Naturally occurring point mutation in the C terminus of the polymerase gene prevents duck hepatitis B virus RNA packaging.

A duck hepatitis B virus (DHBV) genome cloned from a domestic duck from the People's Republic of China has been sequenced and exhibits no variation in sequences known to be important in viral replication or generation of gene products. Intrahepatic transfection of a dimer of this viral genome into ducklings did not result in viremia or any sign of virus infection, indicating that the genome was defective. Functional analysis of this mutant genome, performed by transfecting the DNA into a chicken hepatoma cell line capable of replicating wild-type virus, indicated that viral RNA is not encapsidated. However, virus core protein is made and can assemble into particles in the absence of encapsidation of viral nucleic acid. Using genetic approaches, it was determined that a change of cysteine to tyrosine in position 711 in the polymerase (P) gene C terminus led to this RNA-packaging defect. By site-directed mutagenesis, it was found that while substitution of Cys-711 with tryptophan also abolished packaging, substitution with methionine did not affect packaging or viral replication. Therefore, Cys-711, which is conserved in all published sequences of DHBV, may not be involved in a disulfide bridge structure essential to viral RNA packaging or replication. Our results, showing that a missense mutation in the region of the DHBV polymerase protein thought to be primarily the RNase H domain results in packaging deficiency, support the previous findings that multiple regions of the complex hepadnaviral polymerase protein may be required for viral RNA packaging.     

Naturally occurring mouse hepatitis virus infection in the nude mouse.

A group of athymic nude mice developed an unusual chronic wasting disease within 1-3 months after their arrival into the laboratory. Affected nude mice had severe, acute-to-chronic, active hepatitis with multinucleated giant hepatocytes and fibrosis. Vascular and central nervous system lesions were frequently present, giant cell peritonitis, ascites, and multinucleated giant cells in the intestinal epithelial villi were less frequently observed. Mouse hepatitis virus was isolated from the livers of three mice with lesions. The virus, when inoculated into nude mice, produced lesions similar to those observed in the natural outbreak.     

Naturally occurring escape mutants of hepatitis B virus with various mutations in the S gene in carriers seropositive for antibody to hepatitis B surface antigen.

Hepatitis B virus (HBV) DNA was extracted from sera of six carriers with hepatitis B e antigen as well as antibody to hepatitis B surface antigen and sequenced within the pre-S regions and the S gene. HBV DNA clones from five of these carriers had point mutations in the S gene, resulting in conversion from Ile-126 or Thr-126 of the wild-type virus to Ser-126 or Asn-126 in three carriers and conversion from Gly-145 to Arg-145 in three of them; clones with Asn-126 or Arg-145 were found in one carrier. All 12 clones from the other carrier had an insertion of 24 bp encoding an additional eight amino acids between Thr-123 and Cys-124. In addition, all or at least some of the HBV DNA clones from these carriers had in-phase deletions in the 5' terminus of the pre-S2 region. These results indicate that HBV escape mutants with mutations in the S gene affecting the expression of group-specific determinants would survive in some carriers after they seroconvert to antibody against surface antigen. Carriers with HBV escape mutants may transmit HBV either by donation of blood units without detectable surface antigen or through community-acquired infection, which would hardly be prevented by current hepatitis B immuneglobulin or vaccines.     

Fidelity of hepatitis B virus polymerase.

Although efficient vaccines are available, chronic hepatitis B (HBV) infection poses a major health problem worldwide, and prolonged treatment of chronically infected HBV patients with nucleoside analogs often results in drug-resistant HBV variants. Therefore, it is critical to evaluate the contribution of the HBV polymerase to mutations. FLAG-tagged wild-type (FPolE) and mutant (FPolE/D551A) HBV polymerases have been expressed in insect cells and purified. The purified FPolE showed DNA polymerase activity, but FPolE/D551A did not, implying that the activity was derived from FPolE. No 3'-->5'exonuclease activity was detected in FPolE. The fidelity of FPolE was investigated and compared with that of HIV-1 RT, which is highly error-prone. The fidelity of HBV polymerase seems to be achieved by increasing the Km for the dNTP being misinserted. The nucleotide misinsertion efficiency of FPolE and HIV-1 RT ranged from 3.59 x 10-4 (C : T) to 1.51 x 10-3 (G : T) and from 1.75 x 10-4 (C : T) to 1.62 x 10-3 (G : T), respectively, and the overall misinsertion efficiency of HIV-1 RT was just 1.04-fold higher than that of FPolE, implying that HBV polymerase is fairly error-prone. Though HBV genetic mutation rate in replication is thought to be between those in RNA and DNA viruses, our data shows that the rate of mutation by HBV polymerase is higher than the rate of genetic mutation in vivo. This may be a result from more overlapping HBV genes in the HBV genome than that of other retroviruses.     

