Evidence that T cell activation is required for HIV-1 entry in CD4+ lymphocytes

The relationship of T cell activation to HIV entry and generation of viral DNA intermediates was studied in freshly isolated CD4+ T lymphocytes. Unstimulated cells exposed to infectious virus for up to 48 h did not synthesize any detectable unintegrated HIV DNA duplex forms or integrated genomic provirus. However, activation of these cells with either PHA or OKT3 (anti-CD3) mAb before viral exposure resulted in the generation of unintegrated HIV DNA after 6 h and integrated copies after 24 h. Cell-to-cell fusion studies showed significantly attenuated fusion between freshly isolated resting T cells and T cells constitutively expressing high levels of HIV envelope glycoprotein (HXB/gpt) compared with T cells first stimulated with either PHA or OKT3 mAb. The baseline fusion observed with resting T cells is believed to be a consequence of allogeneic stimulation by the HXB/gpt cell line. These results provide evidence that HIV entry and HIV envelope-dependent cell-to-cell fusion require T cell activation.     

Vaccinia virus l1 protein is required for cell entry and membrane fusion

Genetic and biochemical studies have provided evidence for an entry/fusion complex (EFC) comprised of at least eight viral proteins (A16, A21, A28, G3, G9, H2, J5, and L5) that together with an associated protein (F9) participates in entry of vaccinia virus (VACV) into cells. The genes encoding these proteins are conserved in all poxviruses, are expressed late in infection, and are components of the mature virion membrane but are not required for viral morphogenesis. In addition, all but one component has intramolecular disulfides that are formed by the poxvirus cytoplasmic redox system. The L1 protein has each of the characteristics enumerated above except that it has been reported to be essential for virus assembly. To further investigate the role of L1, we constructed a recombinant VACV (vL1Ri) that inducibly expresses L1. In the absence of inducer, L1 synthesis was repressed and vL1Ri was unable to form plaques or produce infectious progeny. Unexpectedly, assembly and morphogenesis appeared normal and the noninfectious virus particles were indistinguishable from wild-type VACV as determined by transmission electron microscopy and analysis of the component polypeptides. Notably, the L1-deficient virions were able to attach to cells but the cores failed to penetrate into the cytoplasm. In addition, cells infected with vL1Ri in the absence of inducer did not form syncytia following brief low-pH treatment even though extracellular virus was produced. Coimmunoprecipitation experiments demonstrated that L1 interacted with the EFC and indirectly with F9, suggesting that L1 is an additional component of the viral entry apparatus.     

Tetraspanins CD9 and CD81 modulate HIV-1-induced membrane fusion

Protein organization on the membrane of target cells may modulate HIV-1 transmission. Since the tetraspanin CD81 is associated to CD4, the receptor of HIV-1 envelope protein (Env; gp120/gp41), we have explored the possibility that this molecule may modulate the initial steps of HIV-1 infection. On the other hand, CD81 belongs to the tetraspanin family, which has been described as organizers of protein microdomains on the plasma membrane. Therefore, the role of CD81 and other related tetraspanin, CD9, on the cell-to-cell fusion process mediated by HIV-1 was studied. We found that anti-tetraspanin Abs enhanced the syncytia formation induced by HIV-1 envelope proteins and viral entry in human T lymphoblasts. In addition, anti-CD81 Abs triggered its clustering in patches, where CD4 and CXCR4 were included. Moreover, the knocking down of CD81 and CD9 expression resulted in an increase in syncytia formation and viral entry. Accordingly, overexpression of CD81 and CD9 rendered cells less susceptible to Env-mediated syncytia formation. These data indicate that CD9 and CD81 have an important role in membrane fusion induced by HIV-1 envelope.     

