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P Faulkner

Publications and source records attributed to P Faulkner.

At least 37 records · Page 2Linked to original sources
PubMed1989

Identification of p74, a gene essential for virulence of baculovirus occlusion bodies.

DNA sequencing of the HindIII-P fragment of the baculovirus Autographa californica nuclear polyhedrosis virus downstream of a major late protein, p10, revealed the presence of an open reading frame (ORF) 1935 nucleotides in length and in opposite polarity to p10. The gene product is considered essential for virus virulence in Trichoplusia ni larvae since infection with occlusion bodies from a mutant, Ac228z, in which portions of adjacent carboxy-termini from peptides p74 and p10 were deleted, failed to kill larvae, whereas virus with deletions in p10 alone were as infectious to larvae as wild-type virus. The ORF has the potential to code for a polypeptide of 645 amino acid residues (Mr 73,819) and was designated p74. Time course analysis of RNA from infected cells using primer extension assays suggested that the gene's promoter was weak and was most active at 16-20 hr postinfection. The transcription initiation site of the RNA was located at -90/-91 bases upstream of the start codon. The p74 gene was cloned into a baculovirus expression vector and a recombinant virus was produced which overexpressed the p74 protein.

Amino Acid Sequence↗
PubMed1989

A cytopathological investigation of Autographa californica nuclear polyhedrosis virus p10 gene function using insertion/deletion mutants.

The role of the Autographa californica nuclear polyhedrosis virus p10 gene in viral cytopathology and morphogenesis was examined using classes of p10 deletion mutants with and without lacZ (beta-galactosidase) gene fusion. Mutant-infected cells did not form the fibrillar cytoplasmic and nuclear structures normally observed late in infection with wild-type (wt) virus, and the cells failed to lyse even at 2 weeks post-infection. Based on wt and mutant cytopathology, we suggest lysis may be facilitated by stepwise exhaustion of the host nuclear membrane, and may require a function resident in the carboxy region of p10; this portion of the molecule is also essential for formation of the p10-rich fibrillar bodies. Additional changes in cytopathology were correlated with the level of p10/LacZ fusion protein expression. The insertional mutant designated Ac229, which encodes 51 N-terminal amino acids of p10 fused to LacZ, caused intranuclear accumulation of granular structures at sites corresponding to the fibrillar bodies of wt viral infections. Occlusion body membranes, which associate with the fibrillar bodies in wt infections, were also formed in mutant virus-infected cells. However, membranes did not associate with occlusion bodies in Ac229 infections, and were aberrantly attached to occlusion bodies in cells infected with mutants having simple p10 deletions (represented by Ac231). Loss of the outer membrane increased sensitivity of the occlusion bodies to disruption by physical stress; a partially attached membrane afforded some protection from disruption.

Animals↗
PubMed1989

Identification and sequence analysis of a gene encoding gp67, an abundant envelope glycoprotein of the baculovirus Autographa californica nuclear polyhedrosis virus.

Monoclonal antibodies with specificity for the abundant envelope surface glycoprotein (gp67) of Autographa californica nuclear polyhedrosis virus (AcMNPV) were used to screen a lambda gt11 expression library of AcMNPV DNA fragments. The gp67 gene was mapped to the left end of the EcoRI H fragment in a right-to-left orientation on the consensus map of AcMNPV. A 2.1-kilobase transcript which hybridized to the region was first detected in cell extracts at 2 h postinfection; it peaked in abundance at 18 h postinfection and thereafter was present at lower levels. The nucleotide sequence of the region was determined, and a 1,590-nucleotide open reading frame flanked by an AT-rich sequence was identified that could encode a polypeptide with 529 amino acid residues (molecular mass of 60,167 daltons). Computer analysis indicated that the peptide possesses two hydrophobic regions near the N and C termini as well as six potential N-linked glycosylation sites. We suggest that following cleavage of a signal peptide, the polypeptide undergoes further processing and becomes anchored at its C terminus in the virus envelope. The final seven amino acid residues at the C terminus contain basic amino acids and may have a role in virion assembly.

