Sulfur Isotope Enrichment during Maintenance Metabolism in the Thermophilic Sulfate-Reducing Bacterium Desulfotomaculum putei

Values of Δ³⁴S (, where δ³⁴S_HS and indicate the differences in the isotopic compositions of the HS⁻ and SO₄²⁻ in the eluent, respectively) for many modern marine sediments are in the range of −55 to −75‰, much greater than the −2 to −46‰ ɛ³⁴S (kinetic isotope enrichment) values commonly observed for microbial sulfate reduction in laboratory batch culture and chemostat experiments. It has been proposed that at extremely low sulfate reduction rates under hypersulfidic conditions with a nonlimited supply of sulfate, isotopic enrichment in laboratory culture experiments should increase to the levels recorded in nature. We examined the effect of extremely low sulfate reduction rates and electron donor limitation on S isotope fractionation by culturing a thermophilic, sulfate-reducing bacterium, Desulfotomaculum putei, in a biomass-recycling culture vessel, or “retentostat.” The cell-specific rate of sulfate reduction and the specific growth rate decreased progressively from the exponential phase to the maintenance phase, yielding average maintenance coefficients of 10⁻¹⁶ to 10⁻¹⁸ mol of SO₄ cell⁻¹ h⁻¹ toward the end of the experiments. Overall S mass and isotopic balance were conserved during the experiment. The differences in the δ³⁴S values of the sulfate and sulfide eluting from the retentostat were significantly larger, attaining a maximum Δ³⁴S of −20.9‰, than the −9.7‰ observed during the batch culture experiment, but differences did not attain the values observed in marine sediments.Dissimilatory SO₄²⁻ reduction is a geologically ancient, anaerobic, energy-yielding metabolic process during which SO₄²⁻-reducing bacteria (SRB) reduce SO₄²⁻ to H₂S while oxidizing organic molecules or H₂. SO₄²⁻ reduction is a dominant pathway for organic degradation in marine sediments (23) and in terrestrial subsurface settings where sulfur-bearing minerals dominate over Fe³⁺-bearing minerals. For example, at depths greater than 1.5 km below land surface in the fractured sedimentary and igneous rocks of the Witwatersrand Basin of South Africa, SO₄²⁻ reduction is the dominant electron-accepting process (3, 26, 46, 48, 61).The enrichment of ³²S in biogenic sulfides, with respect to the parent SO₄²⁻, imparted by SRB, is traceable through the geologic record (10, 54). The magnitude of the Δ³⁴S (= δ³⁴S_pyrite − δ³⁴S_{barite/gypsum}, where δ³⁴S_pyrite and δ³⁴S_{barite/gypsum} are the isotopic compositions of pyrite and barite or gypsum) increases from −10‰ in the 3.47-billion-year-old North Pole deposits to −30‰ in late-Archaean deposits (55), to −75‰ in Neoproterozoic to modern sulfide-bearing marine sediments (13).The kinetic isotopic enrichment, ɛ³⁴S, deduced from trends in the δ³⁴S values of SO₄²⁻ and HS⁻ in batch culture microbial SO₄²⁻ reduction experiments using the Rayleigh relationship, ranges from −2‰ to −46‰ (6, 7, 11, 17, 22, 27, 28, 30, 31, 38, 39). The variation in ɛ³⁴S values has been attributed to the SO₄²⁻ concentration, the type of electron donor and its concentration, the SO₄²⁻ reduction rate per cell (csSRR) (22), temperature, and species-specific isotope enrichment effects. In these laboratory experiments, doubling times are on the order of hours and csSRRs range from to 0.1 to 18 fmol cell⁻¹ h⁻¹ (7, 12, 17, 22, 30, 32, 39, 40).Experiments performed during the 1960s found that the magnitude of ɛ³⁴S was inversely proportional to the csSRR for organic electron donors (16, 31, 38, 39) when SO₄²⁻ was not limiting. More-recent batch culture experiments on 3 psychrophilic (optimum growth temperature, <20°C) and mesophilic (optimum growth temperature, between 20°C and 45°C) SRB strains (7) and on 32 psychrophilic to thermophilic SRB strains (22), however, have failed to reproduce such a relationship. In 2001, Canfield (11) reported an inverse correlation between ɛ³⁴S and reduction rate using a flowthrough sediment column and demonstrated that ɛ³⁴S values of approximately −35 to −40‰ were produced when organic substrates added by way of amendment were limited with respect to SO₄²⁻. Because it was not possible to readily evaluate changes in biomass in the sediment column with changes in temperature or substrate provision rate, it was inferred that changes in ɛ³⁴S were related