The host structural maintenance of chromosomes 5/6 complex (Smc5/6) suppresses hepatitis B virus (HBV) transcription. HBV counters this restriction by expressing the X protein (HBx), which redirects the cellular DNA damage-binding protein 1 (DDB1)-containing E3 ubiquitin ligase to target Smc5/6 for degradation. However, the details of how HBx modulates the interaction between DDB1 and Smc5/6 remain to be determined. In this study, we performed biophysical analyses of recombinant HBx and functional analysis of HBx mutants in HBV-infected primary human hepatocytes (PHH) to identify key regions and residues that are required for HBx function. We determined that recombinant HBx is soluble and exhibits stoichiometric zinc binding when expressed in the presence of DDB1. Mass spectrometry-based hydrogen-deuterium exchange and cysteine-specific chemical footprinting of the HBx:DDB1 complex identified several HBx cysteine residues (located between amino acids 61 and 137) that are likely involved in zinc binding. These cysteine residues did not form disulfide bonds in HBx expressed in human cells. In line with the biophysical data, functional analysis demonstrated that HBx amino acids 45 to 140 are required for Smc6 degradation and HBV transcription in PHH. Furthermore, site-directed mutagenesis determined that C61, C69, C137, and H139 are necessary for HBx function, although they are likely not essential for DDB1 binding. This CCCH motif is highly conserved in HBV as well as in the X proteins from various mammalian hepadnaviruses. Collectively, our data indicate that the essential HBx cysteine and histidine residues form a zinc-binding motif that is required for HBx function.IMPORTANCE The structural maintenance of chromosomes 5/6 complex (Smc5/6) is a host restriction factor that suppresses HBV transcription. HBV counters this restriction by expressing HBV X protein (HBx), which redirects a host ubiquitin ligase to target Smc5/6 for degradation. Despite this recent advance in understanding HBx function, the key regions and residues of HBx required for Smc5/6 degradation have not been determined. In the present study, we performed biochemical, biophysical, and cell-based analyses of HBx. By doing so, we mapped the minimal functional region of HBx and identified a highly conserved CCCH motif in HBx that is likely responsible for coordinating zinc and is essential for HBx function. We also developed a method to produce soluble recombinant HBx protein that likely adopts a physiologically relevant conformation. Collectively, this study provides new insights into the HBx structure-function relationship and suggests a new approach for structural studies of this enigmatic viral regulatory protein.
- MeSH
- aminokyselinové motivy MeSH
- aminokyseliny MeSH
- DNA vazebné proteiny metabolismus MeSH
- hepatitida B metabolismus virologie MeSH
- interakce hostitele a patogenu MeSH
- lidé MeSH
- rekombinantní fúzní proteiny MeSH
- sekvence aminokyselin MeSH
- trans-aktivátory chemie metabolismus MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- virus hepatitidy B fyziologie MeSH
- zinek metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
We describe here the anti-HBV activity of natural and synthetic retinoids in primary human hepatocytes (PHHs). The most potent compounds inhibited HBsAg, HBeAg, viral RNA and DNA production by HBV infected cells with EC50 values ranging from 0.4 to 2.6 μM. The activity was independent of PHH donor and HBV genotype used in testing. 13-cis retinoic acid (Accutane) was selected for further evaluation in the PXB chimeric mouse model of HBV infection at doses allowing to achieve Accutane peak serum concentrations near its antiviral EC90 and exposures ∼5-fold higher than a typical clinical dose. While these supraclinical exposures of 100 mg/kg/day were well-tolerated by regular Balb/c mice, PXB mice were more sensitive and even a lower those of 60 mg/kg/day led to significant weight loss. Despite dosing at this maximal tolerated dose for 28 days, Accutane failed to show any anti-HBV activity. RAR target engagement was verified using transcriptome analysis of liver samples from treated versus vehicle groups. However, gene expression changes in PXB liver samples were vastly muted when compared to the in vitro PHH system. When comparing transcriptional changes associated with the conditioning of fresh hepatocytes toward enabling HBV infection, we also observed a large number of changes. Noticeably, a significant number of genes that were up- or down-regulated by the conditioning process were down- or up-regulated by HBV infected PHH treatment with Accutane, respectively. While the lack of efficacy in the PXB model may have many explanations, the observed, opposing transcriptional changes upon conditioning PHH and treating these cultured, HBV-infected PHH with Accutane allow for the possibility that the PHH system may yield artificial anti-HBV hits.
