The main goal of the proposed research is to isolate efficacious RNA aptamers against one HIV-1 enzyme and two accessory proteins: PR, Vif and Nef, and to delineate their efficacy and mode of action in blocking viral replication. SELEX experiments will be performed to generate aptamers against these currently unexplored HIV targets. Aptamers will be cloned, sequenced and subjected to additional screens for optimal expression levels and Compartmentalization in cells susceptible to HIV infection. Experiments will be performed in vitro to determine their dissociation constant and ability to inhibit functions of their protein targets. Six of the tightest binding aptamers in each case will be expressed via U6+1 promoter on the retroviral vector MMP-eGFP and used to tranduce CEM x 174 cell lines. These cells will be subjected to virus challenge experiments. In addition, several target-specific experiments will be performed to delineate the mode and efficacy of inhibition employed by these aptamers. Selection experiments will also be performed to identify functional mutations in the target proteins that can escape aptamer pressure. In addition, several experiments will be conducted in an attempt to further optimize these RNA aptamers. ? ? ?
| Ramalingam, Dhivya; Duclair, Sonald; Datta, Siddhartha A K et al. (2011) RNA aptamers directed to human immunodeficiency virus type 1 Gag polyprotein bind to the matrix and nucleocapsid domains and inhibit virus production. J Virol 85:305-14 |
