Biomedical subjects
Publications and source records attributed to M D Summers.
A virus that replicates in the ovary of a parasitoid wasp is injected into the parasitoid's host during oviposition. Successful development of th parasitoid egg within the host depends on the presence of th virus, which acts to suppress the host's immune response (encapsulation) toward the egg. This is an example of obligatory mutualism between a virus and a eukaryotic organism.
Immunological comparisons were made of baculovirus structural proteins by using a modification of the radioimmunological techniques described by Renart et al. (Proc. Natl. Acad. Sci. U.S.A. 76: 3116-3120, 1979) and Towbin et al. (Proc. Natl. Acad. Sci. U.S.A. 76: 4350-4354, 1979). Viral proteins were electrophoresed in polyacrylamide gels, transferred to nitrocellulose, and incubated with viral antisera, and the antibodies were detected with (125)I-labeled Staphylococcus aureus protein A. Antisera were prepared to purified and intact virions from five baculoviruses: Autographa californica, Porthetria dispar, Trichoplusia ni, and Heliothis zea nuclear polyhedrosis viruses (NPVs) and T. ni granulosis virus (GV). These antisera were tested against the virion structural polypeptides of 17 different species of baculoviruses. Specific multiple-nucleocapsid NPV (MNPV), single-nucleocapsid NPV (SNPV), and GV virion polypeptides were shown to have similar antigenic determinants and thus be immunologically related. The molecular weights of the virion polypeptides with cross-reacting antigenic determinants were identified. Antisera prepared to purified A. californica and H. zea MNPV polyhedrin (the occlusion body protein from NPVs) recognized antigenic determinants on all the polyhedrins and granulins (occlusion body protein from GVs) that were tested. No immunological relationship was detected between A. californica MNPV polyhedrin and any of the A. californica MNPV virion structural polypeptides present on either the virus isolated from occlusion bodies or A. californica MNPV extracellular virus from infected-cell cultures.
We isolated polyadenylated RNA from the cytoplasm of cells infected with Autographa californica nuclear polyhedrosis virus late after infection (21 h postinfection). At that time intracellular protein synthesis was directed almost exclusively toward infected cell-specific proteins. The polyadenylic acid-containing RNA sequences in the cytoplasm at 21 h postinfection were radiolabeled in vitro and hybridized to A. californica nuclear polyhedrosis virus DNA restriction fragments. The polyadenylic acid-containing RNA was derived from regions representing the entire viral genome. Translation in a reticulocyte cell-free protein-synthesizing system of cytoplasmic RNA selected by hybridization to viral DNA and polyadenylic acid-containing RNA produced almost identical polypeptide patterns, suggesting that late after infection almost all of the cytoplasmic polyadenylic acid-containing RNA present in infected cells was of viral origin. Polyhedrin protein (molecular weight, 33,000) and a number of virion structural proteins were among the translation products which were identified by immunoprecipitation and by comparing molecular weights. In addition, some tentative nonstructural infected cell-specific proteins were also detected. Using the hybridization selection technique, we determined that sequences complementary to the message coding for polyhedrin were located on EcoRI fragment I of A. californica nuclear polyhedrosis virus DNA, whereas sequences coding for a putative nonstructural protein (molecular weight, 39,000) were on EcoRI fragment J.
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The restriction sites of Rachiplusia ou nuclear polyhedrosis virus (RoMNPV) DNA were mapped for the endonucleases SmaI, KpnI, BamHI, SacI, XhoI, and EcoRI. Of the 60 DNA restriction sites of RoMNPV, 35 mapped in similar positions as compared to the restriction sites of Autographa californica nuclear polyhedrosis virus (AcMNPV) DNA. Two plaque-purified viruses, obtained from randomly picked plaques of a wild-type isolate of RoMNPV, were recombinants of RoMNPV and AcMNPV. The recombinants were shown to have RoMNPV and AcMNPV restriction fragments as well as structural polypeptides from each parental virus. Both recombinant viruses had a major RoMNPV capsid protein but were occluded in the AcMNPV polyhedrin protein.
