Phenotypic variability related to dominant UCHL1 mutations: about three families with optic atrophy and ataxia.

INTRODUCTION: Ubiquitin C-terminal hydrolase L1 (UCHL1) has been associated with a severe, complex autosomal recessive spastic paraplegia (HSP79) [1] [2] [3] [4]. More recently, UCHL1 loss of function (LoF) variants have been associated to an autosomal dominant disease characterized by late-onset spastic ataxia, neuropathy, and frequent optic atrophy [5]. METHODS: Routine clinical care whole-genome (WGS) and exome (ES) sequencing. RESULTS: We present three families with autosomal dominant UCHL1-related disorder. The clinical phenotype mainly associated optic atrophy, mixed cerebellar and sensory ataxia, and possible hearing loss. We delineated two major phenotypes, even within the same family: (1) juvenile severe optic atrophy followed by a later-onset ataxia, or (2) late-onset ataxia with asymptomatic or mild optic atrophy. The families harboured three novel heterozygous variants in UCHL1: two loss of function (p.Lys115AsnfsTer40; c.171_174 + 7del11), and one missense (p.Asp176Asn) involving the catalytic site of the protein and potentially altering the adjacent splice site. DISCUSSION: We confirm the existence of dominantly inherited UCHL1 pathogenic variants. We describe a considerable intrafamilial phenotypic variability, with two main phenotypes. Optic atrophy was consistently present, but with varying degrees of severity. Neither delayed motor or intellectual development, nor dysmorphic features were part of the dominant phenotype in comparison with the autosomal recessive form. The molecular mechanism appears to be haploinsufficiency. UCHL1 monoallelic variants should therefore be considered in any case of early-onset optic atrophy or in late-onset complex ataxic syndrome with asymptomatic optic atrophy.

[SAMHD1 acts at stalled replication forks to prevent interferon induction]. / SAMHD1 agit sur les fourches de réplication bloquées pour empêcher l'induction d'interféron.

DNA replication is an extremely complex process, involving thousands of replication forks progressing along chromosomes. These forks are frequently slowed down or stopped by various obstacles, such as secondary DNA structures, chromatin-acting proteins or a lack of nucleotides. This slowing down, known as replicative stress, plays a central role in tumour development. Complex processes, which are not yet fully understood, are set up to respond to this stress. Certain nucleases, such as MRE11 and DNA2, degrade the neo-replicated DNA at the level of blocked forks, allowing the replication to restart. The interferon pathway is a defense mechanism against pathogens that detects the presence of foreign nucleic acids in the cytoplasm and activates the innate immune response. DNA fragments resulting from genomic DNA metabolism (repair, retrotransposition) can diffuse into the cytoplasm and activate this pathway. A pathological manifestation of this process is the Aicardi-Goutières syndrome, a rare disease characterized by chronic inflammation leading to neurodegenerative and developmental problems. In this encephalopathy, it has been suggested that DNA replication may generate cytosolic DNA fragments, but the mechanisms involved have not been characterized. SAMHD1 is frequently mutated in the Aicardi-Goutières syndrome as well as in some cancers, but its role in the etiology of these diseases was largely unknown. We show that cytosolic DNA accumulates in SAMHD1-deficient cells, particularly in the presence of replicative stress, activating the interferon response. SAMHD1 is important for DNA replication under normal conditions and for the processing of stopped forks, independent of its dNTPase activity. In addition, SAMHD1 stimulates the exonuclease activity of MRE11 in vitro. When SAMHD1 is absent, degradation of neosynthesized DNA is inhibited, which prevents activation of the replication checkpoint and leads to failure to restart the replication forks. Resection of the replication forks is performed by an alternative mechanism which releases DNA fragments into the cytosol, activating the interferon response. The results obtained show, for the first time, a direct link between the response to replication stress and the production of interferons. These results have important implications for our understanding of the Aicardi-Goutières syndrome and cancers related to SAMHD1. For example, we have shown that MRE11 and RECQ1 are responsible for the production of DNA fragments that trigger the inflammatory response in cells deficient for SAMHD1. We can therefore imagine that blocking the activity of these enzymes could decrease the production of DNA fragments and, ultimately, the activation of innate immunity in these cells. In addition, the interferon pathway plays an essential role in the therapeutic efficacy of irradiation and certain chemotherapeutic agents such as oxaliplatin. Modulating this response could therefore be of much wider interest in anti-tumour therapy.

