An adenovirus-based fluorescent reporter vector to identify and isolate HIV-infected cells.
A procedure is described that allows the simple identification and sorting of live human cells that transcribe actively the HIV virus, based on the detection of GFP fluorescence in cells. Using adenoviral vectors for gene transfer, an expression cassette including the HIV-1 LTR driving the reporter gene GFP was introduced into cells that expressed stably either the Tat transcriptional activator, or an inactive mutant of Tat. Both northern and fluorescence-activated cell sorting (FACS) analysis indicate that cells containing the functional Tat protein presented levels of GFP mRNA and GFP fluorescence several orders of magnitude higher than control cells. Correspondingly, cells infected with HIV-1 showed similar enhanced reporter gene activation. HIV-1-infected cells of the lymphocytic line Jurkat were easily identified by fluorescence-activated cell sorting (FACS) as they displayed a much higher green fluorescence after transduction with the reporter adenoviral vector. This procedure could also be applied on primary human cells as blood monocyte-derived macrophages exposed to the adenoviral LTR-GFP reporter presented a much higher fluorescence when infected with HIV-1 compared with HIV-uninfected cells. The vector described has the advantages of labelling cells independently of their proliferation status and that analysis can be carried on intact cells which can be isolated subsequently by fluorescence-activated cell sorting (FACS) for further culture. This work suggests that adenoviral vectors carrying a virus-specific transcriptional control element controlling the expressions of a fluorescent protein will be useful in the identification and isolation of cells transcribing actively the viral template, and to be of use for drug screening and susceptibility assays.
Richman
Larry
L
Swiss Institute for Experimental Cancer Research (ISREC), Chemin des Boveresses 155, CH-1066, Epalinges, Switzerland.
Meylan
Pascal R A
PR
Munoz
Miguel
M
Pinaud
Stéphane
S
Mirkovitch
Jovan
J
D000256:Adenoviridae / Q000235:genetics*; D005434:Flow Cytometry / Q000379:methods*; D015967:Gene Expression Regulation, Viral; D015696:Gene Products, tat; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D015658:HIV Infections / Q000821:virology*; D016325:HIV Long Terminal Repeat / Q000235:genetics; D015497:HIV-1 / Q000235:genetics / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D019169:Jurkat Cells; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D015533:Transcriptional Activation; D014779:Virus Replication; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D008164:Luminescent Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D049452:Green Fluorescent Proteins