Splicing Is Required for Transactivation by the Immediate Early Gene 1 of theLymantria disparMultinucleocapsid Nuclear Polyhedrosis Virus

Abstract

A region of theLymantria disparmultinucleocapsid nuclear polyhedrosis virus (LdMNPV) genome containing the homolog of the baculovirusie-1 gene was identified using a series of overlapping cosmids and individual plasmids in a transient transcriptional expression assay. Sequence analysis of the active region identified two ORFs, one of which is 32% identical to AcMNPV ORF141 (ie-0) and contains a putative splice donor site and the other of which is 29% identical to AcMNPVie-1 and contains a highly conserved splice acceptor consensus sequence. Plasmids containing the LdMNPV ORF141 andie-1 regions were able to stimulate expression of a GUS reporter gene, while plasmids containing theie-1 region alone were inactive, suggesting that only the spliced, IE-0 form of the gene product is an active transactivator. Primer extension analysis confirmed the presence of splicedie-0 mRNA transcripts starting at 6 hr and continuing throughout the time course of viral infection of theL. disparcell line Ld652Y. Using a plasmid containing theie-0 spliced form of the gene as a transactivator,hr4, one of the eight homologous regions of LdMNPV, was shown to act as a transcriptional enhancer. In contrast, a reporter plasmid containing the AcMNPVhr5 enhancer did not show increased activity when cotransfected with LdMNPVie-0, suggesting that these enhancer sequences are viral specific. In a transient replication assay system, LdMNPVie-0 acted as an essential replication gene, but LdMNPVie-1 was inactive. These results indicate that splicing is required to obtain an active gene product in LdMNPV in the Ld652Y cell line.