Molecular Characterization of Integrase-RNA Interactions and Their Role in the Replication of HIV-1 and Other Retroviruses
Date of Award
Doctor of Philosophy (PhD)
HIV-1 integrase (IN) enzyme has an emerging non-catalytic role in particle maturation, whichinvolves its binding to the viral genome in virions. Allosteric integrase inhibitors (ALLINIs) and class II integrase substitutions inhibit the binding of IN to the viral genome and cause formation of eccentric non-infectious HIV-1 particles. These viruses are characterized by the mislocalization of the viral ribonucleoprotein complexes between the translucent conical CA lattice and the viral lipid envelope. We have previously demonstrated that IN binding to the viral genome is mediated by basic residues within the C-terminal domain of IN. In the first chapter, we show how basic residues of the IN CTD mediate RNA binding. We report that we have isolated secondary site suppressors of a class II IN mutant (R269A/K273A) which directly inhibits IN binding to the viral genome. Full-genome deep sequencing revealed the sequential emergence of D256N and D270N mutations within three passages. Reintroduction of these substitutions nearly fully restored the replication defect of the R269A/K273A virus, restored the ability of IN to bind RNA and led to the formation of particles viii with mature morphology. Furthermore, we found that D256R and D256R/270R substitutions also increased the infectivity of R269A/K273A as well as R262A/R263A IN viruses. The nature of these suppressor mutations suggests that IN-RNA binding is partly dictated electrostatic interactions between IN CTD basic residues and RNA. Though these findings imply some level of non-specificity towards gRNA binding, CLIP-seq and in-vitro binding experiments (reported in the third chapter) revealed a striking preference of IN for binding to purine-rich sequences on the viral genome. Taken together, our findings suggest that a combination of electrostatic interactions and semi-specific binding to the viral genome underlies the non-catalytic role of IN in virion maturation. Additional preliminary findings reported in the third chapter show that INRNA binding is a conserved property of retroviruses, further reaffirming this characteristic would be an archetypical target for new antiretroviral agents. In the fourth chapter, we report how we used our expertise of RNA viruses to create an assay that has been usefully in screening for antiviral agents for the SARS-Cov-2 virus during the COVID-19 pandemic. Overall, this dissertation illustrates how the basic molecular properties of integrase and viral genomic RNA that underlie their binding. Furthermore, it shows how these properties could be potentially targeted by the next generation of antiretroviral agents.
Chair and Committee
Sebla B. Kutluay
Shema Mugisha, Christian, "Molecular Characterization of Integrase-RNA Interactions and Their Role in the Replication of HIV-1 and Other Retroviruses" (2022). Arts & Sciences Electronic Theses and Dissertations. 2659.