Replication of Orgyia pseudotsugata baculovirus DNA: lef-2 and ie-1 are essential and ie-2, p34, and Op-iap are stimulatory genes.
A transient DNA replication assay was used to identify genes located within m.u. 90.5-7.0 of the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV) genome that influenced replication of a reporter plasmid containing an OpMNPV origin of replication, when cotransfected into uninfected Lymantria dispar cells. The viral transactivator ie-1 and a 2.4-kb subclone were found to be essential for replication. The 2.4-kb region was sequenced and open reading frames were identified. Replication assays using subclones from this region identified a gene called late expression factor 2 (lef-2), as the essential replication gene. The OpMNPV lef-2 gene encodes a protein with a predicted molecular weight of 22.7 kDa (204 amino acids) and exhibits 54.7% amino acid sequence identity with its homolog in the genome of the Autographa californica MNPV. Transcriptional mapping using both Northern blot and S1 nuclease protection assays demonstrated that OpMNPV lef-2 was expressed at both early and late times postinfection as a transcript of about 1.6 kb. The early transcript initiated approximately 30 nt downstream of a TAATA box, whereas the late transcript initiated from within a late promoter consensus motif. In addition, we identified three genes stimulatory for DNA replication including two OpMNPV transcriptional activators (ie-2 and p34) and Op-iap, which is the functional analog of the AcMNPV p35 gene that inhibits apoptosis in AcMNPV-infected Spodoptera frugiperda cells.
Ahrens
C H
CH
Genetics Program, Oregon State University, Corvallis 97331-7301, USA.
Rohrmann
G F
GF
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D004261:DNA Replication / Q000235:genetics*; D005786:Gene Expression Regulation; D017344:Genes, Insect / Q000235:genetics*; D007313:Insecta / Q000821:virology; D008969:Molecular Sequence Data; D017924:Nucleopolyhedroviruses / Q000235:genetics / Q000502:physiology*; D016366:Open Reading Frames / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D012367:RNA, Viral / Q000096:biosynthesis / Q000235:genetics; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera; D015534:Trans-Activators / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics; D014779:Virus Replication / Q000235:genetics*
D012333:RNA, Messenger; D012367:RNA, Viral; D015534:Trans-Activators; D014764:Viral Proteins; C096108:late expression factor 2, Baculovirus