L for optimal viral infectivity [46]. The generation of an immature/ aberrant
L for optimal viral infectivity [46]. The generation of an immature/ aberrant HIV-1 core is a phenotype that is frequently observed with NC mutants [9?1] and more recent evidence strongly suggest that the proper uncoating of the virus is an essential step at the early stage of infection of target cells [47]. The A1752 appears to have induced an increase in the unprocessed CA-NC precursor as seen in Fig. 6, which might have caused incomplete viral maturation resulting in a deficiency in the functionality of the HIV-1 core formation [48]. Therefore, we further examined the viral core abnormality induced by the A1752 and found indeed that it was much more refractory compared with the wild types in releasing the RT proteins in core (Fig. 7). This would have led to the failure of the reverse transcription as shown by our detection of the significant suppression of the synthesis of early RT products (-ss) DNA (Fig. 3d). Therefore, it could be deduced that the modification of the core stability induced by the A1752 might also have inhibited the normal uncoating process in the modified viral infected cells. Furthermore, based on the inhibition of the NC-mediated cTAR destabilization by the A1752 (Fig. 2d), a similar inhibition of the chaperone activities of the NC by the A1752 could also have contributed to defects in the RT process post infection.and exhibits good antiviral activity. The A1752 strongly binds to NC and thereby interferes with the chaperone functions of NC as well as PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/25609842 the Gag processing. Consequently, this PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28494239 action produces non-infectious and abnormal viruses that are defective in uncoating and viral reverse transcription in cells following infection. Therefore, these results suggest that these unique properties of A1752 support the proposition that it is a functional NC DM-3189 web inhibitor and could serve as an excellent novel candidate molecule for the development of a new anti-HIV drug.MethodsInfection, transfection, and reinfectionThe MT4 and 293FT cells were maintained in RPMI and DMEM, respectively, and the media contained 10 FBS (Hyclone, Logan, UT), penicillin, and streptomycin sulfate (GIBCO, Carlsbad, CA). The MT4 cells were infected with the NL4-3 isolate-derived HIV-1, which had the nef gene replaced with EGFP and were treated simultaneously with the inhibitors as indicated. The 293FT cells were typically transfected with 1 g the NL43/ EGFP-proviral DNA using lipofectamine2000 (Invitrogen, Carlsbad, CA). Following a 6-h transfection, the media were changed and cells were treated with the test compounds. Post infection day 3 or post transfection 36 h, the viral supernatants were collected for further analysis and the remaining cells were analyzed for GFP expression. For the reinfection assay, the viral supernatant produced in the presence of the compounds was first centrifuged at 900 for 10 min, filtered through a 0.45-m filter to remove the cell debris. The viruses were then further purified and concentrated by centrifugation at 28,500 for 3 h to remove any remaining inhibitors used in infection assay. The resulting pellet was resuspended in serum-free fresh RPMI to yield virus stocks. An equivalent amount (typically 2.5 ng) of virion was used to re-infect fresh MT4 cells. Post infection at 48 h, the expression of GFP was examined followed by a FACS analysis for quantification.HIV1 p24 ELISA assayThe amounts of virus particles of each sample were determined using an HIV-1 p24 ELISA kit as recommended by the manufacture.