RESUMEN
The Sendai virus C proteins, C', C, Y1, and Y2, are a nested set of four independently initiated carboxy-coterminal proteins encoded on the P mRNA from an alternate reading frame. Together the C proteins have been shown to inhibit viral transcription and replication in vivo and in vitro and C' binds the Sendai virus L protein, the presumed catalytic subunit of the viral RNA polymerase. To identify amino acids within the C' protein that are important for binding L, site-directed mutagenesis of the gstC' gene was used to change conserved charged amino acids to alanine, generating nine mutants. Additionally, a tenth natural mutant, gstF170S, was also constructed. Six of the gstC' mutants, primarily in the C-terminal half of C', exhibited a defect in the ability to bind L protein. The mutants were assayed for their effect on in vitro transcription and replication from the antigenomic promoter, and the data suggest in all but one case a direct correlation between the ability of C to bind L and to inhibit these steps in RNA synthesis. Further studies with two nonfusion C mutants showed that this correlation was specifically due to the C' portion, and not the gst portion, of the fusion proteins. To study their individual functions, each of the four C proteins was fused downstream of glutathione S-transferase. The gstC', gstC, gstY1, and gstY1 fusion proteins were all able to bind L protein and to inhibit viral mRNA and (+)-leader RNA synthesis, and antigenome replication in vitro. In addition, the nonfusion C, Y1, and Y2 proteins all inhibited transcription. The inhibition of (+)-leader RNA and mRNA synthesis by wt C proteins (nonfusion) showed nearly identical dose-response curves, suggesting that inhibition occurs early in RNA synthesis.
Asunto(s)
ARN Viral/genética , Respirovirus/genética , Respirovirus/metabolismo , Transcripción Genética , Proteínas Virales/metabolismo , Alanina , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Secuencia de Bases , Sitios de Unión , Humanos , Neoplasias Pulmonares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Sondas de Oligonucleótidos , Unión Proteica , ARN Viral/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transfección , Células Tumorales Cultivadas , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
The measles virus RNA-dependent RNA polymerase consists of two virus-encoded subunits, the phosphoprotein (P) and the large (L) protein. The P mRNA also codes for a C protein in the +1 reading frame relative to P. The activities of the measles P and C proteins from the vaccine strain, EdB, a wild-type CM strain, and an SSPE P4 strain were investigated using a CAT reporter minigenome assay. CAT is synthesized following replication and transcription of a DI-CAT minigenome supported by individual P, L, and N plasmids expressed in a mammalian expression system. As measured by CAT activity, CMP1 and P4P1 stimulate transcription and replication four- to six- and six- to eightfold, respectively, better than EdP. There are 10 and 16 amino acid changes in the P protein and three and four changes in C in CMP1 and P4P1, respectively, relative to EdP. By constructing chimeric P genes we showed that mutations throughout P4P1 were required for enhanced polymerase activity, while only mutations in the 5'-terminal portion, encompassing the C ORF, of the CMP1 gene mediated stimulation. Abrogation of C expression from the Ed and CM P genes resulted in an increase in RNA synthesis of twofold for CMP1S and four- to fivefold for EdPS. With the addition of C protein expressed from a separate plasmid that contains only the C ORF, EdC reduces viral RNA synthesis more strongly than CMC. These data suggest that EdC and CMC proteins give a differential inhibition that accounts for most of the differences in RNA synthesis by EdP and CMP1.
Asunto(s)
Proteínas Portadoras/genética , Virus del Sarampión/metabolismo , ARN Viral/biosíntesis , Línea Celular , Cloranfenicol O-Acetiltransferasa/metabolismo , Vacuna Antisarampión , Virus del Sarampión/química , Virus del Sarampión/genética , Mutación , Proteínas de la Nucleocápside/genética , Fosfoproteínas/genética , Recombinación Genética , Panencefalitis Esclerosante Subaguda/virología , Transcripción Genética , Regulación hacia ArribaRESUMEN
The Sendai virus L and P proteins comprise the viral RNA-dependent RNA polymerase. The L subunit is thought to be responsible for all the catalytic activities necessary for viral RNA synthesis. Sequence alignment of the L proteins of negative-stranded RNA viruses revealed six regions of good conservation, domains I-VI, which are thought to correspond to functional domains of the protein. Domain V, amino acids 1129-1378, has no recognizable motifs, and to date its function is unknown. Site-directed mutagenesis was used to construct mutations across domain V. The mutant L proteins were all stably expressed and were tested for activity in several aspects of RNA synthesis. One set of mutants could synthesize more le+ RNA than mRNA, while two mutants showed the opposite phenotype, synthesizing more mRNA than le+ RNA. The majority of the mutants could synthesize mRNA, but not genome RNA in vitro, thus uncoupling transcription and replication. Several mutants could replicate in vivo, but not in vitro, at nearly wildtype L levels, suggesting the importance of the intact host cell for replication in some instances. One L mutant, SS24, was virtually inactive in all viral RNA synthesis. SS24 L was able to form a polymerase complex that recognized the nucleocapsid template, and thus these amino acids are essential for the initiation of RNA synthesis.
