Hepatitis B virus capsid assembly is enhanced by naturally occurring mutation F97L

In chronic hepatitis B virus (HBV) infections, one of the most common mutations to the virus occurs at amino acid 97 of the core protein, where leucine replaces either phenylalanine or isoleucine, depending on strain. This mutation correlates with changes in viral nucleic acid metabolism and/or secretion. We hypothesize that this phenotype is due in part to altered core assembly, a process required for DNA synthesis. We examined in vitro assembly of empty HBV capsids from wild-type and F97L core protein assembly domains. The mutation enhanced both the rate and extent of assembly relative to those for the wild-type protein. The difference between the two proteins was most obvious in the temperature dependence of assembly, which was dramatically stronger for the mutant protein, indicating a much more positive enthalpy. Since the structures of the mutant and wild-type capsids are essentially the same and the mutation is not involved in the contact between dimers, we suggest that the F97L mutation affects the dynamic behavior of dimer and capsid.     

A frequent, naturally occurring mutation (P130T) of human hepatitis B virus core antigen is compensatory for immature secretion phenotype of another frequent variant (I97L)

A frequent mutation at codon 97 of human hepatitis B virus core antigen has been shown to cause an "immature secretion" phenotype, featuring nonselective and excessive secretions of virions containing immature viral genome. Our current study demonstrates that this abnormality can be efficiently offset by another frequent core mutation, P130T.     

Low-level secretion of human hepatitis B virus virions caused by two independent, naturally occurring mutations (P5T and L60V) in the capsid protein

The functional significance of naturally occurring variants of human hepatitis B virus (HBV) remains largely unknown. Previously, we reported an immature secretion phenotype caused by a highly frequent mutation at amino acid 97 of the HBV core (capsid) protein (HBcAg). This phenotype is characterized by a nonselective and excessive secretion of virions containing an immature genome of single-stranded viral DNA. To extend our study of virion secretion to other naturally occurring variants, we have characterized mutations at HBcAg codons 5, 38, and 60 via site-directed mutagenesis. Although the phenotype of the mutation at codon 38 is nearly identical to that for the wild-type virus, our study reveals that a single mutation at codon 5 or 60 exhibits a new extracellular phenotype with significantly reduced virion secretion yet maintains normal intracellular viral DNA replication. A complementation study indicates that the mutant core protein alone is sufficient for the "low-secretion" phenotype. Furthermore, the low-secretion phenotype of the codon 5 mutant appears to be induced by the loss of a parental proline residue, rather than by the gain of a new amino acid. Our study underscores the core protein as another crucial determinant in virion secretion, in addition to the known envelope proteins. Our present results suggest that a very precise structure of both alpha-helical and nonhelical loop regions of the entire HBcAg molecule is important for virion secretion. The low-secretion variants may contribute to the phenomenon of gradually decreasing viremia in chronic carriers during the late phase of persistent infection.     

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.     

Replication of naturally occurring woodchuck hepatitis virus deletion mutants in primary hepatocyte cultures and after transmission to naive woodchucks

Woodchuck hepatitis virus (WHV) mutants with core internal deletions (CID) occur naturally in chronically WHV-infected woodchucks, as do hepatitis B virus mutants in humans. We studied the replication of WHV deletion mutants in primary woodchuck hepatocyte cultures and in vivo after transmission to naive woodchucks. By screening 14 wild-caught, chronically WHV-infected woodchucks, two woodchucks, WH69 and WH70, were found to harbor WHV CID mutants. Consistent with previous results, WHV CID mutants from both animals had deletions of variable lengths (90 to 135 bp) within the middle of the WHV core gene. In woodchuck WH69, WHV CID mutants represented a predominant fraction of the viral population in sera, normal liver tissues, and to a lesser extent, in liver tumor tissues. In primary hepatocytes of WH69, the replication of wild-type WHV and CID mutants was maintained at least for 7 days. Although WHV CID mutants were predominant in fractions of cellular WHV replicative intermediates, mutant covalently closed circular DNAs (cccDNAs) appeared to be a small part of cccDNA-enriched fractions. Analysis of cccDNA-enriched fractions from liver tissues of other woodchucks confirmed that mutant cccDNA represents only a small fraction of the total cccDNA pool. Four naive woodchucks were inoculated with sera from woodchuck WH69 or WH70 containing WHV CID mutants. All four woodchucks developed viremia after 3 to 4 weeks postinoculation (p.i.). They developed anti-WHV core antigen (WHcAg) antibody, lymphoproliferative response to WHcAg, and anti-WHV surface antigen. Only wild-type WHV, but no CID mutant, was found in sera from these woodchucks. The WHV CID mutant was also not identified in liver tissue from one woodchuck sacrificed in week 7 p.i. Three remaining woodchucks cleared WHV. Thus, the presence of WHV CID mutants in the inocula did not significantly change the course of acute self-limiting WHV infection. Our results indicate that the replication of WHV CID mutants might require some specific selective conditions. Further investigations on WHV CID mutants will allow us to have more insight into hepadnavirus replication.     

