A genetic screen identifies cellular factors involved in retroviral -1 frameshifting.
To identify cellular factors that function in -1 ribosomal frameshifting, we have developed assays in the yeast Saccharomyces cerevisiae to screen for host mutants in which frameshifting is specifically affected. Expression vectors have been constructed in which the mouse mammary tumor virus gag-pro frameshift region is placed upstream of the lacZ gene or the CUP1 gene so that the reporters are in the -1 frame relative to the initiation codon. These vectors have been used to demonstrate that -1 frameshifting is recapitulated in yeast in response to retroviral mRNA signals. Using these reporters, we have isolated spontaneous host mutants in two complementation groups, ifs1 and ifs2, in which frameshifting is increased 2-fold. These mutants are also hypersensitive to antibiotics that target the 40S ribosomal subunit. We have cloned the IFS1 gene and shown that it encodes a previously undescribed protein of 1091 aa with clusters of acidic residues in the carboxyl-terminal region. Haploid cells lacking 82% of the IFS1 open reading frame are viable and phenotypically identical to ifs1-1 mutants. This approach could help identify potential targets for antiretroviral agents.
Lee
S I
SI
Department of Microbiology, University of California, San Francisco 94143, USA.
Umen
J G
JG
Varmus
H E
HE
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002352:Carrier Proteins; D003001:Cloning, Molecular; D016368:Frameshift Mutation; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000235:genetics; D005800:Genes, Fungal; D005814:Genes, Viral; D015750:Genes, gag; D005816:Genetic Complementation Test; D015698:Genomic Library; D005838:Genotype; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics / Q000378:metabolism*; D008668:Metallothionein / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016366:Open Reading Frames; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators
D048868:Adaptor Proteins, Signal Transducing; C500769:CUP1-1 protein, S cerevisiae; D002352:Carrier Proteins; D005656:Fungal Proteins; C092470:NMD2 protein, S cerevisiae; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; C007121:copper thionein; D008668:Metallothionein