Structural insights into <scp>SARS</scp> ‐ <scp>CoV</scp> ‐2 nonstructural protein 4 (nsp4) biogenesis

The SARS-CoV-2 virus-responsible for the COVID-19 pandemic-requires a replication-transcription complex (RTC) for efficient RNA synthesis and viral propagation. One critical RTC component is nonstructural protein 4 (nsp4), a multipass transmembrane (TM) protein implicated in endoplasmic reticulum (ER) membrane rearrangements and double-membrane vesicle (DMV) formation. The membrane topology and functional role of nsp4 in SARS-CoV-2 remain unclear. Here we determined that SARS-CoV-2 nsp4 contains a partially cleaved signal peptide, three TM segments, an extracellularly oriented N-terminus (towards the ER lumen in human cells), and a cytoplasm-facing C-terminus. The non-canonical glycosylation sequon (N¹³¹IC) in nsp4 is not glycosylated in mammalian cells. Molecular dynamics simulations based on these findings refined the structural predictions and folding of individual nsp4 molecules in lipid bilayers, elucidating its membrane disposition. This study advances our understanding of nsp4 membrane topology and its contributions to the formation of DMV and double-membrane-spanning pores, which are essential for viral RNA transport.