Journal: PLOS Pathogens

Article Title: Disrupting the OTUD4-USP7 deubiquitinase complex to suppress herpesvirus replication: a novel antiviral strategy

doi: 10.1371/journal.ppat.1013052

Figure Lengend Snippet: (A) iSLK cells were induced with Dox (1 µg/mL) in the presence of indicated peptide (100 µM). WCLs were analyzed by immunoblotting 48 h post-induction. Densitometry analysis of the bands was performed with ImageJ. (B) SLK.iBAC-GFP cells transduced with control shRNA, shRNA targeting OTUD4, or USP7 were induced with Dox (1 µg/mL) and sodium butyrate (0.5 mM) in the presence of the indicated peptide (100 µM). KSHV infectious units were quantified 48 h post-induction. Data are mean ± s.d. of N = 3 independent biological replicates. (C) SLK.iBAC-GFP cells stably transduced with vector control or OTUD4 were induced with Dox (1 µg/mL) and sodium butyrate (0.5 mM) in the presence of the indicated peptide (100 µM). KSHV infectious units were quantified 48 h post-induction. Data are mean ± s.d. of N = 3 independent biological replicates. (D) SLK.iBAC-K-RTA-WT or SLK.iBAC-K-RTA-F689A/R690A/D691A (3A) cells were induced with Dox (1 µg/mL) and sodium butyrate (0.5 mM) in the presence of the indicated peptide (100 µM). KSHV infectious units were quantified 48 h post-induction. Data are mean ± s.d. of N = 3 independent biological replicates. (E) SLK.iBAC-GFP cells were induced with Dox (1 µg/mL) and sodium butyrate (0.5 mM) in the presence of the indicated peptide (100 µM). Cell viability was assessed by measuring ATP levels 48 h post-induction. Data are mean ± s.d. of N = 3 independent biological replicates.

Article Snippet: MHV68 ORF50 was subcloned into pEF-FLAG-N or pEF-HA-N using standard molecular biology techniques; pHAGE-OTUD4 WT and the C45A mutant were kindly provided by Dr. Bo Zhong (Wuhan University) [ ]; mOTUD4 WT was subcloned into pHAGE-vector and pEF-Myc-N, and the C45A and a DUB deletion mutants (Δ34-155 aa) were generated by site-directed mutagenesis; pCMV-FLAG/Myc-USP7, FLAG-MDM2, and HA-p53 were kindly provided by Dr. Jinfang Zhang (Wuhan University), and USP7 was subcloned into the pEF-HA-N vector [ ]; HSV-1 ICP0 was subcloned into pEF-FLAG-N; The TRAF domain of human and murine USP7 (1-208 aa) was subcloned into pGEX-6p-1 (GE Healthcare), and the D164A and W165A mutants (murine are D165A and W166A) were introduced by site-directed mutagenesis; HA-Ub was kindly provided by Dr. Bo Zhong (Wuhan University); Short hairpin RNAs (shRNA) targeting OTUD4 and USP7 were constructed into pLKO.1 (Addgene).

Techniques: Western Blot, Transduction, Control, shRNA, Stable Transfection, Plasmid Preparation