Thioredoxin (Trx1) regulates CD4 membrane domain localization and is required for efficient CD4-dependent HIV-1 entry

BackgroundCD4 is a glycoprotein expressed on the surfaces of certain immune cells. On lymphocytes, an important function of CD4 is to co-engage Major Histocompatibility Complex (MHC) molecules with the T Cell Receptor (TCR), a process that is essential for antigen-specific activation of T cells. CD4 localizes dynamically into distinct membrane microdomains, an important feature of its immunoregulatory function that has also been shown to influence the efficiency of HIV replication. However, the mechanism by which CD4 localization is regulated and the biological significance of this is incompletely understood.MethodsIn this study, we used confocal microscopy, density-gradient centrifugation and flow cytometry to analyze dynamic redox-dependent effects on CD4 membrane domain localization.ResultsBlocking cell surface redox exchanges with both a membrane-impermeable sulfhydryl blocker (DTNB) and specific antibody inhibitors of Thioredoxin-1 (Trx1) induces translocation of CD4 into detergent-resistant membrane domains (DRM). In contrast, Trx1 inactivation does not change the localization of the chemokine receptor CCR5, suggesting that this effect is targeted. Moreover, DTNB treatment and Trx1 depletion coincide with strong inhibition of CD4-dependent HIV entry, but only moderate reductions in the infectivity of a CD4-independent HIV pseudovirion.ConclusionsChanges in the extracellular redox environment, potentially mediated by allosteric consequences of functional disulfide bond oxidoreduction, may represent a signal for translocation of CD4 into DRM clusters, and this sequestration, another potential mechanism by which the anti-HIV effects of cell surface oxidoreductase inhibition are exerted.General significanceExtracellular redox conditions may regulate CD4 function by potentiating changes in its membrane domain localization.     

CD4 receptor localized to non-raft membrane microdomains supports HIV-1 entry. Identification of a novel raft localization marker in CD4

Despite the preferential localization of CD4 to lipid rafts, the significance and role of these microdomains in HIV-1 entry is still controversial. The possibility that CD4, when localized to non-raft domains, might be able to support virus entry cannot be excluded. Because disintegration of rafts by extraction of cellular cholesterol with methyl-beta-cyclodextrin suffers from various adverse effects, we investigated molecular determinants controlling raft localization of the CD4 receptor. Extensive mutagenesis of the receptor showed that a raft-localizing marker, consisting of a short sequence of positively charged amino acid residues, RHRRR, was present in the membrane-proximal cytoplasmic domain of CD4. Substitution of the RHRRR sequence with alanine residues abolished raft localization of the CD4 mutant, RA5, as determined biochemically using solubilization in nonionic detergents and by confocal microscopy. The possible inhibitory effect of the introduced mutations on the adjacent CVRC palmitoylation site was ruled out because wild type (wt) CD4 and RA5, but not a palmitoylation-deficient mutant, were efficiently palmitoylated. Nonetheless, the RA5 mutant supported productive virus entry to levels equivalent to that of wild type (wt) CD4. Sucrose gradient analysis of Triton X-100 virus lysates showed that Gag and envelope gp120 proteins accumulated in low buoyant, high-density fractions. This pattern was changed after virus incubation with cells. Whereas Gag proteins localized to lipid rafts in cells expressing wt CD4 and RA5, gp120 accumulated in rafts in cells expressing wt CD4 but not RA5. We propose that raft localization of CD4 is not required for virus entry, however, post-binding fusion/entry steps may require lipid raft assembly.     

CCL2 increases X4-tropic HIV-1 entry into resting CD4+ T cells

During human immunodeficiency virus type 1 (HIV-1) infection, there is a strong positive correlation between CCL2 levels and HIV viral load. To determine whether CCL2 alters HIV-1 infection of resting CD4(+) T cells, we infected purified resting CD4(+) T cells after incubation with CCL2. We show that CCL2 up-regulates CXCR4 on resting CD4(+) T cells in a CCR2-dependent mechanism, and that this augmentation of CXCR4 expression by CCL2 increases the ability of these cells to be chemoattracted to CXCR4 using gp120 and renders them more permissive to X4-tropic HIV-1 infection. Thus, CCL2 has the capacity to render a large population of lymphocytes more susceptible to HIV-1 late in the course of infection.     