La réplication de l'ADN est un processus extrêmement complexe, impliquant des milliers de fourches de réplication progressant le long des chromosomes. Ces fourches sont fréquemment ralenties ou arrêtées par différents obstacles, tels que des structures secondaires de l'ADN, des protéines agissant sur la chromatine ou encore un manque de nucléotides. Ce ralentissement, qualifié de stress réplicatif, joue un rôle central dans le développement tumoral. Des processus complexes, qui ne sont pas encore totalement connus, sont mis en place pour répondre à ce stress. Certaines nucléases, comme MRE11 et DNA2, dégradent l'ADN néorépliqué au niveau des fourches bloquées, ce qui permet le redémarrage des réplisomes. La voie interféron est un mécanisme de défense contre les agents pathogènes qui détecte la présence d'acides nucléiques étrangers dans le cytoplasme et active la réponse immunitaire innée. Des fragments d'ADN issus du métabolisme de l'ADN génomique (réparation, rétrotransposition) peuvent diffuser dans le cytoplasme et activer cette voie. Une manifestation pathologique de ce processus est le syndrome d'Aicardi-Goutières, une maladie rare caractérisée par une inflammation chronique générant des problèmes neurodégénératifs et développementaux. Dans le cadre de cette encéphalopathie, il a été suggéré que la réplication de l'ADN pouvait générer des fragments d'ADN cytosoliques, mais les mécanismes impliqués n'avaient pas été caractérisés. SAMHD1 est fréquemment muté dans le syndrome d'Aicardi-Goutières ainsi que dans certains cancers, mais son rôle dans l'étiologie de ces maladies était jusqu'à présent largement inconnu. Nous montrons que de l'ADN cytosolique s'accumule dans les cellules déficientes pour SAMHD1, particulièrement en présence de stress réplicatif, activant la réponse interféron. Par ailleurs, SAMHD1 est important pour la réplication de l'ADN en conditions normales et pour le processing des fourches arrêtées, indépendamment de son activité dNTPase. De plus, SAMHD1 stimule l'activité exonucléase de MRE11 in vitro. Lorsque SAMHD1 est absent, la dégradation de l'ADN néosynthétisé est inhibée, ce qui empêche l'activation du checkpoint de réplication et entraine un défaut de redémarrage des fourches de réplication. De plus, la résection des fourches de réplication est réalisée par un mécanisme alternatif qui libère des fragments d'ADN dans le cytosol, activant la réponse interféron. Les résultats obtenus montrent, pour la première fois, un lien direct entre la réponse au stress réplicatif et la production d'interférons. Ces résultats ont des conséquences importantes dans notre compréhension du syndrome d'Aicardi Goutières et des cancers liés à SAMHD1. Par exemple, nous avons démontré que MRE11 et RECQ1 sont responsables de la production des fragments d'ADN qui déclenchent la réponse inflammatoire dans les cellules déficientes pour SAMHD1. Nous pouvons donc imaginer que bloquer l'activité de ces enzymes pourrait diminuer la production des fragments d'ADN et, in fine, l'activation de l'immunité innée dans ces cellules. Par ailleurs, la voie interférons joue un rôle essentiel dans l'efficacité thérapeutique de l'irradiation et de certains agents chimiothérapiques comme l'oxaliplatine. Moduler cette réponse pourrait donc avoir un intérêt beaucoup plus large en thérapie anti-tumorale.

XPC is an RNA polymerase II cofactor recruiting ATAC to promoters by interacting with E2F1.

