A Basic Patch on α-Adaptin Is Required for Binding of Human Immunodeficiency Virus Type 1 Nef and Cooperative Assembly of a CD4-Nef-AP-2 Complex

A critical function of the human immunodeficiency virus type 1 Nef protein is the downregulation of CD4 from the surfaces of infected cells. Nef is believed to act by linking the cytosolic tail of CD4 to the endocytic machinery, thereby increasing the rate of CD4 internalization. In support of this model, weak binary interactions between CD4, Nef, and the endocytic adaptor complex, AP-2, have been reported. In particular, dileucine and diacidic motifs in the C-terminal flexible loop of Nef have been shown to mediate binding to a combination of the α and σ2 subunits of AP-2. Here, we report the identification of a potential binding site for the Nef diacidic motif on α-adaptin. This site comprises two basic residues, lysine-297 and arginine-340, on the α-adaptin trunk domain. The mutation of these residues specifically inhibits the ability of Nef to bind AP-2 and downregulate CD4. We also present evidence that the diacidic motif on Nef and the basic patch on α-adaptin are both required for the cooperative assembly of a CD4-Nef-AP-2 complex. This cooperativity explains how Nef is able to efficiently downregulate CD4 despite weak binary interactions between components of the tripartite complex.CD4, a type I transmembrane glycoprotein that serves as a coreceptor for major histocompatibility complex class II (MHC-II) molecules, is expressed on the surfaces of helper T lymphocytes and cells of the monocyte/macrophage lineage (8). Primate immunodeficiency viruses gain access to these cells by virtue of the interaction of the viral envelope glycoprotein (Env) with a combination of CD4 and a chemokine receptor (63). This interaction causes a conformational change within the Env protein that promotes the fusion of the viral envelope with the plasma membrane. Upon the delivery of the viral genetic material into the cytoplasm of the host cells, one of the first virally encoded proteins to be expressed is Nef, an accessory factor that modulates specific signal transduction and protein-trafficking pathways in a manner that optimizes the intracellular environment for viral replication (reviewed in references 21, 39, and 65). Perhaps the best characterized function of Nef is the downregulation of CD4 from the surfaces of the host cells (6, 22, 29, 45). CD4 downregulation prevents superinfection (6, 41) and enhances virion release (19, 38, 48, 66, 76), thereby contributing to the establishment of a robust infective state (24, 72).The mechanism used by the Nef protein of human immunodeficiency virus type 1 (HIV-1) to downregulate CD4 has been the subject of extensive study, but only recently have the molecular details of this process begun to be unraveled. It is generally acknowledged that HIV-1 Nef accelerates the internalization of CD4 from the plasma membrane by linking the cytosolic tail of the receptor to the clathrin-associated endocytic machinery (1, 12, 20, 34, 40, 64). In support of this model, a hydrophobic pocket comprising W57 and L58 on the folded core domain of Nef binds with millimolar affinity to the cytosolic tail of CD4 (28) (all residues and numbers correspond to the NL4-3 variant of HIV-1 Nef used in this study). In addition, a dileucine motif (ENTSLL, residues 160 to 165) (10, 16, 26) and a diacidic motif (D174 and D175) (2) on the C-terminal flexible loop of Nef mediate an interaction of micromolar affinity with the clathrin-associated, heterotetrameric (α-β2-μ2-σ2) adaptor protein 2 (AP-2) complex (12, 20, 40, 49). These interactions draw CD4 into clathrin-coated pits that eventually bud inwards as clathrin-coated vesicles (11, 27). Internalized CD4 is subsequently delivered to endosomes and then to lysosomes for degradation (3, 23, 59, 64).Despite progress in the understanding of the mechanism of Nef-induced CD4 downregulation, several important aspects remain to be elucidated. Previous studies have shown that the Nef dileucine and diacidic motifs interact with a combination of the α and σ2 subunits of AP-2 (referred to as the α-σ2 hemicomplex) (12, 20, 40, 49), but the precise location of the Nef binding sites is unknown. It also remains to be determined whether Nef can actually bind CD4 and AP-2 at the same time. Indeed, the formation of a tripartite CD4-Nef-AP-2 complex in which Nef links the cytosolic tail of CD4 to AP-2 has long been hypothesized but has never been demonstrated experimentally. Given the relatively weak affinity of Nef for the CD4 tail (28) and AP-2 (12, 40), it is unclear how such a complex could assemble and function in CD4 downregulation.In this study, we have addressed these issues by using a combination of yeast hybrid, in vitro binding, and in vivo CD4 downregulation assays. We report the identification of a candidate binding site for the Nef diacidic motif on the AP-2 complex. This site, a basic patch comprising K297 and R340 on α-adaptin, is specifically required for Nef binding and Nef-induced CD4 downregulation. We also show that the Nef diacidic motif and the α-adaptin basic patch are required for the cooperative assembly of a tripartite complex composed of the CD4 cytosolic tail, Nef, and the α-σ2 hemicomplex. The cooperative manner in which this complex is formed explains how Nef is able to efficiently downregulate CD4 from the plasma membrane despite weak binary interactions between the components of this complex.     