Substitutions in a homologous region of extracellular loop 2 of CXCR4 and CCR5 alter coreceptor activities for HIV-1 membrane fusion and virus entry

CXCR4 and CCR5 are the principal coreceptors for human immunodeficiency virus type-1 (HIV-1) infection. Previously, mutagenesis of CXCR4 identified single amino acid changes that either impaired CXCR4's coreceptor activity for CXCR4-dependent (X4) isolate envelope glycoproteins (Env) or expanded its activity, allowing it to serve as a functional coreceptor for CCR5-dependent (R5) isolates. The most potent of these point mutations was an alanine substitution for the aspartic acid residue at position 187 in extracellular loop 2 (ecl-2), and here we show that this mutation also permits a variety of primary R5 isolate Envs, including those of other subtypes (clades), to employ it as a coreceptor. We also examined the corresponding region of CCR5 and demonstrate that the substitution of the serine residue in the homologous ecl-2 position with aspartic acid impairs CCR5 coreceptor activity for isolates across several clades. These results highlight a homologous and critical element in ecl-2, of both the CXCR4 and CCR5 molecules, for their respective coreceptor activities. Charge elimination expands CXCR4 coreceptor activity, while a similar charge introduction can destroy the coreceptor function of CCR5. These findings provide further evidence that there are conserved elements in both CXCR4 and CCR5 involved in coreceptor function.     

Restricted lateral mobility of plasma membrane CD4 impairs HIV-1 envelope glycoprotein mediated fusion

We investigated the effect of receptor mobility on HIV-1 envelope glycoprotein (Env)-triggered fusion using B16 mouse melanoma cells that are engineered to express CD4 and CXCR4 or CCR5. These engineered cells are resistant to fusion mediated CD4-dependent HIV-1 envelope glycoprotein. Receptor mobility was measured by fluorescence recovery after photobleaching (FRAP) using either fluorescently-labeled antibodies or transient expression of GFP-tagged receptors in the cells. No significant differences between B16 and NIH3T3 (fusion-permissive) cells were seen in lateral mobility of CCR5 or lipid probes. By contrast CD4 mobility in B16 cells was about seven-fold reduced compared to its mobility in fusion-permissive NIH3T3 cells. However, a CD4 mutant (RA5) that localizes to non-raft membrane microdomains exhibited a three-fold increased mobility in B16 cells as compared with WT-CD4. Interestingly, the B16 cells expressing the RA5 mutant (but not the wild type CD4) and coreceptors supported HIV-1 Env-mediated fusion. Our data demonstrate that the lateral mobility of CD4 is an important determinant of HIV-1 fusion/entry.     

pH-independent HIV entry into CD4-positive T cells via virus envelope fusion to the plasma membrane

CD4 functions as the cell-surface receptor for human immunodeficiency virus (HIV); however, the mechanism of virus entry into susceptible cells is unknown. To explore this question we used a human T lymphoblastic cell line (VB) expressing high levels of surface CD4. Neutralization of endosomal compartments (pH greater than 6.4) with lysosomotropic agents did not effectively inhibit HIV nucleocapsid entry into the cytoplasm, and virus treated at low pH (5.5) failed to induce rapid cell-to-cell fusion in uninfected cells. Electron microscopy of VB cells acutely exposed to HIV at neutral pH revealed direct fusion of the virus envelope with the plasma membrane within minutes at 4 degrees C. No endocytosed virions were visualized upon rewarming the HIV-exposed cells to 37 degrees C for as long as 60 min. These results indicate that HIV penetrates CD4-positive T cells via pH-independent membrane fusion.     

Conformation of trimeric envelope glycoproteins: the CD4-dependent membrane fusion mechanism of HIV-1

