Peter Faulkner

A Plaque Assay for Nuclear Polyhedrosis Viruses using a Solid Overlay

Summary The nuclear polyhedrosis viruses of Trichoplusia ni and Autographa californica produce plaques in monolayers of Spodoptera frugiperda cells under a solid overlay containing agarose as the solidifying agent. Plaques are visible macroscopically after staining the cells with neutral red or with the tetrazolium salt, INT, and a linear dose response is observed. The sensitivity of the assay is less than that obtained using an end-point dilution technique; however, plaque formation does provide a simple means of cloning virus.

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.

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.

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.

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 genes 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.

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.

Morphogenesis of nuclear polyhedrosis virus under conditions of prolonged passage in vitro

An ultrastructural description is given of nuclear polyhedrosis virus (NPV) morphogenesis in vitro, with emphasis on changes occurring during long-term serial passage of infectious material. The morphogenesis and infectivity of early passage virus is similar to that which obtains in vivo. The infectivity of polyhedra prepared from late passage infected cultures is greatly diminished; this is paralleled by both a decrease in the total number of polyhedra produced and a decrease in the number of morphologically normal occluded virus particles. Late passage viral morphogenesis is profoundly changed. New observations concerning the acquisition of membranes by NPV nucleocapsids and polyhedron formation are discussed.

Cytological Changes and Viral Morphogenesis during Baculovirus Infection

The family Baculoviridae encompasses a group of arthropod-specific DNA viruses that are characterized by a circular DNA genome packaged within a rod-shaped capsid and enclosed by a lipid envelope (Volkman et al., 1995). At some stage of their life cycle all baculoviruses exist as an occluded form in which virions are embedded within a proteinaceous matrix (Miller, 1996). The family is divided into the genera nucleopolyhedrovirus [nuclear polyhedrosis viruses (NPV)] and granulovirus [granulosis viruses (GV)], which differ in the morphology of their occluded virion form as well as the cytopathology they induce. The occlusion matrix proteins polyhedrin (NPV) and granulin (GV) are closely related and highly conserved, and have been used to assist in the taxonomy of baculoviruses (Rohrmann, 1986a, b; Zanotto et al., 1993). NPV have polyhedral-shaped occlusion bodies, 0.5–15μm in diameter, which contain many virions embedded either as single nucleocapsids per envelope (SNPV) or multiple nucleocapsids per envelope (MNPV), and morphogenesis occurs within the nucleus of infected cells. In contrast, GV produce much smaller ovicylindrical occlusions (capsules, 0.16–0.30 μm × 0.30–0.50 μm) that contain one or occasionally two virions. Replication of GV begins in the cell nucleus but induces apparent breakdown of the nuclear membrane, such that morphogenesis is completed in the cytoplasm or hybrid cellular compartment (Crook, 1991; Granados and Williams, 1986).

Neutralization of budded Autographa californica NPV by a monoclonal antibody: Identification of the target antigen

Abstract A neutralizing monoclonal antibody of the budded phenotype of Autographa californica nuclear polyhedrons virus did not react with the occluded form of the virus as determined by neutralization, ELISA, and indirect immunoperoxidase staining. The antibody did react with the surface of infected cells in the prepolyhedra stage of cytopathic effect, the period of active virus budding. Immunoelectron microscopy indicated the antigen(s) reactive with the neutralizing antibody was present all around the viral envelope, but was more highly concentrated at the end with peplomers. Four proteins were immunoprecipitated from solubilized, radiolabeled budded virus preparations with the monoclonal antibody. The major protein had a molecular weight of approximately 64,000, while the other three were approximately 127,000, 59,000, and 49,000. All four proteins could be labeled with N-acetyl-d-[1-3H]glucosamine. This glycosylation reaction could be inhibited by tunicamycin.

Factors affecting the yield of virus in a cloned cell line of Trichoplusia ni infected with a nuclear polyhedrosis virus

Abstract The TN-368 tissue culture line of the cabbage looper, Trichoplusia ni , has been cloned. The doubling times of three clones at 27°C were 27.6 ± 3.4 hr, 21.9 ± 1.7 hr, and 27.4 ± 5.9 hr and that of the uncloned culture was 15.8 ± 1.5 hr. Growth of cells in all cultures was arrested after infection with a nuclear polyhedrosis virus of T. ni . There was little difference in the yield of polyhedra from cultures of uncloned or cloned cells infected at a multiplicity of infection (m.o.i) = 4. Yields of polyhedra were about the same when a m.o.i. was in the range of 0.01–4.0, but the yield tripled in the range m.o.i. = 20–30. At higher multiplicities, up to m.o.i. = 500 the yield of polyhedra progressively fell. It is concluded that the observed variation in numbers of polyhedra borne by individual cells in culture is not due to genetic variability among cells, nor can it be accounted for as a consequence of differing m.o.i. by virus. It is postulated that variation in polyhedra yield among cells in culture may be due to such factors as (1) strain differences in the virus, (2) the stage in the cell cycle at which a particular cell is present when infected.