From darkness to light: Genetic manipulation of an atypical plant virus unveils key insights into kitavirus biology, highlighting capsid protein and eIF4A engagement to drive viral infection.

Kitaviridae, a newly recognized virus family, includes plant viruses infecting crops of great global importance, notably citrus. Despite its significant impact on citrus agricultural production, the molecular mechanisms underlying kitavirus infections remain largely unknown. Here, we engineered a recombinant citrus leprosis virus C (CiLV-C, genus Cilevirus) expressing green fluorescent protein (GFP) and demonstrated its feasibility for studying the biology of cilevirus. Genetic manipulation of rCiLV-C-GFP revealed that vRNA1 is essential for replication and can self-replicate independently, while vRNA2 is crucial for movement. The intergenic region between the polymerase and capsid protein (CP) acts as a promoter for CP gene expression. Frameshift and deletion analyses provided key insights into replication, movement, and morphogenesis. We reported that CP is critical for viral RNA accumulation, while movement protein (p32) facilitates viral spread. The putative glycoprotein (p61) is not structurally essential, as its deletion did not affect virion assembly, whereas the putative matrix protein (p24) is critical for morphogenesis, likely acting as a structural protein. Deletion of the RNA silencing suppressor (RSS, p15) and p15-p61 attenuated symptoms, implicating them as virulence factors. Additional analyses revealed that CP enhances vRNA accumulation through a mechanism independent of RSS. CP exhibits RNA-binding properties and interacts with eukaryotic initiation factor 4A (eIF4A), suggesting a role in translation. Overexpression of eIF4A increased CiLV-C RNA accumulation, while eIF4A knockdown reduced it, indicating that CP may recruit eIF4A to promote replication. Similar results were observed with turnip crinkle virus (TCV), and notably, the TCV CP efficiently restored RNA accumulation of a CP-defective CiLV-C, suggesting the existence of a conserved, CP-dependent, replication-related mechanism shared across distinct virus families. Our findings support the proposal of an initial model that elucidates the mechanism through which the CPs drive the production of high levels of vRNA manipulating host eIFs.