Macropinocytosis-like HIV-1 internalization in macrophages is CCR5 dependent and leads to efficient but delayed degradation in endosomal compartments.

HIV-1 endocytosis by a macropinocytosis-like mechanism has been shown to lead to productive infection in macrophages. However, little is known of this pathway. In this study, we examined HIV-1 endocytosis using biochemical approaches and imaging techniques in order to better understand the mechanisms that allow for productive infection of these cells via the endosomal pathway. We show here that this macropinocytosis-like mechanism is not the sole pathway involved in HIV-1 endocytosis in macrophages. However, this pathway specifically requires CCR5 engagement at the cell surface, which in turn suggests that the virus and its coreceptor are present in the endosomal environment simultaneously. Furthermore, although we observed efficient viral degradation following endocytosis, analyses of HIV-1 transport through the endolysosomal pathway revealed that viral degradation is delayed following endosomal internalization, possibly allowing the virus to complete its fusion.

Regulation of host gene expression by HIV-1 TAR microRNAs.

BACKGROUND: The transactivating response (TAR) element of human immunodeficiency virus type 1 (HIV-1) is the source of two functional microRNAs (miRNAs), miR-TAR-5p and miR-TAR-3p. The objective of this study was to characterize the post-transcriptional regulation of host messenger RNAs (mRNAs) relevant to HIV-1 pathogenesis by HIV-1 TAR miRNAs. RESULTS: We demonstrated that TAR miRNAs derived from HIV-1 can incorporate into host effector Argonaute protein complexes, which is required if these miRNAs are to regulate host mRNA expression. Bioinformatic predictions and reporter gene activity assays identified regulatory elements complementary and responsive to miR-TAR-5p and miR-TAR-3p in the 3' untranslated region (UTR) of several candidate genes involved in apoptosis and cell survival. These include Caspase 8, Aiolos, Ikaros and Nucleophosmin (NPM)/B23. Analyses of Jurkat cells that stably expressed HIV-1 TAR or contained a full-length latent HIV provirus suggested that HIV-1 TAR miRNAs could regulate the expression of genes in T cells that affect the balance between apoptosis and cell survival. CONCLUSIONS: HIV-1 TAR miRNAs may contribute to the replication cycle and pathogenesis of HIV-1, by regulating host genes involved in the intricate balance between apoptosis and infected cell, to induce conditions that promote HIV-1 propagation and survival.

Differential HIV-1 endocytosis and susceptibility to virus infection in human macrophages correlate with cell activation status.

HIV-1 is an enveloped virus that enters target cells by fusion either directly at the plasma membrane or at the endosomal membrane. The latter mechanism follows a rapid engulfment of HIV-1 after its receptor engagement at the cell surface, and its scale depends on cellular endocytosis/degradation rates and virus fusion kinetics. HIV-1 has recently been shown to exploit a novel Pak1-dependent macropinocytosis mechanism as a way to productively infect macrophages. However, macrophages are highly heterogeneous cells that can adapt functionally to their changing environment, and their endosomal/lysosomal pathway is highly regulated upon cell activation. These changes might impact the ability of HIV-1 to exploit endocytosis as a way to productively infect macrophages. In this study, we compared HIV-1 endocytosis/degradation rates in nonactivated, M1-activated, and M2a-activated monocyte-derived macrophages (MDMs). We found that the rate of HIV-1 endocytosis was increased in M1-activated but decreased in M2a-activated MDMs. However, both M1 and M2a activations of MDMs led specifically to a greater clathrin-mediated endocytosis of HIV-1, which was independent of CD4 and CCR5 binding. Furthermore, clathrin-mediated endocytosis is unlikely to result in productive HIV-1 infection, given that it leads to increased viral degradation. Therefore, we suggest that viral fusion following endocytosis is restricted in activated macrophages.

Fluorescent ligands of the bradykinin B1 receptors: pharmacologic characterization and application to the study of agonist-induced receptor translocation and cell surface receptor expression.

Unlike the widely distributed and preformed B(2) receptors, the bradykinin B(1) receptors exhibit a highly regulated expression and minimal agonist-induced endocytosis. To evaluate the potential usefulness of fluorescent B(1) receptor probes applicable to live cell microscopy and cytofluorometry, combined chemical synthesis and pharmacologic evaluation have been conducted on novel 5(6)-carboxyfluorescein [5(6)CF]-containing peptides. Representative agents are the antagonist B-10376 [5(6)CF-epsilon-aminocaproyl-Lys-Lys-[Hyp(3), CpG(5), D-Tic(7), CpG(8)]des-Arg(9)-bradykinin] and the agonist B-10378 [5(6)CF-epsilon-aminocaproyl-Lys-des-Arg(9)-bradykinin]. B-10376 has a K(i) of 10 to 20 nM to displace [(3)H]Lys-des-Arg(9)-bradykinin from rabbit or human recombinant B(1) receptors expressed in human embryonic kidney (HEK) 293 cells and is a surmountable antagonist in the rabbit aorta contractility assay (pA(2), 7.49). B-10378 was a full agonist at the naturally expressed B(1) receptor (rabbit aorta contraction, calcium transients in human smooth muscle cells) and had a binding competition K(i) of 19 or 89 nM at the recombinant rabbit or human receptor, respectively. Both fluorescent probes can label with specificity human or rabbit B(1) receptors expressed in HEK 293 cells (epifluorescence or confocal microscopy), but the agonist was associated with discontinuous plasma membrane labeling, which coincided with that of a red-emitting caveolin-1 conjugate. Cytofluorometry with B-10376 was applied to recombinant and, in human vascular smooth muscle cells, to naturally expressed B(1) receptors. In all fluorescent applications, the specific labeling was reduced by an excess of a B(1) receptor nonpeptide antagonist. Despite the loss of affinity determined by the introduction of a fluorophore in B(1) receptor agonist or antagonist peptides, the resulting agents allow original applications (imaging in live cells, cytofluorometry).