The HIV-1 envelope glycoproteins are assembled by the trimeric gp120s and gp41s proteins. The gp120 binds sequentially to CD4 and coreceptor for initiating virus entry. Because of noncovalent interaction and heavy glycosylation for envelope glycoproteins, it is highly difficult to determine entire envelope glycoproteins structure now. Such question extremely limits our good understanding of HIV-1 membrane fusion mechanism. Here, a novel and reasonable assembly model of trimeric gp120s and gp41s was proposed based on the conformational dynamics of trimeric gp120-gp41 complex and gp41, respectively. As for gp41, the heptad repeat sequences in the gp41 C-terminal is of enormous flexibility. On the contrary, the heptad repeat sequences in the gp41 N-terminal likely present stable three-helical bundle due to strong nonpolar interaction, and they were predicted to associate three alpha1 helixes from the non-neutralizing face of the gp120 inner domain, which is quite similar to gp41 fusion core structure. Such interaction likely leads to the formation of noncovalent gp120-gp41 complex. In the proposed assembly of trimeric gp120-gp41 complex, three gp120s present not only perfectly complementary and symmetrical distribution around the gp41, but also different flexibility degree in the different structural domains. Thus, the new model can well explain numerous experimental phenomena, present plenty of structural information, elucidate effectively HIV-1 membrane fusion mechanism, and direct to further develop vaccine and novel fusion inhibitors.     

Distinct kinetics of Gag-specific CD4+ and CD8+ T cell responses during acute HIV-1 infection

HIV infection is characterized by a gradual deterioration of immune function, mainly in the CD4 compartment. To better understand the dynamics of HIV-specific T cells, we analyzed the kinetics and polyfunctional profiles of Gag-specific CD4(+) and CD8(+) T cell responses in 12 subtype C-infected individuals with different disease-progression profiles, ranging from acute to chronic HIV infection. The frequencies of Gag-responsive CD4(+) and CD8(+) T cells showed distinct temporal kinetics. The peak frequency of Gag-responsive IFN-γ(+)CD4(+) T cells was observed at a median of 28 d (interquartile range: 21-81 d) post-Fiebig I/II staging, whereas Gag-specific IFN-γ(+)CD8(+) T cell responses peaked at a median of 253 d (interquartile range: 136-401 d) and showed a significant biphasic expansion. The proportion of TNF-α-expressing cells within the IFN-γ(+)CD4(+) T cell population increased (p = 0.001) over time, whereas TNF-α-expressing cells within IFN-γ(+)CD8(+) T cells declined (p = 0.005). Both Gag-responsive CD4(+) and CD8(+) T cells showed decreased Ki67 expression within the first 120 d post-Fiebig I/II staging. Prior to the disappearance of Gag-responsive Ki67(+)CD4(+) T cells, these cells positively correlated (p = 0.00038) with viremia, indicating that early Gag-responsive CD4 events are shaped by viral burden. No such associations were observed in the Gag-specific CD8(+) T cell compartment. Overall, these observations indicated that circulating Gag-responsive CD4(+) and CD8(+) T cell frequencies and functions are not synchronous, and properties change rapidly at different tempos during early HIV infection.     

Role of glycosphingolipid microdomains in CD4-dependent HIV-1 fusion

The fusion of HIV-1 with the plasma membrane of CD4+ cells is triggered by the interaction of HIV-1 surface envelope glycoprotein gp120 with the CD4 receptor, and requires coreceptors (CCR5 and CXCR4). Recent advances in the study of HIV-1 entry into CD4+ cells suggest that glycosphingolipids (GSL) may also participate in the fusion process. GSL are organized in functional microdomains which are associated with specific membrane proteins such as CD4. GSL-enriched microdomains were purified from human lymphocytes and reconstituted as a monomolecular film at the air-water interface of a Langmuir film balance. Surface pressure measurements allowed to characterize the sequential interaction of GSL with CD4 and with gp120. Using this approach, we identified globotriaosylceramide (Gb3) and ganglioside GM3 as the main lymphocyte GSL recognized by gp120. In both cases, the interaction was saturable and dramatically increased by CD4. We propose that GSL microdomains behave as moving platforms allowing the recruitment of HIV-1 coreceptors after the initial interaction between the viral particle and CD4. According to this model, the GSL microdomain may: i) stabilize the attachment of the virus with the cell surface through multiple low affinity interactions between the V3 domain of gp120 and the carbohydrate moiety of GSL, and ii) convey the virus to an appropriate coreceptor by moving freely in the outer leaflet of the plasma membrane. This model can be extrapolated to all envelope viruses (e.g. influenza virus) that use cell surface GSL of the host cells as receptors or coreceptors.     

