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.

Although it is understood that viral replicase interaction with host membranes is a requirement for successful coronavirus infection, it has not yet been made clear which viral proteins are involved in double-membrane vesicle formation and the nature of the cellular organelles that are compromised. In this study, we used immunofluorescence and electron microscopy to examine the ability of the three membrane-spanning nonstructural proteins of the SARS coronavirus to induce double-membrane vesicles via transfection. We found that exogenous nsp3 alone, both full length and the C-terminal transmembrane-containing region, was capable of inducing DMB as well as regions of MGV, suggesting a role for the C terminus of nsp3 in membrane production or expansion of existing membranes. In immunofluorescence time course experiments, nsp3 induced hollow accumulations that grew larger in size as time progressed posttransfection, eventually producing patterns much larger than the nsp3 signal observed in SARS-infected cells. These enlarged accumulations further support the idea of a role for nsp3 in membrane proliferation. It is interesting to note that the addition of either nsp4 or nsp6 with nsp3 reduces the appearance of the MGV phenotype but not the DMB phenotype, suggesting a regulatory role of these two nsps on nsp3's membrane proliferation ability.