Amino Acid Sequence↗
PubMed1984

Time Course Analysis and Mapping of Autographa californica Nuclear Polyhedrosis Virus Transcripts.

To study the expression of the Autographa californica nuclear polyhedrosis virus (AcNPV) genome, intracellular virus-specific proteins and mRNAs were pulsed-labeled, extracted, and analyzed at 6-h intervals during the replicative cycle. Most RNAs were detected between 12 and 24 h postinfection (p.i.), but many continued to be synthesized until late in infection. Polyhedrin and p8 mRNAs were the two most abundant late viral RNA transcripts, and they were synthesized at high rates until late in the infection cycle (60 h p.i.). The abundancy control of polyhedrin and p8 polypeptides was considered to be at the level of transcription. Two other major mRNAs in infected cells were 0.6-kilobase RNA, which was synthesized at its highest rate 12 to 18 h p.i., and 2.8-kilobase RNA, which was synthesized from 12 h p.i. until 48 h p.i. Cytoplasmic polyadenylic acid-containing RNA was isolated at 6-h intervals and was analyzed by Northern blot hybridization. At least 50 virus RNA transcripts were recognized, sized, and mapped onto the genome. Six RNAs hybridized to EcoRI-H, -I, and -J, and HindIII-Q AcNPV DNA restriction fragments, seven RNAs hybridized to EcoRI-B and -D DNA fragments, five RNAs hybridized to EcoRI-A and -E regions of the genome, four RNAs hybridized to EcoRI-C and -N DNA fragments, and one RNA species hybridized to EcoRI-O AcNPV DNA. A transcription map of the AcNPV genome was constructed, and the data were correlated with previously published translation maps.

Journal Article↗
PubMed1983

Monoclonal antibodies to baculovirus structural proteins: determination of specificities by Western blot analysis.

Conventional mouse hybridoma technology was utilized to produce a panel of monoclonal antibodies which reacted with baculovirus proteins. Using an enzyme-linked immunosorbent assay (ELISA), the hybridomas which were raised against polyhedrin from Autographa californica nuclear polyhedrosis virus (AcNPV) and Choristoeura fumiferana nuclear polyhedrosis virus (CfNPV) were found to cross-react differentially with polyhedrins and granulins from several species of baculoviruses. Hybridoma antibodies which reacted against the nonoccluded form (NOV) of AcNPV in an ELISA test expressed different specificities for the occluded form of the virus (OV), a mutant strain of AcNPV, and CfNPV. Four hybridoma clones produced antibody which neutralized the infectivity of AcNPV NOV. One hybridoma antibody reacted strongly with the uninfected Spodoptera frugiperda host cell line. Using Western blot analysis, it was shown that hybridoma antibodies against polyhedrin reacted differentially with the complete polypeptide and protease-generated fragments of polyhedrin. The polypeptide specificity of 19 of 28 hybridoma antibodies which reacted with OV and NOV of AcNPV was assigned using Western blot analysis.

Animals↗
PubMed1983

Analysis of a mutant of Autographa california nuclear polyhedrosis virus with a defect in the morphogenesis of the occlusion body macromolecular lattice.

A mutant (m-29) of Autographa californica nuclear polyhedrosis virus (AcMNPV) grew in Spodoptera frugiperda and Trichoplusia ni cells but did not form typical intranuclear occlusion bodies (OB); instead, small particles (95 to 180 nm diam.) were produced in copious amounts within nuclei. Ultrastructural studies showed that the particles did not occlude enveloped nucleocapsids and that they lacked a macromolecular paracrystalline lattice and a structure equivalent to the occlusion body envelope. The particles within nuclei stained in an immunofluorescence test with antipolyhedrin antibody and when extracted from cells the major polypeptide of a particle preparation was indistinguishable from polyhedrin when examined on SDS--polyacrylamide gels and had an identical peptide pattern following proteolysis with V8 protease. Other elements believed to be implicated in OB morphogenesis such as a proliferation of intranuclear membranes, enveloped bundles of nucleocapsids, patches of fibrous material and fibrous sheets were present in normal amounts. No alteration in the synthesis or processing of polypeptides was seen in mutant-infected cells. Analysis of m-29 DNA with BamHI, EcoRI and HindIII restriction endonucleases revealed that the HindIII restriction site at the F/V junction of viral DNA was absent in the mutant. No other modifications in the restriction patterns were detected. It is proposed that an alteration in the amino acid sequence of polyhedrin towards the--NH2 terminus of the polypeptide may account for the growth characteristics of the mutant.