Asunto(s)
ARN Polimerasas Dirigidas por ADN , Fosfoproteínas/genética , ARN Polimerasa Dependiente del ARN/genética , Respirovirus/metabolismo , Proteínas Virales/genética , Humanos , Mutagénesis Sitio-Dirigida , Fosfoproteínas/metabolismo , ARN Mensajero/biosíntesis , ARN Viral/biosíntesis , ARN Polimerasa Dependiente del ARN/metabolismo , Respirovirus/genética , Transcripción Genética , Células Tumorales Cultivadas , Proteínas Virales/metabolismo , Replicación ViralRESUMEN
The L subunit of the RNA-dependent RNA polymerase of negative strand RNA viruses is believed to possess all the enzymatic activities necessary for viral transcription and replication. Mutations in the L proteins of human parainfluenza virus type 3 (PIV3) and vesicular stomatitis virus (VSV) have been shown to confer temperature sensitivity to the viruses; however, their specific defects have not been determined. Mutant PIV3 L proteins expressed from plasmids were tested for temperature sensitivity in transcription and replication in a minigenome reporter system in cells and for in vitro transcription from purified PIV3 template. The single L mutants, Y942H and L992F, were temperature sensitive (ts) in both assays, although viral RNA synthesis was not completely abolished at the nonpermissive temperature. Surprisingly, the T1558I L mutant was not ts, although its cognate virus was ts. Thus the ts defect in this virus may be due to the abrogation of an essential interaction of the mutant polymerase with a host cell component, which is not measured by the RNA synthesis assays. Most of the combinations of the PIV3 L mutations were not additive and did not show temperature sensitivity in in vitro transcription. Since they were ts in the minigenome assay in vivo, replication appears to be specifically defective. The ts mutations in PIV3 and VSV L proteins were also substituted into the Sendai L protein to compare the defects in related systems. Only Sendai Y942H L was ts in both transcription and replication. One Sendai L mutant, L992F, gave much better replication than transcription. Several other mutants could transcribe but not replicate in vitro, while replication in vivo was normal.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/fisiología , Mutación , Virus de la Parainfluenza 3 Humana/enzimología , Respirovirus/enzimología , Secuencia de Aminoácidos , Células Cultivadas , Humanos , Datos de Secuencia Molecular , Subunidades de Proteína , ARN Viral/biosíntesis , Temperatura , Transcripción GenéticaRESUMEN
The Sendai virus RNA polymerase is a complex of two virus-encoded proteins, the phosphoprotein (P) and the large (L) protein. When aligned with amino acid sequences of L proteins from other negative-sense RNA viruses, the Sendai L protein contains six regions of good conservation, designated domains I-VI, which have been postulated to be important for the various enzymatic activities of the polymerase. To directly address the roles of domains IV and VI, 14 site-directed mutations were constructed either by changing clustered charged amino acids to ala or by substituting selected Sendai L amino acids with the corresponding sequence from measles virus L. Each mutant L protein was tested for its ability to transcribe and replicate the Sendai genome. The series of mutations created a spectrum of phenotypes, from those with significant, near wild-type, activity to those being completely defective for all RNA synthesis. The inactive L proteins, however, were still able to bind P protein and form a polymerase capable of binding the nucleocapsid template. The remainder of the mutations reduced, but did not abolish, enzymatic activity and included one mutant with a specific defect in the synthesis of the leader RNA compared with mRNA, and three mutants that replicated genome RNA much more efficiently in vivo than in vitro. Together, these data suggest that even within a domain, the function of the Sendai L protein is likely to be very complex. In addition, SS3 and SS10 L in domain IV and SS13 L in domain VI were shown to be temperature-sensitive. Both SS3 and SS10 gave significant, although not wild-type, activity at 32 degrees C; however, each was completely inactivated for all RNA synthesis at 37 and 39.6 degrees C. SS13 was completely inactive only when synthesized at the higher temperature. Each polymerase synthesized at 32 degrees C could only be partially heat inactivated in vitro at 39.6 degrees C, suggesting that inactivation involves both thermal lability of the protein and temperature sensitivity for its synthesis.