Edward M. Dougherty

Evidence for integration of Glyptapanteles indiensis polydnavirus DNA into the chromosome of Lymantria dispar in vitro.

Polydnaviruses replicate within calyx cells of the female ovaries of certain species of parasitic wasps and are required for the successful parasitization of lepidopteran hosts. These viruses, which have unusual double-stranded circular DNA segmented genomes, are integrated as proviruses into the genomes of their associated wasp hosts and are believed to be transmitted vertically through germline tissue. Here, by combined Southern hybridization, polymerase chain reaction (PCR) assays and viral sequence analyses we provide evidence that DNA originating from two distinct double-stranded circular segments of the polydnavirus genome from the braconid Glyptapanteles indiensis (GiPDV) integrates in vitro into the genome of cells derived from the natural host, Lymantria dispar. The G. indiensis polydnavirus DNA, as a result of its unique ability to be integrated in part into the chromosome of cells derived from its lepidopteran host, has potential to be developed as an in vitro cell transformation system.

TRANSFORMATION OF LEPIDOPTERAN AND COLEOPTERAN INSECT CELL LINES BY GLYPTAPANTELES INDIENSIS POLYDNAVIRUS DNA

SummaryRecently investigators showed that polydnavirus DNA from the parasitic wasp Glyptapanteles indiensis could transform gypsy moth L. dispar cell lines in vitro (McKelvey et al., 1996). Here we show GiPDV DNA is capable of transforming in vitro to varying degrees lepidopteran (IPLB-TN-R2, IPLB-SF-21, IAL-PID2, IPLB-HvT1) and coleopteran (IPLB-DU182E) insect cell lines derived from various somatic tissue types. An insect cell line derived from dipteran Aedes albopictus (C7/10) could not be transformed with G. indiensis polydnavirus.

Changes in macromolecular synthesis of gypsy moth cell line IPLB-Ld652Y induced by Autographa californica nuclear polyhedrosis virus infection

The aberrant replication of the Autographa californica multiple-enveloped nuclear polyhedrosis virus (AcMNPV) in the Lymantria dispar cell line IPLB-Ld652Y was used as a model system for the investigation of factors regulating baculovirus host specificity. A previous study of this system indicates that viral gene expression in infected cells is extremely attenuated and subsequently all cellular and viral protein synthesis is inhibited. In the present study, infection of IPLB-Ld652Y cells with AcMNPV photochemically inactivated in situ resulted in a rapid reduction in cell mitotic indices and cell growth, as well as inducing a series of distinct morphological changes in these cells. At the molecular level, infection with inactivated virus, followed by pulse labelling with [3H]thymidine, resulted in a rapid [0 to 2 h post-infection (p.i.)] and permanent inhibition of host cellular DNA synthesis. Assays of cellular DNA polymerases in isolated IPLB-Ld652Y nuclei confirmed the reduction in cellular DNA synthesis observed in intact cells and indicated an initial (0 to 2 h p.i.) reduction in the activity of aphidicolin-sensitive DNA polymerases. Activity of all cellular DNA polymerases was inhibited at later times p.i. Host cell protein synthesis was completely inhibited after 48 h p.i. Treatment of inactivated virus and virus-infected cells with various chemical and physical factors (i.e. pH and temperature) or lysosomotropic agents revealed that virus entry into cells and fusion of endocytic vesicles (containing virus) with lysosomes were essential for suppression of cellular macromolecular synthesis. The possible involvement of structural components of the AcMNPV virion in these effects is discussed.

The use of commercial serum replacements in media for the in vitro replication of nuclear polyhedrosis virus

Abstract Two commercially available fetal bovine serum replacements, previously used in media for the culture of vertebrate and insect cells, were evaluated in comparison with fetal bovine serum (FBS) for suitability as medium supplements for the production of the Autographa californica nuclear polyhedrosis virus in fall armyworm cell cultures. The overall patterns of viral protein synthesis were similar in infected cells in each of the media supplements, but some differences were noted in quantities of individual proteins produced and in the temporal regulation of the synthesis. The release of extracellular budded virions (ECV) in all media began at about the same time and the rate of release was similar. However, the final concentration of ECV was consistently lower in medium containing either CPSR-1 or CPSR-3 than that in FBS supplemented medium. The number of occlusion bodies (OB) per cell was also less in the CPSR-1 medium. In the CPSR-3 medium the number OBs per cell produced was not significantly different from those in the FBS medium. The LC 50 value of the OBs bioassayed in neonate Spodoptera frugiperda produced in the CPRS-3 medium was significantly lower than that of OBs from FBS medium but the LC 70 and LC 90 values were not. Abnormal OBs, that is OBs that contained few or no occluded virions, were observed in cells in media with all three supplements.

