Abstract
Flow cytometric analysis is a reliable and convenient method for investigating molecules at the single cell level. Previously, recombinant human immunodeficiency virus type 1 (HIV-1) strains were constructed that express a fluorescent reporter, either enhanced green fluorescent protein, or DsRed, which allow the monitoring of HIV-1-infected cells by flow cytometry. The present study further investigated the potential of these recombinant viruses in terms of whether the HIV-1 fluorescent reporters would be helpful in evaluating viral replication based on fluorescence intensity. When primary CD4+ T cells were infected with recombinant viruses, the fluorescent reporter intensity measured by flow cytometry was associated with the level of CD4 downmodulation and Gag p24 expression in infected cells. Interestingly, some HIV-1-infected cells, in which CD4 was only moderately downmodulated, were reporter-positive but Gag p24-negative. Furthermore, when the activation status of primary CD4+ T cells was modulated by T cell receptor-mediated stimulation, we confirmed the preferential viral production upon strong stimulation and showed that the intensity of the fluorescent reporter within a proportion of HIV-1-infected cells was correlated with the viral replication level. These findings indicate that a fluorescent reporter encoded within HIV-1 is useful for the sensitive detection of productively infected cells at different stages of infection and for evaluating cell-associated viral replication at the single cell level.
Highlights
Human immunodeficiency virus type 1 (HIV-1) interacts with its primary receptor, CD4, and a co-receptor, usually CCR5 or CXCR4, to infect T cells, macrophages, and dendritic cells (Mcclure et al, 1987; Berger et al, 1999; Tsunetsugu-Yokota, 2008)
Because the transcriptional activity of HIV-1 can be quantitatively monitored in indicator cells according to the fluorescence intensity of an enhanced green fluorescent protein (EGFP) reporter driven by the HIV-1 long terminal repeats (LTR) (Dorsky et al, 1996; Gervaix et al, 1997; Kar-Roy et al, 2000), we investigated whether the HIV-1-expressing fluorescent reporters EGFP and DsRed would allow the quantitative evaluation of viral replication using a flow cytometer
To investigate the correlation between the level of CD4 downmodulation and the HIV-1 fluorescent reporter intensity, primary CD4+ T cells infected with HIV-1NL-E, HIV-1NL-D, HIV-1NLAD8-E, or HIV1NLAD8-D followed by T cell receptors (TCR)-stimulation for 1 day and cultivation for a further 4 days were analyzed by flow cytometry
Summary
Human immunodeficiency virus type 1 (HIV-1) interacts with its primary receptor, CD4, and a co-receptor, usually CCR5 or CXCR4, to infect T cells, macrophages, and dendritic cells (Mcclure et al, 1987; Berger et al, 1999; Tsunetsugu-Yokota, 2008). To monitor HIV-1 infection, a recombinant HIV-1 encoding a reporter luciferase (Luc) gene, or indicator cells transduced with enzymatic reporters such as Luc, β-galactosidase, alkaline phosphatase, and chloramphenicol acetyl transferase, incorporated downstream of the HIV-1 long terminal repeats (LTR) have been widely used (Kar-Roy et al, 2000). These reporters require additional substrates or co-factors, and lysis or fixation of cells is required to show reporter activity, which makes the experimental process more complex. We previously constructed nef-intact, replicationcompetent, recombinant HIV-1 strains encoding either EGFP or DsRed, and showed that CXCR4-tropic X4 and CCR5-tropic R5 viruses replicate differently in CD4+ T cells simultaneously infected with X4 HIV-1 encoding EGFP and R5 HIV-1 encoding www.frontiersin.org
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