RESUMEN
Infectious bronchitis virus (IBV), a γ-coronavirus, causes the economically important poultry disease infectious bronchitis. Cellular stress response is an effective antiviral strategy that leads to stress granule (SG) formation. Previous studies suggested that SGs were involved in the antiviral activity of host cells to limit viral propagation. Here, we aimed to delineate the molecular mechanisms regulating the SG response to pathogenic IBV strain infection. We found that most chicken embryo kidney (CEK) cells formed no SGs during IBV infection and IBV replication inhibited arsenite-induced SG formation. This inhibition was not caused by changes in the integrity or abundance of SG proteins during infection. IBV nonstructural protein 15 (Nsp15) endoribonuclease activity suppressed SG formation. Regardless of whether Nsp15 was expressed alone, with recombinant viral infection with Newcastle disease virus as a vector, or with EndoU-deficient IBV, the Nsp15 endoribonuclease activity was the main factor inhibiting SG formation. Importantly, uridine-specific endoribonuclease (EndoU)-deficient IBV infection induced colocalization of IBV N protein/dsRNA and SG-associated protein TIA1 in infected cells. Additionally, overexpressing TIA1 in CEK cells suppressed IBV replication and may be a potential antiviral factor for impairing viral replication. These data provide a novel foundation for future investigations of the mechanisms by which coronavirus endoribonuclease activity affects viral replication. IMPORTANCE Endoribonuclease is conserved in coronaviruses and affects viral replication and pathogenicity. Infectious bronchitis virus (IBV), a γ-coronavirus, infects respiratory, renal, and reproductive systems, causing millions of dollars in lost revenue to the poultry industry worldwide annually. Mutating the viral endoribonuclease poly(U) resulted in SG formation, and TIA1 protein colocalized with the viral N protein and dsRNA, thus damaging IBV replication. These results suggest a new antiviral target design strategy for coronaviruses.
Asunto(s)
Infecciones por Coronavirus , Endorribonucleasas , Virus de la Bronquitis Infecciosa , Gránulos de Estrés , Replicación Viral , Animales , Antivirales/farmacología , Embrión de Pollo , Pollos , Infecciones por Coronavirus/veterinaria , Endorribonucleasas/genética , Virus de la Bronquitis Infecciosa/enzimología , Virus de la Bronquitis Infecciosa/fisiología , Enfermedades de las Aves de Corral/virología , ARN BicatenarioRESUMEN
Coronavirus papain-like proteases (PLPs) can act as proteases that process virus-encoded large replicase polyproteins and also as deubiquitinating (DUB) enzymes. Like the PLPs of other coronaviruses (CoVs), the avian infectious bronchitis virus (IBV) PLP catalyzes proteolysis of Gly-Gly dipeptide bonds to release mature cleavage products. However, the other functions of the IBV PLP are not well understood. In this study, we found that IBV exhibits strong global DUB activity with significant reductions of the levels of ubiquitin (Ub)-, K48-, and K63-conjugated proteins. The DUB activity exhibited a clear time dependence, with stronger DUB activity in the early stage of viral infection. Furthermore, the IBV replicase-encoded PLP, including the downstream transmembrane (TM) domain, is a DUB enzyme and dramatically reduced the level of Ub-conjugated proteins, while processing both K48- and K63-linked polyubiquitin chains. By contrast, PLP did not cause any reduction of haemagglutinin (HA)-Ub-conjugated proteins. In addition, mutations of the catalytic residues of PLP-TM, Cys1274Ser and His1437Lys, reduced DUB activity against Ub-, K48- and K63- conjugated proteins, indicating that the DUB activity of the PLP-TM wild-type protein is not completely dependent on its catalytic activity. Overall, these results demonstrate that the IBV-encoded PLP-TM functions as a DUB enzyme and suggest that IBV may interfere with the activation of host antiviral signaling pathway by degrading polyubiquitin-associated proteins.