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/genética , Mutación , Virus de la Parainfluenza 1 Humana/genética , ARN Viral/biosíntesis , Proteínas Virales/genética , Secuencia de Aminoácidos , Secuencia Conservada , ARN Polimerasas Dirigidas por ADN/química , Electroforesis en Gel de Poliacrilamida , Datos de Secuencia Molecular , Fenotipo , ARN Mensajero/biosíntesis , TemperaturaRESUMEN
Functional analysis of the primary constitutive phosphorylation of Sendai virus P and V proteins was performed using both in vitro and in vivo systems. Sendai virus minigenome transcription and replication in transfected cells were not significantly affected in the presence of primary phosphorylation deficient P protein (S249A, S249D, P250A) as measured by either the luciferase activity or the Northern blot analysis. Similarly, recombinant Sendai viruses lacking the primary phosphorylation in P grew to titers close to the wild-type virus in cell cultures and in the natural host of Sendai virus, the mouse. Mutant viruses showed no altered pathogenesis in mice lungs. Oligomerization of P by binding WT P or mutant P to GST-P (WT) Sepharose beads revealed that the primary phosphorylation was not crucial for P protein oligomerization. Similar to P protein primary phosphorylation, the V protein primary phosphorylation at serine249 was not essential for minigenome transcription and replication, as both WT and mutant V proteins were found equally inhibitory to the minigenome replication. These results show that the primary phosphorylation of P protein has no essential role in Sendai virus transcription, replication, and pathogenesis.
Asunto(s)
Fosfoproteínas/metabolismo , Infecciones por Respirovirus/virología , Respirovirus/fisiología , Proteínas Virales/metabolismo , Replicación Viral , Animales , Northern Blotting , Línea Celular , Genoma Viral , Humanos , Pulmón/virología , Enfermedades Pulmonares/virología , Masculino , Ratones , Ratones Endogámicos ICR , Fosforilación , ARN Viral/biosíntesis , Respirovirus/genética , Infecciones por Respirovirus/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética , Células Tumorales CultivadasRESUMEN
The large (L) protein of Sendai virus complexes with the phosphoprotein (P) to form the active RNA-dependent RNA polymerase. The L protein is believed to be responsible for all of the catalytic activities of the polymerase associated with transcription and replication. Sequence alignment of the L proteins of negative-strand RNA viruses has revealed six conserved domains (I-VI) thought to be responsible for the enzymatic activities. Charged-to-alanine mutagenesis was carried out in a highly charged, conserved region (amino acids 533-569) within domain II to test the hypothesis of Müller et al. [J. Gen. Virol. 75, 1345-1352 (1994)] that this region may contribute to the template binding domain of the viral RNA polymerase. The mutant proteins were tested for expression and stability, the ability to synthesize viral RNA in vitro and in vivo, and protein-protein interactions. Five of the seven mutants were completely defective in all viral RNA synthesis, whereas two mutants showed significant levels of both mRNA and leader RNA synthesis. One of the transcriptionally active mutants also gave genome replication in vitro although not in vivo. The other mutant was defective in all the replication assays and thus the mutation uncoupled transcription and replication. Because the completely inactive L mutants can bind to the P protein to form the polymerase complex and the polymerases bind to the viral nucleocapsid template, these amino acids are essential for the activity of the L protein.
