Detection of a genome-linked protein (VPg) of hepatitis A virus and its comparison with other picornaviral VPgs.

The nucleotide sequence corresponding to the P3 region of the hepatitis A virus (HAV) polyprotein genome was determined from cloned cDNA and translated into an amino acid sequence. Comparison of the amino acid sequences of the genome-linked proteins (VPgs) of other picornaviruses with the predicted amino acid sequence of HAV was used to locate the primary structure of a putative VPg within the genome of HAV. The sequence of HAV VPg, like those of other picornaviral VPg molecules, contains a tyrosine residue as a potential binding site for HAV RNA in position 3 from its N terminus. The potential cleavage sites to generate VPg from a putative HAV polyprotein are between glutamic acid and glycine at the N terminus and glutamic acid and serine or glutamine and serine at the C terminus. A synthetic peptide corresponding to 10 amino acids of the predicted C terminus of HAV VPg induced anti-peptide antibodies in rabbits when it was conjugated to thyroglobulin as a carrier. These antibodies were specific for the peptide and precipitated VPg, linked to HAV RNA, from purified HAV and from lysates of HAV-infected cells. The precipitation reaction was blocked by the synthetic peptide (free in solution or coupled to carrier proteins) and prevented by pretreatment of VPg RNA with protease. Thus, our predicted amino acid sequence is colinear with the nucleotide sequence of the VPg gene in the HAV genome. From our results we concluded that HAV has the typical organization of picornavirus genes in this part of its genome. Similarity among hydrophobicity patterns of amino acid sequences of different picornaviral VPgs was revealed in hydropathy plots. Thus, the VPg of HAV appears to be closely related to VPg1 and VPg2 of foot-and-mouth disease virus. In contrast, HAV VPg has a unique isoelectric point (pI = 7.15) among the picornavirus VPgs.     

Complete nucleotide sequence of wild-type hepatitis A virus: comparison with different strains of hepatitis A virus and other picornaviruses.

The complete nucleotide sequence of wild-type hepatitis A virus (HAV) HM-175 was determined. The sequence was compared with that of a cell culture-adapted HAV strain (R. Najarian, D. Caput, W. Gee, S.J. Potter, A. Renard, J. Merryweather, G.V. Nest, and D. Dina, Proc. Natl. Acad. Sci. USA 82:2627-2631, 1985). Both strains have a genome length of 7,478 nucleotides followed by a poly(A) tail, and both encode a polyprotein of 2,227 amino acids. Sequence comparison showed 624 nucleotide differences (91.7% identity) but only 34 amino acid differences (98.5% identity). All of the dipeptide cleavage sites mapped in this study were conserved between the two strains. The sequences of these two HAV strains were compared with the partial sequences of three other HAV strains. Most amino acid differences were located in the capsid region, especially in VP1. Whereas changes in amino acids were localized to certain portions of the genome, nucleotide differences occurred randomly throughout the genome. The most extensive nucleotide homology between the strains was in the 5' noncoding region (96% identity for cell culture-adapted strains versus wild type; greater than 99% identity among cell culture-adapted strains). HAV proteins are less homologous with those of any other picornavirus than the latter proteins are when compared with each other. When the sequences of wild-type and cell culture-adapted HAV strains are compared, the nucleotide differences in the 5' noncoding region and the amino acid differences in the capsid region suggest areas that may contain markers for cell culture adaptation and for attenuation.     

Genetic Relatedness among Hepatitis A Virus Strains Associated with Food-Borne Outbreaks

