Abstract
Interaction in vitro between cells infected with human immunodeficiency virus (HIV) and surrounding, uninfected, target cells often leads to cell fusion and the formation of multinucleated cells, called syncytia. The presence in HIV-infected individuals of virus strains able to induce syncytia in cultures of T cells is associated with disease progression and AIDS. Even in the asymptomatic stage of infection, multinucleated cells have been observed in different organs, indicating that fused cells may be generated and remain viable in the tissues of patients. We used lymphocytic cells transfected for the expression of the HIV-envelope (Env) glycoproteins to develop a method for the direct quantification of fusion events by flow cytometry (Huerta et al., 2006, J. Virol. Methods 138, 17–23; López-Balderas et al., 2007, Virus Res. 123, 138–146). The method involves the staining of fusion partners with lipophilic probes and the use of fluorescence resonance energy transfer (FRET) to distinguish between fused and aggregated cells. We have shown that such a flow-cytometry assay is appropriate for the screening of compounds that have the potential to modulate HIV-Env–mediated cell fusion. Even those syncytia that are small or few in numbers can be detected. Quantitative analysis of the fusion products was performed with this technique; the results indicated that the time of reaction and initial proportion of fusion partners determine the number, relative size, and average cellular composition of syncytia. Heterogeneity of syncytia generated by HIV-Env–mediated cell-cell fusion may result in a variety of possible outcomes that, in turn, may influence the biological properties of the syncytia and surrounding cells, as well as replication of virus. Given the myriad immune abnormalities leading to AIDS, the full understanding of the extent, diverse composition, and role of fused cells in the pathogenesis of, and immune response to, HIV infection is an important, pending issue.
Highlights
Cell-cell fusion is a form of cell organization that occurs in physiological and pathological conditions, such as the formation of muscle[1] and placenta[2], development in metazoa[3], organ repair by stem cells[4,5,6,7], and malignant transformation[8,9,10]
That syncytia may originate from the fusion of monocytes, lymphocytes, and/or dendritic cells has been evidenced by both the anatomical location and the phenotypic markers of the multinucleated cells[52,54,56]
A few hours after Env+ cells fused in vitro with target cells expressing the CD4 molecule and the proper coreceptor, a variety of fusion products can be detected (Fig. 2). Because these cells provided an appropriate system for the study of human immunodeficiency virus (HIV)-Env syncytia formation, they were used in the experiments described in this article
Summary
Given the requirements for close contact and specific binding between cells, cell-cell fusion may roughly follow the principles of mass action. Given that changes in the proportion of cells remaining nonfused do not directly reflect the actual numbers of cells forming syncytia[84], the following simple expression was derived in order to calculate the CD4+/Env+ ratio of cells incorporated into syncytia, or fusion stoichiometry coefficient (here designated as r)[84], using the percentage data provided by most of FACS equipment available: ri = R0 − (1 + R0 ){cd 4}i fi. This study indicates that fusion-inhibiting antibodies predominate in the early stages of the disease, while loss of inhibition and the presence of fusion-enhancing antibodies appear during the late stages This underscores the point that the effect of antibodies on syncytia formation should be considered in order to understand fully the role of the immune response in the control of the different forms of viral spreading and cell damage
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