Asunto(s)
Secuencia Conservada/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Mutación , ARN Viral/biosíntesis , Respirovirus/enzimología , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Secuencia de Bases , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/genética , Genoma Viral , Humanos , Datos de Secuencia Molecular , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Unión Proteica , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Viral/genética , Respirovirus/genética , Moldes Genéticos , Transcripción Genética/genética , Transfección , Células Tumorales Cultivadas , Proteínas Virales/química , Proteínas Virales/genética , Replicación Viral/genéticaRESUMEN
The Sendai virus P protein is an essential component of the viral RNA polymerase (P-L complex) required for RNA synthesis. To identify amino acids important for P-L binding, site-directed mutagenesis of the P gene changed 17 charged amino acids, singly or in groups, and two serines to alanine within the L binding domain from amino acids 408 to 479. Each of the 10 mutants was wild type for P-L and P-P protein interactions and for binding of the P-L complex to the nucleocapsid template, yet six showed a significant inhibition of in vitro mRNA and leader RNA synthesis. To determine if binding was instead hydrophobic in nature, five conserved hydrophobic amino acids in this region were also mutated. Each of these P mutants also retained the ability to bind to L, to itself, and to the template, but two gave a severe decrease in mRNA and leader RNA synthesis. Since all of the mutants still bound L, the data suggest that L binding occurs on a surface of P with a complex tertiary structure. Wild-type biological activity could be restored for defective polymerase complexes containing two P mutants by the addition of wild-type P protein alone, while the activity of two others could not be rescued. Gradient sedimentation analyses showed that rescue was not due to exchange of the wild-type and mutant P proteins within the P-L complex. Mutants which gave a defective RNA synthesis phenotype and could not be rescued by P establish an as-yet-unknown role for P within the polymerase complex, while the mutants which could be rescued define regions required for a P protein function independent of polymerase function.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/fisiología , Fosfoproteínas/fisiología , Respirovirus/metabolismo , Transcripción Genética , Proteínas Virales/metabolismo , Proteínas Virales/fisiología , Animales , Embrión de Pollo , Chlorocebus aethiops , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Mutagénesis , Nucleocápside/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Respirovirus/genética , Células Vero , Proteínas Virales/genéticaRESUMEN
Alanine substitution mutations in the Sendai virus nucleocapsid (NP) protein have defined highly conserved hydrophobic and charged residues from amino acids (aa) 362 to 371 that are essential for function of the protein in RNA replication. Mutant NP362, which had the change F362A, was incapable of supporting in vitro RNA replication. NP362 expressed alone formed extended oligomers which exhibited an abnormal morphology and density suggesting that these particles were not associated with any RNA. Mutant NP364, which had changes L362A and G365A, was also inactive in RNA replication; however, this was because the protein was unstable and did not form NP-NP complexes. Mutant NP370 mutant, which had changes K370A and D371A, was inactive in in vitro replication, although it could form the required NP0-P and NP-NP protein complexes. The self-assembled nucleocapsid-like particles formed by NP370 alone had a morphology like that of wild-type NP and banded in CsCl as ribonucleoprotein particles, suggesting that they contained cellular RNA. These data suggest that the replication defect of NP370 may be in the ability to specifically encapsidate Sendai virus genome RNA. Mutant NP373, where nonconserved charged residues at aa 373 and 375 were substituted with alanine, gave a wild-type phenotype. Thus these amino acids are not required for either protein-protein interactions or in vitro Sendai virus RNA replication.
Asunto(s)
Genoma Viral , Nucleoproteínas , ARN Viral/metabolismo , Respirovirus/genética , Proteínas del Núcleo Viral/metabolismo , Replicación Viral , Secuencia de Aminoácidos , Centrifugación por Gradiente de Densidad , Microscopía Electrónica , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas de la Nucleocápside , ARN Viral/genética , Respirovirus/química , Respirovirus/metabolismo , Proteínas del Núcleo Viral/química , Proteínas del Núcleo Viral/genéticaRESUMEN
Interferons and chemokines play a critical role in regulating the host response to viral infection. Measles virus, a member of the Paramyxoviridae family, induces RANTES expression by astrocytes. We have examined the mechanism of this induction in U373 cells derived from a human astrocytoma. RANTES was induced in a dose- and time-dependent manner by measles virus infection. Inhibition of receptor binding by the anti-CD46 antibody TRA-2.10 and of virus-membrane fusion by the tripeptide X-Phe-Phe-Gly reduced RANTES expression. Formalin-inactivated virus, which can bind but not fuse, and extensively UV-irradiated virus, which can bind and fuse, were both ineffective. Therefore, virus binding to the cellular receptor CD46 and subsequent membrane fusion were necessary, but not sufficient, to induce RANTES. UV irradiation of virus for less than 10 min proportionally inhibited viral transcription and RANTES expression. RANTES induction was decreased in infected cells treated with ribavirin, which inhibits measles virus transcription. However, RANTES mRNA was superinduced by measles virus in the presence of cycloheximide. These data suggest that partial transcription of the viral genome is sufficient and necessary for RANTES induction, whereas viral protein synthesis and replication are not required. This hypothesis was supported by the fact that RANTES was induced through transient expression of the measles virus nucleocapsid gene but not by measles genes encoding P or L proteins or by leader RNA in A549 cells. Thus, transcription of specific portions of measles virus RNA, such as the nucleocapsid gene, appears able to generate the specific signaling required to induce RANTES gene expression.
