Biomedical subjects

David Rekosh

Publications and source records attributed to David Rekosh.

9 recordsLinked to original sources

PubMed2006

The envelope gene is a cytopathic determinant of CCR5 tropic HIV-1.

Late stage AIDS associated CCR5 tropic HIV-1 clones (R5-AIDS HIV-1) exhibit greater cytopathic effects (CPE) than earlier isolates from the same patients. In this study, envelopes from a series of three biological clones derived from the same patient were evaluated as a cytopathic determinant of R5-AIDS HIV-1 for thymocytes. In a single round of replication in thymocytes, the AIDS associated clone mediated greater initiation of reverse transcription. This enhancement was not due to broadened coreceptor tropism, as all clones studied were exclusively R5 tropic. The full-length R5-AIDS env mediated greater infectivity than R5 pre-AIDS env when used to pseudotype a reporter virus. R5-AIDS env pseudotypes were more resistant to TAK-779 and showed more rapid infection kinetics but similar resistance to a CD4 blocking mAb. We conclude that the enhanced thymic replication and CPE shown by the R5-AIDS clone is due to enhanced efficiency of Env-mediated entry via CCR5.

Amides↗

PubMed2006

An intron with a constitutive transport element is retained in a Tap messenger RNA.

Alternative splicing is a key factor contributing to genetic diversity and evolution. Intron retention, one form of alternative splicing, is common in plants but rare in higher eukaryotes, because messenger RNAs with retained introns are subject to cellular restriction at the level of cytoplasmic export and expression. Often, retention of internal introns restricts the export of these mRNAs and makes them the targets for degradation by the cellular nonsense-mediated decay machinery if they contain premature stop codons. In fact, many of the database entries for complementary DNAs with retained introns represent them as artefacts that would not affect the proteome. Retroviruses are important model systems in studies of regulation of RNAs with retained introns, because their genomic and mRNAs contain one or more unspliced introns. For example, Mason-Pfizer monkey virus overcomes cellular restrictions by using a cis-acting RNA element known as the constitutive transport element (CTE). The CTE interacts directly with the Tap protein (also known as nuclear RNA export factor 1, encoded by NXF1), which is thought to be a principal export receptor for cellular mRNA, leading to the hypothesis that cellular mRNAs with retained introns use cellular CTE equivalents to overcome restrictions to their expression. Here we show that the Tap gene contains a functional CTE in its alternatively spliced intron 10. Tap mRNA containing this intron is exported to the cytoplasm and is present in polyribosomes. A small Tap protein is encoded by this mRNA and can be detected in human and monkey cells. Our results indicate that Tap regulates expression of its own intron-containing RNA through a CTE-mediated mechanism. Thus, CTEs are likely to be important elements that facilitate efficient expression of mammalian mRNAs with retained introns.

Alternative Splicing↗

PubMed2006

The Wilms' tumor 1 (WT1) gene (+KTS isoform) functions with a CTE to enhance translation from an unspliced RNA with a retained intron.

The Wilms' tumor 1 (WT1) gene plays an important role in mammalian urogenital development, and dysregulation of this gene is observed in many human cancers. Alternative splicing of WT1 RNA leads to the expression of two major protein isoforms, WT1(+KTS) and WT1(-KTS). Whereas WT1(-KTS) acts as a transcriptional regulator, no clear function has been ascribed to WT1(+KTS), despite the fact that this protein is crucial for normal development. Here we show that WT1(+KTS) functions to enhance expression from RNA possessing a retained intron and containing either a cellular or viral constitutive transport element (CTE). WT1(+KTS) expression increases the levels of unspliced RNA containing a CTE and specifically promotes the association of this RNA with polyribosomes. These studies provide further support for links between different steps in RNA metabolism and for the existence of post-transcriptional operons.

Amino Acid Sequence↗

PubMed2005

Mutations in tap uncouple RNA export activity from translocation through the nuclear pore complex.

