Hepatitis A virus (HAV) proteinase 3C inhibits HAV IRES-dependent translation and cleaves the polypyrimidine tract-binding protein.

Hepatitis A virus (HAV) infection is still an important issue worldwide. A distinct set of viruses encode proteins that enhance viral cap-independent translation initiation driven by an internal ribosome entry site (IRES) and suppress cap-dependent host translation. Unlike cytolytic picornaviruses, replication of HAV does not cause host cell shut off, and it has been questioned whether HAV proteins interfere with its own and/or host translation. HAV proteins were coexpressed in Huh-7 cells with reporter genes whose translation was initiated by either cap-dependent or cap-independent mechanisms. Among the proteins tested, HAV proteinase 3C suppressed viral IRES-dependent translation. Furthermore, 3C cleaved the polypyrimidine tract-binding protein (PTB) whose interaction with the HAV IRES had been demonstrated previously. The combined results suggest that 3C-mediated cleavage of PTB might be involved in down-regulation of viral translation to give way to subsequent viral genome replication.

Experimental hepatitis A virus infection in guinea pigs.

Although many of the properties of hepatitis A virus (HAV) are known, several aspects of HAV pathogenesis are still not understood, such as the mechanism underlying the hepatotropism or HAV replication in extrahepatic sites. Detailed studies of these aspects were hampered mostly by the lack of accessible animal models, since only nonhuman primates are susceptible to experimental infections. An alternative animal model would also be of interest to assess the primary replication site and for the evaluation of the safety and efficacy of vaccines. A study was undertaken to determine whether HAV can infect guinea pigs and whether they are useful as a model for studying aspects of HAV pathogenesis and for the evaluation of vaccines. HAV variants adapted to primate or guinea pig tissue culture were used to inoculate guinea pigs intraperitoneally and by the oral route. The animals were observed for clinical disease, shedding of HAV in stools, viremia, seroconversion, evidence for liver damage by biochemical liver function tests, virus presence in the liver, development of hepatic histopathological changes, and occurrence of HAV in extrahepatic organs. The animals developed an active, clinically inapparent infection with specific histopathological changes in the liver. Although virus replication occurred, as shown by RT-PCR and isolation of infectious virus from feces and serum, it seems unlikely that guinea pigs are suitable for studying the clinical features of hepatitis A, because the clinical and laboratory parameters remained normal. However, guinea pigs appear useful for studying some aspects of HAV pathogenesis and for testing the safety of vaccines.

A new G-tailing method for the determination of the poly(A) tail length applied to hepatitis A virus RNA.

To study the role of the poly(A) tail length during the replication of poly(A)-containing plus-strand RNA virus, we have developed a simple reverse transcription polymerase chain reaction (RT-PCR)-based method that substantially improves the previously reported PAT [poly(A) test] assay. In contrast to the PAT assay, the new method is based on the enzymatic 3' elongation of mRNA with guanosine residues, thus immediately preserving the 3' end of the RNA and creating a unique poly(A)-oligo(G) junction. The oligo(G)-protected full-length poly(A) tail is reverse transcribed using the universal anti-sense primer oligo(dC(9)T(6)) and amplified by PCR with a gene-specific sense primer. After sequencing the resulting RT-PCR product the length of the poly(A) tail was unequivocally deduced from the number of adenosine residues between the oligo(G) stretch and the sequence upstream of the poly(A) tail. The efficiency and specificity of the newly developed assay was demonstrated by analysing the poly(A) tail length of the hepatitis A virus (HAV) RNA. We show here that the poly(A) tail of HAV RNA rescued after transfection of in vitro transcripts was elongated in the course of HAV replication.

Perinuclear accumulation of hepatitis A virus proteins, RNA, and particles and ultrastructural alterations in infected cells.

The exact intracellular site of hepatitis A virus (HAV) production is unknown, possibly due to its usually slow and inefficient replication. Using immunocytochemistry and in-situ RT-PCR, we show that in cells infected with the rapidly replicating HAV strain HAS-15, viral proteins and RNA are scattered throughout the cytoplasm and accumulate in the perinuclear cytoplasmic area. Various ultrastructural alterations were found in infected cells, such as large polyribosomes, swelling of the perinuclear space and the ER, and dilatation of Golgi cisternae. In addition, HAV infection induced the formation of large arrays of annulate lamellae. Direct visualization of HAV particles was scarce. The various ultrastructural alterations described here might represent different phases of the replicative cycle of HAV that is asynchronous in the infected cell layer.

