Responses of insect cells to baculovirus infection: protein synthesis shutdown and apoptosis.

Protein synthesis is globally shut down at late times postinfection in the baculovirus Autographa californica M nuclear polyhedrosis virus (AcMNPV)-infected gypsy moth cell line Ld652Y. A single gene, hrf-1, from another baculovirus, Lymantria dispar M nucleopolyhedrovirus, is able to preclude protein synthesis shutdown and ensure production of AcMNPV progeny in Ld652Y cells (S. M. Thiem, X. Du, M. E. Quentin, and M. M. Berner, J. Virol. 70:2221-2229, 1996; X. Du and S. M. Thiem, Virology 227:420-430, 1997). AcMNPV contains a potent antiapoptotic gene, p35, and protein synthesis arrest was reported in apoptotic insect cells induced by infection with AcMNPV lacking p35. In exploring the function of host range factor 1 (HRF-1) and the possible connection between protein synthesis shutdown and apoptosis, a series of recombinant AcMNPVs with different complements of p35 and hrf-1 were employed in apoptosis and protein synthesis assays. We found that the apoptotic suppressor AcMNPV P35 was translated prior to protein synthesis shutdown and functioned to prevent apoptosis. HRF-1 prevented protein synthesis shutdown even when the cells were undergoing apoptosis, but HRF-1 could not functionally substitute for P35. The DNA synthesis inhibitor aphidicolin could block both apoptosis and protein synthesis shutdown in Ld652Y cells infected with p35 mutant AcMNPVs but not the protein synthesis shutdown in wild-type AcMNPV-infected Ld652Y cells. These data suggest that protein synthesis shutdown and apoptosis are separate responses of Ld652Y cells to AcMNPV infection and that P35 is involved in inducing a protein synthesis shutdown response in the absence of late viral gene expression in Ld652Y cells. A model was developed for these responses of Ld652Y cells to AcMNPV infection.     

Identification of baculovirus gene that promotes Autographa californica nuclear polyhedrosis virus replication in a nonpermissive insect cell line.

A gene that promotes Autographa californica M nuclear polyhedrosis virus (AcMNPV) replication in IPLB-Ld652Y cells, a cell line that is nonpermissive for AcMNPV, was identified in Lymantria dispar M nuclear polyhedrosis virus (LdMNPV). Cotransfection of AcMNPV DNA and a plasmid carrying the LdMNPV gene into IPLB-Ld652Y cells results in AcMNPV replication. The gene maps between 43.3 and 43.8 map units on the 162-kbp genome of LdMNPV. It comprises a 218-codon open reading frame and encodes a polypeptide with a predicted molecular mass of 25.7 kDa. The predicted polypeptide is glutamic acid and valine rich and negatively charged, with a pI of 4.61. No protein sequence motifs were identified, and no matches with known nucleotide or peptide sequences were found in the AcMNPV genome or database searches that suggest how this gene might function. A recombinant AcMNPV bearing the LdMNPV gene overcomes a block in protein synthesis observed in AcMNPV-infected IPLB-Ld652Y cells. Using Southern blotting techniques, we were unable to identify a homolog in Orgyia pseudotsugata M nuclear polyhedrosis virus, a baculovirus that is routinely propagated in IPLB-Ld652Y cells. This suggests that the LdMNPV host range is unique among the baculoviruses studied to date. We named this gene hrf-1 (for host range factor 1).     

Role of early and late replication events in induction of apoptosis by baculoviruses.

