טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentMeytal Galilee
SubjectStructure-Function Studies Reveal Novel Hot-Spots in
Proteins Crucial for HIV-1 Replication
DepartmentDepartment of Biology
Supervisor Dr. Alian Akram
Full Thesis textFull thesis text - English Version


Abstract

HIV-1 virus, the causative agent of AIDS, continues to escape therapeutic intervention and immune restriction, necessitating the discovery of novel and unexploited targets in the virus lifecycle. The ability of the virus to readily mutate and gain resistance to traditional drugs, which specifically target the viral encoded proteins, has driven the attention to the importance of considering not only fresh targets within the viral proteins but also crucial host-virus interactions. Therefore, I focused my research efforts on providing new insights and potentially targetable hotspots within one virally encoded protein, the integrase (IN), which is crucial for viral integration and successful replication, and a representative family member of the host-cell restriction factors, the APOBEC3 cytidine deaminases, which target and hypermutate the HIV-1 genome leading to its degradation or the formation of defective viral proteins. Nevertheless, the virus has evolved to counteract the activity of APOBEC3s via proteosomal degradation dependent and independent pathways.

To highlight novel intervention targets within the HIV-1 IN, I employed crossspecies comparisons underscoring common and distinct functional features crucial for IN activity. Using the feline Immunodeficiency virus (FIV) as a comparative model system, I found that a single Phe187 amino acid potentially hinges IN dimerization and dimer reorganization upon DNA binding, which can be considered a fragile hotspot in future targeting of IN functional multimerization. I further discovered that this novel hinge region could be extended to comprise a helix-turn-helix (HTH) docking-cleft that crucially mediates IN functional tetramerization. I exploited an antigen-binding fragment (FAB) to target this region and by assessing IN activity and virus infectivity I showed total inhibition of both. To characterize this fragile hotspot for novel intervention strategies, I further determined the crystal structure of IN-FAB complex and used this structure to rationally develop short peptide-derivatives targeting this region and inhibiting IN activity.

Using cross-family comparisons approach, I also compared the cellular anti-HIV restriction factors, APOBEC3 (A3) cytidine deaminases that deaminate polynucleotides, with their ancestral deoxycytidylate deaminases (dCD), which act on cytidine monophosphate in the pyrimidine synthesis pathway. Reminiscent to their dCD ancestrals, I revealed a novel A3 allosteric regulatory mechanism employing a loop surrounding the catalytic site that can regulate A3 deaminase activity. I propose that HIV-1 may exploit this allosteric regulatory feature of A3s to counteract their restriction activity. This may provide an explanation to a reported non-degradative pathway inhibition of A3s by HIV-1 virus infectivity factor (Vif).

Taken together, differences revealed by cross-species comparisons, exploring how orthologous proteins and related viruses coevolved in their natural environment can indeed expose overlooked targets and, perhaps, resistance-mechanisms potentially accessible by mutational adaptation of challenged viruses. Here, I identified a targetable hotspot within the HIV-1 IN protein, targeting of which inhibited virus replication. Short-peptides and small-molecules targeting this region are currently being developed in our lab. I also highlighted a potential counteracting mechanism that HIV-1 Vif may exploit to inhibit the deleterious mutational effect of A3s, a hypothesis that is pending further validation.