The genetic characterization of hepatitis A virus (HAV) strains is commonly accomplished by sequencing subgenomic regions, such as the VP1/P2B junction. HAV genome is not extensively variable, thus presenting opportunity for sharing sequences of subgenomic regions among genetically unrelated isolates. The degree of misrepresentation of phylogenetic relationships by subgenomic regions is especially important for tracking transmissions. Here, we analyzed whole-genome (WG) sequences of 101 HAV strains identified from 4 major multi-state, food-borne outbreaks of hepatitis A in the Unites States and from 14 non-outbreak-related HAV strains that shared identical VP1/P2B sequences with the outbreak strains. Although HAV strains with an identical VP1/P2B sequence were specific to each outbreak, WG were different, with genetic diversity reaching 0.31% (mean 0.09%). Evaluation of different subgenomic regions did not identify any other section of the HAV genome that could accurately represent phylogenetic relationships observed using WG sequences. The identification of 2–3 dominant HAV strains in 3 out of 4 outbreaks indicates contamination of the implicated food items with a heterogeneous HAV population. However, analysis of intra-host HAV variants from eight patients involved in one outbreak showed that only a single sequence variant established infection in each patient. Four non-outbreak strains were found closely related to strains from 2 outbreaks, whereas ten were genetically different from the outbreak strains. Thus, accurate tracking of HAV strains can be accomplished using HAV WG sequences, while short subgenomic regions are useful for identification of transmissions only among cases with known epidemiological association.     

Characterization of recombinant hepatitis A virus genomes containing exogenous sequences at the 2A/2B junction

Hepatitis A virus (HAV) differs from other members of the family Picornaviridae in that the cleavage of the polyprotein at the 2A/2B junction, commonly considered to be the primary polyprotein cleavage by analogy with other picornaviruses, is mediated by 3C(pro), the only proteinase encoded by the virus. However, it has never been formally demonstrated that the 2A/2B junction is the site of primary cleavage, and the actual function of the 2A sequence, which lacks homology with sequence of other picornaviruses, remains unknown. To determine whether 2A functions in cis as a precursor with the nonstructural proteins, we constructed dicistronic HAV genomes in which a heterologous picornaviral internal ribosome entry site was inserted at the 2A/2B junction. Transfection of permissive FRhK-4 cells with these dicistronic RNAs failed to result in the rescue of infectious virus, indicating a possible cis replication function spanning the 2A/2B junction. However, infectious virus was recovered from recombinant HAV genomes containing exogenous protein-coding sequences inserted in-frame at the 2A/2B junction and flanked by consensus 3C(pro) cleavage sites. The replication of these recombinants was less efficient than that of the parent virus but was variable and not dependent upon the length of the inserted sequence. An HAV recombinant containing a 420-nt insertion encoding the bleomycin resistance protein Zeo was stable for up to five passages in cell culture. Inserted sequences were deleted from replicating viruses, but this did not result from homologous recombination at the flanking 3C(pro) cleavage sites, since the 5' and 3' segments of the inserted sequence were retained in the deletion mutants. These results indicate that the HAV polyprotein can tolerate an insertion at the 2A/2B junction and that the 2A polypeptide does not function in cis as a 2AB precursor. Recombinant HAV genomes containing foreign protein-coding sequences inserted at the 2A/2B junction are novel and potentially useful protein expression vectors.     

Identification of a cadherin cell adhesion recognition sequence

The molecular mechanisms by which the cadherins interact with one another to promote cell adhesion have not been elucidated. In particular, the amino acid sequences of the cadherin cell adhesion recognition sites have not been determined. Here we demonstrate that synthetic peptides containing the sequence HAV, which is common to all of the cadherins, inhibit two processes (compaction of eight-cell-stage mouse embryos and rat neurite outgrowth on astrocytes) that are known to be mediated by cadherins. The data suggest that the tripeptide HAV is a component of a cadherin cell adhesion recognition sequence.     

Characterization of a simian hepatitis A virus (HAV): antigenic and genetic comparison with human HAV.

PA21, a strain of hepatitis A virus (HAV) recovered from a naturally infected captive owl monkey, is indistinguishable from human HAV in polyclonal radioimmunoassays and cross-neutralization studies. However, cDNA-RNA hybridization has suggested a significant difference at the genomic level between PA21 and a reference human virus, HM175. Further characterization of this unique HAV was undertaken in an effort to determine the extent of genetic divergence from human HAV and its relation to the conserved antigenic structure of the virus. The close similarity between PA21 and HM175 antigens was confirmed with an extended panel of 18 neutralizing murine monoclonal antibodies: a reproducible difference in binding to the two viruses was detected with only one antibody (B5-B3). The nucleotide sequence of the P1 region of the PA21 genome had only 83.2% identity with HM175 virus, a difference approximately twice as great as that found between any two human strains. Most nucleotide changes were in third base positions, and the amino acid sequences of the capsid proteins were largely conserved. Amino acid replacements were clustered in the carboxy terminus of VP1 and the amino-terminal regions of VP2 and VP1. These data indicate that PA21 virus represents a unique genotype of HAV and suggest the existence of an ecologically isolated niche for HAV among feral owl monkeys.     