Asunto(s)
Regulación Enzimológica de la Expresión Génica/fisiología , Regulación Viral de la Expresión Génica/fisiología , Virus de la Bronquitis Infecciosa/enzimología , Péptido Hidrolasas/metabolismo , Proteínas Virales/metabolismo , Animales , Células Cultivadas , Embrión de Pollo , Clonación Molecular , Virus de la Bronquitis Infecciosa/genética , Virus de la Bronquitis Infecciosa/metabolismo , Riñón/citología , Mutagénesis Sitio-Dirigida , Péptido Hidrolasas/genética , Ubiquitinación , Proteínas Virales/genéticaRESUMEN
Papain-like protease (PLpro) of coronaviruses (CoVs) carries out proteolytic maturation of non-structural proteins that play a role in replication of the virus and performs deubiquitination of host cell factors to scuttle antiviral responses. Avian infectious bronchitis virus (IBV), the causative agent of bronchitis in chicken that results in huge economic losses every year in the poultry industry globally, encodes a PLpro. The substrate specificities of this PLpro are not clearly understood. Here, we show that IBV PLpro can degrade Lys(48)- and Lys(63)-linked polyubiquitin chains to monoubiquitin but not linear polyubiquitin. To explain the substrate specificities, we have solved the crystal structure of PLpro from IBV at 2.15-Å resolution. The overall structure is reminiscent of the structure of severe acute respiratory syndrome CoV PLpro. However, unlike the severe acute respiratory syndrome CoV PLpro that lacks blocking loop (BL) 1 of deubiquitinating enzymes, the IBV PLpro has a short BL1-like loop. Access to a conserved catalytic triad consisting of Cys(101), His(264), and Asp(275) is regulated by the flexible BL2. A model of ubiquitin-bound IBV CoV PLpro brings out key differences in substrate binding sites of PLpros. In particular, P3 and P4 subsites as well as residues interacting with the ß-barrel of ubiquitin are different, suggesting different catalytic efficiencies and substrate specificities. We show that IBV PLpro cleaves peptide substrates KKAG-7-amino-4-methylcoumarin and LRGG-7-amino-4-methylcoumarin with different catalytic efficiencies. These results demonstrate that substrate specificities of IBV PLpro are different from other PLpros and that IBV PLpro might target different ubiquitinated host factors to aid the propagation of the virus.
Asunto(s)
Infecciones por Coronavirus/veterinaria , Infecciones por Coronavirus/virología , Virus de la Bronquitis Infecciosa/enzimología , Papaína/química , Poliubiquitina/metabolismo , Enfermedades de las Aves de Corral/virología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Infecciones por Coronavirus/enzimología , Cristalografía por Rayos X , Virus de la Bronquitis Infecciosa/química , Virus de la Bronquitis Infecciosa/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Papaína/metabolismo , Aves de Corral , Enfermedades de las Aves de Corral/enzimología , Conformación Proteica , Alineación de Secuencia , Especificidad por Sustrato , Ubiquitina/metabolismoRESUMEN
Coronavirus (CoV) 3C-like proteinase (3CLpro), located in nonstructural protein 5 (nsp5), processes the replicase polyproteins 1a and 1ab (pp1a and pp1ab) at 11 specific sites to produce 12 mature nonstructural proteins (nsp5 to nsp16). Structural and biochemical studies suggest that a conserved Gln residue at the P1 position is absolutely required for efficient cleavage. Here, we investigate the effects of amino acid substitution at the P1 position of 3CLpro cleavage sites of infectious bronchitis virus (IBV) on the cleavage efficiency and viral replication by in vitro cleavage assays and reverse genetic approaches. Our results demonstrated that a P1-Asn substitution at the nsp4-5/Q2779, nsp5-6/Q3086, nsp7-8/Q3462, nsp8-9/Q3672, and nsp9-10/Q3783 sites, a P1-Glu substitution at the nsp8-9/Q3672 site, and a P1-His substitution at the nsp15-16/Q6327 site were tolerated and allowed recovery of infectious mutant viruses, albeit with variable degrees of growth defects. In contrast, a P1-Asn substitution at the nsp6-7/Q3379, nsp12-13/Q4868, nsp13-14/Q5468, and nsp14-15/Q5989 sites, as well as a P1-Pro substitution at the nsp15-16/Q6327 site, abolished 3CLpro-mediated cleavage at the corresponding position and blocked the recovery of infectious viruses. Analysis of the effects of these lethal mutations on RNA synthesis suggested that processing intermediates, such as the nsp6-7, nsp12-13, nsp13-14, nsp14-15, and nsp15-16 precursors, may function in negative-stranded genomic RNA replication, whereas mature proteins may be required for subgenomic RNA (sgRNA) transcription. More interestingly, a mutant 3CLpro with either a P166S or P166L mutation was selected when an IBV infectious cDNA clone carrying the Q6327N mutation at the nsp15-16 site was introduced into cells. Either of the two mutations was proved to enhance significantly the 3CLpro-mediated cleavage efficiency at the nsp15-16 site with a P1-Asn substitution and compensate for the detrimental effects on recovery of infectious virus.