to changes in the csSRRs. More recently, Canfield et al. (12) observed a 6‰ variation in ɛ³⁴S values related to the temperature of the batch culture experiments relative to the optimum growth temperature. The few early experiments that were performed using H₂ as the electron donor yielded ɛ³⁴S values ranging from −3 to −19‰ (22, 38, 39), which appear to correlate with the csSRR (39). Hoek et al. (32) also found that the ɛ³⁴S values for the thermophilic SO₄²⁻ reducer Thermodesulfatator indicus increased from between −1.5‰ and −10‰ in batch cultures with high H₂ concentrations to between −24‰ and −37‰ in batch cultures grown under H₂ limitation with respect to SO₄²⁻. Detmers et al. (22) found that the average ɛ³⁴S of SRB that oxidize their organic carbon electron donor completely to CO₂ averaged −25‰, versus −9.5‰ for SRB that release acetate during their oxidation of their organic carbon electron donor. Detmers et al. (22) speculated that the greater free energy yield per mole of SO₄²⁻ from incomplete carbon oxidation relative to that for complete carbon oxidation promotes complete SO₄²⁻ reduction and hinders isotopic enrichment due to isotopic exchange of the intracellular sulfur species pools.None of these experiments, however, have yielded ɛ³⁴S factors capable of producing the Δ³⁴S values of −55 to −75‰ observed in the geological record from ∼1.0 billion years ago to today. Various schemes have been hypothesized, and observations that involve either the disproportionation of S₂O₃²⁻ (36), the disproportionation of S⁰ produced by oxidation of either H₂S or S₂O₃²⁻ (15), or the disproportionation of SO₃²⁻ (29) have been made. Attribution of the increasing Δ³⁴S values recorded for Achaean to Neoproterozoic sediments to the increasing role of H₂S oxidative pathways makes sense in the context of increasing O₂ concentrations in the atmosphere (14) but is not consistent with the lack of significant fractionation observed during oxidative reactions (29). To explain the Δ³⁴S values of −55 to −77‰ reported to occur in interstitial pore waters from 100- to 300-m-deep, hypersulfidic ocean sediments (51, 64, 67), where the presence of a S-oxidative cycle is unlikely, an alternative, elaborate model of the SO₄²⁻-reducing pathway has been proposed by Brunner and Bernasconi (9). This model attributes the large Δ³⁴S values to a multistep, reversible reduction of SO₃²⁻ to HS⁻ involving S₃O₆²⁻ and S₂O₃²⁻ (20, 25, 41, 42, 52, 66). The conditions under which the maximum ɛ³⁴S values might be expressed are a combination of elevated HS⁻ concentrations, electron donor limitations, nonlimiting SO₄²⁻ concentrations, and a very low csSRR. The csSRR for subsurface environments has been estimated from biogeochemical-flux modeling to be 10⁻⁶ to 10⁻⁷ fmol cell⁻¹ h⁻¹ (23), with a corresponding cell turnover rate greater than 1,000 years (37).Batch and chemostat culture systems, despite low growth rates, cannot completely attain a state of zero growth with constant substrate provision and therefore do not accurately reflect the in situ nutritional states of microbes in many natural settings. Retentostats, or recycling fermentor vessels, recycle 100% of biomass to the culturing vessel, allowing experimenters to culture microbial cells to a large biomass with a constant nutrient supply rate until the substrate supply rate itself becomes the growth-limiting factor and cells enter a resting state in which their specific growth rate approaches zero and they carry on maintenance metabolism (1, 47, 53, 58, 59, 62, 63). Utilizing this approach, Colwell et al. (18) were able to obtain a cell-specific respiration rate of 7 × 10⁻⁴ fmol of CH₄ cell⁻¹ h⁻¹ for a mesophilic marine methanogen, a rate that is comparable to that estimated for methanogenic communities in deep marine sediments off the coast of Peru (49).In this study, the conditions that Brunner and Bernasconi (9) hypothesized would lead to the large Δ³⁴S values seen in nature were recreated in the laboratory by limiting the electron donor supply rate with respect to the SO₄²⁻ supply rate in a retentostat vessel. The S isotopic enrichment by a resting culture of Desulfotomaculum putei at an extremely low csSRR was compared to that of a batch culture experiment to determine whether the ɛ³⁴S values produced under the former conditions approach the Δ³⁴S seen in nature.     

