Induction of apoptosis by iridovirus virion protein extract.

Chilo iridescent virus (CIV; IIV-6) is the type member of the genus Iridovirus (family Iridoviridae, large icosahedral cytoplasmic DNA viruses). CIV induces death and deformity in the cotton boll weevil, Anthonomus grandis, replicates productively in larvae of the cotton boll weevil, and significantly reduces laboratory populations of the cotton aphid, Aphis gossypii. CIV virion protein extract (CVPE) shuts down host protein synthesis in several insect cell lines and induces mortality in neonate boll weevil larvae. We report here that CVPE induces apoptosis in spruce budworm and boll weevil cell lines, as detected by blebbing, DNA fragmentation, and TUNEL assay. Tissue culture toxicity dose assays (TCTD(50)) showed that spruce budworm cells were eight times more sensitive to CVPE than boll weevil cells. Pancaspase inhibitor suppressed apoptosis but had marginal effect on inhibition of host protein synthesis. Moreover, the CVPE dose for apoptosis was 1000-fold lower than the dose for shutdown of host synthesis. We also detected protein kinase activity in CVPE. Heating CVPE at 60 degrees C for 30 min destroyed all three activities. Our results suggest that one or more polypeptides in CIV induce apoptosis. This is the first study demonstrating apoptosis induction by a member of the genus Iridovirus and by virion extracts of a member of the family Iridoviridae.

Transcriptional mapping in Chilo iridescent virus infections.

Chilo iridescent virus (CIV) belongs to the family Iridoviridae, which are icosahedral cytoplasmic DNA viruses with large, linear, and circularly permuted genomes. Previous studies on infected-cell-specific polypeptides suggested temporal regulation of CIV gene expression. Recently, we demonstrated three temporal classes at the transcriptional level, in CIV infections of a spruce budworm cell line. We also demonstrated a transcriptional cascade with positive and negative control. In this paper, we assign all detectable viral transcripts into respective temporal classes and map them using restriction fragments from a genomic library. More than 90 percent of the genome is transcriptionally active with at least four major clusters of immediate-early transcription and at least three delayed-early clusters. Late transcripts were observed throughout the genome. There was at least one exclusive region in the genome for each of the three temporal classes. We correlated transcribed regions with ORFs on the CIV genome and showed that known ORFs in the exclusive regions are generally consistent with phase-specific requirements of large DNA viruses. Our data also suggest the presence of 5' or 3' coterminal transcripts. This is the first complete transcription map for a member of the genus Iridovirus.

Replication of Chilo iridescent virus in the cotton boll weevil, Anthonomus grandis, and development of an infectivity assay.

The boll weevil, Anthonomus grandis Boheman, is a devastating pest of cotton. Chemical pesticides are problematic due to relative lack of target specificity and resistance. Microbial pesticides may provide viable alternatives because of their narrow host range. Chilo iridescent virus (CIV) is the type species for genus Iridovirus, family Iridoviridae: large, icosahedral cytoplasmic viruses containing a double-stranded DNA genome. Earlier work suggested that CIV replicated in the boll weevil; however, efficiency or production of infectious virus was not established. We showed that CIV undergoes a productive cycle in A. grandis. CIV DNA levels in boll weevil pupae increased significantly from 0 to 3 days post infection. Moreover, virogenic stromata and complete virus particles were observed in the cytoplasm by 7 days. An endpoint dilution assay using viral DNA replication as indicator suggested a 10(5)-fold increase in infectious virus titer over 7 days. This is the first such demonstration in larval infections with genus Iridovirus. Our study establishes that CIV undergoes a productive cycle in the boll weevil and provides an important and useful model system for replication at the organismal level. These results have important implications for the potential of CIV and its components in boll weevil control.

Transcription and temporal cascade in Chilo iridescent virus infected cells.

