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A Portela

Publications and source records attributed to A Portela.

At least 37 records · Page 2Linked to original sources
PubMed1996

The 5' ends of Thogoto virus (Orthomyxoviridae) mRNAs are homogeneous in both length and sequence.

Thogoto (THO) virus is a tick-borne member of the Orthomyxoviridae whose genome consists of six segments of linear, negative sense, single-stranded RNA. To gain insight into the mechanism by which viral mRNA transcripts are initiated, poly(A)+ RNA isolated from THO virus-infected cells was characterized by (i) primer extension experiments, (ii) immunoprecipitation studies with an anticap monoclonal antibody, (iii) direct sequencing analysis of the isolated RNA, and (iv) cloning and sequencing of individual mRNA molecules. The results indicated that THO virus mRNAs are capped and homogeneous in both length and sequence at their 5' end. These findings contrast with the situation found in all other segmented, negative sense or ambisense, single-stranded RNA viruses so far analyzed in which the 5' ends of viral mRNAs are heterogeneous in length and sequence. These results are discussed in terms of the mechanism used by THO virus to initiate mRNA synthesis.

Poly A↗
PubMed1996

Serine 3 is critical for phosphorylation at the N-terminal end of the nucleoprotein of influenza virus A/Victoria/3/75.

The influenza A virus nucleoprotein (NP) is a phosphoprotein that encapsidates the viral genomic RNA. To map the in vivo phosphorylation site(s) of this protein, 32P-labeled NP was purified from cell cultures infected with influenza virus A/Victoria/3/75 by immunoaffinity chromatography. The purified protein was then subjected to chemical digestion with formic acid, which cleaves proteins at Asp-Pro bonds, and the resulting products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two of the phosphorylated products obtained were identified as fragments corresponding to the N-terminal 88 amino acids and to the C-terminal 196 residues of the NP. To identify the phosphate acceptor site(s) at the N-terminal phosphorylated region of NP, each of the seven serines within this region was individually changed to alanine by site-directed mutagenesis. The mutant proteins were then transiently expressed in mammalian cells and analyzed for their phosphorylation state. It was observed that the S-to-A mutation at position 3 drastically reduced the amount of 32P label incorporated into NP, whereas the other substitutions did not have a discernible effect on the phosphorylation level of the protein. In addition, all serine-altered proteins were tested for their functionality in an artificial system in which expression of a synthetic chloramphenicol acetyl-transferase RNA molecule is driven by influenza virus proteins synthesized from cloned genes. The results obtained demonstrate that all mutant proteins were competent to cooperate with the subunits of the viral polymerase for expression of the synthetic virus-like chloramphenicol acetyltransferase RNA in vivo. These data are discussed regarding the possible roles of NP phosphorylation for the viral replicative cycle.

Amino Acid Sequence↗
PubMed1996

Rescue of a synthetic chloramphenicol acetyltransferase RNA into influenza virus-like particles obtained from recombinant plasmids.

We have shown previously that COS-1 cells infected with a vaccinia virus recombinant (vTF7-3) which expresses the T7 RNA polymerase gene and then transfected with four pGEM-derived plasmids encoding the influenza A virus core proteins (nucleoprotein, PB1, PB2, and PA polypeptides) can express a synthetic influenza virus-like chloramphenicol [correction of chloramphenical] acetyltransferase (CAT) RNA (I. Mena, S. de la Luna, C. Albo, J. Martín, A. Nieto, J. Ortín, and A. Portela, J. Gen. Virol. 75:2109-2114, 1994). Here we demonstrate that by supplying the vTF7-3-infected cells with plasmids containing cDNAs of all 10 influenza virus-encoded proteins, the transfected CAT RNA can be expressed and rescued into particles that are budded into the supernatant fluids. The released particles can transfer the enclosed CAT RNA to MDCK cultures and resemble true influenza virions in that they require trypsin treatment to deliver the RNA to fresh cells and are neutralized by a monoclonal antibody specific for the influenza A virus hemagglutinin. Moreover, analysis by electron microscopy showed that the culture medium harvested from the transfected cells contained vesicles that could be labeled with an anti-HA monoclonal antibody and that were similar in size and morphology to authentic influenza virus particles. It is also shown that detection of recombinant particles capable of transmitting the CAT RNA does not require expression of the influenza virus nonstructural protein NS1. All of these data indicate that influenza virus-like particles enclosing a synthetic virus-like RNA can be assembled in cells expressing all viral structural components from recombinant plasmids.

