HLA Class I Subtype-Dependent Expansion of KIR3DS1+ and KIR3DL1+ NK Cells during Acute Human Immunodeficiency Virus Type 1 Infection

NK cells are critical in the early containment of viral infections. Epidemiological and functional studies have shown an important role of NK cells expressing specific killer immunoglobulin-like receptors (KIRs) in the control of human immunodeficiency virus type 1 (HIV-1) infection, but little is known about the mechanisms that determine the expansion of these antiviral NK cell populations during acute HIV-1 infection. Here we demonstrate that NK cells expressing the activating receptor KIR3DS1⁺ and, to a lesser extent, the inhibitory receptor KIR3DL1⁺ specifically expand in acute HIV-1 infection in the presence of HLA-B Bw480I, the putative HLA class I ligand for KIR3DL1/3DS1. These data demonstrate for the first time the HLA class I subtype-dependent expansion of specific KIR⁺ NK cells during an acute viral infection in humans.NK cells are cytotoxic effector cells that play a vital role in the innate immune response to viral infections (9, 12, 33). The critical role of NK cells in acute viral infections has been best characterized in acute murine cytomegalovirus (MCMV) infection (14, 28). While several murine lab strains are resistant to MCMV infection, others are highly susceptible. Resistance to MCMV infection was mapped to a gene encoding an activating NK cell receptor, Ly49H, which has been shown to be critical in the early recognition and control of MCMV infection via the direct recognition of a viral product (M157) expressed on infected cells (28). Remarkably, MCMV-infected mice exhibit a dramatic expansion of NK cells during acute infection, but this expansion is restricted to the specific accumulation of Ly49H⁺ NK cells (16). Data from these studies suggest that the antiviral activity of the Ly49H⁺ NK cells is linked to their ability to expand early in infection, prior to the development of adaptive antiviral immunity.While the critical role of Ly49H⁺ NK cells in MCMV infection has been well established, very little is known about the clonal composition of NK cells that expand in human viral infections, and the NK cell receptors that mediate their antiviral activity. Unlike T cells and B cells, the specificity of NK cells is not determined by a single NK cell receptor (8); rather, NK cells express an array of activating and inhibitory receptors that regulate their activity. While the expression of these receptors is stochastic, the random combinations of different receptors on the surface of a given NK cell clone determine its ability to respond to a specific target cell (26, 27). It has been suggested that individual NK cell populations expressing a specific array of receptors may respond differentially to diverse viral infections (7). This has been further supported by epidemiological studies associating the expression of individual activating or inhibitory NK cell receptors in combination with their HLA class I ligands with better or worse disease outcomes in viral infections such as hepatitis C virus (22), human immunodeficiency virus (HIV) (29, 30), human papillomavirus (11), and CMV (7). The functional basis for this protective immunity mediated by NK cells in human viral infections remains largely unknown.Similar to MCMV infection, highly functional NK cells expand rapidly in acute HIV-1 infection, prior to the induction of adaptive immune responses (2). One particular activating killer immunoglobulin-like NK cell receptor (KIR3DS1), in combination with its putative ligand, an HLA-B allele with isoleucine at position 80 (HLA-B Bw480I), has been shown to be associated with slower HIV-1 disease progression (29). We have recently shown that KIR3DS1⁺ NK cells can effectively suppress HIV-1 replication in HLA-B Bw480I⁺ target cells in vitro (1). Furthermore, a subset of inhibitory alleles from the same locus, KIR3DL1, that show high cell surface expression levels have similarly been associated with slower disease progression toward AIDS in the presence of their ligand, HLA-B Bw480I (30). These data suggest that both KIR3DS1⁺ and KIR3DL1⁺ NK cells may play a critical role in the control of natural HIV-1 infection, depending on the interaction with their ligand on infected cells (4). However, the mechanisms underlying their protective role are not understood.Given the critical role of NK cells in acute viral infections and the described expansion of NK cells overall during acute HIV-1 infection (16), we assessed clonal NK cell expansions during acute HIV-1 infection by quantitative PCR and flow cytometric analysis. Here we report an HLA class I subtype-dependent specific expansion of KIR3DS1⁺ and KIR3DL1⁺ NK cells during acute HIV-1 infection. These data demonstrate for the first time the impact of the HLA class I ligands on clonal NK cell expansions during an acute human viral infection.     

Role of Natural Killer Cells in a Cohort of Elite Suppressors: Low Frequency of the Protective KIR3DS1 Allele and Limited Inhibition of Human Immunodeficiency Virus Type 1 Replication In Vitro

Natural killer (NK) cells are associated with the innate immune response and are important in many viral infections. Recent studies indicate that NK cells can control human immunodeficiency virus type 1 (HIV-1) replication. We studied the effect of NK cells on HIV-1 replication in a subpopulation of HIV-1-infected individuals termed elite suppressors (ES) or elite controllers. These patients maintain a clinically undetectable viral load without treatment and thus provide a fascinating cohort in which to study the immunological response to HIV-1. Using an autologous system, we analyzed the effects of NK cells and CD8⁺ T cells on viral replication in CD4⁺ T lymphoblasts. Although we had postulated that NK cells of ES would be highly effective at controlling viral replication, we found that NK cells from some, but not all, ES were capable of inhibiting replication in the presence of interleukin-2, and the inhibition was less robust than that mediated by CD8⁺ T cells. Additionally, we examined whether particular alleles of the KIR receptors, specifically KIR3DS1 and KIR3DL1, or allele-ligand combinations correlated with the control of HIV-1 replication by NK cells and whether any specific KIR alleles were overrepresented in ES. Our ES cohort did not differ from the general population with respect to the frequency of individual KIR. However, of the eight ES studied, the four exhibiting the most NK cell-mediated control of viral replication also had the fewest activating KIR and were haplotype A. Thus, the strong NK cell-mediated inhibition of viral replication is not necessary for the immunological control of HIV-1 in all ES.A small subset of untreated, human immunodeficiency virus type 1 (HIV-1)-infected individuals referred to as elite suppressors (ES) control viremia to levels that are undetectable by ultrasensitive commercial assays while maintaining high CD4⁺ T-cell counts (13, 37). While defective virus has been shown to account for the control of virus in some patients, examining multiple host factors in ES with replication-competent virus (9) already has provided critical information on the immune response to HIV-1 and may yield important insights into future therapies and vaccine development.Research on ES suggests that CD8⁺ T cells play a crucial role in an effective response to HIV-1. CD8⁺ T cells from ES are capable of controlling viral replication in autologous CD4⁺ T cells significantly better than CD8⁺ T cells from progressors (36), and only the former proliferate (29) and secrete multiple cytokines (8) in response to HIV-1 antigens. Furthermore, certain class I HLA alleles, such as HLA-B*27 and HLA-B*57, which appear to be important in the cytotoxic T-lymphocyte (CTL) response, are overrepresented in ES (15, 19, 21, 30, 32). A second, less well studied cytotoxic cell also may play a role in the control of HIV-1. Natural killer (NK) cells are part of the innate immune system and are an important component of the host response to many viral infections. They act on target cells via cytokine release and cytolysis in response to the integration of signals from inhibitory and activating receptors.The striking propensity of HIV-1 to evolve rapidly in response to immunologic or pharmacologic pressure suggests that the virus has the capability to evade the NK cell response, and indeed selection for evasive measures seems to have occurred. The virus-induced downregulation of HLA-A and -B molecules on infected cells provides some protection against the CTL response; at the same time, however, HLA-C molecules are not downregulated upon infection (12). NK cell interaction with HLA-C can inhibit NK cytotoxic effects, and thus the retention of HLA-C on infected cells can provide some protection against the NK cell response. Additionally, a variety of alterations in NK cell function have been observed during HIV-1 infection. NK cells of patients with chronic HIV-1 have altered phenotypes and effector capabilities: NK cells from viremic patients have an increased expression of inhibitory receptors, and there is an expansion of the defective CD56⁻ NK cells compared to the levels in patients on highly active antiretroviral therapy or in ES (7, 27). These changes may be due to alterations in the cytokine environment during infection, which can affect the activation of the NK cells (39); they also may be due to direct interactions between HIV-1 gene products and the NK cells (20). Although the precise cause is unknown, the result is the development of defective NK cells that express an altered receptor and NK cell marker phenotype.Studies specifically examining a role for NK cells in the response to HIV-1 have yielded conflicting results. During acute HIV-1 infection, the NK cell population is activated and expands, particularly the cytotoxic CD56^dim population (2, 3). This activation declines in the chronic phase, and at least one study suggests that the drop in the viral load (VL) of patients during acute infection occurs before the CD8⁺ T-cell response is fully activated; this could be attributed to the effect of NK cells (2). At the same time, the study of exposed, uninfected individuals shows a correlation between resistance to acquiring HIV-1 infection and NK cell activation levels, cytokine release, and cytotoxicity to NK cell-sensitive cell lines (33, 38). Additionally, a recent whole-genome association study identified three single-nucleotide polymorphisms that appear to be important for the host control of HIV-1 (16). Two of these may have an impact on NK cell function, one that is associated with HLA-B*57 and a second that correlates with higher HLA-C mRNA expression. Taken together, such data suggest that NK cells are important for preventing HIV-1 infection and/or reducing the magnitude of viral replication in acute infection, thereby contributing to the ability of ES to control viremia.In this study, we provide the first characterization of NK cells in patients who naturally control HIV-1 infection. Considering that the effectiveness of CD8⁺ T cells against viral replication is well documented, we directly compared the effect of NK cells to that of CD8⁺ T cells from ES on viral replication to put the effect of NK cells in perspective. We studied the NK cell response by measuring the change in p24 production when autologous effector cell populations were coincubated with infected CD4⁺ lymphoblasts with and without the addition of interleukin-2 (IL-2). Additionally, we examined the killer immunoglobulin-like receptors (KIR) and KIR ligand genotype of ES patients to determine whether any KIR are overrepresented in ES and whether KIR-ligand combinations correlated with the HIV-1 inhibitory activity of the NK cells from specific patients. Previous studies have identified correlations between the expression of certain KIR and progression to AIDS in chronic progressors (25, 26); however, a connection between KIR, KIR ligands, and the control of HIV-1 has yet to be identified in ES. The results of these studies significantly advance the understanding of the nature of NK cells and of their potential role in reducing HIV-1 replication.     

