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Biomedical subjects

P Palese

Publications and source records attributed to P Palese.

At least 109 records · Page 6Linked to original sources
PubMed1988

The E3 protein of bovine coronavirus is a receptor-destroying enzyme with acetylesterase activity.

In addition to members of the Orthomyxoviridae and Paramyxoviridae, several coronaviruses have been shown to possess receptor-destroying activities. Purified bovine coronavirus (BCV) preparations have an esterase activity which inactivates O-acetylsialic acid-containing receptors on erythrocytes. Diisopropyl fluorophosphate (DFP) completely inhibits this receptor-destroying activity of BCV, suggesting that the viral enzyme is a serine esterase. Treatment of purified BCV with [3H]DFP and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the proteins revealed that the E3 protein was specifically phosphorylated. This finding suggests that the esterase/receptor-destroying activity of BCV is associated with the E3 protein. Furthermore, treatment of BCV with DFP dramatically reduced its infectivity in a plaque assay. It is assumed that the esterase activity of BCV is required in an early step of virus replication, possibly during virus entry or uncoating.

Acetylesterase↗
PubMed1988

Two nuclear location signals in the influenza virus NS1 nonstructural protein.

The NS1 protein of influenza A virus has been shown to enter and accumulate in the nuclei of virus-infected cells independently of any other influenza viral protein. Therefore, the NS1 protein contains within its polypeptide sequence the information that codes for its nuclear localization. To define the nuclear signal of the NS1 protein, a series of recombinant simian virus 40 vectors that express deletion mutants or fusion proteins was constructed. Analysis of the proteins expressed resulted in identification of two regions of the NS1 protein which affect its cellular location. Nuclear localization signal 1 (NLS1) contains the stretch of basic amino acids Asp-Arg-Leu-Arg-Arg (codons 34 to 38). This sequence is conserved in all NS1 proteins of influenza A viruses, as well as in that of influenza B viruses. NLS2 is defined within the region between amino acids 203 and 237. This domain is present in the NS1 proteins of most influenza A virus strains. NLS1 and NLS2 contain basic amino acids and are similar to previously defined nuclear signal sequences of other proteins.

Amino Acid Sequence↗
PubMed1988

Determination of the mutation rate of a retrovirus.

The mutation rate of Rous sarcoma virus (RSV) was measured. Progeny descended from a single virion were collected after one replication cycle, and seven regions of the genome were analyzed for mutations by denaturing-gradient gel electrophoresis. In all, 65,250 nucleotides were screened, yielding nine mutations, and the RSV mutation rate was calculated as 1.4 x 10(-4) mutations per nucleotide per replication cycle. These results indicate that RSV is an extremely mutable virus. We speculate that the mutation rate of a virus may correlate inversely with the effectiveness of vaccination against a given virus and suggest that prevention of retrovirus-mediated disease via vaccination may prove difficult.

Animals↗
PubMed1988

Evidence that the matrix protein of influenza C virus is coded for by a spliced mRNA.

In contrast to influenza A and B viruses, which encode their matrix (M) proteins via an unspliced mRNA, the influenza C virus M protein appears to be coded for by a spliced mRNA from RNA segment 6. Although an open reading frame in RNA segment 6 of influenza C/JJ/50 virus could potentially code for a protein of 374 amino acids, a splicing event results in an mRNA coding for a 242-amino-acid M protein. The message for this protein represents the major M gene-specific mRNA species in C virus-infected cells. Despite the difference in coding strategies, there are sequence homologies among the M proteins of influenza A, B, and C viruses which confirm the evolutionary relationship of the three influenza virus types.

Amino Acid Sequence↗
PubMed1987

Synthesis and cellular location of the ten influenza polypeptides individually expressed by recombinant vaccinia viruses.

