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.

The nsps, also referred to as the replicase proteins, localize to the DMVs and CMs (33) . These vesicles, together with their localized proteins, are referred to as the "replication-transcription complex" (RTC). It has been seen for another group of the Nido-virales, the arteriviruses, that two nonstructural proteins alone were sufficient to induce double-membrane vesicles (40) (41) (42) . The two arterivirus nsps responsible for membrane rearrangement are related to SARS-CoV nsp3 and nsp4, which contain transmembrane domains. Additionally, SARS-CoV has a third integral membrane nonstructural protein, nsp6 (43, 44) . SARS-CoV nsp3 is a 215-kDa, transmembrane, glycosylated, multidomain protein that has been shown to interact with numerous other proteins involved in replication and transcription and, as such, may serve as a scaffolding protein for these processes (45) (46) (47) (48) (49) . nsp4 has been shown to cause aberrant DMV formation upon mutation, leading to a loss of nsp4 glycosylation (50) (51) (52) (53) (54) . nsp6 has been shown to activate autophagy, inducing vesicles containing Atg5 and LC3-II (55) . Expression of a construct encoding the last one-third of nsp3 with nsp4 suggested interaction of these two proteins via their ability to relocalize each other in immunofluorescence imaging (31) . In these coexpressing cells, nsp6 was also relocalized (14) . nsp6 has also been shown to interact with a truncated N-terminal region of nsp3 via yeast two-hybrid assays (46) .