RESUMEN
The large protein L of negative-sense RNA viruses is a multifunctional protein involved in transcription and replication of genomic RNA. It also possesses enzymatic activities involved in capping and methylation of viral mRNAs. The pathway for mRNA capping followed by the L protein of the viruses in the Morbillivirus genus has not been established, although it has been speculated that these viruses may follow the unconventional capping pathway as has been shown for some viruses of Rhabdoviridae family. We had earlier shown that the large protein L of Rinderpest virus expressed as recombinant L-P complex in insect cells as well as the ribonucleoprotein complex from purified virus possesses RNA triphosphatase (RTPase) and guanylyltransferase activities, in addition to RNA dependent RNA polymerase activity. In the present work, we demonstrate that RTPase as well as nucleoside triphosphatase (NTPase) activities are exhibited by a subdomain of the L protein in the C terminal region (a.a. 1640-1840). The RTPase activity depends absolutely on a divalent cation, either magnesium or manganese. Both the RTPase and NTPase activities of the protein show dual metal specificity. Two mutant proteins having alanine mutations in the glutamic acid residues in motif-A of the RTPase domain did not show RTPase activity, while exhibiting reduced NTPase activity suggesting overlapping active sites for the two enzymatic functions. The RTPase and NTPase activities of the L subdomain resemble those of the Vaccinia capping enzyme D1 and the baculovirus LEF4 proteins.
Asunto(s)
Ácido Anhídrido Hidrolasas/metabolismo , Caperuzas de ARN , ARN Viral/metabolismo , Virus de la Peste Bovina/química , Proteínas Virales/metabolismo , Ácido Anhídrido Hidrolasas/química , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Proteínas Virales/químicaRESUMEN
The V proteins of paramyxoviruses are composed of two evolutionarily distinct domains, the N-terminal 75â% being common to the viral P, V and W proteins, and not highly conserved between viruses, whilst the remaining 25â% consists of a cysteine-rich V-specific domain, which is conserved across almost all paramyxoviruses. There is evidence supporting a number of different functions of the V proteins of morbilliviruses in blocking the signalling pathways of type I and II IFNs, but it is not clear which domains of V are responsible for which activities and whether all these activities are required for effective blockade of IFN signalling. We have shown here that the two domains of rinderpest virus V protein have distinct functions: the N-terminal domain acted to bind STAT1, whilst the C-terminal V-specific domain interacted with the IFN receptor-associated kinases Jak1 and Tyk2. Effective blockade of IFN signalling required the intact V protein.
Asunto(s)
Interferones/metabolismo , Virus de la Peste Bovina/metabolismo , Peste Bovina/metabolismo , Transducción de Señal , Proteínas Virales/química , Proteínas Virales/metabolismo , Animales , Línea Celular , Humanos , Interferones/genética , Janus Quinasa 1/genética , Janus Quinasa 1/metabolismo , Fosforilación , Estructura Terciaria de Proteína , Peste Bovina/enzimología , Peste Bovina/genética , Peste Bovina/virología , Virus de la Peste Bovina/química , Virus de la Peste Bovina/genética , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/metabolismo , Proteínas Virales/genéticaRESUMEN
The paramyxovirus P protein is an essential component of the transcriptase and replicase complex along with L protein. In this article, we have examined the functional roles of different domains of P proteins of two closely related morbilliviruses, Rinderpest virus (RPV) and Peste des petits ruminants virus (PPRV). The PPRV P protein physically interacts with RPV L as well as RPV N protein when expressed in transfected cells, as shown by co-immunoprecipitation. The heterologous L-P complex is biologically active when tested in a RPV minigenome replication/transcription system, only when used with PPRV N protein but not with RPV N protein. Employing chimeric PPRV/RPV cDNAs having different coding regions of P protein in the minigenome replication/transcription system, we identified a region between 290 and 346 aa in RPV P protein necessary for transcription of the minigenome.
