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.

Nsp6 induces single-membrane vesicles around microtubule organizing centers. Transfection of nsp6, either alone or along with nsp3, yielded the presence of a large amount of smoothwalled single-membrane spherical vesicles approximately 280 Ï® 60 nm in diameter (Fig. 5C ). While this microtubule organizing center vesiculation (MTOCV) phenotype was not exclusive to nsp6 transfections, it was far more prevalent in nsp6-transfected cells (Table 1) . This was consistent with what we observed in immunofluorescence with single nsp6 transfections, where the nsp6 signals clustered perinuclearly in one area of the cell. Interestingly, when nsp6 was coexpressed with nsp4, the MTOCV phenotype was lost (Fig. 5D ) and regions surrounding the MTOC instead looked like the equivalent areas in untransfected or nsp4 singly transfected cells (Fig. 5A and B) (Table 1) . Nsp3, nsp4, and nsp6 together induce a pattern of doublemembrane vesicles similar to that seen in SARS-CoV-infected cells. A triple transfection of nsp3 and nsp4 and nsp6 yielded double-membrane vesicles (Fig. 6C to E) with connections to convoluted membranes of morphology similar to that of those induced in SARS coronavirus-infected cells (Fig. 6A and B) . Whereas SARS-CoV-induced DMVs tend to remain approximately 210 Ï® 30 nm in diameter, the DMVs induced by nsp3, nsp4, and nsp6 triple transfection exhibited a smaller average diameter of 120 Ï® 40 nm. Both infection-induced and transfectioninduced DMVs showed an approximate 20-nm separation between apposing membranes. As is the case for SARS-induced DMVs, the triple transfection induced interconnected DMVs that appeared perinuclear, showed contiguity with the ER, and exhibited dark membrane staining. In addition to the DMVs induced by the triple transfection, regions of MLB and MTOCV appearing in the same cell were found three times as frequently as DMVs were found. Interestingly, a triple transfection of nsp3C with nsp4 and nsp6 yielded regions of DMB, MGV, and MTOCV but never maze-like bodies, double-membrane vesicles, or any additional novel structures (Table 1) .