The potential role of a late gene expression factor, lef2, from Bombyx mori nuclear polyhedrosis virus in very late gene transcription and DNA replication.
Several late gene expression factors (Lefs) have been implicated in fostering high levels of transcription from the very late gene promoters of polyhedrin and p10 from baculoviruses. We cloned and characterized from Bombyx mori nuclear polyhedrosis virus a late gene expression factor (Bmlef2) that encodes a 209-amino-acid protein harboring a Cys-rich C-terminal domain. The temporal transcription profiles of lef2 revealed a 1.2-kb transcript in both delayed early and late periods after virus infection. Transcription start site mapping identified the presence of an aphidicolin-sensitive late transcript arising from a TAAG motif located at -352 nucleotides and an aphidicolin-insensitive early transcript originating from a TTGT motif located 35 nucleotides downstream to a TATA box at -312 nucleotides, with respect to the +1 ATG of lef2. BmLef2 trans-activated very late gene expression from both polyhedrin and p10 promoters in transient expression assays. Internal deletion of the Cys-rich domain from the C-terminal region abolished the transcriptional activation. Inactivation of Lef2 synthesis by antisense lef2 transcripts drastically reduced the very late gene transcription but showed little effect on the expression from immediate early promoter. Decrease in viral DNA synthesis and a reduction in virus titer were observed only when antisense lef2 was expressed under the immediate early (ie-1) promoter. Furthermore, the antisense experiments suggested that lef2 plays a direct role in very late gene transcription.
Sriram
S
S
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, 560 012, India.
Gopinathan
K P
KP
D000595:Amino Acid Sequence; D000818:Animals; D016590:Aphidicolin / Q000494:pharmacology; D001483:Base Sequence; D012831:Bombyx / Q000821:virology*; D003545:Cysteine; D004261:DNA Replication / Q000187:drug effects; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D015967:Gene Expression Regulation, Viral / Q000187:drug effects; D005814:Genes, Viral / Q000235:genetics; D008969:Molecular Sequence Data; D017924:Nucleopolyhedroviruses / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D016372:RNA, Antisense / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion / Q000235:genetics; D014158:Transcription, Genetic / Q000187:drug effects; D015533:Transcriptional Activation / Q000187:drug effects; D014162:Transfection; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014779:Virus Replication / Q000187:drug effects
D016372:RNA, Antisense; D012333:RNA, Messenger; D014764:Viral Proteins; C096108:late expression factor 2, Baculovirus; D016590:Aphidicolin; C020102:endodeoxyribonuclease DpnI; D015252:Deoxyribonucleases, Type II Site-Specific; D003545:Cysteine