RESUMO
Hepatitis B virus (HBV) expresses co-terminal large (L), middle (M), and small (S) envelope proteins containing preS1/preS2/S, preS2/S, and S domain alone, respectively. S and preS1 domains mediate sequential virion attachment to heparan sulfate proteoglycans and sodium taurocholate cotransporting polypeptide (NTCP), respectively, which can be blocked by anti-S and anti-preS1 antibodies. How anti-preS2 antibodies neutralize HBV infectivity remains enigmatic. The late stage of chronic HBV infection often selects for mutated preS2 translation initiation codon to prevent M protein expression, or in-frame preS2 deletions to shorten both L and M proteins. When introduced to infectious clone of genotype C or D, both M-minus mutations and most 5' preS2 deletions sustained virion production. Such mutant progeny viral particles were infectious in NTCP-reconstituted HepG2 cells. Neutralization experiments were performed on the genotype D clone. Although remaining susceptible to anti-preS1 and anti-S neutralizing antibodies, M-minus mutants were only partially neutralized by two anti-preS2 antibodies tested while preS2 deletion mutants were resistant. By infection experiments using viral particles with lost versus increased M protein expression, or a neutralization escaping preS2 deletion only present on L or M protein, we found that both full-length L and M proteins contributed to virus neutralization by the two anti-preS2 antibodies. Thus, immune escape could be a driving force for the selection of M-minus mutations, and especially preS2 deletions. The fact that both L and M proteins could mediate neutralization by anti-preS2 antibodies may shed light on the underlying molecular mechanism.IMPORTANCEThe large (L), middle (M), and small (S) envelope proteins of hepatitis B virus (HBV) contain preS1/preS2/S, preS2/S, and S domain alone, respectively. The discovery of heparan sulfate proteoglycans and sodium taurocholate cotransporting polypeptide (NTCP) as the low- and high-affinity HBV receptors could explain neutralizing potential of anti-S and anti-preS1 antibodies, respectively, but how anti-preS2 neutralizing antibodies work remains enigmatic. In this study, we found two M-minus mutants in the context of genotype D partially escaped two anti-preS2 neutralizing antibodies in NTCP-reconstituted HepG2 cells, while several naturally occurring preS2 deletion mutants escaped both antibodies. By point mutations to eliminate or enhance M protein expression, and by introducing preS2 deletion selectively to L or M protein, we found binding of anti-preS2 antibodies to both L and M proteins contributed to neutralization of wild-type HBV infectivity. Our finding may shed light on the possible mechanism(s) whereby anti-preS2 antibodies neutralize HBV infectivity.
RESUMO
Hepatitis B virus (HBV) large (L) envelope protein is translated from 2.4-kb RNA. It contains preS1, preS2, and S domains and is detected in Western blotting as p39 and gp42. The 3.5-kb pregenomic RNA produces core and polymerase (P) proteins. We generated L-minus mutants of a genotype A clone and a genotype D clone from 1.1-mer or 1.3-mer construct, with the former overproducing pregenomic RNA. Surprisingly, mutating a preS1 ATG codon(s) or introducing a nonsense mutation soon afterwards switched secreted p39/gp42 to a p41/p44 doublet, with its amount further increased by a nonsense mutation in the core gene. A further-downstream preS1 nonsense mutation prevented p41/p44 production. Tunicamycin treatment confirmed p44 as the glycosylated form of p41. In this regard, splicing of 3.5-kb RNA to generate a junction at nucleotides (nt) 2447 to 2902 for genotype D enables translation of p43, with the N-terminal 47 residues of P protein fused to the C-terminal 371 residues of L protein. Indeed p41/p44 were detectable by an antibody against the N terminus of P protein and eliminated by a nonsense mutation at the 5' P gene or a point mutation to prevent that splicing. Therefore, lost L (and core) protein expression from the 1.1-mer or 1.3-mer construct markedly increased p41/p44 (p43), the P-L fusion protein. Cotransfection with an expression construct for L/M proteins reversed high extracellular p41/p44 associated with L-minus mutants, suggesting that L protein retains p43 in wild-type HBV to promote its intracellular degradation. Considering that p43 lacks N-terminal preS1 sequence critical for receptor binding, its physiological significance during natural infection and therapeutic potential warrant further investigation. IMPORTANCE The large (L) envelope protein of hepatitis B virus (HBV) is translated from 2.4-kb RNA and detected in Western blotting as p39 and gp42. Polymerase (P) protein is expressed at a low level from 3.5-kb RNA. The major spliced form of 3.5-kb RNA will produce a fusion protein between the first 47 residues of P protein and a short irrelevant sequence, although also at a low level. Another spliced form has the same P protein sequence fused to L protein missing its first 18 residues. We found that some point mutations to eliminate L and core protein expression from overlength HBV DNA constructs converted p39/gp42 to p41/gp44, which turned out to be the P-L fusion protein. Thus, the P-L fusion protein can be expressed at extremely high level when L protein expression is prevented. The underlying mechanism and functional significance of this variant form of L protein warrant further investigation.