Molecular evolution of hepatitis A virus: a new classification based on the complete VP1 protein

Hepatitis A virus (HAV) is a positive-stranded RNA virus in the genus Hepatovirus in the family Picornaviridae So far, analysis of the genetic variability of HAV has been based on two discrete regions, the VP1/2A junction and the VP1 N terminus. In this report, we determined the nucleotide and deduced amino acid sequences of the complete VP1 gene of 81 strains from France, Kosovo, Mexico, Argentina, Chile, and Uruguay and compared them with the sequences of seven strains of HAV isolated elsewhere. Overall strain variation in the complete VP1 gene was found to be as high as 23.7% at the nucleotide level and 10.5% at the amino acid level. Different phylogenetic methods revealed that HAV sequences form five distinct and well-supported genetic lineages. Within these lineages, HAV sequences clustered by geographical origin only for European strains. The analysis of the complete VP1 gene allowed insight into the mode of evolution of HAV and revealed the emergence of a novel variant with a 15-amino-acid deletion located on the VP1 region where neutralization escape mutations were found. This could be the first antigenic variant of HAV so far identified.     

Analysis of 5' nontranslated region of hepatitis A viral RNA genotype I from South Korea: comparison with disease severities

The aim of the study was to analyze genotype I hepatitis A virus (HAV) 5' nontranslated region (NTR) sequences from a recent outbreak in South Korea and compare them with reported sequences from Japan. We collected a total of 54 acute hepatitis A patients' sera from HAV genotype I [27 severe disease (prothrombin time INR ≥ 1.50) and 27 mild hepatitis (prothrombin time INR <1.00)], performed nested RT-PCR of 5' NTR of HAV directly sequenced from PCR products (～ 300 bp), and compared them with each other. We could detect HAV 5'NTR sequences in 19 of the 54 (35.1%) cases [12 of 27 severe cases (44.4%) and 7 of 27 self-limited cases (25.9%)], all of which were subgenotype IA. Sequence analysis revealed that sequences of severe disease had 93.6%-99.0% homology and of self-limited disease 94.3%-98.6% homology, compared to subgenotype IA HAV GBM wild-type IA sequence. In this study, confirmation of the 5'NTR sequence differences between severe disease and mild disease was not carried out. Comparison with Japanese HAV A10 revealed (222)C to G or T substitution in 8/12 cases of severe disease and (222)C to G or T and (392)G to A substitutions in 5/7 and 4/7 cases of mild disease, respectively, although the nucleotide sequences in this study showed high homology (93.6%-100%). In conclusion, HAV 5'NTR subgenotype IA from Korea had relatively high homology to Japanese sequences previously reported from Japan, and this region would be considered one of the antiviral targets. Further studies will be needed.     

Insertion of short hepatitis virus A amino acid sequences into poliovirus antigenic determinants results in viable progeny

In an infectious poliovirus cDNA construct, the determinant encoding antigenic epitope N-Ag1 (in a loop located between two beta-strands in poly-peptide VP1) was altered by site-directed mutagenesis, to be partially similar with the determinants for presumptive epitopes in polypeptides VP1 or VP3 of hepatitis A virus (HAV). The modified constructs proved to be infectious. However, another construct, in which the same locus encoded a 'nonsense' and a relatively hydrophobic amino acid sequence, exhibited no infectivity. These data showed the feasibility of the insertion of foreign sequences in a specific antigenically active locus of the poliovirus icosahedron, and suggest some limitations with respect to the sequences to be 'transplanted'.     

Construction of recombinant DNA molecules by the use of a single stranded DNA generated by the polymerase chain reaction: its application to chimeric hepatitis A virus/poliovirus subgenomic cDNA.

