Labeling HIV-1 virions with two fluorescent proteins allows identification of virions that have productively entered the target cell.
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.
Campbell
Edward M
EM
Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Ward 8-140, 303 East Chicago Avenue, Chicago, IL 60611, USA.
Perez
Omar
O
Melar
Marta
M
Hope
Thomas J
TJ
D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics / Q000502:physiology*; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008561:Membrane Fusion; D008856:Microscopy, Fluorescence; D010587:Phagocytosis; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods; D014771:Virion / Q000737:chemistry*; D053586:Virus Internalization; D000097573:Red Fluorescent Protein
D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein