Impact of nonnucleoside reverse transcriptase (RT) inhibitors on the enzyme-substrate interactions and intramolecular dynamics of wild-type and drug resistant HIV-1 RT

HIV-1 Reverse Transcriptase (RT) is entirely responsible for conversion of viral RNA into dsDNA in the host cell cytoplasm, making it an attractive drug target. Nonnucleoside RT Inhibitors (NNRTIs) are highly effective in the treatment and prevention of HIV, yet their mechanism of action remains unknown. Furthermore, NNRTI resistance mutations arise from therapy, complicating treatment, yet the mechanism(s) of how these mutations inhibit polymerization by RT is unclear. In Chapter 2, we examine the role of NNRTI on the dynamics between RT and its Template/Primer (T/P) substrate using a combination of single-molecule and bulk fluorescence techniques which provide an unprecedented glimpse into the dynamics of RT-T/P interaction as well as the intramolecular conformation of RT itself while bound to its substrate. We show that efavirenz, an NNRTI, causes RT to relinquish its grip on the T/P substrate via “molecular arthritis," accompanied by increased shuttling on the substrate, reducing time spent in a polymerase-competent configuration. The K103N mutation relieves the arthritis in the fingers and thumb sub-domains of RT, enabling the efavirenz-bound enzyme to form a stable polymerase-competent complex. We demonstrate that relief of molecular arthritis is likely caused by disruption of a salt bridge between K101 and E138, residues at a key hinge site in the RT heterodimer. The data suggests a unique mechanism of resistance that is mediated by interplay between intramolecular conformational changes in RT and intermolecular dynamics of the RT-template/primer-dNTP complex. In chapter 3, we discuss ongoing experiments to probe the intramolecular dynamics of RT on the T/P substrate in response to NNRTIs. Combining time-resolved measurements of interdomain distances within RT as it binds and responds to NNRTIs in real time with atomistic molecular dynamics simulations of RT, we are attempting to characterize on-pathway structural intermediates of NNRTI-induced conformational change