Structural Domains within the 3′ Untranslated Region of Turnip Crinkle Virus

Abstract

The genomes of positive-strand RNA viruses undergo conformational shifts that complicate efforts to equate structures with function. We have initiated a detailed analysis of secondary and tertiary elements within the 3 end of Turnip crinkle virus (TCV) that are required for viral accumulation in vivo. MPGAfold, a massively parallel genetic algorithm, suggested the presence of five hairpins (H4a, H4b, and previously identified hairpins H4, H5, and Pr) and one H-type pseudoknot (⌿ 3 ) within the 3-terminal 194 nucleotides (nt). In vivo compensatory mutagenesis analyses confirmed the existence of H4a, H4b, ⌿ 3 and a second pseudoknot (⌿ 2 ) previously identified in a TCV satellite RNA. In-line structure probing of the 194-nt fragment supported the coexistence of H4, H4a, H4b, ⌿ 3 and a pseudoknot that connects H5 and the 3 end (⌿ 1 ). Stepwise replacements of TCV elements with the comparable elements from Cardamine chlorotic fleck virus indicated that the complete 142-nt 3 end, and subsets containing ⌿ 3 , H4a, and H4b or ⌿ 3 , H4a, H4b, H5, and ⌿ 2 , form functional domains for virus accumulation in vivo. A new 3-D molecular modeling protocol (RNA2D3D) predicted that H4a, H4b, H5, ⌿ 3 , and ⌿ 2 are capable of simultaneous existence and bears some resemblance to a tRNA. The related Japanese iris necrotic ring virus does not have comparable domains. These results provide a framework for determining how interconnected elements participate in processes that require 3 untranslated region sequences such as translation and replication.Replication of plus-strand RNA viruses initially requires translation of the genomic RNA to produce the virus-encoded, RNA-dependent RNA polymerase (RdRp) and any auxiliary viral proteins necessary for transcription. In a process that is poorly defined but likely dictated by viral and/or cellular factors, translation is terminated and the genomic RNA becomes available for reiterative synthesis of complementary strands, a process that requires membrane association (1, 2, 26). For some viruses, subsequent viral plus-strand synthesis occurs in virus-specific membrane invaginations known as spherules, which contain a limited number of minus-sense genomes and whose formation is induced by specific viral proteins (1,15,26). Although the process of producing viral progeny has been extensively studied using many different viral systems, it remains poorly understood. For example, fundamental questions, such as the role that conformational shifts in RNA structure play in switching the template from translation to replication, the proteins required to enact such events, and if cis-acting core promoters, enhancers, and repressors are organized into functional, interacting modules, remain virtually unanswered.Recent reports that portions of RNA viral genomes undergo conformational shifts to execute different functions (10, 13, 21, 28) complicate efforts to assign biological roles to groups of cis-acting elements that may not structurally coexist. The ability of viral RNAs to assume multiple conformation...

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

“…Briefly, 100 g of raw wheat germ (Bob's Mill) was ground to powder with 100 g of alumina in a cold mortar and resuspended in extraction buffer (20 mM HEPES-KOH, pH 7.6, 100 mM KCl, 1 mM Mg(OAc) 2 , 2 mM CaCl 2 , and 5 mM 2-mercaptoethanol) followed by centrifugation for 10 min at 15,000 ϫ g. Subsequent steps involved purification of the supernatant using a (3 ϫ 25-cm) G-25 column and centrifugation of 150 A 260 units/ml G-25 fractions for 3 h at 170,000 ϫ g. Further purification was performed as described by Spremulli et al (38). Ribosomes were salt-washed using a method used for the preparation of yeast ribosomes, as described previously (39,40). Plant 40S and 60S ribosomal subunits were isolated from purified plant 80S ribosomes by sucrose gradient centrifugation as described previously for the isolation of yeast ribosomal subunits (41).…”

The 3′ proximal translational enhancer of Turnip crinkle virus binds to 60S ribosomal subunits

“…(B) Structure of the 39 region of the TCV 39 UTR. All secondary and tertiary structures have been previously confirmed using single and compensatory mutations (Zhang et al 2006c;McCormack et al 2008). (LSL) large symmetrical loop.…”

“…C 2 , originally identified in the preactive structure of satC (Zhang et al 2006b), pairs sequence in the loop of H4b with residues adjacent to the 39 side of H5, and C 1 , which links 39 terminal residues with the 39 side of the large symmetrical loop (LSL) of H5 (Zhang et al 2006c). Subsets of elements from the related carmovirus Cardamine chlorotic fleck virus were able to functionally replace analogous TCV elements, suggesting that some elements interact to form at least three structural domains: H4 / 39 end, C 3 / H4b, and C 3 / C 2 ; the latter domain is predicted by the three-dimensional (3D) molecular modeling program RNA2D3D (Shapiro et al 2007;Martinez et al 2008) to fold into an internal T-shaped structure (TSS) (McCormack et al 2008).…”

“…1B): H4 along with flanking adenylates functions as a transcriptional enhancer in vitro (Sun and Simon 2006); H4a and adjacent H4b, which are required for viral accumulation in vivo (McCormack et al 2008); H5, which is suggested to be an RdRp chaperone (McCormack and Simon 2004); and Pr, identified as a core promoter in the related subviral RNA satC (Song and Simon 1995). In addition to the hairpins, three pseudoknots have been identified (Zhang et al 2006b;McCormack et al 2008). H-type pseudoknot C 3 connects the loop of H4a and upstream adjacent sequence known as the ''DR'' (de-repressor).…”

Mimicry of molecular pretenders: The terminal structures of satellites associated with plant RNA viruses

“…9 In vivo evidence shows that the randomized sequences of Pr and H5 regions of satC are selected functionally in host plants and maintain similar structures to effect of the terminal structures of satellites on their accumulation in vitro and in vivo. Several well-characterized satellite/helper virus systems, representatives of non-coding satRNAs, mRNA-type satRNAs, circular satRNAs, and satellite viruses listed in Tables 1 and 2 are used as models to compare and contrast the mimicry adopted by different types of satellites.…”