TN-368 cells were infected simultaneously with the closely related Autographa california (AcMNPV) and Rachiplusia ou (RoMNPV) nuclear polyhedrosis viruses. Progeny viral isolates were plaque purified, and their DNAs were analyzed with restriction endonucleases. Of 100 randomly cloned plaques, 7 were AcMNPV and RoMNPV recombinants, 5 were RoMNPV, and 88 were AcMNPV. The recombinants contained DNA sequences derived from both parental genomes. By comparing the restriction cleavage patterns of parental and recombinant DNAs, the crossover sites were mapped. A single double crossover was detected in each of the seven recombinant genomes. In addition, six of the seven recombinants revealed a crossover site mapping between 78 and 89% of the genome. The structural polypeptides of the seven recombinants and two parental viruses were analyzed by polyacrylamide gel electrophoresis, and their polyhedrins were identified by tryptic peptide mapping. An analysis of the segregation of three enveloped nucleocapsid proteins and of the polyhedrins among the recombinants located the DNA sequences coding for AcMNPV structural polypeptides with molecular weights of 37,000 (a capsid polypeptide), 56,000, and 90,000 and the RoMNPV structural polypeptides with molecular weights of 36,000 (a capsid polypeptide), 56,000, and 91,000. The AcMNPV and RoMNPV polypeptides of molecular weights 37,000 and 36,000, respectively, mapped within 78 to 89% or 1 to 29%, the polypeptides of molecular weights 55,000 and 56,000 mapped within 78 to 29%, and the polypeptides of molecular weights 90,000 and 91,000 mapped within 19 to 56% of the genome. The region of the parental DNAs that codes for polyhedrin was located within 70 to 89% of the genome.
The restriction sites of Autographa californica nuclear polyhedrosis virus (AcMNPV) E2 DNA were mapped for the endonucleases SmaI, KpnI, BamHI, SacI, XhoI, and EcoRI. The restriction maps of four other AcMNPV variants, Trichoplusia ni (TnMNPV), and Galleria mellonella (GmMNPV) genomes were determined and compared to the endonuclease cleavage maps of AcMNPV E2 DNA. The viral structural polypeptides of AcMNPV variants S3, E2, S1, M3, and R9 were the same when analyzed by polyacrylamide gel electrophoresis. The major structural polypeptides of GmMNPV and TnMNPV had the same pattern in polyacrylamide gels as did AcMNPV structural polypeptides. GmMNPV and TnMNPV had several minor structural protein differences as compared with AcMNPV. AcMNPV variants, TnMNPV, and GmMNPV were distinct but with very similar genomes and protein structures.
Polyhedrin obtained from Autographa californica nuclear polyhedrosis virus (AcMNPV) was apparently not modified in terms of primary structure after passage through alternate host systems, both in vivo and in vitro, as investigated by two-dimensional, high-voltage electrophoresis of tryptic digests of this protein as well as amino acid analysis. N-terminal analyses were conducted using an improved technique which showed proline to be the N-terminal amino acid. In addition, passage of enveloped nucleocapsids through alternate hosts as studied by SDS-PAGE showed the polypeptide compositions to be very similar.
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The sequence of events in the infection of TN-368-10 and TN-368-13 cells by Autographa californica nuclear polyhedrosis virus (AcMNPV) was investigated by using the indirect immunoperoxidase technique. Antisera raised against enveloped nucleocapsids detected homologous antigens at 6 to 8 h post infection which was about 2 h before the appearance of both intracellular and extracellular infectious virus. Similar tests using polyhedrin antiserum showed that polyhedrin is first synthesized at 12 h post infection, 2 to 4 h after the appearance of infectious nonoccluded virus. The immunoperoxidase technique was also applied to four other invertebrate cell lines after inoculation with AcMNPV. The most significant result was that 90% of AcMNPV-inoculated Bombyx mori 5 cells produced enveloped nucleocapsid antigens and infectious virus but only 1% or less of the cells produced polyhedrin. This disparity emphasizes the need for assays for NPV infection that are independent of polyhedron production.
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Electron microscopic examination and buoyant density profiles of nonoccluded Rachiplusia ou and Autographa californica nuclear polyhedrosis viruses purified from both infectious insect hemolymph and cell culture medium revealed that the viruses are enveloped, single nucleocapsids. The envelopes exhibited variation in the amount and degree of fit with regard to the nucleocapsids. This was determined by: (i) electron microscopic observations of virus budding from the surface of infected cells; (ii) electron microscopic observations of negatively stained preparations of pelleted, highly purified, nonoccluded enveloped particles; and (iii) the resolution and density distributions of nonoccluded virus in sucrose gradients after centrifugation to equilibrium; all were compared with virus extracted from polyhedra. Peplomers, ovserved on the surface of enveloped nucleocapsids of nonoccluded virus, are not associated with polyhedra-derived virus. Density gradient analysis indicated that virus from insect hemolymph and culture medium exhibited similar densities of approximately 1.17 to 1.18 g/ml. This is significantly different from the buoyant density of an alkali-liberated, enveloped single nucleocapsid (1.20 g/ml). Results of this study show that the nonoccluded forms of two nuclear polyhedrosis viruses from two different sources, hemolymph and cell culture, are similar with regard to several morphological and biophysical characteristics but are quite different from the alkali-liberated, polyhedra-derived form of the virus.
