biostudies-literatureAcosta-Caceres JMDeutsche ForschungsgemeinschaftSpanish Ministry of Science and InnovationGeneralitat Valencianae70355https://www.ebi.ac.uk/biostudies/studies/S-EPMC12542298biostudies-literatureUnknown34(11)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 simulProtein science : a publication of the Protein SocietyStructural insights into SARS-CoV-2 nonstructural protein 4 (nsp4) biogenesis.PMC12542298PID2023-152568NB-I00SFB-1638/1-511488495-Z01PID2020-119111GB-I00CIPROM/2022/062Acosta-Caceres JMMingarro IGarcia-Murria MJMartinez-Gil LOrtiz-Mateu JGadea-Salom LLolicato FBelda DfalseStructural insights into SARS-CoV-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 simul2025-01-01T00:00:00Z2025 Nov2026-07-15T07:00:36.322Z2026-07-02T03:08:24.561ZS-EPMC125422984112341510.1002/pro.70355