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Biomedical subjects

P Palese

Publications and source records attributed to P Palese.

At least 127 records · Page 7Linked to original sources
PubMed1985

Expression of influenza virus NS2 nonstructural protein in bacteria and localization of NS2 in infected eucaryotic cells.

The nonstructural NS2 protein of influenza A/PR/8/34 virus was efficiently expressed in bacteria, and monospecific antisera were prepared against the bacterially synthesized polypeptide. These antisera were cross-reactive among the NS2 proteins of various influenza A viruses. However, they did not react with the NS2 of influenza B/Lee/40 virus nor with other proteins of influenza A viruses such as NS1. Antisera against NS2 were used to determine that the NS2 protein is localized in the cell nucleus during influenza virus infection, as shown by immunofluorescence microscopy. Cells infected with simian virus 40 recombinants containing the influenza virus NS gene revealed that both the NS1 and NS2 proteins appeared in the nucleus, even in the absence of expression of other influenza virus-specific components.

Animals↗
PubMed1985

Influenza C virus RNA 7 codes for a nonstructural protein.

The complete nucleotide sequence of RNA segment 7 of influenza C/California/78 virus was determined by using cloned cDNA derived from viral RNA. The gene is 934 nucleotides long and possesses a long open reading frame which can code for a protein of 286 amino acids. Hybrid arrest translation experiments with the cloned cDNA fragment and poly(A)-containing RNA isolated from virus-infected cells showed that a 28,500-molecular-weight protein is coded for by RNA 7. Comparison of the proteins induced in the cell-free system and in virus-infected cells with those found in purified virus suggests that the 28,500-molecular-weight protein is a nonstructural protein.

Amino Acid Sequence↗
PubMed1984

Synthesis of an ochre suppressor tRNA gene and expression in mammalian cells.

We have used site-specific mutagenesis to change the anticodon of a Xenopus laevis tyrosine tRNA gene so that it would recognize ochre codons. This tRNA gene is expressed when amplified in monkey cells as part of a SV40 recombinant and efficiently suppresses termination at both the ochre codon separating the adenovirus 2 hexon gene from a 23-kd downstream gene and the ochre codon at the end of the NS1 gene of influenza virus A/Tex/1/68. Termination at an amber codon of a NS1 gene of another influenza virus strain was not suppressed by the (Su+) ochre gene suggesting that in mammalian cells amber codons are not recognized by ochre suppressor tRNAs. Finally, microinjection into mammalian cells of both (Su+) ochre tRNA genes and selectible genes containing ochre nonsense mutations gives rise to colonies under selective conditions. We conclude that it should be possible to isolate a wide assortment of mammalian cell lines with ochre suppressor activity.

Amino Acid Sequence↗
PubMed1984

Recognition of influenza A virus nucleoprotein by an H-2-restricted cytotoxic T-cell clone.

Cytotoxic T-cell clones raised against X-31 (H3N2) influenza virus in C57BL/6 mice can be directed against an influenza A virus subtype specific determinant (1). A representative T-cell clone (A3.1) has been used in combination with a set of genetically typed recombinant viruses, to show that the A/PR/8/34 nucleoprotein can be responsible for cytotoxic T-lymphocyte recognition of infected target cells.

Animals↗
PubMed1984

Complete nucleotide sequence of the influenza C/California/78 virus nucleoprotein gene.

The complete nucleotide sequence of RNA segment 5 of the influenza C/California/78 (C/Cal/78) virus was determined by using cloned cDNA derived from viral RNA. The gene contains 1809 nucleotides and can code for a protein of 565 amino acids with a molecular weight of 63 525. The RNA 5 protein of the influenza C/Cal/78 virus possesses two short regions which share a high degree (60-83%) of sequence homology with the nucleoproteins of influenza A and B viruses. These and other structural features of the RNA 5 protein suggest that RNA 5 of influenza C viruses codes for the nucleoprotein. The data also suggest that influenza C viruses are orthomyxoviruses, but that they are more distantly related to either type A or type B viruses than are influenza A and B viruses to each other.

Amino Acid Sequence↗
PubMed1984

Analysis of an influenza A virus mutant with a deletion in the NS segment.