Effect of Killer Immunoglobulin-Like Receptors in the Response to Combined Treatment in Patients with Chronic Hepatitis C Virus Infection

Killer immunoglobulin-like receptors (KIRs) are related to the activation and inhibition of NK cells and may play an important role in the innate response against infection with viruses such as hepatitis C virus (HCV). We examined whether the different combinations of KIRs with their HLA class I ligands influenced the response to combined treatment (pegylated alpha interferon and ribavirin) of patients infected by HCV. A total of 186 consecutive patients diagnosed with chronic HCV infection were analyzed. Seventy-seven patients exhibited HCV RNA levels at 6 months posttreatment and were called nonresponders (NR), while 109 cleared viral RNA and were named sustained viral responders (SVR). Patients were typed for HLA-B, HLA-Cw, KIR genes, and HCV genotype. In our study, the frequency of the KIR2DL2 allele was significantly increased in NR (P < 0.001; odds ratio [OR] = 1.95), as was the frequency of the KIR2DL2/KIR2DL2 genotype (P < 0.005; OR = 2.52). In contrast, the frequencies of the KIR2DL3 genotype (P < 0.001) and KIR2DL3/KIR2DL3 genotype (P < 0.05; OR = 0.54) were significantly increased in the SVR. Different combinations of KIR2DL2 and KIR2DL3 alleles with their ligands were analyzed. The frequency of the KIR2DL2/KIR2DL2-HLA-C1C2 genotype was significantly increased in the NR (P < 0.01; OR = 3.15). Additionally, we found a higher frequency of the KIR2DL3/KIR2DL3-HLA-C1C1 genotype in the SVR group (P < 0.05; OR = 0.33). These results were not affected by the HCV genotype. In conclusion, patients who carried the KIR2DL2/KIR2DL2-HLA-C1C2 genotype were less prone to respond to treatment. However, the KIR2DL3/KIR2DL3-HLA-C1C1 genotype clearly correlated with a satisfactory response to treatment, defined by the clearance of HCV RNA.Hepatitis C virus (HCV) infection is a common chronic disease affecting over 170 million people worldwide (48). Around 80% of these individuals evolve to chronic infection, and 10 to 20% of patients develop cirrhosis over a 20-year period. A minority (2%) progresses to hepatocellular carcinoma annually (18). Several host factors including age, body mass index (BMI), gender, fibrosis, cirrhosis, or the absence of cirrhosis and several viral factors including viral genotype and viral load can influence the response to treatment (6, 32, 42). Pegylated alpha interferon (Peg-IFN-α) plus ribavirin (combined therapy) constitute the most effective therapy for the treatment of chronic HCV infection (13). Since this treatment carries serious side effects, it is necessary to identify those patients who can clear HCV infection in order to reduce the period of this aggressive therapy.Natural killer (NK) cells are lymphocytes that play an important role in the host defense against HCV infection (14). NK cell activity is determined by the balance of different signals received and the equilibrium between inhibitory and activating receptors (3). Some receptors are specific for human leukocyte antigen (HLA) class I molecules (4). NK cells check the surface of the surrounding cells, detect the presence of their HLA class I molecules, and then discriminate between healthy, infected, or transformed cells (10). When NK cells contact target cells, the resulting interactions of their receptors produce either activating or inhibitory signals. If the expression of HLA class I molecules on the target cell is absent or reduced, the inhibitory signal is not generated (25).Killer cell immunoglobulin-like receptors (KIRs) are members of a group of regulatory molecules expressed on NK cells and a subset of T cells (30). This family of polymorphic genes is located on chromosome 19 (19q13.4), within the leukocyte receptor complex. The leukocyte receptor complex also encodes a number of genetically and functionally related genes. KIRs with long cytoplasmic tails are inhibitors, based on the presence of immunoreceptor tyrosine-based inhibition motifs in their cytoplasmic domains. KIRs with short tails interact with adaptor molecules such as DAP-12 (DNAX activation protein), which contain immunoreceptor tyrosine-based activation motifs and transmit activating signals (24). Several inhibitory KIRs have been well defined. KIR2DL1 binds the subset of HLA-Cw molecules with lysine at position 80 of the heavy chain (HLA-C2 group). KIR2DL2 and KIR2DL3 bind the subset of HLA-Cw molecules with asparagine at position 80 (HLA-C1 group) (34).Studies that have associated KIR genotypes with diseases have identified mainly viral infections and autoimmune diseases (22, 45). The importance of NK cells in the resolution of viral infections has prompted studies that correlate KIRs and their ligands with outcomes (12). Some studies identified a relationship between KIR genotypes and outcomes with several infectious agents such as human immunodeficiency virus (27, 29), cytomegalovirus (7), hepatitis B virus (28), and HCV (21, 26).Recently, a protective association of the inhibitory receptor KIR2DL3 with HLA-C^Asn80 (HLA-C1) and its effect on the course of HCV infection were described (21). The prevalence of KIR2DL3 and its ligand HLA-C1 is increased in individuals who eliminate HCV spontaneously, in contrast to those who remain chronically infected. The protective effect of KIR2DL3/HLA-C^Asn80 was observed only among individuals who carried both homozygous genes and had received a low HCV exposure dose. Recently, we found that the frequency of HLA-Bw4^I80 ligand and the activating receptor KIR3DS1 was increased healthy in HCV carriers compared to patients who had developed hepatocellular carcinoma (26).The aim of this study was to investigate the influence of KIR genes and KIR-HLA combinations on the response to combined therapy with Peg-IFN-α-2b and ribavirin in a group of patients with HCV infection.     

Human Immunodeficiency Virus Type 1 Nef Protein Targets CD4 to the Multivesicular Body Pathway

