Calcium Flux between the Endoplasmic Reticulum and Mitochondrion Contributes to Poliovirus-Induced Apoptosis

We show that poliovirus (PV) infection induces an increase in cytosolic calcium (Ca²⁺) concentration in neuroblastoma IMR5 cells, at least partly through Ca²⁺ release from the endoplasmic reticulum lumen via the inositol 1,4,5-triphosphate receptor (IP₃R) and ryanodine receptor (RyR) channels. This leads to Ca²⁺ accumulation in mitochondria through the mitochondrial Ca²⁺ uniporter and the voltage-dependent anion channel (VDAC). This increase in mitochondrial Ca²⁺ concentration in PV-infected cells leads to mitochondrial dysfunction and apoptosis.Poliovirus (PV), the prototype member of the Picornaviridae family, is the etiological agent of paralytic poliomyelitis (26, 27). This acute human disease of the central nervous system results from the destruction of motor neurons associated with PV replication. In PV-infected mice, motor neurons die through apoptosis (16). However, the mechanisms involved are poorly understood (5).Apoptosis is an active cell death process triggered by various stimuli, including viral infections (18). This process leads to DNA fragmentation and is triggered by two main pathways (22): (i) the extrinsic pathway, mediated by the activation of cell surface death receptors such as Fas/CD95, and (ii) the intrinsic pathway, characterized notably by mitochondrial membrane permeabilization (MMP). In many models, this process implies a loss of mitochondrial transmembrane potential (Δψ_m) and the release of proapoptotic molecules, including cytochrome c, from the mitochondrial intermembrane space into the cytosol. The apoptotic program initiated by PV infection has been shown to involve mitochondrial dysfunction in several cell lines (2-4, 17).The intrinsic pathway also can originate from the endoplasmic reticulum (ER) (30). The ER participates in protein synthesis and folding, cellular responses to stress, and intracellular calcium (Ca²⁺) homeostasis. Nevertheless, under stress conditions, it may induce apoptosis via several different mechanisms, one of which involves ER cross-talk with mitochondria, mediated by Ca²⁺ release from ER stores through the inositol 1,4,5-triphosphate receptor (IP₃R) and ryanodine receptor (RyR) channels (7, 12, 15). Several recent studies have identified Ca²⁺ signaling as a key cellular target for viral infection (for a review, see reference 8). Upon PV infection, cells display an increase in cytosolic Ca²⁺ concentration (20). Phospholipase C also is activated, leading to an increase in IP₃ concentration in PV-infected cells (19), potentially accounting for the observed increase in cytosolic Ca²⁺ concentration. However, the role of Ca²⁺ efflux from the ER in PV-induced apoptosis has yet to be studied.Here, we postulated that an increase in cytosolic Ca²⁺ following PV infection can have an impact on cell fate and investigated the cellular response in terms of mitochondrial function and apoptosis in neuroblastoma IMR5 cells.     

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.     

Highly Pathogenic H5N1 Avian Influenza Virus Induces Extracellular Ca2+ Influx,Leading to Apoptosis in Avian Cells

