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.

Triple transfection of nsp3, nsp4, and nsp6 produced formations that looked very similar to the DMVs seen in coronavirusinfected cells, with double-membrane vesicles surrounding a central convoluted membrane structure (29, 33, 35) . nsp6 appears to either break up or prevent the formation of the elongated stretches of double-membrane walls seen in the nsp3-nsp4 cotransfection mazes, leaving double-membrane vesicles and regions of convoluted membrane that are consistent with SARS-CoV-infected DMVs. Triply transfected cells containing both MLB and MTOCV were about three times as frequent as cells containing DMVs (Table 1) . Additionally, all cells from the triple transfection containing DMVs also contained evidence of MLBs and MTOCVs. This suggests that DMV formation from expressed nsp3, nsp4, and nsp6 is not particularly efficient. The presence of the MLBs and MTOCVs in DMV-containing cells further suggests that nsp3 and nsp4 interact more readily in this expression system than nsp4 and nsp6, which would result in loss of the MTOCV phenotype. Complementation studies using temperaturesensitive mutants of MHV have suggested that nsp4 through nsp10 may have functions in polyprotein forms prior to cleavage or that they are assembled into the RTC and then cleaved (18) . The polyprotein may have a role in keeping nsp4 and nsp6 in close proximity, allowing more efficient DMV formation than in our expression system. Note that even though our nsp3C produced phenotypes very similar to those seen with the full-length nsp3 in single transfections, a triple transfection of nsp3C with nsp4 and nsp6 was unable to produce double-membrane vesicles. One explanation for the differences observed when using full-length nsp3 versus nsp3C is that the transmembrane domains, or domains C terminal to the transmembrane domains, may be responsible for membrane proliferation and convolution but some domain N terminal to the first transmembrane region of nsp3 is required for membrane pairing and regulation for the formation of DMVs. Since nsp6 was previously shown to interact with an N-terminal truncation of nsp3 via yeast two-hybrid screen, it is possible that this interaction is the critical missing link for DMV formation in the nsp3C-nsp4-nsp6 transfection (46) .