A bulge structure in HIV-1 TAR RNA is required for Tat binding and Tat-mediated trans-activation

The Tat protein of human immunodeficiency virus type 1 (HIV-1) trans-activates viral gene expression and is obligatory for virus replication. Tat function is mediated through a sequence termed TAR that comprises part of the 5'-noncoding region of all HIV-1 mRNAs. This region forms a stable stem-loop structure in vitro. Recent evidence indicates that Tat binds directly to the TAR RNA sequence, and this binding is independent of the nucleotide sequence in the loop but dependent on the integrity of the upper stem. We used the electrophoretic mobility-shift assay to identify the sequence and structure specificity of this interaction and its correlation with Tat trans-activation. We show that a 3-nucleotide bulge structure (positions +23 to +25) in TAR RNA is important for both Tat interaction with TAR RNA and Tat-mediated trans-activation of gene expression. Single base substitutions at position +23 that impair Tat-mediated trans-activation in vivo also reduce binding of Tat to TAR in vitro, suggesting that the first uridine residue in the bulge is the critical base for both functions. In contrast, mutations in the loop (positions +31 to +34) and the stem (positions +9 to +12 and +49 to +52), which reduce Tat-mediated trans-activation, had no effect on Tat binding. We also show that a Tat peptide that includes the basic region required for nucleolar localization binds to TAR RNA with the same specificity as the full-length protein. We conclude that Tat binding to TAR is necessary but not sufficient by itself to account for trans-activation.     

A novel RNA motif that binds efficiently and specifically to the Ttat protein of HIV and inhibits the trans-activation by Tat of transcription in vitro and in vivo

BACKGROUND: To find a novel RNA that would bind efficiently and specifically to Tat protein but not to other cellular factors, we used an in vitro selection method and isolated a novel aptamer RNATat, a 37-mer RNA oligomer, that binds efficiently to the Tat protein of HIV-1. In the present study, we analysed various properties of aptamer RNATat, including binding kinetics, identification of functional groups for Tat binding, and inhibition of Tat function. RESULTS: The binding affinity of the isolated aptamer RNATat to Tat-1 was 133 times higher than that of authentic TAR-1 RNA. RNATat is composed of inverted repeats of two TAR-like motifs, and even though RNATat had two Tat-binding core elements, the interaction with Tat took place at a molar ratio of 1 : 1. Several functional groups of aptamer RNATat responsible for Tat binding were identified. The selected aptamer RNATat competed effectively for binding to Tat even in the presence of a large excess of TAR-1 or TAR-2 RNA in vitro, and specifically prevented Tat-dependent trans-activation both in vitro and in vivo. CONCLUSIONS: Our results indicate that a novel aptamer, RNATat, retained strong affinity for Tat even in the presence of a large excess of HIV TAR. RNATat binds efficiently to Tat proteins or peptides derived from either HIV-1 or HIV-2. Unlike TAR RNA, RNATat affinity does not depend upon cellular proteins such as cyclin T1, thus RNATat has the potential for use as a molecular recognition element in biosensors.     

Differential role of MyD88 in macrophage-mediated responses to opportunistic fungal pathogens

Toll-like receptors mediate macrophage recognition of microbial ligands, inducing expression of microbicidal molecules and cytokines via the adapter protein MyD88. We investigated the role of MyD88 in regulating murine macrophage responses to a pathogenic yeast (Candida albicans) and mold (Aspergillus fumigatus). Macrophages derived from bone marrow of MyD88-deficient mice (MyD88(-/-)) demonstrated impaired phagocytosis and intracellular killing of C. albicans compared to wild-type (MyD88(+/+)) macrophages. In contrast, ingestion and killing of A. fumigatus conidia was MyD88 independent. Cytokine production by MyD88(-/-) macrophages in response to C. albicans yeasts and hyphae was substantially decreased, but responses to A. fumigatus hyphae were preserved. These results provide evidence that MyD88 signaling is involved in phagocytosis and killing of live C. albicans, but not A. fumigatus. The differential role of MyD88 may represent one mechanism by which macrophages regulate innate responses specific to different pathogenic fungi.     

Mapping of HIV-1 Tat protein sequences required for binding to Tar RNA

We have utilized a gel retardation assay to study the binding of chemically synthesized domains of the HIV-1 Tat protein to radiolabeled trans-activating response element (Tar) RNA. As with recombinant Tat protein, synthetic Tat specifically binds to Tar RNA and not to a defective Tar RNA or to anti-sense Tar RNA. The 6 amino acid portion of the basic region containing five arginines is sufficient to confer Tar binding to overlapping Tat protein fragments; Tat fragments that lack the basic region do not bind Tar. In addition, the basic region alone can also bind Tar RNA; however, binding of the basic region is non specific since defective Tar RNA is bound as well as wild type Tar RNA. Binding specificity for wild type Tar RNA can be conferred by the addition of a minimum of 8 random amino acids to either end of the basic region.     

