Leaky scanning and scanning-independent ribosome migration on the tricistronic S1 mRNA of avian reovirus.
The S1 genome segments of avian and Nelson Bay reovirus encode tricistronic mRNAs containing three sequential partially overlapping open reading frames (ORFs). The translation start site of the 3'-proximal ORF encoding the sigmaC protein lies downstream of two ORFs encoding the unrelated p10 and p17 proteins and more than 600 nucleotides distal from the 5'-end of the mRNA. It is unclear how translation of this remarkable tricistronic mRNA is regulated. We now show that the p10 and p17 ORFs are coordinately expressed by leaky scanning. Translation initiation events at these 5'-proximal ORFs, however, have little to no effect on translation of the 3'-proximal sigmaC ORF. Northern blotting, insertion of upstream stop codons or optimized translation start sites, 5'-truncation analysis, and poliovirus 2A protease-mediated cleavage of eIF4G indicated sigmaC translation derives from a full-length tricistronic mRNA using a mechanism that is eIF4G-dependent but leaky scanning- and translation reinitiation-independent. Further analysis of artificial bicistronic mRNAs failed to provide any evidence that sigmaC translation derives from an internal ribosome entry site. Additional features of the S1 mRNA and the mechanism of sigmaC translation also differ from current models of ribosomal shunting. Translation of the tricistronic reovirus S1 mRNA, therefore, is dependent both on leaky scanning and on a novel scanning-independent mechanism that allows translation initiation complexes to efficiently bypass two functional upstream ORFs.
Racine
Trina
T
Department of Microbiology and Immunology, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia B3H 1X5, Canada.
Barry
Chris
C
Roy
Kenneth
K
Dawe
Sandra J
SJ
Shmulevitz
Maya
M
Duncan
Roy
R
D000818:Animals; D002460:Cell Line; D018387:Codon, Initiator / Q000235:genetics / Q000378:metabolism*; D039603:Eukaryotic Initiation Factor-4G / Q000235:genetics / Q000378:metabolism; D005796:Genes / Q000502:physiology*; D016679:Genome, Viral; D008954:Models, Biological; D016366:Open Reading Frames / Q000502:physiology; D030101:Orthoreovirus, Avian / Q000235:genetics / Q000378:metabolism*; D010442:Peptide Chain Initiation, Translational / Q000502:physiology*; D017955:Poliovirus / Q000235:genetics / Q000378:metabolism; D011784:Quail; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*; D014764:Viral Proteins / Q000096:biosynthesis*
D018387:Codon, Initiator; D039603:Eukaryotic Initiation Factor-4G; D012367:RNA, Viral; D014764:Viral Proteins