B. Natural History of Untreated Disease

Following initial infection by HIV, there is evidence that a partially effective immune response reduces viral levels to a viral setpoint, the magnitude of which has prognostic significance with respect to how rapidly disease progression occurs (Mellors et al., 1996). HIV preferentially infects and destroys activated CD4⁺ T lymphocytes, including those that are HIV-specific, which eventually leads to a defective anti-HIV immune response (Douek et al., 2002). Once the total CD4⁺ T cell count reaches <200 cells per microliter of blood, the clinical definition of AIDS, the immune system is functionally impaired and HIV infected individuals become susceptible to opportunistic infections.

A. Disruption of Antigen Presentation to Cytotoxic T Lymphocytes

CD8⁺ cytotoxic T lymphocytes (CTLs) are important for the control of chronic viral infections. CTLs bear receptors capable of distinguishing “self” from “non-self” peptide antigens presented by MHC-I on the cell surface (Figure 2). Normal cellular peptides typically do not activate a CTL response. However, in a virally infected cell, MHC-I molecules also present peptides derived from viral proteins (“non-self” peptides). Once the T cell receptor (TCR) on CD8⁺ CTLs recognize a “non-self” signal presented by MHC-I, the CTL releases perforins and granzymes which kill the virally infected cell preventing further spread of the virus (reviewed in (Berke, 1995)).

Open in a separate window

Figure 2

Antigen presentation by class-I major histocompatibility complexes (MHC-I).

Intracellular peptides (antigens) are produced through protein synthesis and subsequent breakdown by proteasomes. The peptides are then transported into the endoplasmic reticulum through the transporter associated with antigen processing (TAP) and loaded onto MHC-I molecules. Complete MHC-I molecules are transported through the Golgi Network out to the plasma membrane (PM) where the antigen is recognized as “self” or “non-self” by the T Cell Receptor (TCR) on a CD8⁺ CTL. Cells expressing MHC-I in complex with “non-self” peptides are lysed to minimize spread of infection.

There is a great deal of evidence that CTLs play an important role in the control of HIV infection (for review see (Collins, 2004)) and recent evidence indicates that individuals mounting a Gag-specific CTL response have improved parameters with regard to controlling disease (Geldmacher et al., 2007; Kiepiela et al., 2007). Despite the efficacy with which CTLs control viral load early in infection, anti-HIV CTLs ultimately fail to prevent progression of disease. There is evidence that antigenic variation, viral effects on CTL differentiation, viability, proliferative capacity and function influence the ability of CTLs to control HIV infection. However, this review will focus on the effect of the HIV Nef protein on antigen presentation by the infected cell.

Studies performed in vitro have detected different degrees of susceptibility of HIV-infected T cells to anti-HIV CTL killing (Collins et al., 1998; Shankar, Xu and Lieberman, 1999; Yang et al., 1996; Yang et al., 1997). These differences are probably due to the use of viral strains that have variable expression of the HIV Nef protein. Under conditions where killing of HIV-infected cells was directly compared plus or minus Nef expression, it was clear that Nef protected infected cells from CTL-mediated lysis (Collins et al., 1998; Lewinsohn et al., 2002; Tomiyama et al., 2002; Yang et al., 2002). Nef has been shown to protect HIV-infected primary T cells from CTL lysis using flow cytometric killing assays (Collins et al., 1998; Lewinsohn et al., 2002), CTL co-culture assays (Yang et al., 2002) and chromium release assays (Tomiyama et al., 2002). Although Nef limits the ability of CTLs to recognize and kill infected cells, it does not appear to abrogate the capacity of CTLs to produce inhibitory cytokines in response to infected cells (Tomiyama et al., 2002). Recent in vivo evidence supports the hypothesis that CTLs may control HIV infection in vivo primarily by the elaboration of inhibitory cytokines, but fail to eradicate the infection because the CTLs cannot efficiently lyse the infected cell source of new virions (Wong et al., 2010).

B. In vivo Evidence for Nef-induced MHC-I Downmodulation

Based on in vivo studies, it is known that an intact nef gene is necessary for the timely development of AIDS in most humans and monkeys (Deacon et al., 1995; Kestler et al., 1991; Kirchoff et al., 1995). However, Nef has multiple functions, therefore these studies do not prove an important role for Nef-mediated MHC-I downmodulation in vivo. Several studies have used SIV systems to demonstrate that the capacity to downmodulate MHC-I is selected for in vivo (Carl et al., 2001; Munch et al., 2001; Swigut et al., 2004). In addition, it was recently demonstrated that the ability of in vivo-derived Nef to down-regulate MHC-I predicted the resistance of HIV-1 to suppression by CTL in vitro (Lewis et al., 2008). Taken together, these data demonstrate that the ability of Nef to down-regulate MHC-I in vivo is maintained by the need of HIV-1 to cope with the antiviral CTL response.

