278 Single-cell Analysis of HIV-1 Infected Primary Human Macrophages A. Brown*, T. Kawano, C. Cheng-Mayer Aaron Diamond AIDS Res Ctr, New York, NY
Background: Monocyte/macrophages play an important role in the transmission of HIV-1 across mucosal surfaces and in the dissemination of virus in lymphoid tissues, and they serve as a latent and/or persistently infected reservoir. Currently available molecular tools to better understand the physiological impact of HIV-1 infection on macrophages are inadequate in distinguishing infected from uninfected cells.
Methods: We have developed an HIV-1SF162 replication competent macrophage-tropic reporter virus that encodes the green fluorescent gene (GFP) and expresses all viral proteins. Virus produced from 293T-cells could productively infect and spread in stimulated human PBMCs and in CEMx174/CD4/CCR5 cells as measured by p24 antigen secretion. The stability of the recombinant viruses was determined by the ability to generate chronically infected CEM/CD4/CCR5 cell lines.
Results: The percentage of GFP+ PBMCs increased during the 10-14 day observation period. Importantly, the difference in infectivity and spread of the Nef+ versus Nef- virus detected by GFP quantitation correlated with the difference observed in p24 antigen production. Nef-mediated down-regulation of both the CD4 receptor and the MHC class I molecule on infected PBMCs was observed demonstrating that Nef was expressed at sufficient levels and that its function is conserved in the recombinant virus. Human monocyte derived macrophages could be productively infected with both the Nef+ and Nef- reporter viruses as detected by the parallel increase in p24 antigen production and the number of GFP positive cells. The marked phenotypic difference in infectivity and spread of the Nef+ versus the Nef- viruses in macrophages that we reported previously was maintained in the reporter viruses.
Conclusions: The Nef+, Nef- and reporter viruses containing defined Nef mutations will be used to assess the direct and indirect effects of HIV-1 infection on macrophage surface antigen expression.
