Tetherin restricts productive HIV-1 cell-to-cell transmission.

The IFN-inducible antiviral protein tetherin (or BST-2/CD317/HM1.24) impairs release of mature HIV-1 particles from infected cells. HIV-1 Vpu antagonizes the effect of tetherin. The fate of virions trapped at the cell surface remains poorly understood. Here, we asked whether tetherin impairs HIV cell-to-cell transmission, a major means of viral spread. Tetherin-positive or -negative cells, infected with wild-type or DeltaVpu HIV, were used as donor cells and cocultivated with target lymphocytes. We show that tetherin inhibits productive cell-to-cell transmission of DeltaVpu to targets and impairs that of WT HIV. Tetherin accumulates with Gag at the contact zone between infected and target cells, but does not prevent the formation of virological synapses. In the presence of tetherin, viruses are then mostly transferred to targets as abnormally large patches. These viral aggregates do not efficiently promote infection after transfer, because they accumulate at the surface of target cells and are impaired in their fusion capacities. Tetherin, by imprinting virions in donor cells, is the first example of a surface restriction factor limiting viral cell-to-cell spread.

HIV-1 Virological Synapse: Live Imaging of Transmission.

A relatively new aspect of HIV-1 biology is the ability of the virus to infect cells by direct cellular contacts across a specialized structure, the virological synapse. This process was recently described through live cell imaging. Together with the accumulated knowledge on cellular and molecular structures involved in cell-to-cell transmission of HIV-1, the visualization of the virological synapse in video-microscopy has brought exciting new hypotheses on its underlying mechanisms. This review will recapitulate current knowledge with a particular emphasis on the questions live microscopy has raised.

Direct relationship between virus load and systemic immune activation in HIV-2 infection.

Immune activation is a hallmark of disease progression in human immunodeficiency virus (HIV) type 1 (HIV-1) and HIV type 2 (HIV-2) infection. However, the relationship between viremia and systemic immune activation is unclear. We assessed the relationship between HIV-2 plasma virus load and immune system activation in a cross-sectional study in a community cohort of HIV-1-positive, HIV-2-positive, and HIV-negative patients, in which many HIV-2-positive patients had nonprogressing infection. HLA-DR and CD38 expression on CD4(+) and CD8(+) T cells was measured, as were plasma beta(2)-microglobulin levels. These markers were related to clinical (virus load and CD4(+) cell count) and immunological (HIV-2-specific interferon gamma secretion) correlates of delayed disease progression. A consistent positive correlation was identified between the level of HIV-2 viremia and immune activation. We propose that increasing virus load may contribute to systemic immune activation in HIV-2 infection.

Downregulation of the T-cell receptor by human immunodeficiency virus type 2 Nef does not protect against disease progression.

Chronic immune activation is thought to play a major role in human immunodeficiency virus (HIV) pathogenesis, but the relative contributions of multiple factors to immune activation are not known. One proposed mechanism to protect against immune activation is the ability of Nef proteins from some HIV and simian immunodeficiency virus strains to downregulate the T-cell receptor (TCR)-CD3 complex of the infected cell, thereby reducing the potential for deleterious activation. HIV type 1 (HIV-1) Nef has lost this property. In contrast to HIV-1, HIV-2 infection is characterized by a marked disparity in the disease course, with most individuals maintaining a normal life span. In this study, we examined the relationship between the ability of HIV-2 Nef proteins to downregulate the TCR and immune activation, comparing progressors and nonprogressors. Representative Nef variants were isolated from 28 HIV-2-infected individuals. We assessed their abilities to downregulate the TCR from the surfaces of CD4 T cells. In the same individuals, the activation of peripheral lymphocytes was evaluated by measurement of the expression levels of HLA-DR and CD38. We observed a striking correlation of the TCR downregulation efficiency of HIV-2 Nef variants with immune activation in individuals with a low viral load. This strongly suggests that Nef expression can influence the activation state of the immune systems of infected individuals. However, the efficiency of TCR downregulation by Nef was not reduced in progressing individuals, showing that TCR downregulation does not protect against progression in HIV-2 infection.