Autographa californica nuclear polyhedrosis virus (AcMNPV) mutants that lack the apoptotic suppressor gene p35 cause apoptosis in Spodoptera frugiperda SF21 cells. To identify a viral signal(s) that induces programmed cell death, we first defined the timing of apoptotic events during infection. Activation of a P35-inhibitable caspase, intracellular fragmentation of host and AcMNPV DNA, and cell membrane blebbing coincided with the initiation of viral DNA synthesis between 9 and 12 h after infection and thus suggested that apoptotic signaling begins at or before this time. Virus entry was required since binding of budded virus to host cell receptors alone was insufficient to induce apoptosis. To therefore determine the contribution of early and late replication events to apoptotic signaling, we used the AcMNPV mutant ts8 with a temperature-sensitive lesion in the putative helicase gene p143. At the nonpermissive temperature at which viral DNA synthesis was conditionally blocked, ts8 caused extensive apoptosis of the SF21 cell line p3576D, which dominantly interferes with anti-apoptotic function of viral P35. Confirming that apoptosis can be induced in the absence of normal viral DNA synthesis, parental SF21 cells also underwent apoptosis when infected with a ts8 p35 deletion mutant at the nonpermissive temperature. However, maximum levels of ts8 p35 deletion mutant-induced apoptosis required a temperature-sensitive event(s) that included the initiation of viral DNA synthesis. Collectively, these data suggested that baculovirus-induced apoptosis can be triggered by distinct early (pre-DNA synthesis) and late replicative events, including viral DNA synthesis or late gene expression.     

The apoptotic suppressor P35 is required early during baculovirus replication and is targeted to the cytosol of infected cells.

The p35 gene of Autographa californica nuclear polyhedrosis virus (AcMNPV) is required to block virus-induced apoptosis. The trans-dominant activity of p35 suppresses premature cell death and facilitates AcMNPV replication in a cell line- and host-specific manner. To characterize the p35 gene product (P35), a specific polyclonal antiserum was raised. As revealed by immunoblot analyses of wild-type AcMNPV-infected cells, P35 appeared early (8 to 12 h) and accumulated through the late stages of infection (24 to 36 h). Biochemical fractionation of cells both early and late in infection and indirect immunochemical staining demonstrated that P35 localized predominantly to the cytosol (150,000 x g supernatant); comparatively minor quantities of P35 were associated with intracellular membranes. The cytoplasmic localization of P35 was independent of virus infection. The functional significance of the early and late synthesis of P35 was examined by constructing recombinant viruses in which the timing and level of p35 expression were altered. Delaying P35 synthesis by placing p35 under exclusive control of a strong, very late promoter failed to suppress intracellular DNA fragmentation and apoptotic blebbing in most cells. Thus, earlier expression of p35 was required to block virus-induced apoptosis. Site-specific mutagenesis of the p35 promoter demonstrated that low levels of P35 were sufficient to block apoptosis, whereas higher levels were required to maintain wild-type virus gene expression. Consistent with an early role in infection, P35 was also detected in the budded form of AcMNPV. Because of the lack of sequence similarity and its cytosolic targeting, P35 may function in a manner that is mechanistically distinct from other apoptotic regulators, including Bcl-2 and the adenovirus E1B 19-kDa protein.     

Infectivity of Anticarsia gemmatalis nucleopolyhedrovirus to different insect cell lines: Morphology, viral production, and protein synthesis

The Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) is currently used as an efficient biological pesticide for the control of the velvetbean caterpillar (A. gemmatalis), an important pest of soybean in Brazil. Until now, production of the virus has been achieved mainly by infection of larvae on local soybean farms. Studies for the development of in vitro systems and the optimization of mass production in insects reared on artificial diets is now important to help to meet the actual demand for the bioinsecticide. We therefore, investigated the infectivity of AgMNPV in cell culture, which might contribute to the selection of suitable cell lines that may be used for in vitro production of this virus. The cytopathic effects induced by the virus, the production of viral particles and the synthesis of viral polypeptides were examined and compared in the cell lines from A. gemmatalis (UFL-AG-286), Trichoplusia ni (BTI-Tn-5B1-4 and TN-368), Spodoptera frugiperda (IPLB-SF-21AE and Sf9), Lymantria dispar (IPLB-LD-652Y), and Bombyx mori (BM-5). Whereas, Tn-5B1-4 and AG-286 cells produced large numbers of occlusion bodies, no polyhedra were visualized in either Ld-652Y or BM-5 cells, although extensive cell lysis was observed in BM-5. Analysis of the kinetics of viral protein synthesis by SDS–PAGE after pulse labeling with [³⁵S]methionine, showed similar protein patterns in most of the cell lines tested. Exceptions were the LD-652Y and BM-5 cells, in which viral polypeptides, including polyhedrin, were not synthesized. In parallel, measurement of viral titers (budded virus) by the endpoint dilution method showed that Tn-5B1-4, AG-286, and SF-21AE cells were highly productive. Their TCID₅₀ values, at 48 h p.i., were about 10⁷ IU/ml. In addition to the lower formation of polyhedra, the viral titers determined in Sf9 and TN-368 cells were about 5 to10-fold lower. As expected, the viral titers obtained in LD-652Y and BM-5 cells were similar to basal levels.     

