The HIV-1 reverse transcriptase (RT) initiation complex consists of three components: RT-tRNAlys3-vRNA, and it has been extensively studied utilizing biochemical approaches to understand the mode of RT binding and the mechanism associated with this early phase of transcription. However, the structure of the HIV-1 initiation ternary complex has yet to be determined. What conformation RT takes with tRNAlys3-vRNA and where the anticodon loop and 5' region of the tRNAlys3 interact with the rest of the RT is still unknown. Here, we propose to utilize x-ray crystallography to determine the structure of this complex by attempting two different strategies. 1) Prior biochemical studies have shown that there is potential for interaction between the different vRNA secondary structural regions with tRNAlys3, so we will perform a large-scale crystal screening experiment for the RT initiation complex using different HIV vRNA isolates with varying vRNA lengths, which will contain different vRNA secondary structures. 2) We will introduce a tether thiol group to the N2 position on the guanosine of HIV-1 vRNA and generate a cysteine mutation to the thumb subdomain in RT that can interact with the vRNA complex. This will stabilize the RT on the tRNAlys3-vRNA complex. We hypothesize that an interaction between RT and the 3' end of tRNAlys3 (which is annealed to the viral primer-binding site of vRNA in the polymerization-competent mode) will then result in the interaction of the anticodon loop and 5' end of tRNAlys3 with a portion of the p51 subunit of RT. Understanding the structure of this complex has important implications for potential new anti-HIV drug design.
The structure of the HIV-1 initiation complex with reverse transcriptase in complex with tRNAlys3 and viral RNA has not been determined. Here, we propose to vary viral RNA lengths and utilize a chemical crosslinking strategy to stabilize reverse transcriptase on this tRNAlys3 and viral RNA duplex and determine the structure using x-ray crystallography.
| Nguyen, Laura A; Wang, Jimin; Steitz, Thomas A (2017) Crystal structure of Pistol, a class of self-cleaving ribozyme. Proc Natl Acad Sci U S A 114:1021-1026 |
