The core promoter mutants of hepatitis B virus (HBV) emerge as the dominant viral population at the late HBeAg and the anti-HBe stages of HBV infection, with the A1762T/G1764A substitutions as the hotspot mutations. The double core promoter mutations were found by many investigators to moderately enhance viral genome replication and reduce hepatitis B e antigen (HBeAg) expression. A much higher replication capacity was reported for a naturally occurring core promoter mutant implicated in the outbreak of fulminant hepatitis, which was caused by the neighboring C1766T/T1768A mutations instead. To systemically study the biological properties of naturally occurring core promoter mutants, we amplified full-length HBV genomes by PCR from sera of HBeAg ؉ individuals infected with genotype A. All 12 HBV genomes derived from highly viremic sera (5 ؋ 10 9 to 5.7 ؋ 10 9 copies of viral genome/ml) harbored wild-type core promoter sequence, whereas 37 of 43 clones from low-viremia samples (0.2 ؋ 10 7 to 4.6 ؋ 10 7 copies/ml) were core promoter mutants. Of the 11 wild-type genomes and 14 core promoter mutants analyzed by transfection experiments in human hepatoma cell lines, 6 core promoter mutants but none of the wild-type genomes replicated at high levels.

Summary Chronic infection with hepatitis B virus (HBV) greatly increases the risk for liver cirrhosis and hepatocellular carcinoma (HCC). HBV isolates worldwide can be divided into 10 genotypes. Moreover, the immune clearance phase selects for mutations in different parts of the viral genome. The outcome of HBV infection is shaped by the complex interplay of the mode of transmission, host genetic factors, viral genotype and adaptive mutations, as well as environmental factors. Core promoter mutations and mutations abolishing HBeAg expression have been implicated in acute liver failure, while genotypes B, C, subgenotype A1, core promoter mutations, preS deletions, C-terminal truncation of envelope proteins, and spliced pregenomic RNA are associated with HCC development. Our efforts to treat and prevent HBV infection are hampered by the emergence of drug resistant mutants and vaccine escape mutants. This paper provides an overview of the HBV life cycle, followed by review of HBV genotypes and mutants in terms of their biological properties and clinical significance.

Background: Cytokines and host factors triggering innate immunity against hepatitis B virus (HBV) are not well understood.Results: IL-1 and TNFα induced cytidine deaminase AID, an anti-HBV host factor, and reduced HBV infection into hepatocytes.Conclusion: IL-1/TNFα reduced host susceptibility to HBV infection through AID up-regulation.Significance: Proinflammatory cytokines modulate HBV infection through a novel innate immune pathway involving AID.

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.The hepatitis B virus (HBV) is an enveloped DNA virus with a tropism for the liver. The 3.2-kb HBV genome harbors genes encoding core protein and its secreted version (called HBeAg), DNA polymerase, the transcriptional transactivator HBx, and envelope proteins. The envelope gene, which is completely overlapped by the polymerase gene, has three in-frame AUG codons that can serve as alternative translation initiation sites. This leads to the expression of three coterminal envelope proteins: large (L), middle (M), and small (S). The sequence unique to the L protein is called the pre-S1 domain, while a downstream sequence shared with the M protein is called the pre-S2 domain. The S domain is present in all three envelope proteins. The S and M proteins are translated from a 2.1-kb subgenomic RNA with a heterogeneous 5Ј end, while the L protein is expressed from a longer (2.4-kb) subgenomic RNA. The S protein is the most abundantly expressed envelope protein. The L and S proteins exist in nonglycosylated and monoglycosylated forms (L protein, p39 and gp42, respectively; S protein, p24 and gp27, respectively) due to a facultative Nlinked glycosylation site (N-X-S/T) at N146 of the S domain. The M protein contains an extra, constitutive N-linked glycosylation site at position 4 in the pre-S2 domain and consequently exists in monoglycosylated (gp33) and diglycosylated (gp36) form...