A complete set of recombinant vaccinia viruses that express each of the influenza virus polypeptides has been constructed. PB1, PB2, PA, HA, NP, M1, and NS1 genes were derived from influenza virus A/PR/8/34, NA from influenza virus A/Cam/46, and M2 and NS2 genes from influenza virus A/Udorn/72. Cells infected with these recombinant viruses synthesize influenza polypeptides that are precipitable with specific antisera and that have electrophoretic mobilities similar to the corresponding influenza virus polypeptides. Indirect immunofluorescence studies have shown that HA, NA, and MS2 proteins migrate to the cell surface; PB2, PB1, PA, NP, and NS1 proteins migrate to the cell nucleus; and M1 and NS2 are distributed throughout the cell, although NS2 accumulates preferentially in nuclei. These transport processes occurred independently of other influenza polypeptides and are therefore attributable to the intrinsic properties of the influenza polypeptides themselves.

Antigens, Viral↗
PubMed1987

The influenza C virus glycoprotein (HE) exhibits receptor-binding (hemagglutinin) and receptor-destroying (esterase) activities.

A cDNA copy of RNA segment 4 of influenza C/Cal/78 virus was cloned into an SV40 vector and expressed in CV-1 cells. The gene product expressed from the SV40 recombinant virus was immunoprecipitated by monoclonal antibodies directed against the influenza C virus glycoprotein. Cells infected with the recombinant virus also exhibited C virus-specific hemagglutinin and O-acetylesterase activity. This suggests that the same C virus protein is associated with receptor-binding as well as receptor-destroying activity. The latter viral activity was measured using as substrates bovine submaxillary mucin or a low molecular weight compound p-nitrophenylacetate. In analogy to the parainfluenza virus HN protein, the influenza C virus glycoprotein was termed HE, because it possesses hemagglutinin and esterase (receptor-destroying) activity.

Antibodies, Monoclonal↗
PubMed1987

Infectious influenza A and B virus variants with long carboxyl terminal deletions in the NS1 polypeptides.

An influenza A virus, A/turkey/Oregon/71, was shown by protein gel analysis to code for an NS1 protein approximately half the size of those of other influenza A viruses. Sequence analysis of the NS gene of this virus revealed a 10 nucleotide deletion resulting in an NS1 protein of only 124 amino acids. This truncated NS1 polypeptide retained its karyophilic pattern as detected by indirect immunofluorescence analysis of virus infected cells. Also, A/turkey/Oregon/71 virus grew to high titer in embryonated chicken eggs comparable to other influenza A viruses. We also identified a laboratory variant of an influenza B virus, clone 201, which codes for a truncated NS1 protein. Sequence analysis revealed a 13 nucleotide deletion resulting in a shortened NS1 protein of only 127 amino acids as compared to other influenza B virus NS1 proteins possessing a length of 281 amino acids. Again as shown for the NS1 proteins of other influenza B viruses the NS1 polypeptide of B virus clone 201 was found to localize in the nucleus of infected cells. It appears that large deletions in the carboxyl terminus of the NS1 proteins of influenza A and B viruses can be tolerated without affecting the functional integrity of the NS1 polypeptide.

Amino Acid Sequence↗
PubMed1987

Genomic RNAs of influenza viruses are held in a circular conformation in virions and in infected cells by a terminal panhandle.

The viral RNA segments in influenza virions were shown to be circular in conformation by using psoralen crosslinking methods. Electron microscopy of purified RNA following treatment of virus with the psoralen reagent 4'-aminomethyltrioxsalen (AMT) revealed circles with lengths corresponding to the individual segments. RNA blot analysis using polyacrylamide gels demonstrated that RNA from AMT-treated virus had a slowed migration, consistent with it being a single-stranded circle. Furthermore, nuclease S1 protection assays indicated that the termini of the RNA segments form an approximately 15-base-pair-long panhandle. This structure is consistent with the partial sequence complementarity that has been observed for the termini of all influenza virus RNAs. By RNA blot analysis, circular structures of viral sense RNA were also found in influenza virus-infected cells at early and late time points. The circular RNA was the predominant species at the time when the major transcription product is message RNA. This finding and the observation that the termination signal for mRNA synthesis directly abuts the panhandle suggest that a panhandle in the template viral RNA is a cis regulatory signal promoting the synthesis of mRNA instead of plus-sense template. Also, since the panhandle is present in high concentration in virions, we suggest that it is required for packaging and that the input RNA after infection is in the proper conformation for synthesis of primary transcripts.

