Clinical isolates of hepatitis B virus genotype C have higher in vitro transmission efficiency than genotype B isolates.

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.

5' preS1 Mutations To Prevent Large Envelope Protein Expression from Hepatitis B Virus Genotype A or Genotype D Markedly Increase Polymerase-Envelope Fusion Protein.

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.

Role of Small Envelope Protein in Sustaining the Intracellular and Extracellular Levels of Hepatitis B Virus Large and Middle Envelope Proteins.

Hepatitis B virus (HBV) expresses co-terminal large (L), middle (M), and small (S) envelope proteins. S protein drives virion and subviral particle secretion, whereas L protein inhibits subviral particle secretion but coordinates virion morphogenesis. We previously found that preventing S protein expression from a subgenomic construct eliminated M protein. The present study further examined impact of S protein on L and M proteins. Mutations were introduced to subgenomic construct of genotype A or 1.1 mer replication construct of genotype A or D, and viral proteins were analyzed from transfected Huh7 cells. Mutating S gene ATG to prevent expression of full-length S protein eliminated M protein, reduced intracellular level of L protein despite its blocked secretion, and generated a truncated S protein through translation initiation from a downstream ATG. Truncated S protein was secretion deficient and could inhibit secretion of L, M, S proteins from wild-type constructs. Providing full-length S protein in trans rescued L protein secretion and increased its intracellular level from mutants of lost S gene ATG. Lost core protein expression reduced all the three envelope proteins. In conclusion, full-length S protein could sustain intracellular and extracellular L and M proteins, while truncated S protein could block subviral particle secretion.

Lost Small Envelope Protein Expression from Naturally Occurring PreS1 Deletion Mutants of Hepatitis B Virus Is Often Accompanied by Increased HBx and Core Protein Expression as Well as Genome Replication.

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.

Naturally occurring 5' preS1 deletions markedly enhance replication and infectivity of HBV genotype B and genotype C.

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.

Expression Level of Small Envelope Protein in Addition to Sequence Divergence inside Its Major Hydrophilic Region Contributes to More Efficient Surface Antigen Secretion by Hepatitis B Virus Subgenotype D2 than Subgenotype A2.

Hepatitis B surface antigen (HBsAg) promotes persistent hepatitis B virus (HBV) infection. It primarily corresponds to small (S) envelope protein secreted as subviral particles. We previously found that genotype D clones expressed less S protein than genotype A clones but showed higher extracellular/intracellular ratio of HBsAg suggesting more efficient secretion. The current study aimed to characterize the underlying mechanism(s) by comparing a subgenotype A2 clone (geno5.4) with a subgenotype D2 clone (geno1.2). Five types of full-length or subgenomic constructs were transfected to Huh7 cells at different dosage. HBsAg was quantified by enzyme linked immunosorbent assay while envelope proteins were detected by Western blot. We found that ratio of extracellular/intracellular HBsAg decreased at increasing amounts of DNA transfected. Conflicting findings from two types of subgenomic construct confirmed stronger secretion inhibitory effect of the genotype D-derived large envelope protein. Chimeric constructs followed by site-directed mutagenesis revealed geno1.2 specific V118/T127 and F161/A168 in the S protein as promoting and inhibitory of HBsAg secretion, respectively. In conclusion, more efficient HBsAg secretion by subgenotype D2 than subgenotype A2 is attributed to lower level of S protein expression in addition to V118 and T127 in S protein, although its F161 and A168 sequences rather reduce HBsAg secretion.

Tracing the evolutionary history of hepadnaviruses in terms of e antigen and middle envelope protein expression or processing.

Hepatitis B virus (HBV) is the prototype of hepadnaviruses, which can be subgrouped into orthohepadnaviruses infecting mammals, avihehepadnaviruses of birds, metahepadnaviruses of fish, and herpetohepadnaviruses of amphibians and reptiles. The middle (M) envelope protein and e antigen are new additions in the evolution of hepadnaviruses. They are alternative translation products of the transcripts for small (S) envelope and core proteins, respectively. For HBV, e antigen is converted from precore/core protein by removal of N-terminal signal peptide followed by furin-mediated cleavage of the basic C-terminus. This study compared old and newly discovered hepadnaviruses for their envelope protein and e antigen expression or processing. The S protein of bat hepatitis B virus (BHBV) and two metahepadnaviruses is probably myristoylated, in addition to two avihepadnaviruses. While most orthohepadnaviruses express a functional M protein with N-linked glycosylation near the amino-terminus, most metahepadnaviruses and herpetohepadnaviruses probably do not. These viruses and one orthohepadnavirus, the shrew hepatitis B virus, lack an open precore region required for e antigen expression. Potential furin cleavage sites (RXXR sequence) can be found in e antigen precursors of orthohepadnaviruses and avihepadnaviruses. Despite much larger precore/core proteins of avihepadnaviruses and their limited sequence homology with those of orthohepadnaviruses, their proximal RXXR motif can be aligned with a distal RXXR motif for orthohepadnaviruses. Thus, furin or another basic endopeptidase is probably the shared enzyme for hepadnaviral e antigen maturation. A precore-derived cysteine residue is involved in forming intramolecular disulfide bond of HBV e antigen to prevent particle formation, and such a cysteine residue is conserved for both orthohepadnaviruses and avihepadnaviruses. All orthohepadnaviruses have an X gene, while all avihepadnaviruses can express the e antigen. M protein expression appears to be the most recent event in the evolution of hepadnaviruses.

