Replication of orthomyxoviruses.
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Base Sequence↗
Biomedical subjects
J Ortín
Publications and source records attributed to J Ortín.
Characterization of influenza virus PB1 protein binding to viral RNA: two separate regions of the protein contribute to the interaction domain.
The interaction of the PB1 subunit of the influenza virus polymerase with the viral RNA (vRNA) template has been studied in vitro. The experimental approach included the in vitro binding of labeled model vRNA to PB1 protein immobilized as an immunoprecipitate, as well as Northwestern analyses. The binding to model vRNA was specific, and an apparent Kd of about 2 x 10(-8) M was determined. Although interaction with the isolated 3' arm of the panhandle was detectable, interaction with the 5' arm was prominent and the binding was optimal with a panhandle analog structure (5'+3' probe). When presented with a panhandle analog mixed probe, PB1 was able to retain the 3' arm as efficiently as the 5' arm. The sequences of the PB1 protein involved in vRNA binding were identified by in vitro interaction tests with PB1 deletion mutants. Two separate regions of the PB1 protein sequence proved positive for binding: the N-terminal 83 amino acids and the C-proximal sequences located downstream of position 493. All mutants able to interact with model vRNA were capable of binding the 5' arm more efficiently than the 3' arm of the panhandle. Taken together, these results suggest that two separate regions of the PB1 protein constitute a vRNA binding site that interacts preferentially with the 5' arm of the panhandle structure.
Animals↗
Mutational analysis of influenza A virus nucleoprotein: identification of mutations that affect RNA replication.
The influenza A virus nucleoprotein (NP) is a multifunctional polypeptide which plays a pivotal role in virus replication. To get information on the domains and specific residues involved in the different NP activities, we describe here the preparation and characterization of 20 influenza A virus mutant NPs. The mutations, mostly single-amino-acid substitutions, were introduced in a cDNA copy of the A/Victoria/3/75 NP gene and, in most cases, affected residues located in regions that were highly conserved across the NPs of influenza A, B, and C viruses. The mutant NPs were characterized (i) in vivo (cell culture) by analyzing their intracellular localization and their functionality in replication, transcription, and expression of model RNA templates; and (ii) in vitro by analyzing their RNA-binding and sedimentation properties. The results obtained allowed us to identify both a mutant protein that accumulated in the cytoplasm and mutations that altered the functionality and/or the oligomerization state of the NP polypeptide. Among the mutations that reduced the NP capability to express chloramphenicol acetyltransferase protein from a model viral RNA (vRNA) template, some displayed a temperature-sensitive phenotype. Interestingly, four mutant NPs, which showed a reduced functionality in synthesizing cRNA molecules from a vRNA template, were fully competent to reconstitute complementary ribonucleoproteins (cRNPs) capable of synthesizing vRNAs, which in turn yielded mRNA molecules. Based on the phenotype of these mutants and on previously published observations, it is proposed that these mutant NPs have a reduced capability to interact with the polymerase complex and that this NP-polymerase interaction is responsible for making vRNPs switch from mRNA to cRNA synthesis.
Amino Acid Sequence↗
A human sequence homologue of Staufen is an RNA-binding protein that is associated with polysomes and localizes to the rough endoplasmic reticulum.
In the course of a two-hybrid screen with the NS1 protein of influenza virus, a human clone capable of coding for a protein with high homology to the Staufen protein from Drosophila melanogaster (dmStaufen) was identified. With these sequences used as a probe, cDNAs were isolated from a lambda cDNA library. The encoded protein (hStaufen-like) contained four double-stranded RNA (dsRNA)-binding domains with 55% similarity and 38% identity to those of dmStaufen, including identity at all residues involved in RNA binding. A recombinant protein containing all dsRNA-binding domains was expressed in Escherichia coli as a His-tagged polypeptide. It showed dsRNA binding activity in vitro, with an apparent Kd of 10(-9) M. Using a specific antibody, we detected in human cells a major form of the hStaufen-like protein with an apparent molecular mass of 60 to 65 kDa. The intracellular localization of hStaufen-like protein was investigated by immunofluorescence using a series of markers for the cell compartments. Colocalization was observed with the rough endoplasmic reticulum but not with endosomes, cytoskeleton, or Golgi apparatus. Furthermore, sedimentation analyses indicated that hStaufen-like protein associates with polysomes. These results are discussed in relation to the possible functions of the protein.
