Tombusvirus recruitment of host translational machinery via the 3′ UTR

Abstract

RNA viruses recruit the host translational machinery by different mechanisms that depend partly on the structure of their genomes. In this regard, the plus-strand RNA genomes of several different pathogenic plant viruses do not contain traditional translation-stimulating elements, i.e., a 59-cap structure and a 39-poly(A) tail, and instead rely on a 39-cap-independent translational enhancer (39CITE) located in their 39 untranslated regions (UTRs) for efficient synthesis of viral proteins. We investigated the structure and function of the I-shaped class of 39CITE in tombusviruses-also present in aureusviruses and carmoviruses-using biochemical and molecular approaches and we determined that it adopts a complex higher-order RNA structure that facilitates translation by binding simultaneously to both eukaryotic initiation factor (eIF) 4F and the 59 UTR of the viral genome. The specificity of 39CITE binding to eIF4F is mediated, at least in part, through a direct interaction with its eIF4E subunit, whereas its association with the viral 59 UTR relies on complementary RNA-RNA base-pairing. We show for the first time that this tripartite 59 UTR/39CITE/eIF4F complex forms in vitro in a translationally relevant environment and is required for recruitment of ribosomes to the 59 end of the viral RNA genome by a mechanism that shares some fundamental features with cap-dependent translation. Notably, our results demonstrate that the 39CITE facilitates the initiation step of translation and validate a molecular model that has been proposed to explain how several different classes of 39CITE function. Moreover, the virus-host interplay defined in this study provides insights into natural host resistance mechanisms that have been linked to 39CITE activity.

Recruitment of the 40S Ribosome Subunit to the 3′-Untranslated Region (UTR) of a Viral mRNA, via the eIF4 Complex, Facilitates Cap-independent Translation

“…4, A (lane 4) and B). Observation of several bands in toeprint analysis (ϳϩ16 to ϩ18) has been reported by others (26,48) and is seen in cell extract (48).…”

“…One possible mechanistic pathway is that the ribosome interacts with 3Ј-BTE first and subsequently is transferred to the 5Ј-end of the message. The second possible mechanism is that the 3Ј-BTE transfers eIF4F to the 5Ј-end to which the 40S subunit is recruited, as predicted for Tombusviridae genera (26). In both models, eIF4F is required for 40S recruitment, and long distance base pairing between the 3Ј-BTE and 5Ј-UTR is required for delivery of host components, either initiation factors or initiation factors and the 40S subunit.…”

“…Other plant viruses utilize various other RNA structures to bind eIF4F and ultimately deliver the ribosome to the 5Ј-end of the genome (26). A tRNA-shaped 3Ј-CITE of TCV (TSS) was shown to bind directly to the 60S subunit and 80S ribosome, but not the 40S subunit, which leaves the question of how and where the 40S subunit is recruited (29).…”

A Distinct Class of Internal Ribosomal Entry Site in Members of the Kobuvirus and Proposed Salivirus and Paraturdivirus Genera of the Picornaviridae

“…There is direct evidence for the recombinational transfer of the ϳ300-nt-long 3= UTR of grapevine chrome mosaic virus RNA-1 to tomato black ring virus RNA-2 (40), and a similar phenomenon could account for the presence of structurally related and functionally exchangeable translational enhancers known as Barley yellow dwarf virus (BYDV)-like translation elements in the 3= UTRs of members of Luteovirus, Dianthovirus, Necrovirus, and Umbravirus genera (50,86), of the sequence-and structurally related 3=-cap-independent translational enhancer (3=CITE) in the 3= UTRs of members of Tombusvirus, Aureusvirus, and Carmovirus genera (53), and of the stem-loop 2-like motif (s2m) in the 3= end of the genome of some species in Astroviridae, Caliciviridae, Coronaviridae, and Picornaviridae (37,77).…”

Identification of Tobacco Proteins Associated with the Stem-Loop 1 RNAs of Potato virus X

“…In this study, we tried to identify host proteins interacting with PVX SL1 RNAs by proteomic approach since only a limited number of host proteins have been shown to interact with viral RNAs (Fujisaki and Ishikawa, 2008;Nicholson et al, 2010). Although we identified 24 host proteins by selecting only the best-matched protein from individual spots on the gels, it follows that the real number of host proteins interacting with viral RNAs is probably more than 24.…”