This application describes an investigation into the mechanisms by which the cis/trans prolyl isomerase Pin1 inhibits HIV-1. The presence of Pin1 in human producer cells significantly diminishes HIV-1 infectivity. siRNA knock-out of Pin1 in human cells produces dramatically more infectious (8-fold) HIV-1. The viral target, the step in replication, and the mechanism of Pin1 inhibition are unknown. However, the fact that Pin1 incorporation into HIV-1 correlates with inhibition, that HIV-1 capsid contains a sequence similar to known Pin1 binding motifs, and that SIV is not inhibited by Pin1 offer important clues to the mechanism of Pin1 inhibition. This application describes the use of PCR based reverse transcription assays used in conditions that either block or enhance Pin1 inhibition of HIV-1 (specific drugs, siRNA, and Pin1 over expression) to comparatively deduce which step (reverse transcription, nuclear import, integration) is targeted. It also describes approaches which exploit the fact that SIV, unlike HIV-1, is not sensitive to Pin1 inhibition, to identify the viral target of inhibition. These include the use of whole SIV/HIV chimeras, SIV/HIV capsid chimeras (which are in hand), as well as direct immunochemical methods to identify the viral target of Pin1 inhibition. This project will identify 1) the viral target of, and 2) the step in the HIV-1 infective cycle inhibited by Pin1. The long term objective is the development of antiviral therapies that target the same weaknesses in HIV-1 that Pin1 targets. Confidence for the successful completion of the specific aims is enhanced by the clear inhibitory phenotype of Pin1, and the straight-forward methodologies employed. Relevance: There is growing evidence that as part of the infective process, HIV-1 overcomes a series of host cell defenses that inhibit specific steps in retroviral replication. There is a critical need to identify the elements of this cellular defense and to understand their mechanism(s) of action. This application strives to understand how a previously unrecognized inhibitor of HIV-1, Pin1, works, with the hope that development of antiviral therapies that target the same weaknesses in HIV-1 that Pin1 targets. ? ? ? ?
