Skip to main content
. 2021 Aug 16;17(8):e1009824. doi: 10.1371/journal.ppat.1009824

Fig 1. HSV-1 pUL21 directly binds host proteins CERT and PP1, promoting CERT dephosphorylation.

Fig 1

(A) SILAC-labelled HEK293T cells were transfected with plasmids expressing GFP-tagged pUL21 or GFP alone. At 24 hours post-transfection the cells were lysed, subjected to immunoprecipitation (IP) using a GFP affinity resin, captured proteins were proteolytically digested and co-precipitated proteins identified using quantitative mass spectrometry. The horizontal axis shows average fold enrichment in IP of pUL21-GFP compared to GFP across three biological replicates and the vertical axis shows significance (two-sided t-test) across the three replicates. Proteins CERT and PP1 were identified as putative binding partners (blue). (B) Unlabelled HEK293T cells were transfected with plasmids expressing GFP-tagged pUL21 or GFP alone and subjected to IP as in (A). Captured proteins were subjected to SDS-PAGE and immunoblotting using the antibodies shown. GAPDH is used as a loading control and Ponceau S (Pon S) staining of the immunoblot membrane before blocking shows all proteins transferred. (C) Purified recombinant pUL21-GST or GST alone were immobilised on GSH resin and incubated with prey proteins PP1(7–300)-H6 or Strep-CERT. After washing, the bound complexes were eluted and visualized by SDS-PAGE (Coomassie). (D) Lysates of HaCaT cells (parental or stably expressing pUL21) were analysed by SDS-PAGE and immunoblotting using the antibodies listed. The upper strip depicts SDS-PAGE where PhosTag reagent was added, retarding the migration of phosphorylated proteins to enhance separation of CERT that is hyper- (CERTP) or hypo-phosphorylated (CERTO). (E) HaCaT cells were infected at MOI = 5 with wild-type HSV-1 or a mutant lacking pUL21 expression (ΔpUL21). Lysates were harvested at 16 hours post-infection (hpi) and subjected to SDS-PAGE plus immunoblotting as in (D). The HSV-1 major capsid protein VP5 is used as a marker of infection. (F) Schematic representation of putative pUL21 activity, recruiting specific substrates for dephosphorylation by the PP1 catalytic subunit.