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.

Nsp4 with nsp3 produces MLBs featuring doublemembrane walls. Transfection of nsp4 alone induced a punc-tate pattern as observed via immunofluorescence microscopy consistent with the localization of nsp4 to the ER (Fig. 2B ), as others have demonstrated (31, 52) . In electron microscopy, cells transfected with nsp4 alone showed no distinct phenotype ( Table 1) . Cotransfection of nsp3 and nsp4 produced a pattern that was distinct from that seen for either nsp3 alone or nsp4 alone in immunofluorescence (Fig. 2C ). In electron microscopy, it was observed that the membranes in this cotransfection form an extensive (typically~2-m diameter) winding mazelike body (MLB), featuring paired membranes interspersed with double-membrane circular structures with an average diameter of~80 nm (Fig. 4) . The apposing walls of the MLB were typically separated from each other by approximately 20 nm. The MLB appear perinuclear, and interconnections with the ER were present (Fig. 4, black arrowheads) .