Asunto(s)
Virus de la Peste de los Pequeños Rumiantes/química , Fosfoproteínas/química , Proteínas Recombinantes de Fusión/química , Virus de la Peste Bovina/química , Animales , Línea Celular , Humanos , Proteínas de la Nucleocápside/genética , Proteínas de la Nucleocápside/metabolismo , Virus de la Peste de los Pequeños Rumiantes/genética , Virus de la Peste de los Pequeños Rumiantes/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Estructura Terciaria de Proteína , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Virus de la Peste Bovina/genética , Virus de la Peste Bovina/metabolismo , SpodopteraRESUMEN
The nucleocapsid (N) protein of rinderpest virus (RPV) is highly conserved, immunogenic, and abundantly expressed during infection. Six antigenic sites (sites A, B, C, D, E and F), defined previously by a competitive binding assay using corresponding monoclonal antibodies (Mabs), have been further localized by immunoassays using deleted N mutants. Five different forms of RPV N protein, containing residues aa 1-79, aa 1-149, aa 1-421, aa 414-525 and aa 1-525, were expressed as glutathione S transferase (GST) fusion proteins (designated as GST-N1-79, GST-N1-149, GST-N1-421, GST-N414-525, and GST-N1-525, respectively) in E.coli BL21 cells. In ELISA using deleted N mutants, Mabs recognizing sites A, B, C, D and E reacted with 3 GST fusion proteins (GST-N1-149, GST-N1-421 and GST-N1-525), indicating that they are located at aa 80-149. Mab recognizing site F reacted with 4 GST fusion proteins (GST-N1-79, GST-N1-149, GST-N1-421 and GST-N1-525), indicating that site F is located at aa 1-79. Identification of the amino-terminal antigenic sites of the N protein would provide antigen basis for developing sensitive and specific diagnostic reagents for RPV, although it remains to be further investigated antigenic sites at the carboxyl-terminus.
Asunto(s)
Proteínas de la Nucleocápside/genética , Virus de la Peste Bovina/genética , Proteínas Virales/genética , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Secuencia de Bases , Chlorocebus aethiops , Clonación Molecular , Cartilla de ADN , Escherichia coli/genética , Datos de Secuencia Molecular , Proteínas de la Nucleocápside/análisis , Proteínas de la Nucleocápside/química , Proteínas Recombinantes/química , Virus de la Peste Bovina/química , Virus de la Peste Bovina/aislamiento & purificación , Alineación de Secuencia , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Células Vero , Proteínas Virales/análisis , Proteínas Virales/químicaRESUMEN
A competitive enzyme-linked immunosorbent assay (C-ELISA) which detects antibodies unique to rinderpest virus (RPV) has been developed. This test can differentiate antibodies against RPV and those against peste des petits ruminants virus. The recombinant RPV hemagglutinin (H)-protein C-ELISA (recH C-ELISA) is based on the ability of a well-characterized monoclonal antibody (MAb) produced with the soluble, secreted form of the H protein (Sec H protein) of RPV made in a baculovirus expression system to compete with the binding of RPV antibodies in the serum of vaccinated or infected, recovered animals to the Sec H protein. The B-cell epitope recognized by the MAb corresponds to amino acids 575 to 583 on the H protein, which is not present on the antigenically closely related peste des petits ruminants virus hemagglutinin-neuraminidase protein. Initially, a positive-negative threshold cutoff value for percent inhibition of 34 was established with 500 known RPV-negative serum samples. The recH C-ELISA was developed with the enzyme immunoassay software of a commercial RPV C-ELISA kit. Comparative analysis of the test results for 700 serum samples obtained with the commercial kit gave a sensitivity of 112.4% and a specificity of 72.4%. Variations in percent inhibition values were observed for the two assay systems. These variations may have been due to the undefined amount of antigen present in the commercial kit as well as the use of a different MAb. The recH C-ELISA detected more positive serum samples compared to the number detected by the commercial kit, with the results confirmed by a virus neutralization test. Thus, recH C-ELISA is a sensitive tool for RPV serosurveillance in disease eradication programs.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Glicoproteínas/análisis , Virus de la Peste Bovina/aislamiento & purificación , Proteínas Virales/análisis , Animales , Anticuerpos Monoclonales/inmunología , Bovinos , Células Cultivadas , Glicoproteínas/inmunología , Hemaglutininas/inmunología , Hemaglutininas Virales , Juego de Reactivos para Diagnóstico , Proteínas Recombinantes/inmunología , Virus de la Peste Bovina/química , Virus de la Peste Bovina/inmunología , Spodoptera , Proteínas Virales/inmunologíaRESUMEN