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.     

Adult AIDS-Like Disease in a Novel Inducible Human Immunodeficiency Virus Type 1 Nef Transgenic Mouse Model: CD4+ T-Cell Activation Is Nef Dependent and Can Occur in the Absence of Lymphophenia

CD4C/HIV^nef transgenic (Tg) mice express Nef in CD4⁺ T cells and in the cells of the macrophage/monocyte/dendritic lineage, and they develop an AIDS-like disease similar to human AIDS. In these mice, Nef is constitutively expressed throughout life. To rule out the contribution of any developmental defects caused by early expression of Nef, we generated inducible human immunodeficiency virus type 1 (HIV-1) Nef Tg mice by using the tetracycline-inducible system. Faithful expression of the Nef transgene was induced in (CD4C/rtTA × TRE/HIV^Nef) or (CD4C/rtTA2^S-M2 × TRE/HIV^Nef) double-Tg mice upon doxycycline (DOX) treatment in drinking water. Long-term treatment of these mice with DOX also led to loss, apoptosis, and activation of CD4⁺ T cells, this latter phenotype being observed even with low levels of Nef. These phenotypes could be transferred by bone marrow (BM) transplantation, indicating a hematopoietic cell autonomous effect. In addition, in mixed Tg:non-Tg BM chimeras, only Tg and not non-Tg CD4⁺ T cells exhibited an effector/memory phenotype in the absence of lymphopenia. Finally, the DOX-induced double-Tg mice developed nonlymphoid organ diseases similar to those of CD4C/HIV^Nef Tg mice and of humans infected with HIV-1. These results show for the first time that adult mice are susceptible to the detrimental action of Nef and that Nef-mediated T-cell activation can be independent of lymphopenia. These Tg mice represent a unique model which is likely to be instrumental for understanding the cellular and molecular pathways of Nef action as well as the main characteristics of immune reconstitution following DOX withdrawal.Small animal models able to express the entire human immunodeficiency virus (HIV) genome or selected HIV genes have provided useful information on the pathogenesis of AIDS and still represent important research tools toward this goal. Among these models, transgenic (Tg) mice containing intact copies of HIV DNA, defective provirus with the gag and pol genes deleted, or individual HIV-1 genes have been reported to develop various pathologies, some of which resemble those found in human AIDS (2, 3, 8, 9, 16, 17, 18, 24, 27, 29, 30, 38, 44, 45, 46, 49, 51, 52). The cell type context in which the HIV-1 transgene is expressed in these Tg mice appears to play an important role in determining the type of pathological lesions. Tg mice generated in our laboratory and expressing the entire coding sequence of HIV-1 (CD4C/HIV^WT) or HIV-1 Nef alone (CD4C/HIV^Nef) in the relevant target cells of HIV-1, namely, CD4⁺ T cells, macrophages, and dendritic cells, develop pathologies very similar to those in human AIDS (17, 18). The AIDS-like disease of CD4C/HIV^Nef Tg mice is characterized by immunodeficiency, loss of CD4⁺ T cells, thymic atrophy, activation of T cells and pathologies in heart, lungs, and kidneys (18, 53). Similarly, expression of simian immunodeficiency virus (SIV) Nef in Tg mice under the control of the same promoter sequences (CD4C) results in an AIDS-like disease (42). These studies demonstrated that Nef plays an important role in the development of the AIDS-like disease induced by HIV-1 or SIV in Tg mice.Among the AIDS-like phenotypes of these models, the T-cell activation observed by a number of groups in Tg mice expressing Nef (3, 33, 44, 53) may be of special interest for its resemblance to that of humans or macaques infected with HIV-1 or SIV, respectively. HIV infection results in a state of chronic immune activation which correlates very closely with disease progression in humans (11, 14, 23). Similarly, SIV-infected macaques which develop AIDS show aberrant immune activation (35), while SIV-infected sooty mangabey monkeys, natural hosts of SIV, do not develop immunopathologies and do not show immune activation either (41). Various factors may contribute to this immune activation, including increased plasma lipopolysaccharide levels due to microbial translocation from the gut (4), impaired regulatory T cell function (32), or the action of the HIV-1 gene products themselves, such as Env gp120 and Nef (10, 12, 43). Consistent with this latter scenario, we reported that in CD4C/HIV^Nef Tg mice the extent of T-cell activation correlates with levels of Nef expression in CD4⁺ T cells, thus suggesting a direct involvement of Nef in this activation (53). In contrast, Koenen and coworkers reported that T-cell activation in CD2/Nef Tg mice is induced indirectly by lymphophenia (26). In that study, chimeric mice, which were generated from a mixture of non-Tg and Nef Tg bone marrow (BM) cells, were not lymphopenic, and the donor-derived Nef-expressing Tg T cells did not show an activated phenotype. However, the donor Nef Tg T cells constituted only 1 to 2% of peripheral T cells of these chimeric mice (26). Clearly, alternative experimental approaches are needed to study this phenotype in a more physiological context.In the previously described CD4C/HIV^Nef Tg mice (18), Nef expression begins early in life and is constitutively expressed throughout the life of the animal. The AIDS-like disease caused by this early expression of Nef best represents a model for pediatric AIDS. However, in these Tg mice, Nef may interfere with normal developmental processes and these latter defects may contribute to some of the phenotypes observed. To assess the effects of Nef in fully mature adult animals, and thus develop a model of adult AIDS, temporal regulation of Nef expression in adult mice using an inducible system is required.In the present study, we chose the tet-On (rtTA and rtTA2^S-M2) system (13, 15, 25, 48) to induce expression of HIV-1 Nef in CD4⁺ T cells and cells of the macrophage/dendritic lineage of mice using the CD4C tissue-specific regulatory elements. These CD4C sequences were previously used to generate the constitutively Nef-expressing CD4C/HIV^Nef Tg mice (18). These inducible adult (TRE/HIV^Nef × CD4C/rtTA) and (TRE/HIV^Nef × CD4C/rtTA2^S-M2) double-Tg (DTg) mice express Nef when treated with doxycycline (DOX) and develop an AIDS-like disease very similar to that seen in constitutively Nef-expressing CD4C/HIV^Nef Tg mice. We took advantage of this novel biological system to reassess the role of Nef in T-cell activation. Using a mixed chimera made with BM cells from these inducible Nef Tg mice and from non-Tg mice, we could document CD4⁺ T-cell activation only in donor-derived Nef-expressing Tg cells, but not in non-Tg cells, in the absence of lymphopenia. This result strongly suggests that this CD4⁺ T-cell activation phenotype is most likely driven by expression of Nef in these cells.     