Nuclear polyhedrosis virus infections of lepidopteran cells often result in the production of both occluded and nonoccluded virus. The characterization of these two different forms has been the subject of several papers. We have divided the nonoccluded virus (NOV) category further into plasma membrane-budded non-occluded virus (PMB-NOV), intracellular NOV, and hemolymph-derived NOV, and have done additional studies investigating the differences between these nonoccluded forms and the alkali-liberated forms from occlusions of the nuclear polyhedrosis viruses of Autographa californica and Rachiplusa ou. The methods used to discern differences and similarities among the forms were serological, biochemical, and visual, all related to their biological acitivity. Neutralization studies revealed that alkali-liberated virus and PMB-NOV had both similar and different antigens. Antisera raised against alkali-liberated virus from occlusions neutralized the alkali-liberated form of the virus, but did not neutralize the intracellular or extracellular nonoccluded forms. Antisera raised against the TN-368-13 PMB-NOV, however, neutralized the alkali-liberated forms as well as all forms of the NOV. Adsorption of this antisera with alkali-liberated virus did not diminish the neutralization titer against the nonoccluded forms, thus confirming the antigenic differences between the alkali-liberated and nonoccluded forms of the virus. Physical-infectious particle ratio calculations indicated that the PMB-NOV of Autographa californica are about 1,900-fold more infectious than the single-nucleocapsid-per-envelope alkali-liberated particles and about 1,700-fold more infectious than the multiple-nucleocapsid-per-envelope particles, as assayed in vitro. In addition, a study of viral growth kinetics monitored concurrently with the appearance of polyhedra showed that PMB-NOV production is shut down with the onset of polyhedron formation.
Trichoplusia ni granulosis virus granulin consists of one major polypeptide component with an estimated molecular weight of 28,000. The protein is phenol soluble, phosphorylated, and acidic. A protease activated by alkaline conditions is also associated with solubilized granulin preparations. If not properly inactivated, the protease will introduce extensive artifact into the protein giving rise to ambiguous and incorrect results as analyzed by SDS-polyacrylamide gel electrophoresis and peptide mapping. Procedures are documented for enzyme inactivation and the preparation of granulin in highly purified form for characterization.
Cloned cell lines from the established Trichoplusia ni line TN-368 appear to differ from one another in their relative capabilities to serve as plaque assay indicator cell lines for Autographa californica nuclear polyhedrosis virus. Although there seems to be little correlation between their relative generation times and their efficiency in supporting plaque formation as indicator cell lines, there does seem to be a relationship within a given line between its capability to serve as an indicator and its phase of growth as a population; i.e., lag, logarithmic, or stationary. Both the parent line and clone 10 were more efficient indicators when they were in the logarithmic phase of growth than when in either the lag or stationary phases. Also, there appears to be a rough correlation between the capability of a given clone to serve as an indicator and the rate at which polyhedra first appear in the nuclei of the infected cells, with the best indicators producing polyhedra first. Increased incubation time has no effect on equalizing the plaque assay results for the less efficient clones. It was observed, also, that those clones that are the least efficient as plaque assay indicators produce the most external PFU per cell.
The protein solubilized from the proteinic crystalline structure surrounding the granulosis virus of Trichoplusia ni by use of a carbonate buffer (pH 10.7) gives a major component, as analyzed by ultracentrifugation, with a molecular weight of 180,000. This protein has heterogeneous subunit structure as demonstrated by estimates of molecular weights by use of gel electrophoresis, amino-, and carboxy-terminal analyses, and peptide mapping of enzyme digests of the protein. The amino acid composition shows that the protein is acidic with a high percentage of amino acids with hydrophobic side groups. Optical rotatory dispersion studies reveal the presence of beta-structure in the protein complex. The conversion of the beta-structure to alpha-helix with sodium lauryl sulfate and to a random coil state with strong alkaline treatment are observed.
The nuclear polyhedrosis virus DNAs characterized and compared in this study consist of the singly-enveloped nucleocapsids (SNPV) of Trichoplusia ni and the bundles of nucleocapsids common to a single envelope (MNPV) from Spodoptera frugiperda and Rachiplusia ou. The SNPV and MNPV DNAs are very similar in hydrodynamic properties and molecular weights. In addition, the NPV DNAs are similar in size to those extracted from the granulosis viruses that infect T. ni and S. frugiperda. As isolated from purified virus or directly from occluded virus, the nuclear polyhedrosis virus DNAs consist of a mixture of about 20 to 30% double-stranded covalently closed molecules and approximately 60% relaxed circles, with less than 10% in linear duplex form. The molecular weights of all nuclear polyhedrosis virus DNAs as compared in this study are slightly smaller than those of T4 bacteriophage DNA and perhaps slightly smaller than those of the granulosis virus DNAs. The best estimates of these molecular weights by neutral sucrose sedimentation for the nuclear polyhedrosis viruses range from 90 to 100 x 10(6) relative to a size of 108 x 10(6) for T4 DNA. The base compositions of the nuclear polyhedrosis viruses that infect T. ni and S. frugiperda are compared with the respective insect host DNAs.