Animals↗
PubMed1983

Location of Homologous DNA Sequences Interspersed at Five Regions in the Baculovirus AcMNPV Genome.

An examination of Autographa californica nuclear polyhedrosis virus DNA revealed the presence of five interspersed regions, rich in EcoRI restriction sites, which shared homologous sequences. These homologous regions (hr), designated hr(1) to hr(5), occur at or near the following EcoRI fragment junctions: hr(1)EcoRI-B-EcoRI-I (0.0 map units); hr(2), EcoRI-A-EcoRI-J (19.8 map units); hr(3), EcoRI-C-EcoRI-G (52.9 map units); hr(4), EcoRI-Q-EcoRI-L (69.8 map units); and hr(5), EcoRI-S-EcoRI-X (88.0 map units). Four of these regions were identified, by cross-blot hybridization of HindIII-restricted A. californica nuclear polyhedrosis virus DNA, to be within the HindIII-A/B, -F, -L, and -Q fragments. The location of these regions and the identification of a fifth homologous region were confirmed, and their characterization was facilitated, by using two plasmids with HindIII-L or -Q fragment insertions, which contained the homologous regions hr(2) and hr(5), respectively. The sizes of the homologous regions were about 800 base pairs for hr(2), 500 base pairs for hr(5), and less than 500 base pairs for hr(1), hr(3), and hr(4). A set of small EcoRI fragments (EcoRI minifragments) which ranged in size from 225 to 73 base pairs were detected in A. californica nuclear polyhedrosis virus DNA and HindIII-L and -Q fragments by polyacrylamide gel analysis. Some of the minifragments in viral DNA were present in extramolar amounts and corresponded in size to some of the minifragments present in HindIII-L and -Q. Clones of some of the EcoRI minifragments were used as probes in hybridizations to digests of viral DNA and of HindIII-L and -Q. The hybridization data, obtained under various levels of stringency, suggested that there was a degree of mismatching between the sequences which were responsible for the homology.

Journal Article↗
PubMed1982

Bromodeoxyuridine-induced mutants of Autographa californica nuclear polyhedrosis virus defective in occlusion body formation.

Twelve temperature-sensitive (ts) and two morphology mutants of Autographa californica nuclear polyhedrosis virus were generated using the mutagen 5-bromo-2'-deoxyuridine (BrdUrd). The ts mutants grew normally at 25 degrees C but exhibited abnormal occlusion body formation when grown at 33 degrees C whereas the morphology mutants produced an abnormal cytopathic effect at 25 degrees C and 33 degrees C. None of the mutants was severely restricted for production of non-occluded virus; thus, with the eight ts mutants and the two morphology mutants incubated at 33 degrees C the drop in titre was 0.5 to 3.0 log10 from that at 25 degrees C. The mutants fell into three groups based on the amount of polyhedrin synthesized at 25 degrees C and 33 degrees C in infected cells. The eight ts mutants were combined with ts mutants constructed in a previous series of experiments and the collection was sorted into complementation groups. The collection comprised ten groups and the new mutants were sorted into seven of these. It is concluded that gene products of these complementation groups have essential roles in occlusion body formation.

Bromodeoxyuridine↗
PubMed1982

Molecular Cloning and Physical Mapping of Restriction Endonuclease Fragments of Autographa californica Nuclear Polyhedrosis Virus DNA.