Animals↗
PubMed1995

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↗
PubMed1995

Identification of an RNA binding region within the N-terminal third of the influenza A virus nucleoprotein.

The influenza A virus nucleoprotein (NP) has been examined with regard to its RNA-binding characteristics. NP, purified from virions and devoid of RNA, bound synthetic RNAs in vitro and interacted with the ribonucleotide homopolymers poly(A), poly(G), poly(U), and poly(C) in a salt-dependent manner, showing higher binding affinity for polypyrimidine homopolymers. To map the NP regions involved in RNA binding, a series of deleted forms of the NP were prepared, and these truncated polypeptides were tested for their ability to bind poly(U) and poly(C) homopolymers linked to agarose beads. Proteins containing deletions at the N terminus of the NP molecule showed reduced RNA-binding activity, indicating that this part of the protein was required to bind RNA. To identify the NP region or regions which directly interact with RNA, proteins having the maltose-binding protein fused with various NP fragments were obtained and tested for binding to radioactively labeled RNAs in three different assays: (i) nitrocellulose filter binding assays, (ii) gel shift assays, and (iii) UV light-induced cross-linking experiments. A maltose-binding protein fusion containing the N-terminal 180 amino acids of NP behaved as an RNA-binding protein in the three assays, demonstrating that the N terminus of NP can directly interact with RNA. This NP region could be further subdivided into two smaller regions (amino acids 1 to 77 and 79 to 180) that also retained RNA-binding activity.

Amino Acid Sequence↗
PubMed1994

Complex structure of the nuclear translocation signal of influenza virus polymerase PA subunit.

The protein regions involved in the nuclear translocation of the influenza virus PA polymerase subunit have been identified by deletion analysis of the protein expressed from a recombinant simian virus 40. Two regions seem to play a role in the process: region I (amino acids 124 to 139) and region II (amino acids 186 to 247). A nucleoplasmin-like nuclear translocation signal (NLS) has been identified in region I and an additional NLS appears to be present in region II, although no consensus targeting sequence can be detected. Alteration in any of the regions identified by short deletions completely prevented nuclear transport, whereas elimination of the regions I or II by large amino- or carboxy-terminal deletions did not prevent nuclear targeting of the truncated protein. In addition, a point mutation at position 154 completely eliminated nuclear transport. A beta-galactosidase fusion protein containing the 280 amino acid terminal region of the PA protein was partially transported to the nucleus and mutant PA proteins with a cytoplasmic phenotype could not be rescued by superinfection with influenza virus. These results suggest that the PA protein contains a functional nuclear targeting region which is required in influenza virus infection, with two independent NLSs, one in region I and the other in region II.

Amino Acid Sequence↗
PubMed1994

Synthesis of biologically active influenza virus core proteins using a vaccinia virus-T7 RNA polymerase expression system.

An in vivo system in which expression of a synthetic influenza virus-like chloramphenicol acetyltransferase (CAT) RNA is driven by influenza virus proteins synthesized from cloned cDNAs has been developed. Expression of the four influenza virus core proteins (nucleoprotein, PA, PB1 and PB2) was performed by transfection of four pGEM recombinant plasmids, each containing one of the four viral genes, into cell cultures previously infected with a vaccinia virus recombinant encoding the T7 RNA polymerase (vTF7-3). When a naked negative-sense influenza virus-like CAT RNA was transfected into cells expressing the four influenza virus proteins, CAT activity was detected in the cell extracts, demonstrating that the expressed proteins had RNA-synthesizing activity. In this system, CAT RNA templates containing additional nucleotides at the 3' end were also expressed, resulting in CAT activity. This showed that the influenza virus polymerase can recognize its promoter when located internally on an RNA template. In influenza virus-infected cells however, CAT activity was detected only when the CAT RNA contained the viral promoter at the exact 3' end and was transfected as in vitro assembled ribonucleoprotein. These results are discussed in terms of the different requirements of the two helper systems for expression of an exogenously added RNA.