Simultaneous cell-to-cell transmission of human immunodeficiency virus to multiple targets through polysynapses.

Human immunodeficiency virus type 1 (HIV-1) efficiently propagates through cell-to-cell contacts, which include virological synapses (VS), filopodia, and nanotubes. Here, we quantified and characterized further these diverse modes of contact in lymphocytes. We report that viral transmission mainly occurs across VS and through "polysynapses," a rosette-like structure formed between one infected cell and multiple adjacent recipients. Polysynapses are characterized by simultaneous HIV clustering and transfer at multiple membrane regions. HIV Gag proteins often adopt a ring-like supramolecular organization at sites of intercellular contacts and colocalize with CD63 tetraspanin and raft components GM1, Thy-1, and CD59. In donor cells engaged in polysynapses, there is no preferential accumulation of Gag proteins at contact sites facing the microtubule organizing center. The LFA-1 adhesion molecule, known to facilitate viral replication, enhances formation of polysynapses. Altogether, our results reveal an underestimated mode of viral transfer through polysynapses. In HIV-infected individuals, these structures, by promoting concomitant infection of multiple targets in the vicinity of infected cells, may facilitate exponential viral growth and escape from immune responses.

Secretory cytotoxic granule maturation and exocytosis require the effector protein hMunc13-4.

Cytotoxic T lymphocytes and natural killer cells exert their cytotoxic activity through the polarized secretion of cytotoxic granules at the immunological synapse. Rab27a and hMunc13-4 are critical effectors of the exocytosis of cytotoxic granules. Here we show that the cytotoxic function of lymphocytes requires the cooperation of two types of organelles: the lysosomal cytotoxic granule and the endosomal 'exocytic vesicle'. Independently of Rab27a, hMunc13-4 mediated the assembly of Rab11(+) recycling and Rab27(+) late endosomal vesicles, constituting a pool of vesicles destined for regulated exocytosis. It also primed cytotoxic granule fusion, possibly through interaction with active Rab27a. Cytotoxic T lymphocyte-target cell recognition induced rapid polarization of both types of organelles, which coalesced near the cell-cell contact area. Our data provide insight into the regulation of the generation and release of cytotoxic granules by effector cytotoxic T lymphocytes and natural killer cells.

Primary hemophagocytic syndromes point to a direct link between lymphocyte cytotoxicity and homeostasis.

Hemophagocytic syndrome (HS) is a severe and often fatal syndrome resulting from potent and uncontrolled activation and proliferation of T-lymphocytes, leading to excessive macrophage activation and multiple deleterious effects. The onset of HS characterizes several inherited disorders in humans. In each condition, the molecular defect impairs the granule-dependent cytotoxic activity of lymphocytes, thus highlighting the determinant role of this function in driving the immune system to a state of equilibrium following infection. It has also been shown that some of the proteins required for lytic granule secretion are required for melanocyte function, leading to associated hypopigmentation in these conditions. This review focuses on several effectors of this secretory pathway, recently identified, because their defects cause these disorders, and discusses their role and molecular interactions in granule-dependent cytotoxic activity.

Severe and progressive encephalitis as a presenting manifestation of a novel missense perforin mutation and impaired cytolytic activity.

Mutations in the perforin gene cause familial hemophagocytic lymphohistiocytosis (FHL). The first symptoms of FHL are usually intractable fever, hepatosplenomegaly, and pancytopenia. Most FHL patients subsequently develop central nervous system (CNS) manifestations due to infiltration of tissues by activated lymphocytes and macrophages. We report 2 FHL patients with an atypical phenotype characterized by isolated severe neurologic symptoms mimicking chronic encephalitis and leading to an early death. Functional and molecular analyses revealed the same novel missense mutation in the perforin gene in both patients; this mutation affected the calcium-binding domain of the protein. This missense mutation did not affect perforin maturation or expression in cytotoxic cells but impaired in vitro cytotoxic activity. Diagnosis was delayed in both patients because of the initial neurologic expression and the normal expression of perforin in circulating lymphocytes. This emphasizes the importance of early diagnosis of this atypical form of FHL, as CNS involvement causes severe, irreversible encephalopathy. This observation also raises the question of the role of some mutations in the neurologic expression of FHL.

