Biomedical subjects

P Palese

Publications and source records attributed to P Palese.

At least 91 records · Page 5Linked to original sources

PubMed1991

Positive Darwinian evolution in human influenza A viruses.

We earlier suggested that type A human influenza virus genes undergo positive Darwinian selection through immune surveillance. This requires more favorable amino acid replacements fixed in antigenic sites among the surviving lineages than among the extinct lineages. We now show that viral hemagglutinins fix proportionately more amino acid replacements in antigenic sites in the trunk of the evolutionary tree (survivors) than in the branches (nonsurvivors), demonstrating that type A human influenza virus is undergoing positive Darwinian evolution. The hemagglutinin gene is evolving 3 times faster than the nonstructural gene and the average age of the sampled nonsurvivors is only 1.6 years, so that extinction is not only common but rapid.

Amino Acid Sequence↗

PubMed1991

An influenza virus containing nine different RNA segments.

The packaging mechanism of segmented RNA viruses has not been well studied. Specifically, it has not been clear whether influenza A viruses package only eight RNA segments or whether virus particles contain more than eight segments. Using a newly developed ribonucleoprotein (RNP) transfection method, we engineered an influenza virus which must contain nine different RNA segments rather than the usual eight in order to survive under the experimental growth conditions. This result is compatible with a mechanism of packaging which allows influenza virus to encapsidate more than eight RNA segments. We also suggest that the virus packages its RNAs randomly and that this random packaging results in infectious viruses with the required ("right") complement of RNA segments.

Animals↗

PubMed1991

High-efficiency formation of influenza virus transfectants.

cDNA-derived RNAs were introduced into the genomes of influenza viruses by using an improved ribonucleoprotein (RNP) transfection protocol. Up to 10(5) viral transfectants with a novel neuraminidase gene could be obtained by using a 35-mm dish (10(6) cells) for RNP transfection. In addition to genes coding for surface proteins (hemagglutinin and neuraminidase), we also exchanged a gene coding for nonsurface proteins. The cDNA-derived influenza A/PR/8/34 virus NS gene was introduced into a temperature-sensitive mutant with a defect in this gene. We suggest that the term influenza virus transfectant be used for those viruses which are made by RNP transfection with cDNA-derived RNA.

Animals↗

PubMed1991

The polyadenylation signal of influenza virus RNA involves a stretch of uridines followed by the RNA duplex of the panhandle structure.

Appropriate RNAs are transcribed and amplified and proteins are expressed after transfection into cells of in vitro-reconstituted RNA-protein complexes and infection with influenza virus as the helper. This system permits us to study the signals involved in transcription of influenza virus RNAs. For the analysis we used a plasmid-derived RNA containing the reporter gene for chloramphenicol acetyltransferase (CAT) flanked by the noncoding sequences of the NS RNA segment of influenza A/WSN/33 virus. Mutations were then introduced into both the 5' and 3' ends, and the resulting RNAs were studied to determine their transcription in vitro and their CAT expression activity in the RNA-protein transfection system. The results reveal that a stretch of uninterrupted uridines at the 5' end of the negative-strand RNA is essential for mRNA synthesis. Also, a double-stranded RNA "panhandle" structure generated by the 5'- and 3'-terminal nucleotides appears to be required for polyadenylation, since opening up of these base pairs diminished mRNA synthesis and eliminated expression of CAT activity by the mutant RNAs. Finally, it was shown that this double-stranded RNA structural requirement is not sequence specific, since a synthetic GC clamp can replace the virus-coded RNA duplex. The data suggest that the viral RNA polymerase adds poly(A) by a slippage (stuttering) mechanism which occurs when it hits the double-stranded RNA barrier next to the stretch of uridines.

Base Composition↗

PubMed1991

Evolutionary relatedness of the predicted gene product of RNA segment 2 of the tick-borne Dhori virus and the PB1 polymerase gene of influenza viruses.