Metabolic Engineering of the Tricarboxylic Acid Cycle for Improved Lysine Production by Corynebacterium glutamicum

In the present work, lysine production by Corynebacterium glutamicum was improved by metabolic engineering of the tricarboxylic acid (TCA) cycle. The 70% decreased activity of isocitrate dehydrogenase, achieved by start codon exchange, resulted in a >40% improved lysine production. By flux analysis, this could be correlated to a flux shift from the TCA cycle toward anaplerotic carboxylation.With an annual market volume of more than 1,000,000 tons, lysine is one of the dominating products in biotechnology. In recent years, rational metabolic engineering has emerged as a powerful tool for lysine production (16, 18, 22). Hereby, different target enzymes and pathways in the central metabolism could be identified and successfully modified to create superior production strains (1, 2, 5, 8, 10, 17-20). The tricarboxylic acid (TCA) cycle has not been rationally engineered so far, despite its major role in Corynebacterium glutamicum (6). From metabolic flux studies, however, it seems that the TCA cycle might offer a great potential for optimization (Fig. ​(Fig.1),1), which is also predicted from in silico pathway analysis (13, 22). Experimental evidence comes from studies with Brevibacterium flavum exhibiting increased lysine production due to an induced bottleneck toward the TCA cycle (21). In the present work, we performed TCA cycle engineering by downregulation of isocitrate dehydrogenase (ICD). ICD is the highest expressed TCA cycle enzyme in C. glutamicum (7). Downregulation was achieved by start codon exchange, controlling ICD expression on the level of translation.Open in a separate windowFIG. 1.Stoichiometric correlation of lysine yield (%), biomass yield (g/mol) and TCA cycle flux (%; entry flux through citrate synthase) determined by ¹³C metabolic flux analysis achieved by paraboloid fitting of the data set (parameters were determined with Y0 = 105.1, a = −1.27, b = 0.35, c = −9.35 × 10⁻³, d = −11.16 × 10⁻³). The data displayed represent values from 18 independent experiments with different C. glutamicum strains taken from previous studies (1-3, 11, 12, 15, 23).     

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.     

Adsorption of Extracellular Chromosomal DNA and Its Effects on Natural Transformation of Azotobacter vinelandii