Expression and characterization of the naturally occurring mutation L394R in human gamma-glutamyl carboxylase

Patients with mutation L394R in gamma-glutamyl carboxylase have a severe bleeding disorder because of decreased biological activities of all vitamin K-dependent coagulation proteins. Vitamin K administration partially corrects this deficiency. To characterize L394R, we purified recombinant mutant L394R and wild-type carboxylase expressed in baculovirus-infected insect cells. By kinetic studies, we analyzed the catalytic activity of mutant L394R and its binding to factor IX's propeptide and vitamin KH(2). Mutant L394R differs from its wild-type counterpart as follows: 1) 110-fold higher K(i) for Boc-mEEV, an active site-specific, competitive inhibitor of FLEEL; 2) 30-fold lower V(max)/K(m) toward the substrate FLEEL in the presence of the propeptide; 3) severely reduced activity toward FLEEL carboxylation in the absence of the propeptide; 4) 7-fold decreased affinity for the propeptide; 5) 9-fold higher K(m) for FIXproGla, a substrate containing the propeptide and the Gla domain of human factor IX; and 6) 5-fold higher K(m) for vitamin KH(2). The primary defect in mutant L394R appears to be in its glutamate-binding site. To a lesser degree, the propeptide and KH(2) binding properties are altered in the L394R mutant. Compared with its wild-type counterpart, the L394R mutant shows an augmented activation of FLEEL carboxylation by the propeptide.     

Coexistence of two distinct secretion mutations (P5T and I97L) in hepatitis B virus core produces a wild-type pattern of secretion

Unlike a Tokyo isolate of hepatitis B virus variants, we found a Shanghai isolate that secretes few virions with an immature genome despite its core I97L mutation. Core mutations P5T and I97L were found to be mutually compensatory in offsetting their respective distinct effects on virion secretion.     

Interaction of wild-type and naturally occurring deleted variants of hepatitis B virus core polypeptides leads to formation of mosaic particles

The simultaneous presence of hepatitis B virus (HBV) genomes carrying wild-type (wt) and in-frame deleted variants of the HBV core gene has been identified as a typical feature of HBV-infected renal transplant patients with severe liver disease. To investigate possible interactions of wt and deleted core polypeptides a two-vector Escherichia coli expression system ensuring their concomitant synthesis has been developed. Co-expression of wt and a mutant core lacking 17 amino acid residues (77-93) within the immunodominant region led to the formation of mosaic particles, whereas the mutant alone was incapable of self-assembly.     

Structural and functional basis for increased HDL-cholesterol levels due to the naturally occurring V19L mutation in human apolipoprotein A-I

Several hereditary point mutations in human apolipoprotein A-I (apoA-I) have been associated with low HDL-cholesterol levels and/or increased coronary artery disease (CAD) risk. However, one apoA-I mutation, the V19L, recently identified in Icelanders, has been associated with increased HDL-cholesterol levels and decreased CAD risk. In an effort to gain mechanistic insight linking the presence of this mutation in apoA-I with the increase of HDL-cholesterol levels we evaluated the effect of V19L mutation on the conformational integrity and functional properties of apoA-I in lipid-free and lipidated form. ApoA-I[V19L] was found to be thermodynamically destabilized in lipid-free form and displays an increased capacity to associate with phospholipids compared to WT apoA-I. When associated to reconstituted HDL (rHDL), apoA-I[V19L] was more thermodynamically stabilized than WT apoA-I. ApoA-I[V19L] displayed normal capacity to promote ABCA1-mediated cholesterol efflux and to activate the enzyme LCAT, in lipid-free and rHDL-associated forms, respectively. Additionally, rHDL-associated apoA-I[V19L] showed normal capacity to promote ABCG1-mediated cholesterol efflux, but 45% increased capacity to promote SR-BI-mediated cholesterol efflux, while the SR-BI-mediated HDL-lipid uptake was normal. Overall, our findings show that the apoA-I V19L mutation does not affect the first steps of HDL biogenesis pathway. However, the increased capacity of apoA-I[V19L] to associate with phospholipids, in combination with the enhanced thermodynamic stability of lipoprotein-associated apoA-I[V19L] and increased capacity of apoA-I[V19L]-containing lipoprotein particles to accept additional cholesterol by SR-BI could account for the increased HDL-cholesterol levels observed in human carriers of the mutation.     