In this study, we show that the highly pathogenic H5N1 avian influenza virus (AIV) (A/crow/Kyoto/53/04 and A/chicken/Egypt/CL6/07) induced apoptosis in duck embryonic fibroblasts (DEF). In contrast, apoptosis was reduced among cells infected with low-pathogenic AIVs (A/duck/HK/342/78 [H5N2], A/duck/HK/820/80 [H5N3], A/wigeon/Osaka/1/01 [H7N7], and A/turkey/Wisconsin/1/66 [H9N2]). Thus, we investigated the molecular mechanisms of apoptosis induced by H5N1-AIV infection. Caspase-dependent and -independent pathways contributed to the cytopathic effects. We further showed that, in the induction of apoptosis, the hemagglutinin of H5N1-AIV played a major role and its cleavage sequence was not critical. We also observed outer membrane permeabilization and loss of the transmembrane potential of the mitochondria of infected DEF, indicating that mitochondrial dysfunction was caused by the H5N1-AIV infection. We then analyzed Ca²⁺ dynamics in the infected cells and demonstrated an increase in the concentration of Ca²⁺ in the cytosol ([Ca²⁺]_i) and mitochondria ([Ca²⁺]_m) after H5N1-AIV infection. Regardless, gene expression important for regulating Ca²⁺ efflux from the endoplasmic reticulum did not significantly change after H5N1-AIV infection. These results suggest that extracellular Ca²⁺ may enter H5N1-AIV-infected cells. Indeed, EGTA, which chelates extracellular free Ca²⁺, significantly reduced the [Ca²⁺]_i, [Ca²⁺]_m, and apoptosis induced by H5N1-AIV infection. In conclusion, we identified a novel mechanism for influenza A virus-mediated cell death, which involved the acceleration of extracellular Ca²⁺ influx, leading to mitochondrial dysfunction and apoptosis. These findings may be useful for understanding the pathogenesis of H5N1-AIV in avian species as well as the impact of Ca²⁺ homeostasis on influenza A virus infection.Avian influenza viruses (AIVs) are classified as highly or low-pathogenic AIVs (HPAIVs or LPAIVs, respectively) based on their pathogenicity in chickens (1). HPAIVs cause systemic infections and high mortality in chickens (28), whereas poultry are asymptomatic or develop mild respiratory problems and/or intestinal illness after LPAIV infection (49). Hemagglutinin (HA) cleavability is a critical determinant of AIV pathogenicity in avian species (61). Other determinants, such as nonstructural (NS) protein and neuraminidase (NA) protein, reportedly regulate the virulence of AIVs (9, 29, 44). However, waterfowl, known as the natural host for AIVs, do not usually have any symptoms during an HPAIV infection (21), whereas they show neurologic symptoms and death after infection with some of the recently emerged HPAIVs, such as the Asian H5N1 virus (11, 46, 62). Thus, the entire mechanism responsible for the pathogenicity of the AIVs is not yet known. Unknown cellular and viral factors probably underlie the pathogenesis of HPAIVs in avian species, especially waterfowl.The alveolar epithelial cells (66) or vascular endothelial cells (32) of human patients and chickens infected by H5N1-AIV show apoptosis. Other reports suggest that apoptosis of these cells is essential for the development of acute lung injury in mice and acute respiratory distress syndrome in humans (39), which is often observed in H5N1-AIV-infected patients. Therefore, it is necessary to evaluate whether apoptosis is critical for the pathogenesis of H5N1-AIV in vivo and to understand the molecular mechanisms of the apoptotic cell death induced by H5N1-AIV infection.Ca²⁺ is a key regulator of cell survival, and the breakdown of Ca²⁺ homeostasis, due to sustained elevations in Ca²⁺ inside cells, triggers programmed cell death involving apoptosis (24). Indeed, disruption of Ca²⁺ homeostasis plays a key role in apoptosis during the pathogenic process of several types of viral infections, including those with human immunodeficiency virus (HIV), hepatitis C virus, and human T-cell leukemia virus type 1 (3, 4, 31, 57). In addition, the HIV gp120 envelope protein induces neuronal cell death through Ca²⁺ dysregulation, even in the absence of viral particles (25).In this study, we used duck embryonic fibroblasts (DEF) to elucidate the molecular mechanisms of the apoptotic cell death induced by H5N1-AIV. We show here that H5N1-AIV infection triggered extracellular Ca²⁺ influx and that this alteration in the concentration of Ca²⁺ inside the cells subsequently induced mitochondrial dysfunction and led to apoptotic cell death. In addition, we demonstrate that H5N1-HA was a critical viral factor for inducing apoptosis.     

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.     

Structure and Distribution of Organelles and Cellular Location of Calcium Transporters in Neurospora crassa