To better understand the influence of environmental conditions on the adsorption of extracellular chromosomal DNA and its availability for natural transformation, the amount and conformation of adsorbed DNA were monitored under different conditions in parallel with transformation assays using the soil bacterium Azotobacter vinelandii. DNA adsorption was monitored using the technique of quartz crystal microbalance with dissipation (QCM-D). Both silica and natural organic matter (NOM) surfaces were evaluated in solutions containing either 100 mM NaCl or 1 mM CaCl₂. The QCM-D data suggest that DNA adsorbed to silica surfaces has a more compact and rigid conformation in Ca²⁺ solution than in Na⁺ solution and that the reverse is true when DNA is adsorbed to NOM surfaces. While the amounts of DNA adsorbed on a silica surface were similar for Ca²⁺ and Na⁺ solutions, the amount of DNA adsorbed on an NOM-coated surface was higher in Ca²⁺ solution than in Na⁺ solution. Transformation frequencies for dissolved DNA and DNA adsorbed to silica and to NOM were 6 × 10⁻⁵, 5 × 10⁻⁵, and 2.5 × 10⁻⁴, respectively. For NOM-coated surfaces, transformation frequencies from individual experiments were 2- to 50-fold higher in the presence of Ca²⁺ than in the presence of Na⁺. The results suggest that groundwater hardness (i.e., Ca²⁺ concentration) will affect the amount of extracellular DNA adsorbed to the soil surface but that neither adsorption nor changes in the conformation of the adsorbed DNA will have a strong effect on the frequency of natural transformation of A. vinelandii.Horizontal gene transfer contributes to microbial evolution and provides mechanisms for the spread of both antimicrobial resistance genes and genetically engineered DNA. While most studies on horizontal gene transfer focus on conjugation, recent reviews on extracellular DNA (16, 27) document the need to consider also natural transformation. The amount of extracellular DNA in the soil is on the order of hundreds of ng/g of dry soil (27). Extracellular DNA adsorbs to many common soil constituents, including sand, clay, and natural organic matter (NOM) (16, 27), and once adsorbed may persist for days or years (16).As first reported by Lorenz et al. (18), adsorbed DNA is available for natural transformation and therefore represents a potential environmental reservoir facilitating horizontal gene transfer. DNA adsorbed on sand surfaces has been successfully transferred to both Gram-positive and Gram-negative soil bacteria, including Bacillus subtilis (18), Pseudomonas stutzeri (19), and Acinetobacter calcoaceticus (4). Other studies have focused on transformation with DNA adsorbed to other surfaces (for example, clay minerals [15], humic acids [6], and intact soils [25, 31]). In most previous studies, adsorbed DNA transformed at a lower frequency than dissolved DNA (see, for example, references 4 and 11). However, higher transformation frequencies for adsorbed DNA than for dissolved DNA have been reported in two studies (18, 19). In addition, Demaneche et al. were unable to detect Pseudomonas fluorescens transformants in a variety of liquid media despite successful transformations in sterile soil columns (8).Several studies have evaluated the influence of nutrients (24, 34) and soil (8, 11, 23, 31) on transformation efficiency. Less information is available on the influence of adsorbed DNA conformation on transformation efficiency. Pietramellara et al. speculated that the decrease in transformation rates they observed upon repeated wetting and drying cycles of adsorbed DNA was due to conformational changes (28). Cai et al. also speculated that differences in the conformation of adsorbed DNA could be responsible for lower transformation efficiencies for DNA bound to kaolinte and inorganic clays (2), based on their previous work characterizing adsorption to different surfaces (3). Detailed characterizations of the conformation of adsorbed DNA only recently became feasible, and the influence of the conformation of adsorbed DNA on transformation frequencies has not, to our knowledge, been systematically investigated.Characterization of the mass and conformation of DNA adsorbed on different surfaces can be accomplished using quartz crystal microbalance with dissipation (QCM-D) (7, 22). QCM-D measurements are based on the shift in frequency (ΔF) and the decay in vibrating energy (ΔD) that occur as molecules adsorb to piezoelectric sensors (29, 33). Viscosity, elastic shear modulus, and effective thickness of the adsorbed material can be determined by fitting the frequency and dissipation data to the viscoelastic Voigt model (7). Based on Nguyen and colleagues'' previous work with plasmid DNA adsorption on silica and NOM-coated surfaces, increasing electrolyte concentrations and the presence of divalent cations favor DNA adsorption (20-22). In addition, inner sphere complexation by Ca²⁺ with DNA phosphate backbone allows the formation of DNA-adsorbed layers that are more compact than the DNA-adsorbed layer formed by charge shielding in solution with a high Na⁺ concentration (20-22).The objective of this study was to investigate both the adsorption of chromosomal DNA to representative soil particle surfaces and the effect of such adsorption on natural transformation. We used QCM-D to characterize the conformation of adsorbed DNA on silica and NOM surfaces under two different solution chemistries (100 mM Na⁺ and 1 mM Ca²⁺). The influences of adsorption and of differences in the conformation of the adsorbed DNA on transformation frequencies were tested in a common soil bacterium, Azotobacter vinelandii. A. vinelandii is naturally competent (26) but had not been previously reported to be transformed with adsorbed DNA.     

