Protein Tyrosine Kinase 6 Directly Phosphorylates AKT and Promotes AKT Activation in Response to Epidermal Growth Factor

Protein tyrosine kinase 6 (PTK6) is a nonmyristoylated Src-related intracellular tyrosine kinase. Although not expressed in the normal mammary gland, PTK6 is expressed in a majority of human breast tumors examined, and it has been linked to ErbB receptor signaling and AKT activation. Here we demonstrate that AKT is a direct substrate of PTK6 and that AKT tyrosine residues 315 and 326 are phosphorylated by PTK6. Association of PTK6 with AKT occurs through the SH3 domain of PTK6 and is enhanced through SH2 domain-mediated interactions following tyrosine phosphorylation of AKT. Using Src, Yes, and Fyn null mouse embryonic fibroblasts (SYF cells), we show that PTK6 phosphorylates AKT in a Src family kinase-independent manner. Introduction of PTK6 into SYF cells sensitized these cells to physiological levels of epidermal growth factor (EGF) and increased AKT activation. Stable introduction of active PTK6 into SYF cells also resulted in increased proliferation. Knockdown of PTK6 in the BPH-1 human prostate epithelial cell line led to decreased AKT activation in response to EGF. Our data indicate that in addition to promoting growth factor receptor-mediated activation of AKT, PTK6 can directly activate AKT to promote oncogenic signaling.Protein tyrosine kinase 6 (PTK6; also known as the breast tumor kinase BRK) is an intracellular Src-related tyrosine kinase (9, 48). Human PTK6 was identified in cultured human melanocytes (32) and breast tumor cells (39), while its mouse orthologue was cloned from normal small intestinal epithelial cell RNA (50). Although PTK6 shares overall structural similarity with Src family tyrosine kinases, it lacks an N-terminal myristoylation consensus sequence for membrane targeting (39, 51). As a consequence, PTK6 is localized to different cellular compartments, including the nucleus (14, 15). PTK6 is expressed in normal differentiated epithelial cells of the gastrointestinal tract (34, 42, 51), prostate (14), and skin (51-53). Expression of PTK6 is upregulated in different types of cancers, including breast carcinomas (6, 39, 54), colon cancer (34), ovarian cancer (47), head and neck cancers (33), and metastatic melanoma cells (16). The significance of apparent opposing signaling roles for PTK6 in normal differentiation and cancer is still poorly understood.In human breast tumor cells, PTK6 enhances signaling from members of the ErbB receptor family (10, 29, 30, 36, 40, 49, 54). In the HB4a immortalized human mammary gland luminal epithelial cell line, PTK6 promoted epidermal growth factor (EGF)-induced ErbB3 tyrosine phosphorylation and AKT activation (29). In response to EGF stimulation, PTK6 promoted phosphorylation of the focal adhesion protein paxillin and Rac1-mediated cell migration (10). PTK6 can be activated by the ErbB3 ligand heregulin and promotes activation of extracellular signal-regulated kinase 5 (ERK5) and p38 mitogen-activated protein kinase (MAPK) in breast cancer cells (40). PTK6 can also phosphorylate p190RhoGAP-A and stimulate its activity, leading to RhoA inactivation and Ras activation and thereby promoting EGF-dependent breast cancer cell migration and proliferation (49). Expression of PTK6 has been correlated with ErbB2 expression in human breast cancers (4, 5, 54).AKT (also called protein kinase B) is a serine-threonine kinase that is activated downstream of growth factor receptors (38). It is a key player in signaling pathways that regulate energy metabolism, proliferation, and cell survival (7, 45). Aberrant activation of AKT through diverse mechanisms has been discovered in different cancers (2). AKT activation requires phosphorylation of AKT on threonine residue 308 and serine residue 473. The significance of phosphorylation of AKT on tyrosine residues is less well understood. Src has been shown to phosphorylate AKT on conserved tyrosine residues 315 and 326 near the activation loop (11). Substitution of these two tyrosine residues with phenylalanine abolished AKT kinase activity stimulated by EGF (11). Use of the Src family inhibitor PP2 impaired AKT activation following IGF-1 stimulation of oligodendrocytes (13). The RET/PTC receptor tyrosine kinase that responds to glial cell-line-derived neurotrophic factor also phosphorylated AKT tyrosine residue 315 promoting activation of AKT (28). AKT tyrosine residue 474 was phosphorylated when cells were treated with the tyrosine phosphatase inhibitor pervanadate, and phosphorylation of tyrosine 474 contributed to full activation of AKT (12). Recently, the nonreceptor tyrosine kinase Ack1 was shown to regulate AKT tyrosine phosphorylation and activation (37).Here we show that AKT is a cytoplasmic substrate of the intracellular tyrosine kinase PTK6. We identify the tyrosine residues on AKT that are targeted by PTK6, and we demonstrate that tyrosine phosphorylation plays a role in regulating association between PTK6 and AKT. In addition, we show that PTK6 promotes AKT activation and cell proliferation in a Src-independent manner.     