The Nef protein of human immunodeficiency virus type 1 downregulates the CD4 coreceptor from the surface of host cells by accelerating the rate of CD4 endocytosis through a clathrin/AP-2 pathway. Herein, we report that Nef has the additional function of targeting CD4 to the multivesicular body (MVB) pathway for eventual delivery to lysosomes. This targeting involves the endosomal sorting complex required for transport (ESCRT) machinery. Perturbation of this machinery does not prevent removal of CD4 from the cell surface but precludes its lysosomal degradation, indicating that accelerated endocytosis and targeting to the MVB pathway are separate functions of Nef. We also show that both CD4 and Nef are ubiquitinated on lysine residues, but this modification is dispensable for Nef-induced targeting of CD4 to the MVB pathway.Primate immunodeficiency viruses infect helper T lymphocytes and cells of the macrophage/monocyte lineage by binding of their viral envelope glycoprotein, Env, to a combination of two host cell-specific surface proteins, CD4 and either the CCR5 or CXCR4 chemokine receptors (reviewed in reference 62). Ensuing fusion of the viral envelope with the host cell plasma membrane delivers the viral genetic material into the cytoplasm. Remarkably, the most highly transcribed viral gene in the early phase of infection does not encode an enzyme or structural protein but an accessory protein named Nef. Early expression of Nef is thought to reprogram the host cell for optimal replication of the virus. Indeed, Nef has been shown to enhance virus production (19, 24, 59, 74) and to promote progression to AIDS (23, 47, 48), making it an attractive candidate for pharmacologic intervention.Nef is an N-terminally myristoylated protein with a molecular mass of 27 kDa for human immunodeficiency virus type 1 (HIV-1) and 35 kDa for HIV-2 and simian immunodeficiency virus (27, 29, 50, 65). Nef has been ascribed many functions, the best characterized of which is the downregulation of the CD4 coreceptor from the surface of infected cells (28, 35, 57). CD4 downregulation is believed to prevent superinfection (8, 52) and to preclude the cellular retention of newly synthesized Env (8, 49), thus allowing the establishment of a robust infection (30, 71).The molecular mechanism by which Nef downregulates CD4 has been extensively studied. A consensus has emerged that Nef accelerates the endocytosis of cell surface CD4 (2, 64) by linking the cytosolic tail of CD4 to the heterotetrameric (α-β2-μ2-σ2) adaptor protein-2 (AP-2) complex (17, 25, 34, 45, 67). Determinants in the CD4 tail bind to a hydrophobic pocket comprising tryptophan-57 and leucine-58 on the folded core domain of Nef (34). On the other hand, a dileucine motif (i.e., ENTSLL, residues 160 to 165) (14, 22, 32) and a diacidic motif (i.e., DD, residues 174 and 175) (3) (residues correspond to the NL4-3 clone of HIV-1) within a C-terminal, flexible loop of Nef bind to the α and σ2 subunits of AP-2 (17, 18, 25, 51). AP-2, in turn, binds to clathrin, leading to the concentration of CD4 within clathrin-coated pits (15, 33). These pits eventually bud from the plasma membrane as clathrin-coated vesicles that deliver internalized CD4 to endosomes. In essence, then, Nef acts as a connector that confers on CD4 the ability to be rapidly internalized in a manner similar to endocytic receptors (75).Unlike typical endocytic recycling receptors like the transferrin receptor or the low-density lipoprotein receptor, however, CD4 that is forcibly internalized by Nef does not return to the cell surface but is delivered to lysosomes for degradation (4, 64, 68). Thus, expression of Nef decreases both the surface and total levels of CD4. What keeps internalized CD4 from returning to the plasma membrane? We hypothesized that Nef might additionally act on endosomes to direct CD4 to lysosomes. This is precisely the fate followed by signaling receptors, transporters, and other transmembrane proteins that undergo ubiquitination-mediated internalization and targeting to the multivesicular body (MVB) pathway (40, 46). This targeting involves the endosomal sorting complex required for transport (ESCRT), including the ESCRT-0, -I, -II, and -III complexes, which function to sort ubiquitinated cargoes into intraluminal vesicles of MVBs for eventual degradation in lysosomes (40, 46). Herein, we show that Nef indeed plays a novel role in targeting internalized CD4 from endosomes to the MVB pathway in an ESCRT-dependent manner. We also show that both Nef and CD4 undergo ubiquitination on lysine residues, but, strikingly, this modification is not required for CD4 targeting to the MVB pathway.     

Nef-Induced CD4 Endocytosis in Human Immunodeficiency Virus Type 1 Host Cells: Role of p56lck Kinase

Human immunodeficiency virus type 1 (HIV-1) Nef interferes with the endocytic machinery to modulate the cell surface expression of CD4. However, the basal trafficking of CD4 is governed by different rules in the target cells of HIV-1: whereas CD4 is rapidly internalized from the cell surface in myeloid cells, CD4 is stabilized at the plasma membrane through its interaction with the p56^lck kinase in lymphoid cells. In this study, we showed that Nef was able to downregulate CD4 in both lymphoid and myeloid cell lines but that an increase in the internalization rate of CD4 could be observed only in lymphoid cells. Expression of p56^lck in nonlymphoid CD4-expressing cells restores the ability of Nef in order to increase the internalization rate of CD4. Concurrent with this observation, the expression of a p56^lck-binding-deficient mutant of CD4 in lymphoid cells abrogates the Nef-induced acceleration of CD4 internalization. We also show that the expression of Nef causes a decrease in the association of p56^lck with cell surface-expressed CD4. Regardless of the presence of p56^lck, the downregulation of CD4 by Nef was followed by CD4 degradation. Our results imply that Nef uses distinct mechanisms to downregulate the cell surface expression levels of CD4 in either lymphoid or myeloid target cells of HIV-1.Besides proteins that are essential for proper virus processing and assembly, the genomes of primate lentiviruses such as human immunodeficiency virus type 1 (HIV-1) encode auxiliary proteins that modulate viral infectivity. The 27-kDa auxiliary protein Nef is a key element in the progression of primary HIV-1 infection toward AIDS. Cases of patients infected with HIV-1 strains harboring a deletion in the nef gene or a defective nef allele have been reported. Some of these patients exhibit asymptomatic or slow progression toward the disease (6, 17, 37). In vitro, Nef facilitates viral replication and enhances the infectivity of viral particles (13, 47, 69). The mechanisms involved in the Nef-induced increase of viral infectivity remain elusive; however, it is a multifactorial process related to the ability of Nef to alter the trafficking of host cell proteins.Indeed, the most documented effect of Nef during the course of viral infection is its ability to disturb the clathrin-dependent trafficking machinery involved in the transport of transmembrane proteins through endosomal compartments. This leads to the modulation of the level of cell surface expression for some receptors, including CD4, which is the primary receptor of HIV-1 (35) and major histocompatibility complex class I (reviewed in references 22 and 27). The downregulation of CD4, which results in the impairment of the immunological synapse (72) and the downregulation of major histocompatibility complex class I molecules (reviewed in reference 16), is believed to contribute to the escape of HIV-1-infected cells from immunosurveillance. Moreover, the downregulation of CD4 helps avoid superinfection of cells, which would be deleterious to the virus (reviewed in reference 21), and has a direct impact on viral fitness by allowing better incorporation of the functional envelope in viral particles produced from CD4-expressing cells (3, 36, 53).Nef-induced cell surface downregulation of CD4 is efficient in all CD4-expressing cells and depends on the integrity of a di-Leu motif at position 164/165 of the C-terminal flexible loop of HIV-1 Nef (2, 9, 25). This di-Leu motif allows for the interaction with clathrin-associated adaptor protein (AP) complexes that participate in the clathrin-dependent vesicular transport within the endocytic pathway. The AP type 2 (AP-2) complex is localized at the plasma membrane and is essential to the assembly and function of clathrin-coated pits involved in the internalization of receptors from the cell surface (59). The interaction of Nef with AP-2 is well delineated and has been proposed to enhance the targeting of CD4 to clathrin-coated pits and its internalization (10, 12, 26, 32, 39).Helper T lymphocytes are the predominant cell type that expresses CD4; however, CD4 is also present at the surfaces of monocytes and macrophages (70), where its function is yet to be elucidated. Whereas cell surface CD4 is rapidly internalized in myeloid cells, CD4 is stabilized at the plasma membrane in lymphoid cells through its interaction with the Src family protein tyrosine kinase p56^lck. Cys residues located at positions 420/422 in the CD4 cytoplasmic tail are essential to the constitutive association with p56^lck (73). Besides its role in signal transduction, this interaction also correlates with an accumulation of CD4 in lipid rafts and enhanced exclusion of CD4 from clathrin-coated pits (50).In T cells, treatment with phorbol esters such as phorbol 12-myristate 13-acetate (PMA) provokes the phosphorylation of Ser residues found in the cytoplasmic tail of CD4. This correlates with a decreased association of p56^lck with CD4 and the internalization of the receptor (24, 32-34, 41, 45, 48, 52, 56, 61, 66-68). Nef-induced CD4 downregulation is known to be independent of Ser phosphorylation (20) and is therefore governed by mechanisms different from those involved in PMA-induced CD4 downregulation. However, the Leu-based sorting motif in the CD4 cytoplasmic tail is critical for both PMA and Nef-induced CD4 downregulation (2, 5, 24, 31, 56, 60, 68), thus indicating that despite being different, the mechanisms involved in Nef- and PMA-induced CD4 downregulation partially overlap.In the present study, we investigated whether the mechanisms used by Nef to downregulate CD4 are cell type-dependent processes. We looked at the trafficking and steady-state expression of CD4 in the main target cells of HIV-1, CD4-positive T lymphocytes, and cells of the monocyte/macrophage lineage. Our results demonstrate that the presence of p56^lck has a direct impact on the mechanisms used by Nef to downregulate CD4 from the cell surface of T lymphocytes. They also reveal that Nef uses distinct pathways to decrease levels of cell surface expression of CD4 in lymphoid or myeloid target cells of HIV-1.     

