The 'Iceberg region of the Grapevine leafroll-associated virus 3 replicase polyprotein contains signals for mitochondrial targeting and outer membrane association

Positive-sense single-stranded (+ss) RNA viruses such as Grapevine leafroll-associated virus 3 (GLRaV3) replicate their genomes within membrane-bound viral replication complexes (VRCs). The biogenesis of such VRCs is driven by viral "replicase" polyproteins containing several replication-related domains. Past electron microscopy evidence suggests that GLRaV3 forms VRCs from the outer mitochondrial membrane of host grapevine plants. Here, we report the subcellular localization of the replicase polyprotein encoded by GLRaV3 ORF1a (PP1a) towards understanding its putative role in VRC formation. Through confocal laser scanning microscopy analysis of distinct EGFP-tagged PP1a domains, interdomain regions and truncations expressed in model plants, we report the molecular signals responsible for the targeting and association of PP1a to the mitochondria. This signal, located in the "Iceberg" region downstream from the methyltransferase-guanylyltransferase (M/GTase) domain, comprises an amphipathic -helix and a downstream transmembrane domain (TMD). Mutagenesis studies suggest that the polar face of the amphipathic -helix functions as the targeting signal, whereas the non-polar face, together with the TMD, act in membrane-anchoring. Microscopy observations are confirmed through mitochondrial isolation via gradient centrifugation and Western blotting. Structure prediction of the GLRaV3 M/GTase domain and its downstream TMD suggests a putative dodecameric oligomeric state. This dodecamer may gate the neck of GLRaV3 VRCs and contribute to their biogenesis, a hypothesis to be tested in follow-up studies. SIGNIFICANCE STATEMENTPositive-sense RNA viruses such as GLRaV3 form specialized membrane-bound viral replication complexes (VRCs) within host cells to sequester viral genome replication. Past evidence suggests that GLRaV3 targets the outer mitochondrial membrane (OMM) of grapevine host cells for VRC formation, however the viral protein responsible remained unknown. Here, we report a newly identified amphipathic -helix and a downstream transmembrane domain in the replicase polyprotein encoded by GLRaV3 ORF1a that are crucial for OMM targeting and membrane association. Since OMM targeting mechanisms in plants are poorly understood, our findings not only shed light on how GLRaV3 assembles VRCs, but also provides insight into OMM targeting more broadly. This work lays the foundation towards elucidating the molecular mechanisms of GLRaV3 replication and host-pathogen interactions.