RESUMEN
Oligonucleotides as short as 6 nt in length have been shown to bind specifically and tightly to proteins and affect their biological function. Yet, sparse structural data are available for corresponding complexes. Employing a recently developed hexanucleotide array, we identified hexadeoxyribonucleotides that bind specifically to the 3C protease of hepatitis A virus (HAV 3C(pro)). Inhibition assays in vitro identified the hexanucleotide 5'-GGGGGT-3' (G(5)T) as a 3C(pro) protease inhibitor. Using (1)H NMR spectroscopy, G(5)T was found to form a G-quadruplex, which might be considered as a minimal aptamer. With the help of (1)H, (15)N-HSQC experiments the binding site for G(5)T was located to the C-terminal ß-barrel of HAV 3C(pro). Importantly, the highly conserved KFRDI motif, which has previously been identified as putative viral RNA binding site, is not part of the G(5)T-binding site, nor does G(5)T interfere with the binding of viral RNA. Our findings demonstrate that sequence-specific nucleic acid-protein interactions occur with oligonucleotides as small as hexanucleotides and suggest that these compounds may be of pharmaceutical relevance.
Asunto(s)
Cisteína Endopeptidasas/química , Desoxirribonucleótidos/química , Virus de la Hepatitis A/enzimología , Proteínas Virales/química , Proteasas Virales 3C , Antivirales/química , Sitios de Unión , Cisteína Endopeptidasas/metabolismo , Desoxirribonucleótidos/metabolismo , Dimerización , G-Cuádruplex , Resonancia Magnética Nuclear Biomolecular , ARN Viral/metabolismo , Proteínas Virales/metabolismoRESUMEN
Hepatitis A virus (HAV) causes acute hepatitis and sometimes leads to fulminant hepatitis. Amantadine is a tricyclic symmetric amine that inhibits the replication of many DNA and RNA viruses. Amantadine was reported to suppress HAV replication, and the efficacy of amantadine was exhibited in its inhibition of the internal ribosomal entry site (IRES) activities of HAV. Interferon (IFN) also has an antiviral effect through the induction of IFN stimulated genes (ISG) and the degradation of viral RNA. To explore the mechanism of the suppression of HAV replication, we examined the effects of the combination of amantadine and IFN-alpha on HAV IRES-mediated translation, HAV replicon replication in human hepatoma cell lines, and HAV KRM003 genotype IIIB strain replication in African green monkey kidney cell GL37. IFN-alpha seems to have no additive effect on HAV IRES-mediated translation inhibition by amantadine. However, suppressions of HAV replicon and HAV replication were stronger with the combination than with amantadine alone. In conclusion, amantadine, in combination of IFN-alpha, might have a beneficial effect in some patients with acute hepatitis A.
Asunto(s)
Amantadina/farmacología , Antivirales/farmacología , Virus de la Hepatitis A/efectos de los fármacos , Virus de la Hepatitis A/fisiología , Interferón-alfa/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Animales , Línea Celular Tumoral , Chlorocebus aethiops , Interacciones Farmacológicas , Células Epiteliales/virología , Hepatocitos/virología , Humanos , Pruebas de Sensibilidad MicrobianaRESUMEN
Proteolytic cleavage of translation initiation factors is a means to interfere with mRNA circularization and to induce translation arrest during picornaviral replication or apoptosis. It was shown that the regulated cleavages of eukaryotic initiation factor (eIF) 4G and poly(A)-binding protein (PABP) by viral proteinases correlated with early and late arrest of host cap-dependent and viral internal ribosome entry site (IRES)-dependent translation, respectively. Here we show that in contrast to coxsackievirus, eIF4G is not a substrate of proteinase 3C of hepatitis A virus (HAV 3C(pro)). However, PABP is cleaved by HAV 3C(pro) in vitro and in vivo, separating the N-terminal RNA-binding domain (NTD) of PABP from the C-terminal protein-interaction domain. In vitro, NTD has a dominant negative effect on HAV IRES-dependent translation and an enhanced binding affinity to the RNA structural element pY1 in the 5' nontranslated region of the HAV RNA that is essential for viral genome replication. The results point to a regulatory role of PABP cleavage in RNA template switching of viral translation to RNA synthesis.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Regulación Viral de la Expresión Génica , Virus de la Hepatitis A/genética , Iniciación de la Cadena Peptídica Traduccional , Proteínas de Unión a Poli(A)/metabolismo , Proteínas Virales/metabolismo , Proteasas Virales 3C , Regiones no Traducidas 5'/química , Regiones no Traducidas 5'/metabolismo , Línea Celular Tumoral , Factor 4G Eucariótico de Iniciación/metabolismo , Virus de la Hepatitis A/enzimología , Humanos , Modelos Genéticos , Proteínas de Unión a Poli(A)/química , Proteínas de Unión a Poli(A)/fisiología , ARN Viral/química , ARN Viral/metabolismo , Eliminación de SecuenciaRESUMEN
The human RNA-binding protein La, is an essential trans-acting factor in IRES-dependent translation initiation of poliovirus, the prototypic picornavirus. For hepatitis A virus (HAV), an unusual member of this virus family, the role of host proteins in its inefficient translation and slow replication is unclear. Using small interfering RNA in vivo and purified La in vitro, we demonstrate for the first time that La suppresses HAV IRES-mediated translation and replication. We show that La binds specifically to distinct parts of the HAV IRES and that-unlike poliovirus-HAV proteinase 3C does not cleave La. The La-mediated suppression of HAV translation and stimulation of poliovirus translation implies unexpected mechanistic differences between viral IRES elements.