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.     

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.     

Quantitative Fluorescence Resonance Energy Transfer Microscopy Analysis of the Human Immunodeficiency Virus Type 1 Gag-Gag Interaction: Relative Contributions of the CA and NC Domains and Membrane Binding

The human immunodeficiency virus type 1 structural polyprotein Pr55^Gag is necessary and sufficient for the assembly of virus-like particles on cellular membranes. Previous studies demonstrated the importance of the capsid C-terminal domain (CA-CTD), nucleocapsid (NC), and membrane association in Gag-Gag interactions, but the relationships between these factors remain unclear. In this study, we systematically altered the CA-CTD, NC, and the ability to bind membrane to determine the relative contributions of, and interplay between, these factors. To directly measure Gag-Gag interactions, we utilized chimeric Gag-fluorescent protein fusion constructs and a fluorescence resonance energy transfer (FRET) stoichiometry method. We found that the CA-CTD is essential for Gag-Gag interactions at the plasma membrane, as the disruption of the CA-CTD has severe impacts on FRET. Data from experiments in which wild-type (WT) and CA-CTD mutant Gag molecules are coexpressed support the idea that the CA-CTD dimerization interface consists of two reciprocal interactions. Mutations in NC have less-severe impacts on FRET between normally myristoylated Gag proteins than do CA-CTD mutations. Notably, when nonmyristoylated Gag interacts with WT Gag, NC is essential for FRET despite the presence of the CA-CTD. In contrast, constitutively enhanced membrane binding eliminates the need for NC to produce a WT level of FRET. These results from cell-based experiments suggest a model in which both membrane binding and NC-RNA interactions serve similar scaffolding functions so that one can functionally compensate for a defect in the other.The human immunodeficiency virus type 1 (HIV-1) structural precursor polyprotein Pr55^Gag is necessary and sufficient for the assembly of virus-like particles (VLPs). Gag is composed of four major structural domains, matrix (MA), capsid (CA), nucleocapsid (NC), and p6, as well as two spacer peptides, SP1 and SP2 (3, 30, 94). Following particle assembly and release, cleavage by HIV-1 protease separates these domains. However, these domains must work together in the context of the full-length Gag polyprotein to drive particle assembly.Previous studies have mapped two major functional domains involved in the early steps of assembly: first, Gag associates with cellular membranes via basic residues and N-terminal myristoylation of the MA domain (10, 17, 20, 35, 39, 87, 91, 106); second, the Gag-Gag interaction domains that span the CA C-terminal domain (CA-CTD) and NC domain promote Gag multimerization (3, 11, 14, 16, 18, 23, 27, 29, 30, 33, 36, 46, 64, 88, 94, 102, 103). Structural and genetic studies have identified two residues (W184 and M185) within a dimerization interface in the CA-CTD that are critical to CA-CA interactions (33, 51, 74, 96). Analytical ultracentrifugation of heterodimers formed between wild-type (WT) Gag and Gag mutants with changes at these residues suggests that the dimerization interface consists of two reciprocal interactions, one of which can be disrupted to form a “half-interface” (22).In addition to the CA-CTD, NC contributes to assembly via 15 basic residues (8, 9, 11, 14, 18, 23, 25, 28, 34, 40, 43, 54, 57, 58, 74, 79, 88, 97, 104, 105), although some researchers have suggested that NC instead contributes to the stability of mature virions after assembly (75, 98, 99). It is thought that the contribution of NC to assembly is due to its ability to bind RNA, since the addition of RNA promotes the formation of particles in vitro (14-16, 37, 46), and RNase treatment disrupts Gag-Gag interactions (11) and immature viral cores (67). However, RNA is not necessary per se, since dimerization motifs can substitute for NC (1, 4, 19, 49, 105). This suggests a model in which RNA serves a structural role, such as a scaffold, to promote Gag-Gag interactions through NC. Based on in vitro studies, it has been suggested that this RNA scaffolding interaction facilitates the low-order Gag multimerization mediated by CA-CTD dimerization (4, 37, 49, 62, 63, 85). Despite a wealth of biochemical data, the relative contributions of the CA-CTD and NC to Gag multimerization leading to assembly are yet to be determined in cells.Mutations in Gag interaction domains alter membrane binding in addition to affecting Gag multimerization. In particular, mutations or truncations of CA reduce membrane binding (21, 74, 82), and others previously reported