Selective Expression of Human Immunodeficiency Virus Nef in Specific Immune Cell Populations of Transgenic Mice Is Associated with Distinct AIDS-Like Phenotypes

We previously reported that CD4C/human immunodeficiency virus (HIV)^Nef transgenic (Tg) mice, expressing Nef in CD4⁺ T cells and cells of the macrophage/dendritic cell (DC) lineage, develop a severe AIDS-like disease, characterized by depletion of CD4⁺ T cells, as well as lung, heart, and kidney diseases. In order to determine the contribution of distinct populations of hematopoietic cells to the development of this AIDS-like disease, five additional Tg strains expressing Nef through restricted cell-specific regulatory elements were generated. These Tg strains express Nef in CD4⁺ T cells, DCs, and macrophages (CD4E/HIV^Nef); in CD4⁺ T cells and DCs (mCD4/HIV^Nef and CD4F/HIV^Nef); in macrophages and DCs (CD68/HIV^Nef); or mainly in DCs (CD11c/HIV^Nef). None of these Tg strains developed significant lung and kidney diseases, suggesting the existence of as-yet-unidentified Nef-expressing cell subset(s) that are responsible for inducing organ disease in CD4C/HIV^Nef Tg mice. Mice from all five strains developed persistent oral carriage of Candida albicans, suggesting an impaired immune function. Only strains expressing Nef in CD4⁺ T cells showed CD4⁺ T-cell depletion, activation, and apoptosis. These results demonstrate that expression of Nef in CD4⁺ T cells is the primary determinant of their depletion. Therefore, the pattern of Nef expression in specific cell population(s) largely determines the nature of the resulting pathological changes.The major cell targets and reservoirs for human immunodeficiency virus type 1 (HIV-1)/simian immunodeficiency virus (SIV) infection in vivo are CD4⁺ T lymphocytes and antigen-presenting cells (macrophages and dendritic cells [DC]) (21, 24, 51). The cell specificity of these viruses is largely dependent on the expression of CD4 and of its coreceptors, CCR5 and CXCR-4, at the cell surface (29, 66). Infection of these immune cells leads to the severe disease, AIDS, showing widespread manifestations, including progressive immunodeficiency, immune activation, CD4⁺ T-cell depletion, wasting, dementia, nephropathy, heart and lung diseases, and susceptibility to opportunistic pathogens, such as Candida albicans (1, 27, 31, 37, 41, 82, 93, 109). It is reasonable to assume that the various pathological changes in AIDS result from the expression of one or many HIV-1/SIV proteins in these immune target cells. However, assigning the contribution of each infected cell subset to each phenotype has been remarkably difficult, despite evidence that AIDS T-cell phenotypes can present very differently depending on the strains of infecting HIV-1 or SIV or on the cells targeted by the virus (4, 39, 49, 52, 72). For example, the T-cell-tropic X4 HIV strains have long been associated with late events and severe CD4⁺ T-cell depletion (22, 85, 96). However, there are a number of target cell subsets expressing CD4 and CXCR-4, and identifying which one is responsible for this enhanced virulence has not been achieved in vivo. Similarly, the replication of SIV in specific regions of the thymus (cortical versus medullary areas), has been associated with very different outcomes but, unfortunately, the critical target cells of the viruses were not identified either in these studies (60, 80). The task is even more complex, because HIV-1 or SIV can infect several cell subsets within a single cell population. In the thymus, double (CD4⁻ CD8⁻)-negative (DN) or triple (CD3⁻ CD4⁻ CD8⁻)-negative (TN) T cells, as well as double-positive (CD4⁺ CD8⁺) (DP) T cells, are infectible by HIV-1 in vitro (9, 28, 74, 84, 98, 99, 110) and in SCID-hu mice (2, 5, 91, 94). In peripheral organs, gut memory CCR5⁺ CD4⁺ T cells are primarily infected with R5 SIV, SHIV, or HIV, while circulating CD4⁺ T cells can be infected by X4 viruses (13, 42, 49, 69, 70, 100, 101, 104). Moreover, some detrimental effects on CD4⁺ T cells have been postulated to originate from HIV-1/SIV gene expression in bystander cells, such as macrophages or DC, suggesting that other infected target cells may contribute to the loss of CD4⁺ T cells (6, 7, 32, 36, 64, 90).Similarly, the infected cell population(s) required and sufficient to induce the organ diseases associated with HIV-1/SIV expression (brain, heart, and kidney) have not yet all been identified. For lung or kidney disease, HIV-specific cytotoxic CD8⁺ T cells (1, 75) or infected podocytes (50, 95), respectively, have been implicated. Activated macrophages have been postulated to play an important role in heart disease (108) and in AIDS dementia (35), although other target cells could be infected by macrophage-tropic viruses and may contribute significantly to the decrease of central nervous system functions (11, 86, 97), as previously pointed out (25).Therefore, because of the widespread nature of HIV-1 infection and the difficulty in extrapolating tropism of HIV-1/SIV in vitro to their cell targeting in vivo (8, 10, 71), alternative approaches are needed to establish the contribution of individual infected cell populations to the multiorgan phenotypes observed in AIDS. To this end, we developed a transgenic (Tg) mouse model of AIDS using a nonreplicating HIV-1 genome expressed through the regulatory sequences of the human CD4 gene (CD4C), in the same murine cells as those targeted by HIV-1 in humans, namely, in immature and mature CD4⁺ T cells, as well as in cells of the macrophage/DC lineages (47, 48, 77; unpublished data). These CD4C/HIV Tg mice develop a multitude of pathologies closely mimicking those of AIDS patients. These include a gradual destruction of the immune system, characterized among other things by thymic and lymphoid organ atrophy, depletion of mature and immature CD4⁺ T lymphocytes, activation of CD4⁺ and CD8⁺ T cells, susceptibility to mucosal candidiasis, HIV-associated nephropathy, and pulmonary and cardiac complications (26, 43, 44, 57, 76, 77, 79, 106). We demonstrated that Nef is the major determinant of the HIV-1 pathogenicity in CD4C/HIV Tg mice (44). The similarities of the AIDS-like phenotypes of these Tg mice to those in human AIDS strongly suggest that such a Tg mouse approach can be used to investigate the contribution of distinct HIV-1-expressing cell populations to their development.In the present study, we constructed and characterized five additional mouse Tg strains expressing Nef, through distinct regulatory elements, in cell populations more restricted than in CD4C/HIV Tg mice. The aim of this effort was to assess whether, and to what extent, the targeting of Nef in distinct immune cell populations affects disease development and progression.     