We wanted to examine the cellular locations of four Neurospora crassa proteins that transport calcium. However, the structure and distribution of organelles in live hyphae of N. crassa have not been comprehensively described. Therefore, we made recombinant genes that generate translational fusions of putative organellar marker proteins with green or red fluorescent protein. We observed putative endoplasmic reticulum proteins, encoded by grp-78 and dpm, in the nuclear envelope and associated membranes. Proteins of the vacuolar membrane, encoded by vam-3 and vma-1, were in an interconnected network of small tubules and vesicles near the hyphal tip, while in more distal regions they were in large and small spherical vacuoles. Mitochondria, visualized with tagged ARG-4, were abundant in all regions of the hyphae. Similarly, we tagged the four N. crassa proteins that transport calcium with green or red fluorescent protein to examine their cellular locations. NCA-1 protein, a homolog of the SERCA-type Ca²⁺-ATPase of animal cells, colocalized with the endoplasmic reticulum markers. The NCA-2 and NCA-3 proteins are homologs of Ca²⁺-ATPases in the vacuolar membrane in yeast or in the plasma membrane in animal cells. They colocalized with markers in the vacuolar membrane, and they also occurred in the plasma membrane in regions of the hyphae more than 1 mm from the tip. The cax gene encodes a Ca²⁺/H⁺ exchange protein found in vacuoles. As expected, the CAX protein localized to the vacuolar compartment. We observed, approximately 50 to 100 μm from the tip, a few spherical organelles that had high amounts of tagged CAX protein and tagged subunits of the vacuolar ATPase (VMA-1 and VMA-5). We suggest that this organelle, not described previously in N. crassa, may have a role in sequestering calcium.All cells maintain intracellular concentrations of calcium at precise levels, typically about 0.1 μM in the cytosol. Calcium is often present at high levels in the environment, significantly above the level that is tolerated within the cell. Nevertheless, high concentrations are maintained in some organelles because calcium has an essential role in signaling physiological processes (3, 7, 29). In root hairs, pollen tubes, and the hyphae of filamentous fungi calcium has been postulated to have a central role in directing the growth at the tips of these cells (30, 32, 34, 38, 41, 49). Investigators have reported that in filamentous fungi the concentration of calcium is highest at the hyphal tip (56, 59). Disruption of the calcium gradient by ionophores inhibits growth (52). Mutations in some genes that affect hyphal morphology, e.g., frost and spray, can be suppressed by raising the concentration of calcium in the medium (4, 16). However, the growth of wild-type strains is not significantly affected by the external concentration of calcium, which suggests that cytosolic calcium is controlled by regulating calcium uptake and release from organelles (36, 55, 59).The proteins that transport calcium into organelles have been studied extensively in Saccharomyces cerevisiae. In this organism, more than 90% of the intracellular calcium is in the vacuole (19, 22), transported there by a protein that facilitates Ca²⁺/H⁺ exchange,Vcx1p, and by a calcium-pumping ATPase, Pmc1p (13, 14, 47). Another calcium-pumping ATPase, Pmr1p, can transport calcium or manganese into the Golgi bodies (1, 51, 57). S. cerevisiae has not been reported to have a calcium-pumping ATPase in the plasma membrane or a SERCA-type ATPase in the endoplasmic reticulum (ER). Pmr1p may have a dual function in Golgi body- and ER-associated processes (20).In plant and animal cells, three types of calcium-pumping ATPases have been described (3, 7, 9, 58). The PMCA type (most closely related to the Pmc1p ATPase of S. cerevisiae) primarily pumps calcium across the plasma membrane, removing excess calcium from the cytosol. The SERCA type, named by its location in the smooth ER, has a major role in transporting calcium in muscle cells but is also present in the ER in many types of cells. S. cerevisiae has no homolog to the SERCA ATPase. The SPCA type (secretory pathway Ca²⁺-ATPases) is found in the Golgi bodies and is homologous to the Pmr1p ATPase of S. cerevisiae (3, 42). The mitochondria also contain a significant share of intracellular calcium. No calcium-pumping ATPase has been identified in this organelle, and transport has been hypothesized to occur through a channel protein, driven by the same electrochemical gradient that drives the synthesis of ATP (18, 35). In addition, calcium is sequestered in small vesicles and in lysosomelike compartments, presumably transported by Ca^2+/H⁺ exchange proteins (29).The availability of the complete genomes for N. crassa and other filamentous fungi has allowed us to assess the number and types of calcium transport proteins in these organisms (62). Focusing on N. crassa, we found that all three types of calcium-pumping ATPases are present. These genes had been identified earlier in a PCR-based search for P-type ATPases (2). The N. crassa gene nca-1 encodes a SERCA type ATPase. The nca-2 and nca-3 genes are closely related to each other and appear to encode PMCA type ATPases. The pmr gene is a SPCA type. We also identified the cax gene as a homolog of VCX1, the gene encoding the Ca²⁺/H⁺ exchanger that plays a key role in vacuolar transport in S. cerevisiae.Our long-term goal is to use these five genes (nca-1, nca-2, nca-3, pmr, and cax) to find out where calcium is localized in cells and how it gets there. We first wanted to determine the intracellular location of each transporter, using proteins tagged with green and red fluorescent proteins (GFP and RFP, respectively). For N. crassa and most other filamentous fungi a comprehensive analysis of the structure and distribution of organelles in living cells is lacking. GFP and fluorescent dyes have been used successfully to examine nuclei and mitochondria (24, 26). Several reports have shown that “the vacuole” is far more dynamic and complex than the textbook presentation of a large spherical organelle (10, 24, 31, 33, 54). Our understanding of the structure and abundance of the ER and the Golgi body is limited.In this report, we have fused GFP and RFP to proteins predicted to be localized to nuclei, mitochondria, the ER, the Golgi body, and the vacuole. Similarly, we have made GFP- and RFP-tagged forms of each of the five calcium transport proteins described above. We have examined the abundance and structures of the organelles and have observed (as have others) that these change with distance from the hyphal tip. We have tried to determine whether each of the calcium transport proteins is associated with a unique organelle or with the plasma membrane. In studies parallel to those reported here, we are measuring the amount of calcium in cell organelles and characterizing the phenotypes of strains in which the calcium transport genes have been deleted.     

Impact of Cytotoxic-T-Lymphocyte Memory Induction without Virus-Specific CD4+ T-Cell Help on Control of a Simian Immunodeficiency Virus Challenge in Rhesus Macaques

