HIV-Envelope–Dependent Cell-Cell Fusion: Quantitative Studies

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.

Paleovirology of ‘ syncytins ’, retroviral env genes exapted for a role in placentation

“…The same phenomenon was found to occur in the case of the enJSRV env genes [22]. In the case of several enveloped viruses, the Env protein synthesized by infected cells and exposed at their surface can confer on them the ability to fuse with neighbouring uninfected cells that express the appropriate receptor, resulting in the formation of multinucleated giant cells called syncytia (figure 2b) [31]. In this context, several placentaspecific genes encoding fusogenic Env proteins co-opted from members of distinct ERV families have been identified within the genome of various mammalian species.…”

Constrained peptide models from phage display libraries highlighting the cognate epitope-specific potential of the anti-HIV-1 mAb 2F5

“…The results of those immunizations were basically negative despite the presentation of the ELDKWAS (or some longer sequence) peptide on various vectors and the inclusion of a fusion peptide sequence [45] and lipids [46] as parts of the viral structures to optimize the 2F5 antigenic function (see [37] for exhaustive critical analysis of this item). Using a cell-cell fusion system, which was proven to be highly sensitive to inhibitors in our previous experiments [31][32][33][34][35] against 2F5 in this study, we found that mouse antiserum to the both the linear and constrained peptides did not affect fusion while the rabbit antiserum to the linear but not to the constrained peptide decreased fusion by approximately 50% and to 20% at 1:10 and 1:100 dilutions, respectively. Inhibition by mAb 2F5 was not observed in the presence of the 15-mer ELDKWAS-containing peptide competitor of the epitope (Fig.…”

“…The cell-cell fusion protocol developed by Huerta et al [31] and subsequently extensively employed by Huerta et al [32], López-Balderas et al [33], Huerta et al [34], Rivera-Toledo et al [35] was used in these experiments to test the anti-mimotope serum antibodies. The protocol involves labeling fusion partner cells with the fluorescent carbocyanines DiI and DiO (Molecular Probes).…”

“…The fusogenic HXBc2 cell line was grown in the presence of 1 g/ml tetracycline and selection antibiotics (200 g/ml G418 and 200 g/ml hygromycin) as described previously [31][32][33][34][35][36]. Expression of the gp120/gp41 viral env protein was induced by removing tetracycline by washing the cells with PBS and incubating in RPMI-10 with selection antibiotics for 3 days.…”

The role of tetraspanins in fusion

“…The life cycle of enveloped viruses necessitates interaction and fusion of the viral lipid envelope with host-cell membranes for virus entry, intracellular trafficking of viral-envelope proteins and viral egress [13]. The functional significance of syncytia in viral pathology is not fully understood but it is likely that they facilitate cellcell spread of virus and may deplete large populations of susceptible cells [15]. In addition, viral-coat proteins can induce the formation of multinucleated syncytia, where a virus-infected cell fuses with uninfected cells.…”