Role of glycosphingolipid microdomains in CD4-dependent HIV-1 fusion

The fusion of HIV-1 with the plasma membrane of CD4⁺ cells is triggered by the interaction of HIV-1 surface envelope glycoprotein gp120 with the CD4 receptor, and requires coreceptors (CCR5 and CXCR4). Recent advances in the study of HIV-1 entry into CD4⁺ cells suggest that glycosphingolipids (GSL) may also participate in the fusion process. GSL are organized in functional microdomains which are associated with specific membrane proteins such as CD4. GSL-enriched microdomains were purified from human lymphocytes and reconstituted as a monomolecular film at the air–water interface of a Langmuir film balance. Surface pressure measurements allowed to characterize the sequential interaction of GSL with CD4 and with gp120. Using this approach, we identified globotriaosylceramide (Gb3) and ganglioside GM3 as the main lymphocyte GSL recognized by gp120. In both cases, the interaction was saturable and dramatically increased by CD4. We propose that GSL microdomains behave as moving platforms allowing the recruitment of HIV-1 coreceptors after the initial interaction between the viral particle and CD4. According to this model, the GSL microdomain may : i) stabilize the attachment of the virus with the cell surface through multiple low affinity interactions between the V3 domain of gp120 and the carbohydrate moiety of GSL, and ii) convey the virus to an appropriate coreceptor by moving freely in the outer leaflet of the plasma membrane. This model can be extrapolated to all envelope viruses (e.g. influenza virus) that use cell surface GSL of the host cells as receptors or coreceptors.     

Maintenance of HIV-specific CD4+ T cell help distinguishes HIV-2 from HIV-1 infection

Unlike HIV-1-infected people, most HIV-2-infected subjects maintain a healthy CD4+ T cell count and a strong HIV-specific CD4+ T cell response. To define the cellular immunological correlates of good prognosis in HIV-2 infection, we conducted a cross-sectional study of HIV Gag-specific T cell function in HIV-1- and HIV-2-infected Gambians. Using cytokine flow cytometry and lymphoproliferation assays, we show that HIV-specific CD4+ T cells from HIV-2-infected individuals maintained proliferative capacity, were not terminally differentiated (CD57-), and more frequently produced IFN-gamma or IL-2 than CD4+ T cells from HIV-1-infected donors. Polyfunctional (IFN-gamma+/IL-2+) HIV-specific CD4+ T cells were found exclusively in HIV-2+ donors. The disparity in CD4+ T cell responses between asymptomatic HIV-1- and HIV-2-infected subjects was not associated with differences in the proliferative capacity of HIV-specific CD8+ T cells. This study demonstrates that HIV-2-infected donors have a well-preserved and functionally heterogeneous HIV-specific memory CD4+ T cell response that is associated with delayed disease progression in the majority of infected people.     

Effects of sustained HIV-1 plasma viremia on HIV-1 Gag-specific CD4+ T cell maturation and function

An in vitro proliferative defect has been observed in HIV-1-specific CD4(+) T cells from infected subjects with high-level plasma HIV-1 viremia. To determine the mechanism of this defect, HIV-1 Gag-specific CD4(+) T cells from treated and untreated HIV-1-infected subjects were analyzed for cytokine profile, proliferative capacity, and maturation state. Unexpectedly high frequencies of HIV-1-specific, IL-2-producing CD4(+) T cells were measured in subjects with low or undetectable plasma HIV-1 loads, regardless of treatment status, and IL-2 frequencies correlated inversely with viral loads. IL-2-producing CD4(+) T cells also primarily displayed a central memory (T(Cm); CCR7(+)CD45RA(-)) maturation phenotype, whereas IFN-gamma-producing cells were mostly effector memory (T(Em), CCR7(-)CD45RA(-)). Among Gag-specific, IFN-gamma-producing CD4(+) T cells, higher T(Em) frequencies and lower T(Cm) frequencies were observed in untreated, high viral load subjects than in subjects with low viral loads. The percentage of HIV-1 Gag-specific CD4(+) T(Cm) correlated inversely with HIV-1 viral load and directly with Gag-specific CD4(+) T cell proliferation, whereas the opposite relationships were observed for HIV-1-specific CD4(+) T(Em). These results suggest that HIV-1 viremia skews Gag-specific CD4(+) T cells away from an IL-2-producing T(Cm) phenotype and toward a poorly proliferating T(Em) phenotype, which may limit the effectiveness of the HIV-1-specific immune response.     

