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R D Possee

Publications and source records attributed to R D Possee.

46 records · Page 3Linked to original sources
PubMed1989

Conservation of polyhedrin gene promoter function between Autographa californica and Mamestra brassicae nuclear polyhedrosis viruses.

The DNA sequence of the polyhedrin gene of the Mamestra brassicae multiple nucleocapsid nuclear polyhedrosis virus (MbMNPV) was determined and compared with the polyhedrin genes of Autographa californica (Ac) and Panolis flammea (Pf) MNPVs. Using this information, a transfer vector was constructed based on the EcoRI I fragment of AcMNPV in which the polyhedrin promoter was replaced by the homologous region extending 481 nucleotides upstream from the MbMNPV polyhedrin coding sequence. The Escherichia coli lacZ gene was also included downstream from the putative MbMNPV promoter. Cotransfection of this transfer vector with wild-type AcMNPV DNA produced stable recombinant viruses expressing the lacZ gene under the control of the MbMNPV polyhedrin promoter. The levels of beta-galactosidase produced by these recombinants in infected cells were 30% lower than the expression level obtained from viruses with the authentic AcMNPV promoter in front of the lacZ gene. The MbMNPV promoter has thus been shown to function efficiently in the genetic environment of AcMNPV. The implications of this finding for the release of genetically manipulated baculovirus insecticides and for the construction of baculovirus multiple expression vectors are discussed.

Amino Acid Sequence↗
PubMed1989

Analysis of the promoter of the Autographa californica nuclear polyhedrosis virus p10 gene.

Functional analyses of the p10 gene promoter from the Autographa californica nuclear polyhedrosis virus (AcNPV) were performed by progressively deleting the 230 nucleotides upstream from the p10 coding sequences towards the ATG codon. Truncated promoter sequences retaining the full 5' non-coding leader of p10 were inserted in front of the chloramphenicol acetyltransferase (CAT) gene, and promoter activity in transfected AcNPV-infected cells was measured using the transient CAT expression assay. The removal of sequences to a position 101 nucleotides upstream from the p10 ATG did not affect the level of CAT expression. Deletion of a further 13 nucleotides reduced CAT expression by three- to fourfold, but the removal of three more nucleotides, which deleted most of the baculovirus very late gene transcription consensus sequence, almost completely abolished activity. The removal of the TATA motif had no effect on the level of transient expression. We conclude that a sequence of about 101 nucleotides upstream from the ATG codon of p10 is sufficient for high level promoter activity in this transient system.

Animals↗
PubMed1989

Analysis of transcription initiation in the Panolis flammea nuclear polyhedrosis virus polyhedrin gene.

The nucleotide sequence of the polyhedrin gene of Panolis flammea multiple nucleocapsid polyhedrosis virus (PfMNPV) has been determined. The coding sequences of this gene shared 82% similarity at the DNA level and 88% similarity at the protein level with the polyhedrin gene from Autographa californica (Ac) MNPV. A single nucleotide deviation from the consensus transcription initiation sequence for baculovirus very late genes was identified in the PfMNPV polyhedrin gene. RNA was prepared from Mamestra brassicae larvae infected with PfMNPV and compared with RNA harvested at 24 h post-infection from AcMNPV-infected Spodoptera frugiperda cells using Northern blotting with an AcMNPV polyhedrin gene-specific probe. The PfMNPV mRNA was estimated to be 1.0 kb compared with a larger size of 1.15 kb for the AcMNPV polyhedrin mRNA. A cDNA copy of the 5' end of the PfMNPV polyhedrin mRNA was made using the technique of primer extension and sequenced to demonstrate that the point of transcription initiation was similar to that of AcMNPV polyhedrin mRNA.

Animals↗
PubMed1988

Functional analysis of the p10 gene 5' leader sequence of the Autographa californica nuclear polyhedrosis virus.

Deletions were made in the 5' non-coding (leader) sequence of the Autographa californica nuclear polyhedrosis virus (AcNPV) p10 gene which progressively removed nucleotides upstream from the ATG translation initiation codon. The effect of these deletions on p10 gene expression was studied using a transient expression assay. Fragments containing the putative promoter and the entire or partly deleted 5' leader sequence of the p10 gene were inserted in front of the chloramphenicol acetyltransferase (CAT) gene in the pSVO-CAT construct. Transfection of AcNPV-infected Spodoptera frugiperda cells with these plasmids resulted in higher CAT expression with increasing representation of the 5' leader sequence. The lowest level of CAT expression was found with a construct containing only 10% of the 5' leader sequence, but this was enhanced on average by 50-fold if the entire 5' leader sequence was retained. The results indicate that the entire 5' leader sequence of the p10 gene is necessary for the high level of expression. The normal transcription initiation site was utilized in the transient expression of CAT. The data are discussed in relation to the strong promoter of the baculovirus polyhedrin gene.

Animals↗
PubMed1987

Analysis of the polyhedrin gene promoter of the Autographa californica nuclear polyhedrosis virus.

The polyhedrin gene promoter of the Autographa californica nuclear polyhedrosis virus was analysed with respect to which sequences are required upstream of the mRNA transcription initiation (CAP) site for efficient promoter activity. Insertions (8, 95 and 785 nucleotides) were made in this region at an EcoR V site between the CAAT- and TATA-like boxes. When these mutations were introduced into the virus they did not affect the activity of the polyhedrin promoter as judged by expression of the beta-galactosidase (lacZ) gene inserted in lieu of the polyhedrin coding sequences. Deletions were made in the promoter which progressively removed sequences upstream from the CAP site. Removal of the TATA motif did not affect lacZ gene expression. A sequence 69 nucleotides upstream to the normal position of the polyhedrin ATG translation initiation codon was sufficient for maximum promoter activity but this was reduced by 90% when only 56 nucleotides upstream remained. The normal CAP site was utilized by each deletion mutant. Promoter activity was undetectable when the CAP site was deleted. The results are discussed in relation to other eukaryotic promoters.