Pregenomic RNA encapsidation analysis of eleven missense and nonsense polymerase mutants of human hepatitis B virus.

We characterized 11 DNA polymerase mutants of human hepatitis B virus (HBV) which contain single missense or nonsense mutations in the various domains within this gene. Except for mutant 738, a tight association between DNA replication and RNA packaging of these missense pol mutants was observed. Further analysis of HBV core particle-associated RNA indicated that only the 3.5-kb core-specific RNA, but not the precore-specific RNA, is selectively packaged in this tissue culture system. Previously, we have demonstrated that only the 3.5-kb core-specific RNA can serve as an efficient template for pol translation. Taken together, our results suggest that selectivity of HBV RNA packaging occurs as a result of selective translation of pol-containing mRNAs. Furthermore, our data suggest that the RNA encapsidation domain of pol overlaps with all of the domains of pol involved in the synthesis of terminal protein, as well as DNA replication. Finally, on the basis of gradient centrifugation analysis, a pol defect appeared to have no negative effect on the assembly or stability of core particles. A new method to assay RNA encapsidation, as well as potential RNase H activity, is reported.     

A missense mutation in the rpoC gene affects chromosomal replication control in Escherichia coli.

An RNA polymerase mutant with a single-base-pair change in the rpoC gene affects chromosome initiation control. The mutation, which is recessive, is a G to A transition leading to the substitution of aspartate for glycine at amino acid residue 1033 in the RNA polymerase beta' subunit. The chromosome copy number is increased twofold in the mutant at semipermissive growth temperatures (39 degrees C). In a delta oriC strain, in which chromosome initiation is governed by an F replicon, chromosome copy number is not affected. Plasmid pBR322 copy number is also increased in the mutant at 39 degrees C. The mutation causes a more than fivefold increased expression of the dnaA gene at 39 degrees C. It is conceivable that it is this high DnaA concentration which causes the high chromosome copy number and that the mutant RNA polymerase beta' subunit exerts its effect by altering the expression of the dnaA gene. However, other factors must be affected as well to explain why the RNA polymerase mutant can grow in a balanced fashion with a high chromosome concentration. This is in contrast to wild-type cells, which exhibit higher origin concentrations when DnaA protein is overproduced, but in which the overall DNA concentration is only moderately affected.     

Defective replication units of hepatitis B virus.

Templates for the synthesis of defective hepatitis B virus RNA pregenomes were constructed. Viral sequences in these constructs were replaced by the neomycin resistance gene. Deletions spanned up to 80% of the genome and did not include the cohesive end region. The size of the defective replication units was reduced up to half of the wild-type unit length. After cotransfection with replication competent wild-type DNA, defective pregenomes became included into the pool of replicating viral nucleic acids. A natural template for a defective pregenome was derived from the integrated state in a hepatocellular carcinoma. Owing to a deletion, this unit was devoid of the hepatitis B virus enhancer.     

High-level hepatitis B virus replication in transgenic mice.

Hepatitis B virus (HBV) transgenic mice whose hepatocytes replicate the virus at levels comparable to that in the infected livers of patients with chronic hepatitis have been produced, without any evidence of cytopathology. High-level viral gene expression was obtained in the liver and kidney tissues in three independent lineages. These animals were produced with a terminally redundant viral DNA construct (HBV 1.3) that starts just upstream of HBV enhancer I, extends completely around the circular viral genome, and ends just downstream of the unique polyadenylation site in HBV. In these animals, the viral mRNA is more abundant in centrilobular hepatocytes than elsewhere in the hepatic lobule. High-level viral DNA replication occurs inside viral nucleocapsid particles that preferentially form in the cytoplasm of these centrilobular hepatocytes, suggesting that an expression threshold must be reached for nucleocapsid assembly and viral replication to occur. Despite the restricted distribution of the viral replication machinery in centrilobular cytoplasmic nucleocapsids, nucleocapsid particles are detectable in the vast majority of hepatocyte nuclei throughout the hepatic lobule. The intranuclear nucleocapsid particles are empty, however, suggesting that viral nucleocapsid particle assembly occurs independently in the nucleus and the cytoplasm of the hepatocyte and implying that cytoplasmic nucleocapsid particles do not transport the viral genome across the nuclear membrane into the nucleus during the viral life cycle. This model creates the opportunity to examine the influence of viral and host factors on HBV pathogenesis and replication and to assess the antiviral potential of pharmacological agents and physiological processes, including the immune response.     

