Labeling HIV-1 virions with two fluorescent proteins allows identification of virions that have productively entered the target cell

Virology. 2007 Apr 10;360(2):286-93. doi: 10.1016/j.virol.2006.10.025. Epub 2006 Nov 22.

Abstract

GFP-Vpr labeled HIV-1 virions have provided a method to visually examine the interactions between the virus and target cell during infection. However, existing methods to discriminate between virions that have been non-specifically endocytosed from those that have productively entered the host cell cytoplasm have remained problematic. Therefore, we examined the ability of a series of membrane-targeted fluorescent fusion protein constructs to be incorporated into virions. We find that a fluorescent protein fusion targeted to the plasma membrane by the addition of the N-terminal 15 amino acid sequence of c-Src (S15) is efficiently packaged into HIV virions. Using fluorescent proteins fused to this sequence, we have generated virions dually labeled with S15-mCherry and GFP-Vpr. Importantly, we can detect the loss of this S15-mCherry membrane signal following fusion. After infection with VSV-g pseudotyped HIV virions, we find a measurable, specific loss of membrane label during infection. This loss of fluorescence is not observed when fusion is prevented using bafilomycin A. This increased ability to discriminate between non-productively endocytosed virions and those actively undergoing steps in the infectious process will facilitate efforts to examine early steps in infection microscopically.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Cell Line
  • Cell Membrane / chemistry
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism*
  • HIV-1 / genetics
  • HIV-1 / physiology*
  • Humans
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism*
  • Membrane Fusion
  • Microscopy, Fluorescence
  • Phagocytosis
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism*
  • Staining and Labeling / methods
  • Virion / chemistry*
  • Virus Internalization*

Substances

  • Luminescent Proteins
  • Recombinant Fusion Proteins
  • red fluorescent protein
  • Green Fluorescent Proteins