Effects of long- and short-term passage of insect cells in different culture media on baculovirus replication

Two insect cell lines that had been maintained in both serum-free (SFM) and serum-containing (SCM) media for over 5 years were each tested for their ability to replicate baculovirus. The gypsy moth cell line, IPLB-LdEIta (Ld), produced similar (not statistically different) amounts of gypsy moth nucleopolyhedrovirus (LdMNPV) occlusion bodies (OBs) in the two media (serum-free Ex-Cell 400 and TC-100 with 9% (v/v) fetal bovine serum, SCM(1)) but produced more of the Autographa californica nucleopolyhedrovirus (AcMNPV) OBs in SFM than in SCM(1). When Ld cells normally grown in SCM(1) were switched to SFM, production of OBs from both viruses improved and, after three passages, reached higher levels of AcMNPV production than in cells normally maintained in that medium. Alternatively, cells switched from SFM to SCM(1) initially produced as much (in the case of LdMNPV) or higher (in the case of AcMNPV) levels of virus OBs than cells normally maintained in SCM(1) but productivity dropped off over subsequent passages such that after five passages in SCM(1), cells produced substantially fewer OBs of both viruses. A fall armyworm cell line (IPLB-SF21AE; Sf) showed slightly different effects from long- and short-term passage in SFM (Ex-Cell 400) or SCM(2) (TMN-FH). Cells maintained in SFM produced about 20 times more AcMNPV OBs than cells maintained long-term in SCM. Sf cells switched from SFM to SCM maintained the level of production of that seen in SFM at the first passage, but quickly dropped off OB production levels to that normally seen in SCM. Alternatively, SCM-maintained Sf cells produced higher levels at the first passage in SFM and, within five passages in SFM, reached levels found in cells maintained for long term in this medium. Under the conditions in which these two cell lines were infected, the highest levels of AcMNPV OB production in Ld cells were about five times that of Sf cells. In a separate series of experiments, cells normally grown in SFM were passaged over five times in Ex-Cell 400 to which serum was added; both cell lines produced as much virus as that in SFM. These results suggest that it is not the serum per se but rather some other components which differ between the SFM and the SCM formulations that are responsible for the varied virus production obtained in these studies. The results of these studies suggest that a maintenance and virus production protocol can be developed with Ld cells which could improve overall efficiency of virus production. These studies also suggest that long-term maintenance of cells in SFM was not detrimental to their ability to produce baculoviruses.     

In Vitro Translation of Autographa californica Nuclear Polyhedrosis Virus Early and Late mRNAs

