated Myc ubiquitination. Next, we defined the Myc-binding region to four residues (VFVL) near the C-terminus of HBx polypeptide. An HBx variant with mutated VFVL consistently failed to not only interact with Myc but also suppress Myc ubiquitination. Importantly, the VFVL mutant lost the ability to transform NIH3T3 cells in concert with Ras, implying that the HBx-Myc interaction is critical for viral oncogenesis. Consistently, immunohistochemistry of liver biopsies revealed that Myc protein is elevated in HBV-infected tissues. We concluded that HBx stabilizes Myc oncoprotein by inhibiting SCF(Skp2) ubiquitin E3 ligase-mediated Myc ubiquitination, and the HBx-mediated Myc stabilization greatly contributes to viral oncogenesis.

Topical administration of the pan-Src kinase inhibitors, dasatinib and LCB 03-0110, prevents allergic contact dermatitis in mice.

BACKGROUND: Allergic contact dermatitis (ACD) is a delayed type of T cell-mediated cutaneous inflammatory response, in which multiple cell types are involved. Dasatinib and LCB 03-0110 are small molecule multityrosine kinase inhibitors, and they share remarkably similar target kinases such as the c-Src family, Btk and Syk, which play key roles in the cell signalling of T cells and other inflammatory cells. OBJECTIVES: To test the anti-ACD activity of dasatinib and LCB 03-0110 and compare it with that of tacrolimus (FK506) and triamcinolone acetonide (a glucocorticoid), which are widely used for topical treatment of ACD, and to examine the two compounds for their capacity to induce skin atrophy, a side-effect. METHODS: ACD was induced on the ears of mice by repeated topical application of oxazolone. Each test compound was then topically applied on the ear. Ear swelling, epidermal thickness and levels of inflammatory cytokines were measured. The skin atrophy induced by the compounds was tested during prolonged application on the dorsal skin of hairless mice, followed by haematoxylin and eosin staining. RESULTS: Dasatinib and LCB 03-0110 suppressed the symptoms of ACD such as ear swelling, increase in epidermal thickness and synthesis of inflammatory cytokines (i.e. interleukin-1ß, tumour necrosis factor-α and interferon-γ) in a dose-dependent manner. The two compounds showed near-equal potency to tacrolimus; however, their potency was lower than that of triamcinolone acetonide. Prolonged treatment with the two compounds did not induce any skin atrophy, whereas use of steroidal agents induced severe atrophy. CONCLUSIONS: Dasatinib and LCB 03-0110 could be used as effective agents for the treatment of ACD without the adverse side-effect of skin atrophy.

Expression of cyclooxygenase-2 (COX-2) in hepatocellular carcinoma and growth inhibition of hepatoma cell lines by a COX-2 inhibitor, NS-398.

Cyclooxygenase-2 (COX-2) has been suggested to be associated with carcinogenesis. In hepatocellular carcinoma (HCC), the expression pattern of COX-2 protein has been well correlated with the differentiation grade, suggesting that abnormal COX-2 expression plays an important role in hepatocarcinogenesis. We investigated the expression pattern and clinical significance of COX-2 in HCC tissues. In addition, we evaluated the efficacy of a selective COX-2 inhibitor, NS-398, in three hepatoma cell lines. Thirty-six HCC tissues, 15 hepatoma cell lines, 1 colorectal cell line (HT-29), and 1 fibroblast cell line (SV80) were included in the study. We evaluated serological tests and histological and radiological evaluations of HCC tissues. Immunohistochemical staining for COX-2 was performed on 36 HCC tissues and 17 cancer cell lines. A cell viability assay for growth inhibition of NS-398 in five cell lines was performed. Immunohistochemically, all six well-differentiated HCCs were positive, whereas 83% (10 of 12) of the poorly differentiated HCCs were negative. There was no significant relationship between the intensity of COX-2 expression and the level of alpha-fetoprotein, tumor size, presence of portal vein thrombosis, tumor capsule and metastasis, Tumor-Node-Metastasis staging, and growth types (P > 0.05). According to the cell viability assay, NS-398 suppressed the growth of all cell lines, independent of the degree of COX-2 expression. The inhibitory effect on each cell line was identified in 10 microM NS-398 and was significantly strong in 100 microM NS-398. All cell lines exhibited apoptosis, which was identified by 4'-6-diamidino-2-phenylindole staining. In conclusion, COX-2 may be a determinant of the differentiation grade of HCC, and the inhibition of COX-2 can induce growth suppression of hepatoma cell lines via induction of apoptosis.