- MeSH
- antivirové látky krev farmakologie MeSH
- DNA virů metabolismus MeSH
- down regulace MeSH
- exprese genu účinky léků MeSH
- hepatitida B - antigeny e účinky léků MeSH
- hepatitida B - antigeny povrchové účinky léků MeSH
- hepatitida B farmakoterapie virologie MeSH
- hepatocyty metabolismus virologie MeSH
- isotretinoin farmakologie MeSH
- lidé MeSH
- modely nemocí na zvířatech MeSH
- myši inbrední BALB C MeSH
- myši MeSH
- replikace viru účinky léků MeSH
- retinoidy krev farmakologie MeSH
- RNA virová metabolismus MeSH
- upregulace MeSH
- viabilita buněk účinky léků MeSH
- virus hepatitidy B účinky léků genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND & AIMS: Robust data on hepatocellular carcinoma (HCC) incidence among HIV/hepatitis B virus (HBV)-coinfected individuals on antiretroviral therapy (ART) are needed to inform HCC screening strategies. We aimed to evaluate the incidence and risk factors of HCC among HIV/HBV-coinfected individuals on tenofovir disoproxil fumarate (TDF)-containing ART in a large multi-cohort study. METHODS: We included all HIV-infected adults with a positive hepatitis B surface antigen test followed in 4 prospective European cohorts. The primary outcome was the occurrence of HCC. Demographic and clinical information was retrieved from routinely collected data, and liver cirrhosis was defined according to results from liver biopsy or non-invasive measurements. Multivariable Poisson regression was used to assess HCC risk factors. RESULTS: A total of 3,625 HIV/HBV-coinfected patients were included, of whom 72% had started TDF-containing ART. Over 32,673 patient-years (py), 60 individuals (1.7%) developed an HCC. The incidence of HCC remained stable over time among individuals on TDF, whereas it increased steadily among those not on TDF. Among individuals on TDF, the incidence of HCC was 5.9 per 1,000 py (95% CI 3.60-9.10) in cirrhotics and 1.17 per 1,000 py (0.56-2.14) among non-cirrhotics. Age at initiation of TDF (adjusted incidence rate ratio per 10-year increase: 2.2, 95% CI 1.6-3.0) and the presence of liver cirrhosis (4.5, 2.3-8.9) were predictors of HCC. Among non-cirrhotic individuals, the incidence of HCC was only above the commonly used screening threshold of 2 cases per 1,000 py in patients aged >45 years old at TDF initiation. CONCLUSIONS: Whereas the incidence of HCC was high in cirrhotic HIV/HBV-coinfected individuals, it remained below the HCC screening threshold in patients without cirrhosis who started TDF aged <46 years old. LAY SUMMARY: We investigated the incidence of hepatocellular carcinoma in HIV/hepatitis B virus-coinfected individuals from a large multi-cohort study in Europe. Over 32,673 patient-years, 60 individuals (1.7%) developed hepatocellular carcinoma. The incidence of hepatocellular carcinoma remained low in patients without cirrhosis, who started on tenofovir disoproxil fumarate when aged <46 years old.
- MeSH
- dospělí MeSH
- hepatitida B - antigeny povrchové analýza MeSH
- hepatitida B farmakoterapie virologie MeSH
- hepatocelulární karcinom epidemiologie MeSH
- HIV * MeSH
- incidence MeSH
- koinfekce farmakoterapie virologie MeSH
- látky proti HIV terapeutické užití MeSH
- lidé středního věku MeSH
- lidé MeSH
- nádory jater epidemiologie MeSH
- následné studie MeSH
- oportunní infekce doprovázející AIDS farmakoterapie virologie MeSH
- prospektivní studie MeSH
- tenofovir terapeutické užití MeSH
- virus hepatitidy B imunologie MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Klíčová slova
- virologické testy,
- MeSH
- genotypizační techniky přístrojové vybavení MeSH
- Hepacivirus genetika izolace a purifikace MeSH
- hepatitida B * krev virologie MeSH
- hepatitida C * krev virologie MeSH
- HIV infekce krev virologie MeSH
- HIV-1 * izolace a purifikace MeSH
- klinické laboratorní techniky * přístrojové vybavení MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- lidé MeSH
- RNA virová MeSH
- virová nálož přístrojové vybavení MeSH
- virus hepatitidy B genetika izolace a purifikace MeSH
- Check Tag
- lidé MeSH
- MeSH
- Hepacivirus genetika izolace a purifikace MeSH
- hepatitida B * diagnóza epidemiologie virologie MeSH
- hepatitida C * diagnóza epidemiologie virologie MeSH
- HIV infekce * diagnóza epidemiologie virologie MeSH
- HIV-1 genetika izolace a purifikace MeSH
- HIV-2 genetika izolace a purifikace MeSH
- lidé MeSH
- virus hepatitidy B genetika izolace a purifikace MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
OBJECTIVES: The aim of this study was to determine the prevalence of HBV infection among pregnant women in districts of Eastern Slovakia with a diverse prevalence of Roma population. METHODS: Overall 59,279 serum samples from 9 regional departments of clinical microbiology from Eastern Slovakia were collected in the period from January 2008 till December 2009 and analysed. RESULTS: The number of HBsAg positive samples overall and during pregnancy was 1.74% and 2.12%, respectively. Comparing districts with higher (> 5%) and lower (< 5%) Roma population, there was no significant difference in the prevalence of HBsAg positive samples overall (1.95% vs.1.62%). However, in the subgroup of pregnant women the prevalence of HBsAg positive samples (2.72% vs. 0.95%) differs significantly (p < 0.01). CONCLUSIONS: The prevalence of HBV infection among pregnant women in Eastern Slovakia did not rapidly exceed the estimated nationwide prevalence. However, in districts with higher Roma population the expected higher prevalence of HBV infection was confirmed. This indicates the need to pay special attention to the prevention of hepatitis B in these districts.
