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W S Robinson

Publications and source records attributed to W S Robinson.

At least 37 records · Page 2Linked to original sources
PubMed1990

The hepatitis B virus-encoded transcriptional trans-activator hbx appears to be a novel protein serine/threonine kinase.

To study the functional mechanism of the hepatitis B virus (HBV) X (hbx) gene product, we have expressed the hbx protein in E. coli and purified it by HPLC. The purified hbx protein was shown to be active in transactivating transcription directed by the LTR sequence of HIV-1. The hbx protein was found to have an intrinsic serine/threonine protein kinase activity. The hbx protein was detected in hepatitis B virions, and tryptic phosphopeptide maps of the hbx protein phosphorylated in the virion and of the in vitro phosphorylated bacterially expressed hbx protein were similar. Inactivation of the hbx protein by heat, protein-denaturing agents, or an ATP affinity analog, p-fluorosulfonylbenzoyl 5'-adenosine, resulted in loss of both protein kinase activity and trans-activation activity. These results suggest that the HBV-encoded trans-activator hbx is a novel protein kinase.

Base Sequence↗
PubMed1990

Hepadnaviruses in cirrhotic liver and hepatocellular carcinoma.

Hepadnaviruses share properties of virion structure, genome structure and replication, epidemiologic behavior, and pathogenic effects, including an association with hepatocellular carcinoma (HCC). Epidemiologic evidence implicating hepadnavirus infection in HCC includes the observation that the geographic distributions of HBV infection and HCC are similar, that the incidence of HCC is much higher in hepadnavirus infected than uninfected hosts, and that viral DNA sequences are integrated in the cellular DNA of most (e.g., 80-90%) but not all hepadnavirus-associated HCC. Cirrhosis further increases the risk of HCC in HBV infected humans. The precise role of hepadnaviruses in development of most HCC is unclear, although the finding of viral integrations within or near protooncogenes in a few cases suggests the possibility that these integrations may play a direct role in these HCC. However, in the great majority of HCC associated with HBV infections, viral integrations are in different cellular DNA sites in different HCC, integrations are not within domains of known protooncogenes, and integrations are not found in some 10-15% hepadnavirus-associated HCC, suggesting that persisting viral sequences are not directly involved in the development of these HCC as viral sequences are for tumors caused by viruses with oncogenes or viruses that act by a "promoter-insertion" mechanism. It is possible, however, that oncogenic mutations could arise via other mutagenic mechanism that may operate in chronic hepatitis B and/or cirrhosis and which do not involve persisting viral integrations. For example, liver regeneration, which is a feature of the cirrhosis associated with chronic HBV infection (and sometimes with chronic hepatitis B) involves proliferation of many cells with HBV integrations, and such integrations have been shown to be unstable and may lead to mutations through post-integration rearrangements of cellular sequences at sites of viral integrations. Viral sequences appear to be lost or deleted at some such sites of rearranged cell DNA. Chronic HBV infection shares pathologic features of liver cell injury and reactive inflammation, liver regeneration, and in man sometimes cirrhosis with other important risk factors for HCC including chronic alcoholic liver disease, chronic non-A, non-B hepatitis, hemochromatosis, and crypogenic cirrhosis, suggesting that this common pathologic process may be carcinogenic by a mechanism that does not depend specifically on the factor which initiates liver cell injury. The pathogenetic role of chronic hepadnavirus infection in such a process would be in causing liver cell injury with reactive inflammation and hepatocyte proliferation (regeneration).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

Epitope-specific antibody response to the surface antigen of duck hepatitis B virus in infected ducks.

In order to investigate the immune response to duck hepatitis B virus (DHBV) infection, newly hatched DHBV DNA negative ducklings were injected with infectious serum of sufficiently low DHBV-DNA titer to allow clearance of viremia. Of 20 injected ducklings, 13 (65%) became viremic. Of these, 6 (46%) cleared virus from the serum 3 to 22 weeks postinjection. The convalescent sera of these 6 animals were tested for an epitope-specific antibody response in a highly specific competitive inhibition assay using a panel of monoclonal antibodies against duck hepatitis B surface antigen (DHBsAg) that had been well-characterized. All 6 animals recovering from DHBV infection developed antibodies to epitopes on the preS and S proteins of DHBV. Antibody responses were highly variable with marked differences between animals in the extent and specificity of the antibody response. The humoral response to DHBsAg was prolonged in some animals but transient in others. No antibody to preS or S was detected in either preimmune sera or sera of control animals from an uninfected flock. Infected animals that did not clear viremia also remained antibody negative. The humoral responses to neutralizing preS epitopes III and V were weak but antibodies to two immunodominant epitopes on the preS region (II and B) were present in all 6 animals. The humoral response to the two epitopes in the S region was transient and of lower titer when compared to the two immunodominant preS epitopes. The two immunodominant preS epitopes may play an important role in clearance of DHBV infection in ducks.

