Proximity Labelling‐Based Proteomics Identifies Antiviral Host Factors Associated With the Potexvirus Replicase

Plant positive‐strand RNA viruses establish viral replication complexes (VRCs) by remodelling intracellular membranes, primarily the endoplasmic reticulum (ER) and recruiting host proteins to these structures. To identify ER‐associated host proteins involved in potexvirus replication, we developed a multi‐control proximity labelling approach using TurboID‐fused constructs that localise to the cytosol, the ER lumen and the ER membrane. This strategy enabled specific identification of host factors that are proximal to the methyltransferase (MET) domain of Plantago asiatica mosaic virus (PlAMV) replicase, distinguishing them from general ER‐associated proteins. Our proximity labelling identified 33 host factors associated with the PlAMV MET domain, 28 of which showed exclusive enrichment compared to all three controls. Subcellular localization prediction revealed that MET‐proximal proteins predominantly associate with chloroplasts, the cytosol and mitochondria rather than the ER. This suggests that the MET domain localises to specialised ER membrane subdomains interfacing with multiple organelles. Functional analysis of three selected candidates, calcyclin‐binding protein (NbCBP), remorin1.5 (NbREM1.5) and calcium‐sensing receptor (NbCAS), revealed their antiviral roles. Virus‐induced gene silencing of these factors significantly enhanced PlAMV accumulation. Co‐immunoprecipitation demonstrated physical interactions between the three host factors and the MET domain, while confocal microscopy revealed their relocalization to specific subcellular sites during co‐expression with MET. These results demonstrate that potexvirus VRCs interact with diverse host factors from multiple organelles, providing new insights into plant–virus interaction networks.