Hepatitis C virus internal ribosome entry site initiates protein synthesis at the authentic initiation codon in yeast.
Hepatitis C virus (HCV) is an important pathogen causing both acute and chronic infections in humans. The HCV polyprotein is synthesized by cap-independent translation initiation after ribosome binding to the highly structured internal ribosome entry site (IRES). The HCV IRES has been shown to have a low requirement for translation initiation factors and the ability to bind directly to the 40S ribosomal subunit. A novel yeast bicistronic reporter system, suitable for sensitive and accurate analysis of IRES activity, has been developed. It employs signal amplification based on the Gal4p transcription factor-mediated activation of a variety of secondary reporter genes. The system has a broad dynamic range and, depending on the nature of the particular secondary reporter, can be used both for precise measurements of IRES activity and for selection and screening for novel IRES variants and IRES trans-acting factors. By using this novel bicistronic system, it was shown that the HCV IRES is functional in yeast cells. Mutational analysis of the IRES loop IV and the adjacent region revealed that, in yeast, as in mammalian cells, translation initiates preferentially at the authentic (342)AUG codon and that disruption of the HCV IRES loop IV abrogates its function, whilst minor positional changes or substitutions of the initiation codon within loop IV are largely tolerated. These findings bring more general insights to translation initiation, but also open the door for utilization of yeast and its sophisticated genetics for searching for new antiviral drugs and HCV IRES trans-acting proteins.
Masek
Tomas
T
Department of Genetics and Microbiology, Faculty of Science, Charles University in Prague, Vinicna 5, 128 44 Prague, Czech Republic.
Vopalensky
Vaclav
V
Department of Genetics and Microbiology, Faculty of Science, Charles University in Prague, Vinicna 5, 128 44 Prague, Czech Republic.
Horvath
Ondrej
O
Institute of Molecular Genetics, Laboratory of Leukocyte Antigens, AS CR, Videnska 1083, 140 00 Prague, Czech Republic.
Vortelova
Lucie
L
Department of Genetics and Microbiology, Faculty of Science, Charles University in Prague, Vinicna 5, 128 44 Prague, Czech Republic.
Feketova
Zuzana
Z
Department of Genetics and Microbiology, Faculty of Science, Charles University in Prague, Vinicna 5, 128 44 Prague, Czech Republic.
Pospisek
Martin
M
Department of Genetics and Microbiology, Faculty of Science, Charles University in Prague, Vinicna 5, 128 44 Prague, Czech Republic.
D001483:Base Sequence; D018387:Codon, Initiator; D004279:DNA, Viral / Q000235:genetics; D005800:Genes, Fungal; D017930:Genes, Reporter; D005814:Genes, Viral; D005822:Genetic Vectors; D016174:Hepacivirus / Q000235:genetics* / Q000472:pathogenicity / Q000502:physiology*; D006801:Humans; D066298:In Vitro Techniques; D010957:Plasmids / Q000235:genetics; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012270:Ribosomes / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D014764:Viral Proteins / Q000096:biosynthesis* / Q000235:genetics
D018387:Codon, Initiator; D004279:DNA, Viral; D011994:Recombinant Proteins; D014764:Viral Proteins