The nucleocapsid (N) protein of rinderpest virus (RPV) is highly conserved, immunogenic, and abundantly expressed during infection. Six antigenic sites (sites A, B, C, D, E and F), defined previously by a competitive binding assay using corresponding monoclonal antibodies (Mabs), have been further localized by immunoassays using deleted N mutants. Five different forms of RPV N protein, containing residues aa 1-79, aa 1-149, aa 1-421, aa 414-525 and aa 1-525, were expressed as glutathione S transferase (GST) fusion proteins (designated as GST-N1-79, GST-N1-149, GST-N1-421, GST-N414-525, and GST-N1-525, respectively) in E.coli BL21 cells. In ELISA using deleted N mutants, Mabs recognizing sites A, B, C, D and E reacted with 3 GST fusion proteins (GST-N1-149, GST-N1-421 and GST-N1-525), indicating that they are located at aa 80-149. Mab recognizing site F reacted with 4 GST fusion proteins (GST-N1-79, GST-N1-149, GST-N1-421 and GST-N1-525), indicating that site F is located at aa 1-79. Identification of the amino-terminal antigenic sites of the N protein would provide antigen basis for developing sensitive and specific diagnostic reagents for RPV, although it remains to be further investigated antigenic sites at the carboxyl-terminus.
Asunto(s)
Animales , Secuencia de Aminoácidos , Anticuerpos Monoclonales , Secuencia de Bases , Chlorocebus aethiops , Clonación Molecular , Cartilla de ADN , Escherichia coli/genética , Datos de Secuencia Molecular , Proteínas de la Nucleocápside/análisis , Proteínas Recombinantes/química , Virus de la Peste Bovina/química , Alineación de Secuencia , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Células Vero , Proteínas Virales/análisisRESUMEN
A major molecular determinant of virus host-range is thought to be the viral protein required for cell attachment. We used a recombinant strain of Rinderpest virus (RPV) to examine the role of this protein in determining the ability of RPV to replicate in rabbits. The recombinant was based on the RBOK vaccine strain, which is avirulent in rabbits, carrying the haemagglutinin (H) protein gene from the lapinized RPV (RPV-L) strain, which is pathogenic in rabbits. The recombinant virus (rRPV-lapH) was rescued from a cDNA of the RBOK strain in which the H gene was replaced with that from the RPV-L strain. The recombinant grew at a rate equivalent to the RPV-RBOK parental virus in B95a cells but at a lower rate than RPV-L. The H gene swap did not affect the ability of the RBOK virus to act as a vaccine to protect cattle against virulent RPV challenge. Rabbits inoculated with RPV-L became feverish, showed a decrease in body weight gain and leukopenia. High virus titres and histopathological lesions in the lymphoid tissues were also observed. Clinical signs of infection were never observed in rabbits inoculated with either RPV-RBOK or with rRPV-lapH; however, unlike RPV-RBOK, both RPV-L and rRPV-lapH induced a marked antibody response in rabbits. Therefore, the H protein plays an important role in allowing infection to occur in rabbits but other viral proteins are clearly required for full RPV pathogenicity to be manifest in this species.
Asunto(s)
Glicoproteínas/fisiología , Hemaglutininas Virales/fisiología , Conejos/virología , Virus de la Peste Bovina , Proteínas Virales/fisiología , Animales , Anticuerpos Antivirales/análisis , Anticuerpos Antivirales/biosíntesis , Bovinos , Línea Celular , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Células Gigantes , Glicoproteínas/genética , Hemaglutininas Virales/genética , Tejido Linfoide/patología , Tejido Linfoide/virología , Necrosis , Recombinación Genética , Peste Bovina/inmunología , Peste Bovina/prevención & control , Virus de la Peste Bovina/química , Virus de la Peste Bovina/patogenicidad , Virus de la Peste Bovina/fisiología , Especificidad de la Especie , Vacunas Sintéticas/administración & dosificación , Proteínas Virales/genética , Vacunas Virales/administración & dosificación , Vacunas Virales/genéticaRESUMEN
The nucleotide sequence of the matrixprotein (M) gene of the lapinized rinderpest virus (RPV-L) was determined. The full-length cDNA of the RPV-L M gene is composed of 1460 base pairs and is supposed to contain an open reading frame of 1005 nucleotides encoding on M protein of 335 amino acids. The homology of the predicted amino acid among congeneric morbilliviruses such as RPV Kabete 'O' strain (wild strain of RPV), RPV RBOK strain (vaccine strain of RPV for cattle), measles virus (MV), and canine distemper virus (CDV), is approximately 94%, 93%, 87% and 77%, respectively. In the present study, all coding regions of the RPV-L strain have been determined.