Asunto(s)
Autoantígenos/fisiología , Virus de la Hepatitis A/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , Ribonucleoproteínas/fisiología , Ribosomas/fisiología , Proteasas Virales 3C , Autoantígenos/metabolismo , Línea Celular Tumoral , Cisteína Endopeptidasas/metabolismo , Humanos , Interferencia de ARN , Ribonucleoproteínas/metabolismo , Proteínas Virales/metabolismo , Antígeno SS-BRESUMEN
The poly(rC)-binding protein PCBP2 has multiple functions in post-transcriptional control of host and viral gene expression. Since it interacts with picornaviral RNA structures, it was proposed that PCBP2 regulates viral genome translation and replication. The hepatitis A virus (HAV), an atypical picornavirus, contains an unusual pyrimidine-rich tract (pY1) with unknown functions. Using in vivo and in vitro assays, we provide direct evidence that PCBP2 interacts with pY1 and that binding is mediated by KH domains 1 and 3. Proteolytic cleavage by the viral protease 3C generates a C-terminally truncated polypeptide with highly reduced RNA affinity. The results suggest that during HAV infection PCBP2 cleavage might specifically down-regulate viral protein synthesis, thereby giving way to viral RNA synthesis.
Asunto(s)
Virus de la Hepatitis A/química , Virus de la Hepatitis A/enzimología , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , ARN/química , ARN/metabolismo , Sitios de Unión , Unión ProteicaRESUMEN
Hepatitis A virus (HAV) protein 2A has the capacity to harbor and expose a short foreign epitope. The chimeric virus, HAV-gp41, bearing seven amino acids of the 2F5 epitope of the HIV glycoprotein gp41, was shown to replicate in cell culture and laboratory animals and to induce a humoral immune response. As an extension of this work, we now investigated the possibility to insert longer epitopes, their impact on genetic stability, and the production of chimeric HAV. Twenty-seven amino acid residues of either HIV gp41, comprising the 2F5 epitope, or of a mimotope (F78) of the hypervariable region 1 of the hepatitis C virus (HCV) envelope protein E2 were inserted near the C-terminus of HAV 2A and viral capsid formation and replication were studied. The genome of the chimeric virus (HAV-F78) had reduced replication ability, yet the sedimentation profile of the chimeric particles was unchanged and the HCV sequence was maintained over serial viral passages. In contrast, no capsids were formed when an extended HIV epitope of 27 residues was inserted, precluding the rescue of infectious chimeric virus. Based on structural analyses, the data suggest that the isoelectric point (pI) and/or the secondary structure of the chimeric proteins are essential determinants that affect HAV particle formation for which protein 2A serves as an assembly signal.