Asunto(s)
Cisteína Endopeptidasas , Virus de la Bronquitis Infecciosa , Proteínas Virales , Proteasas Virales 3C , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Humanos , Virus de la Bronquitis Infecciosa/enzimología , Virus de la Bronquitis Infecciosa/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Especificidad por Sustrato/genética , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Avian infectious bronchitis virus (IBV) encodes 15 nonstructural proteins (nsps) which play crucial roles in RNA transcription and genome replication. One of them, nsp3, contains an ADRP (adenosine diphosphate-ribose-1'-phosphatase) domain which was revealed in recent studies to have ADP-ribose-1'-monophosphatase (Appr-1'-pase) activity. Appr-1'-pase catalyzes the conversion of ADP-ribose-1'-monophosphate (Appr-1'-p) to ADP-ribose in the tRNA-splicing pathway. The gene segment encoding the IBV nsp3 ADRP domain has been cloned and expressed in Escherichia coli. The protein has been crystallized and the crystals diffracted to 1.8 A resolution. They belonged to space group P1, with unit-cell parameters a = 41.1, b = 43.2, c = 48.9 A, alpha = 78.0, beta = 80.0, gamma = 73.6 degrees . Each asymmetric unit contains two molecules.
Asunto(s)
Aves/virología , ARN Polimerasas Dirigidas por ADN/química , Virus de la Bronquitis Infecciosa/enzimología , Proteínas de la Membrana/química , Monoéster Fosfórico Hidrolasas/química , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/aislamiento & purificación , Adenosina Difosfato Ribosa/análogos & derivados , Adenosina Difosfato Ribosa/química , Animales , Cristalización , Cristalografía por Rayos X , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/aislamiento & purificación , Humanos , Virus de la Bronquitis Infecciosa/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/aislamiento & purificación , Perilipina-2 , Estructura Terciaria de Proteína/genética , Proteínas no Estructurales Virales/genéticaRESUMEN
Infectious bronchitis virus (IBV) is the prototype of the genus Coronavirus. It causes a highly contagious disease which affects the respiratory, reproductive, neurological and renal systems of chickens, resulting great economic losses in the poultry industry worldwide. The coronavirus (CoV) main protease (M(pro)), which plays a pivotal role in viral gene expression and replication through a highly complex cascade involving the proteolytic processing of replicase polyproteins, is an attractive target for antiviral drug design. In this study, IBV M(pro) was overexpressed in Escherichia coli. Crystals suitable for X-ray crystallography have been obtained using microseeding techniques and belong to space group P6(1)22. X-ray diffraction data were collected in-house to 2.7 A resolution from a single crystal. The unit-cell parameters were a = b = 119.1, c = 270.7 A, alpha = beta = 90, gamma = 120 degrees. Three molecules were predicted to be present in the asymmetric unit from a calculated self-rotation function.