Despite many efforts to develop AIDS vaccines eliciting virus-specific T-cell responses, whether induction of these memory T cells by vaccination before human immunodeficiency virus (HIV) exposure can actually contribute to effective T-cell responses postinfection remains unclear. In particular, induction of HIV-specific memory CD4⁺ T cells may increase the target cell pool for HIV infection because the virus preferentially infects HIV-specific CD4⁺ T cells. However, virus-specific CD4⁺ helper T-cell responses are thought to be important for functional CD8⁺ cytotoxic-T-lymphocyte (CTL) induction in HIV infection, and it has remained unknown whether HIV-specific memory CD8⁺ T cells induced by vaccination without HIV-specific CD4⁺ T-cell help can exert effective responses after virus exposure. Here we show the impact of CD8⁺ T-cell memory induction without virus-specific CD4⁺ T-cell help on the control of a simian immunodeficiency virus (SIV) challenge in rhesus macaques. We developed a prophylactic vaccine by using a Sendai virus (SeV) vector expressing a single SIV Gag_241-249 CTL epitope fused with enhanced green fluorescent protein (EGFP). Vaccination resulted in induction of SeV-EGFP-specific CD4⁺ T-cell and Gag_241-249-specific CD8⁺ T-cell responses. After a SIV challenge, the vaccinees showed dominant Gag_241-249-specific CD8⁺ T-cell responses with higher effector memory frequencies in the acute phase and exhibited significantly reduced viral loads. These results demonstrate that virus-specific memory CD8⁺ T cells induced by vaccination without virus-specific CD4⁺ T-cell help could indeed facilitate SIV control after virus exposure, indicating the benefit of prophylactic vaccination eliciting virus-specific CTL memory with non-virus-specific CD4⁺ T-cell responses for HIV control.Virus-specific T-cell responses are crucial for controlling human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication (3, 4, 12, 20, 28, 36, 37). Therefore, a great deal of effort has been exerted to develop AIDS vaccines eliciting virus-specific T-cell responses (23, 27, 30, 47), but whether this approach actually results in HIV control remains unclear (1, 6). It is important to determine which T-cell responses need to be induced by prophylactic vaccination for HIV control after virus exposure.Because HIV preferentially infects HIV-specific CD4⁺ T cells (5), induction of HIV-specific memory CD4⁺ T cells by vaccination may increase the target cell pool for HIV infection and could enhance viral replication (42). However, CD4⁺ helper T-cell responses are important for functional CD8⁺ cytotoxic-T-lymphocyte (CTL) induction (11, 40, 43, 46), and it has remained unknown whether HIV-specific memory CD8⁺ T cells induced by vaccination with non-virus-specific CD4⁺ T-cell help (but without HIV-specific CD4⁺ T-cell help) can exert effective responses after virus exposure. Indeed, the real impact of prophylactic induction of CTL memory itself on HIV replication has not been well documented thus far.We previously developed a prophylactic AIDS vaccine consisting of DNA priming followed by boosting with a recombinant Sendai virus (SeV) vector expressing SIVmac239 Gag (26). Evaluation of this vaccine''s efficacy against a SIVmac239 challenge in Burmese rhesus macaques showed that some vaccinees contained SIV replication whereas unvaccinated animals developed AIDS (15, 27). In particular, vaccination consistently resulted in control of SIV replication in those animals possessing the major histocompatibility complex class I (MHC-I) haplotype 90-120-Ia. Gag_206-216 (IINEEAADWDL) and Gag_241-249 (SSVDEQIQW) epitope-specific CD8⁺ T-cell responses were shown to be involved in SIV control in these vaccinated macaques (14, 16).In the present study, focusing on CD8⁺ T-cell responses directed against one of these epitopes, we have evaluated the efficacy of a vaccine expressing the Gag_241-249 epitope fused with enhanced green fluorescent protein (EGFP) against a SIVmac239 challenge in 90-120-Ia-positive rhesus macaques. The animals exhibited this single-epitope-specific CD8⁺ T-cell response and SeV-EGFP-specific CD4⁺ T-cell responses after vaccination and showed rapid, dominant induction of potent secondary Gag_241-249-specific CD8⁺ T-cell responses after a SIV challenge. Plasma viral loads in these vaccinees were significantly reduced compared to those of naive controls. These results indicate that induction of CD8⁺ T-cell memory without virus-specific CD4⁺ T-cell help by prophylactic vaccination can result in effective CD8⁺ T-cell responses after virus exposure.     

