Natural killer T cell dysfunction in CD39-null mice protects against concanavalin A-induced hepatitis

Concanavalin A (Con A)-induced injury is an established natural killer T (NKT) cell-mediated model of inflammation that has been used in studies of immune liver disease. Extracellular nucleotides, such as adenosine triphosphate, are released by Con A-stimulated cells and bind to specific purinergic type 2 receptors to modulate immune activation responses. Levels of extracellular nucleotides are in turn closely regulated by ectonucleotidases, such as CD39/NTPDase1. Effects of extracellular nucleotides and CD39 on NKT cell activation and upon hepatic inflammation have been largely unexplored to date. Here, we show that NKT cells express both CD39 and CD73/ecto-5'-nucleotidase and can therefore generate adenosine from extracellular nucleotides, whereas natural killer cells do not express CD73. In vivo, mice null for CD39 are protected from Con A-induced liver injury and show substantively lower serum levels of interleukin-4 and interferon-gamma when compared with matched wild-type mice. Numbers of hepatic NKT cells are significantly decreased in CD39 null mice after Con A administration. Hepatic NKT cells express most P2X and P2Y receptors; exceptions include P2X3 and P2Y11. Heightened levels of apoptosis of CD39 null NKT cells in vivo and in vitro appear to be driven by unimpeded activation of the P2X7 receptor. CONCLUSION: CD39 and CD73 are novel phenotypic markers of NKT cells. Deletion of CD39 modulates nucleotide-mediated cytokine production by, and limits apoptosis of, hepatic NKT cells providing protection against Con A-induced hepatitis. This study illustrates a further role for purinergic signaling in NKT-mediated mechanisms that result in liver immune injury.

Fatal hepatitis mediated by tumor necrosis factor TNFalpha requires caspase-8 and involves the BH3-only proteins Bid and Bim

Apoptotic death of hepatocytes, a contributor to many chronic and acute liver diseases, can be a consequence of overactivation of the immune system and is often mediated by TNFalpha. Injection with lipopolysaccharide (LPS) plus the transcriptional inhibitor D(+)-galactosamine (GalN) or mitogenic T cell activation causes fatal hepatocyte apoptosis in mice, which is mediated by TNFalpha, but the effector mechanisms remain unclear. Our analysis of gene-targeted mice showed that caspase-8 is essential for hepatocyte killing in both settings. Loss of Bid, the proapoptotic BH3-only protein activated by caspase-8 and essential for Fas ligand-induced hepatocyte killing, resulted only in a minor reduction of liver damage. However, combined loss of Bid and another BH3-only protein, Bim, activated by c-Jun N-terminal kinase (JNK), protected mice from LPS+GalN-induced hepatitis. These observations identify caspase-8 and the BH3-only proteins Bid and Bim as potential therapeutic targets for treatment of inflammatory liver diseases.

Hepatitis B virus infection is associated with impaired immunological recovery during antiretroviral therapy in the Swiss HIV cohort study

Hepatitis B virus (HBV) infection is a major cause of morbidity and mortality in human immunodeficiency virus (HIV)-infected patients worldwide. It is unclear whether HIV-related outcomes are affected by HBV coinfection. We compared virological suppression and immunological recovery during antiretroviral therapy (ART) of patients of different HBV serological status in the Swiss HIV Cohort Study. CD4 cell recovery during ART was significantly impaired in hepatitis B surface antigen-positive patients and in those with anti-hepatitis B core antigen alone compared with HBV-uninfected patients, despite similar virological efficacy of ART. CD4 increase in patients with resolved HBV infection was similar to that in HBV-uninfected individuals.