C. Natural killer cells

To counteract the effects of viral pathogens on MHC-I expression, natural killer (NK) cells monitor the overall surface levels of MHC-I. Low expression of MHC-I can activate NK cells to lyse target cells. There are three classical MHC-I genes expressed by all nucleated cells; HLA-A, HLA-B and HLA-C. These genes are highly polymorphic and hundreds of alleles of each have been identified. HLA-A and HLA-B are the primary allotypes that present antigens to CTLs, whereas HLA-C may function primarily to regulate NK cell function. In addition, a non-classical MHC-I called HLA-E, which does not commonly present antigens to CTLs also inhibits NK cell function (reviewed in (Natarajan et al., 2002)).

Nef has been shown to directly interact with an amino acid sequence (Y₃₂₀SQAASS₃₂₆) present in the cytoplasmic tail of HLA-A and HLA-B molecules (Williams et al., 2002). This region of the MHC-I cytoplasmic tail is also necessary for Nef-dependent downmodulation of MHC-I molecules (Cohen et al., 1999; Le Gall et al., 1998; Williams et al., 2002). In contrast, HLA-C and HLA-E have amino acid variations within this domain (Cohen et al., 1999; Le Gall et al., 1998; Williams et al., 2002) and thus remain unaffected by Nef. Therefore it has been proposed that Nef selectively downmodulates a subset of MHC-I molecules to evade CTL killing without activating NK cell lysis. However, recent evidence demonstrating that HLA-C is expressed at very low levels on primary T cells suggests that additional mechanisms may be necessary to fully explain HIV evasion of NK cells (Schaefer, 2007).

B. β-COP

COP-I and COP-II coatomers are needed for normal protein trafficking within the Golgi and ER (Rothman, 1994; Schmid, 1997). More recently, COP-I coatomers have been found associated with low pH endosomes in an ARF-1-dependent manner (Aniento et al., 1996; Gu and Gruenberg, 2000). These COP-I coatomers are implicated in recycling endosome function (Daro et al., 1997) and in transport from the endolysosomal network into multivesicular bodies (Gu et al., 1997).

The COP-I subunit β-COP was first identified as a binding partner for Nef in a yeast two-hybrid screen (Benichou et al., 1994). A diacidic motif (EE_{155, 156}) in the C-terminal loop domain of Nef mediates this interaction and targets internalized CD4 to degradative compartments. (Faure et al., 2004; Piguet et al., 1999; Schaefer et al., 2008). The interaction between Nef and βCOP requires ARF-1 but interestingly ARF-1 does not need to be in the activated, GTP bound state (Faure et al., 2004). β-COP binding to a separate region in the N-terminal alpha helical domain of Nef has also been implicated in targeting MHC-I for degradation (Schaefer et al., 2008).

C. PACS proteins

There is evidence that Nef interacts with phosphofurin acidic cluster sorting proteins (PACS-1 and PACS-2) through its acidic cluster E_62–65 (Piguet et al., 2000). PACS 1 and 2 were originally discovered by studying proteins that bind to the phosphorylated cytoplasmic tail of furin (Wan et al., 1998). Models for PACS-1 and PACS-2 function propose that these proteins help recruit AP-1 or AP-3 to cargo with acidic clusters (Crump et al., 2001). While it has yet to be found in coated vesicles (cited as data not shown in (Youker et al., 2009)), PACS has been shown to recruit AP-1 to a protein important for vesicle-membrane fusion, the SNARE vesicle-associated membrane protein (VAMP)-4 (Hinners et al., 2003). Antisense to hPACS-1a increases steady state MHC-I surface expression in Nef-expressing cells by about 20% and redistributes the intracellular localization of MHC-I and a CD4-Nef fusion protein in A7 astrocytic cells. Based on these data a model was proposed in which Nef physically recruits MHC-I and links it to a PACS-1 based TGN retrieval pathway (Piguet et al., 2000). This model was later modified to indicate that PACS-2 was more important for localizing Nef to the TGN and that PACS-1 played a greater role in recycling in mouse embryonic fibroblasts and HeLa cells (Atkins et al., 2008). Another group confirmed that knockdown of PACS-1 inhibited Nef-induced MHC-I downmodulation in HeLa cells but not Jurkat T cells (Yi et al., 2010). Arguing against an important role for PACS proteins is data from other investigators who reported no effect of knocking down PACS-1 on Nef-induced downregulation of MHC-I HLA-A2 or on the localization of other proteins containing acidic cluster motifs in HeLa cells (Lubben et al., 2007). Additionally, Baugh et al was unable to demonstrate a significant interaction between the acidic cluster in Nef and the PACS-1 furin binding region (Baugh, Garcia and Foster, 2008). Moreover, mutating three of the four glutamates in the acidic cluster only decreased Nef’s effects on MHC-I by about 50% (Baugh, Garcia and Foster, 2008).