The complete nucleotide sequence of the second largest RNA segment of Dhori/India/1313/61 virus was determined and the deduced amino acid sequence was compared with the polymerase (P) proteins of influenza A, B, and C viruses. RNA segment 2 (2224 nucleotides) of Dhori virus contains a single long open reading frame that can encode a 716-amino acid polypeptide (81.3 kDa). The predicted polypeptide shares between 27 and 31% sequence identities with the PB1 polypeptides of influenza A, B, and C viruses. Among the regions most highly conserved are the sequences around the Asp-Asp motif common to many RNA polymerases. In spite of the high level of sequence identity between the Dhori RNA segment 2 gene product and the influenza A, B, and C virus PB1 proteins the amino acid composition of the Dhori protein indicates an acidic charge feature at pH 7.0 in contrast to the basic nature of the PB1 proteins of the influenza viruses. We suggest that the Dhori PB1-like protein be designated the P alpha protein of this virus.

Amino Acid Sequence↗

PubMed1990

Introduction of site-specific mutations into the genome of influenza virus.

We succeeded in rescuing infectious influenza virus by transfecting cells with RNAs derived from specific recombinant DNAs. RNA corresponding to the neuraminidase (NA) gene of influenza A/WSN/33 (WSN) virus was transcribed in vitro from plasmid DNA and, following the addition of purified influenza virus RNA polymerase complex, was transfected into MDBK cells. Superinfection with helper virus lacking the WSN NA gene resulted in the release of virus containing the WSN NA gene. We then introduced five point mutations into the WSN NA gene by cassette mutagenesis of the plasmid DNA. Sequence analysis of the rescued virus revealed that the genome contained all five mutations present in the mutated plasmid. The ability to create viruses with site-specific mutations will allow the engineering of influenza viruses with defined biological properties.

Animals↗

PubMed1990

Inhibition of influenza virus replication by phosphorothioate oligodeoxynucleotides.

Oligodeoxynucleotides (ODNs) were synthesized and tested for their antiviral activity against influenza viruses. ODNs corresponded to the polymerase PB1 gene of either influenza A/WSN/33 virus or influenza C/JJ/50 virus. All compounds were 20 nucleotides long, including control ODNs containing mismatches. The phosphodiester ODNs (O-ODNs) failed to inhibit replication of influenza A and influenza C viruses at concentrations up to 80 microM, possibly due to intracellular nuclease digestion of the unmodified oligomers. By contrast, the phosphorothioate derivatives (S-ODNs) were found to inhibit replication of both influenza A and influenza C virus. The antiviral effect of S-ODNs against influenza A virus was found at concentrations as low as 1.25 microM and was present with mismatched oligomers. In the case of influenza C virus, the S-ODN complementary to the 3' end of the viral RNA of the PB1 gene revealed a sequence-specific antiviral activity at a concentration of 20 microM. (At the same concentration, S-ODNs with one or three mismatches showed little or no antiviral activity.) Reduction in plaque number reached six logarithms when this sequence-specific S-ODN was used at a concentration of 80 microM.

Animals↗

PubMed1990

Determination of influenza virus proteins required for genome replication.

An artificial vaccinia virus vector-driven replication system for influenza virus RNA has been developed. In this system, a synthetic NS-like gene is replicated and expressed by influenza virus proteins supplied through infection with vaccinia virus recombinant vectors. The minimum subset of influenza virus proteins needed for specific replication and expression of the viral ribonucleoprotein was found to be the three polymerase proteins (PB1, PB2, and PA) and the nucleoprotein.

Cloning, Molecular↗

PubMed1989

Amplification, expression, and packaging of foreign gene by influenza virus.

A system is described that allows use of recombinant DNA technology to modify the genome of influenza virus, a negative-strand RNA virus, and to engineer vectors for the expression of foreign genes. Recombinant RNA is expressed from plasmid DNA in which the coding sequence of the influenza A virus NS gene is replaced with that of the chloramphenicol acetyltransferase gene. When transfected with purified influenza A virus polymerase proteins--in the presence of helper virus--the recombinant RNA is amplified, expressed, and packaged into virus particles, which can be passaged several times. The data indicate that the 22 5' terminal and the 26 3' terminal bases of the influenza A virus RNA are sufficient to provide the signals for RNA transcription, RNA replication, packaging of RNA into influenza virus particles.

Animals↗

PubMed1989

Comparison of the three large polymerase proteins of influenza A, B, and C viruses.

