Biomedical subjects

A Portela

Publications and source records attributed to A Portela.

At least 19 recordsLinked to original sources

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↗

PubMed1990

Location of a highly conserved neutralizing epitope in the F glycoprotein of human respiratory syncytial virus.

Trypsin digestion of the purified F protein from human respiratory syncytial virus (Long strain) generated a set of fragments in the amino-terminal third of the F1 subunit which contained the epitope 47F involved in neutralization. Sequencing of five escape mutant viruses selected with monoclonal antibody 47F allowed us to map precisely two amino acid residues (262 and 268) of the F1 subunit which are essential for the integrity of this important epitope. The results are discussed in terms of the mechanisms involved in virus neutralization and the design of potential synthetic vaccines.

Amino Acid Sequence↗

PubMed1990

Frame shift mutations as a novel mechanism for the generation of neutralization resistant mutants of human respiratory syncytial virus.

The genetic characterization of four previously reported mutants of human respiratory syncytial (RS) virus resistant to monoclonal antibody 63G is described. Sequences of the G protein genes were obtained from: (i) mRNA derived cDNA recombinants, (ii) direct mRNA sequencing and (iii) amplified vRNA derived cDNAs. The results obtained indicate that the original escape mutants, recovered from individual plaques, contained heterogeneous viral populations. This heterogeneity affected the number of adenosine residues present after nucleotides 588 or 623 of the G protein gene. Mutant viruses recovered after a second plaque purification step generated homogeneous sequences but contained single adenosine insertions or deletions at those two sites compared with the Long sequence. These genetic alterations introduced frameshift changes which are reflected in both the antigenic and structural properties of the mutant G proteins. The origin and importance of frameshift mutations in the RS virus G protein gene are discussed.

Amino Acid Sequence↗

PubMed1988

Nucleotide sequence of the fusion and phosphoprotein genes of human respiratory syncytial (RS) virus Long strain: evidence of subtype genetic heterogeneity.

The nucleotide and deduced amino acid sequences of the fusion (F) and phosphoprotein (P) genes of the Long strain of human respiratory syncytial (RS) virus have been determined from cDNA copies cloned into pBSV9 shuttle vector. Comparison of these sequences with their counterparts of other strains reveals genetic heterogeneity within the same subtype. The percentage of nucleotide and amino acid changes occurring in both proteins is similar. Thus, the Long F and P proteins share 97.9% and 98.3% amino acid identity, respectively, with their homologs of the A2 strain. Nevertheless the F2 subunit of the fusion protein accumulates 3.1 times more amino acid changes than the F1 subunit. In addition, the percentage of nucleotide changes in the 3' extracistronic sequences is 6 times higher in the P than in the F gene. These results are discussed in terms of selective pressures operating in the evolution of RS virus in nature.

Amino Acid Sequence↗

PubMed1987

Permanent cell lines established from ts-COS cells that regulate by temperature the amplification and expression of cloned genes.

Temperature-sensitive COS cells have been transformed at restrictive temperature with SV40 replicons containing the neo or pac markers. Puromycin-resistant cell clones maintained at the restrictive temperature contain the pac gene integrated into the cell DNA. However, when the cells are shifted to the permissive temperature the pac gene is amplified in episomal forms up to 2-4 X 10(4) copies per cell. Concomitant with this, an induction of 35-300 fold in the levels of puromycin acetyl transferase activity is observed, leading to the accumulation of the enzyme up to 10-60 mU/mg of total cell protein. A band of apparent molecular weight 26,500 daltons is observed by polyacrylamide gel electrophoresis of induced culture extracts, that accounts for approximately 3% of the newly synthesized protein. The expression of non-selectable genes can also be regulated, as shown by the induction of influenza virus nucleoprotein synthesis in transformed cells. These results indicate that the ts-COS cells can be used as a highly efficient, regulable mammalian expression system.

Acetyltransferases↗

PubMed1986

Expression in mammalian cells of a gene from Streptomyces alboniger conferring puromycin resistance.

The gene encoding a puromycin N-acetyl transferase from Streptomyces alboniger has been cloned next to the SV40 early promoter in a mammalian cells-Escherichia coli shuttle vector. When this construction was introduced into VERO cells it expressed the relevant enzymic activity. Moreover, the puromycin N-acetyl transferase gene has been used as a dominant marker for the selection of transformed mammalian cells able to grow in the presence of the antibiotic.

Acetyltransferases↗

PubMed1986

Construction of cell lines that regulate by temperature the amplification and expression of influenza virus non-structural protein genes.