Asunto(s)
Virus de la Peste Bovina/genética , Proteínas de la Matriz Viral/genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN Viral/química , ADN Viral/genética , Genes Virales , Datos de Secuencia Molecular , Filogenia , ARN Viral/química , ARN Viral/genética , ARN Viral/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Peste Bovina/virología , Virus de la Peste Bovina/química , Homología de Secuencia de Aminoácido , Proteínas de la Matriz Viral/químicaRESUMEN
Haemagglutinin (HA) and fusion (F) proteins of peste-des-petits-ruminants virus (PPRV) and rinderpest virus (RPV) were purified by immunoaffinity chromatography. The purified proteins were characterized by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). Rabbit hyperimmune sera were raised against the purified HA and F proteins and assayed by enzyme-linked immunosorbent assay (ELISA), haemagglutination-inhibition (HAI) and virus neutralization (VN) tests. The immunized animals were challenged with a virulent lapinized (rabbit-adapted) strain of RPV. Both HA and F proteins of PPRV protected rabbits against a lethal challenge with lapinized RPV. As expected, RPV HA and F proteins also conferred a similar protection against the homologous challenge. The postchallenge antibody responses were of a true anamnestic type.
Asunto(s)
Virus de la Peste de los Pequeños Rumiantes/inmunología , Virus de la Peste Bovina/inmunología , Peste Bovina/prevención & control , Proteínas Virales de Fusión/inmunología , Vacunas Virales/inmunología , Animales , Anticuerpos Monoclonales , Anticuerpos Antivirales/sangre , Chlorocebus aethiops , Cromatografía de Afinidad , Femenino , Hemaglutininas Virales/inmunología , Hemaglutininas Virales/aislamiento & purificación , Masculino , Virus de la Peste de los Pequeños Rumiantes/química , Conejos , Peste Bovina/inmunología , Peste Bovina/virología , Virus de la Peste Bovina/química , Factores de Tiempo , Células Vero/virología , Proteínas Virales de Fusión/aislamiento & purificaciónRESUMEN
We have cloned several cDNAs derived from the P gene of rinderpest virus. One of these, derived from a bicistronic N-P mRNA, has been sequenced in its entirety. Sequencing of a section of the others, and comparison with the genome sequence, showed that P gene transcripts, as for other morbilliviruses, were variable; non-templated Gs could be added at a site resembling the normal stop transcription site. Primer extension analysis showed that about half the transcripts were edited. Sequences of the P, C and V proteins encoded by the normal and edited transcripts were compared with those of other morbilliviruses and with those of the more distantly related paramyxoviruses.
Asunto(s)
Genes Virales/genética , Fosfoproteínas/genética , Virus de la Peste Bovina/genética , Proteínas Virales/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Virus del Sarampión/genética , Datos de Secuencia Molecular , Paramyxoviridae/genética , Fosfoproteínas/química , ARN Viral/genética , Virus de la Peste Bovina/química , Homología de Secuencia de Aminoácido , Proteínas Virales/químicaRESUMEN
The peste des petits ruminants (PPR) is proving to be a disease which has an increasingly significant economic impact on a number of countries in Africa and the Middle East, and possibly also on the Indian sub-continent. The antigenic relationships which exist between the PPR and rinderpest viruses pose problems for diagnosis which complicates rinderpest control and eradication programmes. Progress has recently been made in regard to diagnosis (specific nucleic probes and monoclonal antibodies), as well as control (homologous vaccine). International legislation remains to be established and epidemiological surveys should be conducted in order to determine the exact geographical distribution of the disease.