In this study, we show that HIV-1 Gag, as well as TSG101, differentially affect the kinetics of downregulation of the HIV-1 co-receptors CXCR4 and CD4. SDF-1-induced CXCR4 downregulation was sharply reduced when TSG101 function is inhibited, while PMA-induced CD4 downregulation was relatively unaffected. Depleting TSG101 using siRNA directed specifically against TSG101 has been shown to result in a reduction in the cellular levels of the other two components of the ESCRT-I complex, Vps28 and hVps37 [11
]. Consequently, TSG101-depleted cells have fewer functional ESCRT-I complexes. Thus, our observations imply that CXCR4, but not CD4, is dependent on the ESCRT-I complex for its lysosomal degradation.
Divergent receptors, such as the Epidermal Growth Factor Receptor (EGFR) and the Toll-like receptor, TLR4, have been shown to be dependent on Hrs, ESCRT-I and Vps4 for their transport from early endosomes to late endosomes to lumenal vesicles in the MVB [3
]. However, this is not the only route into the MVB. For example, sorting and lysosomal degradation of the Delta Opioid Receptor (DOR), a GPCR, is Hrs and Vps4 dependent but does not require TSG101 (ESCRT-I) [21
]. Another recent study showed that lysosomal sorting of the GPCR, PAR-1 (Protease-activated receptor-1) does not require Hrs or TSG101 [39
]. Moreover, MVB sorting of the melanosome protein Pmel17 is completely independent of any of the members of the 'Vps' family of proteins [40
]. It is also important to note that not all ESCRT components are essential for HIV-1 budding and release. For example, depletion of ESCRT-II components or of AIP1/Alix has minimal effects on HIV-1 budding [6
]. These studies clearly indicate that protein sorting and MVB biogenesis are complex processes involving multiple points of entry and regulation.
Besides modulating the levels of receptors at the cell surface, the cellular endocytic pathway plays a major role in the attenuation of ligand-induced receptor-mediated signaling [42
]. For EGFR and other Receptor Tyrosine Kinases, sequestration of the activated receptor into the internal vesicles of the MVB is required to shut down signaling. This process occurs 2–3 hours after ligand binding. Activated EGFR remains in early and late endosomes during this time and is capable of mediating continued intracellular signaling [43
]. We previously showed that expression of HIV-1 Gag increases the amount of EGFR that remains in endosomes after EGF addition [14
]. As a consequence, Gag expressing cells exhibit hyperactivated and prolonged MAP kinase signaling. Increased MAP kinase activation is advantageous to the virus as it has been shown to enhance HIV-1 replication and infectivity [44
In contrast, GPCR-mediated signaling is attenuated within minutes after ligand binding. CXCR4, like most GPCRs, is desensitized rapidly through the action of GPCR kinases (GRKs), which phosphorylate the receptor on several C terminal Ser/Thr residues [19
]. Phosphorylation of CXCR4 promotes binding of β-arrestins, which sterically hinder the association of heterotrimeric G proteins with the receptor and thereby block signal transduction [46
]. The increase in intracellular CXCR4 induced by expression of HIV-1 Gag did not result in a change in SDF-1 mediated CXCR4 signaling, as judged by MAP kinase activation (Fig ). This finding is consistent with known GPCR biology and confirms that internalized CXCR4 in Gag expressing cells is desensitized and does not signal.
β-arrestin binding to GPCRs also serves to recruit components of the endocytic machinery including clathrin and AP-2, thereby mediating the internalization of the receptor [47
]. Following internalization, CXCR4 colocalizes with Hrs-positive endosomes [20
]. While Hrs and Vps4 have been implicated in CXCR4 downregulation [20
], no role for TSG101 or ESCRTs had been established in this process until now. Our data (Figs , ) strongly suggest that SDF-1 induced CXCR4 downregulation is TSG101 and ESCRT-I dependent. Given that HIV-1 Gag competes with Hrs for TSG101 in vitro
], and that overexpression of TSG101-binding regions of Hrs inhibits HIV-1 release [49
], we hypothesized that expression of Gag would compete for TSG101 binding and function in vivo
. Our observations that TSG101/ESCRT-I dependent downregulation of CXCR4 (Figs , ) and EGFR [14
] are attenuated in HIV-1 Gag expressing cells indicate that Gag functionally depletes the ESCRT complexes, thereby interrupting other ESCRT-dependent pathways in the cell.