Virus Entry via the Alternative Coreceptors CCR3 and FPRL1 Differs by Human Immunodeficiency Virus Type 1 Subtype

Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding to CD4 and a chemokine receptor, most commonly CCR5. CXCR4 is a frequent alternative coreceptor (CoR) in subtype B and D HIV-1 infection, but the importance of many other alternative CoRs remains elusive. We have analyzed HIV-1 envelope (Env) proteins from 66 individuals infected with the major subtypes of HIV-1 to determine if virus entry into highly permissive NP-2 cell lines expressing most known alternative CoRs differed by HIV-1 subtype. We also performed linear regression analysis to determine if virus entry via the major CoR CCR5 correlated with use of any alternative CoR and if this correlation differed by subtype. Virus pseudotyped with subtype B Env showed robust entry via CCR3 that was highly correlated with CCR5 entry efficiency. By contrast, viruses pseudotyped with subtype A and C Env proteins were able to use the recently described alternative CoR FPRL1 more efficiently than CCR3, and use of FPRL1 was correlated with CCR5 entry. Subtype D Env was unable to use either CCR3 or FPRL1 efficiently, a unique pattern of alternative CoR use. These results suggest that each subtype of circulating HIV-1 may be subject to somewhat different selective pressures for Env-mediated entry into target cells and suggest that CCR3 may be used as a surrogate CoR by subtype B while FPRL1 may be used as a surrogate CoR by subtypes A and C. These data may provide insight into development of resistance to CCR5-targeted entry inhibitors and alternative entry pathways for each HIV-1 subtype.Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding first to CD4 and then to a coreceptor (CoR), of which C-C chemokine receptor 5 (CCR5) is the most common (6, 53). CXCR4 is an additional CoR for up to 50% of subtype B and D HIV-1 isolates at very late stages of disease (4, 7, 28, 35). Many other seven-membrane-spanning G-protein-coupled receptors (GPCRs) have been identified as alternative CoRs when expressed on various target cell lines in vitro, including CCR1 (76, 79), CCR2b (24), CCR3 (3, 5, 17, 32, 60), CCR8 (18, 34, 38), GPR1 (27, 65), GPR15/BOB (22), CXCR5 (39), CXCR6/Bonzo/STRL33/TYMSTR (9, 22, 25, 45, 46), APJ (26), CMKLR1/ChemR23 (49, 62), FPLR1 (67, 68), RDC1 (66), and D6 (55). HIV-2 and simian immunodeficiency virus SIVmac isolates more frequently show expanded use of these alternative CoRs than HIV-1 isolates (12, 30, 51, 74), and evidence that alternative CoRs other than CXCR4 mediate infection of primary target cells by HIV-1 isolates is sparse (18, 30, 53, 81). Genetic deficiency in CCR5 expression is highly protective against HIV-1 transmission (21, 36), establishing CCR5 as the primary CoR. The importance of alternative CoRs other than CXCR4 has remained elusive despite many studies (1, 30, 70, 81). Expansion of CoR use from CCR5 to include CXCR4 is frequently associated with the ability to use additional alternative CoRs for viral entry (8, 16, 20, 63, 79) in most but not all studies (29, 33, 40, 77, 78). This finding suggests that the sequence changes in HIV-1 env required for use of CXCR4 as an additional or alternative CoR (14, 15, 31, 37, 41, 57) are likely to increase the potential to use other alternative CoRs.We have used the highly permissive NP-2/CD4 human glioma cell line developed by Soda et al. (69) to classify virus entry via the alternative CoRs CCR1, CCR3, CCR8, GPR1, CXCR6, APJ, CMKLR1/ChemR23, FPRL1, and CXCR4. Full-length molecular clones of 66 env genes from most prevalent HIV-1 subtypes were used to generate infectious virus pseudotypes expressing a luciferase reporter construct (19, 57). Two types of analysis were performed: the level of virus entry mediated by each alternative CoR and linear regression of entry mediated by CCR5 versus all other alternative CoRs. We thus were able to identify patterns of alternative CoR use that were subtype specific and to determine if use of any alternative CoR was correlated or independent of CCR5-mediated entry. The results obtained have implications for the evolution of env function, and the analyses revealed important differences between subtype B Env function and all other HIV-1 subtypes.     

Transitions to and from the CD4-Bound Conformation Are Modulated by a Single-Residue Change in the Human Immunodeficiency Virus Type 1 gp120 Inner Domain

Binding to the primary receptor CD4 induces conformational changes in the human immunodeficiency virus type 1 (HIV-1) gp120 envelope glycoprotein that allow binding to the coreceptor (CCR5 or CXCR4) and ultimately trigger viral membrane-cell membrane fusion mediated by the gp41 transmembrane envelope glycoprotein. Here we report the derivation of an HIV-1 gp120 variant, H66N, that confers envelope glycoprotein resistance to temperature extremes. The H66N change decreases the spontaneous sampling of the CD4-bound conformation by the HIV-1 envelope glycoproteins, thus diminishing CD4-independent infection. The H66N change also stabilizes the HIV-1 envelope glycoprotein complex once the CD4-bound state is achieved, decreasing the probability of CD4-induced inactivation and revealing the enhancing effects of soluble CD4 binding on HIV-1 infection. In the CD4-bound conformation, the highly conserved histidine 66 is located between the receptor-binding and gp41-interactive surfaces of gp120. Thus, a single amino acid change in this strategically positioned gp120 inner domain residue influences the propensity of the HIV-1 envelope glycoproteins to negotiate conformational transitions to and from the CD4-bound state.Human immunodeficiency virus type 1 (HIV-1), the cause of AIDS (6, 29, 66), infects target cells by direct fusion of the viral and target cell membranes. The viral fusion complex is composed of gp120 and gp41 envelope glycoproteins, which are organized into trimeric spikes on the surface of the virus (10, 51, 89). Membrane fusion is initiated by direct binding of gp120 to the CD4 receptor on target cells (17, 41, 53). CD4 binding creates a second binding site on gp120 for the chemokine receptors CCR5 and CXCR4, which serve as coreceptors (3, 12, 19, 23, 25). Coreceptor binding is thought to lead to further conformational changes in the HIV-1 envelope glycoproteins that facilitate the fusion of viral and cell membranes. The formation of an energetically stable six-helix bundle by the gp41 ectodomain contributes to the membrane fusion event (9, 10, 79, 89, 90).The energy required for viral membrane-cell membrane fusion derives from the sequential transitions that the HIV-1 envelope glycoproteins undergo, from the high-energy unliganded state to the low-energy six-helix bundle. The graded transitions down this energetic slope are initially triggered by CD4 binding (17). The interaction of HIV-1 gp120 with CD4 is accompanied by an unusually large change in entropy, which is thought to indicate the introduction of order into the conformationally flexible unliganded gp120 glycoprotein (61). In the CD4-bound state, gp120 is capable of binding CCR5 with high affinity; moreover, CD4 binding alters the quaternary structure of the envelope glycoprotein complex, resulting in the exposure of gp41 ectodomain segments (27, 45, 77, 92). The stability of the intermediate state induced by CD4 binding depends upon several variables, including the virus (HIV-1 versus HIV-2/simian immunodeficiency virus [SIV]), the temperature, and the nature of the CD4 ligand (CD4 on a target cell membrane versus soluble forms of CD4 [sCD4]) (30, 73). For HIV-1 exposed to sCD4, if CCR5 binding occurs within a given period of time, progression along the entry pathway continues. If CCR5 binding is impeded or delayed, the CD4-bound envelope glycoprotein complex decays into inactive states (30). In extreme cases, the binding of sCD4 to the HIV-1 envelope glycoproteins induces the shedding of gp120 from the envelope glycoprotein trimer (31, 56, 58). Thus, sCD4 generally inhibits HIV-1 infection by triggering inactivation events, in addition to competing with CD4 anchored in the target cell membrane (63).HIV-1 isolates vary in sensitivity to sCD4, due in some cases to a low affinity of the envelope glycoprotein trimer for CD4 and in other cases to differences in propensity to undergo inactivating conformational transitions following CD4 binding (30). HIV-1 isolates that have been passaged extensively in T-cell lines (the tissue culture laboratory-adapted [TCLA] isolates) exhibit lower requirements for CD4 than primary HIV-1 isolates (16, 63, 82). TCLA viruses bind sCD4 efficiently and are generally sensitive to neutralization compared with primary HIV-1 isolates. Differences in sCD4 sensitivity between primary and TCLA HIV-1 strains have been mapped to the major variable loops (V1/V2 and V3) of the gp120 glycoprotein (34, 42, 62, 81). Sensitivity to sCD4 has been shown to be independent of envelope glycoprotein spike density or the intrinsic stability of the envelope glycoprotein complex (30, 35).In general, HIV-1 isolates are more sensitive to sCD4 neutralization than HIV-2 or SIV isolates (4, 14, 73). The relative resistance of SIV to sCD4 neutralization can in some cases be explained by a reduced affinity of the envelope glycoprotein trimer for sCD4 (57); however, at least some SIV isolates exhibit sCD4-induced activation of entry into CD4-negative, CCR5-expressing target cells that lasts for several hours after exposure to sCD4 (73). Thus, for some primate immunodeficiency virus envelope glycoproteins, activated intermediates in the CD4-bound conformation can be quite stable.The HIV-1 envelope glycoprotein elements important for receptor binding, subunit interaction, and membrane fusion are well conserved among different viral strains (71, 91). Thus, these elements represent potential targets for inhibitors of HIV-1 entry. Understanding the structure and longevity of the envelope glycoprotein intermediates along the virus entry pathway is relevant to attempts at inhibition. For example, peptides that target the heptad repeat 1 region of gp41 exhibit major differences in potency against HIV-1 strains related to efficiency of chemokine receptor binding (20, 21), which is thought to promote the conformational transition to the next step in the virus entry cascade. The determinants of the duration of exposure of targetable HIV-1 envelope glycoprotein elements during the entry process are undefined.To study envelope glycoprotein determinants of the movement among the distinct conformational states along the HIV-1 entry pathway, we attempted to generate HIV-1 variants that exhibit improved stability. Historically, labile viral elements have been stabilized by selecting virus to replicate under conditions, such as high temperature, that typically weaken protein-protein interactions (38, 39, 76, 102). Thus, we subjected HIV-1 to repeated incubations at temperatures between 42°C and 56°C, followed by expansion and analysis of the remaining replication-competent virus fraction. In this manner, we identified an envelope glycoprotein variant, H66N, in which histidine 66 in the gp120 N-terminal segment was altered to asparagine. The resistance of HIV-1 bearing the H66N envelope glycoproteins to changes in temperature has been reported elsewhere (37). Here, we examine the effect of the H66N change on the ability of the HIV-1 envelope glycoproteins to negotiate conformational transitions, either spontaneously or in the presence of sCD4. The H66N phenotype was studied in the context of both CD4-dependent and CD4-independent HIV-1 variants.     