Interactions between transport receptors and phenylalanine-glycine (FG) repeats on nucleoporins drive the translocation of receptor-cargo complexes through nuclear pores. Tap, a transport receptor that mediates nuclear export of cellular mRNAs, contains a UBA-like and NTF2-like folds that can associate directly with FG repeats. In addition, two nuclear export sequences (NESs) within the NTF2-like region can also interact with nucleoporins. The Tap-RNA complex was shown to bind to three nucleoporins, Nup98, p62, and RanBP2, and these interactions were enhanced by Nxt1. Mutations in the Tap-UBA region abolished interactions with all three nucleoporins, whereas the effect of point mutations within the NTF2-like domain of Tap known to disrupt Nxt1 binding or nucleoporin binding were nucleoporin dependent. A mutation in any of these Tap domains was sufficient to reduce RNA export but was not sufficient to disrupt Tap interaction with the NPC in vivo or its nucleocytoplasmic shuttling. However, shuttling activity was reduced or abolished by combined mutations within the UBA and either the Nxt1-binding domain or NESs. These data suggest that Tap requires both the UBA- and NTF2-like domains to mediate the export of RNA cargo, but can move through the pores independently of these domains when free of RNA cargo.

ATP-Binding Cassette Transporters↗

PubMed2004

Human immunodeficiency virus type 1 Nef associates with lipid rafts to downmodulate cell surface CD4 and class I major histocompatibility complex expression and to increase viral infectivity.

Lipid rafts are membrane microdomains that are functionally distinct from other membrane regions. We have shown that 10% of human immunodeficiency virus type 1 (HIV-1) Nef expressed in SupT1 cells is present in lipid rafts and that this represents virtually all of the membrane-associated Nef. To determine whether raft targeting, rather than simply membrane localization, has functional significance, we created a Nef fusion protein (LAT-Nef) containing the N-terminal 35 amino acids from LAT, a protein that is exclusively localized to rafts. Greater than 90% of the LAT-Nef protein was found in the raft fraction. In contrast, a mutated form, lacking two cysteine palmitoylation sites, showed less than 5% raft localization. Both proteins were equally expressed and targeted nearly exclusively to membranes. The LAT-Nef protein was more efficient than its nonraft mutant counterpart at downmodulating both cell surface CD4 and class I major histocompatibility complex (MHC) expression, as well as in enhancing first-round infectivity and being incorporated into virus particles. This demonstrates that targeting of Nef to lipid rafts is mechanistically important for all of these functions. Compared to wild-type Nef, LAT-Nef downmodulated class I MHC nearly as effectively as the wild-type Nef protein, but was only about 60% as effective for CD4 downmodulation and 30% as effective for infectivity enhancement. Since the LAT-Nef protein was found entirely in rafts while the wild-type Nef protein was distributed 10% in rafts and 90% in the soluble fraction, our results suggest that class I MHC downmodulation by Nef may be performed exclusively by raft-bound Nef. In contrast, CD4 downmodulation and infectivity enhancement may require a non-membrane-bound Nef component as well as the membrane-bound form.

Animals↗

PubMed2003

Tap and NXT promote translation of unspliced mRNA.

Tap has been proposed to play a role in general mRNA export and also functions in expression of RNA with retained introns that contain the MPMV CTE (constitutive transport element). Tap forms a functional heterodimer with NXT/p15. We have previously demonstrated that unspliced intron-containing CTE RNA is efficiently exported to the cytoplasm in mammalian cells. Here we show that Tap and NXT proteins function together to enhance translation of proteins from the exported CTE RNA. Pulse chase experiments show that Tap/NXT significantly increases the rate of protein synthesis. Sucrose gradient analysis demonstrates that Tap and NXT efficiently shift the unspliced RNA into polyribosomal fractions. Furthermore, Tap, but not NXT is detected in polyribosomes. Taken together, our results indicate that Tap and NXT serve a role in translational regulation of RNA after export to the cytoplasm. They further suggest that Tap/NXT may play a role in remodeling of cytoplasmic RNP complexes, providing a link between export pathways and cytoplasmic fate.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2003

Sam68 enhances the cytoplasmic utilization of intron-containing RNA and is functionally regulated by the nuclear kinase Sik/BRK.

