Elucidating the mechanisms and dynamics of drug-mediated inhibition and latency reversal in HIV-1 (HIV-1)

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] The Human Immunodeficiency Virus Type-1 (HIV-1) is the etiological agent of Acquired Immunodeficiency Syndrome, a disease that causes the host to succumb to secondary infections. There is currently no cure for HIV-1 infection, but Highly Active Anti-Retroviral Therapy (HAART) can bring the viral load in patients down to undetectable levels in the blood (less than 50 copies/mL). Furthermore, when the minimal limit of detection has been reached and the patient stops HAART, the viral load in the blood increases at an exponential rate due to the reactivation of latent HIV-1 infected cells that evaded HAART. Ongoing efforts focus on the eradication of HIV-1 by the development of potent latency reversing agents (LRAs) that can successfully reactivate latently infected cells, and of antivirals that can effectively inhibit re-establishment of infection post reactivation. This dissertation focuses on the evaluations of 2 classes of HIV-1 drugs, Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs), and LRAs, to better understand the mechanisms by which each drug class inhibits and reactivates HIV-1 replication respectively, to aid in the effort towards the development of antivirals that will lead to HIV-1 eradication. Chapter II describes the inhibitory mechanisms of NNRTIs using biochemical methods, which may further explain the differences in potency among drugs of this class. In addition, we explain how changes in the position of HIV-1 RT in the DNA substrate sequence, and the nucleotide terminating the primer 3'-end have a significant effect on the polymerase properties of the enzyme. We demonstrate that there are NNRTI- and site-dependent differences in the potency of NNRTIs, which is demonstrated by the repositioning, or lack there of, of the primer 3'-end of DNA/DNA substrates from the polymerase active site. This is further supported by the efficiency of dNTP or NRTI incorporation in the presence of NNRTI with multiple DNA/DNA substrates, which are representative of different sites in the template sequence. We also show that there are site-dependent differences in the polymerase properties of RT, which is demonstrated by rate of dNTP incorporation and incorporation efficiency at different sites in the template sequence. Chapter III describes the various effects of different types of LRAs, such as histone deacetylase inhibitors and histone methyltransferase inhibitors, on the dynamics of HIV-1 latency reversal in latent cell lines. Here, we demonstrate the use of branched DNA in situ hybridization in combination with immunocytochemistry to study the kinetics and dynamics of latency reversal in various latent cell lines. This technique is augmented with the use of automated screening using microscopy and flow cytometry to quickly detect different populations of latent and reactivated proviruses in thousands of cells in a short amount of time. Understanding the mechanisms by which a drug affects a biological process is important for establishing drug efficacy. Such information can influence what modifications are added to, or removed from drugs, which can cause a change in drug potency. This dissertation outlines assays used to evaluate the mechanisms of various drugs, and the influence of these drugs on the dynamics of HIV-1 replication. It is our hope that the work presented here will help progress efforts to eradicate HIV-1 infection.