Improving proteolytic cleavage at the 3A/3B site of the hepatitis A virus polyprotein impairs processing and particle formation, and the impairment can be complemented in trans by 3AB and 3ABC.

The orchestrated liberation of viral proteins by 3C(pro)-mediated proteolysis is pivotal for gene expression by picornaviruses. Proteolytic processing is regulated either by the amino acid sequence at the cleavage site of the substrate or by cofactors covalently or noncovalently linked to the viral proteinase. To determine the role of the amino acid sequence at cleavage sites 3A/3B and 3B/3C that are essential for the liberation of 3C(pro) from its precursors and to assess the function of the stable processing intermediates 3AB and 3ABC, we studied the effect of cleavage site mutations on hepatitis A virus (HAV) polyprotein processing, particle formation, and replication. Using the recombinant vaccinia virus system, we showed that the normally retarded cleavage at the 3A/3B junction can be improved by altering the amino acid sequence at the scissile bond such that it matches the preferred HAV 3C cleavage sites. In contrast to the processing products of the wild-type polyprotein, 3ABC was no longer detectable in the mutant. VP0 and VP3 were generated less efficiently, implying that processing of the structural protein precursor P1-2A depends on the presence of stable 3ABC and/or 3AB. In addition, cleavage of 2BC was impaired in 3AB/3ABC-deficient mutants. Formation of HAV particles was not affected in mutants with blocked 3A/3B and/or 3B/3C cleavage sites. However, 3ABC-deficient mutants produced small numbers of HAV particles, which could be augmented by coexpressing 3AB or 3ABC. The hydrophobic domain of 3A that has been proposed to mediate membrane anchorage of the replication complex was crucial for restoration of defective particle formation. In vitro transcripts of the various cleavage site mutants were unable to initiate an infectious cycle, and no progeny viruses were obtained even after blind passages. Taken together, the data suggest that accumulation of uncleaved HAV 3AB and/or 3ABC is pivotal for both viral replication and efficient particle formation.

Mapping of protein domains of hepatitis A virus 3AB essential for interaction with 3CD and viral RNA.

The small hydrophobic protein 3AB of the picornaviruses, encompassing the replication primer 3B, has been suggested to anchor the viral replication complex to membranes. For hepatitis A virus (HAV) 3AB, we have previously demonstrated its ability to form stable homodimers, to bind to membranes, and to interact specifically with RNA, implicating its multiple involvement in viral replication. In the present report, we show that HAV 3AB additionally interacts with HAV protein 3CD, a feature also described for the corresponding polypeptide of poliovirus. By assessing the interactions of three deletion mutants, distinct domains of HAV 3AB were mapped. The hydrophobic domain and the 3B moiety were found to be essential for the 3AB interaction with 3CD. Both electrostatic and hydrophobic forces are involved in this interaction. The cluster of charged amino acid residues at the C terminus of 3A seems to determine the specificity of 3AB interaction with RNA structures formed at either terminus of the HAV genome. Furthermore, our data implicate that 3A can interact with HAV RNA. Compared with poliovirus 3AB, which by itself is a nonspecific RNA-binding protein, HAV 3AB specifically recognizes HAV RNA structures that might be of relevance for initiation of viral RNA replication.

Intrinsic signals for the assembly of hepatitis A virus particles. Role of structural proteins VP4 and 2A.

Capsid assembly is the final event of virus replication, and its understanding is pivotal for the design of empty capsid-based recombinant vaccines and drug delivery systems. Although the capsid structure of several members of the picornavirus family has been elucidated, little is known about the structural elements governing the assembly process that is tightly associated with proteolytic processing of the viral polyprotein. Among the picornaviruses, hepatitis A virus (HAV) is unique in that it contains VP1-2A as a structural component and the small structural protein VP4, which argues for an assembly pathway different from that proposed for other picornaviruses. Using a recombinant system we show here that proteolytic processing of the HAV capsid proteins' precursor P1-2A is independent of the terminal domains 2A and VP4 of the substrate. However, both terminal domains play distinct roles in the assembly of viral particles. 2A as part of P1-2A is a primary signal for the assembly of pentameric structures which only further aggregate to empty viral capsids when VP4 is present as the N terminus of the precursor. Particle formation in the hepatovirus genus is thus regulated by two intrinsic signals that are distinct from those described for other picornaviruses.