Presence of HIV-1 Gag-specific IFN-gamma+IL-2+ and CD28+IL-2+ CD4 T cell responses is associated with nonprogression in HIV-1 infection

HIV immunity is likely CD4 T cell dependent. HIV-specific CD4 T cell proliferative responses are reported to correlate inversely with virus load and directly with specific CD8 responses. However, the phenotype and cytokine profile of specific CD4 T cells that correlate with disease is unknown. We compared the number/function of Gag p24-specific CD4 T cells in 17 HIV-infected long-term nonprogressors (LTNPs) infected for a median of 14.6 years with those of 16 slow progressors (SPs), also HIV infected for a median of 14 years but whose CD4 count had declined to <500 cells/ micro l. Compared with SPs, LTNPs had higher numbers of specific CD4s that were double positive for IFN-gamma and IL-2 as well as CD28 and IL-2. However, CD4 T cells that produced IL-2 alone (IL-2(+)IFN-gamma(-)) or IFN-gamma alone (IFN-gamma(+)IL-2(-)) did not differ between LTNPs and SPs. The decrease in p24-specific CD28(+)IL-2(+) cells with a concomitant increase of p24-specific CD28(-)IL-2(+) cells occurred before those specific for a non-HIV Ag, CMV. p24-specific CD28(-)IL-2(+) cells were evident in LTNPs and SPs, whereas the CMV-specific CD28(-)IL-2(+) response was confined to SPs. The difference between LTNPs and SPs in the Gag p24 IFN-gamma(+)IL-2(+) response was maintained when responses to total Gag (p17 plus p24) were measured. The percentage and absolute number of Gag-specific IFN-gamma(+)IL-2(+) but not of IFN-gamma(+)IL-2(-) CD4s correlated inversely with virus load. The Gag-specific IFN-gamma(+)IL-2(+) CD4 response also correlated positively with the percentage of Gag-specific IFN-gamma(+) CD8 T cells in these subjects. Accumulation of specific CD28(-)IL-2(+) helpers and loss of IFN-gamma(+)IL-2(+) CD4 T cells may compromise specific CD8 responses and, in turn, immunity to HIV.     

Physical membrane displacement: Reconstitution in a cell-free system and relationship to cell growth+

Summary Physical membrane displacement is a process common to all forms of vesicle budding as well as cell enlargement and pleomorphic shape changes. Cell-free reconstitution of membrane budding has been achieved with transitional endoplasmic reticulum fractions from both plants and animals where 50 to 70 nm transition vesicles have been observed to bud from the part-rough, part-smooth membrane elements that define transitional endoplasmic reticulum. This budding phenomenon requires ATP, is facilitated by cytosol and guanine nucleotides, and is both time- and temperature-dependent. The transitional endoplasmic reticulum buds that form when concentrated by preparative free-flow electrophoresis will attach specifically to cis Golgi apparatus membranes immobilized on nitrocellulose as an acceptor compartment. Golgi apparatus membranes derived from the trans compartment do not serve as an efficient acceptor compartment. Transfer of the vesicles once formed is rapid, nearly complete and no longer dependent upon added ATP. Transfer shows a strict temperature dependency corresponding to that of the intact cell where at temperatures of 16°C or below, vesicles form but do not attach to cis Golgi whereas at temperatures of greater than 16°C, vesicles both form and fuse. The principle ATPase of transitional endoplasmic reticulum which may be involved in the budding process has been identified, characterized and isolated. A 38 kDa cis Golgi apparatus associated protein also has been identified as a potential candidate as a docking protein. Transfer between trans Golgi apparatus and the plasma membrane also has been studied by cell-free analysis. Here, transfer has been found to be stimulated by NADH or NADH plus ascorbate. The role of NADH is unknown but the ability of plant and Golgi apparatus to oxidize NADH is inhibited by brefeldin A, a compound known to block membrane trafficking even at the level of the trans Golgi network. NADH oxidase activity of plasma membranes also has been described and is inhibited as well by brefeldin. Recent observations suggest that brefeldin A may block both the formation of vesicles at the trans Golgi apparatus as well as auxin hormone-stimulated cell elongation in plants. This once again raises the possibility of whether or not plant cell elongation is obligatorily mediated by membrane input from the Golgi apparatus. The latter seems unlikely based on two additional lines of evidence. The first is that auxin-induced cell elongation in plants shows no sharp temperature transition over the range of 4 to 24°C, whereas production of secretory vesicles from the trans Golgi apparatus appears to be largely prevented at temperatures of 18°C or less. Secondly, the sodium selective ionophore, monensin, which effectively blocks the formation of functional secretory vesicles at the trans Golgi apparatus, is also largely without effect on auxin-induced cell elongation for periods of 4 h or longer. Taken together the findings suggest that the action of brefeldin A on vesicle budding at the Golgi apparatus and cell enlargement, are not directly correlated but may represent a common action of the drug on some constituent essential to membrane displacement mechanisms.Abbreviations BFA brefeldin A - IAA indole-3-acetic acid; 2, 4-D 2, 4-dichlorophenoxyacetic acid - NSF N-ethylmaleimide-sensitive factor Much of the information summarized in this report was presented as a plenary lecture at the XV International Botanical Congress Tokyo, Yokohama, Japan, August 28–September 3, 1993.     

