Glyptapanteles indiensis, a species of braconid parasitic wasp, infects its host Lymantria dispar (gypsy moth) with a polydnavirus (GiPDV) to suppress the host immune system during parasitization. Here it is shown that GiPDV can infect L. dispar cell lines and that a portion of the GiPDV genome is stably maintained in infected cells. Results of Southern hybridization analyses suggested that this portion of the GiPDV genome is integrated into the L. dispar cellular genome. This is the first report of an insect viral DNA molecule that can apparently integrate into lepidopteran insect cells.

Infection of two gypsy moth cell lines (IPLB-Ld652Y and IPLB-LdFB) by the Autographa californica multiple-enveloped nuclear polyhedrosis virus (AcMNPV) is characterized by extremely attenuated viral protein synthesis followed by a total arrest of all viral and cellular protein production. In this study, AcMNPV- and host cell-specific transcription were examined. Overall levels of viral RNAs in infected gypsy moth cells were, at most measured times, comparable to RNA levels from an infected cell line (TN-368) permissive for AcMNPV replication. Northern blot (RNA) analyses using viral and host gene-specific probes revealed predominantly normal-length virus- and cell-specific transcripts postinfection. Transport of viral RNAs from the nucleus to the cytoplasm and transcript stability in infected gypsy moth cells also appeared normal compared with similar parameters for AcMNPV-infected TN-368 cells. Host cellular and viral mRNAs extracted from gypsy moth and TN-368 cells at various times postinfection and translated in vitro yielded similar spectra of host and viral proteins. Treatment of infected gypsy moth cells with the DNA synthesis inhibitor aphidicolin eliminated the total protein synthesis shutoff in infected IPLB-LdFB cells but had no effect on protein synthesis inhibition in infected IPLB-Ld652Y cells. The apparent selective block in the translation of viral transcripts early in infection and the absence of normal translation or transcription of host cellular genes at later times is discussed.

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.

Thirteen different insect cell lines representing three different orders were infected with Autographa californica nuclear polyhedrosis virus (AcMNPV) whose genome had been inactivated in 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.

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 LC50 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 LC70 and LC90 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.

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.

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.

Summary Earlier studies have suggested that successful parasitism by certain braconid parasitoids may depend on the presence in host insect larvae of both polydnavirus and venom. We have shown that venom from the braconid parasitoid, Cotesia melanoscela, was required for in vivo persistence of polydnavirus DNA in host larvae. In parallel studies using an in vitro system, we observed that in the presence of venom nucleocapsids were released into the cytoplasm and subsequently uncoated at nuclear pores; in the absence of venom, this sequence of events was not observed.

Cellular responses to the introduction of foreign objects into the haemocoele of both control and parasitized tussock moth larvae were examined. In normal larvae, the response to large foreign objects such as Hyposoter fugitivus eggs and Sephadex beads was encapsulation, accompanied by a rapid and sustained increase in the total haemocyte count. Smaller objects such as yeast cells were cleared into nodules within a matter of minutes; nodulation too was accompanied by an increased total haemocyte count.

The icheneumonid wasp, Hyposoter fugitivus, is an habitual parasitoid of tent caterpillar (Malacosoma disstria) larvae. Successful parasitism is associated with an apparent behavioural transformation of certain host haemocytes, and is in addition characterized by a suppression of immunity to foreign objects such as parasitoid eggs, yeast, and Sephadex beads; it is suggested that transformation of haemocytes renders them incapable of participating in normal immune responses. Inhibition of some aspects of cellular immunity in host insects can also be induced by manual injection of a polydnavirus isolated from the ovaries of the parasitoid. It was observed that haemolymph from parasitized larvae does not melanize in vitro; this phenomenon may be associated with a greatly increased stability of oencytoids.

Orgyia leucostigmahaemocytes were able to encapsulate and destroy eggs ofHyposoterspecies, thereby preventing successful parasitism. When host larvae had previously been parasitized byCotesia melanoscela, however, this response was not observed, and 3 differentHyposoterspecies could be reared to maturity in the normally non-permissive host. Appropriate injection experiments established thatC. melanoscelavirus and venom were both required in order for successful parasitism byHyposoterto occur. Covalent cross-linking ofC. melanoscelaviral DNA led to an immune response againstHyposoterlarvae, but not eggs, suggesting that an active viral genome may be required for complete development ofHyposoterspecies in tussock moth larvae.