Requirement for Host RNA-Silencing Components and the Virus-Silencing Suppressor when Second-Site Mutations Compensate for Structural Defects in the 3' Untranslated Region.
Turnip crinkle virus (TCV) contains a structured 3' region with hairpins and pseudoknots that form a complex network of noncanonical RNA:RNA interactions supporting higher-order structure critical for translation and replication. We investigated several second-site mutations in the p38 coat protein open reading frame (ORF) that arose in response to a mutation in the asymmetric loop of a critical 3' untranslated region (UTR) hairpin that disrupts local higher-order structure. All tested second-site mutations improved accumulation of TCV in conjunction with a partial reversion of the primary mutation (TCV-rev1) but had neutral or a negative effect on wild-type (wt) TCV or TCV with the primary mutation. SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) structure probing indicated that these second-site mutations reside in an RNA domain that includes most of p38 (domain 2), and evidence for RNA:RNA interactions between domain 2 and 3'UTR-containing domain 1 was found. However, second-site mutations were not compensatory in the absence of p38, which is also the TCV silencing suppressor, or in dcl-2/dcl4 or ago1/ago2 backgrounds. One second-site mutation reduced silencing suppressor activity of p38 by altering one of two GW motifs that are required for p38 binding to double-stranded RNAs (dsRNAs) and interaction with RNA-induced silencing complex (RISC)-associated AGO1/AGO2. Another second-site mutation substantially reduced accumulation of TCV-rev1 in the absence of p38 or DCL2/DCL4. We suggest that the second-site mutations in the p38 ORF exert positive effects through a similar downstream mechanism, either by enhancing accumulation of beneficial DCL-produced viral small RNAs that positively regulate the accumulation of TCV-rev1 or by affecting the susceptibility of TCV-rev1 to RISC loaded with viral small RNAs.


OBJECTIVE
Genomes of positive-strand RNA viruses fold into high-order RNA structures. Viruses with mutations in regions critical for translation and replication often acquire second-site mutations that exert a positive compensatory effect through reestablishment of canonical base pairing with the altered region. In this study, two distal second-site mutations that individually arose in response to a primary mutation in a critical 3' UTR hairpin in the genomic RNA of turnip crinkle virus did not directly interact with the primary mutation. Although different second-site changes had different attributes, compensation was dependent on the production of the viral p38 silencing suppressor and on the presence of silencing-required DCL and AGO proteins. Our results provide an unexpected connection between a 3' UTR primary-site mutation proposed to disrupt higher-order structure and the RNA-silencing machinery.
Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, USA.
Maitreyi
M
Chattopadhyay
Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, USA.
Vera A
VA
Stupina
Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, USA.
Feng
F
Gao
Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, USA.
Christine R
CR
Szarko
Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, USA.
Micki M
MM
Kuhlmann
Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, USA.
Xuefeng
X
Yuan
Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, USA.
Kerong
K
Shi
Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, USA simona@umd.edu.
Anne E
AE
Simon
D020413:3' Untranslated Regions / Q000235:genetics*; D017360:Arabidopsis / Q000821:virology; D029681:Arabidopsis Proteins / Q000235:genetics; D060565:Argonaute Proteins / Q000235:genetics; D036022:Capsid Proteins / Q000235:genetics; D019184:Carmovirus / Q000235:genetics*; D054884:Host-Pathogen Interactions / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D016366:Open Reading Frames / Q000235:genetics; D059370:RNA Folding / Q000235:genetics*; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D014026:Nicotiana / Q000821:virology
D020413:3' Untranslated Regions; C117457:AGO1 protein, Arabidopsis; D029681:Arabidopsis Proteins; D060565:Argonaute Proteins; D036022:Capsid Proteins; D012330:RNA, Double-Stranded; D034741:RNA, Small Interfering; D012367:RNA, Viral; C559855:argonaute 2 protein, Arabidopsis