Processing of proteinase precursors and their effect on hepatitis A virus particle formation.

Proteolytic processing of the picornaviral polyprotein mediated by the differential action of virus-encoded proteinase(s) is pivotal to both RNA genome replication and capsid formation. Possibly to enlarge the array of viral proteins, picornaviral polyprotein processing results in intermediate and mature products which apparently have distinct functions within the viral life cycle. For hepatitis A virus (HAV), we report here on the autoproteolysis of precursor polypeptides comprising the only viral proteinase, 3Cpro, and on their role in viral particle formation. Following transient expression of a nested set of 3Cpro-containing proteins (P3, 3ABC, 3BCD, 3CD, 3BC, and 3C) in eukaryotic cells, the extent of processing was determined by analyzing the cleavage products. The 3C/3D site was more efficiently cleaved than those at the 3A/3B and 3B/3C sites, leading to the accumulation of the intermediate product 3ABC. In the absence of 3A from the precursor, cleavage at the 3B/3C site was further reduced and a switch to an alternative 3C/3D site was observed. Coexpression of various parts of P3 with the precursor of the viral structural proteins P1-2A showed that all 3C-containing intermediates cleaved P1-2A with almost equal efficiency; however, viral particles carrying the neutralizing epitope form much more readily in the presence of the complete P3 domain than with parts of it. These data support the notion that efficient liberation of structural proteins from P1-2A is necessary but not sufficient for productive HAV capsid formation and suggest that the polypeptides flanking 3Cpro promote the assembly of viral particles.

Membrane association and RNA binding of recombinant hepatitis A virus protein 2C.

The direct function of hepatitis A virus (HAV) protein 2C, a putative NTPase, is not known, yet genetic evidence obtained from chimeric viruses carrying the 2C genomic region of different HAV variants indicates that it plays a pivotal role in viral replication. In a first assessment of its potential function(s), membrane and RNA binding properties of HAV 2C were studied after expressing the protein in various recombinant systems. In contrast to poliovirus 2C, expression of HAV 2C was inhibitory to the growth and protein synthesis of bacteria. Deletion of the N-terminal amphipathic helix of 2C abrogated this effect and the ability of 2C to associate with eukaryotic membranes. Both, purified 2C and the N-terminally truncated protein were shown to bind RNA in vitro. Our data taken together suggest that HAV 2C is a multifunctional protein.

Membrane permeability induced by hepatitis A virus proteins 2B and 2BC and proteolytic processing of HAV 2BC.

The ability to rearrange membranes is a unique feature of nonstructural proteins 2B, 2C, and 2BC of some picornaviruses. To analyze in detail membrane binding of the respective proteins of hepatitis A virus (HAV), they were transiently expressed in the vaccinia/T7 system, and their effect on membrane permeability was studied using beta-galactosidase as reporter. Although 2C had no effect, the significantly increased reporter activity observed in the extracellular space of 2B- and 2BC-expressing cells points to a specific effect of HAV proteins 2B and 2BC on membrane permeability. In biochemical fractionation studies, HAV 2C and 2BC showed properties of intregral membrane proteins, whereas 2B was associated with membranes as a peripheral protein. Proteinase 3C-mediated cleavage of precursor 2BC in vivo was most efficient when the enzyme was coexpressed in its precursor forms P3 or 3ABC, which both include the membrane-anchoring domain 3A. 3ABC showed the same solubility pattern as 2BC, suggesting that colocalization of 2BC and 3ABC might be required for the efficient liberation of 2B and 2C and occurs on membranes that have been proposed as the site of viral RNA replication.

In vitro and ex vivo inhibition of hepatitis A virus 3C proteinase by a peptidyl monofluoromethyl ketone.