Isolation of Basal Bodies with C-Ring Components from the Na+-Driven Flagellar Motor of Vibrio alginolyticus

To investigate the Na⁺-driven flagellar motor of Vibrio alginolyticus, we attempted to isolate its C-ring structure. FliG but not FliM copurified with the basal bodies. FliM proteins may be easily dissociated from the basal body. We could detect FliG on the MS ring surface of the basal bodies.The basal body, which is the part of the rotor, is composed of four rings and a rod that penetrates them. Three of these rings, the L, P, and MS rings, are embedded in the outer membrane, peptidoglycan layer and in the inner membrane, respectively (1), while the C-ring of Salmonella species is attached to the cytoplasmic side of the basal body (3). The C-ring is composed of the proteins FliG, FliM, and FliN (25), and genetic evidence indicates that the C-ring is important for flagellar assembly, torque generation, and regulation of rotational direction (33, 34). FliG, 26 molecules of which are incorporated into the motor, appears to be the protein that is most directly involved in torque generation (15). Mutational analysis suggests that electrostatic interactions between conserved charged residues in the C-terminal domain of FliG and the cytoplasmic domain of MotA are important in torque generation (14), although this may not be the case for the Na⁺-type motor of Vibrio alginolyticus (32, 35, 36). FliM interacts with the chemotactic signaling protein CheY in its phosphorylated form (CheY-P) to regulate rotational direction (30). It has been reported that 33 to 35 copies of FliM assemble into a ring structure (28, 29). FliN contributes mostly to forming the C-ring structure (37). The crystal structure of FliN revealed a hydrophobic patch formed by several well-conserved hydrophobic residues (2). Mutational analysis showed that this patch is important for flagellar assembly and rotational switching (23, 24). The association state of FliN in solution was studied by analytical ultracentrifugation, which provided clues to the higher-level organization of the protein. Thermotoga maritima FliN exists primarily as a dimer in solution, and T. maritima FliN and FliM together formed a stable FliM₁-FliN₄ complex (2). The spatial distribution of these proteins in the C-ring of Salmonella species was investigated using three-dimensional reconstitution analysis with electron microscopy (28). However, the correct positioning has still not been clarified.The Na⁺-driven motor requires two additional proteins, MotX and MotY, for torque generation (19-21, 22). These proteins form a unique ring structure, the T ring, located below the LP ring in the polar flagellum of V. alginolyticus (9, 26). It has been suggested that MotX interacts with MotY and PomB (11, 27). Unlike peritrichously flagellated Escherichia coli and Salmonella species, V. alginolyticus has two different flagellar systems adapted for locomotion under different circumstances. A single, sheathed polar flagellum is used for motility in low-viscosity environments such as seawater (18). As described above, it is driven by a Na⁺-type motor. However, in high-viscosity environments, such as the mucus-coated surfaces of fish bodies, cells induce numerous unsheathed lateral flagella that have H⁺-driven motors (7, 8). We have been focusing on the Na⁺-driven polar flagellar motor, since there are certain advantages to studying its mechanism of torque generation over the H⁺-type motor: sodium motive force can be easily manipulated by controlling the Na⁺ concentration in the medium, and motor rotation can be specifically inhibited using phenamil (10). Moreover, its rotation rate is surprisingly high, up to 1,700 rps (compared to ∼200 rps and ∼300 rps for Salmonella species flagella and E. coli flagella, respectively) (12, 16, 17).Although understanding the C-ring structure and function is essential for clarifying the mechanism of motor rotation, there is no information about the C-ring of the polar flagellar motor of Vibrio species or the flagella of any genus other than Salmonella. Since Vibrio species have all of the genes coding for C-ring components, we would expect its location to be on the cytoplasmic side of the MS ring, as in Salmonella species. In this study, we attempted to isolate the polar flagellar basal body with the C-ring attached and investigate whether it is organized similarly to the H⁺-driven flagellar motor of Salmonella enterica serovar Typhimurium.     