that mutations or truncations of NC affect membrane binding (13, 78, 89, 107). These findings are consistent with a myristoyl switch model of membrane binding in which Gag can switch between high- and low-membrane-affinity states (38, 71, 76, 83, 86, 87, 92, 95, 107). Many have proposed, and some have provided direct evidence (95), that Gag multimerization mediated by CA or NC interactions promotes the exposure of the myristoyl moiety to facilitate membrane associations.Gag membrane binding and multimerization appear to be interrelated steps of virus assembly, since membrane binding also facilitates Gag multimerization. Unlike betaretroviruses that fully assemble prior to membrane targeting and envelopment (type B/D), lentiviruses, such as HIV, assemble only on cellular membranes at normal Gag expression levels (type C), although non-membrane-bound Gag complexes exist (45, 58, 60, 61, 65). Consistent with this finding, mutations that reduce Gag membrane associations cause a defect in Gag multimerization (59, 74). Therefore, in addition to their primary effects on Gag-Gag interactions, mutations in Gag interaction domains cause a defect in membrane binding, which, in turn, causes a secondary multimerization defect. To determine the relative contributions of the CA-CTD and the NC domain to Gag-Gag interactions at the plasma membrane, it is essential to eliminate secondary effects due to a modulation of membrane binding.Except for studies using a His-tag-mediated membrane binding system (5, 46), biochemical studies of C-type Gag multimerization typically lack membranes. Therefore, these studies do not fully represent particle assembly, which occurs on biological membranes in cells. Furthermore, many biochemical and structural approaches are limited to isolated domains or truncated Gag constructs. Thus, some of these studies are perhaps more relevant to the behavior of protease-cleaved Gag in mature virions. With few exceptions (47, 74), cell-based studies of Gag multimerization have typically been limited to measuring how well mutant Gag is incorporated into VLPs when coexpressed or not with WT Gag. Since VLP production is a complex multistep process, effects of mutations on other steps in the process can confound this indirect measure. For example, NC contributes to VLP production by both promoting multimerization and interacting with the host factor ALIX to promote VLP release (26, 80). To directly assay Gag multimerization in cells, several groups (24, 45, 52, 56) developed microscopy assays based on fluorescence resonance energy transfer (FRET). These assays measure the transfer of energy between donor and acceptor fluorescent molecules that are brought within ∼5 nm by the association of the proteins to which they are attached (41, 48, 90). However, these microscopy-based Gag FRET assays have not been used to fully elucidate several fundamental aspects of HIV-1 Gag multimerization at the plasma membrane of cells, such as the relative contributions of the CA-CTD and NC and the effect of membrane binding on Gag-Gag interactions. In this study, we used a FRET stoichiometry method based on calibrated spectral analysis of fluorescence microscopy images (41). This algorithm determines the fractions of both donor and acceptor fluorescent protein-tagged Gag molecules participating in FRET. For cells expressing Gag molecules tagged with donor (cyan fluorescent protein [CFP]) and acceptor (yellow fluorescent protein [YFP]) molecules, this method measures the apparent FRET efficiency, which is proportional to the mole fraction of Gag constructs in complex. By measuring apparent FRET efficiencies, quantitative estimates of the mole fractions of interacting proteins can be obtained.Using this FRET-based assay, we aim to answer two questions: (i) what are the relative contributions of CA-CTD and NC domains to Gag multimerization when secondary effects via membrane binding are held constant, and (ii) what is the effect of modulating membrane binding on the ability of Gag mutants to interact with WT Gag?Our data demonstrate that the CA-CTD dimerization interface is essential for Gag multimerization at the plasma membrane, as fully disrupting the CA-CTD interaction abolishes FRET, whereas a modest level of FRET is still detected in the absence of NC. We also present evidence that the CA-CTD dimerization interface consists of two reciprocal interactions, allowing the formation of a half-interface that can still contribute to Gag multimerization. Notably, when Gag derivatives with an intact CA-CTD were coexpressed with WT Gag, either membrane binding ability or NC was required for the Gag mutants to interact with WT Gag, suggesting functional compensation between these factors.     