Molecular evolution of primate immunodeficiency viruses and hepatitis delta virus

Primate immunodeficiency viruses, or lentiviruses (HIV-1, HIV-2, and SIV), and hepatitis delta virus (HDV) are RNA viruses characterized by rapid evolution. Infection by primate immunodeficiency viruses usually results in the development of acquired immunodeficiency syndrome (AIDS) in humans and AIDS-like illnesses in Asian macaques. Similarly, hepatitis delta virus infection causes hepatitis and liver cancer in humans. These viruses are heterogeneous within an infected patient and among individuals. Substitution rates in the virus genomes are high and vary in different lineages and among sites. Methods of phylogenetic analysis were applied to study the evolution of primate lentiviruses and the hepatitis delta virus. The following results have been obtained: (1) The substitution rate varies among sites of primate lentivirus genes according to the two parameter gamma distribution, with the shape parameter $\alpha$ being close to 1. (2) Primate immunodeficiency viruses fall into species-specific lineages. Therefore, viral transmissions across primate species are not as frequent as suggested by previous authors. (3) Primate lentiviruses have acquired or lost their pathogenicity several times in the course of evolution. (4) Evidence was provided for multiple infections of a North American patient by distinct HIV-1 strains of the B subtype. (5) Computer simulations indicate that the probability of committing an error in testing HIV transmission depends on the number of virus sequences and their length, the divergence times among sequences, and the model of nucleotide substitution. (6) For future investigations of HIV-1 transmissions, using longer virus sequences and avoiding the use of distant outgroups is recommended. (7) Hepatitis delta virus strains are usually related according to the geographic region of isolation. (8) Evolution of HDV is characterized by the rate of synonymous substitution being lower than the nonsynonymous substitution rate and the rate of evolution of the noncoding region. (9) There is a strong preference for G and C nucleotides at the third codon positions of the HDV coding region. ^

Seroprevalence of hepatitis E virus in domestic pigs and wild boars in Switzerland.

Hepatitis E is considered an emerging human viral disease in industrialized countries. Studies from Switzerland report a human seroprevalence of hepatitis E virus (HEV) of 2.6-21%, a range lower than in adjacent European countries. The aim of this study was to determine whether HEV seroprevalence in domestic pigs and wild boars is also lower in Switzerland and whether it is increasing and thus indicating that this zoonotic viral infection is emerging. Serum samples collected from 2,001 pigs in 2006 and 2011 and from 303 wild boars from 2008 to 2012 were analysed by ELISA for the presence of HEV-specific antibodies. Overall HEV seroprevalence was 58.1% in domestic pigs and 12.5% in wild boars. Prevalence in domestic pigs was significantly higher in 2006 than in 2011. In conclusion, HEV seroprevalence in domestic pigs and wild boars in Switzerland is comparable with the seroprevalence in other countries and not increasing. Therefore, prevalence of HEV in humans must be related to other factors than prevalence in pigs or wild boars.

Carriage of the PNPLA3 rs738409 C >G polymorphism confers an increased risk of non-alcoholic fatty liver disease associated hepatocellular carcinoma.

BACKGROUND & AIMS Subtle inter-patient genetic variation and environmental factors combine to determine disease progression in non-alcoholic fatty liver disease (NAFLD). Carriage of the PNPLA3 rs738409 c.444C >G minor allele (encoding the I148M variant) has been robustly associated with advanced NAFLD. Although most hepatocellular carcinoma (HCC) is related to chronic viral hepatitis or alcoholic liver disease, the incidence of NAFLD-related HCC is increasing. We examined whether rs738409 C >G was associated with HCC-risk in patients with NAFLD. METHODS PNPLA3 rs738409 genotype was determined by allelic discrimination in 100 European Caucasians with NAFLD-related HCC and 275 controls with histologically characterised NAFLD. RESULTS Genotype frequencies were significantly different between NAFLD-HCC cases (CC=28, CG=43, GG=29) and NAFLD-controls (CC=125, CG=117, GG=33) (p=0.0001). In multivariate analysis adjusted for age, gender, diabetes, BMI, and presence of cirrhosis, carriage of each copy of the rs738409 minor (G) allele conferred an additive risk for HCC (adjusted OR 2.26 [95% CI 1.23-4.14], p=0.0082), with GG homozygotes exhibiting a 5-fold [1.47-17.29], p=0.01 increased risk over CC. When compared to the UK general population (1958 British Birth Cohort, n=1476), the risk-effect was more pronounced (GC vs. CC: unadjusted OR 2.52 [1.55-4.10], p=0.0002; GG vs. CC: OR 12.19 [6.89-21.58], p<0.0001). CONCLUSIONS Carriage of the PNPLA3 rs738409 C >G polymorphism is not only associated with greater risk of progressive steatohepatitis and fibrosis but also of HCC. If validated, these findings suggest that PNPLA3 genotyping has the potential to contribute to multi-factorial patient-risk stratification, identifying those to whom HCC surveillance may be targeted.