Chilo iridescent virus (CIV) is the type species for genus Iridovirus, and belongs to the family Iridoviridae. Members of this family are large, isometric, cytoplasmic DNA viruses. Our laboratory has established that CIV replicates productively in the cotton boll weevil, Anthonomus grandis. Given the economic importance of this host and the dearth of knowledge on this virus, we have initiated host-virus interaction and molecular studies on CIV. This report focuses on regulation of transcription in CIV infections. We carried out northern analyses on total cellular RNA from infections of IPRI-CF-124T cells, using a complete genomic library of CIV and several putative gene-specific probes. Our data show a temporal cascade based on analysis of 137 detectable transcripts comprising 38 immediate-early (IE), 34 delayed-early (DE), and 65 late (L) transcripts. Analysis with gene-specific probes supported the cascade pattern. Both helicase and RNA polymerase were immediate-early; major capsid protein was late. The CIV gene expression cascade appears to operate primarily at the transcriptional level. Temporal classes observed are consistent with earlier studies at the polypeptide level and with transcriptional patterns in frog virus 3, genus Ranavirus in the Iridoviridae. Our results provide an important basis for understanding mechanisms driving the CIV temporal cascade.

Host-specific transcription of nucleopolyhedrovirus gene homologues in productive and abortive Anticarsia gemmatalis MNPV infections.

In a previous report, we showed that Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) infections of Choristoneura fumiferana IPRI-CF-124T and Bombyx mori BM-5 cell lines are abortive, whereas A. gemmatalis UFL-AG-286 cells efficiently produce infectious virus and polyhedral inclusion bodies (PIBs). In the present study, we explored transcription patterns in these infections using representative temporal classes of Autographa californica MNPV (AcMNPV) genes. Northern analyses were carried out using internal fragments of AcMNPV genes that hybridized strongly with AgMNPV genomic DNA. The results showed that ie-1 (immediate-early) homologue, but not dnapol (delayed-early) homologue of AgMNPV was transcribed efficiently in the abortive infections. Transcription of gp67 (late) and polh (very late) homologues was minimal in C. fumiferana cells and undetectable in B. mori cells. Transcription patterns in AgMNPV-infected A. gemmatalis cells were similar to those reported for productive AcMNPV infections. These data are consistent with our previous observation that early cytopathic effect, but not infectious virus or PIBs are detected in abortive infections with AgMNPV. Our results suggest that C. fumiferana and B. mori cells restrict AgMNPV infections at the transcriptional level and that this block likely occurs between immediate-early and delayed-early phases of the NPV cycle. Our data do not preclude the possibility of additional restrictions at other stages.

Replication of Anticarsia gemmatalis nuclear polyhedrosis virus in four lepidopteran cell lines.

Anticarsia gemmatalis nuclear polyhedrosis virus (AgNPV; family Baculoviridae) is pathogenic for larvae of the velvetbean caterpillar, Anticarsia gemmatalis Hübner-an important pest of soybean. AgNPV is a viable alternative to chemical control of A. gemmatalis in Brazil, where its use as a pesticide has brought significant economic and environmental benefits. Although a significant amount of information is available on the ecological and biological control aspects of AgNPV, very little is known about the replication cycle and host specificity of this virus. We examined the susceptibility of four lepidopteran cell lines to AgNPV. Infections of the A. gemmatalis UFL-AG-286 cell line were highly productive. Ninety percent of infected cells had polyhedral inclusion bodies by 48 hr after infection, and the infectious virus titer was 10(8) IU/ml. Viral DNA replication was efficient, and the maximal rate of synthesis was between 6 and 12 hr after infection. Infections of the Spodoptera frugiperda IPLB-SF-21 cell line were productive but less efficient. Infections of Choristoneura fumiferana IPRI-CF-124T cells with this virus were poor, with only 5% of the cells forming polyhedra and an infectious virus titer of 10(6) IU/ml. The level of viral DNA replication was low, suggesting that this system was predominantly abortive. Infections of Bombyx mori BM-5 cell lines were abortive, and cells had apoptosis-like morphology. No polyhedra or increase in infectious levels were observed, and there was little or no replication of viral DNA. Our data suggest that restriction of AgNPV replication in abortive cell lines is due mainly to inability of viral DNA to replicate efficiently. The possible causes of low DNA replication are discussed. Our results suggest that the cell lines utilized in this study can provide an important model for studying mechanisms of AgNPV host specificity.