Activation of the Inositol (1,4,5)-Triphosphate Calcium Gate Receptor Is Required for HIV-1 Gag Release

The structural precursor polyprotein, Gag, encoded by all retroviruses, including the human immunodeficiency virus type 1 (HIV-1), is necessary and sufficient for the assembly and release of particles that morphologically resemble immature virus particles. Previous studies have shown that the addition of Ca²⁺ to cells expressing Gag enhances virus particle production. However, no specific cellular factor has been implicated as mediator of Ca²⁺ provision. The inositol (1,4,5)-triphosphate receptor (IP3R) gates intracellular Ca²⁺ stores. Following activation by binding of its ligand, IP3, it releases Ca²⁺ from the stores. We demonstrate here that IP3R function is required for efficient release of HIV-1 virus particles. Depletion of IP3R by small interfering RNA, sequestration of its activating ligand by expression of a mutated fragment of IP3R that binds IP3 with very high affinity, or blocking formation of the ligand by inhibiting phospholipase C-mediated hydrolysis of the precursor, phosphatidylinositol-4,5-biphosphate, inhibited Gag particle release. These disruptions, as well as interference with ligand-receptor interaction using antibody targeted to the ligand-binding site on IP3R, blocked plasma membrane accumulation of Gag. These findings identify IP3R as a new determinant in HIV-1 trafficking during Gag assembly and introduce IP3R-regulated Ca²⁺ signaling as a potential novel cofactor in viral particle release.Assembly of the human immunodeficiency virus (HIV) is determined by a single gene that encodes a structural polyprotein precursor, Gag (71), and may occur at the plasma membrane or within late endosomes/multivesicular bodies (LE/MVB) (7, 48, 58; reviewed in reference 9). Irrespective of where assembly occurs, the assembled particle is released from the plasma membrane of the host cell. Release of Gag as virus-like particles (VLPs) requires the C-terminal p6 region of the protein (18, 19), which contains binding sites for Alix (60, 68) and Tsg101 (17, 37, 38, 41, 67, 68). Efficient release of virus particles requires Gag interaction with Alix and Tsg101. Alix and Tsg101 normally function to sort cargo proteins to LE/MVB for lysosomal degradation (5, 15, 29, 52). Previous studies have shown that addition of ionomycin, a calcium ionophore, and CaCl₂ to the culture medium of cells expressing Gag or virus enhances particle production (20, 48). This is an intriguing observation, given the well-documented positive role for Ca²⁺ in exocytotic events (33, 56). It is unclear which cellular factors might regulate calcium availability for the virus release process.Local and global elevations in the cytosolic Ca²⁺ level are achieved by ion release from intracellular stores and by influx from the extracellular milieu (reviewed in reference 3). The major intracellular Ca²⁺ store is the endoplasmic reticulum (ER); stores also exist in MVB and the nucleus. Ca²⁺ release is regulated by transmembrane channels on the Ca²⁺ store membrane that are formed by tetramers of inositol (1,4,5)-triphosphate receptor (IP3R) proteins (reviewed in references 39, 47, and 66). The bulk of IP3R channels mediate release of Ca²⁺ from the ER, the emptying of which signals Ca²⁺ influx (39, 51, 57, 66). The few IP3R channels on the plasma membrane have been shown to be functional as well (13). Through proteomic analysis, we identified IP3R as a cellular protein that was enriched in a previously described membrane fraction (18) which, in subsequent membrane floatation analyses, reproducibly cofractionated with Gag and was enriched in the membrane fraction only when Gag was expressed. That IP3R is a major regulator of cytosolic calcium concentration (Ca²⁺) is well documented (39, 47, 66). An IP3R-mediated rise in cytosolic Ca²⁺ requires activation of the receptor by a ligand, inositol (1,4,5)-triphosphate (IP3), which is produced when phospholipase C (PLC) hydrolyzes phosphatidylinositol-4,5-bisphosphate [PI(4,5)P₂] at the plasma membrane (16, 25, 54). Paradoxically, PI(4,5)P₂ binds to the matrix (MA) domain in Gag (8, 55, 59), and the interaction targets Gag to PI(4,5)P₂-enriched regions on the plasma membrane; these events are required for virus release (45). We hypothesized that PI(4,5)P₂ binding might serve to target Gag to plasma membrane sites of localized Ca²⁺ elevation resulting from PLC-mediated PI(4,5)P₂ hydrolysis and IP3R activation. This idea prompted us to investigate the role of IP3R in Gag function.Here, we show that HIV-1 Gag requires steady-state levels of IP3R for its efficient release. Three isoforms of IP3R, types 1, 2, and 3, are encoded in three independent genes (39, 47). Types 1 and 3 are expressed in a variety of cells and have been studied most extensively (22, 39, 47, 73). Depletion of the major isoforms in HeLa or COS-1 cells by small interfering RNA (siRNA) inhibited viral particle release. Moreover, we show that sequestration of the IP3R activating ligand or blocking ligand formation also inhibited Gag particle release. The above perturbations, as well as interfering with receptor expression or activation, led to reduced Gag accumulation at the cell periphery. The results support the conclusion that IP3R activation is required for efficient HIV-1 viral particle release.     