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 Nef Inhibits Ruffles,Induces Filopodia,and Modulates Migration of Infected Lymphocytes

The HIV-1 Nef protein is a pathogenic factor modulating the behavior of infected cells. Nef induces actin cytoskeleton changes and impairs cell migration toward chemokines. We further characterized the morphology, cytoskeleton dynamics, and motility of HIV-1-infected lymphocytes. By using scanning electron microscopy, confocal immunofluorescence microscopy, and ImageStream technology, which combines flow cytometry and automated imaging, we report that HIV-1 induces a characteristic remodeling of the actin cytoskeleton. In infected lymphocytes, ruffle formation is inhibited, whereas long, thin filopodium-like protrusions are induced. Cells infected with HIV with nef deleted display a normal phenotype, and Nef expression alone, in the absence of other viral proteins, induces morphological changes. We also used an innovative imaging system to immobilize and visualize living individual cells in suspension. When combined with confocal “axial tomography,” this technique greatly enhances three-dimensional optical resolution. With this technique, we confirmed the induction of long filopodium-like structures in unfixed Nef-expressing lymphocytes. The cytoskeleton reorganization induced by Nef is associated with an important impairment of cell movements. The adhesion and spreading of infected cells to fibronectin, their spontaneous motility, and their migration toward chemokines (CXCL12, CCL3, and CCL19) were all significantly decreased. Therefore, Nef induces complex effects on the lymphocyte actin cytoskeleton and cellular morphology, which likely impacts the capacity of infected cells to circulate and to encounter and communicate with bystander cells.Human immunodeficiency virus type 1 (HIV-1) mostly replicates in T-cell areas of secondary lymphoid organs (SLOs) and induces pathological changes in their architecture. Such changes are likely due to a combination of events, including destruction of T cells, chronic immune activation, and alteration of T-cell motility toward and inside the SLOs (27, 37, 50, 53). Indeed, to fulfill their immune surveillance role, T cells continuously circulate in and out of blood, lymph nodes (LNs), and tissues (60).Lymphocyte recruitment from the bloodstream into LNs depends on three distinct processes, i.e., attachment to high endothelial venules (HEVs), extravasation, and cell migration (10, 60). Adhesion to the endothelium and extracellular matrix (ECM) is a crucial step, regulated in part by β1 integrins, α4β1 (VLA-4) and α5β1, that bind VCAM-1 and/or fibronectin (56). Chemokines and their Gαi-protein-coupled receptors are key regulators of lymphocyte trafficking (32). For instance, CCL19 and CCL21 are constitutively produced by HEVs and by fibroblastic reticular cells of T-cell areas of LNs (21, 28, 29). These two chemokines share the receptor CCR7, expressed by naïve T cells and a fraction of memory T cells (47). They play a major role in lymphocyte homing to LNs, in steady state as well as under conditions of inflammation, and may control T-cell positioning within defined functional compartments (1, 17, 18, 47). CXCR4 and its ligand CXCL12/SDF-1 also contribute to T-cell entry into LNs (5, 23, 40). In addition, effector and memory T cells express a broad range of receptors binding inflammatory chemokines, such as the CCR5 ligands CCL3 (MIP1α), CCL4 (MIP1β), and CCL5 (Rantes).Efficient accomplishment of lymphocyte migration and immune functions requires tight regulation of the cellular cytoskeleton (59). This is mediated by the small GTPases of the Rho subfamily, such as Rho, Rac, and Cdc42 (11, 58). They activate specific actin filament assembly factors to generate sheet-like protrusive structures (such as lamellipodia and ruffles) and finger-like protrusions (such as filopodia and microvilli) (6). These structures have different functions. Lamellipodia and ruffles are formed during crawling cell motility and spreading. Filopodia protrude from the leading edges of many motile cells. They appear to perform sensory and exploratory functions to steer cells, depending on cues from the environment (42). Moreover, filopodia, or other thin structures called tunneling nanotubes, have been shown to form intercellular bridges, allowing viruses to spread through remote contacts between infected cells and targets (44, 48, 49, 52).HIV-1 hijacks cytoskeleton dynamics in order to ensure viral entry and transport within and egress from target cells (34; reviewed in reference 13). In particular, the viral protein Nef modifies actin remodeling in various cell systems. In T cells, Nef alters actin rearrangements triggered by activation of T-cell (TCR) or chemokine receptors (22, 54). Nef inhibits immunological synapse formation, a dynamic process involving rapid actin modifications (57). Nef also affects plasma membrane plasticity, inducing secretion of microvesicle clusters (33). In macrophages, Nef induces the extension of long intercellular conduits allowing its own transfer to B cells (61). A number of studies have reported that Nef affects T-cell chemotaxis (generally to CXCL12) through the modulation of Rho-GTPase-regulated signaling pathways (7, 24, 39, 54). Migration studies have generally been performed using Nef-expressing cells, and rarely in the context of HIV-1 infection (54). From a molecular standpoint, it has recently been proposed that Nef acts in part by deregulating cofilin, an actin-depolymerizing factor that promotes actin turnover and subsequent cell motility (54).In the present study, our goal was to gain further insights into the effect of HIV-1 infection on cytoskeleton dynamics. We used a panel of innovative techniques allowing analysis of cell shape, adhesion, and motility. We report that in HIV-infected lymphocytes, Nef promotes filopodium-like formation while it inhibits membrane ruffling. Nef impairs cell adhesion on the extracellular matrix and decreases intrinsic cell motility. Lymphocyte migration toward various chemokines (CXCL12, CCL3, and CCL19) is also inhibited. Our results suggest that Nef may facilitate viral spread and contribute to AIDS pathogenesis by manipulating the migration of lymphocytes.     

Analysis of Human Immunodeficiency Virus Type 1 Viremia and Provirus in Resting CD4+ T Cells Reveals a Novel Source of Residual Viremia in Patients on Antiretroviral Therapy