Elimination of racial/ethnic Hepatitis B vaccination disparities in the US in the 2007--2008 National Health and Nutrition Examination Survey

Viral hepatitis is a significant public health problem worldwide and is due to viral infections that are classified as Hepatitis A, B, C, D, and E. Hepatitis B is one of the five known hepatic viruses. A safe and effective vaccine for Hepatitis B was first developed in 1981, and became adopted into national immunization programs targeting infants since 1990 and adolescents since 1995. In the U.S., this vaccination schedule has led to an 82% reduction in incidence from 8.5 cases per 100,000 in 1990 to 1.5 cases per 100,000 in 2007. Although there has been a decline in infection among adolescents, there is still a large burden of hepatitis B infection among adults and minorities. There is very little research in regards to vaccination gaps among adults. Using the National Health and Nutrition Examination Survey (NHANES) question "{Have you/Has SP (Study Participant)} ever received the 3-dose series of the hepatitis B vaccine?" the existence of racial/ethnic gaps using a cross-sectional study design was explored. In this study, other variables such as age, gender, socioeconomic variables (federal poverty line, educational attainment), and behavioral factors (sexual practices, self-report of men having sex with men, and intravenous drug use) were examined. We found that the current vaccination programs and policies for Hepatitis B had eliminated racial and ethnic disparities in Hepatitis B vaccination, but that a low coverage exists particularly for adults who engage in high risk behaviors. This study found a statistically significant 10% gap in Hepatitis B vaccination between those who have and those who do not have access to health insurance.^

Heterogeneous nuclear ribonucleoprotein A1 binds to the transcription-regulatory region of mouse hepatitis virus RNA

A cellular protein, previously described as p35/38, binds to the complementary (−)-strand of the leader RNA and intergenic (IG) sequence of mouse hepatitis virus (MHV) RNA. The extent of the binding of this protein to IG sites correlates with the efficiency of the subgenomic mRNA transcription from that IG site, suggesting that it is a requisite transcription factor. We have purified this protein and determined by partial peptide sequencing that it is heterogeneous nuclear ribonucleoprotein (hnRNP) A1, an abundant, primarily nuclear protein. hnRNP A1 shuttles between the nucleus and cytoplasm and plays a role in the regulation of alternative RNA splicing. The MHV(−)-strand leader and IG sequences conform to the consensus binding motifs of hnRNP A1. Recombinant hnRNP A1 bound to these two RNA regions in vitro in a sequence-specific manner. During MHV infection, hnRNP A1 relocalizes from the nucleus to the cytoplasm, where viral replication occurs. These data suggest that hnRNP A1 is a cellular factor that regulates the RNA-dependent RNA transcription of the virus.

Localization of the N terminus of hepatitis B virus capsid protein by peptide-based difference mapping from cryoelectron microscopy

Recently, cryoelectron microscopy of isolated macromolecular complexes has advanced to resolutions below 10 Å, enabling direct visualization of α-helical secondary structure. To help correlate such density maps with the amino acid sequences of the component proteins, we advocate peptide-based difference mapping, i.e., insertion of peptides, ≈10 residues long, at targeted points in the sequence and visualization of these peptides as bulk labels in cryoelectron microscopy-derived difference maps. As proof of principle, we have appended an extraneous octapeptide at the N terminus of hepatitis B virus capsid protein and determined its location on the capsid surface by difference imaging at 11 Å resolution. Hepatitis B virus capsids are icosahedral particles, ≈300 Å in diameter, made up of T-shaped dimers (subunit Mr, 16–21 kDa, depending on construct). The stems of the Ts protrude outward as spikes, whereas the crosspieces pack to form the contiguous shell. The two N termini per dimer reside on either side of the spike-stem, at the level at which it enters the shell. This location is consistent with formation of the known intramolecular disulfide bond between the cysteines at positions 61 and −7 (in the residual propeptide) in the “e-antigen” form of the capsid protein and has implications for why this clinically important antigen remains unassembled in vivo.