A molecular breadboard: Removal and replacement of subunits in a hepatitis B virus capsid

Hepatitis B virus (HBV) core protein is a model system for studying assembly and disassembly of icosahedral structures. Controlling disassembly will allow re‐engineering the 120 subunit HBV capsid, making it a molecular breadboard. We examined removal of subunits from partially crosslinked capsids to form stable incomplete particles. To characterize incomplete capsids, we used two single molecule techniques, resistive‐pulse sensing and charge detection mass spectrometry. We expected to find a binomial distribution of capsid fragments. Instead, we found a preponderance of 3 MDa complexes (90 subunits) and no fragments smaller than 3 MDa. We also found 90‐mers in the disassembly of uncrosslinked HBV capsids. 90‐mers seem to be a common pause point in disassembly reactions. Partly explaining this result, graph theory simulations have showed a threshold for capsid stability between 80 and 90 subunits. To test a molecular breadboard concept, we showed that missing subunits could be refilled resulting in chimeric, 120 subunit particles. This result may be a means of assembling unique capsids with functional decorations.     

Cold sensitivity in rice (Oryza sativa L.) is strongly correlated with a naturally occurring I99V mutation in the multifunctional glutathione transferase isoenzyme GSTZ2

GSTZs [Zeta class GSTs (glutathione transferases)] are multifunctional enzymes that belong to a highly conserved subfamily of soluble GSTs found in species ranging from fungi and plants to animals. GSTZs are known to function as MAAIs [MAA (maleylacetoacetate) isomerases], which play a role in tyrosine catabolism by catalysing the isomerization of MAA to FAA (fumarylacetoacetate). As tyrosine metabolism in plants differs from animals, the significance of GSTZ/MAAI is unclear. In rice (Oryza sativa L.), a major QTL (quantitative trait locus) for seedling cold tolerance has been fine mapped to a region containing the genes OsGSTZ1 and OsGSTZ2. Sequencing of tolerant (ssp. japonica cv. M-202) and sensitive (ssp. indica cv. IR50) cultivars revealed two SNPs (single nucleotide polymorphisms) in OsGSTZ2 that result in amino acid differences (I99V and N184I). Recombinant OsGSTZ2 containing the Val99 residue found in IR50 had significantly reduced activity on MAA and DCA (dichloroacetic acid), but the Ile184 residue had no effect. The distribution of the SNP (c.295A>G) among various rice accessions indicates a significant association with chilling sensitivity in rice seedlings. The results of the present study show that naturally occurring OsGSTZ2 isoforms differ in their enzymatic properties, which may contribute to the differential response to chilling stress generally exhibited by the two major rice subspecies.     

Phosphorylation of hepatitis B virus core C-terminally truncated protein (Cp149) by PKC increases capsid assembly and stability

The HBV (hepatitis B virus) core is a phosphoprotein whose assembly, replication, encapsidation and localization are regulated by phosphorylation. It is known that PKC (protein kinase C) regulates pgRNA (pregenomic RNA) encapsidation by phosphorylation of the C-terminus of core, which is a component packaged into capsid. Neither the N-terminal residue phosphorylated by PKC nor the role of the C-terminal phosphorylation have been cleary defined. In the present study we found that HBV Cp149 (core protein C-terminally truncated at amino acid 149) expressed in Escherichia coli was phosphorylated by PKC at Ser(106). PKC-mediated phosphorylation increased core affinity, as well as assembly and capsid stability. In vitro phosphorylation with core mutants (S26A, T70A, S106A and T114A) revealed that the Ser(106) mutation inhibited phosphorylation of core by PKC. CD analysis also revealed that PKC-mediated phosphorylation stabilized the secondary structure of capsid. When either pCMV/FLAG-Cp149[WT (wild-type)] or pCMV/FLAG-S106A Cp149 was transfected into Huh7 human hepatoma cells, mutant capsid level was decreased by 2.06-fold with the S106A mutant when compared with WT, although the same level of total protein was expressed in both cases. In addition, when pUC1.2x and pUC1.2x/S106A were transfected, mutant virus titre was decreased 2.31-fold compared with WT virus titre. In conclusion, PKC-mediated phosphorylation increased capsid assembly, stability and structural stability.     

