Co-transfection with the previously characterized HIV derivative pCHIVeGFP which allows the efficient detection of Gag using fluorescence microscopy techniques was employed to investigate SNAP-tag staining specificity and intracellular localization of Gag.SNAP. The TMR-Star reactive Gag protein co-localized with Gag.eGFP in virus expressing cells , indicating that Gag.SNAP showed the correct intracellular localization and that Gag.SNAP molecules arranged in Gag assemblies are accessible to the staining procedure. Specificity of TMR-Star staining for transfected cells was demonstrated by lack of signal in non transfected cells . To further test the applicability of the tag in a more physiological context, the T-cell line C8166 was EX 527 customer reviews infected with wt HIV or HIVSNAP, respectively, and stained with the SNAP-tag reactive dye TMR-Star at 15 days post infection, i.e. after several rounds of virus replication. To visualize infected cells, we performed WZ8040 counterstaining with antiserum raised against HIV-1 CA. No unspecific staining by TMR-Star was detected in cells infected with wt HIV . In contrast, C8166 cells infected with HIVSNAP virions displayed specific staining of Gag.SNAP by the fluorescent SNAP substrate , while staining was absent in cells not reactive to CA antiserum . The staining pattern of TMR-Star differed somewhat from the pattern revealed by immunolabeling, with the TMR-Star label displaying a higher number of distinct punctuate signals at the plasma membrane of virus producing cells . This is explained by the impaired antibody accessibility of epitopes within CA in assembled viral structures, which is known to affect detection of intracellular Gag assemblies by immunostaining and immunoprecipitation approaches . In contrast, assembled Gag.SNAP appeared to be accessible for specific staining, yielding a more faithful representation of intracellular Gag distribution. We conclude that HIVSNAP variants are suitable for live-cell imaging analyses in infected T-cells. The presence of the SNAP-tag, which can be labeled with a variety of synthetic dyes, also makes the fusion protein amenable to visualization by super-resolution fluorescence microscopy techniques.