A restriction fragment library containing Autographa californica nuclear polyhedrosis virus (AcNPV) DNA was constructed by using the pBR322 plasmid as a vector. The library, which is representative of more than 95% of the viral genome, consists of 2 of the 7 BamHI fragments, 12 of the 24 HindIII fragments, and 23 of the 24 EcoRI fragments. The cloned fragments were characterized and used to generate physical maps of the genome by hybridizing nick-translated recombinant plasmid to Southern blots of AcNPV DNA digested with SmaI, BamHI, XhoI, PstI, HindIII, and EcoRI restriction endonucleases. This information was used to define our strain of AcNPV (HR3) with respect to other strains for which physical maps have been previously published. The hybridization data also indicate that reiteration of DNA sequences occurs at the HindIII-L and -Q regions of the genome.

Journal Article↗
PubMed1979

Genetic Analysis of a Baculovirus, Autographa californica Nuclear Polyhedrosis Virus I. Isolation of Temperature-Sensitive Mutants and Assortment into Complementation Groups.

Temperature-sensitive (ts) mutants were isolated from the baculovirus Autographa californica (alfalfa looper) MNPV, grown in Spodoptera frugiperda (fall armyworm) cells in the presence of N-methyl-N'-nitro-N-nitrosoguanidine. Of 567 plaque isolates screened, 27 were temperature sensitive (ts), representing a mutation frequency of 4.8%. Ten ts mutants were studied in detail: six failed to yield nonoccluded virus at 33 degrees C (NOV mutants), whereas the other four produced nonoccluded virus but were restricted in formation of polyhedra at 33 degrees C (Poly mutants). One of the six NOV mutants failed to synthesize viral DNA. Reversion and leak frequencies were determined, and the mutants were assorted into complementation groups based on the yield of polyhedrin synthesis in cells coinfected with pairs of mutants at 33 degrees C, as measured by radioimmunoassay. For NOV mutants, complementation indexes were also based on virus yield and were consistent with those based on polyhedrin synthesis. Nine mutants were assorted into five complementation groups. One mutant remained unclassified.

Journal Article↗
PubMed1978

Comparison of solid-phase radioimmunoassays for baculoviruses.

The sensitivity and cross-reaction of four solid-phase radioimmunoassays (RIA) for Trichoplusia ni nuclear polyhedrosis virus containing singly enveloped virions were investigated. The detection limits of each assay were as follows: Indirect RIA, 5 ng of dissolved polyhedron antigen; direct RIA, 50 ng; indirect sandwich RIA, 200 ng; and direct sandwich RIA, 300 ng. The indirect and indirect sandwich RIAs showed considerable cross-reaction with other baculovirus antigens, but the direct and direct sandwich RIAs showed cross-reaction with only one closely related baculovirus. When microtiter plates used for the solid phase were pretreated with bovine serum albumin, nonspecific binding of labeled antibodies was reduced to a minimum. Antibodies prepared by an immunoadsorption procedure showed greater specific binding than antibodies prepared by ammonium sulfate precipitation of the immunoglobulin fraction. Highly contaminated antigen could not be detected by the indirect RIA, but the direct sandwich RIA was unaffected by antigen contamination. Antigen making up 0.0025% (wt/wt) of a sample of bird droppings could be detected by the direct sandwich RIA.

Journal Article↗
PubMed1978

Plaque assay of nuclear polyhedrosis viruses in cell culture.

The nuclear polyhedrosis virus of Autographa californica has been titrated in Spodoptera frugiperda cells by the plaque method, using a solid overlay which does not require either the use of modified culture medium or expensive purified agarose or the addition of culture medium as a liquid layer above the solid agarose. This assay is more sensitive than that using a viscous methyl cellulose overlay but less sensitive than the end-point dilution technique. Neither Trichoplusia ni nor Bombyx mori cells were satisfactory as indicators for the assay as described, since they failed to form a stable monolayer. Manduca sexta cells could be utilized for assay of A. californica nuclear polyhedrosis virus, but the sensitivity was lower than with S. frugiperda cells.