Enhanced HIV-1 neutralization by a CD4-VH3-IgG1 fusion protein

HIV-1 gp120 is an alleged B cell superantigen, binding certain VH3+ human antibodies. We reasoned that a CD4-VH3 fusion protein could possess higher affinity for gp120 and improved HIV-1 inhibitory capacity. To test this we produced several human IgG1 immunoligands harboring VH3. Unlike VH3-IgG1 or VH3-CD4-IgG1, CD4-VH3-IgG1 bound gp120 considerably stronger than CD4-IgG1. CD4-VH3-IgG1 exhibited ≈1.5-2.5-fold increase in neutralization of two T-cell laboratory-adapted strains when compared to CD4-IgG1. CD4-VH3-IgG1 improved neutralization of 7/10 clade B primary isolates or pseudoviruses, exceeding 20-fold for JR-FL and 13-fold for Ba-L. It enhanced neutralization of 4/8 clade C viruses, and had negligible effect on 1/4 clade A pseudoviruses. We attribute this improvement to possible pairing of VH3 with CD4 D1 and stabilization of an Ig Fv-like structure, rather than to superantigen interactions. These novel findings support the current notion that CD4 fusion proteins can act as better HIV-1 entry inhibitors with potential clinical implications.     

Unstimulated primary CD4+ T cells from HIV-1-positive elite suppressors are fully susceptible to HIV-1 entry and productive infection

Elite controllers or suppressors (ES) are a group of HIV-1-infected individuals who maintain viral loads below the limit of detection of commercial assays for many years. The mechanisms responsible for this remarkable control are under intense study, with the hope of developing therapeutic vaccines effective against HIV-1. In this study, we addressed the question of the intrinsic susceptibility of ES CD4(+) T cells to infection. While we and others have previously shown that CD4(+) T cells from ES can be infected by HIV-1 isolates in vitro, these studies were confounded by exogenous activation and in vitro culture of CD4(+) T cells prior to infection. In order to avoid the changes in chemokine receptor expression that have been associated with such exogenous activation, we infected purified CD4(+) T cells directly after isolation from the peripheral blood of ES, viremic patients, and uninfected donors. We utilized a green fluorescent protein (GFP)-expressing proviral construct pseudotyped with CCR5-tropic or CXCR4-tropic envelope to compare viral entry using a fluorescence resonance energy transfer-based, single-round virus-cell fusion assay. The frequency of productive infection was also compared by assessing GFP expression. CD4(+) T cells from ES were as susceptible as or more susceptible than cells from viremic patients and uninfected donors to HIV-1 entry and productive infection. The results of this physiological study strongly suggest that differences in HIV-1 entry and infection of CD4(+) T cells alone cannot explain the elite control of viral replication.