Asunto(s)
Epítopos/inmunología , Virus de la Hepatitis A/fisiología , Ensamble de Virus , Replicación Viral , Animales , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/inmunología , Proteína gp41 de Envoltorio del VIH/genética , Proteína gp41 de Envoltorio del VIH/inmunología , Virus de la Hepatitis A/inmunología , Punto Isoeléctrico , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología , Proteínas Virales/genética , Proteínas Virales/inmunologíaRESUMEN
Its stable particle structure combined with its high immunogenicity makes the hepatitis A virus (HAV) a perfect carrier to expose foreign epitopes to the host immune system. In an earlier report [Beneduce, F., Kusov, Y., Klinger, M., Gauss-Müller, V., Morace, G., 2002. Chimeric hepatitis A virus particles presenting a foreign epitope (HIV gp41) at their surface. Antiviral Res. 55, 369-377] chimeric virus-like particles (HAV-gp41) were described that carried at their surface the dominant gp41 epitope 2F5 (2F5e) of the human immunodeficiency virus HIV-1. Extending this work, we now report that chimeric virus HAV-gp41 replicates in HAV-susceptible cells as well as in non-human primates. Infected marmosets developed both an anti-HAV and anti-2F5 epitope immune response. Furthermore, an HIV-neutralizing antibody response was elicited in guinea pigs immunized with HAV-gp41 chimeric particles. The results demonstrate that the replication-competent chimeric HAV-gp41 can serve as either a live or a subunit vaccine for eliciting of antibodies directed against a foreign antigenic epitope.
Asunto(s)
Proteína gp41 de Envoltorio del VIH/inmunología , VIH-1/inmunología , Virus de la Hepatitis A/inmunología , Vacunas contra el SIDA/inmunología , Vacunas contra el SIDA/farmacología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Callithrix , Línea Celular Tumoral , Epítopos/genética , Epítopos/inmunología , Cobayas , Proteína gp41 de Envoltorio del VIH/genética , VIH-1/genética , Vacunas contra la Hepatitis A/inmunología , Vacunas contra la Hepatitis A/farmacología , Virus de la Hepatitis A/genética , Virus de la Hepatitis A/fisiología , Humanos , Datos de Secuencia Molecular , Replicación ViralRESUMEN
Proteinase 3C of hepatitis A virus (HAV) plays a key role in the viral life cycle by generating mature viral proteins from the precursor polyprotein. In addition to its proteolytic activity, 3C binds to viral RNA, and thus influences viral genome replication. In order to investigate the interplay between proteolytic activity and RNA binding at the molecular level, we subjected HAV 3C and three variants carrying mutations of the cysteine residues [C24S (Cys-24-->Ser), C172A and C24S/C172A] to proteolysis assays with peptide substrates, and to surface plasmon resonance binding studies with peptides and viral RNA. We report that the enzyme readily forms dimers via disulphide bridges involving Cys-24. Dissociation constants (K(D)) for peptides were in the millimolar range. The binding kinetics for the peptides were characterized by k(on) and k(off) values of the order of 10(2) M(-1) x s(-1) and 10(-2) to 10(-1) s(-1) respectively. In contrast, 3C binding to immobilized viral RNA, representing the structure of the 5'-terminal domain, followed fast binding kinetics with k(on) and k(off) values beyond the limits of the kinetic resolution of the technique. The affinity of viral RNA depended strongly on the dimerization status of 3C. Whereas monomeric 3C bound to the viral RNA with a K(D) in the millimolar range, dimeric 3C had a significantly increased binding affinity with K(D) values in the micromolar range. A model of the 3C dimer suggests that spatial proximity of the presumed RNA-binding motifs KFRDI is possible. 3C binding to RNA was also promoted in the presence of substrate peptides, indicating co-operativity between RNA binding and protease activity. The data imply that the dual functions of 3C are mutually dependent, and regulate protein and RNA synthesis during the viral life cycle.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Virus de la Hepatitis A/enzimología , ARN Viral/metabolismo , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/virología , Línea Celular Tumoral , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/genética , Dimerización , Enzimas Inmovilizadas , Virus de la Hepatitis A/genética , Humanos , Hidrólisis , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/virología , Modelos Moleculares , Mutación/genética , Resonancia Magnética Nuclear Biomolecular/métodos , Fragmentos de Péptidos/metabolismo , Unión Proteica/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Especificidad por Sustrato/genéticaRESUMEN
The replication-deficient vaccinia virus (VV) MVA-T7 produces large amounts of T7 RNA polymerase and permits efficient protein expression from cDNA of T7-promoted genes. Yet, unlike recombinant VV vTF7-3, (VV) MVA-T7 produces no cytopathic effect in primate cells, thus allowing the study of processes with slow kinetics. We have applied MVA-T7 to aid genome expression of HAV, a representative of the Picornaviridae family that is well known for its inefficient replication in mammalian cell cultures. After cDNA transfection and MVA-T7 infection, empty capsids and mature HAV particles were formed with different kinetics and were characterized by their morphology, protein content, and infectivity. The data suggests that HAV genome replication is initiated from RNA, which was transcribed in vivo by the MVA-T7-encoded T7 RNA polymerase. HAV genome replication was also demonstrated in a recombination assay. After co-expression of two subgenomic HAV cDNAs, both by themselves unable to complete the viral life cycle, infectious HAV was rescued, indicating that replication-dependent genetic recombination has occurred. We propose that the high-level genome expression mediated in vivo by the VV-encoded T7 RNA polymerase augments the amount of viral RNA, such that replication of viruses poorly replicating in cell cytoplasm is detectable.