An application of gene comparative image for predicting the effect on replication ratio by HBV virus gene missense mutation

Hepatitis B viruses (HBVs) show instantaneous and high-ratio mutations when they are replicated, some sorts of which significantly affect the efficiency of virus replication through enhancing or depressing the viral replication, while others have no influence at all. The mechanism of gene expression is closely correlated with its gene sequence. With the rapid increase in the number of newly found sequences entering into data banks, it is highly desirable to develop an automated method for simulating the gene regulating function. The establishment of such a predictor will no doubt expedite the process of prioritizing genes and proteins identified by genomics efforts as potential molecular targets for drug design. Based on the power of cellular automata (CA) in treating complex systems with simple rules, a novel method to present HBV gene image has been introduced. The results show that the images thus obtained can very efficiently simulate the effects of the gene missense mutation on the virus replication. It is anticipated that CA may also serve as a useful vehicle for many other studies on complicated biological systems.     

Modulation of hepatitis B virus secretion by naturally occurring mutations in the S gene

Alteration in hepatitis B virus (HBV) secretion efficiency may have pathological consequences. Naturally occurring mutations that regulate virion secretion have not been defined. We recently identified HBV genomes displaying high (4B), substantially reduced (3.4), or negative (4C) virion secretion. In the present study, the underlying mutations were mapped. A T552C point mutation in the 4B genome was responsible for its enhanced virion secretion, whereas a G510A mutation in 3.4 and G660C in 4C impaired virus secretion. The three point mutations generate M133T, G119E, and R169P substitutions in the S domains of viral envelope proteins, respectively, without modifying the coding capacity of the overlapping polymerase gene. The mutated residues are predicted to lie in the luminal side of the endoplasmic reticulum (ER) or to be embedded in the ER membrane and thus are not involved in contact with core particles during envelopment. Of the two mutations inhibitory of virion secretion, G510A greatly reduced small envelope protein (hepatitis B surface antigen [HBsAg]) levels both inside cells and in culture medium, whereas G660C specifically abolished HBsAg secretion. Surprisingly, a T484G mutation in the 4B genome, generating an I110M substitution in the S domain, could also reduce HBsAg secretion and block virion secretion. However, its inhibitory effect was suppressed in the 4B genome by the T552C mutation, the enhancer of virion secretion. T552C can also override the inhibitory G510A mutation, but not the G660C mutation. These findings suggest a hierarchy in the regulation of virion secretion and a close link between defective virion secretion and impaired HBsAg formation or secretion.     

Naturally occurring macrolide-lincosamide-streptogramin B resistance in Bacillus licheniformis.

Resistance to the macrolide-lincosamide-streptogramin B (MLS) group of antibiotics is widespread and of clinical importance. B. Weisblum and his coworkers have demonstrated that this resistance is associated with methylation of the 23S ribosomal ribonucleic acid of the large ribosomal subunit which results in a diminished affinity of this organelle for these antibiotics (Lai et al, J. Mol. Biol. 74:67-72, 1973). We report that 10 of 15 natural isolates of Bacillus licheniformis, a common soil organism, are resistant to the MLS antibiotics. The properties of this resistance (high level of tolerance for erythromycin, broad cross-resistance spectrum, and inducibility) suggest that resistance is conferred as described above. The resistance determinant from one of these strains was cloned onto a B. subtilis plasmid vector, and the resulting hybrid plasmid (pBD90) was used to prepare radioactive probe deoxyribonucleic acid for hybridization studies. All of the resistance B. licheniformis strains studied exhibited homology with the pBD90 insert. Plasmid pBD90 showed no homology to the following staphylococcal and streptococcal MLS-resistance plasmids: pE194, pE5, pAM77, pI258. Plasmids pE194 and pE5, on the other hand, carry homologous MLS genes but showed no detectable homology to one another in their replication genes. pBD90 specified a 35,000-dalton erythromycin-inducible protein, detectable in minicells, which therefore appears different from the 29,000-dalton inducible resistance protein specified by pE194. We conclude that there are at least three distinct MLS resistance determinants to be found among gram-positive bacteria.     

