Open AccessExpression of the Totivirus Helminthosporium victoriae 190S Virus RNA-Dependent RNA Polymerase from Its Downstream Open Reading Frame in Dicistronic Constructs
Author(s) -
Ana I. Soldevila,
Said A. Ghabrial
Publication year - 2000
Publication title -
journal of virology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.617
H-Index - 292
eISSN - 1070-6321
pISSN - 0022-538X
DOI - 10.1128/jvi.74.2.997-1003.2000
Subject(s) - biology , open reading frame , orfs , rna , rna silencing , rna polymerase , green fluorescent protein , microbiology and biotechnology , genetics , virology , rna interference , gene , peptide sequence
The undivided double-stranded RNA (dsRNA) genome ofHelminthosporium victoriae 190S virus (Hv190SV) (genusTotivirus ) consists of two large overlapping open reading frames (ORFs). The 5′-proximal ORF encodes a capsid protein (CP), and the downstream, 3′-proximal ORF encodes an RNA-dependent RNA polymerase (RDRP). Unlike the RDRPs of some other totiviruses, which are expressed as a CP-RDRP (Gag-Pol-like) fusion protein, the Hv190SV RDRP is detected only as a separate, nonfused polypeptide. In this study, we examined the expression of the RDRP ORF fused in frame to the coding sequence of the green fluorescent protein (GFP) in bacteria andSchizosaccharomyces pombe cells. The GFP fusions were readily detected in bacteria transformed with the monocistronic construct RDRP:GFP; expression of the downstream RDRP:GFP from the dicistronic construct CP-RDRP:GFP could not be detected. However, fluorescence microscopy and Western blot analysis indicated that RDRP:GFP was expressed at low levels from its downstream ORF in the dicistronic construct inS. pombe cells. No evidence that the RDRP ORF was expressed from a transcript shorter than the full-length dicistronic mRNA was found. A coupled termination-reinitiation mechanism that requires host or eukaryotic cell factors is proposed for the expression of Hv190SV RDRP.