Animals↗
PubMed1994

Monoclonal antibodies against influenza virus PB2 and NP polypeptides interfere with the initiation step of viral mRNA synthesis in vitro.

Two panels of monoclonal antibodies (MAbs) specific for the influenza A virus PA and PB2 polypeptides have been obtained from mice immunized with denatured proteins produced in Escherichia coli. All MAbs (13 specific for the PA polypeptide and 8 specific for the PB2 protein) reacted to the corresponding influenza virus protein in Western blotting (immunoblotting), immunoprecipitation, and immunofluorescence assays. To gain information about the roles of the nucleoprotein (NP) and PB2 and PA proteins during viral mRNA synthesis, the 21 anti-P antibodies and 3 anti-NP antibodies (J. A. López, M. Guillen, A. Sánchez-Fauquier, and J. A. Melero, J. Virol. Methods 13:255-264, 1986) were purified and tested for their ability to inhibit the transcriptase activity associated with viral cores purified from virions. Four of the antibodies (one anti-PB2 and the three anti-NP MAbs) inhibited transcription by more than 50% compared with unrelated control antibodies. The inhibitory effect was not due to a nonspecific effect of the antibody preparations, because these MAbs did not inhibit transcription when tested on influenza B virus nucleocapsids, which are not recognized by the antibodies. To determine whether the antibodies were acting on an early transcription step, transcription reactions were carried out in the presence of globin mRNA (a mixture of alpha- and beta-globin chains) and only one labeled nucleoside triphosphate (either GTP or CTP). The results obtained showed that MAbs to the PB2 and NP polypeptides interfered with the initiation step of mRNA-primed transcription. The implications of these results regarding initiation of viral mRNA synthesis are discussed.

Animals↗
PubMed1993

Influenza virus naked RNA can be expressed upon transfection into cells co-expressing the three subunits of the polymerase and the nucleoprotein from simian virus 40 recombinant viruses.

The functionality of the influenza virus polymerase subunits and the nucleoprotein expressed from simian virus 40 (SV40) recombinants has been tested by their ability to direct the in vivo expression of influenza virus-like RNAs. These RNAs, which contained either the chloramphenicol acetyltransferase (CAT) or haemagglutinin (HA) genes, were synthesized and reconstituted in vitro into viral ribonucleoproteins with a polymerase/nucleoprotein mixture purified from influenza virus-infected cells. Only the coinfection with SV40 recombinant viruses expressing the three polymerase subunits and the nucleoprotein allowed the expression of the transfecting CAT or HA RNAs, confirming that this set of viral genes is the minimal requirement for viral gene expression. Unexpectedly, transfection of the corresponding naked RNAs into SV40 recombinant-infected cells was as effective in directing the synthesis of CAT or HA proteins as the standard reconstituted ribonucleoprotein transfection. These results may be important for the genetic analysis of trans-acting factors involved in influenza virus transcription and replication and may open the way to rescuing influenza viruses in the absence of a helper virus.

Animals↗
PubMed1992

Nuclear transport of influenza virus polymerase PA protein.

