John Kuzio

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.

Analysis of p74, a PDV envelope protein of Autographa californica nucleopolyhedrovirus required for occlusion body infectivity in vivo.

In nature, nuclear polyhedrosis viruses (NPV) are transmitted when susceptible insect larvae ingest viral occlusion bodies (OB). These dissociate in the alkaline environment of the midgut and release encapsulated virions (PDV) which bind to midgut epithelial cells and initiate an infection. A previous study showed that expression of the Autographa californica NPV (AcMNPV) p74 gene during replication is essential for the production of infectious OB. A set of p74 deletion and overexpression recombinants was used for the production and screening of monoclonal antibodies, and for an investigation of gross cytopathology and localization of p74. No differences in virus structure or morphogenesis were observed in infected cells when the p74 gene of AcMNPV was deleted, even though the infectivity of OB harvested from the cells was abolished when they were fed to Trichoplusia ni larvae. Mutant OB released virus particles and degraded insect peritrophic membrane as in infections with wild-type virus; in addition, virions purified from mutant OB were infectious when injected into the haemocoel of T. ni larvae. Western blot analysis confirmed that p74 was associated with the PDV and could not be detected in the budded form virion phenotype. The polypeptide was readily degraded by treatment of purified PDV with proteinase K, in the presence and absence of detergent, and could be extracted from PDV by a non-ionic detergent treatment. The data are consistent with p74 being a structural polypeptide of the PDV phenotype, most probably as a component associated with the outside surface of the virion envelope. Its presence is shown to be essential for primary infection of midgut cells of insect larvae.

The genome of choristoneura fumiferana nuclear polyhedrosis virus: Molecular cloning and mapping of the EcoRI, BamHI, SmaI, XbaI and Bg1II restriction sites

Abstract The DNA genome of Choristoneura fumiferana nuclear polyhedrosis virus (CfMNPV) was extracted from purified virus preparations and analyzed with restriction endonucleases EcoRI, BamHI, HindIII, SmaI, XbaI and Bg1II. The sizes of the fragments were calculated with respect to mobilities of standard adenovirus-2 DNA fragments. The total size of the genome was calculated to be about 125 kb. Most of the CfMNPV genome was then cloned as EcoRI fragments in the plasmid vector pBR322. The clones were authenticated by electrophoretic analysis in the presence of EcoRI-digested CfMNPV DNA and by hybridization of 32 P nick-translated clones to Southern blots of different enzyme digests of the genome. Data from such hybridization experiments complemented with other mapping procedures were used to construct restriction maps for BamHI, EcoRI, XbaI, Bg1II and SmaI enzymes.

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.

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.

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.

Genomic variants of a temperature-sensitive mutant of Autographa californica nuclear polyhedrosis virus containing specific reiterations of viral DNA

Restriction endonuclease analysis of DNA from isolates of a temperature-sensitive mutant, ts.8, of Autographa californica nuclear polyhedrosis virus revealed the presence of genomic variants. ts8 type A plaque isolate DNA had altered restriction patterns while ts8 type B plaque isolate DNA restriction patterns were similar to wild-type viral DNA. The alterations in ts8 type A included an extra 1.35 kilobase pair (kbp) EcoRI fragment, and a series of extra Hind111 and XhoI restriction fragments present in decreasing molar ratios with increasing molecular weight.Southern blot and DNA-DNA cross-blot analysis indicated that the ts8A extra restriction fragments were homologous to viral sequences from the 90% region of the genome. The t&A extra restriction fragments appeared to result from an insertion of repeated viral DNA sequence at the EcoRI P-B junction. The higher molecular weight ts8A Hind111 and XhoI submolar fragments appeared to have increasing copy numbers of the 1.35 kbp EcoRI-EcoRI repeat-unit flanked by authentic EcoRI fragment P and B sequences. Approximately 57% of the ts8 plaques isolates analyzed were type A and 43% were type B genomic variants. Although type A and type B genomic variants appeared to be stable in high multiplicity of infection passage experiments, lo-20% of plaque isolates in stock homogeneity experiments converted from one type to another. The genomic alterations did not appear to have any observable effect on the temperature-sensitive phenotype of the mutant.

Paucity of the Sau3AI recognition sequence (GATC) in the genome of Methanococcus voltae

SummaryHigh molecular weight genomic DNA isolated from the archaebacterium Methanococcus voltae by alkaline-SDS lysis was not effectively digested with the restriction enzyme Sau3AI, which recognizes the base sequence GATC. Mc. voltae DNA was also resistant to digestion by MboI and BamHI which recognize sites containing the same GATC sequence. Examination of a Mc. voltae genomic library prepared in Escherichia coli JM83 with a pUC vector revealed that the 5–10 kb inserts were still resistant to Sau3AI digestion, indicating a likely lack of the GATC sequence in Mc. voltae DNA.