SPLICING IS REQUIRED FOR TRANSACTIVATION BY THE IMMEDIATE-EARLY GENE-1 OF THE LYMANTRIA-DISPAR MULTINUCLEOCAPSID NUCLEAR POLYHEDROSIS-VIRUS

A region of the Lymantria dispar multinucleocapsid nuclear polyhedrosi s virus (LdMNPV) genome containing the homolog of the baculovirus ie-1 gene was identified using a series of overlapping cosmids and individ ual 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 don or site and the other of which is 29% identical to AcMNPV ie-1 and con tains a highly conserved splice acceptor consensus sequence. Plasmids containing the LdMNPV ORF141 and ie-1 regions were able to stimulate e xpression of a GUS reporter gene, while plasmids containing the ie-1 r egion alone were inactive, suggesting that only the spliced, IE-0 form of the gene product is an active transactivator. Primer extension ana lysis confirmed the presence of spliced ie-0 mRNA transcripts starting at 6 hr and continuing throughout the lime course of viral infection of the L, dispar cell line Ld652Y. Using a plasmid containing the ie-0 spliced form of the gene as a transactivator, hr4, one of the eight h omologous regions of LdM NPV, was shown to act as a transcriptional en hancer. In contrast, a reporter plasmid containing the AcMNPV hr5 enha ncer did not show increased activity when cotransfected with LdMNPV ie -0, suggesting that these enhancer sequences are viral specific. In a transient replication assay system, LdMNPV ie-0 acted as an essential replication gene, but LdMNPV ie-1 was inactive. These results indicate that splicing is required to obtain an active gene product in LdMNPV in the Ld652Y cell line. (C) 1997 Academic Press.