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

Journal Article · Tue Apr 10 04:00:00 EDT 2007 · Virology

DOI:https://doi.org/10.1016/j.virol.2006.10.025· OSTI ID:20977008

Campbell, Edward M ^[1]; Perez, Omar ^[2]; Melar, Marta ^[1]; Hope, Thomas J ^[1]

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1. Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Ward 8-140, 303 East Chicago Avenue, Chicago, IL 60611 (United States)
2. Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, IL 60612 (United States)

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.

• OSTI ID: 20977008
• Journal Information: Virology, Journal Name: Virology Journal Issue: 2 Vol. 360; ISSN VIRLAX; ISSN 0042-6822
• Country of Publication: United States
• Language: English

Related Subjects

60 APPLIED LIFE SCIENCES
AIDS VIRUS
AMINO ACID SEQUENCE
CELL MEMBRANES
CYTOPLASM
FLUORESCENCE
LABELLING
MICROSCOPY
PROTEINS