The perlecan heparan sulfate proteoglycan mediates cellular uptake of HIV-1 Tat through a pathway responsible for biological activity

Cell surface heparan sulfate proteoglycans (HSPGs) mediate internalization of HIV-1 Tat. Herein, we report that human WiDr cells, which express perlecan but no other HSPGs, can internalize 125I-labeled Tat with minimal lysosomal degradation. Pre-treatment of cells with heparitinase almost completely abolished 125I-Tat surface binding, while the use of an HIV-1 long terminal repeat (LTR) promoter-reporter construct demonstrated that transactivation was potently blocked by pretreatment of cells with heparitinase, indicating an essential role for perlecan in the biologic effects of Tat. We conclude that the perlecan mediates Tat uptake and is required for HIV-1 LTR-directed transactivation in this human cell type.     

The C-type lectin DC-SIGN (CD209) is an antigen-uptake receptor for Candida albicans on dendritic cells

Dendritic cells (DC) that express the type II C-type lectin DC-SIGN (CD209) are located in the submucosa of tissues, where they mediate HIV-1 entry. Interestingly, the pathogen Candida albicans, the major cause of hospital-acquired fungal infections, penetrates at similar submucosal sites. Here we demonstrate that DC-SIGN is able to bind C. albicans both in DC-SIGN-transfected cell lines and in human monocyte-derived DC. The binding was shown to be time- as well as concentration-dependent, and live as well as heat-inactivated C. albicans were bound to the same extent. Moreover, in immature DC, DC-SIGN was able to internalize C. albicans in specific DC-SIGN-enriched vesicles, distinct from those containing the mannose receptor, the other known C. albicans receptor expressed by DC. Together, these results demonstrate that DC-SIGN is an exquisite pathogen-uptake receptor that captures not only viruses but also fungi.     

Dysregulation of the IgE/Fc epsilon RI network in HIV-1 infection

Serum IgE levels are increased in adults and children with HIV-1 infection and could be a marker of poor prognosis. Allergic reactions and adverse reactions to drugs are also increased in HIV-1-infected individuals. An imbalance between a T(H)1-like and a T(H)2-like cytokine profile has been documented in HIV-1 infection. We have found that HIV-1 gp120 from different clades is a potent stimulus for histamine and cytokine (IL-4 and IL-13) release from basophils. Gp120 acts as a viral superantigen, interacting with the V(H)3 region of IgE to induce mediator release from human Fc epsilon RI(+) cells. Human basophils and mast cells express the chemokine receptor CCR3, which binds the chemokines eotaxin and RANTES. By interacting with the CCR3 receptor on Fc epsilon RI(+) cells, HIV-1 Tat protein is a potent chemoattractant for human basophils and lung mast cells. Tat protein also induced IL-4 and IL-13 release from basophils. Preincubation of basophils with Tat protein upregulated the surface expression of the CCR3 receptor. Extracellular Tat can influence the directional migration of human Fc epsilon RI(+) cells, the expression of chemokine receptor CCR3, and the release of T(H)2 cytokines. Because Tat protein is actively released by HIV-1-infected cells, our results indicate a novel mechanism by which Fc epsilon RI(+) cells are rendered more susceptible to infection with CCR3-tropic HIV-1 isolates; that is, two HIV-1 proteins, gp120 and Tat, trigger the release of cytokines critical for T(H)2 polarization from Fc epsilon RI(+) cells, and Tat upregulates beta-chemokine receptor CCR3 on these cells.     

Beta(3)-mediated engulfment of apoptotic tumor cells by dendritic cells is dependent on CAMKII: inhibition by HIV-1 Tat

In this paper, we show that the engulfment of apoptotic tumor cells by DC requires the activation of the calcium-calmodulin kinase II (CAMKII). Indeed, DC phagocytosis of apoptotic lymphoma cells is consistently inhibited by KN62 and KN93, two blockers of CAMKII, but not by the inactive compound KN92. Wortmannin and LY294002, two inhibitors of the phosphatidyl-inositol-3 kinase, slightly decrease the phagocytosis of apoptotic cells, at variance with PD98059, an inhibitor of the mitogen-activated protein kinase. It is interesting that the addition of synthetic HIV-1 Tat, which we demonstrated to inhibit phagocytosis and calcium influx in DC, blocks the activation of CAMKII elicited via beta(3) integrin, which is involved in apoptotic body engulfment by DC. Experiments performed with Tat-derived peptides showed that this inhibition is mediated by the C-terminal domain of Tat. Finally, pertussis toxin can prevent HIV-1 Tat-mediated inhibition, suggesting the involvement of a guanosine triphosphate-binding (G) protein in DC-mediated phagocytosis.     