Cultivation of Fastidious Bacteria by Viability Staining and Micromanipulation in a Soil Substrate Membrane System

Soil substrate membrane systems allow for microcultivation of fastidious soil bacteria as mixed microbial communities. We isolated established microcolonies from these membranes by using fluorescence viability staining and micromanipulation. This approach facilitated the recovery of diverse, novel isolates, including the recalcitrant bacterium Leifsonia xyli, a plant pathogen that has never been isolated outside the host.The majority of bacterial species have never been recovered in the laboratory (1, 14, 19, 24). In the last decade, novel cultivation approaches have successfully been used to recover “unculturables” from a diverse range of divisions (23, 25, 29). Most strategies have targeted marine environments (4, 23, 25, 32), but soil offers the potential for the investigation of vast numbers of undescribed species (20, 29). Rapid advances have been made toward culturing soil bacteria by reformulating and diluting traditional media, extending incubation times, and using alternative gelling agents (8, 21, 29).The soil substrate membrane system (SSMS) is a diffusion chamber approach that uses extracts from the soil of interest as the growth substrate, thereby mimicking the environment under investigation (12). The SSMS enriches for slow-growing oligophiles, a proportion of which are subsequently capable of growing on complex media (23, 25, 27, 30, 32). However, the SSMS results in mixed microbial communities, with the consequent difficulty in isolation of individual microcolonies for further characterization (10).Micromanipulation has been widely used for the isolation of specific cell morphotypes for downstream applications in molecular diagnostics or proteomics (5, 15). This simple technology offers the opportunity to select established microcolonies of a specific morphotype from the SSMS when combined with fluorescence visualization (3, 11). Here, we have combined the SSMS, fluorescence viability staining, and advanced micromanipulation for targeted isolation of viable, microcolony-forming soil bacteria.     

Distinct Molecular Pathways to X4 Tropism for a V3-Truncated Human Immunodeficiency Virus Type 1 Lead to Differential Coreceptor Interactions and Sensitivity to a CXCR4 Antagonist

During the course of infection, transmitted HIV-1 isolates that initially use CCR5 can acquire the ability to use CXCR4, which is associated with an accelerated progression to AIDS. Although this coreceptor switch is often associated with mutations in the stem of the viral envelope (Env) V3 loop, domains outside V3 can also play a role, and the underlying mechanisms and structural basis for how X4 tropism is acquired remain unknown. In this study we used a V3 truncated R5-tropic Env as a starting point to derive two X4-tropic Envs, termed ΔV3-X4A.c5 and ΔV3-X4B.c7, which took distinct molecular pathways for this change. The ΔV3-X4A.c5 Env clone acquired a 7-amino-acid insertion in V3 that included three positively charged residues, reestablishing an interaction with the CXCR4 extracellular loops (ECLs) and rendering it highly susceptible to the CXCR4 antagonist AMD3100. In contrast, the ΔV3-X4B.c7 Env maintained the V3 truncation but acquired mutations outside V3 that were critical for X4 tropism. In contrast to ΔV3-X4A.c5, ΔV3-X4B.c7 showed increased dependence on the CXCR4 N terminus (NT) and was completely resistant to AMD3100. These results indicate that HIV-1 X4 coreceptor switching can involve (i) V3 loop mutations that establish interactions with the CXCR4 ECLs, and/or (ii) mutations outside V3 that enhance interactions with the CXCR4 NT. The cooperative contributions of CXCR4 NT and ECL interactions with gp120 in acquiring X4 tropism likely impart flexibility on pathways for viral evolution and suggest novel approaches to isolate these interactions for drug discovery.For human immunodeficiency virus type I (HIV-1) to enter a target cell, the gp120 subunit of the viral envelope glycoprotein (Env) must engage CD4 and a coreceptor on the cell surface. Although numerous coreceptors have been identified in vitro, the two most important coreceptors in vivo are the CCR5 (3, 11, 19, 22, 24) and CXCR4 (27) chemokine receptors. HIV-1 variants that can use only CCR5 (R5 viruses) are critical for HIV-1 transmission and predominate during the early stages of infection (86, 90). The importance of CCR5 for HIV-1 transmission is underscored by the fact that individuals bearing a homozygous 32-bp deletion in the CCR5 gene (ccr5-Δ32) are largely resistant to HIV-1 infection (15, 49, 84). Although R5 viruses typically persist into late disease stages, viruses that can use CXCR4, either alone (X4 viruses) or in addition to CCR5 (R5X4 viruses), emerge in approximately 50% of individuals infected with subtype B or D viruses (12, 39, 44). Although not required for disease progression, the appearance of X4 and/or R5X4 viruses is associated with a more rapid depletion of CD4⁺ cells in peripheral blood and faster progression to AIDS (12, 44, 77, 86). However, it remains unclear whether these viruses are a cause or a consequence of accelerated CD4⁺ T cell decline (57). The emergence of CXCR4-using viruses has also complicated the use of small-molecule CCR5 antagonists as anti-HIV-therapeutics as these compounds can select for the outgrowth of X4 or R5X4 escape variants (93).Following triggering by CD4, gp120 binds to a coreceptor via two principal interactions: (i) the bridging sheet, a four-stranded antiparallel beta sheet that connects the inner and outer domains of gp120, together with the base of the V3 loop, engages the coreceptor N terminus (NT); and (ii) more distal regions of V3 interact with the coreceptor extracellular loops (ECLs) (13, 14, 36-38, 43, 59, 60, 78, 79, 88). Although both the NT and ECL interactions are important for coreceptor binding and entry, their relative contributions vary among different HIV-1 strains (23). For example, V3 interactions with the ECLs, particularly ECL2, serve a dominant role in CXCR4 utilization (7, 21, 50, 63, 72), while R5 viruses exhibit a more variable use of CCR5 domains, with the NT interaction being particularly important (4, 6, 20, 67, 83). Although V3 is the primary determinant of coreceptor preference (34), it is unclear how specificity for CCR5 and/or CXCR4 is determined, and, in particular, it is unknown how X4 tropism is acquired. Several reports have shown that the emergence of X4 tropism correlates with the acquisition of positively charged residues in the V3 stem (17, 29, 87), particularly at positions 11, 24, and 25 (8, 17, 28, 29, 42, 75), raising the possibility that these mutations directly or indirectly mediate interactions with negatively charged residues in the CXCR4 ECLs. However, Env domains outside V3, including V1/V2 (9, 32, 45, 46, 61, 64, 65, 80, 95) and even gp41 (40), can also contribute to coreceptor switching, and it is unclear mechanistically or structurally how X4 tropism is determined.We previously derived a replication-competent variant of the R5X4 HIV-1 clone R3A that contained a markedly truncated V3 loop (47). This Env was generated by introducing a mutation termed ΔV3(9,9), which deleted the distal 15 amino acids of V3. The ΔV3(9,9) mutation selectively ablated X4 tropism but left R5 tropism intact, consistent with the view that an interaction between the distal half of V3 and the ECLs is critical for CXCR4 usage (7, 21, 43, 50, 59, 60, 63, 72). This V3-truncated virus provided a unique opportunity to address whether CXCR4 utilization could be regained on a background in which this critical V3-ECL interaction had been ablated and, if so, by what mechanism. Here, we characterize two novel X4 variants of R3A ΔV3(9,9) derived by adapting this virus to replicate in CXCR4⁺ CCR5⁻ SupT1 cells. We show that R3A ΔV3(9,9) could indeed reacquire X4 tropism but through two markedly different mechanisms. One X4 variant, designated ΔV3-X4A, acquired changes in the V3 remnant that reestablished an interaction with the CXCR4 ECLs; the other, ΔV3-X4B, acquired changes outside V3 that engendered interactions with the CXCR4 NT. These divergent evolutionary pathways led to profound differences in sensitivity to the CXCR4 antagonist AMD3100, with ΔV3-X4A showing increased sensitivity relative to R3A and with ΔV3-X4B becoming completely resistant. These findings demonstrate the contributions that interactions with distinct coreceptor regions have in mediating tropism and drug sensitivity and illustrate how HIV''s remarkable evolutionary plasticity in adapting to selection pressures can be exploited to better understand its biological potential.     

