Sec1/Munc18-family (SM) proteins are required for SNARE-mediated membrane fusion, but their mechanism(s) of action remain controversial. Using single-molecule force spectroscopy, we found that the SM protein Munc18-1 catalyzes step-wise zippering of three synaptic SNAREs (syntaxin, VAMP2, and SNAP-25) into a four-helix bundle. Catalysis requires formation of an intermediate template complex in which Munc18-1 juxtaposes the N-terminal regions of the SNARE motifs of syntaxin and VAMP2, while keeping their C-terminal regions separated. SNAP-25 binds the templated SNAREs to induce full SNARE zippering. Munc18-1 mutations modulate the stability of the template complex in a manner consistent with their effects on membrane fusion, indicating that chaperoned SNARE assembly is essential for exocytosis. Two other SM proteins, Munc18-3 and Vps33, similarly chaperone SNARE assembly via a template complex, suggesting that SM protein mechanism is conserved.

Porcine reproductive and respiratory syndrome virus (PRRSV) and transmissible gastroenteritis virus (TGEV) are two highly infectious and lethal viruses causing major economic losses to pig production. Here, we report generation of double-gene-knockout (DKO) pigs harboring edited knockout alleles for known receptor proteins CD163 and pAPN and show that DKO pigs are completely resistant to genotype 2 PRRSV and TGEV. We found no differences in meat-production or reproductive-performance traits between wild-type and DKO pigs, but detected increased iron in DKO muscle. Additional infection challenge experiments showed that DKO pigs exhibited decreased susceptibility to porcine deltacoronavirus (PDCoV), thus offering unprecedented in vivo evidence of pAPN as one of PDCoV receptors. Beyond showing that multiple gene edits can be combined in a livestock animal to achieve simultaneous resistance to two major viruses, our study introduces a valuable model for investigating infection mechanisms of porcine pathogenic viruses that exploit pAPN or CD163 for entry.

20Sec1/Munc18-family (SM) proteins are required for SNARE-mediated membrane fusion, 21 but their mechanism(s) of action remain controversial. Using single-molecule force 22 spectroscopy, we found that the SM protein Munc18-1 catalyzes step-wise zippering of 23 three synaptic SNAREs (syntaxin, VAMP2, and SNAP-25) into a four-helix bundle. 24 Catalysis requires formation of an intermediate template complex in whichMunc18-1 25 juxtaposes the N-terminal regions of the SNARE motifs of syntaxin and VAMP2, while 26 keeping their C-terminal regions separated. Next, SNAP-25 binds the templated SNAREs 27to form a partially-zippered SNARE complex. Finally, full zippering displaces Munc18-1. 28 Munc18-1 mutations modulate the stability of the template complex in a manner consistent 29with their effects on membrane fusion, indicating that chaperoned SNARE assembly is 30 essential for exocytosis. Two other SM proteins, Munc18-3 and Vps33, similarly chaperone 31 SNARE assembly via a template complex, suggesting that SM protein mechanism is 32 conserved. 33 34 lymphocytes to kill cancerous or infected cells (Cote et al., 2009) and for glucose uptake (Bryant 43 and Gould, 2011), respectively. Consequently, dysfunctions of SM proteins are associated with 44 neurological and immunological disorders, cancers, diabetes, and other diseases (Bryant and 45

Highlights Inhibition of polyamine synthesis suppresses PRRSV proliferation PRRSV infection relies on spermidine and spermine PRRSV decreases the levels of intracellular polyamines PRRSV infection increases the mRNA level of SAT1 SAT1 inhibits PRRSV propagation