HIV-1 gp120 Determinants Proximal to the CD4 Binding Site Shift Protective Glycans That Are Targeted by Monoclonal Antibody 2G12

HIV-1 R5 envelopes vary considerably in their capacities to exploit low CD4 levels on macrophages for infection and in their sensitivities to the CD4 binding site (CD4bs) monoclonal antibody (MAb) b12 and the glycan-specific MAb 2G12. Here, we show that nonglycan determinants flanking the CD4 binding loop, which affect exposure of the CD4bs, also modulate 2G12 neutralization. Our data indicate that such residues act via a mechanism that involves shifts in the orientation of proximal glycans, thus modulating the sensitivity of 2G12 neutralization and affecting the overall presentation and structure of the glycan shield.The trimeric envelope (Env) spikes on HIV-1 virions are comprised of gp120 and gp41 heterodimers. gp120 is coated extensively with glycans (9, 11, 15) that are believed to protect the envelope from neutralizing antibodies. The extents and locations of glycosylation are variable and evolving (15). Thus, while some glycans are conserved, others appear or disappear in a host over the course of infection. Such changes may result in exposure or protection of functional envelope sites and can result from selection by different environmental pressures in vivo, including neutralizing antibodies.We previously reported that HIV-1 R5 envelopes varied considerably in tropism and neutralization sensitivity (3, 4, 12-14). We showed that highly macrophage-tropic R5 envelopes were more frequently detected in brain than in semen, blood, and lymph node (LN) samples (12, 14). The capacity of R5 envelopes to infect macrophages correlated with their ability to exploit low levels of cell surface CD4 for infection (12, 14). Determinants within and proximal to the CD4 binding site (CD4bs) were shown to modulate macrophage infectivity (3, 4, 5, 12, 13) and presumably acted by altering the avidity of the trimer for cell surface CD4. These determinants include residues proximal to the CD4 binding loop, which is likely the first part of the CD4bs contacted by CD4 (1). We also observed that macrophage-tropic R5 envelopes were frequently more resistant to the glycan-specific monoclonal antibody (MAb) 2G12 than were non-macrophage-tropic R5 Envs (13).Here, we investigated the envelope determinants of 2G12 sensitivity by using two HIV-1 envelopes that we used previously to map macrophage tropism determinants (4), B33 from brain and LN40 from lymph node tissue of an AIDS patient with neurological complications. While B33 imparts high levels of macrophage infectivity and is resistant to 2G12, LN40 Env confers very inefficient macrophage infection and is 2G12 sensitive (12-14).     

Antibody Specificities Associated with Neutralization Breadth in Plasma from Human Immunodeficiency Virus Type 1 Subtype C-Infected Blood Donors

Defining the specificities of the anti-human immunodeficiency virus type 1 (HIV-1) envelope antibodies able to mediate broad heterologous neutralization will assist in identifying targets for an HIV-1 vaccine. We screened 70 plasmas from chronically HIV-1-infected individuals for neutralization breadth. Of these, 16 (23%) were found to neutralize 80% or more of the viruses tested. Anti-CD4 binding site (CD4bs) antibodies were found in almost all plasmas independent of their neutralization breadth, but they mainly mediated neutralization of the laboratory strain HxB2 with little effect on the primary virus, Du151. Adsorption with Du151 monomeric gp120 reduced neutralizing activity to some extent in most plasma samples when tested against the matched virus, although these antibodies did not always confer cross-neutralization. For one plasma, this activity was mapped to a site overlapping the CD4-induced (CD4i) epitope and CD4bs. Anti-membrane-proximal external region (MPER) (r = 0.69; P < 0.001) and anti-CD4i (r = 0.49; P < 0.001) antibody titers were found to be correlated with the neutralization breadth. These anti-MPER antibodies were not 4E10- or 2F5-like but spanned the 4E10 epitope. Furthermore, we found that anti-cardiolipin antibodies were correlated with the neutralization breadth (r = 0.67; P < 0.001) and anti-MPER antibodies (r = 0.6; P < 0.001). Our study suggests that more than one epitope on the envelope glycoprotein is involved in the cross-reactive neutralization elicited during natural HIV-1 infection, many of which are yet to be determined, and that polyreactive antibodies are possibly involved in this phenomenon.The generation of an antibody response capable of neutralizing a broad range of viruses remains an important goal of human immunodeficiency virus type 1 (HIV-1) vaccine development. Despite multiple efforts in the design of immunogens capable of inducing such humoral responses, little progress has been made (18, 20, 39). The sequence variability of the virus, as well as masking mechanisms exhibited by the envelope glycoprotein, has further hindered this pursuit (6, 22). It is known that while the majority of HIV-infected individuals mount a strong neutralization response against their own virus within the first 6 to 12 months of infection, breadth is observed in only a few individuals years later (5, 10, 15, 26, 33, 40, 41). However, very little is known about the specificities of the antibodies that confer this broad cross-neutralization. It is plausible that broadly cross-neutralizing (BCN) plasmas contain antibodies that target conserved regions of the envelope glycoprotein, as exemplified by a number of well-characterized broadly neutralizing monoclonal antibodies (MAbs). The b12 MAb recognizes the CD4 binding site (CD4bs), and 2G12 binds to surface glycans (7, 42, 44, 56). The 447-52D MAb recognizes the V3 loop, and 17b, E51, and 412d bind to CD4-induced (CD4i) epitopes that form part of the coreceptor binding site (13, 21, 51, 54). Finally, the MAbs 2F5, 4E10, and Z13e1 recognize distinct linear sequences in the gp41 membrane-proximal external region (MPER) (36, 57). The targets of these neutralizing MAbs provide a rational starting point for examining the complex nature of polyclonal plasma samples.Several groups have addressed the need to develop methodologies to elucidate the presence of certain neutralizing-antibody specificities (1, 8, 9, 29, 30, 43, 55). A number of these studies reported that the BCN antibodies in plasma can in some cases be adsorbed using gp120 immobilized on beads (1, 9, 29, 30, 43). Furthermore, the activities of some of these anti-gp120 neutralizing antibodies could be mapped to the CD4bs, as the D368R mutant gp120 failed to adsorb them (1, 29, 30, 43).Antibodies to CD4i epitopes are frequently found in HIV-1-infected individuals and are thought to primarily target the coreceptor binding site, which includes the bridging sheet and possibly parts of the V3 region. Decker and colleagues (8) showed that MAbs to HIV-1 CD4i epitopes can neutralize HIV-2 when pretreated with soluble CD4 (sCD4), indicating that the CD4i epitope is highly conserved among different HIV lineages. The poor accessibility of CD4i epitopes, however, has precluded this site from being a major neutralizing-antibody target (24), although a recent study suggested that some of the cross-neutralizing activity in polyclonal sera mapped to a CD4i epitope (30).Another site that has attracted considerable attention as a target for cross-neutralizing antibodies is the MPER, a linear stretch of 34 amino acids in gp41. Anti-MPER antibodies have been detected in the plasma of HIV-infected individuals by using chimeric viruses with HIV-1 MPER grafted into a simian immunodeficiency virus or an HIV-2 envelope glycoprotein (15, 55). These studies concluded that 2F5- and 4E10-like antibodies were rarely found in HIV-1-infected plasmas; however, other specificities within the MPER were recognized by around one-third of HIV-1-infected individuals (15). More recently, 4E10-like and 2F5-like antibodies (30, 43), as well as antibodies to novel epitopes within the MPER (1), have been shown to be responsible for neutralization breadth in a small number of plasma samples. The anti-MPER MAb 4E10 has been shown to react to autoantigens, leading to the suggestion that their rarity in human infection is due to the selective deletion of B cells with these specificities (17, 35). Furthermore, a recent study found an association between anti-MPER and anti-cardiolipin (CL) antibodies, although an association with neutralization was not examined (31).A recent study by Binley and coworkers used an array of methodologies to determine the antibody specificities present in subtype B and subtype C plasma samples with neutralization breadth (1). While antibodies to gp120, some of which mapped to the CD4bs, and to MPER were identified, most of the neutralizing activity in the BCN plasma could not be attributed to any of the known conserved envelope epitopes. Furthermore, it is not clear how common these specificities are among HIV-1-positive plasmas and whether they are only associated with BCN activity.In this study, we investigated a large collection of HIV-1-infected plasmas obtained from the South African National Blood Services. We aimed to determine if there is a relationship between the presence of certain antibody specificities, such as those against CD4i epitopes, MPER, or the CD4bs, and the neutralizing activities present in these plasmas. Furthermore, we evaluated the presence of various autoreactive antibodies and analyzed whether they might be associated with neutralization breadth.     

