The DMV pore-forming TM2–Y region of SARS-CoV-2 nsp3 exhibits structural conservation beyond the coronavirus family

Positive-strand RNA viruses remodel host intracellular membranes to form replication organelles in the cytoplasm. SARS-CoV-2 and other coronaviruses, members of the Nidovirales order, replicate within double-membrane vesicles (DMVs) derived from the endoplasmic reticulum that protect newly synthesized viral RNA (vRNA). A recently discovered SARS-CoV-2 molecular pore complex composed of non-structural proteins 3 and 4 (nsp3 and nsp4) connects the DMV interior with the cytoplasm, enabling vRNA export for subsequent packaging into virions. The C-terminal region of nsp3, the Y domain, oligomerizes to form the base of the cytosolic crown of this pore, while the preceding transmembrane domain 2 (TM2) anchors the pore in the DMV membrane. However, the molecular mechanisms underlying pore formation and whether similar structures are conserved across other Nidovirales members remain unclear. Here, we combined AlphaFold structure prediction, structure-informed multiple sequence alignments, and molecular dynamics simulations to investigate the conservation and potential function of the C-terminal TM2-Y region of nsp3 across nidoviruses. We show that despite minimal sequence identity, all modeled TM2-Y regions adopt similar domain folds. In particular, the wedge-shaped TM2, the Zn-binding Y1 subdomain, and its adjacent Y2 subdomain constitute a conserved structural module present in most vertebrate-infecting Nidovirales families, including Coronaviridae, Tobaniviridae, Gresnaviridae, Olifoviridae, and Arteriviridae. Across these viruses, Zn(II) is coordinated with a distinctive octahedral geometry involving two water molecules. This conserved architecture likely represents an evolutionarily maintained mechanism for DMV pore formation, in which the Zn-binding region engages the membrane surface to induce local lipid bilayer destabilization that may precede DMV pore assembly.IMPORTANCEOver the last 25 years, several coronaviruses, including highly pathogenic SARS-CoV, MERS-CoV, and SARS-CoV-2, have emerged in humans through zoonotic transmission. With nearly 100 coronaviruses known, further spillovers remain possible. Coronaviruses and other nidoviruses replicate within specialized replication organelles formed from host membranes; however, how they form remains poorly understood. This study reveals that coronaviruses and distantly related tobaniviruses and arteriviruses might share a conserved molecular framework for assembling membrane pores that connect the DMV interior, where vRNA is synthetized, and the cytoplasm, where virions are assembled. By identifying conserved structural elements in the C-terminal region of nsp3, including wedge-shaped TM2 domain and Zn-binding Y1/Y2 tandem, we show that vertebrate-infecting nidoviruses likely share structural mechanism for DMV pore formation. These findings provide insight into how diverse nidoviruses remodel host membranes and highlight the C-terminal region of nsp3 as a potential target for broad-spectrum antivirals.