Although ADAP acts as an important mediator of T-cell signaling and function [29–32, 34, 37, 41], its role in HIV-1 infection of T-cells had yet to be explored. In this study, we showed that ADAP was a potent regulator of two central events needed for HIV-1 infection, namely, the HIV-1 LTR transcription and viral transfer at the synapses of T-T or DC-T conjugates. Further, the two functions were regulated by two different co-receptors, CD28 in the case of HIV-1 transcription, and LFA-1 in the case of cell-cell transmission. Expression of M12 or the down-regulation of ADAP by siRNA effectively suppressed the propagation of HIV-1. Our findings therefore identify ADAP and the SLP-76/ADAP signaling module as new potential targets for the repression of HIV-1 infection.
Our studies have demonstrated that ADAP regulates two distinct events during HIV-1 infection of T-cells. While NF-κB drives the replication of the long terminal repeat (LTR) , the identity of the full range of upstream regulators of NF-κB-LTR is unknown. A variety of pro-inflammatory stimuli such as TNF-α and IL-1 as well as viral proteins and stress inducers are potent activators . In T-cells, protein kinase Cθ (PKCθ) and PKCα activate NF-κB following CD3/CD28 ligation [43–45]. Phorbol ester activation of PKCs can reactivate HIV-1 in cell lines and importantly, in primary quiescent T cells [46, 47]. More recently, members of the LAT signalosome including ADAP have been found to be needed for optimal NF-κB activation [41, 48]. However, given the different members of the NF-κB family that can be affected by upstream mediators, it has been unclear whether ADAP is needed for HIV-1 LTR transcription. Our findings showed a significant loss of anti-CD3/CD28 induced HIV-1 transcripts in JDAP cells, indicating that ADAP is needed for LTR activation. This in turn was reflected by a lack of detectable IκBα degradation in ADAP deficient JDAP cells. This regulatory event was linked further upstream to SLP-76, since a loss of binding to SLP-76 by the M12 mutant impaired LTR activity in Jurkat and primary human T-cells. It is important to note that overexpression of SLP-76 into JDAP cells did not rescue the defective HIV-1 LTR transcription. This observation suggests that ADAP is the downstream effector of SLP-76 to regulate HIV-1 transcription. Overexpression of SLP-76 increased HIV-1 LTR transcription in WT and SLP-76 deficient J14 Jurkat cells. This effect of SLP-76 on transcription differs from a previous study . The basis of this difference is unclear; however, different results might be caused by different methods used in these studies. Those authors examined the amount of full-length or sliced HIV transcripts by qRT-PCR after J14 or wild type cells were infected with HIV-1 IIIB virus. We used anti-CD3/CD28 to activate J14 or wild type cells and the readout was based on the HIV LTR luciferase reporter assay. The dependency of NF-κB activation on CD28 expression and its engagement in our studies might explain the differences in results. In either case, our findings are consistent with a scenario of SLP-76 upstream regulation of ADAP that in turn is the effector in the regulation of NF-κB transcription.
Further, we observed that the inhibition of Src kinase and PLCγ1 activity blocked ADAP potentiation of HIV-1 LTR transcription in response to anti-CD3/CD28 stimulation. This finding is consistent with the observation that p59fyn can bind and phosphorylate ADAP, while p56lck is potentially involved in NF-κB activation . Consistent with other reports, PLCγ1 activity is required in guanine nucleotide exchange factor Vav-1 induced activation of NF-κB . Overall, our data indicate for the first time that ADAP and SLP-76 are needed for anti-CD3/CD28-induced NF-κB binding to the HIV-1 LTR and optimal HIV-1 transcription.
Our second major observation was that ADAP regulated HIV-1 transmission between DC-T or T-T cells. Evidence has accumulated over the years showing efficient viral spread by direct cell-cell contact . In our study, while the blocking of LFA-1 had no effect on the NF-κB-driven HIV-1 LTR transcription, it nevertheless effectively impaired HIV-1 infection. This observation underscored the distinct nature of the two steps affected by ADAP. JDAP cells and human primary CD4+ T cells with reduced ADAP expression by siRNA formed markedly reduced numbers of T-DC conjugates and showed decreased HIV-1-GFP VLP localization at the VS interface. We observed that the M12 mutant also inhibited T-T conjugate formation, while the remaining conjugates showed a reduced size of the interface at VS. Both events would be expected to interfere with the optimal viral spread between cells. Finally, in agreement, the de novo HIV DNA synthesis as measured by levels of HIV pol in T-cell cultures confirmed a significant reduction in viral spread.
The identity of other signaling mediators other than src kinases and phospholipase C that cooperate with ADAP to regulate the VS formation and cell-to-cell viral spread remains to be determined. ITK and ZAP-70 are needed for viral cell-cell transmission [53, 54], whereas ADAP has additional binding sites for vasodilator-stimulated phosphoprotein (VASP), a regulator of actin branching . LFA-1 ligation can re-model actin in T-cells [31, 56, 57] and T cells require actin polymerization for HIV-1polarization at the cell-cell contact area. This in turn is needed for the proper formation of the VS between T-cells, as well as the efficient entry of HIV-1 into activated CD4+ T cells . In agreement, we observed reduced cell spreading in JDAP cells, as well as a reduced interface between HIV-1 infected T cells and non-infected M12 cells. The inside-out pathway is linked ADAP with the downstream SKAP-1, which is needed for the RapL-Rap1 complex formation and binding of this complex to the cytoplasmic tail of LFA-1 [32, 33, 35, 36, 58]. In this context, LFA-1 also determines the preferential infection of memory CD4+ T cells by HIV-1 . Together, ADAP and the SLP-76-ADAP complex represent exciting novel targets for reducing two steps of HIV-1 infection.