Changing all three cysteine residues to alanines exerted no negative effects on E release or its capability to interact with N or VLP production. (A-B) 293 T cells were transfected with SARS-CoV HA-E expression vectors alone, or co-transfected with HA-E plus M and/or N expression vectors. EC3A denotes alanine replacement of all three cysteine residues at E (C40, C43 and C44). Supernatants and cells were collected at 48 h post-transfection and analyzed by Western immunoblotting. Aliquots of cell and supernatant samples containing E proteins (panel A, lanes 1 to 3 and 10 to 12) were prepared without reducing reagent (β-mercaptoethanol). SARS-CoV E, M, and N were respectively probed with anti-HA, anti-M and anti-N antibodies. Arrowheads indicate the position of HA-E dimer.