Ctamase-Vpr plasmid using order Tirabrutinib previously established calcium phosphate methods and fresh media
Ctamase-Vpr plasmid using previously established calcium phosphate methods and fresh media was added 6 h post transfection. At 48 h post transfection, cells were incubated with media alone or media containing 10 M chaetocin for 4 h. Cells were subsequently treated with CCF2-AM for 2 h at room temperature, washed, and incubated overnight in CO2independent media containing probenecid. The following day, cells were trypsinized, stained with live/dead nearIR viability dye and analyzed for evidence of substrate cleavage.Phosphoproteomic analysis of HIVexposed CD4+ T cellsMemory CD4+ T cells were purified by negative selection from a leukapheresis pack using custom RosetteSep kits (STEMCELL Technologies). Briefly, equal populations of 150 ? 106 cells were exposed to 20 g/ml p24 equivalent AT2-inactivated HIV-1 THRO or protein equivalent concentrations of non-viral microvesicles as control. HIV-1 THRO is a transmitted/founder virus isolated from a subject PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28212752 with early HIV infection that was found to be CCR5-tropic as evidenced by inhibition by TAK779 but not AMD3100 on TZM-bl cells and by its inability to replicate in PBMCs from a patient with the delta32-ccr5 mutation [29]. The AT2-inactivated viruses were derived from a cell clone of THRO (THRO CL.29)Data presented as mean values with standard error of the mean unless stated otherwise. All differences with a p value of <0.05 were considered statistically significant, correcting for multiple comparisons when appropriate. Statistical analyses were performed using student t-tests within GraphPad Prism v7.0.ResultsViral fusion is not affected by compounds in an epigenetic / posttranslational modification screening libraryThe importance of post-translational modifications (PTMs) on HIV replication has been extensively studied in the context of HIV-1 reactivation from latency, where PTMs regulate the accessibility of host transcription factors and the RNA polymerase machinery to the HIV-1 LTR promoter. Most significantly, the histone deacetylase (HDAC) family of enzymes has become a leading targetLucera et al. Retrovirology (2017) 14:Page 4 ofof pharmacological inhibition in efforts to eliminate viral reservoirs from infected individuals by promoting reactivation of HIV PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/25681438 from latency [13]. Recently, we reported this class of drugs has previously unknown effects on early post-entry events in HIV infection, increasing the kinetics and efficiency of reverse transcription and integration [16]. Surprisingly, this enhancement was not due to alterations of histone acetylation but rather to inhibition of the cytoplasmic HDAC6, which regulates tubulin acetylation and microtubule stability [16?9]. To probe for additional epigenetic or post-translational modifications regulating HIV-1 replication, we employed the use of a small molecule inhibitor epigenetic / post-translational modification (PTM) screening library (Additional file 1: Table 1) with our combination reporter virus system that measures both viral fusion and LTR-driven EGFP expression [24]. Viruses bearing a CXCR4-tropic HIV Env were chosen based on the expression of this receptor on a greater percentage of primary CD4+ T cells when compared to the CCR5 co-receptor [30?3]. Compounds within this library have previously been reported to have multiple targets including–but not limited to–histone acetylation and methylation, kinase signaling, and bromodomain and extraterminal domain (BET) protein family members. All compounds screened were prepared.