An intriguing question remains regarding the pleiotropic
Een IN and TNPO3. An intriguing question remains regarding the pleiotropic effects of the mutation W131A on HIV-1 replication. Similar to our observations with HIV-1 W131A, a defect of viral cDNA synthesis was also described for the VSVgpseudotyped HIV-1 W131A and HIV-1 W131D viruses [15,17]. IN mutants have been reported to perturb the reverse transcription step, possibly by altering the overall conformation of the RTC [80]. A direct interaction between IN and RT was reported and could account for these effects [81]. In addition, HIV-1 viruses carrying IN deletion or the C130S substitution were recently described to decrease CypA-CA interaction leading to the destabilization of the viral core [82]. Future experiments would be required to assess the kinetic of CA uncoating of IN mutated viruses depending of their mechanism of entry (i.e. fusion vs. endocytosis).described as a HIV-1 dependency factor able to bind HIV-1 IN, and RNAi studies indicated that TNPO3 participates in the import of the PIC into the nucleus [53,55,62]. Our site-directed mutagenesis analysis aimed at exploring the PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/11834444 contribution of the interaction between IN and TNPO3 to the early stages of the PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/8086425 viral lifecycle. The present study indicates Methyl 5-amino-2,4-difluorobenzoate that IN mutants W131A and Q168L previously shown to be defective for LEDGF/p75 were also impaired for TNPO3 binding, albeit to a lesser extent. However, none of these mutations significantly reduced the level of the 2-LTR circles, arguing against a role of IN-TNPO3 interaction in nuclear import.MethodsCells, viruses and infectionsHeLa and 293T cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10 of fetal calf serum and antibiotics (100 units/ml penicillin G, 100 mg/ml streptomycin; GIBCO, 4 Invitrogen). SupT1 and LEDGF-depleted SupT1 TL34 cells [71] were grown in RPMI 1640 supplemented with 10 of fetal calf serum and antibiotics (100 units/ml penicillin G, 100 mg/ml streptomycin; GIBCO, Invitrogen).Viral productionBru WT or IN mutant virus stocks were generated by transfecting 293T cells with pBru-derived molecular clones using PEI (Polyethylenimine, Polysciences). Cells were washed 16 h after transfection, and supernatants collected 24 h thereafter were filtered through 0.45 filters and aliquoted. Viral stocks were quantified by measuring the CAp24 antigen using the Innotest HIV Antigen mAb kit, accordingly to manufacturer instructions (Innogenetics).Viral replicationSupT1 or Sup T1 TL34 cells were infected with viral doses corresponding to 100 ng of HIV-1 CAp24 antigen per 2.106 cells, viral replication of each mutant viruses compare to the WT was followed by monitoring the HIV-1 CAp24 in the producer cells supernatants as described above.Plasmids and mutagenesis His6-tagged integrasesMutated recombinant IN Q168L, IN W131A, IN K188Q, IN Y194E, IN Y194F were obtained by sitedirected mutagenesis 3-(2,4-Dichlorophenoxy)azetidine of the pINSD.His plasmid [83,84]. IN mutations were introduced into the pBru molecular clone by site-directed mutagenesis.Expression and purification of recombinant proteinsConclusion Interactions between IN and host cellular factors control different stages of HIV replication. TNPO3 was recentlyGST-LEDGF/p75 encoding sequence was introduced in the pGGWA plasmid using the gateway system (Invitrogen, Stratagene). Recombinant GST proteins wereCribier et al. Retrovirology 2011, 8:104 http://www.retrovirology.com/content/8/1/Page 11 ofproduced in Escherichia coli BL21. E. coli transformed with GST-TNPO3 or GST-LEDGF/p75.