A preliminary translational map of the Autographa californica genome was constructed. Eighteen viral DNA restriction fragments were either purified from agarose gels or obtained from pBR322 recombinant DNA plasmids to locate specific gene products. The DNAs were immobilized on nitrocellulose filters and used to select viral mRNAs isolated from RNA obtained from the cytoplasm of infected Spodoptera frugiperda cells at 21 h postinfection. The fragment-specific mRNAs were translated in vitro in the presence of l-[(3)H]leucine by using a rabbit reticulocyte lysate system and analyzed on sodium dodecyl sulfate-polyacrylamide gels. The approximate locations of 19 A. californica nuclear polyhedrosis virus (AcMNPV) gene products were mapped. The genes for mRNAs present late in viral infection were mapped to DNA fragments that represent nearly the entire genome. The molecular weights of many of these proteins were similar to those present in purified AcMNPV extracellular virus and to proteins being made in infected cells at 18 to 21 h postinfection. Cytoplasmic RNA was isolated at 4 h postinfection from infected cells, a time early in the viral infection cycle, and hybridized to AcMNPV DNA immobilized on nitrocellulose filters. AcMNPV-specific early RNA was translated in vitro into at least six polypeptides, the most abundant having a molecular weight of 39,000. Viral polypeptides were detected in cells pulse-labeled with l-[(3)H]leucine at 3 to 6 h postinfection, with molecular weights similar to those of polypeptides made in vitro from early AcMNPV mRNA.     

Molecular characterization of the insect immune protein hemolin and its high induction during embryonic diapause in the gypsy moth,Lymantria dispar

During the embryonic (pharate first instar) diapause of the gypsy moth, Lymantria dispar, a 55 kDa protein is highly up-regulated in the gut. We now identify that protein as hemolin, an immune protein in the immunoglobulin superfamily. We isolated a gypsy moth hemolin cDNA and demonstrated a high degree of similarity with hemolins from three other moth species. Hemolin mRNA levels increased at the time of diapause initiation and remained high throughout the mandatory period of chilling required to terminate diapause in this species, and then dropped in late diapause. This mRNA pattern reflects the pattern of protein synthesis. These results suggest that hemolin is developmentally up-regulated in the gut during diapause. Diapause in this species can be prevented using KK-42, an imidazole derivative known to inhibit ecdysteroid biosynthesis, and gypsy moths treated in this manner failed to elevate hemolin mRNA. Conversely, this diapause appears to be initiated and maintained by the steroid hormone, 20-hydroxyecdysone, and the addition of 20-hydroxyecdysone to the culture medium elevated hemolin mRNA in the gut. Our results thus indicate a role for 20-hydroxyecdysone in the elevation of hemolin mRNA during diapause. Presumably, hemolin functions to protect the gypsy moth from microbial infection during its long, overwintering diapause.     

Comparison of cell line maintenance procedures on insect cells used for producing baculoviruses

Summary A gypsy moth cell line, IPLB-LdEIta, maintained under various conditions was tested for susceptibility to and productivity of two baculoviruses, the Autographa californica nucleopolyhedrovirus (AcMNPV) and Lymantria dispar nucleopolyhedrovirus (LdMNPV). The results suggest that cells maintained in serum-containing medium (modified TC100) were more susceptible (on the basis of titers in an endpoint assay) to LdMNPV than cells maintained in a serum-free medium (ExCell™ 400). Such a difference was not apparent with AcMNPV. Similarly, little difference existed in the proportion of cells containing occlusion bodies (OBs) a wk after inoculation with AcMNPV (i.e., the percent infected) in any LdEIta strains, although one combination of cells and medium (cells maintained in ExCell 400 but infected in TC100) showed a lower percent infection with LdMNPV. Even though the percentage of cells infected varied little, the number of OBs produced varied by 3 logs with AcMNPV and 11/2 logs with LdMNPV. In each case, cells normally grown in ExCell 400 and infected in the same medium produced the lowest number of OBs. However, productivity was improved when cells normally grown in ExCell 400 were infected in TC100. Even more interesting was that cells normally grown in TC100 produced more AcMNPV OBs when infected in ExCell 400 medium. This suggests that changing culture medium (regardless of the normal maintenance medium) can stimulate virus production. In addition to examining virus productivity in LdEIta cells in both serum-containing and serum-free media, I also tested a strain maintained at low temperature (17° C) for over a yr. This maintenance protocol was not detrimental for LdMNPV productivity and was slightly stimulatory for production of AcMNPV.     