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

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

Derivation and Characterization of a Simian Immunodeficiency Virus SIVmac239 Variant with Tropism for CXCR4

Like human immunodeficiency virus type 1 (HIV-1), most simian immunodeficiency virus (SIV) strains use CCR5 to establish infection. However, while HIV-1 can acquire the ability to use CXCR4, SIVs that utilize CXCR4 have rarely been reported. To explore possible barriers against SIV coreceptor switching, we derived an R5X4 variant, termed 239-ST1, from the R5 clone SIVmac239 by serially passaging virus in CD4⁺ CXCR4⁺ CCR5⁻ SupT1 cells. A 239-ST1 env clone, designated 239-ST1.2-32, used CXCR4 and CCR5 in cell-cell fusion and reporter virus infection assays and conferred the ability for rapid, cytopathic infection of SupT1 cells to SIVmac239. Viral replication was inhibitable by the CXCR4-specific antagonist AMD3100, and replication was abrogated in a novel CXCR4⁻ SupT1 line. Surprisingly, parental SIVmac239 exhibited low-level replication in SupT1 cells that was not observed in CXCR4⁻ SupT1 cells. Only two mutations in the 239-ST1.2-32 Env, K47E in the C1 domain and L328W in the V3 loop, were required for CXCR4 use in cell-cell fusion assays, although two other V3 changes, N316K and I324M, improved CXCR4 use in infection assays. An Env cytoplasmic tail truncation, acquired during propagation of 239-ST1 in SupT1 cells, was not required. Compared with SIVmac239, 239-ST1.2-32 was more sensitive to neutralization by five of seven serum and plasma samples from SIVmac239-infected rhesus macaques and was approximately 50-fold more sensitive to soluble CD4. Thus, SIVmac239 can acquire the ability to use CXCR4 with high efficiency, but the changes required for this phenotype may be distinct from those for HIV-1 CXCR4 use. This finding, along with the increased neutralization sensitivity of this CXCR4-using SIV, suggests a mechanism that could select strongly against this phenotype in vivo.Simian immunodeficiency viruses (SIVs) share many structural and biological features with human immunodeficiency virus (HIV), including target cell entry via interactions of the viral envelope glycoprotein (Env) with CD4 and a chemokine coreceptor. For HIV, the most important coreceptors in vivo are CCR5 (2, 13, 19, 21, 22) and CXCR4 (30). HIV type 1 (HIV-1) strains that use only CCR5 (R5 viruses) predominate during the early stages of infection and are critical for transmission (84, 90), as evidenced by the finding that individuals lacking a functional CCR5 protein due to a homozygous 32-bp deletion in the CCR5 gene (ccr5-Δ32) are largely resistant to HIV-1 infection (16, 54, 82). Although R5 viruses generally persist in late-stage disease, viruses that can use CXCR4, either exclusively (X4 viruses) or in addition to CCR5 (R5X4 viruses), emerge in approximately 50% of subtype B-infected individuals (15, 43). This coreceptor switch is associated with a more rapid decline in peripheral blood CD4⁺ T cells and a faster progression to AIDS (15, 43, 77), although it is unclear if CXCR4-using viruses are a cause or a consequence of progressing immunodeficiency. Like HIV, the vast majority of SIVs use CCR5 to establish infection (11, 12, 45). However, although CXCR4-using SIVs have been reported (47, 52, 65, 68, 69), their occurrence is rare, especially in models of pathogenic infection, where only one CXCR4-using SIV has been identified (17, 60, 71).This paucity of CXCR4-using SIVs is surprising for several reasons. First, SIV Envs tend to be more promiscuous than HIV-1 Envs and frequently use alternative coreceptors in addition to CCR5, including GPR1, GPR15, CXCR6, and CCR8 (20, 27, 29, 80, 81, 92) but not CXCR4. Second, HIV-2, which is more closely related to SIVmac than to HIV-1 (56, 57), commonly uses CXCR4 in vitro and in vivo (3, 28, 33, 58, 59, 67). Third, rhesus CXCR4 is ∼98% identical to human CXCR4 in amino acid sequence and can function as a coreceptor for HIV-1 in vitro (12). Finally, chimeric simian-human immunodeficiency viruses (SHIVs) that contain X4 HIV Envs on an SIV core can replicate to high levels in vivo and cause disease in rhesus macaques (39, 86). Moreover, it was recently shown that coreceptor switching can occur in rhesus macaques infected with an R5 SHIV (35). Thus, there does not appear to be any block per se against the use of rhesus CXCR4 as an entry coreceptor either in vitro or in vivo, suggesting that SIV is less capable of adapting to use CXCR4 and/or that mutations required for CXCR4 utilization may lead to a virus that is less fit and/or more susceptible to immune control in this host.For HIV-1, the Env determinants for CXCR4 use have been well documented and often involve the acquisition of positively charged amino acids in the V3 loop (18, 32, 87), particularly at positions 11, 24, and 25 (6, 18, 31, 32, 38, 75). Although the SIVmac239 V3 loop is a critical determinant for Env-coreceptor interactions (44, 63, 72), attempts to create an X4 SIVmac239 by introducing positively charged residues into the V3 loop (63) or by inserting a V3 loop from X4 HIV-1 (44) have been unsuccessful. SIVmac155T3, the only CXCR4-using variant of SIVmac that has been identified to date, was isolated from a rhesus macaque with advanced disease and contains additional positively charged residues in V3, although the determinants for CXCR4 use have not been determined (60, 71).Given questions concerning the possible determinants for and/or barriers to coreceptor switching in SIV, we sought to derive a CXCR4-using variant of the well-characterized pathogenic R5 SIV clone SIVmac239. Here we show that SIVmac239 could indeed acquire CXCR4 utilization when it was adapted in vitro for high-efficiency replication in the CXCR4⁺ CCR5⁻ human SupT1 cell line. An env clone from this virus could use CXCR4 in cell-cell fusion and reporter virus infection assays and conferred CXCR4 tropism to a replication-competent SIV. Although V3 mutations were important for CXCR4 use, an L328W change at the V3 crown rather than the acquisition of positively charged residues was required, as was an unusual K47E mutation in the conserved C1 domain of gp120. These changes also caused the highly neutralization-resistant SIVmac239 strain to become more neutralization sensitive to sera and plasmas from SIVmac239-infected animals, and particularly to soluble CD4. These results indicate that mutations distinct from those typically seen for HIV-1 may be required for SIVmac to gain CXCR4 utilization and suggest that these changes render this virus more susceptible to humoral immune control. Collectively, our findings indicate that there are likely to be strong viral and host selection pressures against CXCR4 use that may contribute to the paucity of X4 coreceptor switching for SIVmac in vivo.     