Highly active antiretroviral therapy (HAART) can reduce human immunodeficiency virus type 1 (HIV-1) viremia to clinically undetectable levels. Despite this dramatic reduction, some virus is present in the blood. In addition, a long-lived latent reservoir for HIV-1 exists in resting memory CD4⁺ T cells. This reservoir is believed to be a source of the residual viremia and is the focus of eradication efforts. Here, we use two measures of population structure—analysis of molecular variance and the Slatkin-Maddison test—to demonstrate that the residual viremia is genetically distinct from proviruses in resting CD4⁺ T cells but that proviruses in resting and activated CD4⁺ T cells belong to a single population. Residual viremia is genetically distinct from proviruses in activated CD4⁺ T cells, monocytes, and unfractionated peripheral blood mononuclear cells. The finding that some of the residual viremia in patients on HAART stems from an unidentified cellular source other than CD4⁺ T cells has implications for eradication efforts.Successful treatment of human immunodeficiency virus type 1 (HIV-1) infection with highly active antiretroviral therapy (HAART) reduces free virus in the blood to levels undetectable by the most sensitive clinical assays (18, 36). However, HIV-1 persists as a latent provirus in resting, memory CD4⁺ T lymphocytes (6, 9, 12, 16, 48) and perhaps in other cell types (45, 52). The latent reservoir in resting CD4⁺ T cells represents a barrier to eradication because of its long half-life (15, 37, 40-42) and because specifically targeting and purging this reservoir is inherently difficult (8, 25, 27).In addition to the latent reservoir in resting CD4⁺ T cells, patients on HAART also have a low amount of free virus in the plasma, typically at levels below the limit of detection of current clinical assays (13, 19, 35, 37). Because free virus has a short half-life (20, 47), residual viremia is indicative of active virus production. The continued presence of free virus in the plasma of patients on HAART indicates either ongoing replication (10, 13, 17, 19), release of virus after reactivation of latently infected CD4⁺ T cells (22, 24, 31, 50), release from other cellular reservoirs (7, 45, 52), or some combination of these mechanisms. Finding the cellular source of residual viremia is important because it will identify the cells that are still capable of producing virus in patients on HAART, cells that must be targeted in any eradication effort.Detailed analysis of this residual viremia has been hindered by technical challenges involved in working with very low concentrations of virus (13, 19, 35). Recently, new insights into the nature of residual viremia have been obtained through intensive patient sampling and enhanced ultrasensitive sequencing methods (1). In a subset of patients, most of the residual viremia consisted of a small number of viral clones (1, 46) produced by a cell type severely underrepresented in the peripheral circulation (1). These unique viral clones, termed predominant plasma clones (PPCs), persist unchanged for extended periods of time (1). The persistence of PPCs indicates that in some patients there may be another major cellular source of residual viremia (1). However, PPCs were observed in a small group of patients who started HAART with very low CD4 counts, and it has been unclear whether the PPC phenomenon extends beyond this group of patients. More importantly, it has been unclear whether the residual viremia generally consists of distinct virus populations produced by different cell types.Since the HIV-1 infection in most patients is initially established by a single viral clone (23, 51), with subsequent diversification (29), the presence of genetically distinct populations of virus in a single individual can reflect entry of viruses into compartments where replication occurs with limited subsequent intercompartmental mixing (32). Sophisticated genetic tests can detect such population structure in a sample of viral sequences (4, 39, 49). Using two complementary tests of population structure (14, 43), we analyzed viral sequences from multiple sources within individual patients in order to determine whether a source other than circulating resting CD4⁺ T cells contributes to residual viremia and viral persistence. Our results have important clinical implications for understanding HIV-1 persistence and treatment failure and for improving eradication strategies, which are currently focusing only on the latent CD4⁺ T-cell reservoir.     

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.     

Proliferation Capacity and Cytotoxic Activity Are Mediated by Functionally and Phenotypically Distinct Virus-Specific CD8 T Cells Defined by Interleukin-7Rα (CD127) and Perforin Expression

