This thesis presents the determination of susceptibility and resistance of HIV integrases (INs) to IN strand transfer inhibitors (INSTIs) and the development of single domain antibody (sdAb) targeting HIV IN. Firstly, the susceptibility of HIV-1 subtype CRF02_AG INs to the latest INSTIs was determined, since previous studies suggested that the significant variations of CRF02_AG IN may have consequential effects on the interaction between the inhibitor and IN. Consistent with in silico study, we found that 3'-processing and strand transfer activity of both HIV-1 subtype B IN and subtype CRF02_AG IN are comparable. The susceptibility of recombinant CRF02_AG INs to employed INSTIs (Raltegravir-RAL, Elevitegravir-EVG and L-731, 988) is similar to that of HIV-1 B IN. Hence, the polymorphism of CRF02_AG IN cannot significantly effect on the susceptibility to INSTIs. Secondly, the resistance of HIV-2 IN to RAL, the unique approved INSTI, has been confirmed in vitro with mutated enzymes harboring resistance mutations. Mutations at positions 155 and 148 played a similar role in HIV-1 and HIV-2, rendering the IN resistant to RAL. The G140S mutation conferred little resistance, but compensated for the catalytic defect due to the Q148R mutation. Conversely, Y143C alone did not confer resistance to RAL unless E92Q is also present. Furthermore, the introduction of the Y143C mutation into the N155H resistant background decreased the resistance level of enzyme containing the N155H mutation, possibly accounting for the lack of detection of these two mutations together in a single genome. Finally, anti-HIV IN sdAb that is endowed with many attractive properties was selected for developing antiretroviral agents. After the selections, we have obtained some sdAbs that specifically recognize a broad range of INs in vitro, including INSTI-resistance mutant G140S/Q148R. However, the inhibition activity of anti-HIV IN sdAbs has not been observed yet. Anti-HIV IN sdAbs can be applied for other application, such as targeting reagents for nanosensor. In future, development of anti-HIV IN sdAbs which are able to block HIV replication remains attractive for obtaining efficient inhibitor of IN.
| Identifer | oai:union.ndltd.org:CCSD/oai:tel.archives-ouvertes.fr:tel-00654080 |
| Date | 30 September 2011 |
| Creators | Ni, Xiaoju |
| Publisher | École normale supérieure de Cachan - ENS Cachan |
| Source Sets | CCSD theses-EN-ligne, France |
| Language | English |
| Detected Language | English |
| Type | PhD thesis |
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