Fluorescence-based quantitative methods for detecting human immunodeficiency virus type 1-induced syncytia

Abstract

Cell fusion induced by human immunodeficiency virus type 1 (HIV-1) is usually assessed by counting multinucleated giant cells (syncytia) visualized by light microscopy. Currently used methods do not allow quantification of syncytia, nor do they estimate the number of cells involved in cell fusion. We describe two fluorescence-based methods for the detection and quantification of HIV-1-induced in vitro syncytium formation. The lymphoblastoid cell lines MT-2 and SupT1 were infected with syncytium-inducing (SI) HIV-1 isolates. Syncytia were detected by DNA staining with propidium iodide using flow cytometry to determine cell size or by two-color cytoplasmic staining of infected cell populations by using fluorescence microscopy. Both methods were able to detect and quantify HIV-induced syncytia. The methods could distinguish between SI and non-SI HIV isolates and could be used with at least two separate types of CD4(+) T-cell lines. Small syncytia can be readily identified by the two-color cytoplasmic staining method. Both methods were also shown to be useful for evaluating antiretroviral compounds, as demonstrated by the accurate assessment of HIV inhibition by azidothymidine (zidovudine), dideoxycytidine (zalcytibine), and hydroxyurea. These fluorescence-based assays allow a rapid and practical method for measuring HIV replication and anti-HIV activity of potential inhibitory compounds.

FIG. 1

Detection and quantitation of HIV-infected MT-2 cells by flow cytometry. HIV-infected MT-2 cells were fixed 72 h postinfection, stained with PI, and analyzed in a set time frame of 100 s by flow cytometry. The analysis of the forward scatter (FSC) versus the DNA content (PI-H) in mock-infected (A) and HIV-infected (C) cultures was compared to the DNA histograms (B and D).

FIG. 2

Syncytia formation correlates with viral replication. Syncytia formation was evaluated by FACS, and HIV-1 replication was determined by measuring p24 antigen concentration in cell culture supernatants.

FIG. 3

Inhibition of syncytia formation by anti-CD4 antibody and antiretroviral drugs. MT-2 cells infected with HIV-1 demonstrated 74% HIV-1 syncytia in this experiment. Prior to infection, CD4 receptors on MT-2 cells were blocked with anti-CD4 antibodies or nonspecific antibodies of the same isotype (anti-CD4 and Isotype, respectively). HIV-1-infected MT-2 cells were also cultured in the presence or absence of the antiviral compounds ddC (20 μM) or ZDV (0.5 μM).

FIG. 4

Mock-infected (panels A to D) or HIV-infected (panels E to H) MT-2 cells were stained with a vital red or green fluorescent dye. Equal numbers of red- and green-stained cells were mixed and incubated for 48 h. HIV-1-induced fusion between red fluorescent (E) and green fluorescent (F) cells can be detected as yellow-stained syncytia (G). Panels D and H show phase contrast microscopy of stained cells.

FIG. 5

Syncytia formation is the major cause of cell loss in infected MT-2 cell cultures. Cell numbers were determined in HIV-infected MT-2 cells and uninfected controls. By using the fluorescent dyes CMFDA and CMTMR, the number of cells present in the syncytia was estimated based on diameter of dually fluorescent cells. The estimated number of cells present in HIV-infected cultures (HIV-Corrected) was determined by adding the number of single fluorescent cells to the estimated number of cell equivalents present in dually fluorescent syncytia. By using a 72-h infection time period, the number of cell equivalents (HIV-Corrected) in the syncytia was approximately equal to the total number of cells present in the mock-infected control.

FIG. 6

Inhibition of syncytium formation by antiviral drugs. Infected MT-2 cells were cultured in the presence or absence of the antiviral compounds ZDV (0.5 μM), ddC (20 μM) (B), or an increasing concentration of HU (A). Syncytium formation was quantitated by confocal microscopy.