Subtype-independent immature secretion and subtype-dependent replication deficiency of a highly frequent, naturally occurring mutation of human hepatitis B virus core antigen

The most frequent mutation of the human hepatitis B virus (HBV) core antigen occurs at amino acid 97. Recently, a phenylalanine (F)-to-leucine (L) mutation at this position (mutant F97L) in HBV surface antigen subtype ayw has been shown to result in an immature secretion phenotype, which is characterized by the nonselective export of an excessive amount of virions containing minus-strand, single-stranded HBV DNA. While subtype ayw mutant F97L has been found in Europe, the major reservoir of HBV resides in Asia and Africa. We report here that the immature secretion phenotype indeed can be found in an HBV strain (subtype adr) prevalent in Asia, changing from an isoleucine (I) to a leucine (mutant I97L). Despite its immature secretion phenotype, the adr variant I97L replicates as well as its parental adr wild-type I97I, supporting the conclusion that the extracellular phenotype of immature secretion is not a consequence of the intracellular HBV DNA replication defect. Further studies demonstrated that it is the acquisition of a leucine, rather than the loss of a wild-type amino acid at codon 97, that is important for immature secretion. We conclude that immature secretion is a subtype-independent phenotype and deficiency in intracellular DNA synthesis is a subtype-dependent phenotype. The former is caused by the trans-acting effect of a mutant core protein, while the latter by a cis-acting effect of a mutated nucleotide on the ayw genome. These immature secretion variants provide an important tool for studying the regulation of HBV virion assembly and secretion.     

Mechanisms for inhibition of hepatitis B virus gene expression and replication by hepatitis C virus core protein

We have demonstrated previously that the core protein of hepatitis C virus (HCV) exhibits suppression activity on gene expression and replication of hepatitis B virus (HBV). Here we further elucidated the suppression mechanism of HCV core protein. We demonstrated that HCV core protein retained the inhibitory effect on HBV gene expression and replication when expressed as part of the full length of HCV polyprotein. Based on the substitution mutational analysis, our results suggested that mutation introduced into the bipartite nuclear localization signal of the HCV core protein resulted in the cytoplasmic localization of core protein but did not affect its suppression ability on HBV gene expression. Mutational studies also indicated that almost all dibasic residue mutations within the N-terminal 101-amino acid segment of the HCV core protein (except Arg(39)-Arg(40)) impaired the suppression activity on HBV replication but not HBV gene expression. The integrity of Arg residues at positions 101, 113, 114, and 115 was found to be essential for both suppressive effects, whereas the Arg residue at position 104 was important only in the suppression of HBV gene expression. Moreover, our results indicated that the suppression on HBV gene expression was mediated through the direct interaction of HCV core protein with the trans-activator HBx protein, whereas the suppression of HBV replication involved the complex formation between HBV polymerase (pol) and the HCV core protein, resulting in the structural incompetence for the HBV pol to bind the package signal and consequently abolished the formation of the HBV virion. Altogether, this study suggests that these two suppression effects on HBV elicited by the HCV core protein likely depend on different structural context but not on nuclear localization of the core protein, and the two effects can be decoupled as revealed by its differential targets (HBx or HBV pol) on these two processes of the HBV life cycle.     

Dynamic changes of hepatitis B virus polymerase gene including YMDD motif in lamivudine-treated patients with chronic hepatitis B

The mutation of YMDD motif of hepatitis B virus (HBV) polymerase gene is the most frequent cause in HBV resistant to lamivudine. The aim of the study was to investigate variation features of HBV polymerase gene in chronic hepatitis B (CHB) patients before and after lamivudine treatment. From the serum samples of five CHB patients before and after 12 months of lamivudine treatment, HBV polymerase gene was amplificated and positive DNA fragments were cloned into JM105 competent cell. Twenty positive clones of every sample were checked with mismatched polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and YMDD variants were sequenced. Among five patients after 12 months of lamivudine treatment, M552I mutations in two patients with HBV DNA rebounding and D553G mutation in one non-responder were detected except two patients with negative HBV DNA consecutively. In summary, D553G mutation is probably one of the reasons that caused non-responders during lamivudine treatment. The mutations of YMDD motif occurred after lamivudine treatment are caused by the induction of lamivudine.     