Asunto(s)
Astrocitoma/virología , Quimiocina CCL5/biosíntesis , Regulación Viral de la Expresión Génica/inmunología , Virus del Sarampión/crecimiento & desarrollo , Activación Viral/inmunología , Astrocitoma/inmunología , Quimiocina CCL5/inmunología , Humanos , Transducción de Señal , Células Tumorales CultivadasRESUMEN
The Sendai virus nested set of C proteins which are expressed in an alternative open reading frame from the P mRNA has been shown to downregulate viral RNA synthesis. Utilizing a glutathione S-transferase (gst) C fusion protein (gstC), we have shown that C protein forms a complex with the L, but not the P, subunit of the viral RNA polymerase. When P, L, and gstC are coexpressed, an oligomer of P, through its interaction with L, is also bound to beads. Since binding of C to L in the P-L complex does not disrupt P binding, the C and P binding sites appear to be different. GstC binding to L occurs only when the proteins are coexpressed in the same cell. The gstC, but not gst, protein inhibits viral transcription in vitro, showing that the fusion protein retains biological function. Pulse-chase experiments of the various complexes show that L protein synthesized alone has a half-life of 1. 2 hr, which is increased 12.5-fold by binding P, but is not significantly increased by binding gstC. Analyses of complex formation with truncations of L protein show that the C-terminal 1333 amino acids of L are not required for binding C. The dose-response curves show that replication of the genomic DI-H RNA is more sensitive to inhibition by C protein than is the synthesis of DI leader RNA, suggesting that the downregulation of RNA synthesis may be more complex than just the inhibition of the initiation of RNA synthesis.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , Regulación Viral de la Expresión Génica , Nucleoproteínas , Proteínas Virales/metabolismo , Animales , Sitios de Unión/genética , Chlorocebus aethiops , Glutatión Transferasa , Humanos , Proteínas de la Nucleocápside , Fosfoproteínas/metabolismo , Plásmidos , ARN Mensajero/análisis , ARN Viral/antagonistas & inhibidores , ARN Viral/biosíntesis , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/fisiología , Transcripción Genética , Transfección , Células Vero , Proteínas del Núcleo Viral/genética , Proteínas Virales/genética , Proteínas Virales/fisiologíaRESUMEN
The nucleocapsid protein (NP) of Sendai virus is an essential component of both the nucleocapsid template and the NP-NP and NP0-P protein complexes required for viral RNA replication. When expressed alone in mammalian cells NP self-assembles into nucleocapsid-like particles which appear to contain cellular RNA. To identify putative NP-NP binding domains, fusions between the monomeric maltose-binding protein (MBP) and portions of NP were constructed. The fusion proteins which contain the central conserved region (CCR) (amino acids 258-357, MBP-NP1) and the N-terminal 255 amino acids (MBP-NP2) of NP both oligomerized, suggesting that these regions contain sequences important for NP-NP self-assembly. In addition, the MBP-NP1 fusion protein can function as an inhibitor of viral RNA replication. Complementary studies involving site-directed mutagenesis of the full-length NP protein have identified specific residues in the CCR which are essential for viral RNA replication in vitro. Two such replication-negative mutants, F324V and F324I, were defective in self-assembly, suggesting that the Phe residue at amino acid 324 is essential for the NP-NP interaction. A third mutant, NP260-1 (Y260D), self-assembled to form aberrant oligomers which exhibit an unusual helical structure and appear to lack any associated RNA. The mutants NP299-5 (L299I and I300V) and NP313-2 (I313F), in contrast, appear to form all the required protein complexes, but were inactive in viral RNA replication, suggesting that interactions specifically with Sendai RNA were disrupted. These data have thus identified specific residues in the CCR of the native NP protein which appear to be important for NP-NP or NP-RNA interactions and for genome replication.