Core gene insertion in hepatitis B virus genotype G functions at both the encoded amino acid sequence and RNA structure levels to stimulate core protein expression.

Hepatitis B virus genotype G possesses a 36-nucleotide (nt) insertion at the 5' end of core gene, adding 12 residues to core protein. The insertion markedly increased core protein level irrespective of viral genotype, with the effect reproducible using CMV-core gene construct. Here we used such expression constructs and transient transfection experiments in Huh7 cells to identify the structural bases. The insertion is predicted to create a stem-loop structure 14nt downstream of core gene AUG. A + 1 or + 2 frameshift into the 36nt mitigated enhancement of core protein level. Point mutations to disrupt or restore the stem-loop had opposite effects on core protein expression. Shifting the translation initiation site downstream or further upstream of the stem-loop rendered it inhibitory or no longer stimulatory of core protein expression. Therefore, both the reading frame and a properly positioned stem-loop structure contribute to marked increase in core protein expression by the 36-nt insertion.

N-Linked Glycosylation Is Not Essential for Sodium Taurocholate Cotransporting Polypeptide To Mediate Hepatitis B Virus Infection In Vitro.

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.

Sequence analysis and functional characterization of full-length hepatitis B virus genomes from Korean cirrhotic patients with or without liver cancer.

This study aimed to identify and characterize mutations in the hepatitis B virus (HBV) genome associated with advanced liver diseases. The 3.2-kb HBV genome of the C2 subgenotype was amplified from sera of 18 cirrhotic Korean patients with (10) or without (8) hepatocellular carcinoma (HCC), and two clones per patient were characterized by transient transfection experiments in human hepatoma cells. While A1762T/G1764A core promoter mutations were highly prevalent in both groups, the G1896A precore mutation to abolish hepatitis B e antigen (HBeAg) expression was more common in HCC clones (55% vs. 20%). High replication capacity was mostly found in HCC clones and associated with core promoter mutations, whereas more non-HCC clones harbored a nonfunctional core gene (34% vs. 8%). Large in-frame deletions in the preS region were found in 60% of HCC clones and 38% of non-HCC clones. They removed the first 11 residues of large envelope protein or impaired small envelope protein expression, or deleted a neutralizing epitope in the preS2 domain. Additional point mutations prevented middle envelope protein expression, or caused nonsense mutations in the preS or S region to truncate large and/or small envelope protein. Consequently, many clones were unable to express or secrete hepatitis B surface antigen (HBsAg). In conclusion, mutations associated with the advanced stage of chronic HBV infection are complex and diverse. Host immune pressure most likely selected for mutations in the HBV genome to abolish or reduce HBeAg or HBsAg production, to enhance genome replication, or to escape neutralizing antibodies. Some of these mutations may contribute to liver cirrhosis or HCC development.

Differential regulation of hepatitis B virus core protein expression and genome replication by a small upstream open reading frame and naturally occurring mutations in the precore region.

Hepatitis B virus (HBV) transcribes two subsets of 3.5-kb RNAs: precore RNA for hepatitis B e antigen (HBeAg) expression, and pregenomic RNA for core and P protein translation as well as genome replication. HBeAg expression could be prevented by mutations in the precore region, while an upstream open reading frame (uORF) has been proposed as a negative regulator of core protein translation. We employed replication competent HBV DNA constructs and transient transfection experiments in Huh7 cells to verify the uORF effect and to explore the alternative function of precore RNA. Optimized Kozak sequence for the uORF or extra ATG codons as present in some HBV genotypes reduced core protein expression. G1896A nonsense mutation promoted more efficient core protein expression than mutated precore ATG, while a +1 frameshift mutation was ineffective. In conclusion, various HBeAg-negative precore mutations and mutations affecting uORF differentially regulate core protein expression and genome replication.

The Envelope Gene of Hepatitis B Virus Is Implicated in Both Differential Virion Secretion and Genome Replication Capacities between Genotype B and Genotype C Isolates.

Chronic infection by hepatitis B virus (HBV) genotype C is associated with a prolonged replicative phase and an increased risk of liver cancer, compared with genotype B infection. We previously found lower replication capacity but more efficient virion secretion by genotype C than genotype B isolates. Virion secretion requires interaction between core particles and ENVELOPE proteins. In the present study, chimeric constructs between genotype B and genotype C clones were generated to identify the structural basis for differential virion secretion. In addition to dimeric constructs, we also employed 1.1mer constructs, where the cytomegalovirus (CMV) promoter drove pregenomic RNA transcription. Through transient transfection experiments in Huh7 cells, we found that exchanging the entire envelope gene or just its S region could enhance virion secretion by genotype B clones while diminishing virion secretion by genotype C. Site-directed mutagenesis established the contribution of genotype-specific divergence at codons 108 and 115 in the preS1 region, as well as codon 126 in the S region, to differential virion secretion. Surprisingly, exchanging the envelope gene or just its S region, but not the core gene or 3' S region, could markedly increase intracellular replicative DNA for genotype C clones but diminish that for genotype B, although the underlying mechanism remains to be clarified.