Combined Gel Probe and Isotope Labeling Technique for Measuring Dissimilatory Nitrate Reduction to Ammonium in Sediments at Millimeter-Level Resolution

Dissimilatory NO₃⁻ reduction in sediments is often measured in bulk incubations that destroy in situ gradients of controlling factors such as sulfide and oxygen. Additionally, the use of unnaturally high NO₃⁻ concentrations yields potential rather than actual activities of dissimilatory NO₃⁻ reduction. We developed a technique to determine the vertical distribution of the net rates of dissimilatory nitrate reduction to ammonium (DNRA) with minimal physical disturbance in intact sediment cores at millimeter-level resolution. This allows DNRA activity to be directly linked to the microenvironmental conditions in the layer of NO₃⁻ consumption. The water column of the sediment core is amended with ¹⁵NO₃⁻ at the in situ ¹⁴NO₃⁻ concentration. A gel probe is deployed in the sediment and is retrieved after complete diffusive equilibration between the gel and the sediment pore water. The gel is then sliced and the NH₄⁺ dissolved in the gel slices is chemically converted by hypobromite to N₂ in reaction vials. The isotopic composition of N₂ is determined by mass spectrometry. We used the combined gel probe and isotopic labeling technique with freshwater and marine sediment cores and with sterile quartz sand with artificial gradients of ¹⁵NH₄⁺. The results were compared to the NH₄⁺ microsensor profiles measured in freshwater sediment and quartz sand and to the N₂O microsensor profiles measured in acetylene-amended sediments to trace denitrification.Nitrate accounts for the eutrophication of many human-affected aquatic ecosystems (19, 21). Sediment bacteria may mitigate NO₃⁻ pollution by denitrification and anaerobic ammonium oxidation (anammox), which produce N₂ (13, 18). However, inorganic nitrogen is retained in aquatic ecosystems when sediment bacteria reduce NO₃⁻ to NH₄⁺ by dissimilatory nitrate reduction to ammonium (DNRA) (5, 12, 16, 39). Hence, DNRA contributes to rather than counteracts eutrophication (23). DNRA may be the dominant pathway of dissimilatory NO₃⁻ reduction in sediments that are rich in electron donors, such as labile organic carbon and sulfide (4, 8, 17, 38, 55). High rates of DNRA are thus found in sediments affected by coastal aquaculture (8, 36) and settling algal blooms (16).DNRA, denitrification, and the chemical factors that control the partitioning between them (e.g., sulfide) should ideally be investigated in undisturbed sediments. The redox stratification of sediments involves vertical concentration gradients of pore water solutes. These gradients are often very steep, and their measurement requires high-resolution techniques, such as microsensors (26, 42) and gel probes (9, 54). If, for instance, the influence of sulfide on DNRA and denitrification is to be investigated, one wants to know exactly the sulfide concentration in the layers of DNRA and denitrification activity, as well as the flux of sulfide into these layers. This information can easily be obtained using H₂S and pH microsensors (22, 43). It is less trivial to determine the vertical distribution of DNRA and denitrification activity in undisturbed sediments. Denitrification activity can be traced using a combination of the acetylene inhibition technique (51) and N₂O microsensors (1). Acetylene inhibits the last step of denitrification, and therefore, N₂O accumulates in the layer of denitrification activity (44). This method underestimates the denitrification activity in sediments with high rates of coupled nitrification-denitrification because acetylene also inhibits nitrification (50).The vertical distribution of DNRA activity in undisturbed sediment has, to the best of our knowledge, never been determined; thus, the microenvironmental conditions in the layer of DNRA activity remain unknown. Until now, the influence of chemical factors on DNRA and denitrification in sediments has been assessed by slurry incubations (4, 12, 30), by flux measurements with sealed sediment cores (7, 47) or flowthrough sediment cores (16, 27, 37), and in one case, in reconstituted sediment cores sliced at centimeter-level resolution (39). Here, we present a new method, the combined gel probe and isotope labeling technique, to determine the vertical distribution of the net rates of DNRA in sediments. The sediments remain largely undisturbed and the NO₃⁻ amendments are within the range of in situ concentrations. The DNRA measurements can be related to the microprofiles of potential influencing factors measured in close vicinity of the gel probe. This allows DNRA activity to be directly linked with the microenvironmental conditions in the sediment.     