Griscelli syndrome: characterization of a new mutation and rescue of T-cytotoxic activity by retroviral transfer of RAB27A gene.

Griscelli syndrome (GS) is caused by mutations in the MYO5A (GS1), RAB27A (GS2), or MLPH (GS3) genes, all of which lead to a similar pigmentary dilution. In addition, GS1 patients show primary neurological impairment, whereas GS2 patients present immunodeficiency and periods of lymphocyte proliferation and activation, leading to their infiltration in many organs, such as the nervous system, causing secondary neurological damage. We report the diagnosis of GS2 in a 4-year-old child with haemophagocytic syndrome, immunodeficiency, and secondary neurological disorders. Typical melanosome accumulation was found in skin melanocytes and pigment clumps were observed in hair shafts. Two heterozygous mutant alleles of the RAB27A gene were found, a C-T transition (C352T) that leads to Q118stop and a G-C transversion on the exon 5 splicing donor site (G467+1C). Functional assays showed increased cellular activation and decreased cytotoxic activity of NK and CD8+ T cells, associated with defective lytic granules release. Myosin-Va expression and localization in the patient lymphocytes were also analyzed. Most importantly, we show that cytotoxic activity of the patient's CD8+ T lymphocytes can be rescued in vitro by RAB27A gene transfer mediated by a recombinant retroviral vector, a first step towards a potential treatment of the acute phase of GS2 by RAB27A transduced lymphocytes.

Molecular basis of the spectral expression of CIAS1 mutations associated with phagocytic cell-mediated autoinflammatory disorders CINCA/NOMID, MWS, and FCU.

NALP proteins are recently identified members of the CATERPILLER (CARD, transcription enhancer, R(purine)-binding, pyrin, lots of LRR) family of proteins, thought to function in apoptotic and inflammatory signaling pathways. Mutations in the CIAS1 gene, which encodes a member of the NALP (NACHT-, LRR-, and PYD-containing proteins) family, the cryopyrin/NALP3/PYPAF1 protein, expressed primarily in phagocytic cells, were recently found to be associated with a spectrum of autoinflammatory disorders. These include chronic infantile neurologic cutaneous and articular (CINCA) syndrome (also known as neonatal-onset multisystem inflammatory disease [NOMID]), Muckle-Wells syndrome (MWS), and familial cold urticaria (FCU). We describe herein 7 new mutations in 13 unrelated patients with CINCA syndrome and identify mutational hotspots in CIAS1 on the basis of all mutations described to date. We also provide evidence of genotype/phenotype correlations. A 3-dimensional model of the nucleotide-binding domain (NBD) of cryopyrin suggested that this molecule is structurally and functionally similar to members of the AAA+ protein family of ATPases. According to this model, most of the mutations known to affect residues of the NBD are clustered on one side of this domain in a region predicted to participate in intermolecular contacts, suggesting that this model is likely to be biologically relevant and that defects in nucleotide binding, nucleotide hydrolysis, or protein oligomerization may lead to the functional dysregulation of cryopyrin in the MWS, FCU, and CINCA/NOMID disorders.

Munc13-4 is essential for cytolytic granules fusion and is mutated in a form of familial hemophagocytic lymphohistiocytosis (FHL3).

