Genomic sequence presentation.
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Base Sequence↗
Biomedical subjects
J Kuzio
Publications and source records attributed to J Kuzio.
Identification and characterization of the v-cath gene of the baculovirus, CfMNPV.
The v-cath gene of the Autographa californica multi-nucleocapsid nuclear polyhedrosis virus (AcMNPV) encodes a cathepsin L-like proteinase which plays a role in the liquefaction of host tissues during a viral infection [1]. We have identified a homologous gene in the spruce budworm virus, Choristoneura fumiferana MNPV (CfMNPV). The CfMNPV v-cath gene is 74% identical to AcMNPV v-cath at the nucleotide sequence level and 80% identical at the level of predicted amino acid sequence. Transcription analysis of the CfMNPV v-cath gene revealed that it is expressed late in infection and that transcription initiates within the consensus baculovirus late-promoter motif.
Amino Acid Sequence↗
Characterization of the baculovirus Choristoneura fumiferana multicapsid nuclear polyhedrosis virus p10 gene indicates that the polypeptide contains a coiled-coil domain.
The DNA sequence and transcription pattern of the p10 gene from the spruce budworm baculovirus Choristoneura fumiferana multicapsid nuclear polyhedrosis virus (CfMNPV) were analysed. The CfMNPV p10 gene codes for a protein 81 amino acids in length: this is the shortest p10 peptide identified thus far. A novel characteristic of the CfMNPV p10 gene is that it contains tandem late initiation sites (TAAG) having different temporal transcription patterns. Comparative analysis of CfMNPV p10 and the amino acid sequences of other p10 gene products showed that they each appear to have a similar N-terminal structure: an amphipathic alpha-helical terminus which condenses as coiled-coil multimers. Another feature of the p10 N terminus is that the central region of the coiled-coil domain resembles a bend or hairpin loop and could fold into a hairpin or form a bent rod structure. The condensation of p10 monomers to coiled-coil multimers is likely to be a step leading to the formation of p10 fibrous bodies in infected cells.
Amino Acid Sequence↗
Characterization of v-cath, a cathepsin L-like proteinase expressed by the baculovirus Autographa californica multiple nuclear polyhedrosis virus.
Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) contains a 966 bp ORF that encodes a papain type cysteine proteinase with cathepsin L-like characteristics. Using Western blot analysis of infected cell extracts we showed that v-cath proteinase has 35.5 kDa and 32 kDa precursor forms which are processed to a 27.5 kDa mature form in a manner characteristic of papain and cathepsin L. V-cath proteinase activity was greatest under acidic conditions (pH 5.0) and was reduced in the presence of the cysteine proteinase inhibitors, leupeptin and E64. Urea, a known enhancer of cathepsin L activity, also enhanced v-cath proteinase activity. AcMNPV v-cath proteinase was detected post-mortem in tissues of insects infected with wild-type (wt) virus. Insects infected with a v-cath deletion mutant did not become flaccid after death as is normally observed with wt AcMNPV infections. These findings indicate a link between v-cath activity and degradation of host tissues during virus pathogenesis.
Amino Acid Sequence↗
The complete DNA sequence of Autographa californica nuclear polyhedrosis virus.
The complete nucleotide sequence of the genome of clone 6 of the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) has been determined. The molecule comprises 133,894 base pairs and has an overall A + T content of 59%. Our analysis suggests that the virus encodes some 154 methionine-initiated, and potentially expressed, open reading frames (ORFs) of 150 nucleotides or greater. These ORFs are distributed evenly throughout the virus genome on either strand. The ORFs are arranged as adjacent, nonoverlapping reading frames separated by short intergenic regions. Based on the primary nucleotide sequence, predictions have been made concerning the functions of certain genes, the sites for initiation of viral DNA replication, the regulation of early and late gene transcription, and factors that may affect the AcNPV gene translational efficiency. The genome sequence data confirm, with minor differences, the information obtained for other AcNPV clones. It is proposed that clone C6 is considered the archetype AcNPV for comparison purposes.
Base Sequence↗
Molecular analysis of archael flagellins: similarity to the type IV pilin-transport superfamily widespread in bacteria.
Ultrastructural, biochemical and genetic evidence has shown that the flagella and flagellin proteins from members of the archaea are distinct from their bacterial counterparts. The most important evidence is the sequence dissimilarity between archael and bacterial flagellins. We report here similarity between archael flagellins and members of the bacterial type IV pilin-transport superfamily. In addition to sequence similarity, the archael flagellins and the type IV pilin-transport superfamily share an unusual signal sequence cleavage site and may have functional parallels. This relationship has important implications for the assembly and biogenesis of archael flagella.
Amino Acid Sequence↗
Nucleotide sequence of the p74 gene of a baculovirus pathogenic to the spruce budworm, Choristoneura fumiferana multicapsid nuclear polyhedrosis virus.
Polypeptide p74 has been found to be essential for production of virulent occlusion bodies of the baculovirus Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV). Hybridization with AcMNPV-derived probes has led to the location of the p74 gene in the spruce budworm virus, Choristoneura fumiferana MNPV. Sequence data indicate that CfMNPV p74 is 73% identical to AcMNPV at the nucleotide level and 77% identical at the amino acid level. Elements of predicted secondary structure are also conserved.
Amino Acid Sequence↗
An overview of the molecular biology and applications of baculoviruses.