Cytotoxicity and proliferation capacity are key functions of antiviral CD8 T cells. In the present study, we investigated a series of markers to define these functions in virus-specific CD8 T cells. We provide evidence that there is a lack of coexpression of perforin and CD127 in human CD8 T cells. CD127 expression on virus-specific CD8 T cells correlated positively with proliferation capacity and negatively with perforin expression and cytotoxicity. Influenza virus-, cytomegalovirus-, and Epstein-Barr virus/human immunodeficiency virus type 1-specific CD8 T cells were predominantly composed of CD127⁺ perforin⁻/CD127⁻ perforin⁺, and CD127⁻/perforin⁻ CD8 T cells, respectively. CD127⁻/perforin⁻ and CD127⁻/perforin⁺ cells expressed significantly more PD-1 and CD57, respectively. Consistently, intracellular cytokine (gamma interferon, tumor necrosis factor alpha, and interleukin-2 [IL-2]) responses combined to perforin detection confirmed that virus-specific CD8 T cells were mostly composed of either perforin⁺/IL-2⁻ or perforin⁻/IL-2⁺ cells. In addition, perforin expression and IL-2 secretion were negatively correlated in virus-specific CD8 T cells (P < 0.01). As previously shown for perforin, changes in antigen exposure modulated also CD127 expression. Based on the above results, proliferating (CD127⁺/IL-2-secreting) and cytotoxic (perforin⁺) CD8 T cells were contained within phenotypically distinct T-cell populations at different stages of activation or differentiation and showed different levels of exhaustion and senescence. Furthermore, the composition of proliferating and cytotoxic CD8 T cells for a given antiviral CD8 T-cell population appeared to be influenced by antigen exposure. These results advance our understanding of the relationship between cytotoxicity, proliferation capacity, the levels of senescence and exhaustion, and antigen exposure of antiviral memory CD8 T cells.Cytotoxic CD8 T cells are a fundamental component of the immune response against viral infections and mediate an important role in immunosurveillance (7, 10, 55), and the induction of vigorous CD8 T-cell responses after vaccination is thought to be a key component of protective immunity (37, 41, 49, 50, 58, 60, 69). Cytotoxic CD8 T cells exert their antiviral and antitumor activity primarily through the secretion of cytotoxic granules containing perforin (pore-forming protein) and several granule-associated proteases, including granzymes (Grms) (5, 15, 20, 44). Several studies have recently advanced the characterization of the mechanism of granule-dependent cytotoxic activity and performed a comprehensive investigation of the content of cytotoxic granules in human virus-specific CD8 T cells (2, 19, 29, 44, 53).Heterogeneous profiles of cytotoxic granules have been identified in different virus-specific memory CD8 T cells and associated with distinct differentiation stages of memory CD8 T cells (2, 19, 29, 44). Furthermore, we have observed a hierarchy among the cytotoxic granules in setting the efficiency of cytotoxic activity and demonstrated that perforin (and to a lesser extent GrmB) but not GrmA or GrmK were associated with cytotoxic activity (29). Recently, a novel mechanism of perforin-dependent granule-independent CTL cytotoxicity has also been demonstrated (45).Major advances in the characterization of antigen (Ag)-specific CD4 and CD8 T cells have been made recently and have aimed at identifying functional profiles that may correlate with protective CD8 T-cell responses (1, 3, 4, 12, 13, 24, 28, 36-38, 40, 41, 49, 50, 56-58, 60, 64, 68). In particular, the functional characterization of antigen-specific T cells was mainly performed on the basis of (i) the pattern of cytokines secreted (i.e., gamma interferon [IFN-γ], tumor necrosis factor