D. ADP-ribosylation factors

In addition to the clathrin-associated adaptor proteins, the small GTPases, ADP-ribosylation factors (ARFs), are important for cellular control of assembly and disassembly of various intracellular trafficking complexes (Balch, Kahn and Schwaninger, 1992; Kahn et al., 1992). ARFs are important for clathrin dependent (Donaldson et al., 1992; Orcl et al., 1993; Palmer et al., 1993; Robinson and Kreis, 1992) and clathrin independent (Taylor, Kahn and Melancon, 1992; Waters, Serafini and Rothman, 1991) trafficking pathways. ARF activation and recruitment to cellular membranes is cyclical and regulated by its GTP binding state. Guanine nucleotide exchange factors (GEFs) are required for the recruitment of GTP to ARF and are necessary for the maintenance of overall Golgi structure ((Helms and Rothman, 1992; Jackson and Casanova, 2000) and reviewed in (Donaldson and Klausner, 1994)). GTPase-activation proteins (GAPs) promote GTP hydrolysis, thus inactivating ARFs (Turner, West and Brown, 2001). ARF-6 localizes to the plasma membrane and is involved in clathrin-independent endocytosis and recycling (Radhakrishna and Donaldson, 1997).

B. AP-1 is a host factor that is required for disruption of antigen presentation by HIV Nef

Because AP-1 was a clathrin adaptor protein that acted at the TGN and because Nef had been reported to interact with AP-1, it was hypothesized that Nef might disrupt post-TGN transport of MHC-I by promoting an interaction between MHC-I and AP-1. Indeed, RNAi directed against AP-1 μ1 inhibited downmodulation of endogenous HLA-A2 in U373mg astrocytoma cells and exogenous HLA-A2 expressed in CEM-SS T cells (Roeth et al., 2004). Recently, siRNA directed against AP-1 μ1 also abolished Nef-induced downmodulation of MHC-I in HeLa and Jurkat cell lines, as well as in primary T Lymphocytes (Yi et al., 2010).

In addition, AP-1 co-precipitated with Nef and endogenous HLA-A2 from lysates made from HIV infected primary T cells (Roeth et al., 2004). In contrast, complexes of Nef-MHC-I and AP-1 were not detected in HeLa cells unless the cells were incubated at room temperature overnight. Further experiments revealed that temperature reduction decreased the rate of MHC-I trafficking sufficiently to allow the Nef-MHC-I-AP-1 complex to form. For unclear reasons, T cells naturally traffic MHC-I at slower rates and lower incubation temperatures do not change the ability of Nef to form this complex (Kasper et al., 2005). These data help explain why investigators that focused on non-T cell lines did not detect this pathway.

C. Nef stabilizes an interaction between the AP-1 tyrosine binding pocket and an extended domain on the MHC-I cytoplasmic tail that includes a tyrosine residue

Yeast two-hybrid interaction assays and microscopic analyses provided evidence that interactions between Nef and the adaptor proteins AP-1 and AP-3 depend on Nef’s dileucine motif (Bresnahan et al., 1998; Craig, Pandori and Guatelli, 1998; Craig et al., 2000; Erdtmann et al., 2000; Greenberg et al., 1998; Janvie ret al., 2003a; Janvier et al., 2003b; Piguet et al., 1998). In contrast, MHC-I downmodulation and AP-1 recruitment in T cell systems did not require these amino acids (Greenberg, Iafrate and Skowronski, 1998; Roeth et al., 2004; Williams et al., 2005). Thus, the complex between Nef-MHC-I and AP-1 most likely occurred independently of the dileucine motif and involved a separate AP-1 binding domain. Indeed, it was demonstrated that the MHC-I cytoplasmic tail mediated a key interaction between the Nef-MHC-I complex and AP-1 (Roeth et al., 2004). Remarkably, the tyrosine in the MHC-I cytoplasmic tail does not form a canonical Yxx AP-1 sorting signal and does not bind AP-1 in T cells in the absence of Nef. However, Nef binding to the cytoplasmic tail provides the necessary elements for this non-canonical tyrosine signal to function as a potent AP-1 binding motif (Roeth et al., 2004).

