A Sequence-Independent, Unstructured Internal Ribosome Entry Site Is Responsible for Internal Expression of the Coat Protein of Turnip Crinkle Virus

Abstract

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 the Turnip 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) both in vitro and in vivo. An 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 region in vitro by 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 accumulated in vivo in the absence of detectable TCV subgenomic RNAs, strongly suggesting that the IRES was active in the gRNA in vivo. Since 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.IMPORTANCE Cap-independent translation is a common strategy among positivesense, 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 protein in vitro and in vivo. Our findings may be applicable to cellular mRNA IRES that also have little or no sequences/structures in common.KEYWORDS IRES, translation, carmovirus, cap-independent translation, Turnip crinkle virus, unstructured RNA, internal ribosome entry site, translational control E ukaryotic mRNAs utilize a cap-dependent translation where the 40S ribosomal subunit is recruited to a 5= 7-methylguanosine (m 7 G) cap with the assistance of eukaryotic initiation factors (eIFs). Translation initiation is largely governed by eIF4F, which consists of eIF4E and eIF4G in plants. eIF4E is responsible for binding to the 5= cap structure, while eIF4G is a scaffolding protein that interacts with eIF4E and poly(A) binding protein (PABP) bound to the 3= poly(A) tail, thus circularizing the mRNA (1). The 43S preinitiation complex, composed of the ribosomal 40S subunit bound to eIF3/eIF5 and eIF2-Met-tRNAi Met , is attracted to the 5= cap via binding of eIF3 to eIF4G and then

Functional 5′ UTR mRNA structures in eukaryotic translation regulation and how to find them

“…Thus, an unstructured element embedded in an RNA structure may actually be the driving force of a structured motif. In the case of IRESs, this notion could provide an explanation for the discovery of short linear poly(U) motifs that harbour IRES activity 144 , for the finding that the ITAF PTB recognizes and binds to a polypyrimidine-rich motif, (CCU) n , in structured cellular IRESs 114 , or for the detection of an unstructured region that is important for the activity of the turnip crinkle virus IRES 190 , despite the fact that viral IRESs are thought to largely be structured. Thus, local mRNA structures as well as unstructured motifs therein can regulate translation.…”

Non-canonical Translation in Plant RNA Viruses

“…However, apparently this observation has not been repeated in other labs (e.g., Fan et al, 2012). A low level of CP translation from genomic RNA of carmoviruses Hibiscus chlorotic ringspot virus (HCRSV) (Koh et al, 2003;Fernández-Miragall and Hernández, 2011), Pelargonium flower break virus (PFBV) (Fernández-Miragall and , and Turnip crinkle virus (TCV) (May et al, 2017) has also been reported to be IRESmediated. Like the crTMV IRES, the TCV IRES seems to require only to be A-rich and lack of structure and its activity is inversely correlated with the size of the RNA.…”

Insights into Structural and Mechanistic Features of Viral IRES Elements

“…Furthermore, enhancement of IRES function in the presence of 3′-poly(A) and the absence of 5′-cap suggest a crosstalk between PABP, the CrTMV IRES and the 3′-poly(A) tail ( Marom et al, 2009 ). Similarly, an unstructured sequence of about 84 nt has been reported to control low levels of translation of the genomic RNA of turnip crinkle virus (TCV) coat protein ( May et al, 2017 ). Surprisingly, in marked contrast to most IRES elements, this A-rich sequence promotes translation irrespectively of the orientation in eIF4G-dependent but in a 4E-independent manner.…”