Replication-Dependent Biogenesis of Turnip Crinkle Virus Long Noncoding RNAs

Area of Science:

• Virology
• Molecular Biology
• RNA Biology

Background:

• Many positive-strand (+) RNA viruses produce long noncoding RNAs (lncRNAs) to enhance infectivity and evade host defenses.
• Existing knowledge suggests viral lncRNAs are primarily generated through host exoribonuclease degradation of viral RNAs.
• The precise mechanisms governing viral lncRNA biogenesis remain incompletely understood.

Purpose of the Study:

• To investigate a novel mechanism for viral long noncoding RNA (lncRNA) production.
• To characterize the biogenesis and function of a specific viral lncRNA from turnip crinkle virus (TCV).
• To explore the role of viral replication machinery in lncRNA production.

Main Methods:

• Overexpression of TCV-encoded RNA-dependent RNA polymerase (RdRp) in infected *Nicotiana benthamiana* cells.
• Analysis of (+) and (-) strand ttsgR production dependent on RdRp functionality.
• Replication assays using short RNA templates and TCV replication proteins (p28 and RdRp) provided *in trans*.
• Mutational analysis of the 5' carmovirus consensus sequence (CCS) in TCV genome and ttsgR.
• Competition assays between wild-type TCV and mutant TCV in *Arabidopsis*.

Main Results:

• A novel viral lncRNA, tiny TCV subgenomic RNA (ttsgR), was identified, produced via a replication-dependent mechanism.
• ttsgR accumulation was dependent on TCV RdRp functionality and could be amplified using short templates.
• A 5' CCS motif was essential for ttsgR replication and accumulation; its presence elsewhere induced new lncRNAs.
• Mutating the 5' CCS of ttsgR resulted in a TCV mutant with reduced competitiveness in *Arabidopsis*.

Conclusions:

• Viral lncRNAs can be generated through replication-dependent mechanisms, in addition to RNA degradation pathways.
• The TCV replication machinery can produce lncRNAs, highlighting a novel viral strategy for RNA management.
• This study provides a framework for discovering low-abundance viral lncRNAs and suggests potential therapeutic targets in human-pathogenic viruses.