Cloning, Molecular↗
PubMed1987

Baculovirus expression vectors: the requirements for high level expression of proteins, including glycoproteins.

The requirements for high level expression of three foreign proteins using the polyhedrin gene promoter of Autographa californica nuclear polyhedrosis virus (AcNPV, Baculoviridae) have been investigated. In Spodoptera frugiperda cells infected with the appropriate recombinant baculoviruses, the synthesis of the two S RNA coded genes of lymphocytic choriomeningitis virus (LCMV; i.e. the nucleoprotein, N, and glycoprotein precursor, GPC), or the haemagglutinin gene of influenza A virus, appears to be related to the degree of integrity of the 5' upstream sequence of the polyhedrin gene. No effect on the level of N protein expression was detected when all the polyhedrin gene coding sequences or some of the immediate 3' downstream sequences were deleted. Using the most efficient expression viruses derived from a new transfer vector, pAcYM1, it has been estimated that LCMV N protein represented approximately 50% of the total cellular protein, an observation consistent with the presence of numerous inclusion bodies in the cytoplasm of infected cells. For recombinant viruses derived from the pAcYM1 transfer vector containing the LCMV GPC gene, the level of synthesis of the arenavirus glycoprotein was equivalent to approximately 20% of the cellular protein. Thin sections of cells infected with the GPC recombinant revealed a highly vacuolated cytoplasm.

Base Sequence↗
PubMed1986

Cell-surface expression of influenza virus haemagglutinin in insect cells using a baculovirus vector.

A baculovirus transfer vector was constructed using a cloned copy of the Autographa californica nuclear polyhedrosis virus (AcNPV) polyhedrin gene. The ATG translation initiation codon and 32 bases of the polyhedrin mRNA 5' leader sequences were deleted and replaced with a cDNA clone of the A/PR/8/34 influenza virus haemagglutinin (HA) gene. The hybrid HA/polyhedrin gene was transferred to AcNPV and polyhedrin-negative recombinants identified which expressed HA activity. The insect cell-derived HA bound a monoclonal antibody specific for the HA gene in immunofluorescence tests and was shown to be plasma membrane-bound by haemadsorption assays. A 65 kDa protein was identified in infected cells by immunoprecipitation. The mRNA from recombinant-infected cells was analysed and revealed a major transcript of 2.95 kilobases consistent with transcription initiation and termination at the authentic polyhedrin sites. Other transcripts of different length were also detected so it was not possible to conclude which was responsible for producing the protein. The results demonstrated that AcNPV can be used to express plasma membrane-bound virus glycoproteins.

Animals↗
PubMed1986

Expression of the S-coded genes of lymphocytic choriomeningitis arenavirus using a baculovirus vector.

A DNA copy of the lymphocytic choriomeningitis virus (LCMV, WE strain)S RNA species has been inserted in both orientations into plasmids containing a 7.1 kb DNA sequence of the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV). The inserts were placed behind the polyhedrin gene promoter of AcNPV and in the place of the nucleotide sequences including and flanking the polyhedrin gene translation initiation codon. The derived plasmids were used to obtain recombinant AcNPV viruses after transfection of Spodoptera frugiperda cells in the presence of infectious AcNPV DNA and the selection of polyhedrin-negative viruses. The expression of the two LCMV S-coded genes, the nucleoprotein and glycoprotein precursor, in S. frugiperda cells by the recombinant baculoviruses is described. Based on the results obtained with three different expression vectors, the site of insertion of the foreign genes in the 5' non-coding region of the polyhedrin gene appears to be an important determinant of the level of expression obtained.

Animals↗
PubMed1982

Studies on the mechanism of neutralization of influenza virus by antibody: evidence that neutralizing antibody (anti-haemagglutinin) inactivates influenza virus in vivo by inhibiting virion transcriptase activity.

Influenza viruses, which had lost up to 99.999% infectivity by incubation with antibody (a) specific for the haemagglutinin (HA) or with monoclonal alpha-HA, attached on to and penetrated chick embryo fibroblast (CEF) cells to the same extent as non-neutralized virus. Neutralized virus was also uncoated efficiently as shown by the accumulation of virion RNA in the nucleus and virion envelope in the cytoplasm. Polyacrylamide gel electrophoresis of virion RNA segments recovered from the nucleus or cytoplasm of cells inoculated with neutralized or non-neutralized virus showed that antibody did not potentiate degradation of RNA. However, these RNAs were not expressed since virus-induced proteins were not detected in cells to which neutralized virus had been added. Assay of virion transcriptase of neutralized virus in vitro showed that its activity was reduced up to sevenfold compared with non-neutralized virus, and annealing studies showed that no detectable transcription took place in vivo with neutralized virus. These studies support the conclusion that antibody directed specifically against the HA protein on the outer surface of the influenza virus particle neutralizes infectivity by inactivating virion transcriptase activity and it is suggested that antibody to HA brings about allosteric rearrangements in the HA molecule which are transmitted across the virus envelope to the interior of the particle.

Animals↗