In order to study the importance of VP4 in picornavirus replication and translation, we replaced the hepatitis A virus (HAV) VP4 with the poliovirus (PV1) VP4. Using a modification of oligonucleotide site directed mutagenesis and the polymerase chain reaction (PCR), we created a subgenomic cDNA chimera of hepatitis A virus in which the precise sequences coding for HAV VP4 capsid protein were replaced by the sequences coding for the poliovirus VP4 capsid protein. The method involved the use of PCR primers corresponding to the 3' and 5' ends of the poliovirus VP4 sequence and that had HAV VP4 3' and 5' flanking sequences on their 5'ends. Single stranded DNA of 240 and 242 nt containing the 204 nt coding for the complete poliovirus VP4 were produced by using a limiting amount of one of the primers in a PCR reaction. These single stranded PCR products were used like mutagenic oligonucleotides on a single stranded phagemid containing the first 2070 bases of the HAV genome. Using this technique, we precisely replaced the HAV VP4 gene by the poliovirus VP4 gene as determined by DNA sequencing. The cDNA was transcribed into RNA and translated in vitro. The resulting protein could be precipitated by antibody to poliovirus VP4 but not to HAV VP4.     

Replication of hepatitis A viruses with chimeric 5' nontranslated regions.

The role of the 5' nontranslated region in the replication of hepatitis A virus (HAV) was studied by analyzing the translation and replication of chimeric RNAs containing the encephalomyocarditis virus (EMCV) internal ribosome entry segment (IRES) and various lengths (237, 151, or 98 nucleotides [nt]) of the 5'-terminal HAV sequence. Translation of all chimeric RNAs, truncated to encode only capsid protein sequences, occurred with equal efficiency in rabbit reticulocyte lysates and was much enhanced over that exhibited by the HAV IRES. Transfection of FRhK-4 cells with the parental HAV RNA and with chimeric RNA generated a viable virus which was stable over continuous passage; however, more than 151 nt from the 5' terminus of HAV were required to support virus replication. Single-step growth curves of the recovered viruses from the parental RNA transfection and from transfection of RNA containing the EMCV IRES downstream of the first 237 nt of HAV demonstrated replication with similar kinetics and similar yields. When FRhK-4 cells infected with recombinant vaccinia virus producing SP6 RNA polymerase to amplify HAV RNA were transfected with plasmids coding for these viral RNAs or with subclones containing only HAV capsid coding sequences downstream of the parental or chimeric 5' nontranslated region, viral capsid antigens were synthesized from the HAV IRES with an efficiency equal to or greater than that achieved with the EMCV IRES. These data suggest that the inherent translation efficiency of the HAV IRES may not be the major limiting determinant of the slow-growth phenotype of HAV.     

Maturation of the Hepatitis A Virus Capsid Protein VP1 Is Not Dependent on Processing by the 3Cpro Proteinase

Most details of the processing of the hepatitis A virus (HAV) polyprotein are known. Unique among members of the family Picornaviridae, the primary cleavage of the HAV polyprotein is mediated by 3Cpro, the only proteinase known to be encoded by the virus, at the 2A/2B junction. All other cleavages of the polyprotein have been considered to be due to 3Cpro, although the precise location and mechanism responsible for the VP1/2A cleavage have been controversial. Here we present data that argue strongly against the involvement of the HAV 3Cpro proteinase in the maturation of VP1 from its VP1-2A precursor. Using a heterologous expression system based on recombinant vaccinia viruses directing the expression of full-length or truncated capsid protein precursors, we show that the C terminus of the mature VP1 capsid protein is located near residue 764 of the polyprotein. However, a proteolytically active HAV 3Cpro that was capable of directing both VP0/VP3 and VP3/VP1 cleavages in vaccinia virus-infected cells failed to process the VP1-2A precursor. Using site-directed mutagenesis of an infectious molecular clone of HAV, we modified potential VP1/2A cleavage sites that fit known 3Cpro recognition criteria and found that a substitution that ablates the presumed 3Cpro dipeptide recognition sequence at Glu764-Ser765 abolished neither infectivity nor normal VP1 maturation. Altered electrophoretic mobility of VP1 from a viable mutant virus with an Arg764 substitution indicated that this residue is present in VP1 and that the VP1/2A cleavage occurs downstream of this residue. These data indicate that maturation of the HAV VP1 capsid protein is not dependent on 3Cpro processing and may thus be uniquely dependent on a cellular proteinase.     

RNA binding specificity of hnRNP A1: significance of hnRNP A1 high-affinity binding sites in pre-mRNA splicing.