Activated ras oncogene collaborates with HBx gene of hepatitis B virus to transform cells by suppressing HBx-mediated apoptosis.

The hepatitis B virus HBx protein is a promiscuous transactivator implicated in the development of hepatocellular carcinoma. The ectopic expression of HBx fails to transform both primary and immortalized rodent cells, but rather induces apoptosis. Furthermore, most transgenic mice harboring HBx do not develop liver tumors. Thus, it remains unclear whether and how HBx contributes to oncogenesis. Here, we show that HBx collaborates with activated H-ras to transform immortalized rodent cells. Indeed, REF52 cells transfected by both HBx and activated H-ras were morphologically transformed and were able to grow in soft agar. Remarkably, nude mice injected with REF52 cells transfected by both HBx and activated H-ras developed tumors, whereas the mice injected with REF52 cells transfected by either gene alone did not. Thus, we concluded that HBx could contribute to neoplastic transformation of cells in collaboration with other oncogenes, such as H-ras, that renders cells to overcome the HBx-mediated apoptosis. Further, we found that HBx mediated apoptosis was suppressed by activated H-ras through activation of the phosphatidylinositol-3 kinase and Akt pathway. Data presented here firmly established the oncogenic potential of HBx during multistage carcinogenesis. Oncogene (2001) 20, 16 - 23.

Evidence that the 5'-end cap structure is essential for encapsidation of hepatitis B virus pregenomic RNA.

Hepatitis B virus (HBV) replicates by reverse transcription of an RNA intermediate, the pregenomic RNA. The first step of HBV genome replication is the encapsidation of the pregenomic RNA encoding the encapsidation signal, termed epsilon, into the core particles, which is preceded by recognition and binding of HBV DNA polymerase to epsilon. The pregenomic RNA contains two identical epsilon elements due to its terminal redundancy: one near the 5' end and another near the 3' end. Despite the fact that both epsilon elements have an identical sequence, only the 5' epsilon, but not the 3' epsilon, is functional for encapsidation. To understand the molecular nature of this position effect, we made a series of lacZ RNA expression plasmids which contain the epsilon element at various positions from the 5' end of the transcripts. Following transfection, the lacZ RNAs in cytoplasmic core particles were measured by RNase protection assay for encapsidation. The results indicated that the lacZ RNAs with epsilon positioned up to 65 nucleotides from the 5' end were encapsidated, whereas the lacZ RNAs with epsilon positioned further downstream were not. Interestingly, the cap-free lacZ RNA transcribed by T7 RNA polymerase was not encapsidated, implying that the 5' cap structure is required for encapsidation of the pregenomic RNA. We hypothesized that HBV DNA polymerase must somehow recognize the cap structure and/or its associated factors, as well as the 5' epsilon, for encapsidation to occur.

Monitoring antibody titers to recombinant Core-NS3 fusion polypeptide is useful for evaluating hepatitis C virus infection and responses to interferon-alpha therapy.