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.     

Trafficking of UL37 Proteins into Mitochondrion-Associated Membranes during Permissive Human Cytomegalovirus Infection

Human cytomegalovirus (HCMV) UL37 proteins traffic sequentially from the endoplasmic reticulum (ER) to the mitochondria. In transiently transfected cells, UL37 proteins traffic into the mitochondrion-associated membranes (MAM), the site of contact between the ER and mitochondria. In HCMV-infected cells, the predominant UL37 exon 1 protein, pUL37x1, trafficked into the ER, the MAM, and the mitochondria. Surprisingly, a component of the MAM calcium signaling junction complex, cytosolic Grp75, was increasingly enriched in heavy MAM from HCMV-infected cells. These studies show the first documented case of a herpesvirus protein, HCMV pUL37x1, trafficking into the MAM during permissive infection and HCMV-induced alteration of the MAM protein composition.The human cytomegalovirus (HCMV) UL37 immediate early (IE) locus expresses multiple products, including the predominant UL37 exon 1 protein, pUL37x1, also known as viral mitochondrion-localized inhibitor of apoptosis (vMIA), during lytic infection (16, 22, 24, 39, 44). The UL37 glycoprotein (gpUL37) shares UL37x1 sequences and is internally cleaved, generating pUL37_NH2 and gpUL37_COOH (2, 22, 25, 26). pUL37x1 is essential for the growth of HCMV in humans (17) and for the growth of primary HCMV strains (20) and strain AD169 (14, 35, 39, 49) but not strain TownevarATCC in permissive human fibroblasts (HFFs) (27).pUL37x1 induces calcium (Ca²⁺) efflux from the endoplasmic reticulum (ER) (39), regulates viral early gene expression (5, 10), disrupts F-actin (34, 39), recruits and inactivates Bax at the mitochondrial outer membrane (MOM) (4, 31-33), and inhibits mitochondrial serine protease at late times of infection (28).Intriguingly, HCMV UL37 proteins localize dually in the ER and in the mitochondria (2, 9, 16, 17, 24-26). In contrast to other characterized, similarly localized proteins (3, 6, 11, 23, 30, 38), dual-trafficking UL37 proteins are noncompetitive and sequential, as an uncleaved gpUL37 mutant protein is ER translocated, N-glycosylated, and then imported into the mitochondria (24, 26).Ninety-nine percent of ∼1,000 mitochondrial proteins are synthesized in the cytosol and directly imported into the mitochondria (13). However, the mitochondrial import of ER-synthesized proteins is poorly understood. One potential pathway is the use of the mitochondrion-associated membrane (MAM) as a transfer waypoint. The MAM is a specialized ER subdomain enriched in lipid-synthetic enzymes, lipid-associated proteins, such as sigma-1 receptor, and chaperones (18, 45). The MAM, the site of contact between the ER and the mitochondria, permits the translocation of membrane-bound lipids, including ceramide, between the two organelles (40). The MAM also provides enriched Ca²⁺ microdomains for mitochondrial signaling (15, 36, 37, 43, 48). One macromolecular MAM complex involved in efficient ER-to-mitochondrion Ca²⁺ transfer is comprised of ER-bound inositol 1,4,5-triphosphate receptor 3 (IP3R3), cytosolic Grp75, and a MOM-localized voltage-dependent anion channel (VDAC) (42). Another MAM-stabilizing protein complex utilizes mitofusin 2 (Mfn2) to tether ER and mitochondrial organelles together (12).HCMV UL37 proteins traffic into the MAM of transiently transfected HFFs and HeLa cells, directed by their NH₂-terminal leaders (8, 47). To determine whether the MAM is targeted by UL37 proteins during infection, we fractionated HCMV-infected cells and examined pUL37x1 trafficking in microsomes, mitochondria, and the MAM throughout all temporal phases of infection. Because MAM domains physically bridge two organelles, multiple markers were employed to verify the purity and identity of the fractions (7, 8, 19, 46, 47).(These studies were performed in part by Chad Williamson in partial fulfillment of his doctoral studies in the Biochemistry and Molecular Genetics Program at George Washington Institute of Biomedical Sciences.)HFFs and life-extended (LE)-HFFs were grown and not infected or infected with HCMV (strain AD169) at a multiplicity of 3 PFU/cell as previously described (8, 26, 47). Heavy (6,300 × g) and light (100,000 × g) MAM fractions, mitochondria, and microsomes were isolated at various times of infection and quantified as described previously (7, 8, 47). Ten- or 20-μg amounts of total lysate or of subcellular fractions were resolved by SDS-PAGE in 4 to 12% Bis-Tris NuPage gels (Invitrogen) and examined by Western analyses (7, 8, 26). Twenty-microgram amounts of the fractions were not treated or treated with proteinase K (3 μg) for 20 min on ice, resolved by SDS-PAGE, and probed by Western analysis. The blots were probed with rabbit anti-UL37x1 antiserum (DC35), goat anti-dolichyl phosphate mannose synthase 1 (DPM1), goat anti-COX2 (both from Santa Cruz Biotechnology), mouse anti-Grp75 (StressGen Biotechnologies), and the corresponding horseradish peroxidase-conjugated secondary antibodies (8, 47). Reactive proteins were detected by enhanced chemiluminescence (ECL) reagents (Pierce), and images were digitized as described previously (26, 47).     