Virus-Bacterium Coupling Driven by both Turbidity and Hydrodynamics in an Amazonian Floodplain Lake

The importance of viruses in aquatic ecosystem functioning has been widely described. However, few studies have examined tropical aquatic ecosystems. Here, we evaluated for the first time viruses and their relationship with other planktonic communities in an Amazonian freshwater ecosystem. Coupling between viruses and bacteria was studied, focusing both on hydrologic dynamics and anthropogenic forced turbidity in the system (Lake Batata). Samples were taken during four hydrologic seasons at both natural and impacted sites to count virus-like particles (VLP) and bacteria. In parallel, virus-infected bacteria were identified and quantified by transmission electron microscopy (TEM). Viral abundance ranged from 0.5 × 10⁷ ± 0.2 × 10⁷ VLP ml⁻¹ (high-water season, impacted site) to 1.7 × 10⁷ ± 0.4 × 10⁷ VLP ml⁻¹ (low-water season, natural site). These data were strongly correlated with the bacterial abundance (r² = 0.84; P < 0.05), which ranged from 1.0 × 10⁶ ± 0.5 × 10⁶ cells ml⁻¹ (high water, impacted site) to 3.4 × 10⁶ ± 0.7 × 10⁶ cells ml⁻¹ (low water, natural site). Moreover, the viral abundance was weakly correlated with chlorophyll a, suggesting that most viruses were bacteriophages. TEM quantitative analyses revealed that the frequency of visibly infected cells was 20%, with 10 ± 3 phages per cell section. In general, we found a low virus-bacterium ratio (<7). Both the close coupling between the viral and bacterial abundances and the low virus-bacterium ratio suggest that viral abundance tends to be driven by the reduction of hosts for viral infection. Our results demonstrate that viruses are controlled by biological substrates, whereas in addition to grazing, bacteria are regulated by physical processes caused by turbidity, which affect underwater light distribution and dissolved organic carbon availability.Viruses are the most abundant and dynamic components of the aquatic microbial community (6, 31, 32). Viruses influence many biogeochemical and ecological processes, including nutrient cycling, system respiration, particle-size distribution, bacterial and algal biodiversity, species distribution, algal blooms, and genetic transfer between microorganisms (21, 49). In addition, viruses play a major role in aquatic microbial food webs by releasing carbon trapped in host cells to the dissolved organic carbon (DOC) pool and ultimately back to the bacterial community (11, 21). The action of viruses is an important mechanism of bacterial regulation in aquatic ecosystems, acting directly on bacterial populations and indirectly on bacterial diversity by decreasing the density of dominant bacterial species (31). Studies based on viral decay rates and electron microscopy analyses have shown that viruses can cause up to 40% of bacterial mortality and more than 10% of phytoplankton mortality in aquatic systems (11, 22, 48, 50, 54, 55). It also has been suggested that viral lysis and protistan grazing cause similar bacterial mortality in aquatic ecosystems (22, 40).Several environmental factors, including solar radiation and temperature, can influence viral abundance. Exposure to solar radiation decreases viral abundance in aquatic ecosystems, while low temperatures decrease their virulence (33, 56). However, the majority of the studies on virus ecology have been performed in temperate or polar regions, where seasonal changes