Two other HIV-1 proteins, Nef and Env, have been shown to interact with or regulate CXCR4. Thus, an important question is whether levels of CXCR4 are altered in the context of an HIV-1 infected cell. Multiple studies have addressed this issue, primarily by quantitating the amount of cell surface CXCR4. A recent study reported that HIV-1 Nef induces downregulation of CXCR4 from the cell surface of infected cells [50
]. The authors propose that Nef-mediated CXCR4 downregulation may protect against superinfection. Superinfection is detrimental to viral replication because the accumulation of unintegrated viral DNA results in the induction of cytopathic effects in the host cell [51
]. However, multiple other studies have shown that HIV-1 Nef does not downregulate cell surface levels of CXCR4 [52
] and that maximal protection from superinfection involves an unidentified mechanism that is independent of CXCR4 downregulation [54
]. Similarly, we observed no change in cell surface levels of CXCR4 in HIV-1 Gag-expressing cells (Fig. ). In contrast, others have, in some cases, seen an upregulation in cell surface expression of CXCR4 in HIV-1 infected CD4+ T cells [55
]. SDF-1 induced CXCR4 signaling could potentially be beneficial to viral replication since it results in the activation of transcription factors such as NFκB [16
], which are known to increase HIV-1 LTR promoter activity [44
]. It is also important to note that HIV-1 Env protein (shed from virus particles or infected cells) can bind to CXCR4 and thereby trigger apoptotic signals. However, CD4 and CXCR4 expression are both required for apoptotic signaling by Env in CD4 T cells [56
]. Since CD4 is efficiently removed from the surface of productively infected cells (see below), only uninfected/bystander CD4 T cells express both CD4 and CXCR4 and are therefore susceptible to Env-induced apoptosis [56
Thus, CXCR4 downregulation might not be essential for HIV-1 replication. We speculate that during the late stages of the viral life cycle when mostly structural proteins such as Gag are expressed, SDF-1 induced CXCR4 downregulation is attenuated resulting in the accumulation of densensitized CXCR4 within intracellular compartments. These receptors could, in the long run, contribute to maintaining or replenishing the cell surface levels of CXCR4 in HIV-1 infected cells.
Unlike SDF-induced CXCR4 downregulation, Gag expression had little to no effect on PMA-induced CD4 downregulation (Fig. ). PMA is a phorbol ester that binds to and activates protein kinase C (PKC) [57
]. PKC is normally activated upon binding of antigen to the T-cell receptor and its associated CD4 [58
]. Activated PKC phosphorylates CD4 on its cytoplasmic tail and induces CD4 internalization and lysosomal degradation [25
]. Several studies have shown that PMA treatment mimics the mechanism of antigen induced CD4 downregulation [23
]. Surprisingly, little is known about how internalized CD4 gets sorted into the internal vesicles of the MVB prior to lysosomal degradation. In the present study, we show that PMA-induced CD4 downregulation can occur efficiently in the absence of functional ESCRT-I and Vps4 (Fig. ) and that expression of HIV-1 Gag has no effect on this process (Fig. ). These findings indicate that Gag affects only ESCRT-dependent processes. We therefore predict that lysosomal degradation of CD4 should not be impeded by Gag in an HIV-1 infected cell. Indeed, loss of cell surface CD4 is a hallmark of HIV-1 infection [51
]. After virus entry, it is essential that HIV-1 efficiently downregulates CD4 for multiple reasons. CD4 downregulation is important to prevent superinfection of the infected cell [61
]. In addition, cross-linking of CD4 in the absence of T cell receptor activation results in the generation of non-proliferative or apoptotic signals [62
]. Viral transcription is also inhibited under these conditions [63
]. Many studies have also reported that cells overexpressing CD4 exhibit a drastic inhibition of virion release [64
]. Moreover, the presence of CD4 at the cell surface appears to significantly reduce the infectivity of released virions [66
]. Exactly how CD4 exerts these effects is unclear, but these observations establish the critical need for HIV-1 to downregulate CD4 in infected cells. Three different viral proteins, Nef, Env and Vpu have evolved to ensure that cell surface CD4 is downregulated soon after entry (by the early protein Nef) and that transport of newly synthesized CD4 to the cell surface at late stages of infection is blocked (by late proteins Env and Vpu) [51
]. Thus, by the time Gag proteins are expressed in an infected cell, most of the surface CD4 has already been downregulated by Nef.