The influenza virus host range mutant CR43-3, derived by recombination from the A/Alaska/6/77 and the cold-adapted and temperature-sensitive A/Ann Arbor/6/60 viruses, has previously been shown to possess a defect in the NS gene. To characterize this defect, nucleotide sequence data were obtained from cloned cDNAs. The CR43-3 NS gene was found to be 854 nucleotides long and to derive from the NS gene of the A/Alaska/6/77 parent virus by an internal deletion of 36 nucleotides. Direct sequencing of RNA 8 of CR43-3 virus confirmed that the deletion in the NS1-coding region was not an artifact that was generated during the cloning procedure. Protein analysis indicated that the NS1 protein of CR43-3 virus was synthesized in equal amounts in the restrictive (MDCK) cells as well as in the permissive (PCK) host cells. Also, indirect immunofluorescence studies of virus-infected cells showed that the NS1 protein of CR43-3 virus, like that of the parent viruses, accumulates in the nuclei of both cell systems. Although no differences in synthesis or localization of the NS1 protein could be detected, a consistent reduction in M1 protein was noted in CR43-3 virus-infected, nonpermissive cells as compared with that of the permissive host. Since analysis of the CR43-3 virus required us to obtain the NS nucleotide sequence of the 1977 isolate A/Alaska/6/77, we were able to compare this sequence with those of corresponding genes of earlier strains. The result of this analysis supports the idea of a common lineage of human influenza A viruses isolated over a 43-year period.

Amino Acid Sequence↗
PubMed1984

Influenza C virus hemagglutinin: comparison with influenza A and B virus hemagglutinins.

The complete nucleotide sequence of the influenza C/California/78 virus RNA 4 was obtained by using cloned cDNA derived from the RNA segment. This gene is 2,071 nucleotides long and can code for a polypeptide of 654 amino acids. Although there are no convincing sequence homologies between RNA 4 and the hemagglutinin genes of influenza A and B viruses, we suggest, on the basis of structural features, that RNA 4 of the influenza C virus codes for the hemagglutinin. The structural features which are common to the hemagglutinins of influenza A, B, and C viruses include (i) a hydrophobic signal peptide, (ii) an arginine cleavage site between the hemagglutinin 1 and 2 subunits, (iii) hydrophobic regions at the amino and carboxyl termini of the hemagglutinin 2 subunit, and (iv) several conserved cysteine residues. Additional evidence that RNA 4 of influenza C virus codes for the hemagglutinin is that the tripeptide Ile-Phe-Gly, known to be present at the amino terminus of the hemagglutinin 2 subunit of influenza C virus, is encoded by RNA 4 at a point immediately adjacent to the presumptive arginine cleavage site. The lack of primary sequence homology between the influenza C virus hemagglutinin and the influenza A or B virus hemagglutinins, which all have similar functions, might be attributed to convergent rather than divergent evolution. However, the structural similarities among the influenza A, B, and C virus hemagglutinins strongly suggest that the three hemagglutinin genes have diverged from a common precursor.

Amino Acid Sequence↗
PubMed1983

Measurement of suppressor transfer RNA activity.

Transfer RNA (tRNA) suppression of nonsense mutations in prokaryotic systems has been widely used to study the structure and function of different prokaryotic genes. Through genetic engineering techniques, it is now possible to introduce suppressor (Su+) tRNA molecules into mammalian cells. A quantitative assay of the suppressor tRNA activity in these mammalian cells is described; it is based on the amount of tRNA-mediated readthrough of a terminating codon in the influenza virus NS1 gene after the cells are infected with virus. Suppressor activity in L cells continuously expressing Su+ (tRNAtyr) was 3.5 percent and that in CV-1 cells infected with an SV40- Su+ (tRNAtyr) recombinant was 22.5 percent.

Animals↗
PubMed1983

Nonsense mutations affecting the lengths of the NS1 nonstructural proteins of influenza A virus isolates.

The proteins from cells infected with influenza A virus field isolates were labeled with [35S]methionine and analyzed by SDS-polyacrylamide gel electrophoresis. By screening more than 100 field isolates, it was found that the NS1 proteins had the greatest mobility differences, far exceeding those observed among other corresponding viral polypeptides. Partial sequence determination of RNA segment eight from 12 viruses revealed the existence of nonsense mutations at six different positions in their NS1 coding regions. The termination codons consisted of opal, ochre, and amber mutations. The sizes predicted from these sequences of 202, 217, 219, 220, 230, and 237 amino acids were in agreement with the observed mobilities of the viral polypeptides on SDS-polyacrylamide gels. The observation of large deletions in the carboxy termini of the NS1 proteins of field virus isolates would suggest that a high degree of variation can be tolerated in this polypeptide without affecting its functional capability.

