VIRALS · Uncovering the biological relevance of the replication-associated protein splicing during viral replication in geminiviruses and other CRESS viruses

Uncovering the biological relevance of the replication-associated protein splicing during viral replication in geminiviruses and other CRESS viruses

Viruses maximize their limited coding capacity by hijacking host processes such as alternative splicing (AS), yet viral transcript splicing remains poorly characterized in plant viruses. Members of the family Geminiviridae are insect-transmitted plant pathogens with small single-stranded DNA genomes that replicate in the nucleus and belong to the broader CRESS DNA phylum. Their central replication-associated protein (Rep) initiates rolling circle replication and exerts multiple regulatory functions. In several CRESS viruses, including certain geminiviruses, Rep is generated from spliced transcripts, with the different spliced products being essential for replication. Recent evidence from the begomovirus (the main genus within the Geminivirdae family) tomato yellow leaf curl virus (TYLCV) shows that Rep associates with the host splicing machinery in the model host Nicotiana benthamiana, and that silencing core spliceosomal components such as U1-70K or SF3B1 severely impairs viral replication. RNA-seq analyses revealed multiple spliced viral transcripts, including five novel Rep isoforms; viruses unable to produce specific variants show defective accumulation. Most Rep spliced variants lack the oligomerization domain required for replication, display altered subcellular localization, and appear replication-incompetent, yet some repress Rep promoter activity, suggesting a regulatory role. Together, these findings indicate that Rep splicing may represent a previously unrecognized layer of viral gene regulation whose mechanisms and functional significance remain to be elucidated, with potential implications for controlling economically important plant viruses. The overarching goal of this project is to determine the biological significance of Rep splicing in geminiviruses and other CRESS DNA viruses, and to establish whether alternative splicing represents a previously unrecognized regulatory layer controlling viral accumulation. To achieve this, we proposed to: 1) Characterize Rep splicing events during infection using next-generation sequencing and molecular approaches. 2) Determine the functional roles of Rep spliced variants, including their impact on viral replication, transcriptional regulation, protein localization, and host factors interactions. 3) Assess the broader relevance of Rep splicing across CRESS viruses, establishing whether this mechanism is conserved and functionally significant beyond geminiviruses.

Data: CORDIS, © European Union