The SARS-CoV-2 NSP4 T492I mutation promotes double-membrane vesicle formation to facilitate transmission.

The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in mutations not only in the spike protein, aiding immune evasion, but also in the NSP3/4/6 proteins, crucial for regulating double-membrane vesicle (DMV) formation. However, the functional consequences of these NSP3/4/6 mutations remain poorly understood. In this study, a systematic analysis was conducted to investigate the evolutionary patterns of NSP3/4/6 mutations and their impact on DMV formation. The findings revealed that the NSP4 T492I mutation, a prevalent mutation found in all Delta and Omicron sub-lineages, notably enhances DMV formation. Mechanistically, the NSP4 T492I mutation enhances its homodimerization, leading to an increase in the size of puncta induced by NSP3/4, and also augments endoplasmic reticulum (ER) membrane curvature, resulting in a higher DMV density per fluorescent puncta. This study underscores the significance of the NSP4 T492I mutation in modulating DMV formation, with potential implications for the transmission dynamics of SARS-CoV-2. It contributes valuable insights into how these mutations impact viral replication and pathogenesis.