The subcellular distribution of influenza polymerase PA subunit has been studied using a SV40-recombinant virus (SVPA76), which allows the expression and accumulation of this protein in COS-1 cells. In contrast to the complete nuclear localization observed for the PA subunit several hours after influenza virus infection, when COS-1 cells were infected with the SVPA76 recombinant, the PA protein accumulated either in the nucleus, in the cytoplasm or was distributed throughout the cell. When cells were infected with the SVPA76 recombinant and superinfected with influenza virus, a clear increase in the proportion of cells showing nuclear localization of the PA protein was observed, suggesting that some trans-factor may be required to allow complete nuclear accumulation of the protein. Double infections using SVPA76 recombinant and either SVPB1 or SVNS recombinant viruses showed a complete correlation between expression of polymerase PB1 subunit or NS1 protein and nuclear localization of polymerase PA subunit. However, no such correlation was observed in the double infections of SVPA76 and SVNP recombinants. These results suggest that polymerase PB1 subunit and the non-structural proteins could be involved in the nuclear targeting or nuclear retention of influenza polymerase PA protein.

Animals↗
PubMed1992

Identification of viral structural polypeptides of Thogoto virus (a tick-borne orthomyxo-like virus) and functions associated with the glycoprotein.

Thogoto (THO) virus is a tick-borne virus which shares morphological and genetic features with members of the Orthomyxoviridae family although the viral glycoprotein appears to be related to gp64 of baculoviruses. Characterization of THO virus was undertaken to clarify its taxonomic position. Purified virus preparations contained at least six virus-encoded polypeptides with apparent M(r) values ranging from 29K to 92K. A 75K polypeptide was identified as an envelope-associated glycoprotein by Triton X-100 and salt dissociation studies, and by proteolytic degradation of the exposed proteins of the virion. By the same criteria, the nucleoprotein and the matrix protein were identified as the 52K and 29K polypeptides, respectively. Immunofluorescence studies using monoclonal antibodies (MAbs) located the glycoprotein on the external cell membrane and the nucleoprotein in the nucleus of infected cells indicating that virus replication involved a nuclear phase. In addition, the virus displayed haemagglutination and haemolytic activities with an optimum at pH 6. These activities are functions of the viral glycoprotein since they were inhibited by anti-glycoprotein MAbs. The data reported here support the notion that THO virus is a member of the Orthomyxoviridae family but that it should be classified in a group distinct from the other influenza viruses.

Animals↗
PubMed1992

In vitro reconstitution of active influenza virus ribonucleoprotein complexes using viral proteins purified from infected cells.

A procedure to obtain RNA-free preparations containing the nucleoprotein (NP) and polymerase (P) proteins from influenza virus-infected cell extracts has been developed. The influenza virus P proteins present in these preparations copied small synthetic RNA molecules derived from plasmid sequences. In addition, RNA molecules encapsidated with the NP and P proteins were amplified and packaged into virus particles when transfected into influenza virus-infected cells. Thus, the preparations of NP and P proteins display features similar to those isolated from purified influenza virions, and represent an alternative for the preparation of active influenza virus P protein.

Animals↗
PubMed1992

[Photoperiod variations of various blood biochemistry constants in the rabbit].

Biochemical blood parameters generally show fluctuations during the day. The aim of this study is to determine the variations of the following parameters: hemoglobin, glucose, triglycerides, ASAT, ALAT, urea and creatinine in New Zealand white (NZW) rabbits, kept under five different circadian rhythms (12/12, 14/10, 10/14, 16/8, 8/16, light/dark). These parameters were analyzed by using a reflection photometer (Reflotron). Most results are found within the physiological range for this species but there are significant differences among cycles. These findings could be explained by the dependence of the biochemical blood parameters on the light/dark cycle. It can be concluded that the most useful cycle to find fewer variations of the biochemical blood parameters in NZW rabbit is 12/12 light/dark cycle.

Alanine Transaminase↗
PubMed1991

Evolution of the G and P genes of human respiratory syncytial virus (subgroup A) studied by the RNase A mismatch cleavage method.