Assuntos
Antígenos de Superfície da Hepatite B , Vírus da Hepatite B , Herpesvirus Cercopitecino 1 , Precursores de Proteínas , Proteínas do Envelope Viral , Proteínas Virais de Fusão , Códon sem Sentido/metabolismo , Genótipo , Hepatite B/virologia , Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/genética , Vírus da Hepatite B/metabolismo , Herpesvirus Cercopitecino 1/genética , Humanos , Mutação , Precursores de Proteínas/genética , Proteínas do Envelope Viral/genética , Proteínas Virais de Fusão/genéticaRESUMO
Serum samples were collected from 54 hepatitis B e antigen (HBeAg)-positive Chinese patients infected with hepatitis B virus (HBV) subgenotype B2 or C2. They were compared for transmission efficiency using same volume of samples or infectivity using same genome copy number. Adding polyethylene glycol (PEG) during inoculation did not increase infectivity of fresh samples but markedly increased infectivity following prolonged sample storage. Differentiated HepaRG cells infected without PEG produced more hepatitis B surface antigen (HBsAg) and higher HBsAg/HBeAg ratio than sodium taurocholate cotransporting polypeptide (NTCP)-reconstituted HepG2 cells infected with PEG. They better supported replication of core promoter mutant in contrast to wild-type (WT) virus by HepG2/NTCP cells. Overall, subgenotype C2 samples had higher viral load than B2 samples, and in general produced more HBeAg, HBsAg, and replicative DNA following same-volume inoculation. Precore mutant was more prevalent in subgenotype B2 and had reduced transmission efficiency. When same genome copy number of viral particles was inoculated, viral signals were not necessarily higher for three WT C2 isolates than four WT B2 isolates. Using viral particles generated from cloned HBV genome, three WT C2 isolates showed slightly reduced infectivity than three B2 isolates. In conclusion, subgenotype C2 serum samples had higher transmission efficiency than B2 isolates in association with higher viral load and lower prevalence of precore mutant, but not necessarily higher infectivity. PEG-independent infection by HBV viremic serum samples is probably attributed to a labile host factor.
Assuntos
Antígenos de Superfície da Hepatite B , Antígenos E da Hepatite B , Vírus da Hepatite B , Hepatite B , Humanos , Genótipo , Antígenos E da Hepatite B/sangue , Antígenos de Superfície da Hepatite B/sangue , Vírus da Hepatite B/genética , Polietilenoglicóis , População do Leste Asiático , Hepatite B/transmissão , Hepatite B/virologia , Células Hep G2RESUMO
Hepatitis B virus (HBV) transcribes coterminal mRNAs of 0.7 to 3.5 kb from the 3.2-kb covalently closed circular DNA, with the 2.1-kb RNA being most abundant. The 0.7-kb RNA produces HBx protein, a transcriptional transactivator, while the 3.5-kb pregenomic RNA (pgRNA) drives core and P protein translation as well as genome replication. The large (L) and small (S) envelope proteins are translated from the 2.4-kb and 2.1-kb RNAs, respectively, with the majority of the S protein being secreted as noninfectious subviral particles and detected as hepatitis B surface antigen (HBsAg). pgRNA transcription could inhibit transcription of subgenomic RNAs. The present study characterized naturally occurring in-frame deletions in the 3' preS1 region, which not only codes for L protein but also serves as the promoter for 2.1-kb RNA. The human hepatoma cell line Huh7 was transiently transfected with subgenomic expression constructs for envelope (and HBx) proteins, dimeric constructs, or constructs mimicking covalently closed circular DNA. The results confirmed lost 2.1-kb RNA transcription and HBsAg production from many deletion mutants, accompanied by increases in other (especially 2.4-kb) RNAs, intracellular HBx and core proteins, and replicative DNA but impaired virion and L protein secretion. The highest intracellular L protein levels were achieved by mutants that had residual S protein expression or retained the matrix domain in L protein. Site-directed mutagenesis of a high replicating deletion mutant suggested that increased HBx protein expression and blocked virion secretion both contributed to the high replication phenotype. Our findings could help explain why such deletions are selected at a late stage of chronic HBV infection and how they contribute to viral pathogenesis. IMPORTANCE Expression of hepatitis B e antigen (HBeAg) and overproduction of HBsAg by wild-type HBV are implicated in the induction of immune tolerance to achieve chronic infection. How HBV survives the subsequent immune clearance phase remains incompletely understood. Our previous characterization of core promoter mutations to reduce HBeAg production revealed the ability of the 3.5-kb pgRNA to diminish transcription of coterminal RNAs of 2.4 kb, 2.1 kb, and 0.7 kb. The later stage of chronic HBV infection often selects for in-frame deletions in the preS region. Here, we found that many 3' preS1 deletions prevented transcription of the 2.1-kb RNA for HBsAg production, which was often accompanied by increases in intracellular 3.5-, 0.7-, and especially 2.4-kb RNAs, HBx and core proteins, and replicative DNA but lost virion secretion. These findings established the biological consequences of preS1 deletions, thus shedding light on why they are selected and how they contribute to hepatocarcinogenesis.
