Severe Acute Respiratory Syndrome Coronavirus Nonstructural Proteins 3, 4, and 6 Induce Double-Membrane Vesicles

Angelini, Megan M.; Akhlaghpour, Marzieh; Neuman, Benjamin W.; Buchmeier, Michael J.

Cotransfection of nsp3 with nsp4 showed a dramatic effect on membrane conformation, creating a perinuclear doublemembrane walled maze-like body. The MLBs in our electron micrographs consist of roughly parallel rows spaced apart by approximately 80 nm and interspersed with double-membrane walled circular structures of about 80-nm diameter, suggesting that the rows and circles are longitudinal and cross sections of closely packed double-membrane walled tubules. Electron tomography studies would prove beneficial in this determination. The MLB produced by SARS nsp3 and nsp4 is distinct from what has been shown for arteriviruses, where the arterivirus homologues of coronavirus nsp3 and nsp4 are sufficient to induce complete DMVs that look like those of arterivirus-infected cells (41) . These results suggest that a biologically meaningful interaction occurs between nsp3 and nsp4, corroborating previously published data showing interactions between nsp3 and nsp4 via mammalian twohybrid assays (Pan et al.) and Venus reporter fluorescence assays (Hagemeijer et al.) (31, 57) . There is immunofluorescence evidence that a truncated protein running from the GSM to nearly the C terminus of mouse hepatitis virus (MHV) nsp3 is able to change the localization of fluorescently tagged nsp4 to form perinuclear protein clusters (31) , which were not investigated further but which may be similar to the nsp3-nsp4 maze-like bodies described here. If that is the case, then the determinants of nsp3-nsp4 interaction that lead to membrane pairing would be expected to lie in the relatively poorly conserved region between the start of the GSM domain and the amino-terminal transmembrane helix of nsp3. Further research is needed to investigate the determinants of nsp3-nsp4 interaction which result in membrane pairing.