in solar radiation and water temperature are more pronounced (26, 30, 32). Viral abundances have been little investigated in tropical aquatic ecosystems (6, 39) and particularly in the Amazonian region, where the abundance and activity of aquatic viruses have not been studied.The greatest watershed in the world is located in the Amazonian region. It is composed of clear-water, black-water, and turbid freshwater ecosystems, which are seasonally influenced by the flood pulse. The hydrologic pulse is characterized by a pronounced change in water level, defining the flood seasons. Nutrient sources and stocks and species dynamics vary according to the water level (25). During the high-water season (flood season), the tight connection between terrestrial and aquatic environments results in an increase in allochthonous DOC input and the dilution of inorganic nutrients and organisms. During the low-water season, there is an increase in nutrient concentrations, organism abundances, and the importance of autochthonous DOC. These seasonal changes differently impact ecosystem functions and aquatic community dynamics (5, 9, 18). For instance, bacterioplankton abundance is less changeable than phytoplankton abundance throughout the hydrological cycle due to the alternative sources of DOC (allochthonous in the high-water period and autochthonous in the low-water period) for bacterial communities (5, 24).Lake Batata is a clear-water Amazonian floodplain lake located in the watershed of the Trombetas River, a tributary of the Amazon River. As a clear-water Amazonian ecosystem, it contains low concentrations of suspended particles and inorganic nutrients (47). Lake Batata is distinct because it was impacted by bauxite tailings for 10 years (1979 to 1989), affecting 30% of the lake''s area. The tailings caused a huge increase in turbidity; large amounts of tailings settled on the sediment surface and often are resuspended by physical mixing or biotic movements (28). The presence of tailings resulted in a clear spatial variation in the lake, forming impacted and natural sites. Furthermore, tailing particles can directly act as a substrate for attaching bacteria and also can adsorb DOC (5).Previous studies on bacterio-, phyto-, and zooplankton communities have shown that flood pulse acts as the primary driver of plankton community structure in Lake Batata (5, 9, 24). Bauxite tailings also affect microbial processes in impacted sites, such as bacterial growth and production (5), photosynthesis rates and primary production (43), or the availability of food for zooplankton (8). However, there still is no evidence indicating a complementary (flood pulse and forced turbidity) effect among these factors in any microbial community. Based on published data, we assume that (i) bacterioplankton abundance is less changeable through the hydrologic cycle than phytoplankton abundance (5, 24), and (ii) tailing particles can act as a substrate for attaching bacteria and also can adsorb organic matter, which is controlled by the flood pulse (5). Therefore, we hypothesized that the relationship between viruses and bacteria in Lake Batata is modulated by a synergistic effect between the hydrological cycle and turbidity.     

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.     

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.     

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

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