To evaluate the clinical feasibility of the antibody titer against a chimeric polypeptide (named Core 518), in which a domain of Core and NS3 of hepatitis C virus (HCV) was fused, ELISA was performed in a total of 76 serum samples. Each serum was serially diluted using two-fold dilution method with distilled water into 10 concentrations. They were all positive for second generation anti-HCV assay (HCV EIA II; Abbott Laboratories). Genotyping RT-PCR, quantitative competitive RT-PCR, and RIBA (Lucky Confirm; LG Biotech) were also assayed. Anti-Core 518 antibody was detected in x 12800 or higher dilutions of sera from 35 of 43 chronic hepatitis C (81.4%) and nine of 16 hepatocellular carcinoma sera (56.3%), one of four cirrhosis (25%), 0 of four acute hepatitis C, and one of nine healthy isolated anti-HCV-positive subjects (p=0.0000). The anti-Core 518 antibody titers were well correlated with the presence of HCV RNA in serum (p=0.002). The anti-Core 518 antibody titers decreased significantly in nine of ten responders to IFN-alpha treatment. Monitoring anti-Core 518 titers may be helpful not only for differentiating the status of HCV infection among patients with various type C viral liver diseases, but also for predicting responses to IFN-alpha treatment.

A novel parvovirus isolated from Manchurian chipmunks.

A novel parvovirus was identified in Manchurian chipmunks inhabiting Korea. Hepatitis B surface antigen (HBsAg) was detected in sera from 4 animals among 62 apparently healthy chipmunks. Electron microscopic examination of the HBsAg-positive sera revealed virus-like spherical particles 20-22 nm in diameter. Extraction of nucleic acid under annealing conditions from the serum samples containing virus-like particles yielded a single species of DNA molecule with the electrophoretic mobility of 5.6-kb double-stranded DNA. Four overlapping clones that encompassed almost the full-length viral genome, except both ends, were obtained. By sequencing these clones, we determined the sequence of 5097 nucleotides of the viral DNA. Two open reading frames were identified, with the left side open reading frame encoding a putative nonstructural protein and the right side open reading frame encoding a putative capsid protein. The nucleotide and amino acid sequences showed significant homology to parvovirus B19 and simian parvovirus, but showed little homology to other mammalian autonomous parvoviruses or adeno-associated viruses. These observations indicate that the virus isolated from Manchurian chipmunks is a novel parvovirus and may be a potentially useful animal model of human B19 infection as a new member of the Erythrovirus genus of the Parvoviridae.

Presentation of the hydrophilic domains of hepatitis C viral E2 envelope glycoprotein on hepatitis B surface antigen particles.

Subviral particles of hepatitis B virus have been used to present foreign epitopes. We attempted to present the hydrophilic domains of E2 envelope protein of hepatitis C virus (HCV) as a fusion protein with hepatitis B virus surface antigen (HBsAg). The five hydrophilic domains of HCV E2 antigen were inserted into HBsAg such that the inserted hydrophilic domains were presented on the outer surface of HBV subviral particles. In addition, a fusion encoding the hypervariable region (HVR) of E2 antigen was also made. Cell lysate and culture medium were analyzed for the synthesis and secretion of the fusion proteins by immunoprecipitation with polyclonal anti-HBsAg antibody using recombinant vaccinia virus system. The results showed that the fusion proteins containing these six E2 domains were made in the cell, but only two out of six fusion proteins were secreted into culture medium. Further, cesium chloride density gradient analysis and electron microscopy revealed that these fusions were secreted into culture media as particles. It will be of interest to test immunogenicity of the HBsAg fusion particles containing the HCV E2 domains in animal model.

Effect of hormone replacement therapy on lipoprotein(a) and lipid levels in postmenopausal women. Influence of various progestogens and duration of therapy.