Differential Susceptibility of RAE-1 Isoforms to Mouse Cytomegalovirus

The NKG2D receptor is one of the most potent activating natural killer cell receptors involved in antiviral responses. The mouse NKG2D ligands MULT-1, RAE-1, and H60 are regulated by murine cytomegalovirus (MCMV) proteins m145, m152, and m155, respectively. In addition, the m138 protein interferes with the expression of both MULT-1 and H60. We show here that one of five RAE-1 isoforms, RAE-1δ, is resistant to downregulation by MCMV and that this escape has functional importance in vivo. Although m152 retained newly synthesized RAE-1δ and RAE-1γ in the endoplasmic reticulum, no viral regulator was able to affect the mature RAE-1δ form which remains expressed on the surfaces of infected cells. This differential susceptibility to downregulation by MCMV is not a consequence of faster maturation of RAE-1δ compared to RAE-1γ but rather an intrinsic property of the mature surface-resident protein. This difference can be attributed to the absence of a PLWY motif from RAE-1δ. Altogether, these findings provide evidence for a novel mechanism of host escape from viral immunoevasion of NKG2D-dependent control.Cytomegaloviruses (CMVs) are ubiquitous pathogens causing morbidity in immune suppressed and immunodeficient hosts (34). Since CMVs are strictly species-specific viruses, the infection of mice with murine CMV (MCMV) represents a widely used model for studying CMV infection and disease (22, 40).Natural killer (NK) cells play a crucial role in the control of many viruses and are among the first cells to sense proinflammatory cytokines, as well as the perturbations in the expression of major histocompatibility complex (MHC) class I molecules and other surface molecules induced by viral infection (13). Both human CMV (HCMV) and MCMV have evolved strategies to compromise innate immunity-mediated by NK cells (20, 49).Although proinflammatory cytokines released during the early stage of MCMV infection induce NK cell activation, this is usually not sufficient for virus control (11). Namely, most mouse strains fail to mount an effector phase of NK cell response against infected cells (42), in spite of the fact that MCMV infection causes the downmodulation of MHC I molecules (17), which should activate NK cells via a “missing-self” mechanism (28). The lack of NK cell activation by MCMV is even more puzzling considering that NK cells possess activating receptors that recognize cellular ligands induced by infection. Among these is the activating receptor NKG2D, a C-type lectinlike receptor encoded by a single gene in humans and rodents (39). Engagement of NKG2D transduces a strong activating signal to promote NK cell stimulation. NKG2D also serves as a costimulatory receptor on CD8⁺ T cells (2). Several NKG2D ligands have been described in mice: MULT-1, H60a, H60b, H60c, and RAE-1α, -1β, -1γ, -1δ, and -1ɛ isoforms (4-6, 10, 14, 32, 35, 44). What prevents the activation of NK cells via the NKG2D receptor during MCMV infection? We and others have characterized four MCMV proteins involved in the downmodulation of NKG2D ligands (15, 23, 24, 26, 29, 30). Furthermore, the deletion of any of the four MCMV inhibitors of NKG2D ligands rendered virus mutants susceptible to NK cell control in vivo. The MCMV immunoevasin of NKG2D described first was the glycoprotein gp40, encoded by the gene m152 (23). Note that m152 also compromises the CD8⁺ T-cell response by downregulation of MHC class I molecules (25, 54). Later, it was noticed that m152 also affects the expression of RAE-1 proteins (29). It is important to point out that mouse strains express different RAE-1 isoforms. Some strains, such as BALB/c, express RAE-1α, -1β, and -1γ, while others, such as C57BL/6, express RAE-1δ and -1ɛ (29). All five RAE-1 isoforms are glycosylphosphatidylinositol (GPI)-linked proteins and contain MHC class I-like α1 and α2 domains (6, 10, 14, 35).Based on our initial observation that there is NKG2D-dependent control of wild type (WT) MCMV in certain mouse strains, we postulated NKG2D ligands that resist virus mediated downmodulation. We show here that the RAE-1 proteins differ in their susceptibility to downregulation by MCMV. In contrast to RAE-1γ, representing the sensitive isoform, surface-resident RAE-1δ remains present on MCMV-infected cells. The differential downmodulation of RAE-1 isoforms during MCMV infection is caused by differences in the stability of the mature RAE-1 molecules associated with a sequence motif absent in RAE-1δ.     

Immunization with Wild-Type or CD4-Binding-Defective HIV-1 Env Trimers Reduces Viremia Equivalently following Heterologous Challenge with Simian-Human Immunodeficiency Virus

We recently reported that rhesus macaques inoculated with CD4-binding-competent and CD4-binding-defective soluble YU2-derived HIV-1 envelope glycoprotein (Env) trimers in adjuvant generate comparable levels of Env-specific binding antibodies (Abs) and T cell responses. We also showed that Abs directed against the Env coreceptor binding site (CoRbs) were elicited only in animals immunized with CD4-binding-competent trimers and not in animals immunized with CD4-binding-defective trimers, indicating that a direct interaction between Env and CD4 occurs in vivo. To investigate both the overall consequences of in vivo Env-CD4 interactions and the elicitation of CoRbs-directed Abs for protection against heterologous simian-human immunodeficiency virus (SHIV) challenge, we exposed rhesus macaques immunized with CD4-binding-competent and CD4-binding-defective trimers to the CCR5-tropic SHIV-SF162P4 challenge virus. Compared to unvaccinated controls, all vaccinated animals displayed improved control of plasma viremia, independent of the presence or absence of CoRbs-directed Abs prior to challenge. Immunization resulted in plasma responses that neutralized the heterologous SHIV challenge stock in vitro, with similar neutralizing Ab titers elicited by the CD4-binding-competent and CD4-binding-defective trimers. The neutralizing responses against both the SHIV-SF162P4 stock and a recombinant virus pseudotyped with a cloned SHIV-SF162P4-derived Env were significantly boosted by the SHIV challenge. Collectively, these results suggest that the capacity of soluble Env trimers to interact with primate CD4 in vivo and to stimulate the production of moderate titers of CoRbs-directed Abs did not influence the magnitude of the neutralizing Ab recall response after viral challenge or the subsequent control of viremia in this heterologous SHIV challenge model.The external glycoprotein gp120 and the membrane-anchored glycoprotein gp41 of human immunodeficiency virus type 1 (HIV-1), collectively referred to as the envelope glycoproteins (Env), mediate viral entry and are the sole virally encoded targets for neutralizing antibodies (NAbs). Prior to binding the primary host cell receptor, CD4, the trimeric Env spike may sample multiple conformations on the surface of the virus. Which of these potential conformations display neutralizing Ab epitopes and are recognized by broadly reactive NAbs is currently unclear. A substantial conformational change occurs when the functional Env spike interacts with CD4, leading to the exposure and the formation of the bridging sheet, a highly conserved and immunogenic structure spanning the inner and outer domains of gp120 that contributes to coreceptor interaction (6, 14, 25, 30). CD4 binding is also thought to lead to the displacement of variable region 3 (V3) from a less exposed conformation in the packed functional spike to a more protruding conformation. Exposure of V3 is necessary for viral entry, as it also contributes to Env interaction with coreceptor (21). Additional or concurrent rearrangements of the functional spike structure may occur upon CD4 binding, as suggested by cryotomography (38), However, these rearrangements are less well understood due to the absence of a high-resolution structure of the static or CD4-liganded trimeric spike.In attempts to elicit broadly reactive NAbs against HIV-1 through vaccination, a range of recombinant Env variants were designed and tested (reviewed in references 15, 26, 49, and 50). The capacity of such immunogens to elicit broadly reactive NAbs is often determined using standardized in vitro neutralization assays (34). However, the ability of HIV-1 Env vaccine-elicited B cell responses to mediate actual protective and functional responses against in vivo virus challenge is evaluated less frequently, since this requires the use of nonhuman primates (NHPs) and infection with chimeric simian-human immunodeficiency viruses (SHIVs). A series of SHIVs was developed, including those based on the HIV-1 Env glycoproteins from SF162 (40), 89.6 (54), ADA (45), BaL (48), DH12 (59), and 1157i (27). So far, few of these models, if any, fully mimic HIV-1 infection in humans. Currently, serially passaged CCR5-using SHIV-SF162 (SHIV-SF162P), which establishes transient or more prolonged viremia in macaques, represent a frequently used model to evaluate the protective effect of Env-based immunogens (2-5, 19, 20, 23, 24, 29, 53, 67). Depending on the number and nature of passages that this virus has been exposed to, the SHIV-SF162P stocks are more or less neutralization resistant (19, 62), allowing one to test the efficacy of a given vaccine candidate against a more or less rigorous form of viral challenge. Protection against mucosal SHIV-SF162P4 challenge after homologous SF162ΔV2 Env protein immunization of rhesus macaques was recently reported (2, 3). However, the nature and specificities of the vaccine-induced immune responses that mediate this effect remain incompletely defined.We recently showed that Abs against the HIV-1 gp120 coreceptor binding site (CoRbs) are elicited as a consequence of in vivo interactions between Env and primate CD4 during immunization with soluble CD4 (sCD4)-binding-competent Env trimers (14). We subsequently showed that rhesus macaques inoculated with CD4-binding competent and CD4-binding defective soluble YU2-derived gp140-F trimers in adjuvant generate comparable levels of Env-specific binding Abs and T cell responses but that CoRbs-directed Abs are elicited only in animals immunized with wild-type (wt) CD4-binding competent Env trimers (13). So far, the impact of Env-CD4 in vivo interactions during Env immunization and the role of CoRbs-directed Abs in protection against SHIV infection remain incompletely understood. A majority of the well-characterized CoRbs-directed monoclonal Abs (MAbs) lack the capacity to neutralize primary viruses in vitro (7, 31). However, it has been suggested that Abs directed against this region may contribute to the neutralizing Ab response seen in some HIV-1-infected individuals (18, 35, 58) and to the protection observed in some SHIV challenge experiments (12).The distinct difference in the capacity of the CD4-binding competent and CD4-binding defective Env trimers to elicit CoRbs-directed Abs described in our previous study presented an opportunity to evaluate the protective effect of CoRbs-directed Abs in the SHIV model. The availability of animals immunized with these Env immunogens also allowed us to ask the more general question about whether in vivo interactions between soluble Env trimers and CD4-expressing host cells would influence the outcome of heterologous SHIV-SF162P4 infection. We show here that Env trimer-immunized animals displayed improved control of SHIV-SF162P4 viremia compared to unimmunized control animals, independent of whether they were inoculated with CD4-binding-competent or CD4-binding-defective trimers. These results suggest that the capacity of soluble Env trimers to interact with CD4 in vivo and to stimulate the production of CoRbs-directed Abs did not measurably influence the protective effect of the vaccine-elicited immune responses in this SHIV challenge model.     