Asunto(s)
Secuencia Conservada , Nucleocápside/metabolismo , Nucleoproteínas , Respirovirus/metabolismo , Proteínas del Núcleo Viral/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Proteínas Portadoras/metabolismo , Humanos , Proteínas de Unión a Maltosa , Virus del Sarampión/metabolismo , Datos de Secuencia Molecular , Nucleocápside/genética , Proteínas de la Nucleocápside , Mutación Puntual , ARN Viral/biosíntesis , Conejos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Respirovirus/genética , Respirovirus/fisiología , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Células Tumorales Cultivadas , Proteínas del Núcleo Viral/genética , Ensamble de VirusRESUMEN
The nucleocapsid protein (NP) of Sendai virus encapsidates the genome RNA, forming a helical nucleocapsid which is the template for RNA synthesis by the viral RNA polymerase. The NP protein is thought to have both structural and functional roles, since it is an essential component of the NP0-P (P, phosphoprotein), NP-NP, nucleocapsid-polymerase, and RNA-NP complexes required during viral RNA replication. To identify domains in the NP protein, mutants were constructed by using clustered charge-to-alanine mutagenesis in a highly charged region from amino acids 107 to 129. Each of the mutants supported RNA encapsidation in vitro. The product nucleocapsids formed with three mutants, NP114, NP121, and NP126, however, did not serve as templates for further amplification in vivo, while NP107, NP108, and NP111 were nearly like wild-type NP in vivo. This template defect in the NP mutants from amino acids 114 to 129 was not due to a lack of NP0-P, NP-NP, or nucleocapsid-polymerase complex formation, since these interactions were normal in these mutants. We propose that amino acids 114 to 129 of the NP protein are required for the nucleocapsid to function as a template in viral genome replication.
Asunto(s)
Regulación Viral de la Expresión Génica , Nucleocápside/genética , ARN Viral/genética , Respirovirus/genética , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Mutación , ARN Viral/biosíntesisRESUMEN
The interactions of Sendai virus proteins required for viral RNA synthesis have been characterized both by the yeast two-hybrid system and through the use of glutathione S-transferase (gst)-viral fusion proteins synthesized in mammalian cells. Using the two-hybrid system we have confirmed the previously identified P-L (RNA polymerase), NPo-P (encapsidation substrate), and P-P complexes and now demonstrate NP-NP and NPo-V protein interactions. Expression of gstP and P proteins and binding to glutathione-Sepharose beads as a measure of complex formation confirmed the P-P interaction. The P-gstP binding occurred only on expression of the proteins in the same cell and was mapped to amino acids 345-411. We also show that full-length and deletion gstV and gstW proteins bound NPo protein when these sets of proteins were coexpressed and have identified one required region from amino acids 78-316. Neither gstV nor gstW bound NP assembled into nucleocapsids. Furthermore, both V and W proteins lacking the N-terminal 77 amino acids inhibited DI-H genome replication in vitro, showing the biological relevance of the remaining region. We propose that the specific inhibition of genome replication by V and W proteins occurs through interference with either the formation or the use of the NPo-P encapsidation substrate.
Asunto(s)
Nucleoproteínas , ARN Viral/biosíntesis , Respirovirus/metabolismo , Proteínas del Núcleo Viral/metabolismo , Proteínas Virales/metabolismo , Animales , Chlorocebus aethiops , Virus Defectuosos/genética , Eliminación de Gen , Genoma Viral , Humanos , Nucleocápside/metabolismo , Proteínas de la Nucleocápside , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Respirovirus/genética , Células Tumorales Cultivadas , Células Vero , Proteínas del Núcleo Viral/genética , Proteínas Virales/genéticaRESUMEN
Multiple epizootics of pneumonia in captive snakes have been attributed to viruses which have been tentatively placed in the family Paramyxoviridae. Viruses isolated from an ill Neotropical rattlesnake (Crotalus durissus terrificus), from an Aruba Island rattlesnake (Crotalus unicolor), and from a bush viper (Atheris sp.) were propagated in Vero cells and characterized. Viral particles produced in Vero cells were pleomorphic, enveloped, and contained helical nucleocapsids. The viruses were sensitive to ether and to acidic and basic pH. Moreover, they had neuraminidase activity and were able to agglutinate erythrocytes from chicken and a variety of species of mammals. Hemagglutination was inhibited with rabbit antiserum raised against each virus. The buoyant densities of the three isolates ranged from 1.13/cm3 to 1.18/cm3, values consistent with that for an enveloped virus. The nucleic acid in the virion was determined to be RNA by [3H]uridine incorporation. Viral proteins characteristic of paramyxoviruses were immunoprecipitated from cells infected with each of the three isolates using rabbit anti-Neotropical virus serum. The morphologic appearance, physico- and biochemical properties, and cytopathologic effects of these snake viruses were consistent with those of certain members of the family Paramyxoviridae.
