Regulation of gene expression and transcription mapping of an insect iridescent virus in cell culture

Our laboratory has established that Chilo iridescent virus (CIV) replicates productively in the cotton boll weevil, Anthonomus grandis. Given the economic impact of this pest, we have initiated host-virus interaction and molecular studies in both cell culture and larval systems. The objectives of the present study were to investigate transcriptional regulation and construct a map of all detectable transcripts for CIV infections in an existing cell culture model (IPRI-CF-124Tcells derived from Choristoneurafumiferana). Another objective was to establish an improved cell culture model using boll weevil cells, BRL-AG-3A (AG3A).

Northern analyses were carried out on total cellular RNA extracted in the presence or absence of metabolic inhibitors, using a complete CIV genomic library as well as gene-specific probes. The data showed that the gene expression cascade in CIV operates primarily at the transcriptional level. Three classes of transcripts and a temporal cascade comprising immediate-early (IE), delayed-early (DE), and late (L) genes were observed. Both positive and negative regulatory patterns were discerned within IE transcripts. Delayed-early transcripts were positi'ely or negatively regulated, or were not subject to regulation. Late transcripts were categorized into three sub-classes based on time of initiation. Using the experimental design described above, the entire (209 kb) viral genome was mapped, localizing a total of 137 transcripts (38 IE, 34 DE, and 65 L). Four discrete regions were identified for IE and DE transcription, respectively. Late transcription was observed throughout the viral genome.

After the above studies had already been initiated, a complete and productive model for replication of CIV in AG3A cells was also established and characterized. This system was utilized to develop the first infectivity assay for CIV based on a cytopathic effect. Virus was serially passaged through AG3A cells and high infectious titers were observed up to at least 24 passages. Dot-blot analyses and electron microscopic examination established production of high levels of progeny DNA and mature virions. Thus, the robust and productive replication of CIV in AG3A cells constitutes an excellent model and should be useful for both basic and applied research on the interaction between Chilo iridescent virus and the cotton boll weevil.