Amino Acid Sequence↗
In vivo reconstitution of active Thogoto virus polymerase: assays for the compatibility with other orthomyxovirus core proteins and template RNAs.
Tick-borne Thogoto virus (THOV), the prototype of a new genus in the Orthomyxoviridae family, contains six single-stranded RNA segments of negative polarity. Four of them encode gene products that correspond to the influenza virus PB1, PB2, PA and NP core proteins. Here we describe an in vivo system in which the expression of a THOV model RNA is driven by THOV core proteins synthesized from cloned cDNAs. Our results demonstrated the biological activity of our cloned genes and showed that the three polymerase subunits and the NP are required for gene expression. For comparison, we also used the in vivo reconstituted systems of the influenza A and B viruses. None of the polymerase or NP proteins was active in a heterologous orthomyxovirus core, indicating a high specificity in core assembly and/or function. Interestingly, the THOV polymerase did not recognize the influenza A virus promoter and vice versa.
Cloning, Molecular↗
The PA influenza virus polymerase subunit is a phosphorylated protein.
The induction of proteolysis by expression of the influenza virus PA polymerase subunit is the only biochemical activity ascribed to this protein. In the course of studying viral protein synthesis by two-dimensional gel electrophoresis, we observed the existence of several PA isoforms with different isoelectric points. These isoforms were also present when the PA gene was singly expressed in three different expression systems, indicating that a cellular activity is responsible for its post-translational modification. In vivo labelling with [32P]orthophosphate, followed by two-dimensional gel electrophoresis, clearly demonstrated the incorporation of phosphate into the PA molecule. Phosphoserine and phosphothreonine epitopes were present in PA, while phosphotyrosine residues were absent, as tested by immunoblotting with specific antibodies. These facts, as well as the presence of multiple consensus sites for casein kinase II (CKII) phosphorylation, prompted us to test the involvement of this kinase in PA covalent modification. PA protein purified by immunoprecipitation could be specifically labelled by the catalytic alpha subunit of human CKII, which was expressed and purified from bacteria. Collectively, these data demonstrate that the PA subunit of the influenza virus RNA polymerase is a phosphoprotein.
Animals↗
The N-terminal half of the influenza virus NS1 protein is sufficient for nuclear retention of mRNA and enhancement of viral mRNA translation.
A collection of C-terminal deletion mutants of the influenza A virus NS1 gene has been used to define the regions of the NS1 protein involved in its functionality. Immunofluorescence analyses showed that the NS1 protein sequences downstream from position 81 are not required for nuclear transport. The capacity of these mutants to bind RNA was studied by in vitro binding tests using a model vRNA probe. These experiments showed that the N-terminal 81 amino acids of NS1 protein are sufficient for RNA binding activity. The collection of mutants also served to map the NS1 sequences required for nuclear retention of mRNA and for stimulation of viral mRNA translation, using the NP gene as reporter. The results obtained indicated that the N-terminal 113 amino acids of NS1 protein are sufficient for nuclear retention of mRNA and stimulation of viral mRNA translation. The possibility that this region of the protein may be sufficient for virus viability is discussed in relation to the sequences of NS1 genes of field isolates and to the phenotype of known viral mutants affected in the NS1 gene.
Animals↗
Cleavage of p220 by purified poliovirus 2A(pro) in cell-free systems: effects on translation of capped and uncapped mRNAs.