Base Sequence↗
PubMed1983

Preparation of influenza virus subviral particles lacking the HA1 subunit of hemagglutinin: unmasking of cross-reactive HA2 determinants.

Acid treatment of influenza A and B virus preparations followed by addition of dithiothreitol (DTT) and centrifugation through a sucrose cushion removes the HA1 subunit of hemagglutinin from virus. Rabbit sera made against these subviral particles and untreated virus were tested in a radioimmune precipitation assay using [35S]cysteine-labeled virus. Conditions of the assay permitted discrimination of discrete HA1- and HA2-specific antibody populations. It was found that (a) sera raised to intact influenza A virus preparations contained both HA1- and HA-2 specific antibodies, (b) sera made to subviral particles of influenza A virus contained HA2-specific antibody but had little or no detectable HA1-specific antibody. (c) the HA2-specific antibodies were partially cross-reactive with the HA2 of an influenza A virus of a different subtype, and (d) sera raised against two strains of untreated influenza B viruses contained antibodies which were cross-reactive with the HA2 as well as the NP of influenza A viruses.

Cross Reactions↗
PubMed1983

Sequential mutations in hemagglutinins of influenza B virus isolates: definition of antigenic domains.

Comparative analysis of the amino acid sequences of hemagglutinins (HAs) of influenza B/Lee/40, B/Md/59, and B/HK/73 viruses has allowed examination of the molecular basis of antigenic variation in type B viruses. As seen with influenza type A viruses, antigenic drift in influenza B viruses proceeds mostly through the accumulation of amino acid substitutions within the HA1 portion of the HA molecule. However, the rate of variation observed among the influenza B virus HAs appears to be significantly lower than the observed rate of variation among influenza A virus HAs. The overall rate of amino acid change in the HA1s of the influenza B viruses studied is 2% per 10 years, whereas the HA1s of H3 influenza A viruses vary by 9.2% per 10 years. The sequences of the influenza B HAs were also examined in relation to the three-dimensional model for the A/Aichi/2/68 HA. When the primary amino acid sequences are compared, it appears that most of the important structural features of the type A HAs--such as the sialic acid binding site, the disulfide linkages, and the stem structure of the trimer--are conserved in the influenza B virus HAs. Regions are also identified where extensive amino acid substitutions have occurred among the three antigenically distinct influenza B virus HAs. The locations of these areas in the B HA structure correspond to antigenic regions proposed for the A virus HAs. In addition, modulation of antigenic regions in B virus HAs may also occur through amino acid deletions and variation in glycosylation sites.

Amino Acid Sequence↗
PubMed1983

Efficient expression of influenza virus NS1 nonstructural proteins in Escherichia coli.

RNA segment 8 of the influenza A virus genome codes for two nonstructural proteins, NS1 and NS2, for which the functions are unknown. Cloned cDNA copies of this gene from three different influenza A virus strains were inserted into an Escherichia coli plasmid expression vector, pAS1, carrying the strong regulatable lambda phage promoter, PL. After induction, the NS1 proteins were overproduced to levels of 20-25% of total cellular protein. This was surprising in that the codon composition for these eukaryotic genes is similar to that for weakly expressed proteins in E. coli. Thus, under the appropriate conditions, it appears that high level expression of genes containing a relatively large proportion of minor codons can be obtained. The NS1 protein produced in bacteria from a cloned cDNA copy of the A/PR/8/34 virus NS gene was purified to apparent homogeneity and used to generate a high-titer monospecific rabbit antiserum. Immunoprecipitation studies showed this antibody to be crossreactive against the NS1 proteins produced by several different influenza A virus strains. Immunofluorescence experiments in Madin-Darby canine kidney cells showed the NS1 proteins to be located in the nucleoplasm early in infection for all strains examined. With some of the strains, NS1-specific immunofluorescence was observed predominantly in the nucleoli later in infection. This technology can be used to obtain other viral proteins in pure form for structural, functional, and immunological studies.

Animals↗
PubMed1983

Sequential mutations in the NS genes of influenza virus field strains.

