17th International AIDS Conference Mexico City, Mexico - August 13 - 18, 2008

QUASISPECIES ANALYSIS BY ULTRA-DEEP PYROSEQUENCING OF HIV-1 DERIVING FROM LYMPHOMONOCYTE SUB-POPULATIONS IN CHRONICALLY INFECTED PATIENTS AT THE MOMENT OF HAART INTERRUPTION

Int Conf AIDS. 2008 Aug 13-18;17 Abstract No. MOAA0305

G. Rozera¹, I. Abbate ¹, A. Bruselles¹, G. Chillemi², C. Vlassi¹, G. D'Offizi¹, P. Narciso¹, M.R. Capobianchi^1
1INMI L.Spallanzani, Rome, Italy, ²CASPUR, Rome, Italy

BACKGROUND: Virus-associated cell membrane proteins acquired during budding may give information on the cellular source of circulating virions. The aim of the study was to characterize viral quasispecies originating from lymphomonocyte subpopulations in patients who interrupted successful HAART by applying the powerful technology of ultra-deep pyrosequencing of clonally amplified amplicons.

METHODS: Five chronically HIV-1 infected patients who underwent therapy interruption after >5 years of successful HAART (T0) were enrolled. HIV quasispecies analysis was applied to cellular DNA at T0, and, after one month (T1), to both proviral DNA and circulating virions. The PBMC subpopulations were sorted by immunomagnetic beads, targeting monocytes (CD36) and lymphocytes (CD26). Circulating virions were sorted with the same MAbs. The analysis has been performed by ultra-deep pyrosequencing of at least 1300 clonally amplified V3 loop regions, using the newest genome sequencer (GS-FLX Roche) technology. Several in-house written scripts have been used to translate, correct and clusterize aminoacid sequences. Distance-based methods were used to construct phylogenetic trees. Co-receptor usage prediction was performed using position-specific scoring matrices.

RESULTS: An higher heterogeneity of V3 loop quasispecies in proviral sequences with respect to circulating virions was observed. In addition, within a large background of viral sequences common to both cell types and to shed virions, phylogenetic trees revealed clusters of sequences specific for any of the two cellular reservoirs in both cells and plasma. Score matrix results for co-receptor analysis suggest biological differences between virions subpopulations arising from CD26 and CD36 cells, with score values for those originating from CD36 in line with a more pronounced CCR-5 co-receptor usage.

CONCLUSIONS: Our data show that ultra-deep pyrosequencing of thousands of clonally amplified PCR products is a powerful tool able to provide information on viral quasispecies dynamics, being able to identify minority variants, and, on the whole, to shed light on viral pathogenesis.

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2008-08-13
MOAA0305

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