Assuntos
Genoma Viral , Antígenos do Núcleo do Vírus da Hepatite B/genética , Vírus da Hepatite B/genética , Transativadores/biossíntese , Proteínas do Envelope Viral/biossíntese , Proteínas do Envelope Viral/genética , Proteínas Virais Reguladoras e Acessórias/biossíntese , Replicação Viral , Linhagem Celular Tumoral , Deleção de Genes , Regulação Viral da Expressão Gênica , Células Hep G2 , Vírus da Hepatite B/metabolismo , Humanos , Regiões Promotoras Genéticas , RNA Viral/metabolismo , Replicação Viral/genéticaRESUMO
BACKGROUND AND AIMS: Deletion of 15-nucleotide or 18-nucleotide (nt) covering preS1 ATG frequently arises during chronic infection with HBV genotypes B and C. Since the second ATG is 33nt downstream, they truncate large (L) envelope protein by 11 residues like wild-type genotype D. This study characterised their functional consequences. METHODS: HBV genomes with or without deletion were amplified from a patient with advanced liver fibrosis and assembled into replication competent 1.1mer construct. Deletion, insertion or point mutation was introduced to additional clones of different genotypes. Viral particles concentrated from transfected HepG2 cells were inoculated to sodium taurocholate cotransporting polypeptide (NTCP)-reconstituted HepG2 (HepG2/NTCP) cells or differentiated HepaRG cells, and HBV RNA, DNA, proteins were monitored. RESULTS: From transfected HepG2 cells, the 15-nt and 18-nt deletions increased HBV RNA, replicative DNA and extracellular virions. When same number of viral particles was inoculated to HepG2/NTCP cells, the deletion mutants showed higher infectivity. Conversely, HBV infectivity was diminished by putting back the 18nt into naturally occurring genotype C deletion mutants and by adding 33nt to genotype D. Infectivity of full-length genotype C clones was also enhanced by mutating the first ATG codon of the preS1 region but diminished by mutating the second in-frame ATG. Removing N-terminal 11 residues from preS1 peptide 2-59 of genotype C potentiated inhibition of HBV infection and enhanced binding to HepG2/NTCP cells. CONCLUSIONS: The 15-nt and 18-nt deletions somehow increase HBV RNA, replicative DNA and virion production. Shortened L protein is more efficient at mediating HBV infection.
Assuntos
Vírus da Hepatite B/genética , Vírus da Hepatite B/patogenicidade , Hepatite B/virologia , Diferenciação Celular , DNA Viral/genética , Regulação Viral da Expressão Gênica , Genótipo , Células Hep G2 , Humanos , Transportadores de Ânions Orgânicos Dependentes de Sódio , Mutação Puntual , RNA Viral/genética , Análise de Sequência de DNA , Análise de Sequência de RNA , Deleção de Sequência , Simportadores , Transfecção , Replicação ViralRESUMO
Sodium taurocholate cotransporting polypeptide (NTCP) has been identified as a hepatitis B virus (HBV) receptor, and its overexpression in HepG2 cell lines leads to efficient secretion of hepatitis B e antigen (HBeAg) following challenge with a large dose of cell culture-derived HBV (cHBV) particles. However, NTCP-reconstituted HepG2 cells are inefficiently infected by patient serum-derived HBV (sHBV) and release very little hepatitis B surface antigen (HBsAg) following cHBV infection, unlike differentiated HepaRG cells, which are naturally susceptible to both cHBV and sHBV particles. Here, we investigated whether NTCP could explain the different behaviors of the two cell types. Endogenous NTCP protein from differentiated HepaRG cells was unglycosylated despite wild-type coding sequence. HepaRG cells stably transfected with an epitope-tagged NTCP expression construct displayed higher sHBV but not cHBV susceptibility than cells transfected with the null mutant. Tagged NTCP introduced to both HepG2 and HepaRG cells was glycosylated, with N5 and N11 being sites of N-linked glycosylation. Mutating N5, N11, or both did not alter cell surface availability of NTCP or its subcellular localization, with both the singly glycosylated and nonglycosylated forms still capable of mediating cHBV infection in HepG2 cells. In conclusion, nonglycosylated NTCP is expressed by differentiated HepaRG cells and capable of mediating cHBV infection in HepG2 cells, but it cannot explain differential susceptibility of HepaRG and HepG2/NTCP cells to cHBV versus sHBV infection and different HBsAg/HBeAg ratios following cHBV infection. The responsible host factor(s) remains to be identified.IMPORTANCE HBV can infect differentiated HepaRG cells and also HepG2 cells overexpressing NTCP, the currently accepted HBV receptor. However, HepG2/NTCP cells remain poorly susceptible to patient serum-derived HBV particles and release very little hepatitis B surface antigen following infection by cell culture-derived HBV. We found differentiated HepaRG cells expressed nonglycosylated NTCP despite a wild-type coding sequence. NTCP introduced to HepG2 cells was glycosylated at two N-linked glycosylation sites, but mutating either or both sites failed to prevent infection by cell culture-derived HBV or to confer susceptibility to serum-derived HBV. Overexpressing NTCP in HepRG cells did not increase infection by cell culture-derived HBV or distort the ratio between the two viral antigens. These findings suggest that host factors unique to HepaRG cells are required for efficient infection by serum-derived HBV, and factors other than NTCP contribute to balanced viral antigen production following infection by cell culture-derived HBV.