Characterization of contrasting features between hepatitis B virus genotype A and genotype D in small envelope protein expression and surface antigen secretion.

Hepatitis B virus (HBV) genotypes A and D are prevalent in many parts of the world and show overlapping geographic distributions. We amplified the entire HBV genome from sera of patients with genotypes A and D and generated overlength constructs for transient transfection into Huh7 or HepG2 cells. Genotype D clones were associated with less HBsAg in culture supernatant and even less intracellular HBsAg. They produced less 2.1-kb RNA due to a weaker SPII promoter. Chimeric promoter constructs identified three divergent positions as most critical, and their exchange reversed extracellular HBsAg phenotype. The S protein of genotype D was more efficient at secretion, while its L protein possessed greater inhibitory effect. Swapping the S gene diminished genotypic difference in intracellular S protein but widened the difference in secreted HBsAg. In conclusion, HBV genotypes A and D differ in S protein expression, secretion and modulation by L protein.

Generation of Replication-Competent Hepatitis B Virus Genome from Blood Samples for Functional Characterization.

Hepatitis B virus (HBV) infection can be associated with a spectrum of clinical outcomes. Transient transfection of the clinical HBV isolates in human hepatoma cell lines can establish their biological properties to shed light on their different pathogenic potentials, yet very few clinical HBV isolates have been functionally characterized so far. The technical challenges include faithful amplification of the full-length HBV genome from clinical samples and conversion into a replication-competent form. We have improved a published method to amplify the full-length HBV genome from blood samples. Two alternative approaches are used to render the cloned HBV genome replication competent: release and circularization of the 3.2-kb HBV genome prior to each transfection experiment or conversion of the monomeric clone into a tandem dimer version.

Identification of Tumor Antigen AF20 as Glycosylated Transferrin Receptor 1 in Complex with Heat Shock Protein 90 and/or Transporting ATPase.

We previously isolated AF20, a murine monoclonal antibody that recognizes a cell surface glycoprotein of approximately 90-110 kDa. The AF20 antigen is specifically expressed in human hepatoma and colon cancer cell lines, and thus could serve as a cancer biomarker. To uncover the molecular identity of the AF20 antigen, a combination of ion-exchange chromatography, immunoprecipitation, and SDS-polyacrylamide gel electrophoresis was employed to purify the AF20 antigen followed by trypsin digestion and mass spectrometry. Surprisingly, three host proteins were thus purified from human hepatoma and colon cancer cell lines: transferrin receptor 1 (TFR1), heat shock protein 90 (HSP90), and Na+/K+ ATPase or Mg++ ATPase. Co-immunoprecipitation followed by Western blot analysis confirmed interaction among the three proteins. However, only the cDNA encoding TFR1 conferred strong cell surface staining by the AF20 antibody following its transient transfection into a cell line lacking endogenous AF20. In support of the molecular identity of AF20 as TFR1, diferric but not iron-free transferrin could prevent AF20 antigen-antibody interaction during immunoprecipitation. Moreover, very similar patterns of AF20 and TFR1 overexpression was documented in colon cancer tissues. In conclusion, AF20 is glycosylated TFR1. This finding could explain the molecular structure of AF20, its cell surface localization, as well as overexpression in cancer cells. Glycosylated TFR1 should serve as a usefulness target for anti-cancer therapy, or a vehicle for delivery of anti-tumor drugs with high affinity and specificity. The biological significance of the complex formation between TFR1, HSP90, and/or transporting ATPase warrants further investigation.

Functional characterization of hepatitis B virus core promoter mutants revealed transcriptional interference among co-terminal viral mRNAs.

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.

Unusual Features of Sodium Taurocholate Cotransporting Polypeptide as a Hepatitis B Virus Receptor.

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.

Two-way molecular ligation for efficient conversion of monomeric hepatitis B virus DNA constructs into tandem dimers.

Replication of the 3.2-kb hepatitis B virus (HBV) genome is driven by the covalently closed circular (ccc) DNA in the nucleus, from which four classes of co-terminal RNAs are transcribed. Genome replication requires just the 3.5-kb pregenomic RNA, which is terminally redundant. Cloning the full-length HBV genome into a vector disrupts its continuity, thus preventing genome replication at the step of pregenomic RNA transcription. This can be overcome by converting the monomeric construct into a tandem dimer, yet the need to ligate two molecules of the HBV genome with vector DNA makes it inefficient and even unsuccessful. To overcome this problem we partially digested the monomeric construct with the unique restriction enzyme used for cloning, and dephosphorylated the linearized monomer before its ligation with another copy of the HBV genome. Alternatively, the monomer was linearized at another unique restriction site inside the HBV genome, followed by its dephosphorylation and ligation with another copy of the HBV genome linearized at the same site. These approaches of two-way molecular ligation greatly improved the efficiency of dimer formation with about 50% of the bacterial colonies screened harboring tandem dimers.