BACKGROUND: Estrogen replacement therapy in postmenopausal women reduces the risk of coronary artery disease. One of the possible mechanisms of this effect is the modification of lipid profiles. However, there is controversy concerning the effects on lipoprotein(a) [Lp (a)] and lipid levels of progestogens administered with estrogen. METHODS: Five hundred fifty-one postmenopausal women were divided into 5 groups: group 1, 0.625 mg of conjugated equine estrogen (CEE) (n = 140); group 2, 0.625 mg of CEE plus 5 mg of medroxyprogesterone acetate (MPA) (n = 97); group 3, 0.625 mg of CEE plus 10 mg of MPA (n = 109); group 4, 2 mg of estradiol valerate plus 0.5 mg of norgestrel (n = 134); and group 5, control (n = 71). The Lp(a) and lipid levels were measured before and 2, 6, and 12 months after hormone replacement therapy. RESULTS: Estrogen replacement therapy for 12 months lowered the Lp(a) level by 37.1%. The addition of progestogen attenuated the Lp(a)-lowering effect of estrogen. The high-density lipoprotein cholesterol (HDL-C) level was markedly increased in group 1 (16.5%), was moderately increased in groups 2 (10.8%) and 3 (11.3%), and was not changed in group 4. The low-density lipoprotein cholesterol level was decreased by 10.9% to 17.6% in all the treatment groups. Estrogen replacement therapy for 2, 6, and 12 months raised the HDL-C level by 7.2%, 17.4%, and 17.8%, respectively. In the group with combined estradiol plus norgestrel therapy, the HDL-C level was decreased after 2 months and was not changed after 6 and 12 months. The groups that received CEE plus MPA showed intermediate effects between the group that received CEE only and the group that received estradiol plus norgestrel. CONCLUSIONS: Combined estrogen and progestogen therapy may have effects on the heart different from those of estrogen therapy alone because of adverse impact of progestogens on Lp(a) and HDL-C levels. The effects of progesterones were dependent on the androgenic potency of progestogen and the duration of therapy.

Changes in Lp(a) lipoprotein and lipid levels after cessation of female sex hormone production and estrogen replacement therapy.

OBJECTIVE: To investigate the serial changes in Lp(a) lipoprotein levels with the loss of female sex hormones by surgical menopause and with estrogen replacement therapy in the same woman. PATIENTS AND METHODS: Forty-four premenopausal women who underwent a transabdominal hysterectomy (TAH) because of benign gynecological disorders were divided into two groups: women who underwent a TAH and unilateral salpingo-oophorectomy (n=31) and women who underwent a TAH and bilateral salpingo-oophorectomy (n=13). In the group of women who underwent a TAH and bilateral salpingo-oophorectomy, 0.625 mg of conjugated equine estrogen was given daily 2 months after the operation. The levels of Lp(a) lipoprotein and lipids were measured before and at 2 and 4 months after the operation. RESULTS: In the group of women who underwent a TAH and bilateral salpingo-oophorectomy, the mean (+/-SD) concentration of Lp(a) lipoprotein was increased by 24.5% from 0.48+/-0.47 mmol/L (18.4+/-18.3 mg/dL) to 0.59+/-0.54 mmol/L (22.9+/-21.0 mg/dL) after 2 months (P<.05), and it was reduced by 30.6% to 0.41+/-0.51 mmol/L (15.9+/-20.1 mg/dL)(P<.005) with therapy with conjugated equine estrogen (Premarin). The Lp(a) lipoprotein levels were not changed in the group of women who underwent a TAH and unilateral salpingo-oophorectomy. In the group of women who underwent a TAH and bilateral salpingo-oophorectomy, the high density lipoprotein cholesterol level showed a trend of increase after 2 months from 1.45+/-0.48 mmol/L (56.1+/-18.5 mg/dL) to 1.58+/-0.309 mmol/L (61.2+/-15.1 mg/dL) without statistical significance, and it revealed a significant elevation to 1.76+/-0.43 mmol/L (68.2+/-16.8 mg/dL) with therapy with conjugated equine estrogen (Premarin) compared with that of the basal level (P<.05). CONCLUSIONS: tHE Lp(a) lipoprotein levels appear to be closely associated with female sex hormones. This association might play a pivotal role in postmenopausal increases of atherosclerotic diseases and cardioprotective effect of estrogen in postmenopausal women.

Hepatitis delta virus mutant: effect on RNA editing.