Caveolin-1 Modulates HIV-1 Envelope-Induced Bystander Apoptosis through gp41

Human immunodeficiency virus (HIV) envelope (Env)-mediated bystander apoptosis is known to cause the progressive, severe, and irreversible loss of CD4⁺ T cells in HIV-1-infected patients. Env-induced bystander apoptosis has been shown to be gp41 dependent and related to the membrane hemifusion between envelope-expressing cells and target cells. Caveolin-1 (Cav-1), the scaffold protein of specific membrane lipid rafts called caveolae, has been reported to interact with gp41. However, the underlying pathological or physiological meaning of this robust interaction remains unclear. In this report, we examine the interaction of cellular Cav-1 and HIV gp41 within the lipid rafts and show that Cav-1 modulates Env-induced bystander apoptosis through interactions with gp41 in SupT1 cells and CD4⁺ T lymphocytes isolated from human peripheral blood. Cav-1 significantly suppressed Env-induced membrane hemifusion and caspase-3 activation and augmented Hsp70 upregulation. Moreover, a peptide containing the Cav-1 scaffold domain sequence markedly inhibited bystander apoptosis and apoptotic signal pathways. Our studies shed new light on the potential role of Cav-1 in limiting HIV pathogenesis and the development of a novel therapeutic strategy in treating HIV-1-infected patients.HIV infection causes a progressive, severe, and irreversible depletion of CD4⁺ T cells, which is responsible for the development of AIDS (9). The mechanism through which HIV infection induces cell death involves a variety of processes (58). Among these processes, apoptosis is most likely responsible for T-cell destruction in HIV-infected patients (33), because active antiretroviral therapy has been associated with low levels of CD4⁺ T-cell apoptosis (7), and AIDS progression was shown previously to correlate with the extent of immune cell apoptosis (34). Importantly, bystander apoptosis of uninfected cells was demonstrated to be one of the major processes involved in the destruction of immune cells (58), with the majority of apoptotic CD4⁺ T cells in the peripheral blood and lymph nodes being uninfected in HIV patients (22).Binding to uninfected cells or the entry of viral proteins released by infected cells is responsible for the virus-mediated killing of innocent-bystander CD4⁺ T cells (2-4, 9, 65). The HIV envelope glycoprotein complex, consisting of gp120 and gp41 subunits expressed on an HIV-infected cell membrane (73), is believed to induce bystander CD4⁺ T-cell apoptosis (58). Although there is a soluble form of gp120 in the blood, there is no conclusive agreement as to whether the concentration is sufficient to trigger apoptosis (57, 58). The initial step in HIV infection is mediated by the Env glycoprotein gp120 binding with high affinity to CD4, the primary receptor on the target cell surface, which is followed by interactions with the chemokine receptor CCR5 or CXCR4 (61). This interaction triggers a conformational change in gp41 and the insertion of its N-terminal fusion peptide into the target membrane (30). Next, a prehairpin structure containing leucine zipper-like motifs is formed by the two conserved coiled-coil domains, called the N-terminal and C-terminal heptad repeats (28, 66, 70). This structure quickly collapses into a highly stable six-helix bundle structure with an N-terminal heptad repeat inside and a hydrophobic C-terminal heptad repeat outside (28, 66, 70). The formation of the six-helix bundle leads to a juxtaposition and fusion with the target cell membrane (28, 66, 70). The fusogenic potential of HIV Env is proven to correlate with the pathogenesis of both CXCR4- and CCR5-tropic viruses by not only delivering the viral genome to uninfected cells but also mediating Env-induced bystander apoptosis (71). Initial infection is dominated by the CCR5-tropic strains, with the CXCR4-tropic viruses emerging in the later stages of disease (20). Studies have shown that CXCR4-tropic HIV-1 triggers more depletion of CD4⁺ T cells than CCR5-tropic strains (36).Glycolipid- and cholesterol-enriched membrane microdomains, termed lipid rafts, are spatially organized plasma membranes and are known to have many diverse functions (26, 53). These functions include membrane trafficking, endocytosis, the regulation of cholesterol and calcium homeostasis, and signal transduction in cellular growth and apoptosis. Lipid rafts have also been implicated in HIV cell entry and budding processes (19, 46, 48, 51). One such organelle is the caveola, which is a small, flask-shaped (50 to 100 nm in diameter) invagination in the plasma membrane (5, 62). The caveola structure, which is composed of proteins known as caveolins, plays a role in various functions by serving as a mobile platform for many receptors and signal proteins (5, 62). Caveolin-1 (Cav-1) is a 22- to 24-kDa major coat protein responsible for caveola assembly (25, 47). This scaffolding protein forms a hairpin-like structure and exists as an oligomeric complex of 14 to 16 monomers (21). Cav-1 has been shown to be expressed by a variety of cell types, mostly endothelial cells, type I pneumocytes, fibroblasts, and adipocytes (5, 62). In addition, Cav-1 expression is evident in immune cells such as macrophages and dendritic cells (38, 39). However, Cav-1 is not expressed in isolated thymocytes (49). Furthermore, Cav-1 and caveolar structures are absent in human or murine T-cell lines (27, 41, 68). Contrary to this, there has been one report showing evidence of Cav-1 expression in bovine primary cell subpopulations of CD4⁺, CD8⁺, CD21⁺, and IgM⁺ cells with Cav-1 localized predominantly in the perinuclear region (38). That report also demonstrated a membrane region staining with Cav-1-specific antibody of human CD21⁺ and CD26⁺ peripheral blood lymphocytes (PBLs). Recently, the expression of Cav-1 in activated murine B cells, with a potential role in the development of a thymus-independent immune response, was also reported (56). It remains to be determined whether Cav-1 expression is dependent on the activation state of lymphocytes. For macrophages, however, which are one of the main cell targets for HIV infection, Cav-1 expression has been clearly documented (38).The scaffolding domain of Cav-1, located in the juxtamembranous region of the N terminus, is responsible for its oligomerization and binding to various proteins (5, 62, 64). It recognizes a consensus binding motif, ΦXΦXXXXΦ, ΦXXXXΦXXΦ, or ΦXΦXXXXΦXXΦ, where Φ indicates an aromatic residue (F, W, or Y) and X indicates any residue (5, 62, 64). A Cav-1 binding motif (WNNMTWMQW) has been identified in the HIV-1 envelope protein gp41 (42, 43). Cav-1 has been shown to associate with gp41 by many different groups under various circumstances, including the immunoprecipitation of gp41 and Cav-1 in HIV-infected cells (42, 43, 52). However, the underlying pathological or physiological functions of this robust interaction between Cav-1 and gp41 remain unclear.Here, we report that the interaction between Cav-1 and gp41 leads to a modification of gp41 function, which subsequently regulates Env-induced T-cell bystander apoptosis. Moreover, we show that a peptide containing the Cav-1 scaffold domain sequence is capable of modulating Env-induced bystander apoptosis, which suggests a novel therapeutic application for HIV-1-infected patients.     