Comparative susceptibilities of insect cell lines to infection by the occlusion-body derived phenotype of baculoviruses

Twelve insect cell lines from six species were tested for susceptibility to baculovirus infection by occlusion-derived virus (ODV) phenotype through the use of a typical endpoint assay procedure. ODV from three nucleopolyhedroviruses were prepared by alkali treatment (sodium carbonate) of occlusion bodies (OBs) and the virus preparations were titered on various cell lines. More than a four-log difference was realized for each of theses viruses between the various cell lines. The TN368 line from Trichoplusia ni was only marginally susceptible to ODV from each virus, showing only 3-6 infectious units (IU) per million OBs while the gypsy moth line, LdEp was most susceptible, realizing more than 100,000 IU/million OBs. The other lines tested showed various levels of susceptibility between these two extremes and also varied between the three viruses tested. In additional tests, the ODV were treated with trypsin prior to application to the cells. With most cell lines, this treatment increased the infectivity of each virus by 2-10-fold. Exceptions to this trend included the gypsy moth LdEp line, on which the trypsinized ODV from two of the viruses were slightly less infectious than each virus without trypsin, and the TN-368 line, on which the trypsinized ODV was 5,000-75,000 times more infectious. The variable results of trypsinized virus on the different lines are probably due to the levels of endogenous protease activity in the various lines, but the mode of action of the trypsin has not been elucidated. Ultimately, the variable response of cell lines to ODV of different viruses, and the variable effects of trypsin on the ODV may lead to an improved understanding of the infection process of this virus phenotype as well as factors relating to baculovirus host range.     

Effect of cytosine arabinoside on viral-specific protein synthesis in cells infected with herpes simplex virus.

The relationship between viral DNA and protein synthesis during herpes simplex virus type 1 (HSV-1) replication in HeLa cells was examined. Treatment of infected cells with cytosine arabinoside (ara-C), which inhibited the synthesis of HSV-1 DNA beyond the level of detection, markedly affected the types and amounts of viral proteins made in the infected cell. Although early HSV-1 proteins were synthesized normally, there was a rapid decline in total viral protein synthesis beginning 3 to 4 h after infection. This is the time that viral DNA synthesis would normally have been initiated. ara-C also prevented the normal shift from early to late viral protein synthesis. Finally, it was shown that the effect of ara-C on late protein synthesis was dependent upon the time after infection that the drug was added. These results suggest that inhibition of progeny viral DNA synthesis by ara-C prevents the "turning on" of late HSV-1 protein synthesis but allows early translation to be "switched off."     

Cell lines from diamondback moth exhibiting differential susceptibility to baculovirus infection and expressing midgut genes

Abstract Six new cell lines were established from embryonic tissues of the diamondback moth, Plutella xylostella (L.). The cell lines showed differential characteristics, including growth in attachment or in suspension, susceptibility to a baculovirus infection and expression of genes involved in the glucosinolate detoxification pathway in R xylostella larvae. Five of the cell lines grew attached to the culture flask and one cell line grew unattached as a suspension cell line. The cell lines had population doubling times ranging from IS to 23 h. Among five of the P. xylostella cell lines examined for infection of a nucleopolyhe. drovirus from Autographa californica, AcMNPV four cell lines were highly susceptible to AcMNPV infection, but one was only semi-permissive to AcMNPV infection. The production of two recombinant proteins, a β-galactosidase of bacterial origin and a secreted alkaline phosphatase of eukaryotic origin, in the R xylostella cell lines was examined in comparison with that in the cell line Sf9 which is commonly used for recombinant protein production. In the P. xylostella cell lines, expression of three important midgut genes involved in the glucosinolate detoxification pathway, including the glucosinolate sulfatase genes GSS1 and GSS2 and the sulfatase modifying factor gene SUMF1、was detected. The R xylostella cell lines developed in this study could be useful in in vitro research systems for studying insec-virus interactions and complex molecular mechanisms in glucosinolate detoxification and insect-plant interactions.     