- MeSH
- hepatitida B - antigeny izolace a purifikace MeSH
- hepatitida B epidemiologie etnologie virologie MeSH
- infekční komplikace v těhotenství etnologie virologie MeSH
- lidé MeSH
- surveillance populace MeSH
- těhotenství MeSH
- Check Tag
- lidé MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Slovenská republika MeSH
- MeSH
- hepatitida B epidemiologie genetika virologie MeSH
- klinické laboratorní techniky MeSH
- lidé MeSH
- Check Tag
- lidé MeSH
- Klíčová slova
- doporučené, povinné,
- MeSH
- břišní tyfus prevence a kontrola MeSH
- cestování MeSH
- cholera epidemiologie patologie prevence a kontrola MeSH
- difterie patologie prevence a kontrola virologie MeSH
- hepatitida A prevence a kontrola virologie MeSH
- hepatitida B prevence a kontrola virologie MeSH
- japonská encefalitida patologie prevence a kontrola virologie MeSH
- lidé MeSH
- meningokoková meningitida epidemiologie prevence a kontrola virologie MeSH
- rabies patologie prevence a kontrola virologie MeSH
- směrnice pro lékařskou praxi jako téma MeSH
- tetanus patologie prevence a kontrola MeSH
- vakcinace klasifikace trendy využití MeSH
- žlutá zimnice epidemiologie patologie prevence a kontrola MeSH
- Check Tag
- lidé MeSH
BACKGROUND: Hepatitis B vaccination in children born to hepatitis B surface antigen (HBsAg)-positive mothers considerably decreases the risk of vertical transmission. However, whether this protection against carriage of hepatitis B virus is maintained into early adulthood is as yet unknown. PATIENTS AND METHODS: A combined passive-active immunization programme for newborns of HBsAg-positive mothers was initiated in the north-eastern part of the Czech Republic in 1988. The number of immunized newborns had reached 665 newborns by the end of 2006. All mothers of immunized infants were HBsAg-positive during pregnancy, and 34 (5%) were also hepatitis B e antigen (HBeAg)-positive. The immunization programme consists of providing newborns with protection at birth with hepatitis B immunoglobulin, followed by three 10-ug doses of plasma-derived or, since 1990, recombinant vaccine administered at 0, 1 and 6months of life. Only 29 children of HBeAg-positive mothers received vaccine at 0, 1 and 2months of life. Blood samples were obtained after immunization, at 2years of age, and biennially thereafter. Samples were tested for HBsAg and hepatitis B surface and core antibodies (anti-HBs, anti-HBc). RESULTS: The immunization schedules were completed in 640 children. A protective anti-HBs level after immunization was proven in 574 of 620 children (93%). Persistence of protective anti-HBs antibodies was detected in 70, 40 and 25% of children at 5, 10 and 15years of age. Vertical transmission with chronic HBsAg carrier status was detected in two infants. Anti-HBc seroconversion was proven in ten children from 3 to 15years of age. Natural boosting with an anti-HBs increase was detected in 38 children (twice in one child). CONCLUSION: Our results show that combined active-passive immunization of newborns against hepatitis B provides persistent protection up to adolescence despite a frequent waning of anti-HBs antibodies, suggesting there is no need for booster vaccination during adolescence.
- MeSH
- čas MeSH
- dítě MeSH
- hepatitida - antigeny imunologie krev MeSH
- hepatitida B - protilátky imunologie krev MeSH
- hepatitida B imunologie prevence a kontrola virologie MeSH
- kojenec MeSH
- lidé MeSH
- mladiství MeSH
- následné studie MeSH
- novorozenec MeSH
- očkovací schéma MeSH
- předškolní dítě MeSH
- těhotenství MeSH
- vakcína proti hepatitidě B * aplikace a dávkování imunologie MeSH
- vakcinace MeSH
- vertikální přenos infekce * prevence a kontrola MeSH
- virus hepatitidy B * imunologie MeSH
- Check Tag
- dítě MeSH
- kojenec MeSH
- lidé MeSH
- mladiství MeSH
- mužské pohlaví MeSH
- novorozenec MeSH
- předškolní dítě MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
