Fig 5.

NEDA is triggered in response to true late viral protein expression. (A) Viral protein expression kinetics in macrophages infected with HSV-1 wt for 2 to 8 h or mock-infected cells, with antibodies for the immediate early proteins (IE) ICP0 and ICP27, the leaky late (LL) protein γ34.5, and the true late (TL) proteins gC and pUL36. Tubulin was used as a loading control. (B) Immunoblot analysis of macrophages infected with the HSV-1 wt, Δγ34.5, Δbeclin-1, or ΔPP1α virus, using a polyclonal anti-HSV-1 antibody and antibodies for VP5, γ34.5, gC, and pUL36. Calnexin and tubulin were used as loading controls. (C to E) Macrophages were infected with HSV-1 wt and treated with DMSO, cycloheximide (CH), or 200 to 400 μM acyclovir at 0 hpi and analyzed at 8 hpi. (C) Immunoblot with antibodies against ICP0, ICP27, γ34.5, pUL36, gC, calnexin (clxn), and LC3 (LC3-I uncleaved, LC3-II cleaved membrane-associated form). (D and E) Immunofluorescence analysis with an antibody against cleaved LC3a. Error bars, SD; ***, P < 0.001 in a two-sided Student t test.