RESUMO
Structural organization of HIV-1 integrase is based on a tetramer formed by two protein dimers. Within this tetramer, the catalytic domain of one subunit of the first dimer interacts with the N-terminal domain of the second dimer subunit. It is the tetrameric structure that allows both ends of the viral DNA to be correctly positioned relative to the cellular DNA and to realize catalytic functions of integrase, namely 3'-processing and strand transfer. However, during the HIV-1 replicative cycle, integrase is responsible not only for the integration stage, it is also involved in reverse transcription and is necessary at the stage of capsid formation of the newly formed virions. It has been suggested that HIV-1 integrase is a structurally dynamic protein and its biological functions depend on its structure. Accordingly, studying interactions between the domains of integrase that provide its tetrameric structure is important for understanding its multiple functions. In this work, we investigated the role of three amino acids of the catalytic domain, I182, R187, and K188, located in the contact region of two integrase dimers in the tetramer structure, in reverse transcription and integration. It has been shown that the R187 residue is extremely important for formation of the correct integrase structure, which is necessary at all stages of its functional activity. The I182 residue is necessary for successful integration and is not important for reverse transcription, while the K188 residue, on the contrary, is involved in formation of the integrase structure, which is important for the effective reverse transcription.
Assuntos
Domínio Catalítico , Integrase de HIV , HIV-1 , Transcrição Reversa , Integração Viral , Integrase de HIV/metabolismo , Integrase de HIV/química , Integrase de HIV/genética , HIV-1/enzimologia , HumanosRESUMO
Integration of HIV-1 genomic cDNA results in the formation of single-strand breaks in cellular DNA, which must be repaired for efficient viral replication. Post-integration DNA repair mainly depends on the formation of the HIV-1 integrase complex with the Ku70 protein, which promotes DNA-PK assembly at sites of integration and its activation. Here, we have developed a first-class inhibitor of the integrase-Ku70 complex formation that inhibits HIV-1 replication in cell culture by acting at the stage of post-integration DNA repair. This inhibitor, named s17, does not affect the main cellular function of Ku70, namely its participation in the repair of double-strand DNA breaks through the non-homologous end-joining pathway. Using a molecular dynamics approach, we have constructed a model for the interaction of s17 with Ku70. According to this model, the interaction of two phenyl radicals of s17 with the L76 residue of Ku70 is important for this interaction. The requirement of two phenyl radicals in the structure of s17 for its inhibitory properties was confirmed using a set of s17 derivatives. We propose to stimulate compounds that inhibit post-integration repair by disrupting the integrase binding to Ku70 KuINins.