HIV-1 Nef compensates for disorganization of the immunological synapse by inducing trans-Golgi network–associated Lck signaling

Abstract

IntroductionReplication of HIV-1 in primary human T lymphocytes is tightly coupled to their activation state. Whereas HIV-1 undergoes early replication events in quiescent CD4 ϩ T lymphocytes, subsequent steps in the viral life cycle require cell activation. 1 T lymphocyte activation is primarily governed by signaling through the TCR complex after engagement in a tight contact with APCs; this is referred to as the immunological synapse (IS).TCR engagement by specific MHC-presented peptides launches highly dynamic and coordinated transport events that recruit specific factors to the IS and exclude others from it. This signal initiation triggers a broad cascade of downstream signaling that include dynamic F-actin remodeling at the IS, tyrosine phosphorylation, release of calcium flux, and activation of transcription. These events increase production of the T-cell survival cytokine IL-2 and are coordinated by the TCR proximal tyrosine kinase Lck, a master switch of TCR signaling. Immediately after TCR engagement, active Lck is recruited to the IS. 2-4 Whereas signal diversification and enhancement occur at the plasma membrane (PM), subsequent TCR signaling is compartmentalized and also occurs at intracellular membranes. An important intracellular arm of the TCR response is regulated by the N-Ras GTPase that is activated at Golgi membranes downstream of Lck. [5][6][7][8][9] T-cell activation is thought to be beneficial to HIV-1 because it allows transcriptional activation of latent provirus and progression of the life cycle. However, activation-induced cell death after TCR engagement runs the risk of limiting the lifespan of productively infected cells and thus the amount of viral progeny produced.Consequently, HIV-1 encodes gene products such as Nef to fine-tune the activation states of infected T lymphocytes. 10,11 Nef is a 25-to 34-kDa myristoylated accessory protein encoded by HIV-1, HIV-2, and SIV. Ex vivo, Nef enhances the single-round infectivity of virus particles and moderately accelerates virus spread over multiple rounds. 12 In vivo, Nef strongly boosts virus replication, particularly during primary infection, when the presence of Nef can elevate virus titers by more than 2 logs, and is critical for rapid disease progression. [13][14][15] This role of Nef as a pathogenicity factor is also revealed in transgenic mice, in which Nef expression induces AIDS-like depletion of CD4 ϩ T lymphocytes. 16 Delineating the mechanisms of Nef action has been hampered by the multitude of interactions with host T-cell proteins suggested to modulate various intracellular transport and signaling pathways. 17,18 This includes modulating exposure of cell-surface receptors such as MHC-I and II, CD4, and chemokine receptors to evade immune recognition and to prevent superinfection of infected cells, respectively (reviewed in Laguette et al 12 ). In addition, Nef affects the basal states of T-cell activation and the responsiveness of T lymphocytes to TCR signaling. [19][20][21][22] Initial studies with overexpression strategies ...

HIV-1 Nef Limits Communication between Linker of Activated T Cells and SLP-76 To Reduce Formation of SLP-76–Signaling Microclusters following TCR Stimulation

“…The expression plasmid for Nef F195I.myc was generated by subcloning the nef orf from an expression plasmid for a Nef F195I.GFP fusion protein (38). Expression plasmids for TCR z.GFP or Unc119.GFP and Unc119.RFP were gifts of M. Davis (Stanford University) or described previously (40), respectively. The proviral constructs used are based on HIV-1NL4-3 that lack Nef expression (HIV ΔNef) or encode Nef from HIV-1SF2 (HIV-wild type [WT]) or its F195I mutant (HIV Nef F195I), as described (41).…”

“…Human PBMC from healthy donors were obtained from the Heidelberg University Hospital Blood Bank and purified by Ficoll gradient centrifugation, as described (40). HEK 293T and Jurkat T cell lines or primary T lymphocytes were cultivated in DMEM and RPMI 1640 medium plus GlutaMAX-I, respectively, supplemented with 10% FCS, 1% L-glutamine, and 1% penicillin-streptomycin (all from Invitrogen).…”

“…The plasmid encoding the Nef protein of HIV-1 SF2 with a C-terminal Myc epitope tag and the corresponding control plasmid encoding just the myc epitope tag were described previously (40). The expression plasmid for Nef F195I.myc was generated by subcloning the nef orf from an expression plasmid for a Nef F195I.GFP fusion protein (38).…”

“…For single-cell intensity quantification of LAT accumulation and subcellular localization, wide-field z-stacks were taken under identical conditions and deconvolved and background subtracted using Huygens software (Scientific Volume Imaging, Hilversum, The Netherlands). Sum intensity projections were made and sum pixel intensities were determined using Fiji, as recently described for Lck (40). Signal intensities in areas of interest were plotted relative to the total per cell signal.…”

“…These morphological alterations at the IS were accompanied by interference with early TCR signaling such as induction of tyrosine phosphorylation (36,37,39). We recently reported that Nef achieves enhanced T cell activation responses despite this disruption of early signaling events by the assembly of active signaling complexes at the trans Golgi network (TGN) (40). This mechanism largely uncouples TCR signaling from the plasma membrane and appears to tailor T cell activation outputs in a manner that particularly favors HIV-1 replication.…”