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134 NFAT Is a Determining Factor in Preferential HIV-1 Replication of CD45RO-Expressing CD4+ T Cells
G. A. Robichaud*1, B. Barbeau1, J.-F. Fortin1, D. M. Rothstein2, and M. J. Tremblay1
1Univ. Laval, Québec, PQ, Canada and 2Yale Univ., New Haven, CT, USA
Background: From the first isolation of the HIV-1, studies have consistently shown that viral pathogenesis in CD4+ T cells is characterized in such that HIV-1 preferably replicates in the memory subset (CD45RO+/CD45RA() in contrast to the naive subset (CD45RA+/CD45RO(). This phenomenon occurs for reasons that still remain unknown. HIV-1 regulation is also greatly dependent upon the enhancer sequence of the 5 LTR region. This site binds the NF-kappaB transcription factor and has shown, as demonstrated recently, to be equally regulated by NFAT. Since the surface phosphatase CD45 is greatly involved in cellular differentiation, signaling, and activation events, we set out to investigate HIV-1 modulation by different isoforms of CD45 in T-cell clones and freshly isolated human CD4+ T-cell subsets.
Methods: We evaluated NF-kappaB, NFAT, and HIV-1 LTR activities through luciferase-based reporter gene assays with the DEAE-dextran protocol in Jurkat-derived cell clones either deficient in CD45 expression or positive for surface expression of mixed CD45+ isoforms (wt), CD45RABC+, or CD45RO+. Band-shift and super-shift assays were also conducted with nuclear extracts from these T-cell clones as well as with negatively selected CD4+ T-cell subsets using labeled probes specific for NF-kappaB, NFAT, and the HIV-1 LTR enhancer sequences.
Results: We demonstrate for the first time that the CD45RO isoform promotes a higher level of NFAT activation without significantly altering that from NF-kappaB following T-cell activation. More importantly, we show that this transcriptional modulation by CD45RO was also reflected by a concomitant rise in HIV-1 LTR activity and viral production. Finally, super-shift assays from these cellular clones and primary CD4+ T-cell subsets from peripheral blood indicate that these events are NFAT-1-dependent and mediated through the HIV-1 LTR enhancer region.
Conclusions: Altogether these results underscore the importance of the NFAT factor in HIV-1 regulation and present for the first time a limiting factor responsible for a selective viral production in the human memory CD4+ T-cell subset. The NFAT factor therefore presents itself as an interesting target molecule for therapeutic intervention.
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