The KtrA and KtrE Subunits Are Required for Na+-Dependent K+ Uptake by KtrB across the Plasma Membrane in Synechocystis sp. Strain PCC 6803

The Na⁺-dependent K⁺ uptake KtrABE system is essential for the adaptation of Synechocystis to salinity stress and high osmolality. While KtrB forms the K⁺-translocating pore, the role of the subunits KtrA and KtrE for Ktr function remains elusive. Here, we characterized the role of KtrA and KtrE in Ktr-mediated K⁺ uptake and in modulating Na⁺ dependency. Expression of KtrB alone in a K⁺ uptake-deficient Escherichia coli strain conferred low K⁺ uptake activity that was not stimulated by Na⁺. Coexpression of both KtrA and KtrE with KtrB increased the K⁺ transport activity in a Na⁺-dependent manner. KtrA and KtrE were found to be localized to the plasma membrane in Synechocystis. Site-directed mutagenesis was used to analyze the role of single charged residues in KtrB for Ktr function. Replacing negatively charged residues facing the extracellular space with residues of the opposite charge increased the apparent K_m for K⁺ in all cases. However, none of the mutations eliminated the Na⁺ dependency of Ktr-mediated K⁺ transport. Mutations of residues on the cytoplasmic side had larger effects on K⁺ uptake activity than those of residues on the extracellular side. Further analysis revealed that replacement of R262, which is well conserved among Ktr/Trk/HKT transporters in the third extracellular loop, by Glu abolished transport activity. The atomic-scale homology model indicated that R262 might interact with E247 and D261. Based on these data, interaction of KtrA and KtrE with KtrB increased the K⁺ uptake rate and conferred Na⁺ dependency.Cyanobacterium Synechocystis sp. strain PCC 6803 contains a number of different K⁺ uptake systems that may contribute to satisfying its requirement of K⁺ (3, 19, 36). Among these systems, Ktr has been shown to have a major role not only in K⁺ uptake but also in adaptation against high-osmolarity stress (3, 19). Inactivation of the ktr gene renders the cells hypersensitive to high concentrations of NaCl and the nonionic compound sorbitol. Ktr-mediated K⁺ uptake depends on the presence of Na⁺ in the medium, which is likely to be an adaptation to salinity stress. A requirement of Na⁺ for K⁺ transport activity has also been found in the homologous protein from Vibrio alginolyticus (21). This dependency on Na⁺ is a unique property of Ktr-type transporters and has not been found in other types of K⁺ transporters or channels (32). The structure and function of Ktr-type transporters have been studied in a number of organisms (3, 6, 7, 9, 11-14, 18-20, 30, 32-34). The Ktr system from Synechocystis consists of three subunits, KtrA, KtrB, and KtrE (19). The KtrE gene and the KtrB gene form a cistron, whereas the KtrA gene resides at a site distant from the KtrEB genes in the Synechocystis genome (19). KtrB, the K⁺-translocating subunit, is a member of the Ktr/Trk/HKT family of K⁺ transporters. These transporters have been proposed to have evolved from two membrane-spanning K⁺ channels (6, 7). According to the model, this type of transporter contains eight transmembrane domains, which consist of a 4-fold-repeated membrane-pore-membrane (M1-P-M2) motif (6, 7, 13, 18). An intramolecular electrostatic interaction of Synechocystis KtrB has been proposed to stabilize the protein in its active configuration (12). In addition, a conserved His in the external region in Synechocystis KtrB has been shown to be crucial for KtrB function (39). The region of the Vibrio Ktr protein responsible for gating of ion permeation has been identified (9). However, not much is known about the mechanism of Na⁺ binding to KtrB in Synechocystis.The KtrA subunit belongs to the family of KTR (K⁺-transport nucleotide binding)/RCK (regulating the conductance of K⁺ channels) proteins, which contain a Rossmann-fold sequence encoding β-α protein structure for NAD⁺/NADH binding (17). Accordingly KtrA has been proposed to regulate the K⁺ transport activity of KtrB by changing its binding from NAD⁺ to NADH through a ligand-mediated conformational switch mechanism (25). It has also been shown that ATP promotes complex formation between KtrA and KtrB and that KtrAB from V. alginolyticus when expressed in Escherichia coli cells requires both ATP and the membrane potential for its activity (17).KtrE is a unique subunit found only in Synechocystis; it is not involved in KtrB-mediated K⁺ transport in V. alginolyticus and Bacillus subtilis (11, 32). The termination codon of ktrE overlaps the initiation codon of ktrB in the same cistron, which has not been found in other bacterial ktrB-related genes. Coexpression of KtrA with KtrB alone does not complement the growth defect of an E. coli K⁺ uptake mutant. However, introduction of KtrE into the same mutant background in addition to KtrA and KtrB complements the mutation of the K⁺ uptake system (19). Interestingly, the KtrE protein has been shown to function as a digalactosyldiacylglycerol (DGDG) synthase (EC 2.4.6.241), an enzyme that produces DGDG from monogalactosyldiacylglycerol (MGDG). KtrE has therefore also been designated DgdA (1). Under nonstress conditions, DGDG is found in the thylakoid membranes, which helps stabilize the photosystem II complex in Synechocystis (29). Under phosphate-limited conditions, DGDG is synthesized instead of phospholipids in Synechocystis (1). However, KtrB functions as a major K⁺-conducting transport pore in the Synechocystis plasma membrane. The subcellular localization of KtrE has not been identified directly. Inactivation of ktrE (also called dgdA) in Synechocystis does not result in sensitivity to osmotic stress imposed by 300 mM sorbitol (1). This may be inconsistent with the requirement of KtrE for KtrB-mediated K⁺ uptake in the presence of KtrA in the E. coli expression system (19).Because of these uncertainties about the roles of the KtrA and KtrE subunits in K⁺ uptake by KtrB in Synechocystis and about the identity of the Na⁺ binding site in KtrB, we examined the subcellular localization and membrane association of KtrA and KtrE, the requirement of these subunits for KtrB-mediated K⁺ uptake, and the primary target for Na⁺ binding in KtrB.     