Poliovirus protease 2A(pro) has been obtained in soluble form as a fusion protein with maltose binding protein (MBP). Addition of MBP-2A(pro) to rabbit reticulocyte cell-free systems gives rise to efficient cleavage of the initiation factor of translation p220 (eIF-4G). Translation of capped mRNA encoding the influenza virus NP protein is severely impaired in lysates in which p220 has been proteolytically cleaved. This inhibition is dependent on the concentration of mRNA added to the lysate. Thus, increasing the concentrations of mRNA substantially overcomes the blockade of NP synthesis after p220 cleavage. Notably, translation of uncapped NP mRNA is also compromised in p220-deficient rabbit reticulocyte lysates, suggesting that p220 participates in the translation of both capped and uncapped NP mRNAs. The effects of p220 proteolysis by poliovirus 2A(pro) have also been assayed on luciferase mRNA translation. Three types of mRNAs encoding for luciferase have been examined: capped, uncapped, and mRNA bearing the poliovirus 5' leader region (leader luc mRNA). Synthesis of luciferase directed by any of these mRNAs was inhibited after cleavage of p220 in rabbit reticulocyte lysates. Interestingly, supplementation of the lysate with HeLa cell extracts stimulates leader luc mRNA translation by poliovirus 2A(pro). These results indicate that activation of translation of mRNAs bearing the poliovirus leader region promoted by this poliovirus protease requires a factor present in HeLa cell extracts. These findings agree well with recent experiments implicating p220 not only in protein synthesis directed by capped mRNAs but also in the translation of naturally uncapped mRNAs.
Carrier Proteins↗
Influenza virus NS1 protein interacts with viral transcription-replication complexes in vivo.
The interaction of influenza virus NS1 protein with other viral products in the infected cell was analysed by co-immunoprecipitation studies. The three subunits of the polymerase and the nucleoprotein, but not M1 protein, were co-immunoprecipitated by NS1-specific serum but not when control serum was used. Such co-immunoprecipitation was not sensitive to RNase treatment of the immunoprecipitates. Co-immunoprecipitation was also obtained when the viral transcription-replication system was reconstituted in vivo by transfection of cDNAs and model vRNA template into vaccinia virus-T7-infected cells. Analysis of the RNA pulled-down in the NS1-specific precipitates indicated the presence of both vRNA and mRNA. These results are discussed in the context of the phenotype of virus temperature-sensitive mutants affected in the NS1 gene.
Animals↗
The influenza A virus PB2 polymerase subunit is required for the replication of viral RNA.
The transcription and replication of influenza virus RNA (vRNA) were reconstituted in vivo. The experimental approach involved the transfection of plasmids encoding the viral subunits of the polymerase and the nucleoprotein into cells infected with a vaccinia virus recombinant virus expressing the T7 RNA polymerase. As templates, one of two model RNAs was transfected: vNSZ or cNSZ RNA. The RNAs were 240 nucleotides in length, contained the terminal sequences of the NS viral segment, and were of negative or positive polarity, respectively. The accumulation of cRNA and mRNA in cells transfected with vNSZ RNA and the accumulation of vRNA and mRNA in cells transfected with cNSZ RNA were determined by RNase protection assays with labeled vNSZ-L or cNSZ-L probes. The patterns of protected bands obtained indicated that both cRNA replication intermediate and mRNA accumulated when the system was reconstituted with vNSZ RNA. Likewise, both vRNA and mRNA accumulated after reconstitution with cNSZ RNA. The reconstitution of incomplete systems in which any of the subunits of the polymerase or the model RNA were omitted was completely negative for the accumulation of cRNA or vRNA, indicating that the presence of the PB2 subunit in the polymerase is required for replication of vRNA.
Animals↗
Identification of two separate domains in the influenza virus PB1 protein involved in the interaction with the PB2 and PA subunits: a model for the viral RNA polymerase structure.