Asunto(s)
Respirovirus , Viperidae/virología , Animales , Anticuerpos Antivirales/inmunología , Línea Celular , Chlorocebus aethiops , Éteres/química , Concentración de Iones de Hidrógeno , Neuraminidasa/metabolismo , ARN Viral/análisis , Respirovirus/genética , Respirovirus/inmunología , Respirovirus/aislamiento & purificación , Respirovirus/ultraestructura , Células Vero , Proteínas Virales/análisisRESUMEN
The nucleocapsid protein (N, 525 amino acids) of measles virus plays a central role in the replication of the viral genomic RNA. Its functions require interactions with itself and with other viral components. The N protein encapsidates genomic RNA, a function reflected in its ability to self-assemble into nucleocapsid-like particles in the absence of other viral proteins. The substrate for the packaging of nascent RNA during RNA replication is a complex between the N and phosphoprotein (P). The domains on the N protein that promote binding to P protein and self-assembly have been identified utilizing a series of N protein deletions. Two noncontiguous regions, amino acids 4-188 and 304-373 of N protein, are required for the formation of the soluble N-P complex, while deletion of amino acids 189-239 did not affect N-P binding. Amino acids 240-303 appear to be necessary for the stability of the protein. The N-terminal 398 amino acids are all required for the formation of organized nucleocapsid-like particles, since deletion of the central region from amino acids 189-373 completely abolished N-N interaction, and deletion of amino acids 4-188 and 374-492 caused the formation of unstructured aggregates.
Asunto(s)
Virus del Sarampión/metabolismo , Nucleoproteínas/metabolismo , Fosfoproteínas , Proteínas Virales/metabolismo , Ensamble de Virus , Animales , Sitios de Unión , Virus del Sarampión/genética , Virus del Sarampión/fisiología , Proteínas de la Nucleocápside , Nucleoproteínas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Virales/genéticaRESUMEN
To begin to map functional domains of the Sendai P-L RNA polymerase complex we wanted to characterize the P binding site on the Sendai L protein. Analysis of in vitro and in vivo P-L polymerase complex formation with carboxyl-truncations of the L protein showed that the N-terminal half of the protein was required. Site-directed mutagenesis of the Sendai virus L gene was employed to change amino acids within a highly conserved region of the N-terminal domain I from amino acids (aa) 348-379 singly or in pairs from the Sendai to the corresponding measles L sequence or to alanine. The mutant L proteins coexpressed with the viral P and NP proteins in mammalian cells were assayed for their ability to form the P-L complex and to synthesize RNA in vitro and showed a variety of defective phenotypes. While most of the mutant L proteins still formed the P-L polymerase complex, a change from serine to arginine at aa 368 and a three-amino-acid insertion at aa 379 virtually abolished both complex formation and RNA synthesis. Changes of aas 370 and 376-377 in the L protein gave only small decreases in viral RNA synthesis. Substitutions at either aas 349-350 or aas 354-355 and a three-amino-acid insertion at aa 348 in the L protein yielded enzymes that catalyzed significant transcription, but were defective in DI RNA replication, thus differentially affecting the two processes. Since DI leader RNA, but not genome RNA, was still synthesized by this class of mutants, the defect in replication appears to be in the ability of the mutant enzyme to package newly synthesized nascent RNA. Single changes at aas 362, 363, and 366 in the L protein gave enzymes with severely decreased overall RNA synthesis, although some leader RNA was synthesized, suggesting that they cannot transcribe or replicate past the leader gene. These studies have identified a region in conserved domain I critical for multiple functions of the Sendai virus L protein.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , Virus de la Parainfluenza 1 Humana/genética , Fosfoproteínas/metabolismo , ARN Viral/biosíntesis , Transcripción Genética , Proteínas Virales/metabolismo , Replicación Viral , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Secuencia Conservada , Cartilla de ADN , ADN Viral/genética , ARN Polimerasas Dirigidas por ADN/genética , Virus Defectuosos/genética , Virus del Sarampión/genética , Datos de Secuencia Molecular , Mutación , Virus de la Parainfluenza 1 Humana/metabolismo , Fosfoproteínas/genética , Proteínas Recombinantes de Fusión/metabolismo , Especificidad de la Especie , Proteínas Virales/genéticaRESUMEN