The G and P genes of human respiratory syncytial viruses (subgroup A), isolated between 1961 and 1989, were analyzed by RNase A one-dimensional fingerprinting, using the Long strain as the reference. Total RNA extracted from cells infected with the different isolates was hybridized to radiolabeled antisense G or P RNA probes of the Long virus. The RNA:RNA heteroduplexes were digested with RNase A and the resistant products analyzed by gel electrophoresis. Comparative analysis of the cleavage patterns revealed extensive genetic heterogeneity in both genes among viruses isolated in different epidemics. In contrast, 13 viruses isolated in Montevideo during a 3-month period showed much more restricted heterogeneity; thus, 11 viruses represented the predominant type of this outbreak and only 2 other viruses generated different RNA cleavage patterns distantly related to the major type. Statistical analysis of the results obtained indicated progressive accumulation of genetic changes with time along cocirculating evolutionary lineages within the same antigenic subgroup of RS virus. The results are discussed in terms of a model for RS virus evolution.

Antigens, Viral↗
PubMed1991

Regulated M1 mRNA splicing in influenza virus-infected cells.

Influenza virus RNA segment 7 generates three poly(A)+ RNAs, M1 mRNA, M2 mRNA and mRNA3, the last of which has almost no coding capacity; M2 mRNA and mRNA3 derive from M1 mRNA by removal of a single intron. The kinetics of M1 and M2 mRNA accumulation in the cytoplasm of productively infected cells were studied by means of a quantitative RNA protection assay; the ratio of M2 mRNA to M1 mRNA increased 2.7-fold during the course of infection. To analyse the basis for this change, the kinetics of M1 and M2 mRNA synthesis and nuclear accumulation, their stability and nucleocytoplasmic transport were studied. Under the experimental conditions used, the synthesis of segment 7-specific RNA showed a peak at 4 h post-infection and continued later at a slower rate. The half-lives of M1 and M2 mRNAs were indistinguishable (2.73 h for M1 mRNA and 2.70 h for M2 mRNA) and the kinetics of nucleocytoplasmic transport in vivo or in vitro showed no preference for either mRNA early or late in infection. Consequently, regulation at the level of mRNA splicing is proposed. Using the mRNA synthesis and stability data, a simulation was performed to predict the change in splicing efficiency required to account for the mRNA accumulation results. The best fit was obtained when splicing efficiency changed about 20 times during a period in which viral gene expression was maximal.

Animals↗
PubMed1991

Premature stop codons in the G glycoprotein of human respiratory syncytial viruses resistant to neutralization by monoclonal antibodies.

Mutants of human respiratory syncytial (RS) virus which escaped neutralization by monoclonal antibodies directed against the G glycoprotein were selected from the Long strain. Most mutants showed drastic antigenic changes, reflected in the lack of reactivity with several anti-G antibodies, including the one used for selection. Sequence analysis revealed the presence of in-frame premature stop codons in the mutated G genes which shortened the G polypeptide by between 11 and 42 amino acids. In contrast, two mutants selected with monoclonal antibody 25G contained two amino acid substitutions (Phe-265----Leu and Leu-274----Pro) and had lost only the capacity to bind the antibody used in their selection. These results demonstrate that the carboxy-terminal end of the G molecule is dispensable for infectivity in tissue culture and indicate the importance of this part of the G protein in determining its antigenicity.

Antibodies, Monoclonal↗
PubMed1990

Analysis of genetic variability in human respiratory syncytial virus by the RNase A mismatch cleavage method: subtype divergence and heterogeneity.

We have applied the RNase A mismatch cleavage method to the analysis of genetic variability among human Respiratory Syncytial (RS) viruses. Antisense RNA probes of the Long strain were hybridized to total RNA extracted from cells infected with other strains. The RNA:RNA heteroduplexes were digested with RNase A and the resistant products analyzed by gel electrophoresis. Each virus generated characteristic band patterns with the different probes. Comparative analyses of the cleavage patterns indicate that antigenic subtypes correlate with genetically distinct viral groups. Viruses within each subtype, however, show substantial genetic heterogeneity and progressive accumulation of genetic changes with time. This heterogeneity is also observed among viruses of the same epidemic outbreak which cannot be distinguished with a panel of monoclonal antibodies. Different genes and gene regions also differ in their rates of change. These results are discussed in terms of RS virus evolution.

Cell Line↗