Pre-mRNA is processed as a large complex of pre-mRNA, snRNPs and pre-mRNA binding proteins (hnRNP proteins). The significance of hnRNP proteins in mRNA biogenesis is likely to be reflected in their RNA binding properties. We have determined the RNA binding specificity of hnRNP A1 and of each of its two RNA binding domains (RBDs), by selection/amplification from pools of random sequence RNA. Unique RNA molecules were selected by hnRNP A1 and each individual RBD, suggesting that the RNA binding specificity of hnRNP A1 is the result of both RBDs acting as a single RNA binding composite. Interestingly, the consensus high-affinity hnRNP A1 binding site, UAGGGA/U, resembles the consensus sequences of vertebrate 5' and 3' splice sites. The highest affinity 'winner' sequence for hnRNP A1 contained a duplication of this sequence separated by two nucleotides, and was bound by hnRNP A1 with an apparent dissociation constant of 1 x 10(-9) M. hnRNP A1 also bound other RNA sequences, including pre-mRNA splice sites and an intron-derived sequence, but with reduced affinities, demonstrating that hnRNP A1 binds different RNA sequences with a > 100-fold range of affinities. These experiments demonstrate that hnRNP A1 is a sequence-specific RNA binding protein. UV light-induced protein-RNA crosslinking in nuclear extracts demonstrated that an oligoribonucleotide containing the A1 winner sequence can be used as a specific affinity reagent for hnRNP A1 and an unidentified 50 kDa protein. We also show that this oligoribonucleotide, as well as two others containing 5' and 3' pre-mRNA splice sites, are potent inhibitors of in vitro pre-mRNA splicing.     

Study of Peptide Mimetics of Hepatitis A Virus Conjugated to Keyhole Limpet Hemocyanin and as Multiple Antigen Peptide System

Mimotopes mimic binding properties of natural antigen epitopes. They could be used for vaccine design, drugs development, and diagnostic assays. We have previously identified four bacteriophages displaying hepatitis A virus (HAV) mimotopes from a phage-display peptide library by affinity selection on serum antibodies from hepatitis A patients. Three of these HAV mimotopes showed similarity in their amino acid sequences with at least one of the VP3 and VP1 antigenic proteins of HAV and the four induced specific anti-HAV antibodies. In the present work, four conjugations were done. In each of them, a linear peptide (46, 53, 54 or 56) containing the amino-acid sequence of the corresponding mimotope was conjugated to keyhole limpet hemocyanin (KLH). Conjugation products were named: 46KLH, 53KLH, 54KLH and 56KLH. A two-arm multiple antigen peptide (MAP) system containing peptide sequence 46, and a second MAP containing two copies of peptide sequence 56 were synthesized and dimerized, to obtain the heterodimeric four-arms MAP (named MAP46-56) containing two copies of peptides 46 and 56. Mice were immunized with peptides conjugated to KLH and MAP46-56 to evaluate the ability of these two forms of mimotope presentation, to elicit antibodies that bind to the original antigen. KLH conjugated peptides rendered the highest levels of anti-peptide antibodies and were the only ones that induced specific anti-HAV antibodies. The results of immunizations showed that for the mimotopes chosen here, conjugation to a carrier protein was the most effective option to induce antibodies that cross-reacted with the natural antigen.     

Neutralizing monoclonal antibodies to hepatitis A virus: partial localization of a neutralizing antigenic site.