Monkey cell lines have been transformed with a mixture of plasmids pSV2neo and pSLVa232N, a derivative of plasmid pSLVa232 (Portela et al., 1985b). Plasmid pSLVa232N contained the influenza virus genes encoding non-structural proteins under the control of the SV40 late promoter in pSLts1 vector that includes the SV40 ori and the tsA209 T-antigen gene. At restrictive temperature, plasmid sequences remained stably integrated in the cell genome, but upon temperature shift-down, defined circular DNA molecules were generated and amplified up to 2000-5000 copies/cell. Restriction analysis, Southern blot hybridization and partial sequencing indicate that one such episome, pC5, was derived from the integrated plasmid sequences by a homologous recombination event that led to deletion of the pBR322 sequences included in pSLVa232N. Concomitant with gene amplification, an induction of 20-65-fold in the expression of NS1 and NS2 proteins was observed after temperature shift-down. Thus, gene cloning into vector pSLts1 and transformation at restrictive temperature of cells permissive for SV40 DNA replication, appears to be a useful strategy for the controlled amplification and expression of cloned genes.

Animals↗

PubMed1985

Regulation of gene amplification and expression in cells that constitutively express a temperature sensitive SV40 T-antigen.

Simian cells have been transformed with SV40 origin-defective recombinant plasmids containing the tsA209 T-antigen gene. These plasmids contain deletions of either 5 or 52 nucleotides that include the BglI site at the SV40 ori, are defective for replication in COS-1 cells but retain a functional SV40 early promoter. Two cell lines transformed with these plasmids, U4 and S7, and their respective clonal derivatives E5 and F11, contain the tsA209 T-antigen gene integrated into the cell DNA and express T-antigen as detected by immunoprecipitation and immunofluorescence. These cells behave as ts-COS cells, since they complement in a temperature dependent manner the replication of an SV40 derived recombinant plasmid. When transfected with recombinant plasmids containing the chloramphenicol acetyl transferase (CAT) gene cloned into SV40 replicons, ts-COS cells were able to regulate the induction of the CAT activity by temperature. The ratios of CAT activity observed at permissive versus restrictive temperature were in the range of 20-400. Thus, these ts-COS cells are useful systems for the regulated expression of cloned genes in simian cells.

Acetyltransferases↗

PubMed1985

A primer vector system that allows temperature dependent gene amplification and expression in mammalian cells: regulation of the influenza virus NS1 gene expression.

Influenza virus RNA segment 8 has been cloned into primer-vector pSLts1. This vector was designed to replicate in simian cells in a temperature dependent fashion by use of the SV40 tsA209 T-antigen gene. The oriented synthesis of cDNA on dT-tailed pSLts1 was performed on in vitro synthesized mRNA, and the second DNA strand was primed with an influenza-specific terminal oligodeoxynucleotide. Recombinant pSLVa232 contained the RNA segment 8 sequence directly fused to the SV40 late promoter contained in pSLts1, and followed by the SV40 polyadenylation signal. Expression of NS1 gene in transfected COS cells took place at a level comparable to that found in infected cells. When VERO cell cultures were transfected with recombinant pSLVa232, expression of the NS1 gene was temperature dependent. Close to one hundred fold increase in the amplification and expression of the cloned gene was observed after shift down of the transfected cells to permissive temperature. Vector pSLts1 and the cloning strategy described may be useful for the specific cloning and regulated expression of mRNAs of known 5'-terminal sequence.

Animals↗

PubMed1985

Oriented synthesis and cloning of influenza virus nucleoprotein cDNA that leads to its expression in mammalian cells.

The influenza virus nucleoprotein gene has been cloned by a procedure that involves direct cDNA synthesis onto the primer-vector pBSV9, a pBR322-SV40 recombinant plasmid. dT-tailed pBSV9 was used to prime the synthesis of cDNA on a template of in vitro synthesized viral mRNA. The synthesis of ds-cDNA was initiated by a specific oligodeoxynucleotide and the resulting recombinant was circularized by intramolecular ligation. Recombinant pSVa963 contained the viral nucleoprotein gene directly fused to the SV40 early promoter region included in pBSV9 and followed by a dA:dT tail and the SV40 polyadenylation signal. When pSVa963 was used to transfect COS-1 cells, the presence of three NP-specific mRNAs of 1600, 1900 and 2500 nucleotides in length could be detected. Pulse labelling experiments of COS-1 transfected cells and immunobinding to a nucleoprotein monoclonal antibody indicated the synthesis of nucleoprotein. This nucleoprotein accumulated in the nucleus of transfected cells at a level similar to that found in infected cells. The vector and method described may be useful for the specific cloning and expression of any mRNA for which a 5'-terminal sequence is known.

Animals↗

PubMed1985

The quasispecies (extremely heterogeneous) nature of viral RNA genome populations: biological relevance--a review.

We review evidence that cloned (or uncloned) populations of most RNA viruses do not consist of a single genome species of defined sequence, but rather of heterogeneous mixtures of related genomes (quasispecies). Due to very high mutation rates, genomes of a quasispecies virus population share a consensus sequence but differ from each other and from the consensus sequence by one, several, or many mutations. Viral genome analyses by sequencing, fingerprinting, cDNA cloning etc. indicate that most viral RNA populations (quasispecies) contain all possible single and double genomic site mutations and varying proportions of triple, quadruple, etc. site mutations. This quasispecies structure of RNA virus populations has many important theoretical and practical implications because mutations at only one or a few sites may alter the phenotype of an RNA virus.

Base Sequence↗