During the replication cycle of hepatitis delta virus (HDV), RNA editing occurs at position 1012 on the 1679-nucleotide RNA genome. This changes an A to G in the amber termination codon, UAG, of the small form of the delta antigen (delta Ag). The resultant UGG codon, tryptophan, allows the translation of a larger form of the delta Ag with a 19-amino-acid C-terminal extension. Using HDV cDNA-transfected cells, we examined the editing potential of HDV RNA mutated from G to A at 1011 on the antigenome, adjacent to normal editing site at 1012. Four procedures were used to study not only the editing of the A at 1012, but also that of the new A at 1011: (i) nucleotide sequencing, (ii) a PCR-based RNA-editing assay, (iii) immunoblot assays, and (iv) immunofluorescence. Five findings are reported. (i) Even after the mutation at 1011, editing still occurred at 1012. (ii) Site 1011 itself now acted as a novel RNA-editing site. (iii) Sites 1011 and 1012 were edited independently. (iv) At later times, both sites became edited, thereby allowing the synthesis of the large form of the delta Ag (delta Ag-L). (v) Via immunofluorescence, such double editing became apparent as a stochastic event, in that groups of cells arose in which the changes had taken place. Evaluation of these findings and of those from previous studies of the stability of the HDV genomic sequence (H.J. Netter et al., J. Virol. 69:1687-1692, 1995) supports both the recent reevaluation of HDV RNA editing as occurring on antigenomic RNA (Casey and Gerin, personal communication) and the interpretation that editing occurs via the RNA-modifying enzyme known as DRADA.

Association of hepatitis C virus particles with immunoglobulin: a mechanism for persistent infection.

The physical properties of hepatitis C virus (HCV) particles were determined by ultracentrifugation on 20-60% isopycnic sucrose density gradients. We report that (i) two populations of HCV particles were found in the sera of patients with chronic HCV infection [at high density (1.186-1.213 g/ml) and at low density (1.099-1.127 g/ml)], (ii) virus particles with high density values were associated with immunoglobulin, and (iii) virus particles with low density values accumulated base changes within a hypervariable region (HVR) of the E2 envelope domain of the RNA genome. The results indicate that base changes within the HVR of E2 lead to the accumulation of immunoglobulin-free virus particles. Therefore, these findings imply that persistent HCV infection is established as a consequence of sequence variation in the E2 envelope domain.

Nucleotide sequence stability of the genome of hepatitis delta virus.

Cultured cells were cotransfected with a fully sequenced 1,679-base cDNA clone of human hepatitis delta virus (HDV) RNA genome and a cDNA for the genome of woodchuck hepatitis virus (WHV). The HDV particles released were able to infect a woodchuck that was chronically infected with WHV. The HDV so produced was passaged a total of six times in woodchucks in order to determine the stability of the HDV nucleotide sequence. During a final chronic infection with such virus, liver RNA was extracted, and the HDV nucleotide sequence for the 352-base region, positions 905 to 1256, was obtained. By means of PCR, we obtained double-stranded cDNA both for direct sequencing and also for molecular cloning followed by sequencing. By direct sequencing, we found that a consensus sequence existed and was identical to the original sequence. From the sequences of 31 clones, we found 32% (10 of 31) to be identical to the original single nucleotide sequence. For the remainder, there were neither insertions nor deletions but there was a small number of single-nucleotide changes. These changes were predominantly transitions rather than transversions. Furthermore, the transitions were largely of just two types, uridine to cytidine and adenosine to guanosine. Of the 40 changes detected on HDV, 35% (14 of 40) occurred within an eight-nucleotide region that included position 1012, previously shown to be a site of RNA editing. These findings may have significant implications regarding both the stability of the HDV RNA genome and the mechanism of RNA editing.

Ribonucleoprotein complexes of hepatitis delta virus.

Human hepatitis delta virus (HDV) is a subviral satellite agent of hepatitis B virus (HBV). The envelope proteins of HDV are provided by the helper virus, HBV, but very little is known about the internal structure of HDV. The particles contain multiple copies of the delta antigen and an unusual RNA genome that is small, about 1,700 nucleotides in length, single stranded, and circular. By using UV cross-linking, equilibrium density centrifugation, and immunoprecipitation, we obtained evidence consistent with the interpretation that delta antigen and genomic RNA form a stable ribonucleoprotein (RNP) complex within the virion. Furthermore, electron-microscopic examination of the purified viral RNP revealed a roughly spherical core-like structure with a diameter of 18.7 +/- 2.5 nm. We also isolated HDV-specific RNP structures from the nuclei of cells undergoing HDV genome replication; both the genome and antigenome (a complement of the genome) of HDV were found to be in such complexes. From the equilibrium density analyses of the viral and nuclear RNPs, we were able to deduce the number of molecules of delta antigen per molecule of HDV RNA. For virions, this number was predominantly ca. 70, which was larger than for the nuclear RNPs, which were more heterogeneous, with an average value of ca. 30.