Coinfecting Prion Strains Compete for a Limiting Cellular Resource

Prion strain interference can influence the emergence of a dominant strain from a mixture; however, the mechanisms underlying prion strain interference are poorly understood. In our model of strain interference, inoculation of the sciatic nerve with the drowsy (DY) strain of the transmissible mink encephalopathy (TME) agent prior to superinfection with the hyper (HY) strain of TME can completely block HY TME from causing disease. We show here that the deposition of PrP^Sc, in the absence of neuronal loss or spongiform change, in the central nervous system corresponds with the ability of DY TME to block HY TME infection. This suggests that DY TME agent-induced damage is not responsible for strain interference but rather prions compete for a cellular resource. We show that protein misfolding cyclic amplification (PMCA) of DY and HY TME maintains the strain-specific properties of PrP^Sc and replicates infectious agent and that DY TME can interfere, or completely block, the emergence of HY TME. DY PrP^Sc does not convert all of the available PrP^C to PrP^Sc in PMCA, suggesting the mechanism of prion strain interference is due to the sequestering of PrP^C and/or other cellular components required for prion conversion. The emergence of HY TME in PMCA was controlled by the initial ratio of the TME agents. A higher ratio of DY to HY TME agent is required for complete blockage of HY TME in PMCA compared to several previous in vivo studies, suggesting that HY TME persists in animals coinfected with the two strains. This was confirmed by PMCA detection of HY PrP^Sc in animals where DY TME had completely blocked HY TME from causing disease.Prions are infectious agents of animals, including humans, which are comprised of PrP^Sc, a misfolded isoform of the noninfectious host encoded protein PrP^C (17, 24, 50, 63). Prion diseases of humans are unique neurodegenerative disorders in that they can have either a sporadic, familial, or infectious etiology. Prions cause disease in economically important domestic and wild animal species such as bovine spongiform encephalopathy in cattle and chronic wasting disease in wild and captive cervids (20, 62). Prion diseases can be zoonotic as illustrated by the transmission of bovine spongiform encephalopathy to humans that resulted in the emergence of variant Creutzfeldt-Jacob disease (14, 19, 22, 23, 46, 61, 68). Prion diseases are inevitably fatal and there are currently no effective treatments (21).Prion strains are defined by a characteristic set of features that breed true upon experimental passage (33, 34). Strain-specific differences have been identified in incubation period, clinical signs, agent distribution, overdominance, host range, neuropathology, and biochemical properties of PrP^Sc (5, 10, 11, 13, 28, 34, 42, 44). Strain-specific conformations of PrP^Sc are hypothesized to encode prion strain diversity; however, it is not understood how these differences result in the distinct strain properties (11, 19, 40, 47, 59, 66).Prion strain interference may be involved in the emergence of a dominant strain from a mixture as could occur during prion adaptation to a new host species or during prion evolution (4, 36, 43, 48, 56). In the natural prion diseases, there are examples where an individual host may be infected with more than one prion strain (15, 25, 55, 57, 58). Experimentally, coinfection or superinfection of prion strains can result in interference where a blocking, long incubation period strain extends the incubation period or completely blocks a superinfecting, short incubation period strain from causing disease (26, 27). Prion interference has been described in experimental studies of mice and hamsters infected with a wide variety of prion strains and routes of inoculation, suggesting it may be a common property of prion disease (3, 27, 52, 53, 60).It has been proposed that prion strains compete for a shared “replication site”; however, mechanistic details are not known, and it is unclear whether the blocking strain destroys or occupies the replication sites required for the superinfecting strain (28). The transport to and relative onset of replication of interfering strains in a common population of neurons is an important factor that can determine which strain will emerge (8). In the present study, we sought to determine whether the blocking strain disables transport and spread of the superinfecting strain or whether prion interference is due to competition for a cellular resource.     

