Splicing is required for transactivation by the immediate early gene 1 of the Lymantria dispar multinucleocapsid nuclear polyhedrosis virus.
A region of the Lymantria disper multinucleocapsid nuclear polyhedrosis virus (LdMNPV) genome containing the homolog of the baculovirus ie-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 AcMNPV ie-1 and contains a highly conserved splice acceptor consensus sequences. Plasmids containing the LdMNPV ORF141 and ie-1 regions were able to stimulate expression of a GUS reporter gene, while plasmids containing the ie-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 spliced ie-0 mRNA transcripts starting at 6 hr and continuing throughout the time 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 homologous regions of LdMNPV, was shown to act as a transcriptional enhancer. In contrast, a reporter plasmid containing the AcMNPV hr5 enhancer 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.
Pearson
M N
MN
Department of Microbiology, Oregon State University, Corvallis 97331-7301, USA. pearsonm@bcc.orst.edu
Rohrmann
G F
GF
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002213:Capsid / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D002460:Cell Line; D003001:Cloning, Molecular; D003360:Cosmids; D017931:DNA Primers; D004261:DNA Replication; D004279:DNA, Viral / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000096:biosynthesis; D016679:Genome, Viral; D017874:Immediate-Early Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D009036:Moths / Q000821:virology*; D017924:Nucleopolyhedroviruses / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D016133:Polymerase Chain Reaction; D012326:RNA Splicing; D012367:RNA, Viral / Q000378:metabolism; D015183:Restriction Mapping; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D015534:Trans-Activators / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D014158:Transcription, Genetic; D015533:Transcriptional Activation
C069183:Ac-IE-1 protein, Autographa californica nucleopolyhedrovirus; D017931:DNA Primers; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D017874:Immediate-Early Proteins; D012367:RNA, Viral; D015534:Trans-Activators