Chromosomal site of hepatitis B virus (HBV) integration in a human hepatocellular carcinoma-derived cell line

The single site of integration of hepatitis B virus in the human hepatocellular carcinoma cell line Hep 3B 2-1/7 was found to segregate with human chromosome 12 in somatic cell hybrids. Analysis of metaphase spreads of Hep 3B 2-1/7 following in situ hybridization with pHBV revealed integration at 12q13----q14, a location that coincides with a fragile site, fra (12q13). The possible significance of this location to the development of hepatocellular carcinomas is discussed.     

Direct infection and replication of naturally occurring hepatitis C virus genotypes 1, 2, 3 and 4 in normal human hepatocyte cultures

Background

Hepatitis C virus (HCV) infection afflicts about 170 million individuals worldwide. However, the HCV life cycle is only partially understood because it has not been possible to infect normal human hepatocytes in culture. The current Huh-7 systems use cloned, synthetic HCV RNA expressed in hepatocellular carcinoma cells to produce virions, but these cells cannot be infected with naturally occurring HCV obtained from infected patients.

Methodology/Principal Findings

Here, we describe a human hepatocyte culture permissible to the direct infection with naturally occurring HCV genotypes 1, 2, 3 and 4 in the blood of HCV-infected patients. The culture system mimics the biology and kinetics of HCV infection in humans, and produces infectious virions that can infect naïve human hepatocytes.

Conclusions/Significance

This culture system should complement the existing systems, and may facilitate the understanding of the HCV life cycle, its effects in the natural host cell, the hepatocyte, as well as the development of novel therapeutics and vaccines.     

Repair and polyadenylation of a naturally occurring hepatitis C virus 3' nontranslated region-shorter variant in selectable replicon cell lines

The 3' nontranslated region (NTR) of the hepatitis C virus (HCV) genome is highly conserved and contains specific cis-acting RNA motifs that are essential in directing the viral replication machinery to initiate at the correct 3' end of the viral genome. Since the ends of viral genomes may be damaged by cellular RNases, preventing the initiation of viral RNA replication, stable RNA hairpin structures in the 3' NTR may also be essential in host defense against exoribonucleases. During 3'-terminal sequence analysis of serum samples of a patient with chronic hepatitis related to an HCV1b infection, a number of clones were obtained that were several nucleotides shorter at the extreme 3' end of the genome. These shorter 3' ends were engineered in selectable HCV replicons in order to enable the study of RNA replication in cell culture. When in vitro-transcribed subgenomic RNAs, containing shorter 3' ends, were introduced into Huh-7 cells, a few selectable colonies were obtained, and the 3' terminus of these subgenomic RNAs was sequenced. Interestingly, most genomes recovered from these colonies had regained the wild-type 3' ends, showing that HCV, like several other positive-stranded RNA viruses, has developed a strategy to repair deleted 3' end nucleotides. Furthermore, we found several genomes in these replicon colonies that contained a poly(A) tail and a short linker sequence preceding the poly(A) tail. After recloning and subsequent passage in Huh-7 cells, these poly(A) tails persisted and varied in length. In addition, the connecting linker became highly diverse in sequence and length, suggesting that these tails are actively replicated. The possible terminal repair mechanisms, including roles for the poly(A) tail addition, are discussed.     

Rab33B and its autophagic Atg5/12/16L1 effector assist in hepatitis B virus naked capsid formation and release

Hepatitis B virus morphogenesis is accompanied by the production and release of non‐enveloped capsids/nucleocapsids. Capsid particles are formed inside the cell cytosol by multimerization of core protein subunits and ultimately exported in an uncommon coatless state. Here, we investigated potential roles of Rab GTPases in capsid formation and trafficking by using RNA interference and overexpression studies. Naked capsid release does not require functions of the endosome‐associated Rab5, Rab7 and Rab27 proteins, but depends on functional Rab33B, a GTPase participating in autophagosome formation via interaction with the Atg5‐Atg12/Atg16L1 complex. During capsid formation, Rab33B acts in conjunction with its effector, as silencing of Atg5, Atg12 and Atg16L1 also impaired capsid egress. Analysis of capsid maturation steps revealed that Rab33B and Atg5/12/16L1 are required for proper particle assembly and/or stability. In support, the capsid protein was found to interact with Atg5 and colocalize with Atg5/12/16L1, implicating that autophagy pathway functions are involved in capsid biogenesis. However, a complete and functional autophagy pathway is dispensable for capsid release, as judged by knockdown analysis of Atg8/LC3 family members and pharmaceutical ablation of canonical autophagy. Experiments aimed at analysing the capsid release‐stimulating activity of the Alix protein provide further evidence for a link between capsid formation and autophagy.