Animals↗
PubMed1990

Transcriptional activation of the human immunodeficiency virus type 1 long terminal repeat by hepatitis B virus X-protein requires de novo protein synthesis.

Human hepatitis B virus (HBV) X-gene, previously shown to be capable of trans-activating heterologous regulatory elements of the human beta-interferon gene, the human immunodeficiency virus type I (HIV-1) long terminal repeat (LTR), the simian virus 40 (SV40), and HBV, has the capacity to code for a 17-kDa polypeptide (designated pX17). We now report that pX17 synthesized in Escherichia coli can activate transcription controlled by the HIV-1 LTR using a protoplast fusion technique. Protoplasts of E. coli-containing presynthesized X-protein were fused with lymphocytic H938 cells harboring an integrated copy of a plasmid with the CAT gene under control of the HIV-1 LTR (HIV-1 LTR CAT) and a marked increase in the steady state expression of the CAT mRNA was observed. When the same fused cells were treated with the protein synthesis inhibitor cyclohexamide, the pX17-dependent activation of the HIV-1 LTR was abolished. This result indicates that the X-protein expressed in E. coli is biologically active and suggests that the HBV X-protein-mediated trans-activation of the HIV-1 LTR in this system requires de novo cellular protein synthesis.

Chloramphenicol O-Acetyltransferase↗
PubMed1989

Expression of immunogenic epitopes of hepatitis B surface antigen with hybrid flagellin proteins by a vaccine strain of Salmonella.

A nonvirulent Salmonella dublin flagellin-negative, aromatic-dependent live vaccine strain has been used to express hepatitis B virus surface antigen epitopes in an immunogenic form. The envelope proteins of the virion are encoded by the S gene, which contains the pre-S1, pre-S2, and S coding regions. Synthetic oligonucleotides corresponding to amino acid residues S-(122-137) and pre-S2-(120-145) were inserted in-frame into the hypervariable region of a cloned Salmonella flagellin gene, and the recombinant plasmids were introduced into a flagellin-negative aroA mutant live vaccine strain of S. dublin, SL5928. The flagellin gene was expressed in bacteria carrying the plasmids as detected by immunoblotting with anti-flagellin (H1-d) serum. Both the S and pre-S2 epitopes were detected in bacteria carrying the relevant plasmid by immunoblotting with anti-HBs (antibody to hepatitis B virus surface antigen) and anti-peptide antisera. Animals immunized intramuscularly or orally with the live recombinant bacteria developed antibodies specific to these hepatitis B virus epitopes as detected by ELISA.

Amino Acid Sequence↗
PubMed1989

Hepatitis B virus X gene activates kappa B-like enhancer sequences in the long terminal repeat of human immunodeficiency virus 1.

The role of the hepatitis B virus (HBV) X gene during virus infection has not been defined. We previously showed that expression of the HBV X gene in the human hepatocellular carcinoma cell line HepG2 trans-activates chloramphenicol acetyltransferase gene expression under control of the human immunodeficiency virus 1 (HIV-1) long terminal repeat and we have now identified a specific sequence in the HIV-1 long terminal repeat that is responsive to the HBV X gene. Plasmid constructs with the chloramphenicol acetyltransferase gene regulated by an isolated and twice-repeated 12-base-pair HIV-1 enhancer sequence homologous to the nucleotide sequence that binds the nuclear transcription factor NF-kappa B (the HIV-1 kappa B-like sequence) were trans-activated by the HBV X gene in HepG2 cells, indicating that the kappa B-like enhancer sequence in the HIV-1 long terminal repeat is responsive to the X gene. When eight copies of the HIV-1 kappa B-like sequence were used to regulate beta-globin gene expression, transcription of this gene was activated by the HBV X gene in HepG2 cells and no beta-globin gene transcription was detected in the absence of the HBV X gene. beta-globin gene expression regulated by the activator protein 2 (AP-2) binding sequence was not activated by the HBV X gene. Treatment of HepG2 cells with phorbol ester resulted in modest activation of the HIV-1 kappa B-like enhancer sequence suggesting that an NF-kappa B-like factor was induced in these cells as it is in T lymphocytes by phorbol ester; however, phorbol ester did not demonstrably enhance the activation of the HIV-1 enhancer observed with the HBV X gene. These experiments indicate that the HIV-1 kappa B-like transcriptional enhancer sequence is activated by the HBV X gene and suggest that the HBV X gene might play a role in regulating transcription of a gene under control of a kappa B-like enhancer during HBV infection. Since such a sequence has not been found in the HBV genome and HBV gene expression appears not to be regulated by the HBV X gene, a cellular gene that plays a role in HBV replication could be the target of the X gene during HBV infection.

