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

Edward M. Campbell, Omar Perez, Marta Melar, Thomas J. Hope*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

101 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)286-293
Number of pages8
JournalVirology
Volume360
Issue number2
DOIs
StatePublished - Apr 10 2007

Keywords

  • Endocytosis
  • Fusion
  • Infection
  • Microscopy
  • Src
  • mCherry

ASJC Scopus subject areas

  • Virology

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