Asunto(s)
Cristalografía por Rayos X/métodos , Cisteína Endopeptidasas/química , Virus de la Bronquitis Infecciosa/enzimología , Animales , Proteasas 3C de Coronavirus , Cisteína Endopeptidasas/aislamiento & purificaciónRESUMEN
Previous studies in vitro of the processing of cloned polyprotein fragments from the coronavirus infectious bronchitis virus (IBV) large open reading frame (ORF1), confirmed the activity of a predicted 3C-like proteinase (3CLP) domain and suggested that the proteinase is released autocatalytically from the polyprotein in the form of a 35 kDa protein, 3CLpro, capable of further cleavages in trans. In order to identify such cleavages within the ORF1 polyprotein mediated by 3CLpro, the proteinase was expressed in bacteria, purified and used in trans cleavage assays with polyprotein fragments lacking the 3CLP domain as targets. The proteinase was expressed as a polyprotein fragment which was able to process during expression in bacterial cells, releasing mature 3CLpro. A histidine (His6) tag was introduced close to the C-terminus of the proteinase to aid purification. Processing demonstrated by the tagged proteinase was indistinguishable from that of the wild-type enzyme indicating that the site chosen for the tag was permissive. From these studies we were able to demonstrate trans cleavages consistent with the use of most of the previously predicted or identified sites within the open reading frame of gene 1. This tentatively completes the processing map for the ORF1 region with respect to 3CLpro.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Histamina/genética , Virus de la Bronquitis Infecciosa/enzimología , Animales , Sitios de Unión , Catálisis , Proteasas 3C de Coronavirus , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/genética , Escherichia coli/genética , Regulación Enzimológica de la Expresión Génica , Histamina/química , Virus de la Bronquitis Infecciosa/genética , Mutagénesis Sitio-Dirigida , Procesamiento Proteico-Postraduccional , Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Coronavirus IBV encodes a piconarvirus 3C-like proteinase. In a previous report, this proteinase was shown to undergo rapid degradation in vitro in reticulocyte lysate due to a posttranslational event involving ubiquitination of the protein. Several lines of evidence presented here indicate that the proteinase itself is stable. Translation of the IBV sequence from nucleotide 8864 to 9787 resulted in the synthesis of a 33 kDa protein, representing the full-length 3C-like proteinase. Pulse-chase and time-course experiments showed that this protein was stable in reticulocyte lysate for up to 2 hours. However, a 45 kDa protein encoded by the IBV sequence from nucleotide 8693 to 9911 underwent rapid degradation in reticulocyte lysate, but was stable in wheat germ extract, suggesting that an ATP-dependent protein degradation pathway may be involved in the turnover of the 45 kDa protein. To identify the IBV sequence responsible for the instability of this 45 kDa protein species, the region from nucleotide 8693 to 9787 was translated both in vitro and in vivo, leading to the synthesis of a stable 43 kDa protein. These results suggest that a destabilising signal may be located in the IBV sequences between the nucleotides 9787 and 9911. Meanwhile, protein aggregation was observed when the product encoded by the IBV sequence from nucleotide 9911 to 10,510 was boiled for 5 minutes before being analysed in SDS-PAGE; when the same product was treated at 37 degrees C for 15 minutes, however, protein aggregation was not detected. Deletion studies indicate that the presence of a hydrophobic domain downstream of the 3C-like proteinase-encoding region may be the cause for the aggregation of the product encoded by this region of ORF 1a.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Virus de la Bronquitis Infecciosa/enzimología , Proteínas Virales/metabolismo , Animales , Chlorocebus aethiops , Proteasas 3C de Coronavirus , Cisteína Endopeptidasas/genética , Expresión Génica , Virus de la Bronquitis Infecciosa/genética , Conejos , Células Vero , Proteínas Virales/genéticaRESUMEN
Our previous studies have shown that two overlapping papain-like proteinase domains (PLPDs) encoded by the IBV sequence from nucleotides 4155 to 5550 is responsible for cleavage of the ORF 1a polyprotein to an 87 kDa protein. In this study, we demonstrate that only the more 5' one of the two domains, PLPD-1 encoded between nucleotides 4155 and 5031, is required for processing to the 87 kDa protein. Site-directed mutagenesis studies have shown that the Cys1274 and His1435 residues are essential for the PLPD-1 activity, suggesting that they may be the components of the catalytic centre of this proteinase. Coexpression and immunoprecipitation studies have further revealed that PLPD can interact with the 87 kDa protein. Meanwhile, data obtained from the construction and expression of a series of deletion mutants have indicated that the 87 kDa protein is encoded by the 5'-most 2600 bp part of ORF1a. further deletion and mutagenesis studies are underway to determine precisely the C-terminal cleavage site of the 87 kDa protein.
