Anti-retroviral drug combinations currently used for the treatment of HIV- 1 infection potently suppress plasma HIV- 1 RNA to below detectable levels. Although this suppression can be maintained over prolonged periods, viral replication rapidly rebounds in individuals who discontinue therapy. The ability of HIV-1 to persist in the face of highly active anti-retroviral therapy (HAART) has been attributed to the presence of a reservoir of latently infected cells that rekindles virus replication following therapy interruption. However, even in well-suppressed patients, there is evidence of viral sequence evolution, which suggests that there may be on going viral replication. The examination of viral reservoirs in suppressed, aviremic patients is hampered by a Jack of assays that are able to discriminate between latent infection and on-going replication. As a consequence, the extent of viral replication that persists in the face of HAART and the reservoir that supports it is poorly understood. Following infection of the cell, viral reverse transcription leads to the synthesis of a full-length linear cDNA that circularize to form episomes containing either one LTR (l-LTR circle) or 2 LTRs (2-LTR circle). We have recently demonstrated that 2-L1'R circles are highly labile and, as such, represent a specific marker for a recent infection event. We have developed a quantitative 2-LTR circle assay and have demonstrated that these circles persist in a large percentage of infected individuals on HAART with sustained undetectable levels of plasma viral RNA. This suggests the presence of a reservoir of "covert" virus replication that may allow the virus to persist in the face of HAART. We propose to use this 2-LTR circle assay to understand the basis for viral persistence in the face of HAART and to identify reservoirs for covert viral replication. Specifically, we propose to Aim 1: Evaluate the half-life of 2-LTR circles in primary cell types in vitro Aim 2: Examine stability of 2-LTR circles in vivo and identify reservoirs for covert viral replication during HAART Aim 3: Longitudinally examine 2-LTR circle copy number in patients on HAART in order to evaluate the decay characteristics of the persistent viral reservoir These studies may shed further light on the underlying mechanism which allows viral persistence in the face of HAART and may provide an approach to allow monitoring of' the effectiveness of antiretroviral suppression when plasma viral RNA assays are negative.