Molecular Characterization of Integrase-RNA Interactions and Their Role in the Replication of HIV-1 and Other Retroviruses

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

Emergence of compensatory mutations reveals the importance of electrostatic interactions between HIV-1 integrase and genomic RNA

HIV-1 integrase (IN) has a noncatalytic function in virion maturation through its binding to the viral RNA genome (gRNA). Class II IN substitutions inhibit IN-gRNA binding and result in the formation of virions with aberrant morphologies marked by mislocalization of the gRNA between the capsid lattice and the lipid envelope. These viruses are noninfectious due to a block at an early reverse transcription stage in target cells. HIV-1 IN utilizes basic residues within its C-terminal domain (CTD) to bind to the gRNA; however, the molecular nature of how these residues mediate gRNA binding and whether other regions of IN are involved remain unknown. To address this, we have isolated compensatory substitutions in the background of a class II IN mutant virus bearing R269A/K273A substitutions within the IN-CTD. We found that the nearby D256N and D270N compensatory substitutions restored the ability of IN to bind gRNA and led to the formation of mature infectious virions. Reinstating the local positive charge of the IN-CTD through individual D256R, D256K, D278R, and D279R substitutions was sufficient to specifically restore IN-gRNA binding and reverse transcription for the IN R269A/K273A as well as the IN R262A/R263A class II mutants. Structural modeling suggested that compensatory substitutions in the D256 residue created an additional interaction interface for gRNA binding, whereas other substitutions acted locally within the unstructured C-terminal tail of IN. Taken together, our findings highlight the essential role of CTD in gRNA binding and reveal the importance of pliable electrostatic interactions between the IN-CTD and the gRNA

A simplified quantitative real-time PCR assay for monitoring SARS-CoV-2 growth in cell culture

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected millions within just a few months, causing severe respiratory disease and mortality. Assays to monitor SARS-CoV-2 growt

Amilorides inhibit SARS-CoV-2 replication in vitro by targeting RNA structures

The SARS-CoV-2 pandemic, and the likelihood of future coronavirus pandemics, emphasized the urgent need for development of novel antivirals. Small-molecule chemical probes offer both to reveal aspects of virus replication and to serve as leads for antiviral therapeutic development. Here, we report on the identification of amiloride-based small molecules that potently inhibit OC43 and SARS-CoV-2 replication through targeting of conserved structured elements within the viral 5′-end. Nuclear magnetic resonance–based structural studies revealed specific amiloride interactions with stem loops containing bulge like structures and were predicted to be strongly bound by the lead amilorides in retrospective docking studies. Amilorides represent the first antiviral small molecules that target RNA structures within the 5′ untranslated regions and proximal region of the CoV genomes. These molecules will serve as chemical probes to further understand CoV RNA biology and can pave the way for the development of specific CoV RNA–targeted antivirals

Amilorides inhibit SARS-CoV-2 replication in vitro by targeting RNA structures

The SARS-CoV-2 pandemic, and the likelihood of future coronavirus pandemics, emphasized the urgent need for development of novel antivirals. Small-molecule chemical probes offer both to reveal aspects of virus replication and to serve as leads for antiviral therapeutic development. Here, we report on the identification of amiloride-based small molecules that potently inhibit OC43 and SARS-CoV-2 replication through targeting of conserved structured elements within the viral 5′-end. Nuclear magnetic resonance–based structural studies revealed specific amiloride interactions with stem loops containing bulge like structures and were predicted to be strongly bound by the lead amilorides in retrospective docking studies. Amilorides represent the first antiviral small molecules that target RNA structures within the 5′ untranslated regions and proximal region of the CoV genomes. These molecules will serve as chemical probes to further understand CoV RNA biology and can pave the way for the development of specific CoV RNA–targeted antivirals

Systematic analysis of SARS-CoV-2 infection of an ACE2-negative human airway cell

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) variants govern transmissibility, responsiveness to vaccination, and disease severity. In a screen for new models of SARS-CoV-2 infection, we identify human H522 lung adenocarcinoma cells as naturally permissive to SARS-CoV-2 infection despite complete absence of angiotensin-converting enzyme 2 (ACE2) expression. Remarkably, H522 infection requires the E484D S variant; viruses expressing wild-type S are not infectious. Anti-S monoclonal antibodies differentially neutralize SARS-CoV-2 E484D S in H522 cells as compared to ACE2-expressing cells. Sera from vaccinated individuals block this alternative entry mechanism, whereas convalescent sera are less effective. Although the H522 receptor remains unknown, depletion of surface heparan sulfates block H522 infection. Temporally resolved transcriptomic and proteomic profiling reveal alterations in cell cycle and the antiviral host cell response, including MDA5-dependent activation of type I interferon signaling. These findings establish an alternative SARS-CoV-2 host cell receptor for the E484D SARS-CoV-2 variant, which may impact tropism of SARS-CoV-2 and consequently human disease pathogenesis

Emergence of compensatory mutations reveals the importance of electrostatic interactions between HIV-1 integrase and genomic RNA

HIV-1 integrase (IN) has a noncatalytic function in virion maturation through its binding to the viral RNA genome (gRNA). Class II IN substitutions inhibit IN-gRNA binding and result in the formation of virions with aberrant morphologies marked by mislocalization of the gRNA between the capsid lattice and the lipid envelope. These viruses are noninfectious due to a block at an early reverse transcription stage in target cells. HIV-1 IN utilizes basic residues within its C-terminal domain (CTD) to bind to the gRNA; however, the molecular nature of how these residues mediate gRNA binding and whether other regions of IN are involved remain unknown. To address this, we have isolated compensatory substitutions in the background of a class II IN mutant virus bearing R269A/K273A substitutions within the IN-CTD. We found that the nearby D256N and D270N compensatory substitutions restored the ability of IN to bind gRNA and led to the formation of mature infectious virions. Reinstating the local positive charge of the IN-CTD through individual D256R, D256K, D278R, and D279R substitutions was sufficient to specifically restore IN-gRNA binding and reverse transcription for the IN R269A/K273A as well as the IN R262A/R263A class II mutants. Structural modeling suggested that compensatory substitutions in the D256 residue created an additional interaction interface for gRNA binding, whereas other substitutions acted locally within the unstructured C-terminal tail of IN. Taken together, our findings highlight the essential role of CTD in gRNA binding and reveal the importance of pliable electrostatic interactions between the IN-CTD and the gRNA