Assembly of hepatitis delta virus particles.

Hepatitis delta virus (HDV) is a subviral satellite of hepatitis B virus (HBV). Since the RNA genome of HDV can replicate in cultured cells in the absence of HBV, it has been suggested that the only helper function of HBV is to supply HBV coat proteins in the assembly process of HDV particles. To examine the factors involved in such virion assembly, we transiently cotransfected cells with various hepadnavirus constructs and cDNAs of HDV and analyzed the particles released into the medium. We report that the HDV genomic RNA and the delta antigen can be packaged by coat proteins of either HBV or the related hepadnavirus woodchuck hepatitis virus (WHV). Among the three co-carboxy-terminal coat proteins of WHV, the smallest form was sufficient to package the HDV genome; even in the absence of HDV RNA, the delta antigen could be packaged by this WHV coat protein. Also, of the two co-amino-terminal forms of the delta antigen, only the larger form was essential for packaging.

Simian virus 40 late transcripts lacking excisable intervening sequences are defective in both stability in the nucleus and transport to the cytoplasm.

Little or no simian virus 40 (SV40) late mRNA accumulates in the cytoplasm when the primary transcript lacks an excisable intervening sequence. To begin to understand why, we analyzed the synthesis, processing, transport, and stability of SV40 late transcripts accumulated in the nucleus and cytoplasm of monkey cells cotransfected with the DNAs of wild-type and mutants of SV40 lacking precisely various introns. The data from these experiments indicated that (i) the presence of excisable intervening sequences in SV40 late transcripts is necessary for efficient accumulation in the cytoplasm of any of the SV40 late RNA species and (ii) SV40 late transcripts lacking excisable intervening sequences are defective in both stability in the nucleus and transport to the cytoplasm but not in stability in the cytoplasm. We hypothesize that SV40 late transcripts need to be processed via a pathway that couples stabilization of the primary transcript within the nucleus, excision of intervening sequences, proper 5'- and 3'-end formation, and transport to the cytoplasm.

The late spliced 19S and 16S RNAs of simian virus 40 can be synthesized from a common pool of transcripts.

The late transcripts from the simian virus 40 (SV40) are alternatively spliced into two classes of spliced RNAs, 19S and 16S in size. We are interested in understanding the precursor-product relationships that result in the excision of different intervening sequences (introns) from the late transcripts. SV40 mutants containing precise deletions of the introns for each of the spliced 19S and 16S RNA species, including a previously undetected doubly spliced 19S RNA species, were isolated. Analysis by S1 mapping and a modified primer extension technique of the viral RNAs made in monkey cells transfected with each of these mutants led to the following conclusions. (i) Spliced late 19S RNA is not an intermediate in the synthesis of the late 16S RNAs. (ii) The 3' splice site used in the synthesis of the late 16S RNAs can join, albeit inefficiently, with alternative 5' splice sites in the absence of the 5' splice site normally used to synthesize 16S RNA. (iii) There is no obligatory order of excision of introns in the formation of the doubly spliced SV40 late 19S and 16S RNA species. A mutant was constructed by site-directed mutagenesis in which the 5'-proximal 3' splice site used in the synthesis of the doubly spliced RNAs is inactive. Cells transfected with this mutant processed transcripts into 19S RNA which, in wild-type-transfected cells, would have become doubly spliced 16S RNA. Therefore, we conclude that some of the spliced late 19S and 16S RNA can be synthesized from a common pool of transcripts.