Biased Hypermutagenesis Associated with Mutations in an Untranslated Hairpin of an RNA Virus

Abstract

The mutation frequency of Turnip crinkle virus can increase 12-fold without inducing error catastrophe. Lesions in a hairpin repressor frequently reverted and led to second-site alterations biased for specific mutations. These results suggest that the hairpin may also function as an RNA chaperone to properly fold the RNA-dependent RNA polymerase.Turnip crinkle virus (TCV), a member of the genus Carmovirus in the family Tombusviridae, has been used as a model for identifying cis-acting elements important for RNA replication. The 4,054-base TCV genome consists of a positive-sense, single-stranded RNA with five overlapping open reading frames (ORF) encoding proteins that function in replication, movement, and packaging (3,8,13) (Fig. 1A). p28 and its readthrough product p88 are required for replication in vivo. p88 contains the RNA-dependent RNA polymerase (RdRp) active site and can by itself promote complementary-strand synthesis from cognate templates in an in vitro (cell-free) assay (16).TCV is also associated with several noncoding satellite RNAs (satRNAs) that range from 194 to 356 bases, with satC (356 bases) sharing its 3Ј-terminal 166 bases with TCV (19). Based on studies using satC, several elements in the 3Ј untranslated region (3Ј UTR) of TCV that are likely important for replication have been identified. These include a core promoter hairpin (Pr) for synthesis of minus strands, which is located at the 3Ј terminus of plus strands and can function independently as a promoter in in vitro assays (2,20,22), and hairpin 5 (H5), a recently identified repressor of minus-strand synthesis (24) (Fig. 1B). Hairpins with similar sequence and/or structural features are located in analogous positions in nearly all carmoviruses (24). H5, which contains a large symmetrical internal loop (LSL), represses minus-strand synthesis in vitro when the 3Ј side of the LSL is base paired with the 3Ј-terminal 4 bases of the RNA (GCCC-OH), thereby sequestering the 3Ј terminus from the RdRp (Fig. 1B). Disruption of the interaction between the satC 3Ј terminus and the LSL substantially enhances synthesis of both full-length and aberrantly initiated complementary strands in vitro. In addition, localized disruption of H5 (and 3Ј-proximal sequences) occurred in satC transcripts containing a deletion of 3Ј-terminal bases. In TCV, compensatory exchanges between the LSL and 3Ј-terminal bases enhanced replication relative to that of virus containing the individual alterations, strongly suggesting that H5 serves an analogous function in TCV (unpublished data). A repressor of minus-strand synthesis with a large asymmetrical internal loop was also recently discovered in Tomato bushy stunt virus, a virus belonging to the same family as TCV (14). In the present study, we have identified an additional feature of H5. Mutations introduced into the TCV LSL resulted, surprisingly, in an increase of as much as 12-fold in second-site mutations scattered throughout the sequenced region; most of these alterations were uridylate-to-cytidylate or adenylate-...

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

“…We have recently determined that mutating these adenylates suppresses specific RdRp binding to the 39 region of the virus, which could account for the more severe effect of m49 on virus accumulation (M. Young and A. E. Simon, unpubl.). While the H5 mutations had no effect on translation and deletion of H5 only reduced F1 ribosome binding by twofold, previous mutations in H5 (McCormack and Simon 2004) and m23 (this report) significantly lowered TCV accumulation in protoplasts. We previously demonstrated that accumulation of nontranslated satC requires maintenance of H5 structure and the LSL adenylates (Zhang et al 2004;Zhang and Simon 2005).…”

“…While mutations in the H5 LSL (Fig. 3A, m23,m38) did not affect translation, virus containing these mutations accumulated to 31% and 88% of wt levels, respectively, reflecting a role for some of the H5 LSL adenylates (e.g., A3978 [m23]) in replication (McCormack and Simon 2004;Zhang et al 2006c). …”

“…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).…”

Conformational changes involved in initiation of minus-strand synthesis of a virus-associated RNA

“…Using in vitro and in vivo approaches, the Pr was previously identified as the satC core promoter (Song and Simon 1995;Carpenter and Simon 1998). H5, also critical for satC and TCV replication (McCormack and Simon 2004;Zhang et al 2004a;Zhang and Simon 2005), contains a large symmetrical internal loop (LSL) whose sequence is highly conserved among related carmoviruses (Zhang et al 2004b). A combination of approaches including single-site mutagenesis, in vivo genetic selection (SELEX), and sequence replacements with analogous segments from the related carmovirus Cardamine chlorotic fleck virus (CCFV) indicates that both specific sequence and structural features throughout H5 are necessary for robust satC accumulation in plants and protoplasts (Zhang et al 2004a;Zhang and Simon 2005).…”

Análisis Funcional De Proteínas Codificadas Por El Virus De La Rotura Del Color De La Flor Del Pelargonium

“…A los elementos cis que intervienen en la replicación se les atribuyen diversas funciones, entre ellas: (1) participar en la formación de los complejos replicativos Wu et al, 2009], (2) actuar como promotores de la replicación promoviendo la síntesis de novo de la cadena complementaria [Kim et al, 2000;Kao et al, 2001], (3) regular la síntesis asimétrica de las cadenas de polaridad positiva y negativa [Ball, 2007], (4) intervenir en la transcripción de los distintos sgRNAs, si procede [Kim & Hemenway, 1999;Grdzelishvili et al, 2005] y, (5) funcionar como chaperonas de RNA [McCormack & Simon, 2004]. como mRNA permitiendo la traducción de las replicasas virales.…”