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The terminal loop of a 3' proximal hairpin plays a critical role in replication and the structure of the 3' region of Turnip crinkle virus.

ABSTRACT: Plus-strand RNA viruses serve as templates for translation and then transcription by newly synthesized RdRp. A ribosome-binding tRNA-shaped structure (TSS) and upstream hairpin H4 in the 3' UTR of Turnip crinkle virus (TCV) play key roles in translation and transcription. Second-site mutations generated to compensate for altering the critical asymmetric internal loop of H4 included a three- to two-base alteration in the terminal loop of a 3' proximal hairpin (Pr) located downstream of the TSS. Unlike the non-deleterious three-base alteration, single mutations in Pr loop were detrimental for RdRp transcription while enhancing translation and RdRp binding. One deleterious mutation in the Pr loop altered the structures of both the TSS and H4. These complex interactions in the 3' UTR support a compact structural arrangement likely permitting RdRp access to a number of residues within a 195-base region including the 3' end that are necessary for efficient transcription initiation.

SUBMITTER: Yuan X

PROVIDER: S-EPMC2891086 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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The terminal loop of a 3' proximal hairpin plays a critical role in replication and the structure of the 3' region of Turnip crinkle virus.

Yuan Xuefeng X Shi Kerong K Young Megan Y L MY Simon Anne E AE

Virology 20100418 2

Plus-strand RNA viruses serve as templates for translation and then transcription by newly synthesized RdRp. A ribosome-binding tRNA-shaped structure (TSS) and upstream hairpin H4 in the 3' UTR of Turnip crinkle virus (TCV) play key roles in translation and transcription. Second-site mutations generated to compensate for altering the critical asymmetric internal loop of H4 included a three- to two-base alteration in the terminal loop of a 3' proximal hairpin (Pr) located downstream of the TSS. U ...[more]

PMID: 20403628

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Project description:Long noncoding RNAs (lncRNAs) of virus origin accumulate in cells infected by many positive-strand (+) RNA viruses to bolster viral infectivity. Their biogenesis mostly utilizes exoribonucleases of host cells that degrade viral genomic or subgenomic RNAs in the 5'-to-3' direction until being stalled by well-defined RNA structures. Here, we report a viral lncRNA that is produced by a novel replication-dependent mechanism. This lncRNA corresponds to the last 283 nucleotides of the turnip crinkle virus (TCV) genome and hence is designated tiny TCV subgenomic RNA (ttsgR). ttsgR accumulated to high levels in TCV-infected Nicotiana benthamiana cells when the TCV-encoded RNA-dependent RNA polymerase (RdRp), also known as p88, was overexpressed. Both (+) and (-) strand forms of ttsgR were produced in a manner dependent on the RdRp functionality. Strikingly, templates as short as ttsgR itself were sufficient to program ttsgR amplification, as long as the TCV-encoded replication proteins p28 and p88 were provided in trans. Consistent with its replicational origin, ttsgR accumulation required a 5' terminal carmovirus consensus sequence (CCS), a sequence motif shared by genomic and subgenomic RNAs of many viruses phylogenetically related to TCV. More importantly, introducing a new CCS motif elsewhere in the TCV genome was alone sufficient to cause the emergence of another lncRNA. Finally, abolishing ttsgR by mutating its 5' CCS gave rise to a TCV mutant that failed to compete with wild-type TCV in Arabidopsis. Collectively, our results unveil a replication-dependent mechanism for the biogenesis of viral lncRNAs, thus suggesting that multiple mechanisms, individually or in combination, may be responsible for viral lncRNA production. IMPORTANCE Many positive-strand (+) RNA viruses produce long noncoding RNAs (lncRNAs) during the process of cellular infections and mobilize these lncRNAs to counteract antiviral defenses, as well as coordinate the translation of viral proteins. Most viral lncRNAs arise from 5'-to-3' degradation of longer viral RNAs being stalled at stable secondary structures. Here, we report a viral lncRNA that is produced by the replication machinery of turnip crinkle virus (TCV). This lncRNA, designated ttsgR, shares the terminal characteristics with TCV genomic and subgenomic RNAs and overaccumulates in the presence of moderately overexpressed TCV RNA-dependent RNA polymerase (RdRp). Furthermore, templates that are of similar sizes as ttsgR are readily replicated by TCV replication proteins (p28 and RdRp) provided from nonviral sources. In summary, this study establishes an approach for uncovering low abundance viral lncRNAs, and characterizes a replicating TCV lncRNA. Similar investigations on human-pathogenic (+) RNA viruses could yield novel therapeutic targets.

Dataset Information

The terminal loop of a 3' proximal hairpin plays a critical role in replication and the structure of the 3' region of Turnip crinkle virus.

Publications

The terminal loop of a 3' proximal hairpin plays a critical role in replication and the structure of the 3' region of Turnip crinkle virus.

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