Identification of cis-acting signals in the giardiavirus (GLV) genome required for expression of firefly luciferase in Giardia lamblia.
Giardiavirus (GLV) is a 6,277-bp double-stranded RNA virus of Giardia lamblia, one of the earliest eukaryotic divergents from the prokaryotes. Our previous success in GLV-mediated transfection of G. lamblia has provided an effective way of monitoring the mechanisms underlining GLV gene replication and mRNA translation in this organism. Here we have investigated the cis-acting signals in the GLV genome that regulate replication, transcription, and translation of an inserted firefly luciferase gene in GLV-infected G. lamblia. By modifying the two terminal regions of a full-length GLV cDNA clone used to flank a luciferase gene, various in vitro chimeric transcripts were generated and introduced into GLV-infected G. lamblia via electroporation. Expression of luciferase (+) strand and (-) strand RNAs in the transfected cells was monitored and the luciferase activity assayed. The results indicated that the 5'-untranslated region (UTR) of 366 nt and the 3'-terminal 2,022 nt of the viral transcript are both needed for optimal expression of the two RNA strands. Although the entire 5'-UTR is needed for the chimeric mRNA synthesis, both the primary sequence and the secondary structure at the 3' end of GLV transcript are essential for the synthesis of (-) strand RNA. When the 5' end of GLV transcript was extended 265 nt into the capsid protein open reading frame and fused with that of luciferase, there was no change in the level of luciferase chimeric RNA, but a 5,000-fold increase of luciferase activity was observed that may be attributed to an enhanced translational efficiency of the chimeric mRNA in G. lamblia.
Yu
D C
DC
Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco 94143-0446, USA. dyu@cgl.ucsf.edu
Wang
C C
CC
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D015967:Gene Expression Regulation, Viral; D016679:Genome, Viral; D016829:Giardia lamblia / Q000821:virology*; D008156:Luciferases / Q000235:genetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012328:RNA Viruses / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid; D014158:Transcription, Genetic; D014162:Transfection
D017931:DNA Primers; D012367:RNA, Viral; D008156:Luciferases