Production of a cellular macromolecular synthesis inhibition factor(s) in gypsy moth cells infected with the Autographa californica nuclear polyhedrosis virus

Abstract The gypsy moth cell lines IPLB-Ld652Y and IPLB-LdFB have been shown to be semipermissive for replication of the Autographa californica multiple-embedded nuclear polyhedrosis virus (AcMNPV). We report here that AcMNPV infection of these cell lines results in the production, by the infected cells, of a proteinaceous viral derived factor(s) which is secreted into the tissue culture medium (IPL-52B). Uninfected IPLB-Ld652Y and IPLB-LdFB cells, when incubated with media from AcMNPV-infected cells, exhibit markedly reduced levels of cell growth, mitosis, DNA, and protein synthesis. The factor(s), which has been designated the macromolecular synthesis inhibition factor (MSIF), is produced and secreted from infected cells between 1 and 30 hr postinfection and is produced in the absence of viral gene activity in infected cells. A preliminary characterization of the MSIF revealed the presence of a heat-labile, proteinaceous, pH sensitive molecule(s) whose activity was neutralized by three different monoclonal antibodies directed against the AcMNPV 64-kDa envelope glycoprotein. Production of the MSIF(s) did not require any new viral or cellular gene activity and was inhibited by treatment of infected cells with the lysosomal protease inhibitor, leupeptin. It is postulated that the MSIF is the AcMNPV 64-kDa glycoprotein or some component or complex of this protein, which is removed from the inoculum virus, and secreted, by the infected cells, into the tissue culture medium.

Effect of anAutographa californica nuclear polyhedrosis virion component(s) on DNA synthesis and growth in several insect cell lines

SummaryThirteen different insect cell lines representing three different orders were infected withAutographa californica nuclear polyhedrosis virus (AcMNPV) whose genome had been inactivatedin situ by photochemical means or by short wave UV irradiation. Changes in rates of cellular DNA synthesis, as measured by [3H]thymidine incorporation, and cell growth were subsequently measured at various times post infection. Seven cell lines exhibited a significant decline in [3H]thymidine incorporation (compared to control levels) during an initial 12 h period post infection, while three cell lines showed substantial declines in [3H]thymidine incorporation over a 4 day period post infection. All cell lines which showed a significant decline in [3H]thymidine over the duration of the experiment (4 days) also exhibited reduced cell growth rates. The role of a putative AcMNPV virion associated factor(s) in influencing these cellular events is discussed.

DNA-binding proteins of baculovirus-infected cells during permissive and semipermissive replication

Certain insect cell lines have been shown to be permissive (TN-368) or semipermissive (IPLB-LD-652Y) for Autographa californica nuclear polyhedrosis virus (AcMNPV) replication (McClintock et al., 1986b). In this report DNA-binding proteins were identified in such cell:virus systems by hybridizing Western blots containing uninfected and infected cell proteins with AcMNPV or host DNA probes. In the AcMNPV-infected TN-368 permissive cell system, 8 virus-induced DNA-binding proteins with molecular weights ranging from 67.5K to 18.75K were observed under the highest conditions of stringency. When these DNA-binding proteins were compared to structural proteins of AcMNPV, several appeared to be similar to those observed in SDS-PAGE protein profiles of nonoccluded virus (NOV) and occlusion body (PIBs) preparations. Using an AcMNPV occlusion negative mutant (L1GP-gal3) and an anti-AcMNPV-polyhedrin monoclonal antibody, a major DNA-binding protein (33.0K), observed in the permissive system, corresponded to polyhedrin and to a comigrating virus-induced DNA-binding protein. In the AcMNPV-infected IPLB-LD-652Y semipermissive cell system, no virus-induced DNA-binding proteins were detected. However, several host DNA-binding proteins were present but their ability to bind DNA decreased significantly following infection.