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.

A possible explanation for DMV formation is that nsp3 is responsible for membrane proliferation that results in enough membrane to form the network of DMVs that is required for RTC formation. The 20-nm distance typically found between the apposing membranes in SARS-CoV-induced DMVs and CMs was the same for nsp3-nsp4-nsp6 transfection-induced DMVs and CMs. This 20-nm distance was also found in nsp3-nsp4 MLBs, suggesting that nsp3 and nsp4 together are responsible for the DMV-like membrane pairing of the triple transfection. nsp3-nsp4 MLBs may represent a more organized version of SARS-CoVinduced convoluted membrane. The role of nsp6 may be to force the double-membrane structures mainly toward the formation of spherical vesicles as opposed to the MLBs seen in the absence of nsp6. These nsp6-induced structures appear to be consistent with what has been shown previously regarding the role of nsp6 in inducing autophagosomes (55) . Since cleavage of nsp3 and nsp4 occurs very rapidly upon polyprotein production and nsp6 cleavage may be comparatively delayed, one possibility for DMV formation could be that the MLBs and MTOCVs form in the cell somewhat independently and then rapidly meet to produce the DMVs (20, 61) . However, the presence of all three at once may directly lead to production of DMVs without any of the intermediate structures. While the DMVs that are produced by nsp3-nsp4-nsp6 transfection are similar in structure and organization to authentic SARS-induced DMVs, they are smaller. This suggests a role for other proteins or the presence of viral RNA in determining DMV size.