Hepatitis B virus X protein and p53 tumor suppressor interactions in the modulation of apoptosis

We have reported previously that the hepatitis B virus oncoprotein, HBx, can bind to the C terminus of p53 and inhibit several critical p53-mediated cellular processes, including DNA sequence-specific binding, transcriptional transactivation, and apoptosis. Recognizing the importance of p53-mediated apoptosis for maintaining homeostasis and preventing neoplastic transformation, here we further examine the physical interaction between HBx and p53 as well as the functional consequences of this association. In vitro binding studies indicate that the ayw and adr viral subtypes of HBx bind similar amounts of glutathione S-transferase-p53 with the distal C terminus of HBx (from residues 111 to 154) being critical for this interaction. Using a microinjection technique, we show that this same C-terminal region of HBx is necessary for sequestering p53 in the cytoplasm and abrogating p53-mediated apoptosis. The transcriptional transactivation domain of HBx also maps to its C terminus; however, a comparison of the ability of full-length and truncated HBx protein to abrogate p53-induced apoptosis versus transactivate simian virus 40- or human nitric oxide synthase-2 promoter-driven reporter constructs indicates that these two functional properties are distinct and thus may contribute to hepatocarcinogenesis differently. Collectively, our data indicate that the distal C-terminal domain of HBx, independent of its transactivation activity, complexes with p53 in the cytoplasm, partially preventing its nuclear entry and ability to induce apoptosis. These pathobiological effects of HBx may contribute to the early stages of hepatocellular carcinogenesis.

Native display of complete foreign protein domains on the surface of hepatitis B virus capsids

The nucleocapsid of hepatitis B virus (HBV), or HBcAg, is a highly symmetric structure formed by multiple dimers of a single core protein that contains potent T helper epitopes in its 183-aa sequence. Both factors make HBcAg an unusually strong immunogen and an attractive candidate as a carrier for foreign epitopes. The immunodominant c/e1 epitope on the capsid has been suggested as a superior location to convey high immunogenicity to a heterologous sequence. Because of its central position, however, any c/e1 insert disrupts the core protein’s primary sequence; hence, only peptides, or rather small protein fragments seemed to be compatible with particle formation. According to recent structural data, the epitope is located at the tips of prominent surface spikes formed by the very stable dimer interfaces. We therefore reasoned that much larger inserts might be tolerated, provided the individual parts of a corresponding fusion protein could fold independently. Using the green fluorescent protein (GFP) as a model insert, we show that the chimeric protein efficiently forms fluorescent particles; hence, all of its structurally important parts must be properly folded. We also demonstrate that the GFP domains are surface-exposed and that the chimeric particles elicit a potent humoral response against native GFP. Hence, proteins of at least up to 238 aa can be natively displayed on the surface of HBV core particles. Such chimeras may not only be useful as vaccines but may also open the way for high resolution structural analyses of nonassembling proteins by electron microscopy.

Hepatitis B virus transactivator protein, HBx, associates with the components of TFIIH and stimulates the DNA helicase activity of TFIIH.

Human hepatitis B virus genome encodes a protein, termed HBx, that is widely recognized as a transcriptional transactivator. While HBx does not directly bind cis-acting transcriptional control elements, it has been shown to associate with cellular proteins that bind DNA. Because HBx transactivated a large number of viral/cellular transcriptional control elements, we looked for its targets within the components of the basal transcriptional machinery. This search led to the identification of its interactions with TFIIH. Here, we show that HBx interacts with yeast and mammalian TFIIH complexes both in vitro and in vivo. These interactions between HBx and the components of TFIIH are supported by several lines of evidence including results from immunoprocedures and direct methods of measuring interactions. We have identified ERCC3 and ERCC2 DNA helicase subunits of holoenzyme TFIIH as targets of HBx interactions. Furthermore, the DNA helicase activity of purified TFIIH from rat liver and, individually, the ERCC2 component of TFIIH is stimulated in the presence of HBx. These observations suggest a role for HBx in transcription and DNA repair.

Costimulatory protein B7-1 enhances the cytotoxic T cell response and antibody response to hepatitis B surface antigen.