The DNA damage sensor XPC is involved in nucleotide excision repair. Here we show that in the absence of damage, XPC co-localizes with RNA polymerase II (Pol II) and active post-translational histone modifications marks on a subset of class II promoters in human fibroblasts. XPC depletion triggers specific gene down-expression due to a drop in the deposition of histone H3K9 acetylation mark and pre-initiation complex formation. XPC interacts with the histone acetyltransferase KAT2A and specifically triggers the recruitment of the KAT2A-containing ATAC complex to the promoters of down-expressed genes. We show that a strong E2F1 signature characterizes the XPC/KAT2A-bound promoters and that XPC interacts with E2F1 and promotes its binding to its DNA element. Our data reveal that the DNA repair factor XPC is also an RNA polymerase II cofactor recruiting the ATAC coactivator complex to promoters by interacting with the DNA binding transcription factor E2F1.

A genome-wide association study of pulmonary tuberculosis in Morocco.

Although epidemiological evidence suggests a human genetic basis of pulmonary tuberculosis (PTB) susceptibility, the identification of specific genes and alleles influencing PTB risk has proven to be difficult. Previous genome-wide association (GWA) studies have identified only three novel loci with modest effect sizes in sub-Saharan African and Russian populations. We performed a GWA study of 550,352 autosomal SNPs in a family-based discovery Moroccan sample (on the full population and on the subset with PTB diagnosis at <25 years), which identified 143 SNPs with p < 1 × 10(-4). The replication study in an independent case/control sample identified four SNPs displaying a p < 0.01 implicating the same risk allele. In the combined sample including 556 PTB subjects and 650 controls these four SNPs showed suggestive association (2 × 10(-6) < p < 4 × 10(-5)): rs358793 and rs17590261 were intergenic, while rs6786408 and rs916943 were located in introns of FOXP1 and AGMO, respectively. Both genes are involved in the function of macrophages, which are the site of latency and reactivation of Mycobacterium tuberculosis. The most significant finding (p = 2 × 10(-6)) was obtained for the AGMO SNP in an early (<25 years) age-at-onset subset, confirming the importance of considering age-at-onset to decipher the genetic basis of PTB. Although only suggestive, these findings highlight several avenues for future research in the human genetics of PTB.

Thrombotic microangiopathy with skin localization secondary to cytarabine-daunorubicin association: report of a case.

The thrombotic microangiopathy is a syndrome characterized by the combination of mechanical hemolytic anemia, peripheral thrombocytopenia, and organ failure of variable severity. In addition to the idiopathic form, several cases are identified as secondary to pregnancy, infections, disease systems, organ transplants, and cancer. Other forms are secondary to drugs including antimitotics. We report the case of a patient followed for acute myelogenous leukemia. She received induction chemotherapy combining daunorubicin and cytarabine, complicated by thrombotic thrombocytopenic purpura.

[Seroprevalence of HBV and HCV in blood donors at the Blood Transfusion Center of Mohammed V Military Teaching Hospital in Rabat Morocco]. / Prévalence des marqueurs sériques des virus de l'hépatite B et C chez les donneurs de sang au Centre de Transfusion Sanguine de l'Hôpital Militaire d'Instruction Mohammed V de Rabat (Maroc).

The purpose of this report is to present the findings of a retrospective study (2008-2009) to determine the seroprevalence of hepatitis B and C virus in blood donors at the Blood Transfusion Center of Military Teaching Hospital Mohammed V in Rabat, Morocco. Samples from 19,801 consecutive blood donors were analyzed by the immuno-enzymatic method (Enzyme Linked Immunosorbent Assay, third generation). The overall seroprevalence of HBV and HCV was 0.8% and 0.2% respectively. A total of 98 units were rejected because of elevated alanine transaminase. No case of co-infection was found. From 1991 to 2010, HBV and HCV seropositivity showed a significant declining trend. In spite of the low prevalence observed, this study confirms that the risk of transfusion transmitted infection exists and thus underlines the need to implement preventive strategies to improve blood transfusion safety.

Interaction between cyclin T1 and SCF(SKP2) targets CDK9 for ubiquitination and degradation by the proteasome.

