Figure 1.

Design and characterization of the bifunctional content/core marker for visualization of pseudovirus fusion. A, schematic depicting the design of the Vpr-based bifunctional marker containing the mCherry/YFP-Vpr tandem separated by a linker made of two cleavage sites, SQNY and IRKVL, for HIV-1 protease. B, HXB2 pseudotyped virus stocks were produced unlabeled or labeled with YFP-Vpr, the bifunctional marker (mCherry-2xCL-YFP-Vpr), or Gag-imCherry/YFP-Vpr. Concentrated stocks were subjected to SDS-PAGE and immunoblotted for mCherry, GFP, or HIV proteins. Percent cleavage products report the fraction of cleaved product released after proteolytic processing found by densitometry measurements. C, HXB2 pseudoviruses labeled with the bifunctional marker were immobilized on coverslips and imaged at high signal-to-noise before and after the addition of saponin to mildly permeabilize the viral membrane and release cleaved mCherry. D, the specific infectivity of viruses in B was determined at 48 hours post-infection (h.p.i.) using TZM-bl cells. Error bars indicate standard error from three independent experiments. E and F, fractions of co-labeled YFP+ particles and co-labeled particles with releasable content (E) and occurrence of YFP-only particles (F) were assessed. Error bars indicate standard error from three independent experiments. *, p < 0.05; ***, p < 0.001.