Hepatitis A virus (HAV) 3C proteinase is the enzyme responsible for the processing of the viral polyprotein. Although a cysteine proteinase, it displays an active site configuration like those of the mammalian serine proteinases (Malcolm, B. A. Protein Science 1995, 4, 1439). A peptidyl monofluoromethyl ketone (peptidyl-FMK) based on the preferred peptide substrates for HAV 3C proteinase was generated by first coupling the precursor, N,N-dimethylglutamine fluoromethylalcohol, to the tripeptide, Ac-Leu-Ala-Ala-OH, and then oxidizing the product to the corresponding peptidyl-FMK (Ac-LAAQ'-FMK). This molecule was found to be an irreversible inactivator of HAV 3C with a second-order rate constant of 3.3 x 10(2) M-1 s-1. 19F NMR spectroscopy indicates the displacement of fluoride on inactivation of the enzyme by the fluoromethyl ketone. NMR spectroscopy of the complex between the 13C-labeled inhibitor and the HAV 3C proteinase indicates that an (alkylthio)methyl ketone is formed. Studies of polyprotein processing, using various substrates generated by in vitro transcription/translation, demonstrated efficient blocking of even the most rapid proteolytic events such as cleavage of the 2A-2B and 2C-3A junctions. Subsequent ex vivo studies, to test for antiviral activity, show a 25-fold reduction in progeny virus production as the result of treatment with 5 microM inhibitor 24 h post-infection.

Proteinase 3C-mediated processing of VP1-2A of two hepatitis A virus strains: in vivo evidence for cleavage at amino acid position 273/274 of VP1.

Two prominent features distinguish hepatitis A virus (HAV) from other members of the picornavirus family. A C-terminally prolonged precursor of the structural protein VP1 is incorporated into assembly intermediates (e.g., the provirion), and a single proteinase is contained within the HAV polyprotein. Using an in vivo expression system, we show that proteolytic liberation of VP1 from its precursors P1-2A and VP1-2A is catalyzed by the virus-encoded proteinase 3Cpro. Among the proposed cleavage sites within VP1-2A, the Glu/Ser pair found at VP1 amino acid position 273/274 of most HAV strains is efficiently processed, whereas proteolysis of the Val/Ser site of the attenuated HM175 strain is protracted. Two mutations within VP1-2A (Lys[297]Arg and Ser[330]Asn) had no effect on 3Cpro-mediated cleavage at this site. Additional sites in this region of VP1-2A can also be utilized as substrates by the proteinase, yet less efficiently, and might give rise to smaller and larger VP1 polypeptides also detected in HAV-infected cells.

In vitro RNA binding of the hepatitis A virus proteinase 3C (HAV 3Cpro) to secondary structure elements within the 5' terminus of the HAV genome.

The secondary structure elements at the 5' nontranslated region (NTR) of the picornaviral RNAs can be divided functionally into two domains, one of which directs cap-independent translation, whereas the other is essential for viral RNA replication. For the latter, the formation of an RNA replication complex that involves particularly viral proteinase-containing polypeptides and cellular proteins has been shown (Andino R, Rieckhof GE, Achacoso PL, Baltimore D, 1993, EMBO J 12:3587-3598; Xiang W et al., 1995, RNA 1:892-904). To initiate studies on the formation of the hepatitis A virus (HAV) RNA replication complex, binding of the HAV proteinase 3Cpro and 3CD to secondary structure elements at the 5' and 3' NTR of the HAV RNA was investigated. Using mobility shift assay, UV crosslinking/ label transfer, and northwestern analysis, we show that both the HAV 3Cpro and the proteolytically inactive mutant bind to in vitro synthesized transcripts, suggesting that the RNA-binding site of the enzyme is separated spatially from its catalytic center. Weak interactions with HAV 3Cpro were found for individual secondary structure elements comprising less than 100 nt. RNA-binding specificity was unambiguous for transcripts comprising at least two stem-loops along with the polypyrimidine tract. Furthermore, competition experiments suggest that the 5' terminus of the HAV genome contains multiple binding sites for HAV 3Cpro. In contrast to poliovirus, binding capacity of HAV 3CD to RNA of the 5' NTR was not improved as compared to 3C. The data imply that, during the viral life cycle, HAV 3Cpro might serve replicative function(s) in addition to proteolysis of the viral polyprotein.

Interaction of hepatitis A virus (HAV) precursor proteins 3AB and 3ABC with the 5' and 3' termini of the HAV RNA.