Assuntos
Antígenos de Superfície da Hepatite B/metabolismo , Vírus da Hepatite B/metabolismo , Hepatite B/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Simportadores/metabolismo , Proteínas Virais/metabolismo , Glicosilação , Células Hep G2 , Hepatite B/genética , Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/genética , Humanos , Mutação , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Simportadores/genética , Proteínas Virais/genéticaRESUMO
UNLABELLED: Cell culture (cc)-derived hepatitis B virus (HBV) can infect differentiated HepaRG cells, but efficient infection requires addition of polyethylene glycol (PEG) during inoculation. Identification of sodium taurocholate cotransporting polypeptide (NTCP) as an HBV receptor enabled ccHBV infection of NTCP reconstituted HepG2 cells, although very little hepatitis B surface antigen (HBsAg) is produced. We found infection by patient serum-derived HBV (sHBV), which required purification of viral particles through ultracentrifugation or PEG precipitation, was PEG independent and much more efficient in HepaRG cells than in HepG2/NTCP cells. In contrast to hepatitis B e antigen (HBeAg), HBsAg was not a reliable marker of productive sHBV infection at early time points. A low HBsAg/HBeAg ratio by ccHBV-infected HepG2/NTCP cells was attributable to dimethyl sulfoxide (DMSO) in culture medium, NTCP overexpression, and HBV genotype D. HepG2/NTCP cells released more viral antigens than HepG2 cells after HBV genome delivery by adeno-associated virus, and stable expression of NTCP in a ccHBV producing cell line increased viral mRNAs, proteins, replicative DNA, and covalently closed circular DNA. NTCP protein expression in HepG2/NTCP cells, despite being driven by the cytomegalovirus promoter, was markedly increased by DMSO treatment. This at least partly explains ability of DMSO to promote ccHBV infection in such cell lines. In conclusion, NTCP appeared inefficient to mediate infection by serum-derived HBV. It could promote HBV RNA transcription while inhibiting HBsAg secretion. Efficient PEG-independent sHBV infection of HepaRG cells permits comparative studies of diverse clinical HBV isolates and will help identify additional factors on virion surface promoting attachment to hepatocytes. IMPORTANCE: Currently in vitro infection with hepatitis B virus (HBV) depends on cell culture-derived HBV inoculated in the presence of polyethylene glycol. We found patient serum-derived HBV could efficiently infect differentiated HepaRG cells independent of polyethylene glycol, which represents a more physiological infection system. Serum-derived HBV has poor infectivity in HepG2 cells reconstituted with sodium taurocholate cotransporting polypeptide (NTCP), the currently accepted HBV receptor. Moreover, HepG2/NTCP cells secreted very little hepatitis B surface antigen after infection with cell culture-derived HBV, which was attributed to NTCP overexpression, genotype D virus, and dimethyl sulfoxide added to culture medium. NTCP could promote HBV RNA transcription, protein expression, and DNA replication in HepG2 cells stably transfected with HBV DNA, while dimethyl sulfoxide could increase NTCP protein level despite transcriptional control by a cytomegalovirus promoter. Therefore, this study revealed several unusual features of NTCP as an HBV receptor and established conditions for efficient serum virus infection in vitro.