Lepidopteran cell lines after long-term culture in alternative media: Comparison of growth rates and baculovirus replication

Summary Three insect cell lines, IPLB-LdFB and IPLB-LdEIta from gypsy moth fat body and embryos and UFL-AG-286 from velvetbean caterpillar embryos, have been concurrently maintained for 1 to 12 yr on two media formulations, modified TC-100 containing 9% fetal bovine serum and Ex-cell 400, a commercial serum-free medium (SFM). Cells grown in each medium were tested for susceptibility to and productivity of various multiply embedded nucleopolyhedroviruses. The three lines chosen for these experiments fall into three categories of relative growth in SFM versus TC-100: LdFB cells grew similarly in each medium, LdEIta grew better in Ex-Cell than in TC-100, and AG-286 grew better in TC-100 than in Ex-Cell. The susceptibility of cells to infection also varies, although without any apparent correlation to which medium was best for supporting growth. Endpoint assays suggested that LdFB cells grown in serum-containing medium are more susceptible to virus infection than their SFM counterparts, while the opposite is true for LdEIta cells. Production of virus, based on numbers of occlusion bodies, showed fewer differences with only AcMNPV production with AG-286 in TC-100 being statistically higher than production of the same virus in Ex-cell 400. These studies suggest that long-term passage in alternative media may impact the ability of cells to support virus infection and replication, but the effects on each cell line and virus system need to be determined.     

Replication of simian virus 40 DNA in normal human fibroblasts and in fibroblasts from xeroderma pigmentosum.

Simian virus 40 infection of semipermissive human diploid fibroblasts (HF), at early passage in cell culture, was compared with that of permissive established monkey cell lines. Viral DNA can be readily detected at 24 to 48 h postinfection at 37 degrees C with a high multiplicity of infection, approaching 10% of that of monkey cells (TC7). The length of time necessary for replication of an average molecule of viral DNA was found to be indistinguishable in HF and TC7 cells. Strand elongation plus termination were assessed by following the accumulation of DNA I at 40 degrees C from replicative intermediates of tsA30 prelabeled at 33 degrees C, obviating isotope pool problems. Combined initiation and elongation of wild-type viral DNA was measured by density shift experiments involving a 5-bromodeoxyuridine chase of prelabeled [3H]thymidine-labeled viral DNA. Determination of accumulation of viral T and V antigens supports the conclusion that the most likely basis for the reduced virus yield in HF cells results from the inefficiency of an early stage in virus infection, before or during uncoating. Similar results were obtained in fibroblasts derived from patients with xeroderma pigmentosum, suggesting that enzymes of UV repair are not required in unirradiated simian virus 40 DNA synthesis.     

Central role of hemocytes in Autographa californica M nucleopolyhedrovirus pathogenesis in Heliothis virescens and Helicoverpa zea

Autographa californica M nucleopolyhedrovirus (AcMNPV) can infect and kill a wide range of larval lepidopteran hosts, but the dosage required to achieve mortal infection varies greatly. Using a reporter gene construct, we identified key differences between AcMNPV pathogenesis in Heliothis virescens and Helicoverpa zea, a fully permissive and a semipermissive host, respectively. Even though there was more than a 1,000-fold difference in the susceptibilities of these two species to mortal infection, there was no significant difference in their susceptibilities to primary infections in the midgut or secondary infections in the tracheal epidermis. Foci of infection within the tracheal epidermis of H. zea, however, were melanized and encapsulated by 48 h after oral inoculation, a host response not observed in H. virescens. Further, H. zea hemocytes, unlike those of H. virescens, were highly resistant to AcMNPV infection; reporter gene expression was observed only rarely even though virus was taken up readily, and nucleocapsids were transported to the nucleus. Collectively, these results demonstrated that hemocytes-by removing virus from the hemolymph instead of amplifying it and by participating in the encapsulation of infection foci-together with the host's melanization response, formed the basis of H. zea's resistance to fatal infection by AcMNPV.