Asunto(s)
Virus de la Bronquitis Infecciosa/enzimología , Papaína/metabolismo , Precursores de Proteínas/metabolismo , Proteínas Virales/metabolismo , Animales , Sitios de Unión , Catálisis , Proteasas Similares a la Papaína de Coronavirus , Mutagénesis Sitio-Dirigida , Papaína/genéticaRESUMEN
We present here evidence demonstrating that four previously predicted Q-S(G) cleavage sites, encoded by the IBV sequences from nucleotide 15,129 to 15,134, 16,929 to 16,934, 18,492 to 18,497, and 19,506 to 19,511, respectively, can be recognised and transcleaved by the 3C-like proteinase. Five mature products with sizes of approximately 100 kDa, 65 kDa, 63 kDa, 42 kDa and 35 kDa are released from the ORF1b polyprotein by the 3C-like proteinase-mediated cleavage at these positions. Meanwhile, expression of plasmids containing only the ORF1b region showed no autocleavage of the polyprotein encoded, suggesting that the 3C-like proteinase may be the sole proteinase involved in processing of the 1b polyprotein. These data may therefore represent a complete processing map of the polyprotein encoded by ORF1b of mRNA1.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Virus de la Bronquitis Infecciosa/enzimología , Procesamiento Proteico-Postraduccional , Proteínas/metabolismo , Proteínas Virales/metabolismo , Anticuerpos/metabolismo , Sitios de Unión , Proteasas 3C de Coronavirus , Cisteína Endopeptidasas/genética , Endopeptidasas , Células Eucariotas , Expresión Génica , Virus de la Bronquitis Infecciosa/genética , Metales/metabolismo , Proteínas/inmunología , ARN Helicasas/metabolismoAsunto(s)
ARN Polimerasas Dirigidas por ADN/genética , Genes Virales , Virus de la Bronquitis Infecciosa/genética , Sistemas de Lectura Abierta , Animales , Secuencia de Bases , Virus de la Bronquitis Infecciosa/enzimología , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación de Ácido Nucleico , Biosíntesis de Proteínas , ARN Mensajero/genética , Ribosomas/metabolismoAsunto(s)
Cisteína Endopeptidasas/metabolismo , Virus de la Bronquitis Infecciosa/enzimología , Proteínas Virales/metabolismo , Animales , Células COS/virología , Chlorocebus aethiops , Proteasas 3C de Coronavirus , Cisteína Endopeptidasas/genética , Virus de la Bronquitis Infecciosa/metabolismo , Células Vero/virología , Proteínas Virales/genéticaAsunto(s)
Virus de la Bronquitis Infecciosa/enzimología , Virus de la Bronquitis Infecciosa/genética , Sistemas de Lectura Abierta , Serina Endopeptidasas/biosíntesis , Proteínas Virales/biosíntesis , Secuencia de Aminoácidos , Animales , Chlorocebus aethiops , Análisis Mutacional de ADN , Electroforesis en Gel de Poliacrilamida , Expresión Génica , Datos de Secuencia Molecular , Peso Molecular , Plásmidos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Eliminación de Secuencia , Serina Endopeptidasas/química , Serina Endopeptidasas/aislamiento & purificación , Transfección , Tripsina , Células Vero , Proteínas Virales/química , Proteínas Virales/aislamiento & purificaciónRESUMEN
We have previously demonstrated that the replacement of the S gene from an avirulent strain (Beaudette) of infectious bronchitis virus (IBV) with an S gene from a virulent strain (M41) resulted in a recombinant virus (BeauR-M41(S)) with the in vitro cell tropism of the virulent virus but that was still avirulent. In order to investigate whether any of the other structural or accessory genes played a role in pathogenicity we have now replaced these from the Beaudette strain with those from M41. The recombinant IBV was in effect a chimaeric virus with the replicase gene derived from Beaudette and the rest of the genome from M41. This demonstrated that it is possible to exchange a large region of the IBV genome, approximately 8.4 kb, using our transient dominant selection method. Recovery of a viable recombinant IBV also demonstrated that it is possible to interchange a complete replicase gene as we had in effect replaced the M41 replicase gene with the Beaudette derived gene. Analysis of the chimaeric virus showed that it was avirulent indicating that none of the structural or accessory genes derived from a virulent isolate of IBV were able to restore virulence and that therefore, the loss of virulence associated with the Beaudette strain resides in the replicase gene.