Assuntos
Vírus da Hepatite B/fisiologia , Hepatócitos/virologia , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Receptores Virais/metabolismo , Simportadores/metabolismo , Ligação Viral , Linhagem Celular , DNA Viral/biossíntese , Antígenos da Hepatite B/biossíntese , Humanos , RNA Mensageiro/biossíntese , RNA Viral/biossíntese , Liberação de VírusRESUMO
Sodium taurocholate cotransporting polypeptide (NTCP) is an entry receptor for hepatitis B virus (HBV) and is regarded as one of the determinants that confer HBV permissiveness to host cells. However, how host factors regulate the ability of NTCP to support HBV infection is largely unknown. We aimed to identify the host signaling that regulated NTCP expression and thereby permissiveness to HBV. Here, a cell-based chemical screening method identified that Ro41-5253 decreased host susceptibility to HBV infection. Pretreatment with Ro41-5253 inhibited the viral entry process without affecting HBV replication. Intriguingly, Ro41-5253 reduced expression of both NTCP mRNA and protein. We found that retinoic acid receptor (RAR) regulated the promoter activity of the human NTCP (hNTCP) gene and that Ro41-5253 repressed the hNTCP promoter by antagonizing RAR. RAR recruited to the hNTCP promoter region, and nucleotides -112 to -96 of the hNTCP was suggested to be critical for RAR-mediated transcriptional activation. HBV susceptibility was decreased in pharmacologically RAR-inactivated cells. CD2665 showed a stronger anti-HBV potential and disrupted the spread of HBV infection that was achieved by continuous reproduction of the whole HBV life cycle. In addition, this mechanism was significant for drug development, as antagonization of RAR blocked infection of multiple HBV genotypes and also a clinically relevant HBV mutant that was resistant to nucleoside analogs. Thus, RAR is crucial for regulating NTCP expression that determines permissiveness to HBV infection. This is the first demonstration showing host regulation of NTCP to support HBV infection.
Assuntos
Vírus da Hepatite B/fisiologia , Hepatócitos/virologia , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Receptores do Ácido Retinoico/metabolismo , Simportadores/metabolismo , Adamantano/análogos & derivados , Adamantano/química , Adamantano/farmacologia , Antivirais/química , Antivirais/farmacologia , Benzoatos/química , Benzoatos/farmacologia , Linhagem Celular Tumoral , Células Cultivadas , Cromanos/química , Cromanos/farmacologia , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Genótipo , Células Hep G2 , Vírus da Hepatite B/genética , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Immunoblotting , Estrutura Molecular , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica , Receptores do Ácido Retinoico/antagonistas & inibidores , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Simportadores/genéticaRESUMO
Hepatitis B virus (HBV) has a 3.2 kb circular DNA genome. It employs four promoters in conjunction with a single polyadenylation signal to generate 3.5, 2.4, 2.1 and 0.7 kb co-terminal RNAs. The 3.5 kb RNA is subdivided into the precore RNA for e-antigen expression and pregenomic RNA for genome replication. When introduced to a genotype A clone, several core promoter mutations markedly enhanced HBV genome replication, but suppressed e-antigen expression through up-regulation of pregenomic RNA at the expense of precore RNA. In this study, we found such mutations also diminished envelope proteins and hepatitis B surface antigen, products of the 2.1 and 2.4 kb subgenomic RNAs. Indeed, Northern blot analysis revealed overall increase in 3.5 kb RNA, but reduction in all subgenomic RNAs. To validate transcriptional interference, we subcloned 1.1×, 0.7× and 0.6× HBV genome, respectively, to a vector with or without a cytomegalovirus (CMV) promoter at the 5' end, so as to produce the pregenomic RNA, 2.4 kb RNA, and 2.1 kb RNA in large excess or not at all. Parallel transfection of the three pairs of constructs into a human hepatoma cell line confirmed the ability of pregenomic RNA to suppress all subgenomic transcripts and established the ability of the 2.4 and 2.1 kb RNAs to suppress the 0.7 kb RNA. Consistent with our findings, pregenomic RNA of the related duck HBV has been reported to interfere with transcription of the subgenomic RNAs. Transcriptional interference might explain why HBV produces so little 0.7 kb RNA and HBx protein despite a strong X promoter.
Assuntos
Vírus da Hepatite B/genética , Hepatite B/virologia , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Viral/genética , Regulação Viral da Expressão Gênica , Antígenos do Núcleo do Vírus da Hepatite B/genética , Antígenos do Núcleo do Vírus da Hepatite B/metabolismo , Antígenos de Superfície da Hepatite B/genética , Antígenos de Superfície da Hepatite B/metabolismo , Antígenos E da Hepatite B/genética , Antígenos E da Hepatite B/metabolismo , Vírus da Hepatite B/metabolismo , Humanos , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Transcrição GênicaRESUMO
Virus infection is restricted by intracellular immune responses in host cells, and this is typically modulated by stimulation of cytokines. The cytokines and host factors that determine the host cell restriction against hepatitis B virus (HBV) infection are not well understood. We screened 36 cytokines and chemokines to determine which were able to reduce the susceptibility of HepaRG cells to HBV infection. Here, we found that pretreatment with IL-1ß and TNFα remarkably reduced the host cell susceptibility to HBV infection. This effect was mediated by activation of the NF-κB signaling pathway. A cytidine deaminase, activation-induced cytidine deaminase (AID), was up-regulated by both IL-1ß and TNFα in a variety of hepatocyte cell lines and primary human hepatocytes. Another deaminase APOBEC3G was not induced by these proinflammatory cytokines. Knockdown of AID expression impaired the anti-HBV effect of IL-1ß, and overexpression of AID antagonized HBV infection, suggesting that AID was one of the responsible factors for the anti-HBV activity of IL-1/TNFα. Although AID induced hypermutation of HBV DNA, this activity was dispensable for the anti-HBV activity. The antiviral effect of IL-1/TNFα was also observed on different HBV genotypes but not on hepatitis C virus. These results demonstrate that proinflammatory cytokines IL-1/TNFα trigger a novel antiviral mechanism involving AID to regulate host cell permissiveness to HBV infection.
