(A) Left panels, HEK-293T cells were transfected with Myc-ubiquitin, Flag-nsp16 and an increasing amount of HA-VHL (1

(A) Left panels, HEK-293T cells were transfected with Myc-ubiquitin, Flag-nsp16 and an increasing amount of HA-VHL (1.0 and 2.0?g). yeast two-hybrid screening. (A) The AH109 competent cells were transformed with the binary plasmids and were plated twice on SD lacking Leu-Trp-Ura-His plate. BD-p53+AD-T shows positive control clone. (B) The single yeast colonies containing these plasmids were tested using the X-Gal assay. The blue color shows positive interaction of MHV nsp16 with candidate proteins. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) 3.2. VHL interacts with nsp16 Among the four nsp16-interacting proteins, we selected VHL for further studies as VHL was previously shown to interact with the integrase MK-5172 hydrate of HIV-1 and promote its degradation to regulate the viral replication cycle [18]. To verify the connection between SARS-CoV nsp16 and VHL, GST pull-down assay was used. The results showed that GST-nsp16 bound to His-VHL in reciprocal pull-down assays, whereas GST control did not (Fig.?2 A). Further cellular reciprocal co-immunoprecipitation assays confirmed that nsp16 could associate with VHL within cells (Fig.?2B). Co-localization experiments showed that eGFP-nsp16 and Red-VHL have the same localization in HeLa cells. In addition, fluorescence imaging showed that Flag-nsp16 and Red-VHL have the same distribution in Vero cells. These results suggested that SARS nsp16 can interact with VHL in cells and the nsp16-VHL interplay may involve in SARS-CoV replication. Open in a separate windowpane Fig.?2 VHL interacts with SARS-CoV nsp16. (A) 6xHis-VHL or 6xHis-nsp16 proteins indicated in BL21 cells were incubated with glutathione beads and the purified GST, GST-nsp16 or GST-VHL proteins. The bound proteins were eluted and recognized by Western blotting with an anti-His or anti-GST antibody. (B) Flag-nsp16 and HA-VHL were transfected into HEK293T cells. After 36?h, the cell components were immunoprecipitated (IP) with an anti-HA or MK-5172 hydrate anti-Flag antibody and analyzed by immunoblotting (IB) with an MK-5172 hydrate anti-Flag or anti-HA antibody (top panel). (C) The eGFP-nsp16 and Red-VHL plasmids were co-transfected into HeLa cells. After 24?h, the cells were fixed with 4% paraformaldehyde and the nuclei were stained with DAPI. The cells were examined by confocal microscopy. (D) After transfection, the Vero cells were fixed with 4% paraformaldehyde, labeled with rabbit anti-Flag antibody (1:100), then probed with FITC-tagged goat anti-rabbit IgG. (For interpretation of the referrals to color with this number legend, the reader MK-5172 hydrate is referred to the STAT2 web version of this article.) 3.3. VHL regulates MK-5172 hydrate SARS nsp16 protein stability, degradation and SARS replication Because earlier studies statement that VHL inhibits protein synthesis [22], we decided to examine whether VHL regulates the protein stability of SARS-CoV nsp16. To answer this question, a CHX chase assay was performed. HEK293T cells were transfected with Flag-nsp16 and Flag-VHL, followed by treatment with the protein translation inhibitor CHX for 4 different time periods (0, 1, 3, and 5?h). We found that the half-life of nsp16 protein became shorter when it was co-transfected with VHL (Fig.?3 A). This result suggested the stability of SARS nsp16 could be affected by VHL. In this experiment, we observed that VHL could promote nsp16 degradation (Fig.?3A lane 1 and 5). To study the mechanism of VHL-promoted nsp16 degradation, HEK293T cells were transfected with Flag-nsp16 and HA-VHL, and then the cells were treated with the 26S proteasome inhibitor MG132 or lysosome inhibitor NH4Cl. The results showed that.