The emergence of HBe-minus hepatitis B virus (HBV) mutants, usually through a UAG nonsense mutation at codon 28 of the precore region, helps the virus to survive the anti-HBe immune response of the host. Host and viral factors that predispose to the emergence of such mutants are not well characterized. The fact that the precore region forms a hairpin structure essential for the packaging of viral pregenomic RNA may explain the extremely high prevalence of the UAG mutation at codon 28. It converts a wobble U-G pair in the packaging signal between nucleotide 3 of codon 15 (CCU) and nucleotide 2 of codon 28 (UGG) into a U-A pair. Since genotype A of HBV has a CCC sequence at codon 15, the UAG mutation would, instead, disrupt a C-G pair present in the wild-type virus. This alteration was shown by transfection experiments to greatly compromise the packaging of pregenomic RNA. The implication of this finding was elucidated by molecular epidemiological studies. Genotype A was found to be the most prevalent genotype in the wild-type virus populations in France but was found in only 1 of the 46 isolates of HBe-minus mutants found there. These mutants were contributed chiefly by genotype D, the second most prevalent genotype in France, which is characterized by a CCU sequence at codon 15. The role of the single nucleotide at codon 15 was confirmed by the finding of the single genotype A isolate in which both wild-type and mutant viruses were present. Interestingly, nearly all of the mutants had a codon 15 sequence of CCU instead of the CCC present in the wild-type viruses. Our results suggest that genotype A of HBV rarely circulates as HBe-minus mutants, probably because of a requirement for a simultaneous sequence change at codon 15. These data, together with the virtual absence of genotype A in the Chinese samples examined, may provide some insights into the uneven prevalence of HBe-minus mutants in the world.

Hepatitis B virus (HBV) contains three coterminal envelope proteins on the virion surface: large (L), middle (M), and small (S). The M and S proteins are also secreted as empty "subviral particles," which exceed virions by at least 1,000-fold. The S protein serves as the morphogenic factor for both types of particles, while the L protein is required only for virion formation. We found that cotransfecting replication constructs with a small dose of the expression construct for the missing L, M, and S proteins reconstituted efficient virion secretion but only 5 to 10% of subviral particles. The L protein inhibited secretion of subviral particles in a dose-dependent manner, whereas a too-high or too-low L/S protein ratio inhibited virion secretion. Consistent with the results of cotransfection experiments, a point mutation at the ؊3 position of the S gene AUG codon reduced HBsAg secretion by 60 to 70% but maintained efficient virion secretion. Surprisingly, ablating M protein expression reduced virion secretion but markedly increased the maturity of virion-associated genomes, which could be reversed by providing in trans both L and M proteins but not just M protein. M protein stability was dependent on the coexpression of S protein. Our findings suggest that efficient HBV virion secretion could be maintained despite drastic reduction in subviral particle production, which supports the recent demonstration of separate secretion pathways adopted by the two types of particles. The M protein appears to facilitate core particle envelopment, thus shortening the window of plus strand DNA elongation.Hepatitis B virus (HBV) is a small enveloped DNA virus that infects only humans and higher primates (17). It is estimated that 2 billion people worldwide have been exposed to HBV and more than 350 million are chronically infected. Inside the 42-nm virion is a nucleocapsid or core particle that shields the relaxed circular double stranded DNA genome. The minus-strand DNA is full length (3.2-kb) and terminally redundant, whereas the plus-strand DNA is incomplete. Upon infection of hepatocytes, the genome is repaired to form the unit-length, fully double-stranded covalently closed circular DNA in the nucleus (54), which serves as the template for transcription. The four major transcripts of 3.5, 2.4, 2.1, and 0.7 kb are termed pre-C/C, pre-S1, preS2/S, and X, respectively, all sharing the same 3Ј end but different 5Ј initiation sites (4). The mRNAs are exported to cytoplasm for protein translation and genome replication. The 3.5-kb transcript is over the genome length and therefore contains all of the genetic information of the viral genome. The longer and less abundant version (preC RNA) directs the expression of hepatitis B e antigen (HBeAg), a secreted viral protein with immunomodulatory function (37). The shorter one or pregenomic RNA (pgRNA) serves as the messenger for core protein and viral polymerase, as well as the template for DNA synthesis. To this end, the nascent core protein assembles into core particle, packaging bot...

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