Base Sequence↗
PubMed1989

Hepatitis B virus X gene can transactivate heterologous viral sequences.

The smallest open reading frame of hepatitis B virus (HBV) has been designated the X gene and its biological function during HBV infection and replication is not known. Experiments described here demonstrate that expression of the HBV X gene in HepG2 cells containing a plasmid with the chloramphenicol acetyltransferase (CAT) gene under control of the human immunodeficiency virus (HIV-1) long terminal repeat (LTR) sequence leads to a marked increase in CAT gene transcription as well as expression of the gene product (CAT). The HIV-1 tatIII gene and the HBV X gene together increased HIV-1 LTR-regulated CAT expression above that observed with either gene alone, suggesting a synergistic effect of the X gene and tat. HBV X gene also stimulated expression of the CAT gene under control of the simian virus 40 enhancer and early promoter but not the visna virus LTR or the human T-cell lymphotropic virus type I (HTLV-I) LTR, indicating that the HBV X gene can transactivate some but not other heterologous viral sequences. Transactivation of the HIV-1 LTR by the HBV X gene varied in different cell lines, suggesting that it may be mediated by a cellular factor(s).

Cell Line↗
PubMed1989

Epitope mapping of neutralizing monoclonal antibodies against duck hepatitis B virus.

In this article we report the first topological mapping of neutralizing epitopes of a hepadnavirus. Duck hepatitis B virus is the only hepadnavirus that can replicate and spread from cell to cell in tissue culture. As a result, it is possible to study hepadnaviral neutralization in vitro with this system. To accomplish this goal, we produced a library of monoclonal antibodies against duck hepatitis B virus and identified 12 neutralizing monoclonal antibodies by using an in vitro neutralization assay. The characteristics of six of the neutralizing monoclonal antibodies were further studied by epitope mapping. From the results of competitive binding studies, three distinct neutralizing epitopes were identified on the pre-S polypeptides and one was identified on the S polypeptide. Our findings suggest that antibodies to both the pre-S and S gene products of duck hepatitis B virus can neutralize viral infection in vitro. The pre-S gene product is at least as important as the S gene product in eliciting neutralizing antibodies.

Animals↗
PubMed1989

Identification of a region within the human immunodeficiency virus type 1 long terminal repeat that is essential for transactivation by the hepatitis B virus gene X.

Hepatitis B virus (HBV) X-gene product activates transcription of the chloramphenicol acetyltransferase (CAT) gene under control of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR). To identify a cis-acting regulatory sequence within the HIV-1 LTR which is responsive to the HBV X-gene trans-activating function, we examined the effects of HBV X-gene expression in cells with a series of LTR/CAT deletion mutants. A region of the HIV-1 LTR containing the previously identified kappa B-like enhancer element was found to be responsive to HBV X-gene activation, and this effect was independent of, and additive with, the effect of the HIV-1 tat-III protein on CAT expression. Since kappa B-like enhancer sequences are known to regulate transcription of a variety of viruses and cellular genes, our results suggest that the X gene could activate such a gene during HBV infection and replication.

Cell Line↗
PubMed1989

Hepadnaviruses and hepatocellular carcinoma (HCC).

Members of the hepadnavirus family share properties of virion structure, genome structure and replication, epidemiologic behavior, and pathogenic effects. Persistent infections with hepatitis B virus (HBV) in man, woodchuck hepatitis virus (WHV) in Marmota monax, ground squirrel hepatitis virus (GSHV) in Spermophilus beecheyi, and duck hepatitis B virus (DHBV) in domestic ducks of China are associated with development of hepatocellular carcinoma (HCC). Epidemiological evidence implicating hepadnavirus infection in HCC includes the observation that the geographic distributions of HBV infection and HCC are similar, that the incidence of HCC is much higher in hepadnavirus-infected than uninfected hosts, and that viral DNA sequences are integrated in the cellular DNA of most (e.g., 80 to 90%), but not all, hepadnavirus-associated HCC. Cirrhosis further increases the risk of HCC in HBV-infected humans. The precise role of hepadnaviruses in development of most HCC is unclear, although the finding of viral integrations within or near protooncogenes in a few cases suggests the possibility that these integrations may play a direct role in these HCC. However, in the great majority of HCC, viral integrations are in different cellular DNA sites in different HCC, integrations are not within domains of known protooncogenes, and integrations are not found in some 10 to 15% hepadnavirus-associated HCC, suggesting that persisting viral sequences are not directly involved in the development of these HCC as viral sequences are for tumors caused by viruses with oncogenes or viruses that act by a "promoter-insertion" mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

State of hepatitis B viral genomes in cirrhotic and hepatocellular carcinoma nodules.