Additional mutational analysis of the MHC-I cytoplasmic tail revealed two other amino acids (A₃₂₄ and D₃₂₇) that were needed for co-precipitation of AP-1 but not Nef. Interestingly, all three MHC-I cytoplasmic tail amino acids necessary for formation of the Nef-MHC-I-AP-1 complex are only found in HLA-A and HLA-B allotypes but not in HLA-C or HLA-E. The fact that these three amino acids (Y₃₂₀, A₃₂₄, and D₃₂₇) are important for co-precipitation of AP-1 suggests that this binding site may have a normal and as yet unidentified function in uninfected cells. Consistent with the fact that this site can be utilized by AP-1 in the absence of Nef, mutation of the cytoplasmic tail to create a somewhat more hydrophobic signal (Y₃₂₀SQV₃₂₃) allowed for AP-1 recruitment in the absence of Nef and significantly enhanced Nef’s ability to downmodulate HLA-A2 and recruit AP-1 (Wonderlich, Williams and Collins, 2008). Providing further support for the model that Nef stabilized an interaction between the AP-1 tyrosine-binding pocket (TBP) and the tyrosine residue in the MHC-I cytoplasmic tail, it was shown that a dominant negative mutant of AP-1 μ1 that had two amino acid substitutions in the tyrosine binding pocket (TBPM) dramatically and specifically inhibited Nef-mediated MHC-I downmodulation (Wonderlich, Williams and Collins, 2008).

D. Nef domains and AP-1-dependent MHC-I trafficking

All of the domains of Nef that are required for MHC-I downmodulation are also required for Nef to interact with the MHC-I cytoplasmic tail (Williams et al., 2005). To determine whether some of these domains might also be important for recruitment of AP-1, a fusion protein between MHC-I and Nef was examined (Roeth et al., 2004). These studies confirmed that the MHC-I cytoplasmic tail tyrosine was required for AP-1 recruitment and that Nef’s dileucine motif was dispensable for this interaction (Roeth et al., 2004; Wonderlich, Williams and Collins, 2008). In this system, the acidic cluster (E_62–65) and polyproline helix (P_72/75/78) of Nef were dispensable for AP-1 recruitment as long as a chemical crosslinker was used (Roeth et al., 2004). However, when a digitonin detergent based buffer that lacked crosslinker was substituted, a requirement for these domains to stabilize the interaction between AP-1 and MHC-I was noted (Wonderlich, Williams and Collins, 2008). In addition, the N-terminal α-helix and specifically M₂₀, were required for AP-1 recruitment under all conditions tested (Roeth et al., 2004; Wonderlich, Williams and Collins, 2008). Therefore, at least three Nef domains are required for AP-1 recruitment and subsequent downmodulation of MHC-I in Nef expressing cells (Roeth et al., 2004; Wonderlich, Williams and Collins, 2008).

E. Binding studies with purified proteins

Experiments using purified Nef-MHC-I cytoplasmic tail fusion proteins and either whole AP-1 complexes from crude lysates or purified μ1 subunit support the conclusion that Nef stabilizes an interaction between the MHC-I cytoplasmic tail and the AP-1 μ-1 subunit. Moreover, these experiments using purified protein provide evidence that the polyproline helix and the acidic domain within Nef are needed for Nef to stabilize the interaction between the AP-1 μ-1 subunit and the MHC-I cytoplasmic tail domain. In the pure protein system formation of a complex between the Nef-MHC-I cytoplasmic tail fusion protein and the AP-1 μ1 subunit also required an intact tyrosine binding pocket in the AP-1 μ1 subunit. However, no role for Nef M₂₀ was identified and thus this amino acid, which is required for Nef-induced MHC-I downmodulation may not be directly involved in protein-protein interactions but may serve another role in intact cells (Singh et al., 2009).

This work was funded by NIH AI046998 and NIH AI051192. E.W. was supported by the Cellular and Molecular Biology training grant from NIH to the University of Michigan and a University of Michigan Rackham Predoctoral Fellowship. J. L. was supported by the Cellular and Molecular Biology training grant and the Molecular Mechanisms in Microbial Pathogenesis training grant from NIH to the University of Michigan