alpha [TNF-α], interleukin-2 [IL-2], or macrophage inflammatory protein 1β [MIP-1β]), (ii) the proliferation capacity, and (iii) the cytotoxic capacity (13, 28, 59). Of note, degranulation activity (i.e., CD107a mobilization following specific stimulation) has been used as a surrogate marker of cytotoxic activity (11, 13).The term “polyfunctional” has been used to define T-cell immune responses that, in addition to typical effector functions such as secretion of IFN-γ, TNF-α, or MIP-1β and cytotoxic activity (measured by the degranulation capacity), comprise distinct T-cell populations able to secrete IL-2 and retain proliferation capacity (13, 28, 49, 50). Some evidence indicates that a hallmark of protective immune responses is the presence of polyfunctional T-cell responses (59). Furthermore, the ability to secrete IL-2 was shown to be linked to proliferation capacity, and both factors have been associated with protective antiviral immunity (13, 28, 49, 50). Although a lack of correlation between degranulation activity and GrmB expression was reported in mice (65), the relationship between degranulation activity and perforin expression has never been comprehensively investigated in mice and in humans.The private α chain of the IL-7 receptor (IL-7Rα, also called CD127) has been suggested to selectively identify CD8 T cells that will become long-lived memory cells (6, 34, 36). Moreover, it was shown in mice (34, 36) and humans (14, 48, 63) that the CD127^high memory-precursor CD8 T cells produced IL-2 in contrast to CD127^low effector CD8 T cells. Of interest, CD127 expression has also been shown to correlate with Ag-specific proliferation capacity in mice (34, 36). A similar correlation was observed in humans, although only for polyclonal stimulations (48). With the exception of studies performed in HIV-1 infection, where an association between CD127 expression and HIV-1 viremia has been shown (21, 22, 42, 48, 54), very limited information is available on the CD127 expression in human virus-specific CD8 T cells other that HIV-1.Although cytotoxic activity and proliferation capacity are key components of the antiviral cellular immune response, the relationship between these functions has been only investigated in nonprogressive HIV-1 infection (46), where these two functions were shown to be related. However, it still remains to be determined whether these functions are mediated by the same or by different T-cell populations.In the present study, we performed a comprehensive characterization of virus-specific CD8 T-cell responses against HIV-1, cytomegalovirus (CMV), Epstein Barr virus (EBV), and influenza virus (Flu) in order to (i) analyze the degree of concordance between degranulation activity and perforin/Grm expression; (ii) identify the relevance of CD127 in identifying virus-specific CD8 T cells endowed with proliferation capacity; (iii) delineate the relationship between proliferation capacity, cytotoxic activity, activation/differentiation stage, and level of exhaustion of CD8 T cells; and (iv) determine the influence of antigen exposure in shaping the functional composition of virus-specific CD8 T cells.Our data indicate that cytotoxic (as defined by perforin expression) and proliferating (as defined by CD127 expression or IL-2 secretion) virus-specific CD8 T cells are contained within distinct CD8 T-cell populations. Furthermore, the proportion of proliferating and cytotoxic T cells within a given virus-specific CD8 T-cell population appears to be influenced by antigen exposure. These results advance our understanding of the relationship between cytotoxicity, proliferative capacity, differentiation stage, and Ag exposure of memory CD8 T cells.     

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).     

