Otein from cell lysates were separated by 4-12 SDS-PAGE and transferred
Otein from cell lysates were separated by 4-12 SDS-PAGE and transferred onto nitrocellulose membranes. TASK and -tubulin proteins PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26866270 were detected by immunoblotting using polyclonal antibodies and visualization by chemilumenescence. C. TASK-1 does not decrease HIV-1 Gag virus-like particle release. 293T cells were co-transfected with plasmid encoding Gag driven by CMV promoter and TASK-1 or empty vector. After 48 hours supernatants were collected, and a p24 ELISA was performed. Data are normalized to the empty vector control (EV). D. TASK-1 expression in transfected cells from (C). 293T cells from (C.) were harvested and lysed with a detergent buffer containing protease inhibitors. Equal protein amounts of cell lysate were separated by 4-12 SDS-PAGE and transferred to nitrocellulose membranes. TASK and -tubulin proteins were revealed by immunoblotting using polyclonal antibodies followed by chemilumenescence detection. (* P < .0001; Student T test was used to determine statistical significance).is not due to inhibition of Gag assembly and release. Furthermore TASK protein expression preferientally suppresses gene transcription driven by the HIV-LTR but not transcription from CMV or SV40 promoters. Since the late stages of virus replication were not affected by TASK protein overexpression we examined the effect of TASK protein expression on the earlier stages of HIV-1 replication. 293T cells were cotransfected with pNL4-3 and TASK-3 or empty vector. After 48 hours intracellular Gag from cell lysates was quantified with a capture p24 antigen ELISA. We found that overexpression of TASK PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28607003 proteins decreased intracellular Gag by over 83 (data not shown).TASK-1 protein suppresses HIV-1 transcriptionTo test this possibility, a reporter virus construct derived from pNL4-3 in which the luciferase gene is substituted for nef (pNL4-3Luc), was cotransfected into 293T cells along with TASK-1. After 48 hours, the transfected cells were lysed and a luciferase assay was performed to measure HIV-1 OPC-8212 web LTR-dependent transcription. Overexpression of TASK-1 significantly inhibited HIV-1 transcription, and this inhibition was dose-dependent (Figure 2a, 2c). Similar results were obtained when cells were co-transfected with TASK-3 and the reporter virus construct (data not shown). Taken together, these results indicate that TASK protein suppresses HIV-1 replication by inhibition of viral transcription.TASK channel function is not required for suppression of HIV-1 transcriptionOne possible explanation for the decrease in both extracellular and intracellular Gag expression in cells overexpressing TASK proteins is suppression of HIV-1 gene transcription.TASK proteins are potassium channels whose ion channel function contributes significantly to maintaining theEmeagwali and Hildreth Virology Journal 2012, 9:277 http://www.virologyj.com/content/9/1/Page 4 ofABEV TASK-1 IB: anti TASK-*IB: anti -tublinpNL4-EV +TASK-1 +CEV 0.5 1.0 2.0 ugIB: anti TASK-***IB: anti -tublinEV0.1.2.0 TASK-Figure 2 TASK proteins suppress HIV-1 transcription. A. 293T cells were transfected with pNL4-3Luc and TASK-1 expression vector or empty vector (EV). After 48 hours cells were lysed and a luciferase assay was performed to determine the effect of TASK-1 on HIV-1 transcription. Data are normalized to the empty vector control (EV). B. Transfected 293T cells were harvested and lysed with a detergent buffer containing protease inhibitors. Equal protein amounts of cell lysate were separated by 4-12.