Asunto(s)
Virus de la Bronquitis Infecciosa/enzimología , Virus de la Bronquitis Infecciosa/genética , Riñón/virología , Glicoproteínas de Membrana/química , ARN Polimerasa Dependiente del ARN/genética , Proteínas del Envoltorio Viral/química , Animales , Línea Celular , Pollos , Chlorocebus aethiops , Cricetinae , Genoma Viral , Glicoproteínas de Membrana/metabolismo , Modelos Genéticos , Enfermedades de las Aves de Corral/genética , Enfermedades de las Aves de Corral/virología , Tráquea/virología , Células Vero , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Ensamble de Virus/genéticaRESUMEN
Infectious bronchitis virus (IBV) produces six subgenomic (sg) mRNAs, each containing a 64 nucleotide (nt) leader sequence, derived from the 5' end of the genome by a discontinuous process. Several putative functional domains such as a papain-like proteinase (PL(pro)), main protease (M(pro)), RNA-dependent RNA polymerase (RdRp), and RNA helicase encoded by the replicase gene are important for virus replication. We have sequenced four regions of the replicase genes corresponding to the 5'-terminal sequence, PL(pro), M(pro), and RdRp domains from 20 heterologous IBV strains, and compared them with previously published coronavirus sequences. All the coronavirus 5'-termini and PL(pro) domains were divergent, unlike the M(pro) and the RdRp domains that were highly conserved with 28% and 48% conserved residues, respectively. Among IBV strains, the 5' untranslated region including the leader sequence was highly conserved (>94% identical); whereas, the N-terminal coding region and the PL(pro) domains were highly variable ranging from 84.6% to 100%, and 77.6% to 100% identity, respectively. The IBV M(pro) and RdRp domains were highly conserved with 82.7% and 92.7% conserved residues, respectively. The BJ strain was the most different from other IBVs in all four regions of the replicase. Phylogeny-based clustering based on replicase genes was identical to the antigen-based classification of coronaviruses into three groups. However, the IBV strain classification based on replicase gene domains did not correlate with that of the type-specific antigenic groups. The replicase gene sequences of many IBVs recovered from infected chickens were identical to those of vaccine viruses irrespective of serotype, suggesting that either there has been an exchange of genetic material among vaccine and field isolates or that there is a convergent evolution to a specific replicase genotype. There was no correlation between the genotype of any region of the replicase gene and pathotype, suggesting that the replicase is not the sole determinant of IBV pathogenicity.
