Spatial Proteomics Using BiFCPL Identifies Regulators of DMV Formation Involved in Coronavirus Replication

Area of Science:

• Virology
• Molecular Biology
• Cell Biology

Background:

• Beta-coronaviruses rely on host cell machinery to replicate.
• Double membrane vesicles (DMVs) are essential replication sites for coronaviruses, but the host factors involved are not fully understood.

Purpose of the Study:

• To identify host proteins involved in SARS-CoV-2 induced double membrane vesicle (DMV) formation.
• To investigate the role of identified host factors in DMV biogenesis and coronavirus replication.

Main Methods:

• Utilized a bimolecular fluorescence complementation-based proximity labeling (BiFCPL) strategy to profile the DMV proteome.
• Co-expressed SARS-CoV-2 non-structural proteins (NSP3 and NSP4) to induce DMV formation.
• Confirmed protein localization and interactions using microscopy and genetic manipulation.

Main Results:

• Identified 62 high-confidence host proteins associated with DMVs, including five endoplasmic reticulum-localized proteins interacting with NSP3/NSP4.
• Demonstrated that GRAMD1B and TEX2 are recruited to DMVs via NSP3/NSP4.
• Showed that depletion of GRAMD1B or TEX2 leads to enlarged DMVs and impaired coronavirus replication.

Conclusions:

• GRAMD1B and TEX2 are novel host factors essential for proper DMV formation and coronavirus replication.
• These findings provide insights into host-virus interactions and potential targets for antiviral therapies.