The three large RNA segments of influenza C virus C/JJ/50 were cloned and sequenced, and the deduced amino acid sequences were compared with those of the polymerase (P) proteins of influenza A and B viruses. The coding strategy of the C virus RNA segments is the same as that for the large A and B virus segments as one long open reading frame is present in each segment. RNA segment 1 of influenza C virus encodes the equivalent of the PB2 protein; it has an approximate 25% sequence identity with the corresponding (cap binding) influenza A and B virus PB2 proteins. The PB1 protein of influenza C virus, coded for by segment 2, has an approximate 40% sequence identity with the corresponding proteins of influenza A and B viruses including the Asp-Asp sequence motif found in many RNA polymerase molecules. The PB1 polymerase is thus the most highly conserved protein among the influenza A, B, and C viruses. Although the protein coded for by RNA 3 of influenza C virus shows an approximate 25% sequence identity with the acid polymerase (PA) proteins of the A and B viruses, its sequence does not display any acid charge features at neutral pH. This protein is thus referred to as the P3 (rather than the PA) protein of influenza C virus.

Amino Acid Sequence↗

PubMed1989

Expression of antisense RNA fails to inhibit influenza virus replication.

Cell lines were constructed which permanently express influenza virus-specific RNA. Two approaches were followed. C127 cells were transformed with bovine papilloma virus (BPV) vectors and the resulting cell lines were found to inhibit the replication of influenza virus at low multiplicity of infection (MOI 0.05). However, examination of cellular RNA using single-stranded probes revealed the presence of both (+)sense and antisense RNA transcripts (45-70 copies per cell). In this BPV-based system the inhibitory activity appeared to be associated with a non-specific, interferon (IFN)-mediated effect. In the second approach, an expression system was used which involved 293 cells, a chimeric human cytomegalovirus (CMV)/human immunodeficiency virus (HIV) promoter, and methotrexate- (Mtx)-mediated gene amplification. Cells were found to express up to 7500 copies of influenza virus-specific RNA per cell at a steady state level. In this system no RNA transcripts of the opposite orientation were found. However, all cell lines permanently expressing either (-)sense or (+)sense viral RNA failed to reduce influenza virus titers in a multi-cycle replication experiment (MOI 0.01).

Animals↗

PubMed1989

Complementation and analysis of an NP mutant of influenza virus.

Cell lines were constructed so as to express the influenza A virus nucleoprotein (NP) at levels approximating 5% of the total NP made throughout virus infection. Two types of cell lines were analyzed. One cell line (NP-5) expresses only the NP while another cell line was constructed which expresses the three viral polymerase proteins in addition to the NP (3PNP-4). Both cell lines were able to complement the growth of an NP mutant, ts56, at the non-permissive temperature. The 3PNP-4 cell line, constructed by transfecting a cell line already expressing the three polymerase proteins, continued to be able to complement viral PB2 mutants. In addition, sequence analysis was performed on the NP gene segment of A/WSN/33 and ts56 viruses. This analysis revealed that the mutant phenotype exhibited by ts56 at non-permissive temperature is due to a single serine to asparagine change (at codon 332) within the protein.

Amino Acid Sequence↗

PubMed1989

Promoter analysis of influenza virus RNA polymerase.

Influenza virus polymerase, which was prepared depleted of viral RNA, was used to copy small RNA templates prepared from plasmid-encoded sequences. Template constructions containing only the 3' end of genomic RNA were shown to be efficiently copied, indicating that the promoter lay solely within the 15-nucleotide 3' terminus. Sequences not specific for the influenza virus termini were not copied, and, surprisingly, RNAs containing termini identical to those from plus-sense cRNA were copied at low levels. The specificity for recognition of the virus sense promoter was further defined by site-specific mutagenesis. It was also found that increased levels of viral protein were required in order to catalyze both the cap endonuclease-primed and primer-free RNA synthesis from these model templates, as well as from genomic-length RNAs. This finding indicates that the reconstituted system has catalytic properties very similar to those of native viral ribonucleoprotein complexes.

Base Sequence↗

PubMed1989

Influenza C virus esterase: analysis of catalytic site, inhibition, and possible function.

The active site serine of the acetylesterase of influenza C virus was localized to amino acid 71 of the hemagglutinin-esterase protein by affinity labeling with 3H-labeled diisopropylfluorophosphate. This serine and the adjacent amino acids (Phe-Gly-Asp-Ser) are part of a consensus sequence motif found in serine hydrolases. Since comparative analysis failed to reveal esterase sequence similarities with other serine hydrolases, we suggest that this viral enzyme is a serine hydrolase constituting a new family of serine esterases. Furthermore, we found that the influenza C virus esterase was inhibited by isocoumarin derivatives, with 3,4-dichloroisocoumarin being the most potent inhibitor. Addition of this compound prevented elution of influenza C virus from erythrocytes and inhibited virus infectivity, possibly through inhibition of virus entry into cells.