Asunto(s)
ADN Complementario/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Virus de la Hepatitis A/fisiología , Virus Vaccinia/genética , Replicación Viral , Bacteriófago T7/genética , ARN Polimerasas Dirigidas por ADN/genética , Vectores Genéticos/genética , Virus de la Hepatitis A/genética , Virus de la Hepatitis A/patogenicidad , Humanos , Microscopía Inmunoelectrónica , ARN Viral/metabolismo , Recombinación Genética , Transfección , Células Tumorales Cultivadas , Virus Vaccinia/metabolismo , Proteínas ViralesRESUMEN
Hepatitis A virus (HAV) protein 2A has been demonstrated to be involved in virus morphogenesis and suggested to be located on the surface of the particle. To determine whether this protein can function as a target structure to harbor and expose foreign epitopes on HAV particles, a full-length HAV cDNA, containing a seven amino acid stretch of human immunodeficiency virus type 1 (HIV-1) envelope protein gp41, was constructed. Following vaccinia virus MVA-T7-mediated expression of the cDNA in COS7 and Huh-T7 cells, chimeric HAV particles, exposing the foreign epitope gp41 on their surface, were produced. These particles were found to be empty capsids (70S), as judged by immunospecific enzyme linked immunosorbent assay (ELISA) on sucrose gradient fractions and immunoelectron microscopy. The immunological detection of VP1-2A harboring the gp41 epitope of HIV suggests that the 2A domain of HAV is suitable to present foreign antigenic epitopes.
Asunto(s)
Antígenos Virales/inmunología , Epítopos/inmunología , Proteína gp41 de Envoltorio del VIH/inmunología , Virus de la Hepatitis A/inmunología , Proteínas Recombinantes de Fusión/inmunología , ADN Complementario , Ensayo de Inmunoadsorción Enzimática , Proteína gp41 de Envoltorio del VIH/genética , Antígenos de Hepatitis A , Virus de la Hepatitis A/genética , Microscopía Inmunoelectrónica , Morfogénesis , Vacunas SintéticasRESUMEN
The initial step during assembly of the hepatitis A virus particle is driven by domain 2A of P1-2A, which is the precursor of the structural proteins. The proteolytic removal of 2A from particulate VP1-2A by an as yet unknown host enzyme presumably terminates viral morphogenesis. Using a genetic approach, we show that a basic amino acid residue at the C-terminus of VP1 is required for efficient particle assembly and that host proteases trypsin and cathepsin L remove 2A from hepatitis A virus particles in vitro. Analyses of insertion mutants in the C-terminus of 2A reveal that this part of 2A is important for liberation of P1-2A from the polyprotein. The data provide the first evidence that the VP1/2A junction is involved in both viral particle assembly and maturation and, therefore, seems to coordinate the first and last steps in viral morphogenesis.