RNA secondary structures within the terminal nontranslated regions of entero- and rhinoviral genomes interact specifically with viral nonstructural proteins and are required in cis for viral RNA replication. Here we show that recombinant hepatitis A virus (HAV) polypeptide 3ABC specifically interacts in vitro with secondary RNA structures formed at both the 5' and 3' terminus of the viral genome. Similar to protein 3AB, HAV 3ABC bound to the 3' terminal RNA structure which did not interact with the mature proteinase 3C. In contrast to 3AB, 3ABC interacted with RNA stem-loop IIa and combinations of individual secondary structure elements of the 5' noncoding region. RNA binding of the precursor polypeptide 3ABC was 50 times stronger than that of 3AB and 3C, implicating a specific role of this stable processing intermediate in viral genome replication.

RNA-protein interactions at the 3' end of the hepatitis A virus RNA.

The regulative cis-acting terminal RNA structures and the proteins involved in the amplification of the hepatitis A virus (HAV) genome are unknown. By UV cross-linking/label transfer experiments, we have analyzed sequences of the 3'-nontranslated region (3'NTR) and preceding domains of the viral genome for their ability to interact with host proteins. A series of cDNA constructs were used to create genomic- and antigenomic-sense transcripts. The results show that the 3'-NTR-poly(A) interacted with host cell proteins with molecular masses of 38, 45, 57, 84, and 110 kDa only weakly, compared with RNA structures also consisting of 3D-coding regions. Protein p38 was most efficiently labeled after interaction with secondary-structure elements located at the 3' end of the HAV RNA, p38 also interacted with a 5'-terminal RNA probe. Optimal RNA binding was found to be dependent on the salt concentration. The specificity of the RNA-protein interaction was proven by competition assays. These data might indicate that a higher-order structure formed at the junction of the 3Dpol-coding sequence and the 3'-NTR of the HAV genome (putative RNA pseudoknot) significantly improves binding of host proteins and thus suggests that this structure might be essential for the formation of the replication complex initiating minus-strand RNA synthesis.

Polyprotein processing in echovirus 22: a first assessment.

The major steps of the polyprotein processing of Echovirus 22 (EV22), a highly unusual member of the picornavirus family, have been characterized for the first time by employing in vitro assay systems. Cell-free expression of a P1-2ABC precursor as well as VP1-2A yielded autoproteolytically inactive proteins, suggesting that the 2A region of the EV22 polyprotein does not contain a proteolytic activity. The intra- and intermolecular cleavage specificity of proteinase 3C, the major proteolytic enzyme in picornaviruses, was studied by expressing the enzyme of EV22 in a bacterial system as well as in the framework of precursor molecules generated by in vitro transcription/translation in a cell-free system. A VP1-2A percursor could very efficiently be cleaved in trans by the recombinant 3C, whereas the junction between P2 and P3 remained uncleaved. Expression of the complete P3-region in the cell-free system led to the autocatalytic release of large amounts of p22, a protein of the predicted molecular weight of the EV22 proteinase 3C that was recognized by an antibody raised against the recombinant enzyme.

Recombinant expression of hepatitis A virus protein 3A: interaction with membranes.

The function of hepatitis A virus (HAV) protein 3A and its structural requirements were studied in vitro and in a bacterial expression system by comparing the polypeptide precursor 3AB derived from a cytopathogenic strain with that of an attenuated strain. Although the precursor polypeptides 3AB of both HAV strains bind to microsomal membranes after translation in vitro they differ in inducing membrane permeability when expression is induced in bacteria. Intake and release of macromolecules was dramatically increased when 3AB of the cytopathogenic strain was expressed. Amino acid sequence alignments suggest that membrane binding might be due to a hydrophobic stretch near the C-terminus of 3A found in all picornaviruses whereas the ability to induce permeability of E. coli membranes is determined by an amphipathic helix formed at the N-terminus of 3A of HAVFG.

Cleavage specificity of purified recombinant hepatitis A virus 3C proteinase on natural substrates.