Asunto(s)
Virus de la Bronquitis Infecciosa/genética , ARN Polimerasa Dependiente del ARN/genética , Regiones no Traducidas 5'/química , Secuencia de Bases , Virus de la Bronquitis Infecciosa/enzimología , Virus de la Bronquitis Infecciosa/patogenicidad , Datos de Secuencia Molecular , FilogeniaRESUMEN
We have previously reported that the 3C-like proteinase of the coronavirus infectious bronchitis virus (IBV) is responsible for processing of the 1a and 1a/1b polyproteins to three mature products of 24, 10, and 100 kDa (Liu et al., 1994, 1997; Ng and Liu, 1998). The C-terminal cleavage site of the 100-kDa protein was defined to be the Q891(1b)-S892(1b) dipeptide bond encoded by nucleotides 15,129 to 15,134 (Liu and Brown, 1995). In this report, other cleavage sites of the 3C-like proteinase in the polyprotein encoded by the ORF 1b region were mapped by coexpression, deletion, and site-directed mutagenesis studies. Using two ORF 1b-specific antisera, V58 and V17, three more Q-S(G) dipeptide bonds, encoded by nucleotides 16,929 to 16,934, 18,492 to 18,497, and 19,506 to 19,511, respectively, were demonstrated to be the cleavage sites of the 3C-like proteinase. Cleavage at these four positions would result in the release of four mature products with molecular masses of approximately 68, 58, 39, and 35 kDa. Among them, the 39- and 35-kDa proteins were specifically identified in IBV-infected cells. Taken together with the 100-kDa protein previously identified, these results suggest that the ORF 1b region of IBV mRNA1 may be able to encode five mature products.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Virus de la Bronquitis Infecciosa/enzimología , Procesamiento Proteico-Postraduccional , Proteínas Virales/metabolismo , Animales , Sitios de Unión , Chlorocebus aethiops , Proteasas 3C de Coronavirus , Mutagénesis Sitio-Dirigida , Sistemas de Lectura Abierta , Precursores de Proteínas/metabolismo , Células VeroRESUMEN
A region of the infectious bronchitis virus (IBV) genome between nucleotide positions 8693 and 10927 which encodes the predicted 3C-like proteinase (3CLP) domain and several potential cleavage sites has been clones into a T7 transcription vector. In vitro translation of synthetic transcripts generated from this plasmid was not accompanied by detectable processing activity of the nascent polypeptide unless the translation was carried out in the presence of microsomal membrane preparations. The processed products so obtained closely resembled in size those expected from cleavage at predicted glutamine-serine (Q/S) dipeptides and included a protein with a size of 35 kDa (p35) that corresponds to the predicted size of 3CLP. Efficient processing was dependent on the presence of membranes during translation; processing was found to occur when microsomes were added posttranslationally, but only after extended periods of incubation. C-terminal deletion analysis of the encoded polyprotein fragment revealed that cleavage activity was dependent on the presence of most but not all of the downstream and adjacent hydrophobic region MP2. Dysfunctional mutagenesis of the putative active-site cysteine residue of 3CLP to either serine or alanine resulted in polypeptides that were impaired for processing, while mutagenesis at the predicted Q/S release sites implicated them in the release of the p35 protein. Processed products of the wild-type protein were active in trans cleavage assays, which were used to demonstrate that the IBV 3CLP is sensitive to inhibition by both serine and cysteine protease class-specific inhibitors. These data reveal the identity of the IBV 3C-like proteinase, which exhibits characteristics in common with the 3C proteinases of picornaviruses.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Virus de la Bronquitis Infecciosa/enzimología , Animales , Sitios de Unión , Catálisis , Clonación Molecular , Proteasas 3C de Coronavirus , Cisteína/metabolismo , Cisteína Endopeptidasas/genética , Dipéptidos/metabolismo , Virus de la Bronquitis Infecciosa/genética , Mutagénesis Sitio-Dirigida , Procesamiento Proteico-PostraduccionalRESUMEN
In order to investigate the mechanisms involved in the processing of infectious bronchitis virus polyproteins, several candidate regions of the genome have been cloned and expressed in vitro. During these studies it was observed that the translation product encoded by one of these clones (pKT205) was poorly expressed. Biochemical and genetic analyses revealed that the basis for the poor expression was a post-translational event involving ubiquitination of the protein and degradation by an ATP-dependent system operating in the reticulocyte lysate used for the in vitro expression. Two independently acting regions which conferred instability were identified, one of which mapped to the predicted 3C protease domain, contained within the 5' end of the clone, while the other, more C-terminal region, was effective in conferring instability upon a heterologous protein to which it had been transferred. These regions may influence the stability of the authentic viral protein(s) in vivo and hence allow for the control of their expression and/or function at the level of proteolysis by cellular protease(s).