The ‘Iceberg’ region of the Grapevine leafroll-associated virus 3 replicase polyprotein contains signals for mitochondrial targeting and outer membrane association
Bibliographic record
Abstract
ABSTRACT 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 α-helix and a downstream transmembrane domain (TMD). Mutagenesis studies suggest that the polar face of the amphipathic α-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 STATEMENT Positive-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 α-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.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.001 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.001 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".