Journal: Journal of Virology
Article Title: HIV-1 Nef Antagonizes SERINC5 Restriction by Downregulation of SERINC5 via the Endosome/Lysosome System
Figure Lengend Snippet: Nef binds to Ser5. (A) Schematic of the BiFC fusion proteins is presented. Venus is divided into an N-terminal region from residues 2 to 173 (VN) and a C-terminal region from residues 154 to 238 (VC). VN and VC were fused to the C terminus of Ser5 or Nef, and each fusion protein also contains an HA, V5, or FLAG epitope. (B) pcDNA3.1-Nef-VN-HA, pcDNA3.1-Nef4D-VN-HA, pcDNA3.1-Ser5-VN-HA, pcDNA3.1-Nef-V5-VC, pcDNA3.1-Nef4D-V5-VC, pcDNA3.1-CD4-V5-VC, and pcDNA3.1-Ser5-FLAG-VC were expressed individually or pairwise in 293T cells as indicated. The mean fluorescence intensities (MFI) of BiFC fluorescent signals were determined by flow cytometry and are presented as relative values to the signal from untransfected cells. Error bars represent the SD from three independent experiments. (C) The vectors in panel B are expressed pairwise in HeLa cells as indicated. Ser5-VN-HA (panels I and II) and Nef-VN-HA (panel IV) fusion proteins were stained with a rabbit anti-HA monoclonal antibody at a dilution of 1:500, followed by an Alexa Fluor 647-conjuated donkey anti-rabbit antibody at a 1:1,000 dilution. CD4-V5-VC (panel III) fusion was stained with a mouse anti-V5 antibody at a 1:250 dilution, followed by an Alexa Fluor 647-conjugated goat anti-mouse antibody at a 1:1,000 dilution. Colocalization of the red and the BiFC green fluorescent signals were visualized by confocal microscopy. (D) Schematic of the HIV-1 NL4-3 Nef protein. The N-terminal flexible anchor domain (AN), core domains, internal flexible loop (FL), and critical residues and motifs for MHC-I (red) and CD4 (green) downregulation are shown. The indicated Nef mutations were created in the pcDNA3.1-Nef-V5-VC vector, and Nef expression was determined by Western blotting with an anti-V5 antibody. (E) pMSCV-Ser5-VN-HA was expressed with pcDNA3.1-Nef-V5-VC vectors expressing the indicated Nef mutants in 293T cells. The BiFC MFI was determined by flow cytometry and are presented as values relative to the signal from untransfected cells. Error bars represent the SD from three independent experiments.
Article Snippet: The Ub K48R, K63R, and K48/63R mutants were created in the pCMV-HA-Ub vector by site-directed mutagenesis. pMSCV-CD4-FLAG expression vector was created by cloning the CD4 cDNA with a C-terminal FLAG into the pMSCVpuro vector after XhoI/EcoRI digestion. pCMV-CD4-FLAG was constructed by cloning CD4 into the pCMV6-Entry vector after BamHI/EcoRV digestion. pcDNA3.1-MHC-I-FLAG expressing the HLA-A gene ( ) from the pcDNA3.1+/C-(K)DYK vector was ordered from GenScript (product no. OHu21196D). pcDNA3.1-SF2Nef-V5 expression vector was created by directly cloning the Nef gene from HIV-1 SF2 strain into the pcDNA3.1D/V5-His-TOPO vector via the TOPO-cloning strategy (Invitrogen).
Techniques: Bimolecular Fluorescence Complementation Assay, FLAG-tag, Fluorescence, Flow Cytometry, Cytometry, Staining, Confocal Microscopy, Plasmid Preparation, Expressing, Western Blot