Involvement of Dcr1 in post-transcriptional regulation of gene expression in Schizosaccharomyces pombe.

The ribonuclease III Dicer (Dcr1) has been shown to be required for chromosome segregation and gene silencing in Schizosaccharomyces pombe. These effects are thought to be transcriptional, mediated by formation and maintenance of heterochromatin, and guided by small RNAs derived from Dcr1 along a process known as RNA interference. In order to get further insights into the gene regulatory role of Dcr1, we performed comparative analyses of dcr1 knockout and wild-type fission yeast strains. Analysis of part of the soluble proteomes identified eight cellular proteins whose expression is under Dcr1 control, three of which are integral constituents of the glycolysis pathway. Further correlations with their respective mRNA transcript levels are compatible with the existence of a post-transcriptional gene regulatory mechanism involving Dcr1 or a Dcr1 complex. Experiments designed to identify components of Dcr1 complexes unveiled two novel Dcr1 interactors, namely the zinc finger protein Byr3 and the ribosomal protein L12. Consistently enriched in Dcr1 immune complexes, Byr3 and L12 may link Dcr1 to the transcriptional and translational machineries, respectively, and contribute to post-transcriptional gene regulation in fission yeast.

Regulatory RNAs: future perspectives in diagnosis, prognosis, and individualized therapy.

With potentially up to 1000 microRNAs (miRNAs) present in the human genome, altogether regulating the expression of thousands of genes, one can anticipate that miRNAs will play a significant role in health and disease. Deregulated protein expression induced by a dysfunctional miRNA-based regulatory system is thus expected to lead to the development of serious, if not lethal, genetic diseases. A relationship among miRNAs, Dicer, and cancer has recently been suggested. Further investigations will help establish specific causal links between dysfunctional miRNAs and diseases. miRNAs of foreign origin, e.g., viruses, may also be used as specific markers of viral infections. In these cases, miRNA expression profiles could represent a powerful diagnostic tool. Regulatory RNAs may also have therapeutic applications, by which disease-causing genes or viral miRNAs could be neutralized, or functional miRNAs be restored. Will bedside miRNA expression profiling eventually assist physicians in providing patients with accurate diagnosis, personalized therapy, and treatment outcome?

MicroRNAs in gene regulation: when the smallest governs it all.

Encoded by the genome of most eukaryotes examined so far, microRNAs (miRNAs) are small approximately 21-nucleotide (nt) noncoding RNAs (ncRNAs) derived from a biosynthetic cascade involving sequential processing steps executed by the ribonucleases (RNases) III Drosha and Dicer. Following their recent identification, miRNAs have rapidly taken the center stage as key regulators of gene expression. In this review, we will summarize our current knowledge of the miRNA biosynthetic pathway and its protein components, as well as the processes it regulates via miRNAs, which are known to exert a variety of biological functions in eukaryotes. Although the relative importance of miRNAs remains to be fully appreciated, deregulated protein expression resulting from either dysfunctional miRNA biogenesis or abnormal miRNA-based gene regulation may represent a key etiologic factor in several, as yet unidentified, diseases. Hence is our need to better understand the complexity of the basic mechanisms underlying miRNA biogenesis and function.

Dicer-derived microRNAs are utilized by the fragile X mental retardation protein for assembly on target RNAs.

In mammalian cells, fragile X mental retardation protein (FMRP) has been reported to be part of a microRNA (miRNA)-containing effector ribonucleoprotien (RNP) complex believed to mediate translational control of specific mRNAs. Here, using recombinant proteins, we demonstrate that human FMRP can act as a miRNA acceptor protein for the ribonuclease Dicer and facilitate the assembly of miRNAs on specific target RNA sequences. The miRNA assembler property of FMRP was abrogated upon deletion of its single-stranded (ss) RNA binding K-homology domains. The requirement of FMRP for efficient RNA interference (RNAi) in vivo was unveiled by reporter gene silencing assays using various small RNA inducers, which also supports its involvement in an ss small interfering RNA (siRNA)-containing RNP (siRNP) effector complex in mammalian cells. Our results define a possible role for FMRP in RNA silencing and may provide further insight into the molecular defects in patients with the fragile X syndrome.