Our long-term goal is to define the catalytic domains of the L protein subunit of the Sendai virus RNA polymerase. An aberrant polyadenylation phenotype in the vesicular stomatitis virus tsG16 L protein mutant has recently been identified as a phenylalanine to serine change at amino acid 1488 (Hunt and Hutchinson, Virology 193, 786-793, 1993). To test if functional domains are conserved in the L proteins of negative-strand RNA viruses, we attempted to create a similar polyadenylation defect in the Sendai virus L protein. Nine different amino acid substitutions at the analogous site in the Sendai L protein (cysteine at amino acid 1571) were constructed by site-directed mutagenesis of the gene. Each mutant L protein was synthesized and bound to the Sendai P protein to form the P-L polymerase complex. While none of these L mutants exhibited a change in polyadenylation, the single amino acid changes yielded a variety of activities in vitro. Mutants containing valine, leucine, or phenylalanine at amino acid 1571, amino acids found naturally in the L proteins of other paramyxoviruses, yielded polymerases that had biological activity equal to or better than the wild-type (WT) polymerase. Serine or threonine substitutions in the L protein at this position also resulted in polymerases with nearly WT synthetic activity. In contrast, a glycine substitution significantly decreased overall polymerase activity, whereas a tyrosine substitution gave decreased transcription, but virtually no DI genome replication in vitro. The tyrosine-substituted polymerase may be unable to carry out the packaging step of replication, since DI leader RNA synthesis was normal in this mutant. Mutant L proteins with basic arginine or histidine substitutions were inactive in all viral RNA synthesis in vitro, although the polymerase complexes could bind the nucleocapsid template.
Asunto(s)
ARN Polimerasas Dirigidas por ADN , Virus de la Parainfluenza 1 Humana/genética , ARN Viral/biosíntesis , Proteínas Virales/genética , Aminoácidos/metabolismo , Secuencia de Bases , Sitios de Unión , Línea Celular , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Virus de la Parainfluenza 1 Humana/metabolismo , Proteínas Virales/metabolismoRESUMEN
It has been postulated that the cellular double-stranded (ds) RNA adenosine deaminase enzyme is responsible for biased hypermutation during persistent SSPE measles infections in humans. As a test of this hypothesis we studied the effect of negative-strand RNA virus infection on enzyme activity. The adenosine deaminase activity was found in nuclear extracts of both uninfected CV-1 and A549 cells and in cytoplasmic extracts of A549, but not CV-1, cells. During measles or Sendai virus infection of either CV-1 or A549 cells the adenosine deaminase activity in the nucleus remained fairly constant up to 24 h post infection, and there was no apparent re-partitioning of the enzyme between the nucleus and the cytoplasm. Transcription complexes of Sendai virus in vitro or measles virus in vivo did not serve as substrates for the enzyme. These data suggest that even though some portion of the adenosine deaminase enzyme may be present in the cytoplasm of at least some cells during virus infection, modification of the viral RNAs by this enzyme, if it occurs at all, must be at a very low level not directly detectable by biochemical analysis.
Asunto(s)
Adenosina Desaminasa/metabolismo , Virus del Sarampión/enzimología , ARN Viral/metabolismo , Adenosina Monofosfato/metabolismo , Línea Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Humanos , Inosina Monofosfato/metabolismo , Virus de la Parainfluenza 1 Humana/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN , Especificidad por Sustrato , Transcripción Genética/fisiología , Replicación Viral/fisiologíaRESUMEN
Sequence analysis of the nucleoprotein and phosphoprotein (P) genes of viruses in the hamster neurotropic lineage of measles virus revealed that the neurotropic variants are quite different from the Philadelphia 26 progenitor strain. In Vero cells persistently infected with the hamster neurotropic strain, predicted changes occur in 5.0% of the nucleoprotein and 8.1% of the P amino acids and some of these changes appear to affect the relative electrophoretic mobility of each protein. To evaluate one aspect of the viral polymerase complex containing these mutations, the distribution of P mRNA editing in each of the three strains was determined by both cloning and sequencing of polymerase chain reaction-amplified DNA fragments which included the editing site and by primer extension analysis of viral mRNA. Editing of P mRNA in the neurotropic strains shows a shift away from the single G insertion product to those with greater than 2 Gs inserted. The altered editing distribution has implications for the role of transcriptional regulation in measles virus persistence.