CDK9 paired with cyclin T1 forms the human P-TEFb complex and stimulates productive transcription through phosphorylation of the RNA polymerase II C-terminal domain. Here we report that CDK9 is ubiquitinated and degraded by the proteasome whereas cyclin T1 is stable. SCF(SKP2) was recruited to CDK9/cyclin T1 via cyclin T1 in an interaction requiring its PEST domain. CDK9 ubiquitination was modulated by cyclin T1 and p45(SKP2). CDK9 accumulated in p45(SKP2-/-) cells, and its expression during the cell cycle was periodic. The transcriptional activity of CDK9/cyclin T1 on the class II major histocompatibility complex promoter could be regulated by CDK9 degradation in vivo. We propose a novel mechanism whereby recruitment of SCF(SKP2) is mediated by cyclin T1 while ubiquitination occurs exclusively on CDK9.

Selective CXCR4 antagonism by Tat: implications for in vivo expansion of coreceptor use by HIV-1.

Chemokines and chemokine receptors play important roles in HIV-1 infection and tropism. CCR5 is the major macrophage-tropic coreceptor for HIV-1 whereas CXC chemokine receptor 4 (CXCR4) serves the counterpart function for T cell-tropic viruses. An outstanding biological mystery is why only R5-HIV-1 is initially detected in new seroconvertors who are exposed to R5 and X4 viruses. Indeed, X4 virus emerges in a minority of patients and only in the late stage of disease, suggesting that early negative selection against HIV-1-CXCR4 interaction may exist. Here, we report that the HIV-1 Tat protein, which is secreted from virus-infected cells, is a CXCR4-specific antagonist. Soluble Tat selectively inhibited the entry and replication of X4, but not R5, virus in peripheral blood mononuclear cells (PBMCs). We propose that one functional consequence of secreted Tat is to select against X4 viruses, thereby influencing the early in vivo course of HIV-1 disease.

Tat protein of human immunodeficiency virus type 1 induces interleukin-10 in human peripheral blood monocytes: implication of protein kinase C-dependent pathway.

The clinical manifestations observed in human immunodeficiency virus type 1 (HIV-1)-infected patients are primarily due to the capacity of the virus and its components to inactivate the immune system. HIV-1 Tat protein could participate in this immune system disorder. This protein is secreted by infected cells of HIV-infected patients and is free in the plasma, where it can interact and be taken up by both infected and noninfected cells. In asymptomatic patients infected by HIV-1, production of interleukin-10 (IL-10), a highly immunosuppressive cytokine, is associated with disease progression to AIDS. In the present work, we tested the capacity of Tat to induce IL-10 production by peripheral blood monocytes of healthy donors. The results show that Tat causes the production of IL-10 in a dose- and stimulation time-dependent manner. Investigations of the mechanisms involved in signal transduction show that (i) the calcium pathway is not or only slightly involved in Tat-induced IL-10 production, (ii) the protein kinase C pathway plays an essential role, and (iii) monocyte stimulation by Tat results in the intranuclear translocation of transcription factor NF-kappaB and in the induction of phosphorylation of the mitogen-activated protein kinases ERK1 and ERK2; activation of these two potential substrates of protein kinase C is required for the production of IL-10. Finally, our results suggest that the effect of Tat is exerted at the membrane level and that the active domain is located within N-terminal residues 1 to 45. This production of IL-10 induced by Tat could participate in the progression of HIV infection to AIDS.

CD4+ T cell surface CCR5 density as a determining factor of virus load in persons infected with human immunodeficiency virus type 1.

The intensity of expression of the chemokine receptor CCR5 is involved in in vitro cell infectability by human immunodeficiency virus (HIV)-1 R5 isolates. Because CCR5 expression varies among individuals, the hypothesis that this expression could determine virus load in HIV-1-infected persons was tested. The mean number of CCR5 molecules per cell was measured on peripheral blood CD4+ T lymphocytes (CCR5 density) from HIV-1-infected, asymptomatic, nontreated adults. There was a strong correlation between HIV RNA plasma level and CCR5 density (P=.009) that was independent of cell activation and was not due to an HIV-induced CCR5 up-regulation. These data are compatible with the hypothesis that CCR5 density is a key factor governing cell infectability and in vivo virus production and explain the protective effect of the Delta32CCR5 deletion, which results in low CCR5 expression. CCR5 density might be of critical predictive value in HIV infection.