BALB/c mice were immunized with purified preparations of hepatitis A virus (HAV) isolated after 21 days of growth in LLC-MK2 cells. The HAV antigen was isolated from CsCl gradients and consisted primarily of the following three proteins as analyzed after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie blue staining: VP-1 at 33,000 daltons, VP-2 at 29,000 to 30,000 daltons, and VP-3 at 27,000 daltons. The spleen cells isolated from two BALB/c mice, immunized with two inoculations of HAV, were fused with SP 2/0 myeloma cells and grown in hypoxanthine-aminopterin-thymidine medium. Of 270 hybridomas initially screened, 72 were positive for binding HAV by a noncompetitive radioimmunoassay. All 72 were tested for the ability to neutralize the infectivity of HAV in an in vitro cell culture assay that was adapted for microtiter plates and that used detergent-treated virus for improved neutralization sensitivity and newborn cynomolgus monkey kidney cells for rapid growth. Eighteen hybridomas were positive for neutralization; 16 remained stable. Of the 16, 9 were able to compete with labeled polyclonal serum for binding to HAV. The nine competing hybridomas could be separated into two groups which appear to be directed towards two different sites on HAV and could complement each other in the competitive radioimmunoassay against polyclonal sera. Of the original 16 neutralizing hybridomas, 4 were subcloned through two cycles of limit dilutions. All four monoclonal antibodies retained their original neutralizing and competitive properties; three were immunoglobulin G2a, and one was immunoglobulin G1. All four monoclonal antibodies readily precipitate whole 125I-labeled HAV but are not able to recognize the disrupted proteins of the virus (as tested by immune precipitations of heat- and detergent-disrupted virions or Western blot analyses). However, the heterobifunctional cross-linking reagent toluene-2,4-diisocyanate was used to cross-link purified Fab fragments of two different monoclonal antibodies (2D2 and 6A5) to HAV before disruption. This reagent demonstrated a specific reaction of the monoclonal antibodies to the VP-1 of HAV, suggesting this major surface protein contains at least one of the major neutralization sites for HAV.     

The refined crystal structure of the 3C gene product from hepatitis A virus: specific proteinase activity and RNA recognition.

The virally encoded 3C proteinases of picornaviruses process the polyprotein produced by the translation of polycistronic viral mRNA. The X-ray crystallographic structure of a catalytically active mutant of the hepatitis A virus (HAV) 3C proteinase (C24S) has been determined. Crystals of this mutant of HAV 3C are triclinic with unit cell dimensions a = 53.6 A, b = 53.5 A, c = 53.2 A, alpha = 99.1 degrees, beta = 129.0 degrees, and gamma = 103.3 degrees. There are two molecules of HAV 3C in the unit cell of this crystal form. The structure has been refined to an R factor of 0.211 (Rfree = 0.265) at 2.0-A resolution. Both molecules fold into the characteristic two-domain structure of the chymotrypsin-like serine proteinases. The active-site and substrate-binding regions are located in a surface groove between the two beta-barrel domains. The catalytic Cys 172 S(gamma) and His 44 N(epsilon2) are separated by 3.9 A; the oxyanion hole adopts the same conformation as that seen in the serine proteinases. The side chain of Asp 84, the residue expected to form the third member of the catalytic triad, is pointed away from the side chain of His 44 and is locked in an ion pair interaction with the epsilon-amino group of Lys 202. A water molecule is hydrogen bonded to His 44 N(delta1). The side-chain phenolic hydroxyl group of Tyr 143 is close to this water and to His 44 N(delta1) and may be negatively charged. The glutamine specificity for P1 residues of substrate cleavage sites is attributed to the presence of a highly conserved His 191 in the S1 pocket. A very unusual environment of two water molecules and a buried glutamate contribute to the imidazole tautomer believed to be important in the P1 specificity. HAV 3C proteinase has the conserved RNA recognition sequence KFRDI located in the interdomain connection loop on the side of the molecule diametrically opposite the proteolytic site. This segment of polypeptide is located between the N- and C-terminal helices, and its conformation results in the formation of a well-defined surface with a strongly charged electrostatic potential. Presumably, this surface of HAV 3C participates in the recognition of the 5' and 3' nontranslated regions of the RNA genome during viral replication.     

中国五省市甲型肝炎病毒基因分型的研究

为了解甲型肝炎（甲肝）病毒（ＨＡＶ）在中国几个城市的基因型分布,选择浙江杭州、江苏启东、安徽铜陵、云南昆明和上海市等的甲肝病人粪便标本或血清标本,以逆转录－套式聚合酶链反应（ＲＴ－ｎＰＣＲ）扩增合成ＨＡＶ ＶＰ１／２Ａ交接区基因区,并进行直接核苷酸序列分析和差异比较。结果表明,从这些城市甲肝病人分离到的１７株ＨＡＶ株均属基因Ⅰ型,为ＩＡ和ＩＢ亚型;所有ＨＡＶ株间核苷酸差异均小于１５％,但约５０％Ｈ