Amino Acid Sequence↗

PubMed1988

Influenza B virus evolution: co-circulating lineages and comparison of evolutionary pattern with those of influenza A and C viruses.

Sequence analyses and comparison of the genes coding for the nonstructural (NS) and hemagglutinin (HA) proteins of different influenza B viruses isolated between 1940 and 1987 reveal that the number of substitutions is not always proportional to the time between isolates. Examination of 14 influenza B virus NS gene and 10 HA gene sequences by the maximum parsimony method suggested that--as with influenza C viruses--there are multiple evolutionary lineages which can coexist for considerable periods of time. Comparison of the sequence divergence among genes of viruses belonging to type A, B, and C virus suggests that, in man, influenza B viruses evolve slower than A viruses and faster than C viruses. We propose an evolutionary model for influenza B viruses that is intermediate between the pattern for human influenza A viruses and that for influenza C viruses.

Amino Acid Sequence↗

PubMed1988

Novel method of detecting single base substitutions in RNA molecules by differential melting behavior in solution.

We have developed a quick and reliable way of detecting point mutations in RNA molecules. This method involves melting RNA-RNA heteroduplexes of varying lengths in a series of tubes containing a stepwise salt or formamide gradient, followed by polyacrylamide gel electrophoresis to distinguish between single- and double-stranded RNA molecules. The manipulations required are technically simple, and the method is sensitive enough to detect destabilization of the highest melting domain of a dsRNA duplex by a single base mismatch. When this method is used in parallel with denaturing gradient gel electrophoresis, which detects point mutations in low-melting domains of duplexes, it should now be possible to rapidly screen for mutations located throughout the length of any RNA molecule whose wild-type sequence is known.

Base Composition↗

PubMed1988

Human and bovine coronaviruses recognize sialic acid-containing receptors similar to those of influenza C viruses.

Human coronavirus OC43 and bovine coronavirus elute from agglutinated chicken erythrocytes when incubated at 37 degrees C, suggesting the presence of a receptor-destroying enzyme. Moreover, bovine coronavirus exhibits an acetylesterase activity in vitro using bovine submaxillary mucin as substrate similar to the enzymatic activity found in influenza C viruses. Furthermore, pretreatment of erythrocytes with either influenza C virus or bovine coronavirus eliminates subsequent binding and agglutination by either coronaviruses or influenza C virus, whereas binding of influenza A virus remains intact. In addition, hemagglutination by coronaviruses can be inhibited by pretreatment of erythrocytes with Arthrobacter ureafaciens or Clostridium perfringens neuraminidase or by addition of sialic acid-containing gangliosides. These results suggest that, like influenza C viruses, human coronavirus OC43 and bovine coronavirus recognize O-acetylated sialic acid or a similar derivative as cell receptor.

Animals↗

PubMed1988

Gaucher disease type 1: cloning and characterization of a cDNA encoding acid beta-glucosidase from an Ashkenazi Jewish patient.

Gaucher disease (GD) type 1 is the most prevalent lysosomal storage disease and the most prevalent genetic disease among the Ashkenazi Jews. The defective activity of acid beta-glucosidase is the enzymatic basis of GD and is inherited as an autosomal recessive trait. To investigate the genetic basis of Ashkenazi Jewish GD type 1, a cDNA encoding acid beta-glucosidase was isolated from a cDNA library constructed using splenic poly(A)+RNA from a patient. The cDNA was sequenced to identify mutations, and the presence of a single missense mutation in the patients' genome was confirmed by selective oligonucleotide hybridization and by restriction endonuclease digestion analyses of amplified genomic sequences. This G----A transition (Arg-119 to Gln-119) was present heterozygously in the index patient and his affected third cousin but was not present in normal non-Jewish individuals. This mutation is the second single base mutation found in Ashkenazi Jewish GD type 1 patients. Furthermore, results obtained with the affected third cousin suggest that at least three mutant alleles may be present in this GD subpopulation.

Child↗