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Animals↗
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↗
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↗
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↗
Structure and functions of Pseudomonas aeruginosa lipopolysaccharide.
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Chromatography, Ion Exchange↗
O-antigen conversion in Pseudomonas aeruginosa PAO1 by bacteriophage D3.
The lysogenization of Pseudomonas aeruginosa PAO by phage D3 results in derivatives which are resistant to superinfection by phage D3c by virtue of the fact that homologous phage cannot adsorb to these cells. The serologically and morphologically unrelated phage E79 showed a markedly decreased adsorption rate to the lysogen PAO(D3). Since both of these phages are lipopolysaccharide specific, these results suggested lysogenic conversion of the phage receptor. The lipopolysaccharide was extracted from strain PAO by the hot phenol-water technique, but this procedure was ineffective with PAO(D3). We developed a technique involving cold trichloroacetic acid extraction, followed by ultracentrifugation, digestion of the high-speed pellet with proteinase K, and ultimate purification on CsCl step gradients. The lipopolysaccharide from the wild type had inactivating activity against D3 and E79, whereas that from PAO(D3) inactivated neither. Chromatographic analysis indicated that the convertant lipopolysaccharide was smooth, and quantitative chemical analyses of the two preparations showed no differences in the level of the major fatty acids, amino compounds, or neutral sugars. On the other hand, sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the side chains had a decreased migration rate through the gel matrix. The application of 1H and 13C nuclear magnetic resonance spectroscopic analysis revealed that the PAO side chain is chemically identical to that of serotype O:2a,d, containing 2,3-(1-acetyl-2-methyl-2-imidazolino-5,4)-2,3-dideoxy-D-mannuronic acid, 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid, and 2-acetamido-2,6-dideoxy-D-galactose (D-fucosamine). The molecular basis of the conversion event was (i) the introduction of an acetyl group into position 4 of the fucosamine residue and a change in the bonding between trisaccharide repeating units from alpha 1 leads to 4 to beta 1 leads to 4.
Antigens, Bacterial↗
Spontaneous release of lipopolysaccharide by Pseudomonas aeruginosa.
Pseudomonas aeruginosa PAO grown in glucose mineral salts medium released lipopolysaccharide which was chemically and immunologically similar to the cellular lipopolysaccharide. In addition, it possessed identical phage E79-inactivating properties. Through neutralization of phage activity and hemolysis inhibition assays, the organism was found to liberate lipopolysaccharide at a constant rate during log-phase growth equivalent to 1.3 to 2.2 ng/10(8) cells over a growth temperature range of 25 to 42 degrees C. At 19 degrees C, a lipopolysaccharide was released which was deficient in phage-inactivating activity but retained its immunological properties. Chemical analysis of lipopolysaccharide extracted from cells grown at 19 degrees C showed a deficiency in the O-side-chain component fucosamine. Gel exclusion chromatography of the polysaccharide fraction derived from lipopolysaccharide isolated from cells grown at 19 degrees C exhibited a decreased content of side-chain polysaccharide as well as a difference in the hexosamine:hexose ratio. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis confirmed these results as well as establishing that an essentially normal distribution of side-chain repeating unit lengths were to be found in the 19 degrees C preparation. These results suggest a decrease in the frequency of capping R-form lipopolysaccharide at 19 degrees C.
Adsorption↗
Chemical and chromatographic analysis of lipopolysaccharide from an antibiotic-supersusceptible mutant of Pseudomonas aeruginosa.
Lipopolysaccharides extracted from Pseudomonas aeruginosa strain K799 and its antibiotic-supersusceptible derivative Z61 were analyzed chemically and chromatographically. The side-chain polysaccharides purified by gel exclusion chromatography were compositionally identical, being composed of fucosamine (2-amino-2,6-dideoxygalactose), quinovosamine (2-amino-2,6-dideoxyglucose), and an unidentified amino sugar. In addition, low amounts of the core-specific components (glucose, rhamnose, alanine, and galactosamine) were found associated with the side chains from both strains. An average molecular weight of 38,000 to 50,000 was calculated for this fraction based on the glucose and rhamnose levels. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the lipopolysaccharides from these two strains were microheterogeneous. Qualitative analysis of the lipopolysaccharide neutral sugars, using a series of single-step revertants of mutant Z61, demonstrated that full revertants showed patterns indistinguishable from those of the wild-type strain K799, whereas partial revertants had intermediate levels and mutant Z61 low levels of neutral sugars. Quantitative analysis revealed that the core oligosaccharide fraction from the wild-type strain had a glucose/rhamnose/galactosamine ratio of 4:1:1, whereas the core from Z61 exhibited major deficiencies in both glucose and rhamnose. The lipid A from both strains contained five fatty acids, namely, 3-hydroxydecanoate, dodecanoate, 2- and 3-hydroxydodecanoate, and hexadecanoate. Whereas the overall fatty acid content was equal, the mutant strain showed markedly lower levels of dodecanoate and hexadecanoate and increased levels of 2-hydroxydodecanoate. Results of whole-cell fatty acid analyses were consistent with this observation. Evidence for an additional alteration of the lipid A of strain Z61 was obtained from acid hydrolysis studies and infrared spectra of isolated lipid A, although the actual chemical basis could not be determined by a variety of techniques. It is suggested that the state of the lipopolysaccharide is able to influence the number of open functional protein F pores in the outer membrane of P. aeruginosa.
Anti-Bacterial Agents↗