There is a need for more effective therapy for chronic virus infections. A principle natural mechanism for elimination of virus-infected host cells is activation of viral antigen-specific cytotoxic T lymphocytes (CTL). In an effort to develop methods of inducing virus-specific CTL responses that might be utilized in therapy of virus infections, we have investigated the effect of B7, a costimulatory factor for T-cell activation. In this study we show that delivery of genes encoding human B7-1 and a viral antigen in the same recombinant viral vector to cells of mice induces a greater viral antigen-specific CTL response than does similar delivery of the viral antigen gene alone. Two recombinant adenovirus vectors were constructed with the foreign genes inserted in the early region 3. One of them (Ad1312) directed expression of the surface antigen gene of hepatitis B virus (HBS); the other (Ad1310) directed coexpression of HBS and human B7-1 (CD80) by means of an internal ribosomal entry site placed between the two coding sequences. When inoculated into BALB/c mice, both vectors induced a viral surface antigen-specific CTL response. The response induced by Ad1310 was stronger than that by Adl312 as measured by a chromium release assay for CTL activity and limiting dilution analysis for CTL precursor frequency, indicating that the B7-1 gene co-delivered with the HBS gene had an enhancing effect on the CTL response against surface antigen. Ad1310 also induced a higher titer of antibody against surface antigen than did Ad1312. This result suggests that expression of a costimulatory protein and a viral antigen in the same cells in vivo induces stronger immune responses than expression of the antigen alone. This could be a novel strategy for development of both preventive and therapeutic vaccines against infectious agents.

A truncated mutant (residues 58-140) of the hepatitis B virus X protein retains transactivation function.

The hepatitis B virus X protein (HBx) sequence (154 aa) has been divided into six regions (A-F) based on its sequence homology with X proteins of other mammalian hepadnaviruses. Regions A, C, and E are more conserved and include all the four conserved cysteines (C7, C61, C69, and C137). To localize the regions of HBx important for transactivation, a panel of 10 deletion mutants (X5-X14) and 4 single point mutants (X1-X4), each corresponding to a conserved cysteine residue, was constructed by site-directed mutagenesis. A HBx-specific monoclonal antibody was developed and used to confirm the expression of mutants by Western blot. Transactivation property of the HBx mutants was studied on Rous sarcoma virus-long terminal repeat (RSV-LTR) in transient transfection assays. We observed that deletion of the most conserved region A or substitution of the N-terminal cysteine (C7) had no effect on transactivation. Deletion of the nonconserved regions B or F also had no deleterious effects. Deletions of regions C and D resulted in a significant loss of function. Substitution of both C61 and C69 present in region C, caused almost 90% loss of activity that could be partially overcome by transfecting more expression plasmid. The fully conserved 9 amino acid segment (residues 132 to 140) within region E including C137 appeared to be crucial for its activity. Finally, a truncated mutant X15 incorporating only regions C to E (amino acids 58-140) was able to stimulate the RSV-LTR quite efficiently, suggesting a crucial role played by this domain in transactivation function.

Viral dynamics in hepatitis B virus infection.

Treatment of chronic hepatitis B virus (HBV) infections with the reverse transcriptase inhibitor lamivudine leads to a rapid decline in plasma viremia and provides estimates for crucial kinetic constants of HBV replication. We find that in persistently infected patients, HBV particles are cleared from the plasma with a half-life of approximately 1.0 day, which implies a 50% daily turnover of the free virus population. Total viral release into the periphery is approximately 10(11) virus particles per day. Although we have no direct measurement of the infected cell mass, we can estimate the turnover rate of these cells in two ways: (i) by comparing the rate of viral production before and after therapy or (ii) from the decline of hepatitis B antigen during treatment. These two independent methods give equivalent results: we find a wide distribution of half-lives for virus-producing cells, ranging from 10 to 100 days in different patients, which may reflect differences in rates of lysis of infected cells by immune responses. Our analysis provides a quantitative understanding of HBV replication dynamics in vivo and has implications for the optimal timing of drug treatment and immunotherapy in chronic HBV infection. This study also represents a comparison for recent findings on the dynamics of human immunodeficiency virus (HIV) infection. The total daily production of plasma virus is, on average, higher in chronic HBV carriers than in HIV-infected patients, but the half-life of virus-producing cells is much shorter in HIV. Most strikingly, there is no indication of drug resistance in HBV-infected patients treated for up to 24 weeks.