Penetration and damage of endothelial cells by Candida albicans.

The mechanisms of phagocytosis of Candida albicans by human vascular endothelial cells and subsequent endothelial cell injury were examined in vitro. Both live and killed C. albicans cells were phagocytized by endothelial cells. This organism specifically induced endothelial cell phagocytosis because neither Candida tropicalis nor Torulopsis glabrata was ingested. Endothelial cell microfilaments polymerized around C. albicans as the organisms were phagocytized. Cytochalasin D inhibited this polymerization of microfilaments around C. albicans and blocked phagocytosis. The blocking of actin depolymerization with phalloidin had no effect on microfilament condensation around the organism, indicating that the microfilaments surrounding C. albicans are formed from a pool of G-actin. Intact microtubules were also necessary for the phagocytosis of C. albicans, since the depolymerizing of endothelial cell microtubules with nocodazole prevented the condensation of actin filaments around the organisms and inhibited phagocytosis. In contrast, microtubule depolymerization was not required for microfilament function because the blocking of microtubule depolymerization with taxol had no effect on microfilament condensation around C. albicans. The phagocytosis of C. albicans was pivotal in the induction of endothelial cell damage, since the blocking of candidal internalization significantly reduced endothelial cell injury. Endothelial cells were not damaged by phagocytosis of dead organisms, indicating that injury was caused by a factor associated with viable organisms. Therefore, C. albicans is uniquely able to induce endothelial cell phagocytosis by comparison with non-albicans species of Candida. Furthermore, at least two components of the endothelial cytoskeleton, microfilaments and microtubules, are necessary for the phagocytosis of C. albicans.     

Multiple levels of PKR inhibition during HIV-1 replication

Recent therapeutic approaches against HIV-1 include IFN in combination therapy for patients with coinfections or as an alternative strategy against the virus. These treatment options require a better understanding of the weak efficacy of the IFN-stimulated genes, such as the protein kinase RNA-activated (PKR), which results in viral progression. Activated PKR has a strong antiviral activity on HIV-1 expression and production in cell culture. However, PKR is not activated upon HIV-1 infection when the virus reaches high levels of replication, due to viral and cellular controls. PKR is activated by low levels of the HIV-1 trans-activation response (TAR) RNA element, but is inhibited by high levels of this double-stranded RNA. The viral Tat protein also counteracts PKR activation by several mechanisms. In addition, HIV-1 replicates only in cells that have a high level of the TAR RNA binding protein (TRBP), a strong inhibitor of PKR activation. Furthermore, increased levels of adenosine deaminase acting on RNA (ADAR1) are observed when HIV-1 replicates at high levels and the protein binds to PKR and inhibits its activation. Finally, the PKR activator (PACT) also binds to PKR during HIV-1 replication with no subsequent kinase activation. The combination of all the inhibiting pathways that prevent PKR phosphorylation contributes to a high HIV-1 production in permissive cells. Enhancing PKR activation by counteracting its inhibitory partners could establish an increased innate immune antiviral pathway against HIV-1 and could enhance the efficacy of the IFN treatment.     

Mechanisms of IgE elevation in HIV-1 infection

Serum IgE levels are high in adults and children with HIV-1 infection and could be a marker of poor prognosis. Allergic reactions and adverse reactions to drugs also tend to increase in HIV-1-infected individuals. An imbalance between a "T(H)1-like" and a "T(H)2-like" cytokine profile has been documented in HIV-1 infection. We have demonstrated that HIV-1 gp 120 from different clades is a stimulus for histamine and cytokine (IL-4 and IL-13) release from basophils. Gp 120 acts as a viral superantigen, interacting with the V(H)3 region of IgE to induce mediator release from human Fc epsilonRI+ cells. Human basophils and mast cells express the chemokine receptor CCR3, which binds the chemokines eotaxin and RANTES. By interacting with the CCR3 receptor on Fc epsilonRI+ cells, HIV-I Tat protein is a potent chemoattractant for human basophils and lung mast cells. Preincubation of basophils with Tat protein upregulates mRNA CCR3 and the surface expression of this chemokine receptor. Tat also induces IL-4 and IL-13 release from basophils. Extracellular Tat can influence the directional migration of human Fc epsilonRI+ cells, the expression of chemokine receptor CCR3, and the release of T(H)2 cytokines. Our results indicate two novel mechanisms by which two HIV-1 proteins, gp120 and Tat, trigger the release of cytokines critical for T(H)2 polarization from human Fc epsilonRI+ cells.