Session 25Oral Abstract Presentations Viral Pathogenesis Session Day and Time: Thursday 10 am - 12:30 pm Presentation Time: 10:45 Room: Ballroom A
123 Systematic Analysis of Host Factors Modifying CD4+ Cell Permissiveness to HIV-1 A. Ciuffi*, G. Bleiber, R. Martinez, C. Suarez, A. Telenti CHUV Lausanne, Switzerland
Background: The genetic background of an individual has been shown to influence pathogenesis of HIV-1. Polymorphisms in several host genes implicated in the HIV life cycle help predict disease progression. We aimed at developing an in vitro model to identify polymorphisms in new gene candidates that restrict HIV-1 replication, and allow repetitive and large scale analysis (e.g., transcriptome and proteome characterization).
Methods: A collection of CD4+ cells from 100 healthy blood donors was established. Cells were stored as native purified CD4+ and after in vitro expansion. Permissiveness phenotype was established by standardized infection with R5 and R4 NL4-3 strains. The collection was genotyped for CCR5 (delta32 and P1), CCR2-64I, and CX3CR1-280M. Fine SNP mapping of a series of gene candidates was performed by SSCP analysis and sequencing of cellular DNA (promoter region, exons, and intron-exon boundaries). New SNPs were analyzed for association with cell permissiveness to infection in vitro. Selected SNPs identified in vitro were then evaluated in a cohort of 700 HIV+ patients as markers for protection or progression to AIDS.
Results: CD4+ cells displayed > 5 log differences in permissiveness to NL4-3 (BaL env) infection. In vitro expansion of hypo- (< 104 ) and hyper-permissive (> 105 ) CD4+ cells maintained the observed phenotype. Cells from these subsets are currently subjected to proteome analysis to identify candidates contributing to hypo- and hyper-permissiveness. In the in vitro system, known polymorphism in CCR5 and CCR2 were correctly identified as modifying permissiveness. SNP mapping identified 17 new and 2 known polymorphisms in Tsg101, PPIA, Ini1, PML, bTRCP, CEM15 and NAF-1.Two (2) novel polymorphisms (in Tsg101 and PPIA) were associated with restriction of HIV-1 in vitro and, in preliminary analysis, in vivo.
Conclusions: An in vitro model of infection of CD4+ cells from healthy donors serves as a screening tool for genetic markers associated with restriction of HIV-1 infection. SNP analysis of the first 7 gene candidates identified 2 potential markers. The possibility to do in vitro expansion of fully characterized cells, while maintaining the permissiveness phenotype, allows for advanced analysis of transcriptome, proteome, abrogation studies, and large scale SNP mapping.