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ADAP, that is necessary for up regulation of LFA 1 adhesion. This pathway is mediated downstream by SKAP1 that regulates the complicated formation in between Rap1 and RapL. Two tyrosine motifs at Y595DDV and Y651DDV of ADAP bind towards the SH2 domain of Fer-1 SLP 76 upon TCR stimulation. A double point mutation in ADAP at Y595F and Y651F is defective in SLP 76 binding and shows lowered LFA 1 adhesion and pSMAC formation. In spite of this, a potential connection in between ADAP and HIV 1 infection has not been explored. In this study, we demonstrate that ADAP and its bin ding to SLP 76 regulate two steps of HIV 1 infection by cooperating differentially with two distinct co receptors. Loss of ADAP plus the SLP 76 ADAP module mar kedly impaired CD28 mediated HIV 1 transcription as well as LFA 1 dependent formation of virological synapse for cell cell viral spread.
These findings iden tify ADAP and its signaling module as important regulators of HIV 1 infection. Results Disruption OAC1 the SLP 76 ADAP signaling module inhibits HIV 1 infection We and others have previously outlined the value with the SLP 76 ADAP SKAP1 pathway within the activation of LFA 1. A mutant of ADAP lacking tyrosine resi dues 595 and 651 is unable to bind to SLP 76 and impairs LFA 1 activation. We assessed irrespective of whether wild form ADAP plus the mutant M12 could regulate HIV 1 infection in Jurkat T cells. Jurkat T cells were stably transduced with retroviral su pernatants encoding ADAP IRES GFP or M12 IRES GFP or with GFP alone. Expression remained stable resulting from inte gration.
The transfectants showed the same expression levels Bafilomycin A1 of CD4, CXCR4, CD3, CD28, B1 and B2 integrins as the control GFP expressing Jurkat cells as measured by flow cytometry. Nucleophilic aromatic substitution We subsequent infected these cells with a single cycle HIV 1 virus carrying a luciferase reporter. The mRNA levels of HIV 1 gag were measured at 72 hours post infection by quantitative RT PCR with specific primers for HIV 1 gag. JK ADAP GFP cells showed three four fold greater levels of HIV 1 gag mRNA when in comparison to JK GFP cells. By contrast, JK M12 GFP cells failed to assistance the enhance of HIV 1 gag mRNA beyond that observed within the JK GFP cells. The level of transfected M12 was similar to ADAP as observed by western blotting. We confirmed that right after HIV 1 infection, overexpression of ADAP GFP or M12 GFP had no effects on CD4 or CXCR4 expression in Jurkat cells. We subsequent stably overexpressed GFP, ADAP or M12 into human C8166 T cells.
These cells were infected with low dose or higher dose of HIV 1. Superna tants were collected and quantified by ELISA for levels of of HIV 1 p24Gag at various occasions post infection. We discovered that at each doses of input Siponimod virus, C8166 M12 cells were impaired in their assistance of HIV 1 replication relative to cells expressing wild form ADAP. When we made use of low dose of virus to infect cells, C8166 ADAP cells Fer-1 plus the control cells supported productive infection, whereas C8166 M12 cells failed to make the detectable levels of p24Gag. More than 95% of C8166 T cells overexpressed GFP, or ADAP GFP or M12 GFP, which had no effect on the expression of surface receptors and showed similar development prices. We additional examined irrespective of whether HIV 1 infection of human major CD4 T cells was dependent on ADAP.
ADAP expression was lowered employing specific siRNAs. qRT PCR showed a 50 60% reduction in ADAP mRNA transcripts over a period of 96 hours post transfection. Similarly, western blotting Siponimod of cells at 48 Fer-1 hours confirmed the signi ficantly lowered ADAP expression right after transfection with siRNA ADAP. siRNA transfected human CD4 T cells were then infected with all the single cycle HIV 1 virus containing luciferase reporter. siRNA for ADAP lowered HIV 1 gag mRNA levels by 30% when assessed at 72 hours post infection. A measurement of luciferase activity confirmed that siRNA for ADAP resulted inside a considerable reduction of HIV 1 infection. The surface expression of CD3, CD4, CD28, CXCR4, B1 B2 integrins and ICAM 1 in human CD4 T cells was not affected by knockdown of ADAP.
Collectively, Siponimod these information indicate that ADAP is necessary for the optimal HIV 1 infection of T cell lines and major human T cells. ADAP and SLP 76 regulates HIV 1 LTR transcription inside a CD28 and NFB dependent manner To uncover the molecular basis of ADAP involvement in HIV 1 infection, we firstly examined its potential ef fects on the induction of HIV 1 LTR transcription. Wild form, SLP 76 deficient Jurkat T cells or ADAP deficient Jurkat T cells were transfected with a pLTR gag3 flag luc reporter plasmid followed by stimulation by means of anti CD3 CD28 ligation for six hours. The pLTR gag3 flag luc plasmid consists of the HIV 1 5 LTR promoter area with two NFB binding internet sites as well as a firefly luciferase open reading frame. HIV 1 transcription was then assessed by a measure of luciferase activity. Anti CD3 CD28 stimula tion induced a two fold enhance in HIV 1 transcription in wild form Jurkat cells, an effect that was not observed in J14 cells. Re expression of SLP 76 into J14 cells restored and enhanced HIV 1 tran scription