Secretion of cytolytic granules content at the immunological synapse is a highly regulated process essential for lymphocyte cytotoxicity. This process requires the rapid transfer of perforin containing lytic granules to the target cell interface, followed by their docking and fusion with the plasma membrane. Defective cytotoxicity characterizes a genetically heterogeneous condition named familial hemophagocytic lymphohistiocytosis (FHL), which can be associated with perforin deficiency. The locus of a perforin (+) FHL subtype (FHL3), observed in 10 patients, was mapped to 17q25. This region contains hMunc13-4, a member of the Munc13 family of proteins involved in vesicle priming function. HMunc13-4 mutations were shown to cause FHL3. HMunc13-4 deficiency results in defective cytolytic granule exocytosis, despite polarization of the secretory granules and docking with the plasma membrane. Expressed tagged hMunc13-4 localizes with cytotoxic granules at the immunological synapse. HMunc13-4 is therefore essential for the priming step of cytolytic granules secretion preceding vesicle membrane fusion.

Griscelli syndrome restricted to hypopigmentation results from a melanophilin defect (GS3) or a MYO5A F-exon deletion (GS1).

Griscelli syndrome (GS) is a rare autosomal recessive disorder that associates hypopigmentation, characterized by a silver-gray sheen of the hair and the presence of large clusters of pigment in the hair shaft, and the occurrence of either a primary neurological impairment or a severe immune disorder. Two different genetic forms, GS1 and GS2, respectively, account for the mutually exclusive neurological and immunological phenotypes. Mutations in the gene encoding the molecular motor protein Myosin Va (MyoVa) cause GS1 and the dilute mutant in mice, whereas mutations in the gene encoding the small GTPase Rab27a are responsible for GS2 and the ashen mouse model. We herein present genetic and functional evidence that a third form of GS (GS3), whose expression is restricted to the characteristic hypopigmentation of GS, results from mutation in the gene that encodes melanophilin (Mlph), the ortholog of the gene mutated in leaden mice. We also show that an identical phenotype can result from the deletion of the MYO5A F-exon, an exon with a tissue-restricted expression pattern. This spectrum of GS conditions pinpoints the distinct molecular pathways used by melanocytes, neurons, and immune cells in secretory granule exocytosis, which in part remain to be unraveled.

Biochemical and functional characterization of Rab27a mutations occurring in Griscelli syndrome patients.

Rab27a is a member of the Rab family of small GTPase proteins, and thus far is the first member to be associated with a human disease (ie, the Griscelli syndrome type 2). Mutations in the Rab27a gene cause pigment as well as cytotoxic granule transport defects, accounting for the partial albinism and severe immune disorder characteristics of this syndrome. So far, 3 Rab27a missense mutations have been identified. They open a unique opportunity to designate critical structural and functional residues of Rab proteins. We show here that the introduction of a proline residue in the alpha 4 (Ala152Pro) or beta 5 (Leu130Pro) loop, observed in 2 of these spontaneous mutants, dramatically affects both guanosine triphosphate (GTP) and guanosine diphosphate (GDP) nucleotide-binding activity of Rab27a, probably by disrupting protein folding. The third mutant, Trp73Gly, is located within an invariant hydrophobic triad at the switch interface, and was previously shown in active Rab3A to mediate rabphilin3A effector interaction. Trp73Gly is shown to display the same nucleotide-binding and GTPase characteristics as the constitutively active mutant Gln78Leu. However, in contrast to Gln78Leu, Trp73Gly mutant construct neither interacts with the Rab27a effector melanophilin nor modifies melanosome distribution and cytotoxic granule exocytosis. Substitutions introduced at the 73 position, including the leucine residue present in Ras, did not restore Rab27a protein functions. Taken together, our results characterize new critical residues of Rab proteins, and identify the Trp73 residue of Rab27a as a key position for interaction with the specific effectors of Rab27a, both in melanocytes and cytotoxic cells.

Functional consequences of perforin gene mutations in 22 patients with familial haemophagocytic lymphohistiocytosis.

