SARS-CoV-2 polyprotein expression and the induction of double-membrane vesicles

Area of Science:

• Virology
• Molecular Biology
• Cell Biology

Background:

• Coronaviruses remodel host cell membranes to create double-membrane vesicles (DMVs). These DMVs are crucial platforms for viral RNA replication and transcription by replication-transcription complexes (RTCs).
• The precise mechanisms of DMV assembly and the recruitment of RTC components are not fully understood.
• Non-structural proteins (nsps) 3 and 4 are known to be essential for initial DMV formation and pore assembly.

Purpose of the Study:

• To develop a surrogate expression system for studying severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) induced double-membrane vesicle (DMV) formation and replication-transcription complex (RTC) recruitment independently of viral replication.
• To dissect the roles of specific SARS-CoV-2 non-structural proteins (nsps) in DMV biogenesis and RTC localization.
• To investigate the function of the membrane-associated element (MAE) of nsp6 in DMV formation.

Main Methods:

• Established a surrogate expression system using SARS-CoV-2 polypeptides to induce DMV formation in host cells.
• Characterized DMVs formed by expressing various combinations of nsps (e.g., nsp3-4, nsp3-8, nsp3-10).
• Assessed the localization and membrane association of RTC components using proteinase K (PK) protection assays after cell permeabilization.

Main Results:

• Expression of nsp3-8 and nsp3-10 polyproteins resulted in larger and more heterogeneous DMVs compared to nsp3-4.
• Nsps 5, 7, and 8 were found associated with DMV-enriched membrane fractions, with partial protection from proteinase K digestion, suggesting localization within or near the DMV interior.
• Mutations in the membrane-associated element (MAE) of nsp6 disrupted nsp5 processing, abrogated DMV formation, and caused endoplasmic reticulum (ER) abnormalities.

Conclusions:

• The surrogate expression system is effective for studying coronavirus-induced membrane remodeling and RTC recruitment.
• Nsp6 plays a critical and essential role in DMV biogenesis, particularly through its MAE.
• The findings provide mechanistic insights into how coronaviruses manipulate host membranes for viral replication.