Journal: Journal of Virology
Article Title: Molecular Determinants Directing HIV-1 Gag Assembly to Virus-Containing Compartments in Primary Macrophages
Figure Lengend Snippet: HIV-1 Gag localization to VCCs requires higher-order multimerization. (A) Schematic illustrations of WT, EE75,76AA, delNC, LZ, and LZ4 Gag-YFP. LZ4 Gag-YFP contains the WM184,185AA CA mutation, which disrupts Gag dimerization, in addition to replacement of NC with a tetramer-forming LZ sequence. (B) MDMs were infected with pseudotyped HIV-1 encoding WT Gag-YFP or Gag-YFP derivatives containing the substitutions shown in panel A. At 48 h postinfection, cells were stained with ConA labeled with Alexa Fluor 594, fixed, immunostained with a mouse monoclonal anti-CD81 antibody and anti-mouse IgG conjugated with Alexa Fluor 647, and analyzed using a confocal microscope. Note that for cells expressing delNC Gag-YFP or LZ4 Gag-YFP, prominent YFP signals were found at the cell surface as detected by ConA (white arrowheads), whereas such surface YFP signals were nearly undetectable in WT Gag-YFP-expressing cells. (C) Pearson's correlation coefficients for colocalization of Gag-YFP with ConA are shown as means and SEM. Twenty to 40 cells were analyzed per condition. *, P
Article Snippet: The cells were immunostained with anti-CD81 antibody (BD Biosciences Pharmingen, San Diego, CA) for 1 h at room temperature, washed twice with PBS, and stained with goat anti-mouse IgG conjugated to Alexa Fluor 647 (Invitrogen).
Techniques: Mutagenesis, Sequencing, Infection, Staining, Labeling, Microscopy, Expressing