Journal Article↗
PubMed1978

Reversible inhibition of Sindbis virus penetration in hypertonic medium.

Under hypertonic conditions, the penetration of baby hamster kidney cells by Sindbis virus was inhibited. Virus became cell associated, but remained susceptible to neutralization by specific antiserum and to removal by elution. When cells were returned to isotonicity, inhibition was reversed.

Adsorption↗
PubMed1978

Modification of Trichoplusia ni nuclear polyhedrosis virus passaged in vivo.

A designated strain of plaque-purified Trichoplusia ni nuclear polyhedrosis virus (NPV) was used to initiate a serial passage series in vivo. The virulence of polyhedra inclusion bodies (PIB) and the distribution of MP (many PIB) and FP (few PIB) strains in hemolymph were monitored. When virus was passaged per os as PIB, there was no major change in virulence after 15 passes in insects. Plaque analysis of hemolymph from the infected insects indicated that there was no selection for the FP strain of virus. There was little overall change in the infectivity of nonoccluded virus (NOV) after 16 passes by injection into T. ni larvae; however, plaque analysis of the hemolymph from the infected insects indicated progressive selection of FP virus. It is postulated that when PIB are the vehicles of virus transmission, there is no progressive accumulation of noninfectious FP PIB; however, when NOV are the vehicles of virus transmission, FP NOV production displaces MP virus synthesis. This may result from a growth advantage of the FP NOV over the MP NOV as shown in vitro and from the noninfectious nature of FP PIB.

Animals↗
PubMed1976

Strain selection during serial passage of Trichoplusia in nuclear polyhedrosis virus.

Two strains of a nuclear polyhedrosis virus (NPV) of Trichoplusia ni were isolated on the basis of plaque morphology. They are designated as MP (having greater than 30 polyhedra per nucleus) and FP (having fewer than 10 polyhedra per nucleus). Serial, undiluted passage of plaque, purified MP nonoccluded. Virus (NOV) in tissue culture led to the production of the FP phenotype detectable at passage 9. With continued serial, undiluted passage, FP became the predominant strain. Comparative growth curves showed that FP NOV are released faster than MP NOV. MP morphology was not observed after 14 serial, undiluted passages of plaque-purified FP. By the plaque neutralization assay, NOV from both strains of virus was neutralized by the homologus and heterologous antisera. The FP phenotype was observed when FP virus was grown in culture at 17, 22, and 27 C. Hence, the FP phenotype was not considered to be the result of temperature-inhibited crystallization of polyhedrin under standard tissue culture conditions. The NOV of both strains killed insects when injected directly into the hemocoele of T. ni larvae. Only MP inclusion bodies were virulent per os. The FP inclusion bodies fed to cabbage looper larvae did not kill, and no infectious agent could be detected in the hemolymph. Electron micrographs of MP polyhedra showed bundles of nucleocapsids of normal length within the polyhedra, whereas FP polyhedra contained heterogeneous, electron-dense material, which could account for their lack of pathogenicity.

Animals↗
PubMed1975

Interfering passages of Sindbis virus: concomitant appearance of interference, morphological variants, and trucated viral RNA.

Serial passage of Sindbis at high multiplicities of infection resulted in cyclical variations in virus titer. Decreases in virus titer were correlated with the appearance of smaller-sized virions, interference and truncated viral RNA. The smaller particles were 37 nm in diameter, exclusive of the hemagglutinin spikes as compared with a diameter of 50 nm for standard virions. Passages which contained 37-nm partilces also interfered with infectious center formation by standard, plaque-purified virus. Polyacrylamide gel analysis of RNA isolated from virions present in interfering passages demonstrated the sequential appearance of three RNA species smaller than standard RNA with approximate molecular weights of 3.3 X 106, 2.7 X 106, and 2.2 X 106. The 3.3 X 106 RNA was evident in passage 5, by passage 8 both the 3.3 X 106 and 2.7 X 106 RNAs were present, and by passage 13 all three were present with the 2.2 X 106 RNA predominating.

Cell Line↗