Assuntos
Citidina Desaminase/biossíntese , Regulação Enzimológica da Expressão Gênica , Vírus da Hepatite B/metabolismo , Hepatite B/metabolismo , Interleucina-1beta/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Citidina Desaminase/genética , Citidina Desaminase/imunologia , DNA Viral/biossíntese , DNA Viral/genética , DNA Viral/imunologia , Células Hep G2 , Hepacivirus/genética , Hepacivirus/imunologia , Hepacivirus/metabolismo , Hepatite B/genética , Hepatite B/imunologia , Vírus da Hepatite B/genética , Vírus da Hepatite B/imunologia , Humanos , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Mutação , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Regulação para Cima/genéticaRESUMO
Hepatitis B virus immune escape mutants have been associated with vaccine failure and reinfection of grafted liver despite immune prophylaxis, but their biological properties remain largely unknown. Transfection of 20 such mutants in a human hepatoma cell line identified many with severe impairment in virion secretion, which can be rescued to various extents by coexpression of wild-type envelope proteins or introduction of a novel glycosylation site. Consistent with their role in maintaining intra- or intermolecular disulfide bonds, cysteine residues within the "a" determinant are critical for virion secretion.
Assuntos
Vírus da Hepatite B/imunologia , Vírus da Hepatite B/fisiologia , Evasão da Resposta Imune , Mutação , Replicação Viral , Linhagem Celular , Vírus da Hepatite B/genética , Hepatócitos/virologia , Humanos , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismoRESUMO
Duck hepatitis B virus (DHBV) is an avian member of the hepatotropic DNA viruses, or hepadnaviridae. It shares with the human hepatitis B virus (HBV) a similar genomic organization and replication strategy via reverse transcription, but is simpler than HBV in lacking the X gene and in expressing just two coterminal envelope proteins: Large (L) and small (S). DHBV has been extensively used as a convenient and valuable animal model for study of the hepadnaviral life cycle, and for drug screening in vitro but also in vivo. Ducks and primary duck hepatocytes (PDHs) are inexpensive, easily accessible, and readily infected with DHBV. The high levels of genome replication and protein expression in duck liver and PDHs also facilitate monitoring of viral life cycle using conventional molecular biology techniques such as Southern blot for replicative DNA and covalently closed circular DNA (cccDNA), Northern blot for viral RNAs, and Western blot for viral proteins.
Assuntos
Patos , Infecções por Hepadnaviridae , Vírus da Hepatite B do Pato , Hepatócitos , Replicação Viral , Animais , Patos/virologia , Hepatócitos/virologia , Hepatócitos/metabolismo , Vírus da Hepatite B do Pato/genética , Infecções por Hepadnaviridae/virologia , Infecções por Hepadnaviridae/veterinária , Modelos Animais de Doenças , Hepatite Viral Animal/virologia , DNA Viral/genética , Células Cultivadas , Cultura Primária de Células/métodos , Técnicas de Cultura de Células/métodosRESUMO
BACKGROUND: HBV infects ~257 million people and can cause hepatocellular carcinoma. Since current drugs are not curative, novel therapies are needed. HBV infects chimpanzee and human livers. However, chimpanzee studies are severely restricted and cost-prohibitive, while transgenic/chimeric mouse models that circumvent the species barrier lack natural HBV infection and disease progression. Thus, in vitro human models of HBV infection are useful in addressing the above limitations. Induced pluripotent stem cell-derived hepatocyte-like cells mitigate the supply limitations of primary human hepatocytes and the abnormal proliferation/functions of hepatoma cell lines. However, variable infection across donors, deficient drug metabolism capacity, and/or low throughput limit iHep utility for drug development. METHODS: We developed an optimal pipeline using combinations of small molecules, Janus kinase inhibitor, and 3',5'-cAMP to infect iHep-containing micropatterned co-cultures (iMPCC) with stromal fibroblasts within 96-well plates with serum-derived HBV and cell culture-derived HBV (cHBV). Polyethylene glycol was necessary for cell-derived HBV but not for serum-derived HBV infection. RESULTS: Unlike iHep monocultures, iMPCCs created from 3 iHep donors could sustain HBV infection for 2+ weeks. Infected iMPCCs maintained high levels of differentiated functions, including drug metabolism capacity. HBV antigen secretion and gene expression patterns in infected iMPCCs in pathways such as fatty acid metabolism and cholesterol biosynthesis were comparable to primary human hepatocyte-MPCCs. Furthermore, iMPCCs could help elucidate the effects of interferons and direct-acting antiviral drugs on the HBV lifecycle and any hepatotoxicity; iMPCC response to compounds was similar to primary human hepatocyte-MPCCs. CONCLUSIONS: The iMPCC platform can enable the development of safe and efficacious drugs against HBV and ultimately help elucidate genotype-phenotype relationships in HBV pathogenesis.