DNA of individual cirrhotic nodules (CN) and hepatocellular carcinoma nodules (HCN) of three hepatitis B surface antigen positive autopsy cases with macronodular cirrhosis were analyzed by Southern blot and slot blot hybridization with a hepatitis B virus (HBV) DNA probe. Evidence of episomal or replicating viral DNA, viral DNA integration at the same cellular DNA site in many cells (clonal integration) and viral integration in different cellular DNA sites in many different cells (non-clonal integration) was found in different cirrhotic nodules of the same liver, indicating heterogeneity in the state of HBV in different cells and in different cirrhotic nodules within each infected liver. Episomal or replicating viral DNA forms were found in all cirrhotic nodules of one liver, in less than 10% of examined nodules of a second liver and in none of the third. Evidence of clonal viral integration was found in CN of all three livers and non-clonal integration in CN of the latter two. Cirrhotic nodules with apparent different integrations in many different cells (non-clonal integration) outnumbered those with the same integration site in many cells (clonal integration), and many cirrhotic nodules in those two livers had no detectable viral DNA. Cirrhotic nodules with a viral integration in the same cellular DNA site in many cells would appear to have been formed by clonal expansion of an original cell containing the viral integration, and cirrhotic nodules with different integrations in many different cells (non-clonal integration) may have been formed by recruitment of many different cells with different viral integrations or by clonal expansion of cells without HBV integrations and subsequent viral integrations occurring integration. In one liver, three different hepatocellular carcinoma nodules appeared to represent metastatic lesions because the clonal pattern of HBV integration was identical in each, and in another liver different HCN appeared to be of different clonal origin, i.e. to have arisen from different cells, because multiple viral integrations (i.e. multiple individual restriction fragments with HBV sequences) were each different in different HCN of that liver.

Blotting, Southern↗
PubMed1987

Antibody to human immunodeficiency virus correlates with decreased T helper lymphocytes in asymptomatic individuals.

To examine the relationship between human immunodeficiency virus (HIV) seropositivity and T lymphocyte subsets in a clinically well population, we assayed HIV antibody and analyzed T lymphocyte subsets in 30 people at increased risk for acquired immunodeficiency syndrome (AIDS) who were clinically well. Seventy-six percent of the HIV-seropositive individuals had abnormally low numbers of T helper lymphocytes, and HIV seropositivity was strongly correlated with an abnormally low number of T helper cells (p less than 0.00002). Among these clinically well subjects at increased risk for AIDS, HIV-sero-positive individuals had a significant decrease in mean T helper lymphocytes and mean T helper:T suppressor ratios as compared to those who were seronegative (483 cells/mm3 vs 915 cells/mm3, p less than 0.002; and 0.80 vs 1.7, p less than 0.002, respectively). Because of the strong correlation of HIV seropositivity and abnormally low numbers of T helper lymphocytes in this asymptomatic population, these findings suggest that asymptomatic seropositive individuals should be followed closely for development of AIDS-related disease and should be considered for future antiviral therapy when it becomes available.

Acquired Immunodeficiency Syndrome↗
PubMed1987

Duck hepatitis B virus replicates in the yolk sac of developing embryos.

Duck hepatitis B virus (DHBV) is the only member of the hepadnavirus family in which nearly 100% vertical transmission from carrier mother to embryo has been reported. Large quantities of maternally transmitted virus particles are present in the yolk prior to incubation of the eggs, and replicative forms of DHBV DNA are detectable in the liver at 6 days of incubation. Since the yolk sac is similar to the liver in its production of serum proteins, we examined the yolk sacs of developing embryos for signs of viral replication. We detected the supercoiled form of DHBV DNA, DHBV RNA transcripts similar to those in the virus-replicating liver, and DNA polymerase activity and viral DNA in corelike particles in extracts of yolk sac tissue of naturally infected eggs. DHBV core antigen was strongly stained in only the endodermal layer of the yolk sac by immunofluorescence. DHBV RNA was detectable in the yolk sac from 4 days of incubation until hatching, and a larger quantity of DHBV RNA was present in the yolk sac than in the liver during all the stages of embryogenesis. Our data indicate that DHBV replicates actively in the yolk sac from an earlier stage than that previously reported in studies of embryonic liver and that replication is limited to the endodermal cell layer, which is ontogenetically and functionally related to the liver. The yolk sac may support the vertical transmission of DHBV.

Animals↗