The domains of the PB1 subunit of the influenza virus polymerase involved in the interaction with the PB2 and PA subunits have been defined by mutational analysis of PB1 protein. The experimental approach included in vivo competition of the PB1 activity, two-hybrid interaction assays and in vitro binding to PB1-specific matrices. Mutants of the PB1 gene including N-terminal, C-terminal and internal deletions and single amino acid insertions were constructed. They were unable to support polymerase activity in a reconstituted transcription-replication system and were tested for their competition activity when expressed in excess over wild-type PB1 protein. The pattern of competition obtained suggested that the N-terminal 78 amino acids and the sequences between positions 506 and 659 in the PB1 protein are involved in the interaction with the other components of the polymerase. We identified the N-terminal region of PB1 protein as responsible for the interaction with the PA subunit by two-hybrid assays in mammalian cells. N- and C-terminal fragments of the PB1 protein were expressed as His-tagged proteins and purified on Ni2+-NTA resin. Such PB1-specific matrices were used in binding assays in vitro with metabolically labelled PB2 and PA proteins and mutants thereof. The results obtained indicated that the N-terminal and the C-terminal regions of PB1 are responsible for binding to PA and PB2 subunits, respectively. With this information and previously published results we propose a preliminary model for the architecture of the influenza virus RNA polymerase.
Animals↗
Mutational analysis of the influenza virus A/Victoria/3/75 PA protein: studies of interaction with PB1 protein and identification of a dominant negative mutant.
The RNA polymerase activity and PB1 binding of influenza virus PA mutants were studied using an in vivo-reconstituted polymerase assay and a two hybrid system. Deletions covering the whole PA protein abolished polymerase activity, but the deletion of the 154 N-terminal amino acids allowed PB1 binding, indicating that the PA protein N terminus is not absolutely required for this interaction. Further internal or C-terminal deletions abolished PB1 interaction, suggesting that most of the protein is involved in this association. As a novel finding we showed that a single amino acid insertion mutant, PAI672, was responsible for a temperature-sensitive phenotype. Mutant PAS509, which had a serine insertion at position 509, bound to PB1 like wild-type PA but did not show any polymerase activity. Over-expression of PAS509 interfered with the polymerase activity of wild-type PA, identifying PAS509 as a dominant negative mutant.
Animals↗
Experiments in a rotating Hele-Shaw cell.
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Journal Article↗
Mutational analysis identifies functional domains in the influenza A virus PB2 polymerase subunit.
A collection of influenza virus PB2 mutant genes was prepared, including N-terminal deletions, C-terminal deletions, and single-amino-acid insertions. These mutant genes, driven by a T7 promoter, were expressed by transfection into COS-1 cells infected with a vaccinia virus encoding T7 RNA polymerase. Mutant proteins accumulated to levels similar to that of wild-type PB2. Immunofluorescence analyses showed that the C-terminal region of the protein is essential for nuclear transport and that internal sequences affect nuclear localization, confirming previous results (J. Mukaijawa and D. P. Nayak, J. Virol. 65:245-253, 1991). The biological activity of these mutants was tested by determining their capacity to (i) reconstitute RNA polymerase activity in vivo by cotransfection with proteins NP, PB1, and PA and a virion-like RNA encoding the cat gene into vaccinia virus T7-infected COS-1 cells and (ii) complete with the wild-type PB2 activity. In addition, when tested at different temperatures in vivo, two mutant PB2 proteins showed a temperature-sensitive phenotype. The lack of interference shown by some N-terminal deletion mutants and the complete interference obtained with a C-terminal deletion mutant encoding only 124 amino acids indicated that this protein domain is responsible for interaction with another component of the polymerase, probably PB1. To further characterize the mutants, their ability to induce in vitro synthesis of viral cRNA or mRNA was tested by using ApG or beta-globin mRNA as a primer. One of the mutants, 1299, containing an isoleucine insertion at position 299, was able to induce cRNA and mRNA synthesis in ApG-primed reactions but required a higher beta-globin mRNA concentration than wild-type PB2 for detection of in vitro synthesis. This result suggested that mutant I299 has diminished cap-binding activity.
Base Sequence↗
The amino-terminal one-third of the influenza virus PA protein is responsible for the induction of proteolysis.