Enhancement of human immunodeficiency virus (HIV)-specific CD8+ T cells in cerebrospinal fluid compared to those in blood among antiretroviral therapy-naive HIV-positive subjects

During untreated human immunodeficiency virus type 1 (HIV-1) infection, virus-specific CD8⁺ T cells partially control HIV replication in peripheral lymphoid tissues, but host mechanisms of HIV control in the central nervous system (CNS) are incompletely understood. We characterized HIV-specific CD8⁺ T cells in cerebrospinal fluid (CSF) and peripheral blood among seven HIV-positive antiretroviral therapy-naïve subjects. All had grossly normal brain magnetic resonance imaging and spectroscopy and normal neuropsychometric testing. Frequencies of epitope-specific CD8⁺ T cells by direct tetramer staining were on average 2.4-fold higher in CSF than in blood (P = 0.0004), while HIV RNA concentrations were lower. Cells from CSF were readily expanded ex vivo and responded to a broader range of HIV-specific human leukocyte antigen class I restricted optimal peptides than did expanded cells from blood. HIV-specific CD8⁺ T cells, in contrast to total CD8⁺ T cells, in CSF and blood were at comparable maturation states, as assessed by CD45RO and CCR7 staining. The strong relationship between higher T-cell frequencies and lower levels of viral antigen in CSF could be the result of increased migration to and/or preferential expansion of HIV-specific T cells within the CNS. This suggests an important role for HIV-specific CD8⁺ T cells in control of intrathecal viral replication.Human immunodeficiency virus type 1 (HIV-1) invades the central nervous system (CNS) early during primary infection (21, 30, 35), and proviral DNA persists in the brain throughout the course of HIV-1 disease (7, 25, 29, 47, 77, 83). Limited data from human and nonhuman primate studies suggest that little or no viral replication occurs in the brain during chronic, asymptomatic infection, based on the absence of demonstrable viral RNA or proteins (8, 85). In contrast, cognitive impairment affects approximately 40% of patients who progress to advanced AIDS without highly active antiretroviral therapy (21, 30, 35, 65). During HIV-associated dementia, there is active HIV-1 replication in the brain (23, 52, 61, 81), and viral sequence differences between cerebrospinal fluid (CSF) and peripheral tissues suggest distinct anatomic compartments of replication (18, 19, 22, 53, 75, 76, 78). Host mechanisms that control viral replication in the CNS during chronic, asymptomatic HIV-1 infection are incompletely understood.Anti-HIV CD8⁺ T cells are present in blood and peripheral tissues throughout the course of chronic HIV-1 infection (2, 14). Multiple lines of evidence support a critical role for these cells in controlling HIV-1 replication. During acute HIV-1 infection, the appearance of CD8⁺ T-cell responses correlates temporally with a decline in viremia (11, 43), and a greater proliferative capacity of peripheral blood HIV-specific CD8⁺ T cells correlates with better control of viremia (36, 54). In addition, the presence of certain major histocompatibility complex class I human leukocyte antigen (HLA) alleles, notably HLA-B*57, predicts slower progression to AIDS and death during chronic, untreated HIV-1 infection (55, 62). Finally, in the simian immunodeficiency virus (SIV) model, macaques depleted of CD8⁺ T cells experience increased viremia and rapid disease progression (39, 51, 67).Little is known regarding the role of intrathecal anti-HIV CD8⁺ T cells in HIV neuropathogenesis. Nonhuman primate studies have identified SIV-specific CD8⁺ T cells in the CNS early after infection (16, 80). Increased infiltration of SIV antigen-specific CD8⁺ T cells and cytotoxic T lymphocytes has been detected only in CSF of slow progressors without neurological symptoms (72). In chronically infected macaques with little or no SIV replication in the brain, the frequency of HIV-specific T cells was higher in CSF than in peripheral blood but did not correlate with the level of plasma viremia or CD4⁺ T-cell counts (56). Although intrathecal anti-HIV CD8⁺ T cells may help control viral replication, a detrimental role in the neuropathogenesis of HIV-1 has also been postulated (38). Immune responses contribute to neuropathogenesis in models of other infectious diseases, and during other viral infections cytotoxic T lymphocytes can worsen disease through direct cytotoxicity or release of inflammatory cytokines such as gamma interferon (IFN-γ) (3, 17, 31, 37, 42, 44, 71).We tested the hypothesis that quantitative and/or qualitative differences in HIV-specific CD8⁺ T-cell responses are present in CSF compared to blood during chronic, untreated HIV-1 infection. We characterized HIV-specific CD8⁺ T-cell responses in CSF among seven antiretroviral therapy-naïve adults with chronic HIV-1 infection, relatively high peripheral blood CD4⁺ T-cell counts, and low plasma HIV-1 RNA concentrations. We show that among these HIV-positive individuals with no neurological symptoms and with little or no HIV-1 RNA in CSF, frequencies of HIV-specific T cells are significantly higher in CSF than in blood. These CSF cells are at a state of differentiation similar to that of T cells in blood and are functionally competent for expansion and IFN-γ production. The higher frequency of functional HIV-specific CD8⁺ T cells in CSF, in the context of low or undetectable virus in CSF, suggests that these cells play a role in the control of intrathecal viral replication.     

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).