The complete nucleotide sequences of the NS genes from three human influenza viruses, A/FM/1/47 (H1N1), A/FW/1/50 (H1N1), and A/USSR/90/77 (H1N1), were determined. Only five single-base differences were found within the sequences of the A/FW/1/50 and A/USSR/90/77 NS genes, thus confirming earlier data suggesting that the 1977 H1N1 viruses are closely related to virus strains that were circulating around 1950. Comparison of all three sequences with those from A/PR/8/34 and A/Udorn/72 viruses illustrates that these genes (with the exception of that of the A/USSR/90/77 strain) evolve through cumulative base changes along a single common lineage. A nucleotide sequence variation of approximately 2.2 to 3.4% per 10 years was determined for the NS gene segments. Extensive size variation was also observed among the NS1 proteins of the various human viruses. The A/FM/1/47 NS1 protein, which consists of 202 amino acids, is 15% shorter than the A/Udorn/72 NS1 protein, which consists of 237 amino acids.

Amino Acid Sequence↗
PubMed1982

Variation of influenza A, B, and C viruses.

Influenza is caused by highly variable RNA viruses belonging to the orthomyxovirus group. These viruses are capable of constantly changing the genes coding for their surface proteins as well as for their nonsurface proteins. The mechanisms responsible for these changes in type A influenza viruses include recombination (reassortment) of genes among strains, deletions and insertions in genes, and, frequently, point mutations. In addition, old strains may reappear in the population. Influenza viruses of types B and C appear to vary to a lesser degree. The mechanisms responsible for changes in these viruses are not well characterized.

Genetic Variation↗
PubMed1982

Oligonucleotide mapping: evaluation of its sensitivity by computer-simulation.

A frequently used method of comparing large RNA molecules employs the two-dimensional display of oligonucleotides generated through the action of specific RNases (oligonucleotide mapping, fingerprinting). Using computer simulations and simple analytic expressions the number of large RNase T1-resistant oligonucleotides obtained from random RNA sequences can be estimated. The computer simulations also permit estimation of the number of large oligonucleotides which remain unchanged as random variations are introduced into a random RNA sequence. In addition, computer analysis also provides a means of estimating statistical confidence limits to be used in a quantitative comparison of fingerprints of different RNA molecules. The model shows that two RNA sequences which differ overall by 1%, 5% or 10% share, on average, only 85%, 50% or 25%, respectively, of their large oligonucleotides. Thus, the use of fingerprint analysis is recommended only when closely related RNAs or regions of RNAs are compared (sequence homology greater than 90%).

Base Sequence↗
PubMed1982

Evolution of influenza A and B viruses: conservation of structural features in the hemagglutinin genes.

The complete nucleotide sequence of the hemagglutinin (HA) gene of a type B influenza virus (B/Lee/40) was obtained by using cloned cDNA derived from the RNA segment. The gene is 1,882 nucleotides long and can code for a protein precursor of 584 amino acids. Structural features common to type A virus HAs are also conserved in the B virus HA. These include a hydrophobic signal peptide, hydrophobic NH2 and COOH termini of the HA2 subunit, and a HA1/HA2 cleavage site involving an arginine residue. The sequence of the B HA gene and its deduced amino acid sequence were compared to those of a type A influenza virus (A/PR/8/34). When these two genes were aligned, it was found that 24% of the amino acids in the HA1 subunits and 39% of the amino acids in the HA2 subunits are conserved. This degree of relatedness between type B virus and type A virus HAs (intertypic comparison) is similar to the homologies observed among certain type A virus HAs (intratypic comparison). A close evolutionary relationship is therefore suggested between the HAs of type A and type B influenza viruses.

Amino Acid Sequence↗
PubMed1981

An avian tumor virus promoter directs expression of plasmid genes in Escherichia coli.

A sequence in the long terminal repeat (LTR) of avian tumor virus (ATV) DNA was shown to contain a promoter acute in Escherichia coli. For this analysis the bacterial promoters for the tetracycline (Tc) and neomycin (Nm) resistance genes were deleted from different plasmids and replaced with various fragments derived from the ATV DNA. Expression of the drug-resistant phenotype in the recombinant plasmids at levels comparable to or greater than those found with parental bacterial promotes was shown to be dependent on the presence of an intact sequence ranging from nucleotide +19 to -23 (relative to the cap site) in the ATV DNA. Comparison of the consensus bacterial promoter with the nucleotide sequence in this region revealed strong similarities.

Avian Sarcoma Viruses↗