HIV-1 tat transcriptional activity is regulated by acetylation.

The human immunodeficiency virus (HIV) trans- activator protein, Tat, stimulates transcription from the viral long-terminal repeats (LTR) through an RNA hairpin element, trans-activation responsive region (TAR). We and others have shown that trans-activator protein (Tat)-associated histone acetyltransferases (TAHs), p300 and p300/CBP-associating factor (PCAF), assist functionally in the activation of chromosomally integrated HIV-1 LTR. Here, we show that p300 and PCAF also directly acetylate Tat. We defined two sites of acetylation located in different functional domains of Tat. p300 acetylated Lys50 in the TAR RNA binding domain, while PCAF acetylated Lys28 in the activation domain of Tat. In support of a functional role for acetylation in vivo, histone deacetylase inhibitor (trichostatin A) synergized with Tat in transcriptional activation of the HIV-1 LTR. Synergism was TAR-dependent and required the intact presence of both Lys28 and Lys50. Mechanistically, acetylation at Lys28 by PCAF enhanced Tat binding to the Tat-associated kinase, CDK9/P-TEFb, while acetylation by p300 at Lys50 of Tat promoted the dissociation of Tat from TAR RNA that occurs during early transcription elongation. These data suggest that acetylation of Tat regulates two discrete and functionally critical steps in transcription, binding to an RNAP II CTD-kinase and release of Tat from TAR RNA.

Activation of integrated provirus requires histone acetyltransferase. p300 and P/CAF are coactivators for HIV-1 Tat.

A unique aspect of the retrovirus life cycle is the obligatory integration of the provirus into host cell chromosomes. Unlike viruses that do not integrate, retroviruses must conserve an ability to activate transcription from a chromatin context. Human immunodeficiency virus (HIV)-1 encodes an unusual and an unusually potent transcriptional transactivator, Tat, which binds to a nascent viral leader RNA, TAR. The action of Tat has been well studied in various reductive model systems; however, the physiological mechanism through which Tat gains access to chromatin-associated proviral long terminal repeats (LTRs) is not understood. We show here that a nuclear histone acetyltransferase activity associates with Tat. Intracellularly, we found that Tat forms a ternary complex with p300 and P/CAF, two histone acetyltransferases (HATs). A murine cell defect in Tat transactivation of the HIV-1 LTR was linked to the reduced abundance of p300 and P/CAF. Thus, overexpression of p300 and P/CAF reconstituted Tat transactivation of the HIV-1 LTR in NIH3T3 cells to a level similar to that observed for human cells. By using transdominant p300 or P/CAF mutants that lack enzymatic activity, we delineated a requirement for the HAT component from the latter but not the former in Tat function. Finally, we observed that Tat-associated HAT is preferentially important for transactivation of integrated, but not unintegrated, HIV-1 LTR.

Interaction of the second coding exon of Tat with human EF-1 delta delineates a mechanism for HIV-1-mediated shut-off of host mRNA translation.

HIV-1 Tat has pleiotropic functions. While its most studied role is to activate transcription from the retroviral long terminal repeat (LTR)-promoter, Tat also has functions as a secretable growth factor, a T-cell activator, and an inducer of cellular apoptosis, amongst others. For its transcriptional function, the first coding exon of Tat appears wholly sufficient; however, lentiviruses (HIVs and SIVs) maintain and conserve a second coding exon for Tat. While the function(s) of the second exon of Tat has remained largely unknown, its integrity in lentiviral genomes suggests biological importance, possibly a role in non-transcriptional activities. To understand better the biology of the second exon of Tat in HIV-1 infection of cells, we have searched for cellular proteins that bind specifically to this protein domain. Here, we report that the human translation elongation factor 1-delta (EF-1 delta) binds to the second exon of HIV-1 Tat. Interaction between Tat and EF-1 delta dramatically reduces the efficiency of the translation of cellular, but not viral, mRNAs. These findings suggest that a non-transcriptional activity of Tat modulates cellular protein synthesis, thereby affecting the metabolism of host cells.

Oncogenic potential of TAR RNA binding protein TRBP and its regulatory interaction with RNA-dependent protein kinase PKR.