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.     

Impact of HLA in Mother and Child on Disease Progression of Pediatric Human Immunodeficiency Virus Type 1 Infection

A broad Gag-specific CD8⁺ T-cell response is associated with effective control of adult human immunodeficiency virus (HIV) infection. The association of certain HLA class I molecules, such as HLA-B*57, -B*5801, and -B*8101, with immune control is linked to mutations within Gag epitopes presented by these alleles that allow HIV to evade the immune response but that also reduce viral replicative capacity. Transmission of such viruses containing mutations within Gag epitopes results in lower viral loads in adult recipients. In this study of pediatric infection, we tested the hypothesis that children may tend to progress relatively slowly if either they themselves possess one of the protective HLA-B alleles or the mother possesses one of these alleles, thereby transmitting a low-fitness virus to the child. We analyzed HLA type, CD8⁺ T-cell responses, and viral sequence changes for 61 mother-child pairs from Durban, South Africa, who were monitored from birth. Slow progression was significantly associated with the mother or child possessing one of the protective HLA-B alleles, and more significantly so when the protective allele was not shared by mother and child (P = 0.007). Slow progressors tended to make CD8⁺ T-cell responses to Gag epitopes presented by the protective HLA-B alleles, in contrast to progressors expressing the same alleles (P = 0.07; Fisher''s exact test). Mothers expressing the protective alleles were significantly more likely to transmit escape variants within the Gag epitopes presented by those alleles than mothers not expressing those alleles (75% versus 21%; P = 0.001). Reversion of transmitted escape mutations was observed in all slow-progressing children whose mothers possessed protective HLA-B alleles. These data show that HLA class I alleles influence disease progression in pediatric as well as adult infection, both as a result of the CD8⁺ T-cell responses generated in the child and through the transmission of low-fitness viruses by the mother.Human immunodeficiency virus (HIV)-specific CD8⁺ T cells play a central role in controlling viral replication (12). It is the specificity of the CD8⁺ T-cell response, particularly the response to Gag, that is associated with low viral loads in HIV infection (7, 17, 34). Although immune control is undermined by the selection of viral mutations that prevent recognition by the CD8⁺ T cells, evasion of Gag-specific responses mediated by protective class I HLA-B alleles typically brings a reduction in viral replicative capacity, facilitating subsequent immune control of HIV (2, 20, 21). The same principle has been demonstrated in studies of simian immunodeficiency virus infection (18, 22).Recent studies showed that the class I HLA-B alleles that protect against disease progression present more Gag-specific CD8⁺ T-cell epitopes and drive the selection of more Gag-specific escape mutations than those alleles that are associated with high viral loads (23). These protective HLA-B alleles not only are beneficial to infected individuals expressing those alleles but also benefit a recipient following transmission, since the transmitted virus carrying multiple Gag escape mutations may have substantially reduced fitness (3, 4, 8). However, there is no benefit to the recipient if he or she shares the same protective allele as the donor because the transmitted virus carries escape mutations in the Gag epitopes that would otherwise be expected to mediate successful immune control in the recipient (8, 11).The sharing of HLA alleles between donor and recipient occurs frequently in mother-to-child transmission (MTCT). The risk of MTCT is related to viral load in the mother, and a high viral load is associated with nonprotective alleles, such as HLA-B*18 and -B*5802. This may contribute in two distinct ways to the more rapid progression observed in pediatric HIV infection (24, 26, 27). First, because infected children share 50% or more of their HLA alleles with the transmitting mother, they are less likely than adults to carry protective HLA alleles (16). Thus, infected children as a group carry fewer protective HLA alleles and more nonprotective HLA alleles. Second, even when the child has a protective allele, such as HLA-B*27, this allele does not offer protection if the maternally transmitted virus carries escape mutations within the key Gag epitopes that are presented by the protective allele (11, 19).However, it is clear that infected children who possess protective alleles, such as HLA-B*27 or HLA-B*57, can achieve durable immune control of HIV infection if the virus transmitted from the mother is not preadapted to those alleles (6, 10). HIV-specific CD8⁺ T-cell responses are detectable from birth in infected infants (32). Furthermore, as in adult infection (3, 8), HIV-infected children have the potential to benefit from transmission of low-fitness viruses in the situation where the mother possesses protective HLA alleles and the child does not share those protective alleles. MTCT of low-fitness viruses carrying CD8⁺ T-cell escape mutations was recently documented (28; J. Prado et al., unpublished data).In this study, undertaken in Durban, South Africa, we set out to test the hypothesis that HIV-infected children are less likely to progress rapidly to disease if either the infected child or the transmitting mother possesses a protective HLA allele that is not shared. The HLA alleles most strongly associated with low viral loads and high CD4 counts in a cohort of >1,200 HIV-infected adults in Durban are HLA-B*57 (-B*5702 and -B*5703), HLA-B*5801, and HLA-B*8101 (16; A. Leslie et al., unpublished data). These four alleles all present Gag-specific CD8⁺ T-cell epitopes, and in each case the escape mutations selected in these epitopes reduce viral replicative capacity (2-4, 8, 21, 23).Analyzing a previously described cohort of 61 HIV-infected children in Durban (24, 26, 32), South Africa, who were all monitored from birth, we first addressed the question of whether possession of any of these four alleles by either mother or child is associated with slower disease progression in the child and then determined whether sharing of protective alleles by mother and child affects the ability of the child to make the Gag-specific CD8⁺ T-cell responses restricted by the shared allele.     

Identification of a Novel Class of Membrane-Bound [NiFe]-Hydrogenases in Thermococcus onnurineus NA1 by In Silico Analysis

In silico analysis of group 4 [NiFe]-hydrogenases from a hyperthermophilic archaeon, Thermococcus onnurineus NA1, revealed a novel tripartite gene cluster consisting of dehydrogenase-hydrogenase-cation/proton antiporter subunits, which may be classified as the new subgroup 4b of [NiFe]-hydrogenases-based on sequence motifs.Hydrogenases are the key enzymes involved in the metabolism of H₂, catalyzing the following chemical reaction: 2H⁺ + 2e⁻ ↔ H₂. Hydrogenases can be classified into [NiFe]-hydrogenases, [FeFe]-hydrogenases, and [Fe]-hydrogenases, based on their distinctive functional core containing the catalytic metal center (11, 17).The genomic analysis of Thermococcus onnurineus NA1, a hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent area, revealed the presence of several distinct gene clusters encoding seven [NiFe]-hydrogenases and one homolog similar to Mbx (membrane-bound oxidoreductase) from Pyrococcus furiosus (1, 6, 8, 12). According to the classification system of hydrogenases by Vignais et al. (17), three hydrogenases (one F₄₂₀-reducing and two NADP-reducing hydrogenases) belong to group 3 [NiFe]-hydrogenases, and four hydrogenases belong to group 4 [NiFe]-hydrogenases. The group 4 hydrogenases are widely distributed among bacteria and archaea (17), with Hyc and Hyf (hydrogenase 3 and 4, respectively) from Escherichia coli (19), Coo (CO-induced hydrogenase) from Rhodospirillum rubrum (4), Ech (energy-converting hydrogenase) from Methanosarcina barkeri (7), and Mbh (membrane-bound hydrogenase) from P. furiosus (6, 10, 12) being relatively well-characterized hydrogenases in this group. One of the four group 4 hydrogenases from T. onnurineus NA1 was found to be similar in sequence to that of P. furiosus Mbh (10).