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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 complex formation between Rap1 and RapL. Two tyrosine motifs at Y595DDV and Y651DDV of ADAP bind for the SH2 domain of OAC1 SLP 76 upon TCR stimulation. A double point mutation in ADAP at Y595F and Y651F is defective in SLP 76 binding and shows decreased LFA 1 adhesion and pSMAC formation. Despite this, a prospective connection between ADAP and HIV 1 infection has not been explored. Within 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 essential regulators of HIV 1 infection. Final results Disruption Fer-1 the SLP 76 ADAP signaling module inhibits HIV 1 infection We and other people have previously outlined the value from the SLP 76 ADAP SKAP1 pathway inside 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 no matter whether wild type 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 exact same expression levels Bafilomycin A1 of CD4, CXCR4, CD3, CD28, B1 and B2 integrins because the control GFP expressing Jurkat cells as measured by flow cytometry. Nucleophilic aromatic substitution We subsequent infected these cells using 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 certain primers for HIV 1 gag. JK ADAP GFP cells showed 3 4 fold larger levels of HIV 1 gag mRNA when compared to JK GFP cells. By contrast, JK M12 GFP cells failed to support the enhance of HIV 1 gag mRNA beyond that observed inside the JK GFP cells. The level of transfected M12 was comparable to ADAP as noticed by western blotting. We confirmed that just 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 high dose of HIV 1. Superna tants were collected and quantified by ELISA for levels of of HIV 1 p24Gag at numerous times post infection. We located that at both doses of input Bafilomycin A1 virus, C8166 M12 cells were impaired in their support of HIV 1 replication relative to cells expressing wild type ADAP. When we utilized low dose of virus to infect cells, C8166 ADAP cells OAC1 plus the control cells supported productive infection, whereas C8166 M12 cells failed to generate the detectable levels of p24Gag. Over 95% of C8166 T cells overexpressed GFP, or ADAP GFP or M12 GFP, which had no impact on the expression of surface receptors and showed comparable growth rates. We additional examined no matter whether HIV 1 infection of human key CD4 T cells was dependent on ADAP.
ADAP expression was decreased utilizing certain siRNAs. qRT PCR showed a 50 60% reduction in ADAP mRNA transcripts more than a period of 96 hours post transfection. Similarly, western blotting Bafilomycin A1 of cells at 48 OAC1 hours confirmed the signi ficantly decreased ADAP expression just after transfection with siRNA ADAP. siRNA transfected human CD4 T cells were then infected together with the single cycle HIV 1 virus containing luciferase reporter. siRNA for ADAP decreased 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 within a significant 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, Bafilomycin A1 these data indicate that ADAP is necessary for the optimal HIV 1 infection of T cell lines and key human T cells. ADAP and SLP 76 regulates HIV 1 LTR transcription within a CD28 and NFB dependent manner To uncover the molecular basis of ADAP involvement in HIV 1 infection, we firstly examined its prospective ef fects on the induction of HIV 1 LTR transcription. Wild type, SLP 76 deficient Jurkat T cells or ADAP deficient Jurkat T cells were transfected using a pLTR gag3 flag luc reporter plasmid followed by stimulation via anti CD3 CD28 ligation for 6 hours. The pLTR gag3 flag luc plasmid includes the HIV 1 five LTR promoter area with two NFB binding websites and 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 type Jurkat cells, an impact that was not noticed in J14 cells. Re expression of SLP 76 into J14 cells restored and enhanced HIV 1 tran scription