Asunto(s)
Virus de la Hepatitis A/fisiología , Precursores de Proteínas/metabolismo , Proteínas Estructurales Virales/metabolismo , Ensamble de Virus/fisiología , Secuencia de Aminoácidos , Línea Celular , Virus de la Hepatitis A/genética , Virus de la Hepatitis A/ultraestructura , Humanos , Datos de Secuencia Molecular , Mutación , Péptido Hidrolasas/metabolismo , Precursores de Proteínas/genética , Estructura Terciaria de Proteína , Proteínas Estructurales Virales/genética , Virión/metabolismo , Replicación ViralRESUMEN
Infection by hepatitis A virus (HAV) can cause acute hepatitis and, rarely, fulminant liver failure, in particular in patients chronically infected with hepatitis C virus. Based on our previous observation that small interfering RNAs (siRNAs) can silence translation and replication of the firefly luciferase-encoding HAV replicon, we now exploited this technology to demonstrate the effect of siRNAs on viral infection in Huh-7 cells. Freshly and persistently infected cells were transfected with siRNAs targeting various sites in the HAV nonstructural genes. Compared to a single application, consecutive siRNA transfections targeting multiple sequences in the viral genome resulted in a more efficient and sustained silencing effect than a single transfection. In most instances, multiple applications of a single siRNA led to the emergence of viral escape mutants with mutated target sites that rendered these genomes resistant to RNA interference (RNAi). Efficient and sustained suppression of the viral infectivity was achieved after consecutive applications of an siRNA targeting a computer-predicted hairpin structure. This siRNA holds promise as a therapeutic tool for severe courses of HAV infection. In addition, the results provide new insight into the structural bases for sequence-specific RNAi.
Asunto(s)
Silenciador del Gen/efectos de los fármacos , Hepacivirus/efectos de los fármacos , Hepatitis/prevención & control , Interferencia de ARN , ARN Interferente Pequeño/farmacología , ARN Viral/biosíntesis , Línea Celular Tumoral , Hepacivirus/genética , Hepatitis/genética , Hepatitis/metabolismo , Humanos , ARN Interferente Pequeño/química , ARN Interferente Pequeño/genética , ARN Viral/genética , Replicación Viral/efectos de los fármacos , Replicación Viral/genéticaRESUMEN
The precise role of the poly(A) tail at the 3' end of the picornavirus RNA genome and the cellular factors that control its homeostasis are unknown. To assess the importance of the poly(A) tail for virus replication, the genome of the slowly replicating hepatitis A virus (HAV) with and without a poly(A) tail was studied after transfection into cells maintained under various conditions. A tailless HAV genome had a shorter half-life than a poly(A)-containing genome and was unable to replicate in quiescent cells. In dividing cells, the tailless RNA gave rise to infectious virus with a restored poly(A) tail of up to 60 residues. Cells arrested at the G(0) and the G(2)/M phase produced lower amounts of infectious HAV than cells in the G(1) phase. These data suggest that the 3' poly(A) tail of HAV can be restored with the help of a cellular and/or viral function that is regulated during the cell cycle.
Asunto(s)
Genoma Viral , Virus de la Hepatitis A/fisiología , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Replicación Viral , Ciclo Celular/fisiología , Células Cultivadas , Virus de la Hepatitis A/genética , Humanos , PicornaviridaeRESUMEN
Small interfering RNAs (siRNAs) targeting the coding region of hepatitis A virus (HAV) were shown to specifically inhibit viral genome replication. Compared to the coding region, the HAV internal ribosomal entry site (IRES) in the 5' non-coding region is highly sequence-conserved and folds into stable secondary structures. Here, we report efficient and sustained RNA interference mediated by both RNase III-prepared siRNA (esiRNA) and vector-derived short hairpin RNAs (shRNAs) that are targeted to various domains of the HAV IRES. Using reporter constructs, and the DNA-based HAV replicon system, we found that shRNAs targeting the HAV IRES domains IIIc and V sustainably suppressed genome translation and replication whereas the IRES domains IIIa and IV were resistant to RNA interference. Our study suggests that some HAV IRES domains might be used as a universal and effective target for specific inhibition of HAV infection.
