A Sequence-Independent, Unstructured Internal Ribosome Entry Site Is Responsible for Internal Expression of the Coat Protein of Turnip Crinkle Virus.
To maximize the coding potential of viral genomes, internal ribosome entry sites (IRES) can be used to bypass the traditional requirement of a 5' cap and some/all of the associated translation initiation factors. Although viral IRES typically contain higher-order RNA structure, an unstructured sequence of about 84 nucleotides (nt) immediately upstream of theTurnip crinkle virus(TCV) coat protein (CP) open reading frame (ORF) has been found to promote internal expression of the CP from the genomic RNA (gRNA) bothin vitroandin vivoAn absence of extensive RNA structure was predicted using RNA folding algorithms and confirmed by selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) RNA structure probing. Analysis of the IRES regionin vitroby use of both the TCV gRNA and reporter constructs did not reveal any sequence-specific elements but rather suggested that an overall lack of structure was an important feature for IRES activity. The CP IRES is A-rich, independent of orientation, and strongly conserved among viruses in the same genus. The IRES was dependent on eIF4G, but not eIF4E, for activity. Low levels of CP accumulatedin vivoin the absence of detectable TCV subgenomic RNAs, strongly suggesting that the IRES was active in the gRNAinvivoSince the TCV CP also serves as the viral silencing suppressor, early translation of the CP from the viral gRNA is likely important for countering host defenses. Cellular mRNA IRES also lack extensive RNA structures or sequence conservation, suggesting that this viral IRES and cellular IRES may have similar strategies for internal translation initiation.IMPORTANCECap-independent translation is a common strategy among positive-sense, single-stranded RNA viruses for bypassing the host cell requirement of a 5' cap structure. Viral IRES, in general, contain extensive secondary structure that is critical for activity. In contrast, we demonstrate that a region of viral RNA devoid of extensive secondary structure has IRES activity and produces low levels of viral coat proteinin vitroandin vivoOur findings may be applicable to cellular mRNA IRES that also have little or no sequences/structures in common.
Department of Cell Biology and Molecular Genetics, University of Maryland-College Park, College Park, Maryland, USA.
Jared
J
May
Department of Cell Biology and Molecular Genetics, University of Maryland-College Park, College Park, Maryland, USA.
Philip
P
Johnson
Department of Cell Biology and Molecular Genetics, University of Maryland-College Park, College Park, Maryland, USA.
Huma
H
Saleem
Department of Cell Biology and Molecular Genetics, University of Maryland-College Park, College Park, Maryland, USA simona@umd.edu.
Anne E
AE
Simon
D036022:Capsid Proteins / Q000096:biosynthesis* / Q000235:genetics; D019184:Carmovirus / Q000235:genetics*; D039561:Eukaryotic Initiation Factor-4E / Q000378:metabolism; D015870:Gene Expression; D000067556:Internal Ribosome Entry Sites; D009690:Nucleic Acid Conformation; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D036022:Capsid Proteins; D039561:Eukaryotic Initiation Factor-4E; D000067556:Internal Ribosome Entry Sites; D012333:RNA, Messenger
IRES; Turnip crinkle virus; cap-independent translation; carmovirus; internal ribosome entry site; translation; translational control; unstructured RNA