Cells normally restrict the nuclear export and expression of intron-containing mRNA. In many cell lines, this restriction can be overcome by inclusion of cis-acting elements, such as the Mason-Pfizer monkey virus constitutive transport element (CTE), in the RNA. In contrast, we observed that CTE-mediated expression from human immunodeficiency virus Gag-Pol reporters was very inefficient in 293 and 293T cells. However, addition of Sam68 led to a dramatic increase in the amount of Gag-Pol proteins produced in these cells. Enhancement of CTE function was not seen when a Sam68 point mutant (G178E) that is defective for RNA binding was used. Additionally, the effect of Sam68 was inhibited in a dose-dependent manner by coexpression of an activated form of the nuclear kinase Sik/BRK that hyperphosphorylated Sam68. RNA analysis showed that cytoplasmic Gag-Pol-CTE RNA levels were only slightly enhanced by the addition of Sam68, compared to a 60- to 70-fold increase in the levels of Gag-Pol protein expression. Thus, in this system, Sam68 functioned to enhance the cytoplasmic utilization of RNA containing the CTE. These results suggest that Sam68 may interact with specific RNAs in the nucleus to provide a "mark" that affects their cytoplasmic fate. They also provide further evidence of links between signal transduction and RNA utilization.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Chondrocyte transplantation into articular cartilage defects with use of calcium alginate: the fate of the cells.

BACKGROUND: The fate of transplanted chondrocytes used to elicit the repair of osteochondral defects is unknown. The objective of this study was to examine the fate and the expression of cartilage-specific genes in chondrocytes when the chondrocyte phenotype was maintained preoperatively by alginate suspension culture, the cells were labeled with enhanced green fluorescent protein, and the chondrocytes in alginate were then implanted into full-thickness osteochondral defects in rabbits. METHODS: To determine the effect of alginate on rabbit chondrocytes in vitro, cells were grown in monolayer or in alginate suspension culture, and gene expression for aggrecan, type-I collagen, and type-II collagen was analyzed by reverse transcription-polymerase chain reaction. Cells were genetically labeled with the gene for enhanced green fluorescent protein, and the effect of transfer of the gene for enhanced green fluorescent protein on chondrocyte phenotype was assessed in vitro. Chondrocytes labeled with enhanced green fluorescent protein that were embedded in alginate were implanted into osteochondral defects in rabbit knees, either immediately after creation of the defects or after the cells had been preconditioned in alginate suspension culture for two weeks. The repair tissue within the osteochondral defects was assessed at one to four weeks. Cells labeled with enhanced green fluorescent protein were quantified by confocal microscopy, and the repair tissue was examined histologically with safranin O. RESULTS: Gene expression by chondrocytes demonstrated a selective upregulation of cartilage-specific genes in alginate suspension culture. This effect was less pronounced in cells that were transduced with enhanced green fluorescent protein. Chondrocytes transplanted in vivo were detected in the repair tissue for the entire period of observation with diminishing cell density over time. At one week, the cell density of the transplanted chondrocytes was 100% of the initial density; at two and three weeks, the cell density was 70%; and, after four weeks, the cell density had decreased to 15%. Safranin-O staining of histological sections indicated cartilage-specific matrix production in vitro and in vivo. Integration of transplanted cells into the host repair tissue was not observed. The two-week period of preconditioning in alginate suspension culture had no apparent influence on the temporal fate of the cells or the histological appearance of the repair tissue. CONCLUSIONS AND CLINICAL RELEVANCE: Alginate promotes expression of cartilage-specific genes and allows delivery of chondrocytes into osteochondral defects. Transgenic chondrocytes labeled with enhanced green fluorescent protein are detectable in the defect, but they do not appear to form repair tissue and they decrease in number with time. In view of the clinical application of cell-based cartilage repair, understanding the fate of transplanted cells becomes increasingly relevant. Transgenic chondrocytes are an effective tool to study the role of transplanted chondrocytes in articular cartilage repair.

Alginates↗

PubMedDate not supplied

Tracing transduced cells in osteochondral defects.

In this study the authors explored the feasibility of using transduced cells for gene therapy to induce healing of osteochondral defects. Both a mouse mesenchymal cell line and mixed rabbit adherent stromal cells were transduced with either liposomal transfection or retroviral transduction using a traceable gene. Transduction efficiency was more than 95% with the retroviral construct and expression was maintained for over 6 months of passage. The liposomal transfection led to a transient expression with an efficiency of 50%. The expression of osteochondral genes was diminished but preserved after transduction in vitro. Transduced rabbit cells were transplanted into osteochondral defects in rabbit femoral condyles. Cells transplanted in vivo could be detected for 4 weeks in the repair tissue. The authors' data demonstrate that mesenchymal cells from bone marrow, stably transduced with a traceable gene product, retain the bone and cartilage phenotype and can be followed in vivo after transplantation into cartilage defects.

Animals↗