Asunto(s)
Virus de la Hepatitis A/efectos de los fármacos , ARN Interferente Pequeño/farmacología , Secuencia de Bases , Vectores Genéticos , Genoma Viral , Virus de la Hepatitis A/genética , Virus de la Hepatitis A/fisiología , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Biosíntesis de Proteínas/efectos de los fármacos , ARN Interferente Pequeño/síntesis química , ARN Interferente Pequeño/genética , ARN Viral/genética , Ribonucleasa III/química , Ribosomas/genética , Proteínas Virales/biosíntesis , Proteínas Virales/genética , Replicación Viral/efectos de los fármacos , Replicación Viral/genéticaRESUMEN
Unlike other picornaviruses, hepatitis A virus (HAV) replicates so inefficiently in cell culture that the study of its RNA biosynthesis presents a major experimental challenge. To assess viral RNA replication independent of particle formation, a subgenomic replicon representing a self-replicating RNA was constructed by replacing the P1 domain encoding the capsid proteins with the firefly luciferase sequence. Although translation of the HAV replicon was as efficient as a similar poliovirus replicon, the luciferase activity derived from replication of the HAV construct was more than 100-fold lower than that of poliovirus. The replication capacity of the HAV replicon was clearly demonstrated by its ability to recombine genetically with a non-viable, full-length HAV genome that served as capsid donor and thus to rescue a fully infectious virus. In contrast to a replication-deficient replicon, co-expression of the genetically marked and replication-competent HAV replicon with several lethally mutated HAV genomes resulted in the successful rescue of infectious HAV with a unique genetic marker. Our data suggest: (i) that autonomous HAV RNA replication does not require sequences for the HAV structural proteins; and (ii) that low-level genome replication can unequivocally be demonstrated by the rescue of infectious virus after co-expression with non-viable genomes.
Asunto(s)
Virus de la Hepatitis A/genética , ARN Viral/biosíntesis , Replicón , Animales , Línea Celular , Genoma Viral , Luciferasas/genética , Recombinación Genética , Proteínas Estructurales Virales/genética , Replicación ViralRESUMEN
The rate of acute liver failure due to hepatitis A virus (HAV) has not decreased, and therapy of severe infections is still of major interest. Using a DNA-based HAV replicon cell culture system, we demonstrate that small interfering RNAs (siRNAs) targeted against viral sequences or a reporter gene contained in the viral genome specifically inhibit HAV RNA replication in HuhT7 cells. Combinations of siRNAs were more effective suppressors of HAV RNA replication. Also, siRNAs targeted against HAV 2C and 3D inhibited the expression of the respective protein. Expressions of endogenous beta-actin and double-stranded-specific RNA-activated serin/threonine kinase (PKR) were unaltered, demonstrating that the siRNA inhibitory effect was not connected to interferon inhibition, but rather was specifically targeted against HAV RNA. These results suggest that RNA interference might ultimately be useful in treatment of severe HAV infection with or without chronic liver diseases.
Asunto(s)
Virus de la Hepatitis A/efectos de los fármacos , ARN Interferente Pequeño/farmacología , Replicación Viral/efectos de los fármacos , Actinas/biosíntesis , Secuencia de Bases , Línea Celular Tumoral , Regulación Viral de la Expresión Génica/efectos de los fármacos , Regulación Viral de la Expresión Génica/genética , Silenciador del Gen , Vectores Genéticos , Virus de la Hepatitis A/fisiología , Humanos , Luciferasas/antagonistas & inhibidores , Luciferasas/genética , Luciferasas/metabolismo , Plásmidos/genética , Proteínas Serina-Treonina Quinasas/biosíntesis , ARN Bicatenario/metabolismo , ARN Interferente Pequeño/genética , ARN Viral/genética , Transfección , Proteínas Virales/antagonistas & inhibidores , Proteínas Virales/biosíntesis , Replicación Viral/genéticaRESUMEN
Among the picornaviridae, hepatitis A virus (HAV) is unique in that its assembly is driven by domain 2A of P1-2A, the precursor of the structural proteins (Probst, C., Jecht, M., and Gauss-Müller, V. (1999) J. Biol. Chem. 274, 4527-4531). Whereas infected individuals excrete in stool mature HAV capsids with VP1 as the major structural protein, its C-terminal extended form VP1-2A is the main component of immature procapsids produced in HAV-infected cells in culture. Obviously, a postassembly proteolytic step is required to remove the primary assembly signal 2A from VP1-2A of procapsids. Mutants of VP1-2A were expressed in COS7 cells to determine the cleavage site in VP1-2A and to test for the cleavage potential of viral and host proteinases (factor Xa and thrombin). Site-specific in vitro cleavage by factor Xa and thrombin occurred in procapsids that contained VP1-2A with engineered cognate cleavage sites for these proteinases. Interestingly, factor Xa but not thrombin liberated mature VP1 also from native procapsids in an assembly-dependent manner. The data show that domain 2A, which is required for pentamerization of its precursor polypeptides and thus for the primary step of HAV assembly, is removed from the surface of immature procapsid by a host proteinase. Moreover, our data open a novel avenue to produce homogeneous HAV particles from recombinant intermediates by in vitro treatment with exogenously added proteases such as factor Xa or thrombin.