Cross-Linking Reagents↗
PubMed1986

Evolution of human influenza A viruses over 50 years: rapid, uniform rate of change in NS gene.

Variation in influenza A viruses was examined by comparison of nucleotide sequences of the NS gene (890 bases) of 15 human viruses isolated over 53 years (1933 to 1985). Changes in the genes accumulate with time, and an evolutionary tree based on the maximum parsimony method can be constructed. The evolutionary rate is approximately 2 X 10(-3) substitution per site per year in the NS genes, which is about 10(6) times the evolutionary rate of germline genes in mammals. This uniform and rapid rate of evolution in the NS gene is a good molecular clock and is compatible with the hypothesis that positive selection is operating on the hemagglutinin (or perhaps some other viral genes) to preserve random mutations in the NS gene.

Base Sequence↗
PubMed1986

Detection of single base substitutions in influenza virus RNA molecules by denaturing gradient gel electrophoresis of RNA-RNA or DNA-RNA heteroduplexes.

Single point mutations in the NS gene of influenza virus were detected by electrophoresis of double-stranded RNA heteroduplexes in denaturing gradient gels. The heteroduplex RNAs were made by hybridization of virion RNA with SP6-derived RNA probes of varying length. Mutations located at different positions along the NS gene (890 nucleotides long) were all detected in a predictable fashion. The method of heteroduplex analysis was also successfully used in detecting single point mismatches in DNA-RNA hybrids.

Base Sequence↗
PubMed1986

Epidemiology of influenza C virus in man: multiple evolutionary lineages and low rate of change.

The nucleotide sequences of nonstructural protein (NS) genes of human influenza C viruses isolated between 1947 and 1983 were determined and compared. Assuming constant evolutionary rates, the extent of nucleotide differences among NS genes does not correspond to the isolation years of the strains. This suggests that more than one gene lineage is present in the population. Furthermore, examination of the eight C virus NS gene sequences by the maximum parsimony method (W. M. Fitch, 1971, Syst. Zool. 20, 406-416) yielded phylogenetic trees that were grossly different from those obtained using the hemagglutinin genes for the same eight isolates. This result is compatible with the idea of reassortment of genes in nature across lineages of influenza C viruses. The sequence analysis also suggests that nucleotide substitutions occur at a lower rate in the C virus NS genes than in influenza A virus NS genes.

Amino Acid Sequence↗
PubMed1986

The influenza C virus NS gene: evidence for a spliced mRNA and a second NS gene product (NS2 protein).

It has previously been shown that the shortest RNA (RNA 7) of influenza C viruses codes for a nonstructural (NSI) protein (Nakada et al. (1985) J. Virol. 56, 221-226). Experiments reported here indicate that RNA 7 also directs the synthesis of a second nonstructural protein via a spliced mRNA. The amino terminal 62 codons of this NS2 protein appear to be shared with the NS1 protein and the carboxyl terminal 59 amino acids are unique (derived from a +1 open reading frame in the mRNA). Although the size of the C virus NS2 protein is comparable to that of the A and B virus NS2 proteins, the overall arrangement of the C virus NS gene is quite different from that of the A and B virus NS genes. The second (+1) open reading frame of the C virus NS gene is completely overlapped by that of the NS1 protein, whereas the second (+1) open reading frame of the A and B virus NS genes extends to the 3' end of the RNA and only partially (or in some strains not at all) overlaps the NS1 open reading frame.

Amino Acid Sequence↗
PubMed1986

Expression of the three influenza virus polymerase proteins in a single cell allows growth complementation of viral mutants.

Transformed cell lines derived from murine C127 cells were constructed that express the influenza virus RNA-dependent RNA polymerase proteins (PA, PB1, and PB2). Cell lines that express only one or all three of the proteins were tested for their ability to complement temperature-sensitive viral mutants incubated at the nonpermissive temperature. Two cell lines were isolated that express all three polymerase genes and complement the growth of PB2 temperature-sensitive mutants at the nonpermissive temperature. One of these lines also complemented PA temperature-sensitive mutants. The viral titers obtained in these two cell lines were 12-fold to 1000-fold higher than the viral titers obtained upon growth of the corresponding temperature-sensitive mutant in C127 cells at the nonpermissive temperature.

