I. ESTABLISHMENT OF A SENSITIVE ASSAY SYSTEM FOR THE DETECTION OF NONSENSE SUPPRESSOR TRANSFER-RNA ACTIVITY IN HIGHER EUKARYOTIC CELLS AND II. ANALYSIS OF INFECTIOUS INFLUENZA A AND B VIRUS VARIANTS WITH LONG CARBOXYL TERMINAL DELETIONS IN THE NS1 POLYPEPTIDES AND III. EXAMINATION OF INFLUENZA VIRUS GENE PRODUCTS EXPRESSED BY VACCINIA VIRUS RECOMBINANTS.
Item
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Title
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I. ESTABLISHMENT OF A SENSITIVE ASSAY SYSTEM FOR THE DETECTION OF NONSENSE SUPPRESSOR TRANSFER-RNA ACTIVITY IN HIGHER EUKARYOTIC CELLS AND II. ANALYSIS OF INFECTIOUS INFLUENZA A AND B VIRUS VARIANTS WITH LONG CARBOXYL TERMINAL DELETIONS IN THE NS1 POLYPEPTIDES AND III. EXAMINATION OF INFLUENZA VIRUS GENE PRODUCTS EXPRESSED BY VACCINIA VIRUS RECOMBINANTS.
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Identifier
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AAI8708308
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identifier
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8708308
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Creator
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NORTON, GERARD PATRICK.
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Contributor
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Peter Palese
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Date
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1987
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Microbiology
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Abstract
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Suppressor tRNA genes were cloned into the late region of SV40 DNA and the recombinant vectors were used to express suppressor tRNA activity in infected CV-1 cells. The amount of readthrough NS1 gene product after superinfection of these cells with influenza viruses indicated that the efficiency of suppression was 31-49%. Permanent CHO cell lines expressing suppressor tRNA activity were also established (Ho et al., 1986). Cells containing an average of 200 copies of the amber suppressor tRNA gene showed a 3% level of suppression. An improved detection of the readthrough NS1 protein by Western blot analysis allowed identification of suppressor tRNA activity of only 1% in cells containing an average of 20 copies of the suppressor tRNA gene. Thus the suppressor tRNA activity in permanently expressing cells correlates with the number of copies of suppressor tRNA genes contained in the cells.;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 reading frame of only 124 amino acids. This truncated NS1 polypeptide retained its karyophilic pattern as detected by immunofluorescence analysis of virus infected cells.;A laboratory variant of an influenza B virus, clone 201, was identified which codes for a truncated NS1 protein. Sequence analysis of the NS gene revealed a 13 nucleotide deletion resulting in a shortened NS1 protein of only 127 amino acids. Two other influenza B virus NS genes which have been sequenced possess NS1 proteins of 281 amino acids. As shown for the NS1 proteins of other influenza B viruses, the truncated 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.;Vaccinia virus recombinants containing the cloned hemag- glutinin (HA) genes of influenza viruses, A/PR/8/34 (H1 subtype) and A/Japan/305/57 (H2 subtype), directed the expression of influenza viral HA proteins in infected CV-1 cells. Western blot analysis revealed that these vaccinia-HA recombinant vectors produced levels of HA proteins approximately 1/2-1/5 of those seen in cells infected with the appropriate influenza viruses. (Abstract shortened with permission of author.).
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
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Program
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Biomedical Sciences