TAR RNA binding protein (TRBP) belongs to an RNA binding protein family that includes the double-stranded RNA-activated protein kinase (PKR), Drosophila Staufen and Xenopus xlrbpa. One member of this family, PKR, is a serine/threonine kinase which has anti-viral and anti-proliferative effects. In this study we show that TRBP is a cellular down-regulator of PKR function. Assaying expression from an infectious HIV-1 molecular clone, we found that PKR inhibited viral protein synthesis and that over-expression of TRBP effectively countered this inhibition. In intracellular and in cell-free assays we show that TRBP directly inhibits PKR autophosphorylation through an RNA binding-independent pathway. Biologically, TRBP serves a growth-promoting role; cells that overexpress TRBP exhibit transformed phenotypes. Our results demonstrate the oncogenic potential of TRBP and are consistent with the notion that intracellular PKR function contributes physiologically towards regulating cellular proliferation.

Mechanism of transdominant inhibition of CCR5-mediated HIV-1 infection by ccr5delta32.

Human chemokine receptor 5 (CCR5) functions as a co-receptor for Human immunodeficiency virus (HIV-1) infection. CCR5 is a seven-transmembrane cell surface receptor. Recently, a naturally occurring mutation of CCR5, ccr5Delta32, has been described. A small number of Caucasians are homozygously ccr5Delta32/ccr5Delta32, while a larger number of individuals are heterozygously CCR5/ccr5Delta32. The ccr5Delta32/ccr5Delta32 genotype has been linked to a phenotype that is "highly" protected from HIV-1 infection. On the other hand, several studies have shown that the CCR5/ccr5Delta32 genotype confers "relative" protection from AIDS with onset of disease being delayed by 2-4 years. Although it is known that peripheral blood lymphocytes from heterozygous individuals (CCR5/ccr5Delta32) support ex vivo HIV-1 replication at a reduced level compared with CCR5/CCR5 cells, the molecular basis for this observation is unknown. Here we report on events that post-translationally modify CCR5. We show that CCR5 progresses through the endoplasmic reticulum prior to appearing on the cell surface. Mature CCR5 can be post-translationally modified by phosphorylation and/or co-translationally by multimerization. By contrast, mutant ccr5Delta32, although retaining the capacity for multimerization, was incapable of being phosphorylated. ccr5Delta32 heterocomplexes with CCR5, and this interaction retains CCR5 in the endoplasmic reticulum resulting in reduced cell surface expression. Thus, co-expression in cells of ccr5Delta32 with CCR5 produces a trans-inhibition by the former of ability by the latter to support HIV-1 infection. Taken together, our findings suggest CCR5/ccr5Delta32 heterodimerization as a molecular explanation for the delayed onset of AIDS in CCR5/ccr5Delta32 individuals.

Inhibition of human immunodeficiency virus type 1 production in infected peripheral blood mononuclear cells by human leukocyte antigen class I-specific antibodies: evidence for a novel antiviral mechanism.

A well-characterized mechanism by which anti-HLA class I monoclonal antibodies (MAb) inhibit human immunodeficiency virus type 1 (HIV-1) propagation in in vitro cell cultures is the neutralization of the virus through interactions with HLA molecules associated with the virion envelope. Yet, the possibility that another mechanism of inhibition might affect a postbinding stage of the virus life cycle has been strongly suggested by our previous investigations. To demonstrate that the interaction of MAb B1-1G6 with the light chain of cell surface-expressed HLA class I molecules inhibits a postbinding step of the HIV-1 life cycle, peripheral blood mononuclear cells (PBMCs) were exposed to viruses grown in HLA class I-negative, CD4-positive cells (these viruses, which did not carry HLA class I molecules, cannot be neutralized by anti-HLA MAb during the first round of infection), and PCR was used at various times postexposure to search for the different forms of HIV-1 DNA and RNA in virus-exposed PBMCs cultured in either the presence or [correction of] absence of MAb B1-1G6. Although viral DNA was found in MAb B1-1G6-treated cells, spliced HIV-1 mRNA could not be detected in those cells. In contrast, HIV-1 gene expression was found in HIV-1-infected PBMCs treated with B9-12-1, another HLA class I-specific MAb which prevents infection of cells by cell-free viruses but which fails to inhibit cell-to-cell transmission of HIV-1. These results highlight a second antiviral mechanism by which anti-HLA MAb inhibit in vitro HIV-1 propagation.