Animals↗
PubMed1986

Rapid RNA sequencing using double-stranded template DNA, SP6 polymerase, and 3'-deoxynucleotide triphosphates.

A simple and efficient nucleic acid sequencing method is described in which RNA transcription by the SP6 polymerase is specifically terminated using 3'-deoxynucleotide triphosphates. Initial difficulties in resolving the RNA ladder were overcome by replacing guanosine triphosphate by inosine triphosphate in the reaction mixture and electrophoresing gels at high temperature (50 degrees C). This method presents advantages over current sequencing techniques: Unprocessed plasmid DNA is the template and preparation of inserts and/or single-stranded templates is unnecessary. Use of the specific promoter for SP6 polymerase removes the need for a primer in sequencing reactions.

Base Sequence↗
PubMed1986

Measurement of the mutation rates of animal viruses: influenza A virus and poliovirus type 1.

Epidemiologic and genetic evidence suggests that influenza A viruses evolve more rapidly than other viruses in humans. Although the high mutation rate of the virus is often cited as the cause of the extensive variation, direct measurement of this parameter has not been obtained in vivo. In this study, the rate of mutation in tissue culture for the nonstructural (NS) gene of influenza A virus and for the VP1 gene in poliovirus type 1 was assayed by direct sequence analysis. Each gene was repeatedly sequenced in over 100 viral clones which were descended from a single virion in one plaque generation. A total of 108 NS genes of influenza virus were sequenced, and in the 91,708 nucleotides analyzed, seven point changes were observed. A total of 105 VP1 genes of poliovirus were sequenced, and in the 95,688 nucleotides analyzed, no mutations were observed. We then calculated mutation rates of 1.5 X 10(-5) and less than 2.1 X 10(-6) mutations per nucleotide per infectious cycle for influenza virus and poliovirus, respectively. We suggest that the higher mutation rate of influenza A virus may promote the rapid evolution of this virus in nature.

Base Sequence↗
PubMed1986

Human acid beta-glucosidase: Northern blot and S1 nuclease analysis of mRNA from HeLa cells and normal and Gaucher disease fibroblasts.

Human acid beta-glucosidase (beta-Glc) mRNA was evaluated by dot blot, Northern blot, and S1 nuclease analyses of extracts of HeLa cells and cultured fibroblasts from normal individuals and Gaucher disease patients. Dot blot quantitation indicated an equal concentration of specific mRNA in all sources. Northern blot analyses demonstrated the presence of three poly (A)+ mRNAs of about 5,600, 2,500, and 2,000 nucleotides in length from all cell extracts. All three mRNAs were present in normal amounts in fibroblast extracts from several subtypes and variants of Gaucher disease. The largest poly (A)+ mRNA species was thought to represent an unspliced nuclear precursor for the two smaller beta-Glc mRNAs. S1 nuclease analyses, using SP6 transcripts of beta-Glc cDNA, indicated that the 2,000 nucleotide mRNA differs from the 2,500 nucleotide form at both the 5' and 3' ends. These results are consistent with the use of an alternate 5' splice site and 3' polyadenylation signal. These results also suggest that the subtype and variants of Gaucher disease result from single base alterations that lead to the synthesis of defective beta-Glc.

Cells, Cultured↗
PubMed1985

Noncumulative sequence changes in the hemagglutinin genes of influenza C virus isolates.

Sequence analysis and comparison of hemagglutinin (HA) genes of different influenza C viruses isolated between 1947 and 1983 reveals that (1) the extent of difference among the HA genes is independent of the year in which these viruses were isolated and that (2) changes in the HA genes do not appear to accumulate with time. These results suggest that epidemiologically dominant variants of influenza C viruses do not emerge successively with time and that C virus variants derived from multiple evolutionary pathways cocirculate at any one time. Thus the epidemiology of influenza C viruses differs markedly from that of influenza A viruses, which is characterized by the emergence of successive variants. Based on the nucleotide sequence data, we propose different evolutionary models for influenza A and influenza C viruses.

Amino Acid Sequence↗