Characterization of Respiratory Syncytial Virus M- and M2-Specific CD4 T Cells in a Murine Model

CD4 T cells have been shown to play an important role in the immunity and immunopathogenesis of respiratory syncytial virus (RSV) infection. We identified two novel CD4 T-cell epitopes in the RSV M and M2 proteins with core sequences M_213-223 (FKYIKPQSQFI) and M2_27-37 (YFEWPPHALLV). Peptides containing the epitopes stimulated RSV-specific CD4 T cells to produce gamma interferon (IFN-γ), interleukin 2 (IL-2), and other Th1- and Th2-type cytokines in an I-A^b-restricted pattern. Construction of fluorochrome-conjugated peptide-I-A^b class II tetramers revealed RSV M- and M2-specific CD4 T-cell responses in RSV-infected mice in a hierarchical pattern. Peptide-activated CD4 T cells from lungs were more activated and differentiated, and had greater IFN-γ expression, than CD4 T cells from the spleen, which, in contrast, produced greater levels of IL-2. In addition, M_209-223 peptide-activated CD4 T cells reduced IFN-γ and IL-2 production in M- and M2-specific CD8 T-cell responses to D^b-M_187-195 and K^d-M2_82-90 peptides more than M2_25-39 peptide-stimulated CD4 T cells. This correlated with the fact that I-A^b-M_209-223 tetramer-positive cells responding to primary RSV infection had a much higher frequency of FoxP3 expression than I-A^b-M2_26-39 tetramer-positive CD4 T cells, suggesting that the M-specific CD4 T-cell response has greater regulatory function. Characterization of epitope-specific CD4 T cells by novel fluorochrome-conjugated peptide-I-A^b tetramers allows detailed analysis of their roles in RSV pathogenesis and immunity.CD4 T lymphocytes play an important role in the resolution of primary viral infections and the prevention of reinfection by regulating a variety of humoral and cellular immune responses. CD4 T cells provide cytokines and other molecules to support the differentiation and expansion of antigen-specific CD8 T cells, which are major effectors for both virus clearance and immunopathology during primary infection with respiratory syncytial virus (RSV) (3, 17, 42, 43). CD4 T-cell help is mandatory for an effective B-cell response (14), which is necessary for producing neutralizing antibodies that prevent secondary RSV infection (12, 18, 21). A concurrent CD4 T-cell response also promotes the maintenance of CD8 T-cell surveillance and effector capacity (9). Previous studies have shown that interleukin 2 (IL-2) from CD4 T cells can restore CD8 T-cell function in lungs (10) and that IL-2 supplementation can increase the production of gamma interferon (IFN-γ) by CD8 T cells upon peptide stimulation in vitro (45).While CD4 T cells are important for providing support to host immunity, they have also been associated with immunopathogenesis by playing a key role in the Th2-biased T-cell response (34, 46), which may be the common mechanism of enhanced lung pathology and other disease syndromes shown in murine studies (2, 16, 17, 19, 35). Earlier studies showed the positive association of formalin-inactivated RSV (FI-RSV) immunization-mediated enhanced illness upon subsequent natural RSV infection with a Th2-biased CD4 T-cell response (19, 44). Th2-orientated CD4 T cells elicit severe pneumonia with extensive eosinophilic infiltrates in the lungs of FI-RSV-immunized mice (13, 24, 48). Patients with severe RSV disease showed an elevated Th2/Th1 cytokine ratio in nasal secretions and peripheral blood mononuclear cells (27, 29, 31, 38). Increased disease severity has also been associated with polymorphisms in Th2-related cytokine genes, such as the IL-4, IL-4 receptor, and IL-13 genes (11, 23, 36). Th2 cytokines from CD4 T cells can also diminish the CD8 T-cell response and delay viral clearance (4, 8).The evaluation of CD4 T-cell responses in viral infection is particularly relevant in the RSV model because of the association of RSV and allergic inflammation, which is largely mediated by CD4 T cells. Understanding the influence of CD4 T cells on CD8 T-cell responses and other immunological effector mechanisms is central to understanding RSV pathogenesis and developing preventive vaccine strategies for RSV. Our lab and others have demonstrated that CD8 T cells target RSV M and M2 proteins with cytolytic effector activities (28, 30, 39). In this study, we found that both RSV M and M2 proteins also contain CD4 T-cell epitopes. These epitopes have 11-mer amino acid core sequences and are associated with the major histocompatibility complex (MHC) class II molecule I-A^b. Fluorochrome-conjugated peptide-I-A^b molecule tetrameric complexes can identify RSV M- and M2-specific CD4 T cells from CB6F1 mice following RSV infection in a hierarchical pattern. Peptides containing the epitopes can stimulate CD4 T cells from RSV M or M2 DNA-immunized and virus-challenged mice and can lead to the production of IFN-γ, IL-2, and other Th1- and Th2-type cytokines that can modulate the CD8 T-cell response to RSV M and M2. We also found that CD4 T cells from the lungs and spleens of immunized mice have different phenotype and cytokine profiles upon in vitro stimulation. These observations suggest a regulatory role for CD4 T cells in the host response to RSV infection. The development of novel MHC class II tetramer reagents allows the characterization of epitope-specific CD4 T-cell responses to RSV and will enable the investigation of basic mechanisms by which CD4 T cells affect pathogenesis and immunity to viral infections.