Dermatan Sulfate Epimerase 1-Deficient Mice Have Reduced Content and Changed Distribution of Iduronic Acids in Dermatan Sulfate and an Altered Collagen Structure in Skin

Dermatan sulfate epimerase 1 (DS-epi1) and DS-epi2 convert glucuronic acid to iduronic acid in chondroitin/dermatan sulfate biosynthesis. Here we report on the generation of DS-epi1-null mice and the resulting alterations in the chondroitin/dermatan polysaccharide chains. The numbers of long blocks of adjacent iduronic acids are greatly decreased in skin decorin and biglycan chondroitin/dermatan sulfate, along with a parallel decrease in iduronic-2-O-sulfated-galactosamine-4-O-sulfated structures. Both iduronic acid blocks and iduronic acids surrounded by glucuronic acids are also decreased in versican-derived chains. DS-epi1-deficient mice are smaller than their wild-type littermates but otherwise have no gross macroscopic alterations. The lack of DS-epi1 affects the chondroitin/dermatan sulfate in many proteoglycans, and the consequences for skin collagen structure were initially analyzed. We found that the skin collagen architecture was altered, and electron microscopy showed that the DS-epi1-null fibrils have a larger diameter than the wild-type fibrils. The altered chondroitin/dermatan sulfate chains carried by decorin in skin are likely to affect collagen fibril formation and reduce the tensile strength of DS-epi1-null skin.Chondroitin sulfate (CS) is an unbranched polymer chain composed of alternating glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc) units (36, 49). In dermatan sulfate (DS), d-glucuronic acid is converted to its epimer l-iduronic acid (IdoA) (25). The extent of this modification, which varies from a few percent of the glucuronic acid being epimerized to a predominant presence of iduronic acid, depends on the variable epimerase activity in tissues and on the core protein attached to the chain in CS/DS proteoglycans (PGs) (41, 47). The same CS/DS PG has a different iduronic acid content, depending on the cell type and tissue of origin (4, 5). The name CS/DS denotes the hybrid GlcA-IdoA nature of the chain. It has long been known that the distribution of iduronic acids within the chain is not random but follows two patterns: either they are clustered together, forming long iduronic acid blocks, or they are isolated, i.e., interspersed among surrounding glucuronic acids (11). DS epimerase 1 (DS-epi1) and DS-epi2, encoded in mouse by the Dse and Dsel (Dse-like) genes, respectively, are present in organisms ranging from Xenopus tropicalis to humans but not in worms and flies (23, 34). During DS biosynthesis, epimerization is followed by the action of eight C-specific O-sulfotranferases, which transfer a sulfate group to C-2 of both IdoA and GlcA and to C-4, C-6, and C-4/C-6 of GalNAc (18). These modification reactions, individually affecting only part of the available substrate, produce structural variability in the CS/DS chain. Considerable efforts have been made to characterize specific sequences in the CS/DS chains responsible for binding to protein and the subsequent mediation of a biological effect (28). For instance, (IdoA-2OS-GalNAc-4OS)₃- and GalNAc-4/6-diOS-containing structures bind and activate heparin cofactor II, which is the major antithrombotic system in the subendothelial layer (48). IdoA/GlcA-2OS-GalNAc-6OS-containing structures bind to pleiotrophin, mediating neuritogenic activity (3, 44). IdoA-GalNAc-4OS-containing structures bind to basic fibroblast growth factor, and the complex has been shown to be active in wound healing (46).CS/DS PGs are mainly found in the extracellular matrix. They belong to four families: lecticans, e.g., versican, aggrecan, brevican, and neurocan; collagens, e.g., collagen IX; basement membrane PGs, e.g., SMC3, collagen XV, and perlecan, containing both heparan sulfate (HS) and CS/DS; and small leucine-rich repeat PGs. Some PGs of the first three groups are referred to as CS PGs. The actual presence of iduronic acid, depending on the tissue examined and on the developmental stage, has been overlooked in many cases (37, 44). The archetypical small leucine-rich repeat PG family members decorin, biglycan, fibromodulin, and lumican bind fibrillar collagens and affect collagen fibril and scaffold formation in connective tissues (15). Decorin and biglycan are substituted with one and two CS/DS chains, respectively. Decorin is involved in collagen type I fibril formation and matrix assembly in a wide range of connective tissues and binds near the C terminus of collagen monomers, delaying their accretion to the growing fibrils. We have identified an SYIRIADTNIT sequence in decorin as essential for binding to collagen (16). The role of the decorin CS/DS chain in vivo has not been explored, although in vitro studies suggest that IdoA promotes the binding of CS/DS to collagen (31) and is required for self-association of CS/DS chains (6, 10, 22).Here the function of DS-epi1 in mice was disrupted. DS-epi1-deficient mice show CS/DS with a marked deficiency in iduronic acid-containing structures. The deletion of DS-epi1 is likely to affect many types of PGs and to result in a complex phenotype. We focus on skin alterations presumably caused by altered decorin/biglycan CS/DS chains.     

Differential Activation of NK Cells by Influenza A Pseudotype H5N1 and 1918 and 2009 Pandemic H1N1 Viruses

Natural killer (NK) cells are the effectors of innate immunity and are recruited into the lung 48 h after influenza virus infection. Functional NK cell activation can be triggered by the interaction between viral hemagglutinin (HA) and natural cytotoxicity receptors NKp46 and NKp44 on the cell surface. Recently, novel subtypes of influenza viruses, such as H5N1 and 2009 pandemic H1N1, transmitted directly to the human population, with unusual mortality and morbidity rates. Here, the human NK cell responses to these viruses were studied. Differential activation of heterogeneous NK cells (upregulation of CD69 and CD107a and gamma interferon [IFN-γ] production as well as downregulation of NKp46) was observed following interactions with H5N1, 1918 H1N1, and 2009 H1N1 pseudotyped particles (pps), respectively, and the responses of the CD56^dim subset predominated. Much stronger NK activation was triggered by H5N1 and 1918 H1N1 pps than by 2009 H1N1 pps. The interaction of pps with NK cells and subsequent internalization were mediated by NKp46 partially. The NK cell activation by pps showed a dosage-dependent manner, while an increasing viral HA titer attenuated NK activation phenotypes, cytotoxicity, and IFN-γ production. The various host innate immune responses to different influenza virus subtypes or HA titers may be associated with disease severity.Influenza is a contagious, acute respiratory disease caused by influenza viruses and has caused substantial human morbidity and mortality over the past century (24, 27). The 1918-1919 pandemic caused by influenza virus type A H1N1 was responsible for an estimated 50 million deaths (21). In recent years, novel subtype influenza viruses, such as H5N1 and the 2009 pandemic H1N1, have been transmitted directly from animals to the human population. These infections were characterized by unusually high rates of severe respiratory disease and mortality among young patients (8, 18). Various genetic shifts have occurred in these viruses, allowing them to evade the host protective effects of specific antihemagglutinin (HA) or antineuraminidase (NA) antibodies (27). Therefore, host innate immunity in the early phase of infection, which includes a variety of pattern recognition molecules, inflammatory cytokines, and immune cells, such as macrophages and natural killer (NK) cells, plays a critical role in host defense.NK cells are bone marrow-derived, large, granular lymphocytes and are key effector cells in innate immunity for host defense against invading infectious pathogens and malignant transformation through cytolytic activity and production of cytokines, such as gamma interferon (IFN-γ) (10, 28, 43, 51). In humans, NK cells account for approximately 10% of all blood lymphocytes and are identified by their expression of the CD56 surface antigen and their lack of CD3. Two distinct subsets of human NK cells have been defined according to the cell surface density of CD56 expression (10). The majority (∼90% in blood) of human NK cells are CD56^dim, and a minor population (∼10% in blood) is CD56^bright. These NK subsets are functionally distinct, with the immunoregulatory CD56^bright cells producing abundant cytokines and the cytotoxic CD56^dim cells probably functioning as efficient effectors of natural and antibody-dependent target cell lysis (11).Many lines of evidence suggest that NK cells can be functionally activated by the interaction between natural cytotoxicity receptors (NCRs) on the cell surface and influenza virus HA protein or stress-induced proteins from infected cells (2, 13, 33, 44, 46). On the other hand, influenza virus is able to evade host immunity by infecting NK cells and triggering cell apoptosis or by attenuating NK cell lysis of H3N2-infected cells, owing to alterations in HA binding properties (35, 39). The infiltration of macrophages and lymphocytes into the lung and strong inflammatory responses were detected in H5N1 and the 1918 and 2009 pandemic H1N1 infections. Nevertheless, little is known about the precise roles of NK cells in these infections.In this study, the responses of NK cells to 1918 H1N1, 2009 H1N1, and H5N1 influenza A viruses were evaluated using three strains of influenza A virus pseudotyped particles (pps). Our findings may aid in understanding the pathogenicity of influenza viruses and its correlation with clinical severity.