Binding of HIV-1 virions or gp120-anti-gp120 immune complexes to HIV-1-infected quiescent peripheral blood mononuclear cells reveals latent infection.

HIV-1-infected quiescent CD4+ cells harbor the virus in an inactive state until subsequent activation. The possibility that HIV-1 itself and the virus envelope glycoprotein 120 (gp120) might be important agents of this activation was investigated. The present data indicate that binding of heat-inactivated HIV-1 (iHIV-1) to infected resting PBMCs was sufficient to activate NF-kappa B and AP-1, to induce transition from the G0/G1 stage of the cell cycle to the S/G2/M stage, to induce cell surface expression of CD25, to stimulate provirus integration, and to commit cells to produce virus. The cumulative amount of HIV-1 produced by iHIV-1-stimulated cells strictly depended on the concentration of p24gag in the virion preparations used for stimulation. Moreover, virus production was not evidenced in infected resting cells exposed to iHIV-1 previously incubated with soluble CD4 (sCD4), indicating that activation requires a contact between HIV-1 envelope glycoproteins and cell surface CD4. Although soluble gp120 did not stimulate virus production, we found that transition to the S/G2/M stage of the cell cycle, cell surface expression of activation Ags, and virus production were stimulated by cross-linking of CD4 by gp120-anti-gp120 immune complexes. Finally, incubation of gp120-anti-gp120 immune complexes with sCD4 inhibited these effects. These findings suggest that virions and gp120 anti-gp120 immune complexes found in infected patients at all times of infection can stimulate virus production in CD4+ cells harboring HIV-1 in an inducible state.

HIV induces activation of phosphatidylinositol 4-kinase and mitogen-activated protein kinase by interacting with T cell CD4 surface molecules.

T cell surface CD4 molecules act as co-receptors that amplify the T cell receptor (TcR)/CD3-induced signal transduction by a mechanism that requires the interaction of CD4 with p56lck tyrosine kinase (Veillette et al.; Nature 1989 338:257). Here, we demonstrate that in the absence of TcR signaling, heat-inactivated HIV-1 (HIV-HI) also elicits a cascade of events generally considered to convey a positive signal, such as protein tyrosine phosphorylation, phosphatidylinositol 4-kinase and mitogen-activated protein kinase activation. These results contribute to understand better the control that HIV may exert on its own replication or on T cell apoptosis by modulating the activation status of its target cells through its interaction with T cell surface CD4 molecules.

Functional epitope analysis of the human CD4 molecule: antibodies that inhibit human immunodeficiency virus type 1 gene expression bind to the immunoglobulin CDR3-like region of CD4.

We recently demonstrated that monoclonal antibody (MAb) 13B8-2, specific for the immunoglobulin (Ig) complementary determining region 3 (CDR3)-like region of the CD4 molecule, inhibits viral transcription in human immunodeficiency virus (HIV)-infected CEM cells and HIV type 1 (HIV-1) promoter activity. Here, we have studied the capacity of several MAb specific for the D1 domain of CD4, including anti-CDR2-like (Leu-3a and ST4) and anti-CDR3-like (13B8-2 and ST40) MAb, and for the D2 domain of CD4 (BL4) to inhibit both provirus transcription in HIV-1LAI-infected CEM cells and transcription of the chloramphenicol acetyltransferase (CAT) gene under control of the HIV-1 long terminal repeat in transiently transfected CEM cells. We found that HIV-1 promoter activity and provirus transcription are inhibited only by MAb that bind to the CDR3-like region in domain 1 of CD4. Moreover, we demonstrated that the Fab fragment of an anti-CDR3-like region-specific anti-CD4 MAb is a powerful inhibitor of HIV-1 promoter activity. These results have implications for understanding the role of the CDR3-like region in CD4 T-cell signaling, which controls provirus transcription.