Regulation of infected-cell-specific protein synthesis in SF IPLB-21 cells productively infected with Spodoptera frugiperda multicapsid nuclear polyhedrosis virus.

In Spodoptera frugiperda (SF IPLB-21) cells productively infected with S. frugiperda multicapsid nuclear polyhedrosis virus (SfMNPV), we observed the synthesis of infected-cell-specific polypeptides (ICSPs) using the protein synthesis inhibitor cycloheximide (CX) and the DAN synthesis inhibitor cytosine arabinoside (Ara-C) in an attempt to assess whether a temporal cascade and viral DNA synthesis are involved in the gene expression program of SfMNPV. Inhibition of protein synthesis with CX at 0 h postinfection resulted in the active synthesis of a group of ICSPs immediately after removal of CX, suggesting that these polypeptides, designated alpha-ICSP, did not require de novo protein synthesis for their production. A second group of ICSPs (designated beta-ICSPs), requiring a prior interval of protein synthesis, was detected when CX was added at later times. A third group of ICSPs also needed an interval of ICSP synthesis, but differed from beta-ICSPs in that they required a longer interval of protein synthesis. Moreover, the synthesis of most of these ICSPs also required DNA synthesis, which was demonstrated by addition of Ara-C before and after viral DNA replication; these ICSPs were therefore designated gamma-ICSPs. In conclusion, two kinds of regulatory processes are involved in SfMNPV infection: the first process controls the sequential synthesis of the tree ICSP groups, that is alpha-->beta-->gamma; a second process represses the synthesis of ICSP groups, first of the alpha-ICSPs and subsequently of beta-ICSPs. Thus, we propose temporally regulated as well as negative control circuits in SfMNPV gene expression.

Host-specific transcription of baculovirus genes.

We examined the differential replication of Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV; Family: Baculoviridae) in Spodoptera frugiperda (SF) and Bombyx mori (BM) cell lines. Our previous studies have shown that infection of BM cells is abortive; infectious progeny is not produced, and although virogenic stroma is produced, complete virions or inclusion bodies are not assembled. We designed the present study to determine the extent of infected-cell-specific polypeptides (ICSPs) and viral DNA synthesis in the abortive infection and to see if restriction of virus replication in BM cells is due to a block at the transcriptional level. Pulse-labeling studies showed that a majority of the ICSPs detected in productive SF cells were not synthesized in BM cells. Many of these ICSPs were virion components. Viral DNA replicated in BM cells but more slowly and at a much lower level than in SF cells. Northern blot analysis showed that i) AcMNPV immediate-early gene (IE-1) was transcribed in BM cells at higher levels than in SF cells; ii) a delayed-early gene (dnapol) and a late gene (cap) were transcribed at much lower levels in the abortive infection; and iii) a very late gene (polh) was transcribed at extremely low levels in BM cells. We conclude that virus replication in BM cells is primarily restricted during or prior to the delayed-early stage and occurs at the transcriptional level. It appears that the late and very late effects observed are consequences of the primary block.

Infected cell specific protein and viral DNA synthesis in productive and abortive infections of Spodoptera frugiperda nuclear polyhedrosis virus.

This study examines viral protein and DNA synthesis in Spodoptera frugiperda multicapsid nuclear polyhedrosis virus (SfMNPV) infections of S. frugiperda and Trichoplusia ni cells. A total of 28 infected cell specific polypeptides (ICSPs) were detected in the productive S. frugiperda cells. Of these, 14 were identified as structural polypeptides. Based on the change in their rate of synthesis during the replication cycle, these ICSPs were grouped into four classes. Only a 97k and a 29k ICSP were detected in SfMNPV infections of T. ni cells. Inhibition of host protein synthesis occurred in productive infections only, beginning at 10 h postinfection (p.i.) and reaching maximal levels by 20 h p.i. The rate of viral DNA synthesis in the productive cells was maximal between 8 to 16 h postinfection, and only low levels of viral DNA were synthesized in T.ni cells. The data suggest that the productive SfMNPV/S.frugiperda cell infection has a gene expression program similar but not identical to that of Autographa californica MNPV infections. The SfMNPV/T.ni cell infection is nonpermissive and is restricted at the earliest phase of the viral gene expression program.