Inhibition of hepatitis B virus replication by cIAP2 involves accelerating the ubiquitin-proteasome-mediated destruction of polymerase

Cellular inhibitor of apoptosis protein 2 (cIAP2) is a potent suppressor of apoptotic cell death. We have shown previously that cIAP2 is involved in the tumor necrosis factor alpha (TNF-α)-induced anti-hepatitis B virus (HBV) response; however, the mechanism for this antiviral effect remains unclear. In the present study, we demonstrate that cIAP2 can significantly reduce the levels of HBV DNA replication intermediates but not the total viral RNA or core protein levels. Domain-mapping analysis revealed that the carboxy-terminal domains of cIAP2 were indispensable for this anti-HBV ability and that an E3 ligase-deficient mutant of cIAP2 (termed cIAP2*) completely lost its antiviral activity. We further identified HBV polymerase as the target of cIAP2. Overexpression of cIAP2 but not cIAP2* reduced polymerase protein levels, while cIAP2 knockdown increased polymerase expression. In addition, we observed that cIAP2 promoted the degradation of the viral polymerase through a proteasome-dependent pathway. Further experiments demonstrated that cIAP2 can bind to polymerase and promote its polyubiquitylation. Finally, we found that cIAP2 downregulated the encapsidation of HBV pregenomic RNA. Taken together, these data reveal a novel mechanism for the inhibition of HBV replication by cIAP2 via acceleration of the ubiquitin-proteasome-mediated decay of polymerase and reduction of the encapsidation of HBV pregenomic RNA, making this mechanism a novel strategy for HBV therapy.     

Interleukin-12 inhibits hepatitis B virus replication in transgenic mice.

Interleukin-12 (IL-12) is a heterodimeric cytokine produced by antigen-presenting cells that has the ability to induce gamma interferon (IFN-gamma) secretion by T and natural killer cells and to generate normal Th1 responses. These properties suggest that IL-12 may play an important role in the immune response to many viruses, including hepatitis B virus (HBV). Recently, we have shown that HBV-specific cytotoxic T lymphocytes inhibit HBV replication in the livers of transgenic mice by a noncytolytic process that is mediated in part by IFN-gamma. In the current study, we demonstrated that the same antiviral response can be initiated by recombinant murine IL-12 and we showed that the antiviral effect of IL-12 extends to extrahepatic sites such as the kidney. Southern blot analyses revealed the complete disappearance of HBV replicative intermediates from liver and kidney tissues at IL-12 doses that induce little or no inflammation in these tissues. In addition, immunohistochemical analysis demonstrated the disappearance of cytoplasmic hepatitis B core antigen from both tissues after IL-12 treatment, suggesting that IL-12 either prevents the assembly or triggers the degradation of the nucleocapsid particles within which HBV replication occurs. Importantly, we demonstrated that although IFN-gamma, tumor necrosis factor alpha, and IFN-alpha/beta mRNA are induced in the liver and kidney after IL-12 administration, the antiviral effect of IL-12 is mediated principally by its ability to induce IFN-gamma production in this model. These results suggest that IL-12, through its ability to induce IFN-gamma, probably plays an important role in the antiviral immune response to HBV during natural infection. Further, since relatively nontoxic doses of recombinant IL-12 profoundly inhibit HBV replication in the liver and extrahepatic sites in this model, IL-12 may have therapeutic value as an antiviral agent for the treatment of chronic HBV infection.     

Characterization of hepatitis B virus DNA polymerase

Hepatitis B virus (HBV) particles were separated from the blood-plasma containing HBe and HBs antigens (subtype adr) and the nature of the endogenous DNA polymerase in the HBV core particles was studied. The HBV endogenous DNA polymerase activity was examined under the conditions used for preparation of HBV vaccine. The endogenous DNA polymerase activity was reduced slowly upon the heat treatment or the formalin treatment. The reductions of the activity were 65% and 70% upon the heat treatment at 60 C for 10 hr and the formalin treatment at 37 C for 90 hr, respectively. Properties of the HBV endogenous DNA polymerase were studied by utilizing specific inhibitors against the eukaryotic DNA polymerases. Our results showed that the HBV endogenous DNA polymerase is resistant to aphidicolin and N-ethylmaleimide, and sensitive to 2',3'-dideoxythymidine 5'-triphosphate, phosphonoformic acid and 9-beta-D-arabinofuranosyladenosine 5'-triphosphate.