HIV-1 integrase (IN) plays a key role in several steps of HIV-1 replication. IN carry out 3’-processing in cytoplasm and then translocate to the nucleus for integration in the formation of pre-integration complex (PIC) with the viral DNA. Translocation of viral IN into nucleus is a critical precondition of integration during the life cycle of HIV-1. This step is complicated and involve in a number of virion-derived and cellular proteins. Specifically inhibiting IN nuclear import would undoubtedly block HIV-1 infection in non-dividing cells, so this critical step of HIV-1 replication is of great prospect as a drug target. In this study, wild-type HIV-1IN was cloned into pEGFP-C1 expression vector to form pEGFP-C1-IN plasmid. pEGFP-C-IN and pEGFP-C1 plasmids were transfected into 293T cells respectively and the fusion proteins EGFP-IN mainly accumulated in the nucleus but EGFP was evenly full of the whole cells by Leica DMI4000B microscopy twenty four hours after transfection. It has reported that D77, one benzoic acid derivative, showed strong inhibition activity against IN-LEDGF/p75 interaction and affected the HIV-1 IN nuclear distribution resulting in inefficient replication. D77 was added at 5h post-transfection and cells were observed 24h later and EGFP-IN appeared diffusely distributed in cytoplasm and no fluorescence could be observed in nucleus. In this work, we screened for inhibitors of HIV-1 IN nuclear import using this method and D77 as positive control. Firstly, the anti-HIV activities and cytotoxicity of compounds were evaluated simultaneously in vitro. For the better candidates, the activities against IN nuclear import was detected and the results showed that some of them like D77 made the EGFP-IN diffusely distribution in cytoplasm and no fluorescence in nucleus. To further investigate the target of action of these compounds on IN translocation , a time-of-drug addition assay was carried out to target identification of potent antiviral compounds. Viral replication was inhibited up to the time point corresponding to the stage of HIV-1 replication targeted by compounds. The results of a time-of-drug addition assay showed that the compounds inhibited the syncytia formation before seven hours. Following reverse transcription completion after 4~6 h of HIV-1 infection, IN and cDNA will transport to the nucleus in the formation of PIC. According this, these compounds maybe target nuclear import of HIV-1 IN. Then a study of molecular docking suggested that these compounds bound to the hydrophobic pocket of IN catalytic core domain (CCD) in complex to the IN-binding domain (IBD) of LEDGF/p75. It has reported that residues Q168, E170, and T174 in chain A of IN, T125 and W131 in chain B of IN as well as I365, D366, F406 and V408 in LEDGF/p75 were responsible for their binding. Our docking studies found that these compounds could formed H-bond with T174 and hydrophobic contacts with Q168 in chain A, E170, T174 in chain A and W131 in chain B, respectively. Obviously, these compounds affected the interaction IN and LEDGF/p75 and resulted in the inhibition of IN nuclear translocation. According to above results, we speculate that these compounds maybe compete binding IN with LEDGF/p75 and inhibit the nuclear import of the HIV-1 IN. Further studies on anti-HIV activities exhibited the inhibition of drug-resistant viral strains (HIV-1A17, HIV-174V and HIV-1AO18) and HIV-2 (HIV-2ROD and HIV-2CBL-20).
修改评论