Hepatitis A virus (HAV) 3C proteinase expressed in Escherichia coli was purified to homogeneity, and its cleavage specificity towards various parts of the viral polyprotein was analyzed. Intermolecular cleavage of the P2-P3 domain of the HAV polyprotein gave rise to proteins 2A, 2B, 2C, 3ABC, and 3D, suggesting that in addition to the primary cleavage site, all secondary sites within P2 as well as the 3C/3D junction are cleaved by 3C. 3C-mediated processing of the P1-P2 precursor liberated 2A and 2BC, in addition to the structural proteins VP0, VP3, and VP1-2A and the respective intermediate products. A clear dependence on proteinase concentration was found for most cleavage sites, possibly reflecting the cleavage site preference of 3C. The most efficient cleavage occurred at the 2A/2B and 2C/3A junctions. The electrophoretic mobility of processing product 2B, as well as cleavage of the synthetic peptide KGLFSQ*AKISLFYT, suggests that the 2A/2B junction is located at amino acid position 836/837 of the HAV polyprotein. Furthermore, using suitable substrates we obtained evidence that sites VP3/VP1 and VP1/2A are alternatively processed by 3C, leading to either VP1-2A or to P1 and 2A. The results with regard to intermolecular cleavage by purified 3C were confirmed by the product pattern derived from cell-free expression and intramolecular processing of the entire polyprotein. We therefore propose that polyprotein processing of HAV relies on 3C as the single proteinase, possibly assisted by as-yet-undetermined viral or host cell factors and presumably controlled in a concentration-dependent fashion.

Proteinase 3C of hepatitis A virus (HAV) cleaves the HAV polyprotein P2-P3 at all sites including VP1/2A and 2A/2B.

Thus far, the only virus-encoded proteinase of hepatitis A virus (HAV) detected is 3C, which was shown to catalyze proteolysis of most of the suggested cleavage sites within the HAV precursor polyprotein. To elucidate whether or not HAV proteinase 3C and its precursors are involved in processing of the yet unidentified sites in the polyprotein P2-P3, the genomic region of 3C including flanking sequences were expressed in a bacterial system and by cell-free translation. In both systems 2A-reactive proteins of 10 (2A) and 16 kDa (delta VP1-2A) were processing products of a polyprotein representing delta VP1-P2-P3* (delta and * denote N- or C-terminally truncated proteins, respectively), thus providing evidence for cleavage at sites VP1/2A and 2A/2B by proteinase 3C. In the cell-free expression system, processing at the P2/P3 junction was rapid and complete, whereas sites 3A/3B, 3B/3C, and 3C/3D were inefficiently cleaved, as evidenced by the accumulation of the stable precursor polypeptides P3* and 3ABC. In contrast to the eukaryotic system, mature 3C was produced in Escherichia coli. Intermolecular cleavage by recombinant 3C occurred at all putative sites within the proteolytically inactive polyprotein P2-P3* mu. The results of this study indicate that proteinase 3C mediates the primary as well as the secondary cleavages of the HAV polyprotein and thus shows an activity profile broader than that of 3C proteinases of other picornaviruses.

Cell-free translation and proteolytic processing of the hepatitis A virus polyprotein.

Virus-encoded proteinase activity of hepatitis A virus (HAV) was studied in vitro. Genomic regions coding for segments of the viral polyprotein were expressed by in vitro transcription and translation in rabbit reticulocyte lysates. Polyproteins translated from synthetic transcripts encoding P1-P2 or delta VP1-P2 were not processed indicating that no proteolytic activity is encoded within P2 of HAV, in contrast to other picornaviruses. Proteinase activity was, however, detected in the genomic region encoding 3C. Mutant transcripts (mu) which encode an alanine in place of the cysteine residue at amino acid position 172 of 3C did not yield proteolytic activity, consistent with the hypothesis that proteinase 3C is a cysteine-containing trypsin-like proteinase. Processing products 3ABC and P3 were identified by immunoprecipitation, providing evidence that proteolytic cleavage occurs at the 2C/3A and less frequently at the 3C/3D junction. For cleavages at either site, the complete 3D moiety was not required. In general, analysis of cleavage products was made difficult by the presence of polypeptides which were translated from internal start sites, predominantly within the P3 region. Since only small amounts of the full-length products P1-P2-P3 or P2-P3 were translated, possible cleavage of P1 and P2 by 3C could not be resolved in this system. Furthermore, no intermolecular cleavage could be detected when in vitro translated polypeptides of the P3 region were incubated with P1, P1-P2 or P2-P3 mu as substrates.