A semipermissive nuclear polyhedrosis virus infection: characterization of infection kinetics and morphogenesis.

An in vitro host-range model system, designated System II, was developed for the multicapsid nuclear polyhedrosis virus Spodoptera frugiperda (SF-MNPV). This system consisted of SF-MNPV-infected S. frugiperda cells (permissive system) and SF-MNPV-infected Trichoplusia ni cells (semipermissive system). Infection kinetic studies revealed that while progeny virus was produced in the semipermissive system, the infectious virus yield was at least 700-fold lower than that in positive controls. Electron microscopy showed that the vast majority of progeny nucleocapsids in semipermissive infections were not enveloped and were found in only 5% of infected T. ni cells; the remaining 95% of the cells exhibited cytopathic effect as well as virogenic stroma. The data suggest that the infection in System II is restricted at or prior to the early stages of virion morphogenesis.

Genomic divergence among single-nucleocapsid nuclear polyhedrosis viruses of plusiine hosts.

The DNAs of five single-nucleocapsid baculoviruses (S-NPVs) from plusiine hosts were compared. Restriction endonuclease fragments of three geographical isolates of Trichoplusia ni S-NPVs and isolates of Pseudoplusia incluens and Syngrapha selects viruses were compared using a battery of enzymes which included EcoRI, BamHI, SalI, SmaI, and HindIII. The results show that T. ni and P. includens S-NPVs are distinct, but related, viruses and that the S. selecta virus is a strain of T. ni S-NPV. Minor differences in geographical isolates of T. ni S-NPVs were detected. The data are generally consistent with the host-range properties of these viruses, suggesting that the combination of DNA restriction profiles and host-range matrices may provide a valuable tool for the identification and cataloging of the plusiine S-NPVs.

Structural polypeptides of Choristoneura biennis entomopoxvirus.

The structural polypeptides of Choristoneura biennis (Free) entomopoxvirus (EPV) were analyzed. The virion contained at least 40 polypeptides ranging from 12,000 to 250,000 in molecular weight. Twelve major proteins constitute 95% of the total virus protein. Core particles generated by treatment with Nonidet P-40, under conditions during which endogenous alkaline protease is active, display a smooth exterior similar to those obtained for orthopoxviruses. The major component of these cores is a polypeptide with a molecular weight of 59,000.

Development of an attached strain from a continuous insect cell line.

A continuous attached cell strain has been developed from the IPRI-CF-124 line of the spruce budworm, Choristoneura fumiferana. This was done by discarding suspended cells at each passage, rinsing attached cells with 0.05% trypsin and using only the strongly attached cells for subculturing. The method is very effective in that the proportion of attached cells increased from 6% in the parent cell line to 97% in the new cell strain after 20 passages. The attachment and growth properties are stable after storage of cells in liquid nitrogen. The new cell strain is designated IPRI-CF-124T and has a population doubling time comparable to that of the parent cell line.

Comparative study of 125 I- and (3H)acetate-labeled antibodies in detecting iridescent viruses.

Radioimmunoassays for detecting cell-associated or released virus are described using either (125)I- or [(3)H]acetate-labeled antibodies. In the first assay system, antigen-antibody complexes were separated from free antibody by centrifugation. Sensitivities of 0.1 mug of iridescent virus could be achieved with either (125)I- or [(3)H]acetate-labeled antibody. In the second assay, the antigen was fixed to cover-slip cell cultures, and then reacted with labeled antibody, unbound radioactivity being removed by repeated washing. Nonspecific binding with this method was 0.5 to 1% of the total radioactivity added and sensitivities of 0.1 or 10 mug were achieved with (125)I and [(3)H]acetate, respectively. Immunoglobulins were labeled at the rate of 1 in 300 for (125)I and 1 in 200 with [(3)H]acetate although there was a 400-fold greater isotopic abundance of (125)I relative to (3)H. The possibility of preparing labeled protein of high specific activity using carrier-free [2-(3)H]iodoacetic acid is discussed.