We have previously described the fact that the individual expression of influenza virus PA protein induced a generalized proteolysis (J.J. Sanz-Ezquerro, S. de la Luna, Ortin, and A. Nieto, J. Virol. 69:2420-2426, 1995). In this study, we have further characterized this effect by mapping the regions of PA protein required and have found by deletion analysis that the first 247 amino acids are sufficient to bring about this activity. PA mutants that were able to decrease the accumulation levels of coexpressed proteins also presented lower steady-state levels due to a reduction in their half-lives. Furthermore, the PA wild type produced a decrease in the stationary levels of different PA versions, indicating that is itself a target for its induced proteolytic process. All of the PA proteins that induced proteolysis presented nuclear localization, being the sequences responsible for nuclear transport located inside the first 247 amino acids of the molecule. To distinguish between the regions involved in nuclear localization and those involved in induction of proteolysis, we fused the nuclear localization signal of the simian virus 40 T antigen to the carboxy terminus of the cytosolic versions of PA. None of the cytosolic PA versions affected in the first 247-amino-acid part of PA, which were now located in the nucleus, were able to induce proteolysis, suggesting that conservation of a particular conformation in this region of the molecule is required for the effect observed. The fact that all of the PA proteins able to induce proteolysis presented nuclear localization, together with the observation that this activity is shared by influenza virus PA proteins from two different type A viruses, suggests a physiological role for this PA protein activity in viral infection.
Base Sequence↗
Open reading frame 5 of porcine reproductive and respiratory syndrome virus as a cause of virus-induced apoptosis.
The gene product of open reading frame 5 (p25) of porcine reproductive and respiratory syndrome (PRRS) virus has been expressed by coinfection of culture cells with vaccinia virus expressing the T7 RNA polymerase and a recombinant vaccinia virus encoding the open reading frame 5 gene under the T7 promoter and the encephalomyocarditis virus internal ribosome entry site. In spite of the reported efficiency of the expression system, very poor accumulation of p25 protein was observed and a strong cytotoxicity was produced in the doubly infected cells. This cell toxicity was shown to occur by induction of apoptosis, as indicated by nucleosome ladder formation, chromatin condensation, and rRNA degradation. Apoptosis induction was also observed after infection of cultured cells with an adapted PRRS virus strain and after infection of swine macrophage cells with a PRRS virus field strain. Contrary to the observations made for other cases of virus-induced apoptosis, we could not prevent p25 protein-induced apoptosis by using a cell line permanently expressing Bcl-2 protein.
Animals↗
Statistics of avalanches in martensitic transformations. I. Acoustic emission experiments.
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Journal Article↗
Epitope mapping of cross-reactive monoclonal antibodies specific for the influenza A virus PA and PB2 polypeptides.
Characterization of the epitopes recognized by 21 monoclonal antibodies (MAbs) specific for the influenza A virus PA (13 MAbs) and PB2 (8 MAbs) polypeptides (Bárcena et al. (1994) J. Virol. 68, 6900-6909) raised against denatured polypeptides produced in E. coli is described. MAbs were characterized by: (1) competitive binding ELISAs; (2) mapping of the protein regions that specify their binding sites; and (3) analyses of their ability to recognize the corresponding viral protein in a number of viral isolates. Five and three non-overlapping antigenic areas were defined by the anti-PA and anti-PB2 MAbs, respectively. Five of the anti-PA MAbs recognized antigenic determinants located within the amino-terminal 157 amino acids of the PA protein, and 6 others reacted strongly with a PA fragment comprising the first 236 amino acids. All 8 anti-PB2 antibodies reacted strongly with a polypeptide fragment containing amino acids 1-113 of the PB2 protein. Analyses of the reactivities of 4 anti-P antibodies with 23 influenza A virus reference strains isolated over a period of 61 years and recovered from humans, pigs, birds and horses, showed that the epitopes were conserved among all viral isolates. The application of these antibodies as research and diagnostic tools is discussed.
Animals↗