Assuntos
Vírus da Hepatite B , Hepatócitos , Células-Tronco Pluripotentes Induzidas , Humanos , Hepatócitos/virologia , Células-Tronco Pluripotentes Induzidas/virologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Vírus da Hepatite B/genética , Vírus da Hepatite B/fisiologia , Vírus da Hepatite B/efeitos dos fármacos , Hepatite B/virologia , Hepatite B/tratamento farmacológico , Técnicas de Cocultura , Inibidores de Janus Quinases/farmacologia , Antivirais/farmacologia , Células CultivadasRESUMO
The pregenomic RNA (pgRNA) of hepatitis B virus (HBV) serves as the messenger for both core and P proteins, with the downstream P gene translated by ribosomal leaky scanning. HBV replication begins with packaging of the pgRNA and P protein into core protein particles, followed by conversion of RNA into DNA. Genotype G has a low replication capacity due to a low pgRNA level. It has a 36-nucleotide (nt) insertion in the 5' end of the core gene, adding 12 residues to the core protein. The insertion is needed to maintain efficient core protein expression and genome replication but causes inefficient virion secretion yet high maturity of virion DNA. In the present study, we confirmed that the 36-nt insertion had similar effects on core protein expression and virion secretion when it was introduced into genotype A and D clones but no impact on virion genome maturity. Surprisingly, the insertion impaired genome replication in both genotypes. Transcomplementation assays suggest that increased efficiency of core protein translation diminishes ribosomal scanning toward the downstream P gene. Indeed, mutating the core gene Kozak sequence restored core protein to lower levels but increased replication of the insertion mutant. Similar mutations impaired replication in genotype G. On the other hand, replacement of the core promoter sequence of genotype G with genotype A sequence increased pgRNA transcription and genome replication, implicating this region in the low replication capacity of genotype G. Why the 36-nt insertion is present in genotype G but absent in other genotypes is discussed.
Assuntos
Expressão Gênica , Antígenos do Núcleo do Vírus da Hepatite B/biossíntese , Vírus da Hepatite B/crescimento & desenvolvimento , Vírus da Hepatite B/genética , Mutagênese Insercional , Liberação de Vírus , Genótipo , Vírus da Hepatite B/classificação , Replicação ViralRESUMO
Infection by hepatitis B virus (HBV) genotype C is associated with a prolonged viremic phase, delayed hepatitis B e antigen (HBeAg) seroconversion, and an increased incidence of liver cirrhosis and hepatocellular carcinoma compared with genotype B infection. Genotype C is also associated with the more frequent emergence of core promoter mutations, which increase genome replication and are independently associated with poor clinical outcomes. We amplified full-length HBV genomes from serum samples from Chinese and U. S. patients with chronic HBV infection and transfected circularized genome pools or dimeric constructs of individual clones into Huh7 cells. The two genotypes could be differentiated by Western blot analysis due to the reactivities of M and L proteins toward a monoclonal pre-S2 antibody and slightly different S-protein mobilities. Great variability in replication capacity was observed for both genotypes. The A1762T/G1764A core promoter mutations were prevalent in genotype C isolates and correlated with increased replication capacity, while the A1752G/T mutation frequently found in genotype B isolates correlated with a low replication capacity. Importantly, most genotype C isolates with wild-type core promoter sequence replicated less efficiently than the corresponding genotype B isolates due to less efficient transcription of the 3.5-kb RNA. However, genotype C isolates often displayed more efficient virion secretion. We propose that the low intracellular levels of viral DNA and core protein of wild-type genotype C delay immune clearance and trigger the subsequent emergence of A1762T/G1764A core promoter mutations to upregulate replication; efficient virion secretion compensates for the low replication capacity to ensure the establishment of persistent infection by genotype C.