Familial haemophagocytic lymphohistiocytosis (FHL), an inherited form of haemophagocytic lymphohistiocytosis (HLH) syndrome, is characterized by the overwhelming activation of T lymphocytes and macrophages invariably leading to death in the absence of treatment. FHL is a heterogeneous autosomal recessive disorder, with one known causative gene which codes for perforin, a cytotoxic effector protein. In this study, we have characterized the genotype and phenotype of 14 unrelated families with perforin deficiency. Four new missense mutations of the perforin gene were identified. In every case, perforin gene mutations led to undetectable intracellular perforin expression within cytotoxic cells, while some residual T-cell cytotoxic activity could be associated with certain missense mutations. Clinical and biological analyses did not differentiate between patients with nonsense or missense mutations, although age at diagnosis, which tended to be similar within members of the same family, was delayed in patients from two families belonging to the second group. In one case, consequences of perforin deficiency, diagnosed at birth, could be assessed prior to onset of clinical manifestations. No evidence for T-cell activation could be shown, suggesting that an exogenous event is required to trigger the disease manifestation. Control assessment of perforin expression and cytotoxic assays by lymphocytes from young children led to the conclusion that perforin content of natural killer cells could be a reliable diagnostic test at any age. Altogether, these data enabled a better characterization of perforin deficiency and its consequences, and defined reliable diagnostic tools.

Selective inhibition of dipeptidyl peptidase I, not caspases, prevents the partial processing of procaspase-3 in CD3-activated human CD8(+) T lymphocytes.

Activation of primary human T cells by anti-CD3 and interleukin-2 resulted in partial processing of procaspase-3 in activated nonapoptotic (Delta Psi(m)high) CD8(+) T cells but not in CD4(+) T cells. Apical caspases-8 and -9 were not activated, and Bid was not processed to truncated Bid. Boc-D.fmk, a broad spectrum caspase inhibitor, did not prevent this process, whereas GF.dmk, a selective inhibitor of dipeptidyl peptidase I, was effective. Dipeptidyl peptidase I is required for the activation of granule-associated serine proteases. It is enriched in the cytolytic granules of cytotoxic lymphocytes, where it promotes the proteolytic activation of progranzymes A and B. Inhibition of granzyme B (GrB)-like serine proteases by Z-AAD.cmk prevented partial processing of procapase-3, whereas inhibition of GrA activity by D-FPR.cmk had no effect. Specific inhibitors of other lysosomal proteases such as cathepsins B, L, and D did not interfere in this event. Patients with Chediak-Higashi syndrome or with perforin deficiency also displayed partial processing of procaspase-3, excluding the involvement of granule exocytosis for the delivery of the serine protease in cause. The p20/p12 processing pattern of procaspase-3 in our model points to GrB, the sole serine protease with caspase activity. Small amounts of GrB were indeed exported from cytolytic granules to the cytosol of a significant fraction of GrB-positive cells.

Chronic infantile neurological cutaneous and articular syndrome is caused by mutations in CIAS1, a gene highly expressed in polymorphonuclear cells and chondrocytes.

Chronic infantile neurological cutaneous and articular (CINCA) syndrome is a severe chronic inflammatory disease of early onset, characterized by cutaneous symptoms, central-nervous-system involvement, and arthropathy. In the present study, we report, in seven unrelated patients with CINCA syndrome, distinct missense mutations within the nucleotide-binding site of CIAS1, a gene encoding cryopyrin and previously shown to cause Muckle-Wells syndrome and familial cold urticaria. Because of the severe cartilage overgrowth observed in some patients with CINCA syndrome and the implications of polymorphonuclear cell infiltration in the cutaneous and neurological manifestations of this syndrome, the tissue-specific expression of CIAS1 was evaluated. A high level of expression of CIAS1 was found to be restricted to polymorphonuclear cells and chondrocytes. These findings demonstrate that CIAS1 missense mutations can result in distinct phenotypes with only a few overlapping symptoms and suggest that this gene may function as a potential inducer of apoptosis.