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.

Expression of SARS-CoV nsp3, nsp4, and nsp6. To determine if any of the three integral membrane nonstructural proteins of the SARS-CoV are capable of inducing double-membrane vesicles, we first validated the expression of our various nsp3, nsp4, and nsp6 constructs via Western blot analysis. Constructs were created (Fig. 1A ) as described previously (56) . Lysates from HEK293T cells transfected with our full-length nsp3 construct, termed nsp3 and featuring a C-terminal hemagglutinin (HA) tag followed by a tobacco etch virus (TEV) cleavage site and a biotinylation signal, yield a pattern similar to that seen with SARS-CoV-infected cell lysates when probed using an anti-nsp3 antibody (Fig. 1B) . A truncated form of nsp3 (N terminus through the group II-specific marker [GSM] domain), called nsp3N, was also detectable using an anti-nsp3 antibody (Fig. 1B) . Our nsp3N-terminal construct, nsp3C-terminal construct (spanning the first transmembrane domain through the C terminus), nsp4 construct, and nsp6 construct, all featuring a C-terminal HA tag followed by a 3Ï« FLAG tag, are detectable using an anti-FLAG antibody (Fig. 1C) . We note here that the nsp3C-terminal construct that we used is distinct from that used by Hagemeijer et al. (32) mentioned in the introduction, which included the GSM domain. Immunofluorescence detection of all constructs was also performed (Fig. 2) .