Assuntos
Vírus da Hepatite B/fisiologia , Regiões Promotoras Genéticas , Vírion/isolamento & purificação , Liberação de Vírus , Replicação Viral , Linhagem Celular , China , Análise Mutacional de DNA , Genótipo , Vírus da Hepatite B/classificação , Vírus da Hepatite B/genética , Vírus da Hepatite B/isolamento & purificação , Hepatite B Crônica/virologia , Hepatócitos/virologia , Humanos , Mutação Puntual , Estados Unidos , Carga ViralRESUMO
Different hepatitis B virus (HBV) genotypes and variants are associated with different clinical outcomes and/or response to antiviral therapy, yet the comparison of the in vitro replication capacity of a large number of clinical isolates remains technically challenging and time-consuming. Although the full-length HBV genome can be amplified from high-titer blood samples by PCR using High Fidelity(plus) DNA polymerase and primers targeting the conserved precore region, the HBV clones thus generated are replication deficient due to the inability to generate the terminally redundant pregenomic RNA essential for genome replication. The transfection experiment is further complicated by PCR errors and the presence of viral quasispecies. A previous study found that the precise removal of non-HBV sequence by SapI digestion led to HBV replication in transfected cells, possibly due to low-level genome circularization by a cellular enzyme. We released HBV genome from the cloning vector using BspQI, an inexpensive isoschizomer of SapI, and increased the efficiency of genome replication by an extra step of in vitro DNA ligation. The uncut plasmid DNA can be used for transfection if the sole purpose is to study envelope protein expression. We found significant PCR errors associated with the High Fidelity(plus) DNA polymerase, which could be greatly diminished using Phusion DNA polymerase or masked by the use of a clone pool. The reduced PCR error and modified enzymatic steps prior to transfection should facilitate a more widespread functional characterization of clinical HBV isolates, while the clone pool approach is useful for samples with significant sequence heterogeneity.
Assuntos
Genoma Viral , Vírus da Hepatite B/crescimento & desenvolvimento , Vírus da Hepatite B/genética , Hepatite B/virologia , Proteínas Virais/biossíntese , Replicação Viral , Clonagem Molecular , Expressão Gênica , Vetores Genéticos , Vírus da Hepatite B/isolamento & purificação , Humanos , Proteínas Virais/genética , Cultura de VírusRESUMO
Mutations in the S region of the hepatitis B virus (HBV) envelope gene are associated with immune escape, occult infection, and resistance to therapy. We previously identified naturally occurring mutations in the S gene that alter HBV virion secretion. Here we used transcomplementation assay to confirm that the I110M, G119E, and R169P mutations in the S domain of viral envelope proteins impair virion secretion and that an M133T mutation rescues virion secretion of the I110M and G119E mutants. The G119E mutation impaired detection of secreted hepatitis B surface antigen (HBsAg), suggesting immune escape. The R169P mutant protein is defective in HBsAg secretion as well and has a dominant negative effect when it is coexpressed with wild-type envelope proteins. Although the S domain is present in all three envelope proteins, the I110M, G119E, and R169P mutations impair virion secretion through the small envelope protein. Conversely, coexpression of just the small envelope protein of the M133T mutant could rescue virion secretion. The M133T mutation could also overcome the secretion defect caused by the G145R immune-escape mutation or mutation at N146, the site of N-linked glycosylation. In fact, the M133T mutation creates a novel N-linked glycosylation site ((131)NST(133)). Destroying this site by N131Q/T mutation or preventing glycosylation by tunicamycin treatment of transfected cells abrogated the effect of the M133T mutation. Our findings demonstrate that N-linked glycosylation of HBV envelope proteins is critical for virion secretion and that the secretion defect caused by mutations in the S protein can be rescued by an extra glycosylation site.
Assuntos
Hepatite B/metabolismo , Mutação/genética , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Vírion/fisiologia , Replicação Viral , Fosfatase Alcalina/metabolismo , Substituição de Aminoácidos , Western Blotting , Replicação do DNA , Genótipo , Glicosilação , Hepatite B/genética , Hepatite B/virologia , Antígenos de Superfície da Hepatite B/metabolismo , Vírus da Hepatite B , Humanos , Mutagênese Sítio-Dirigida , Fenótipo , Precursores de Proteínas/metabolismoRESUMO
The entry mechanism of hepatitis B virus (HBV) has not been defined, and this impedes development of antiviral therapies aimed at an early step in the viral life cycle. HBV infection has both host and tissue specificities. For the related duck hepatitis B virus (DHBV), duck carboxypeptidase D (DCPD) has been proposed as the species-specific docking receptor, while glycine decarboxylase (DGD) may serve as a tissue-specific cofactor or secondary receptor. DGD binds to several truncated versions of the viral large envelope protein but not to the full-length protein, suggesting a need for proteolytic cleavage of the envelope protein by a furin-like proprotein convertase. In the present study, we found that transfected DCPD could confer DHBV binding to non-duck cell lines but that this was followed by rapid virus release from cells. Coexpression of furin led to DCPD cleavage and increased virus retention. Treatment of DHBV particles with endosome prepared from duck liver led to cleavage of the large envelope protein, and such viral preparation could generate a small amount of covalently closed circular DNA in LMH cells, a chicken hepatoma cell line resistant to DHBV infection. A furin inhibitor composed of decanoyl-RVKR-chloromethylketone blocked endosomal cleavage of the large envelope protein in vitro and suppressed DHBV infection of primary duck hepatocytes in vivo. These findings suggest that furin or a furin-like proprotein convertase facilitates DHBV infection by cleaving both the docking receptor and the viral large envelope protein.