Biomedical subjects

D Rekosh

Publications and source records attributed to D Rekosh.

At least 19 recordsLinked to original sources

PubMed2001

RNA export mediated by tap involves NXT1-dependent interactions with the nuclear pore complex.

Nuclear export of ribonucleoprotein complexes requires cis-acting signals and recognition by receptors that mediate translocation through the nuclear pore complex. Translocation is likely to involve a series of physical interactions between the ribonucleoprotein complex and nucleoporins within the nuclear pore complex. Here, we have characterized the function of NXT1 in the context of the Tap-dependent RNA export pathway. Tap has been implicated in the nuclear export of RNA transcripts derived from Mason-Pfizer monkey virus that contain the constitutive transport element. We demonstrate that NXT1 stimulates binding of a Tap-RNA complex to nucleoporins in vitro, and we provide mutational analysis that shows these interactions are necessary for nuclear export of an intron-containing viral mRNA in vivo. Tap contains separate domains for binding to nucleoporins and NXT1, both of which are critical for its export function. RNA export is mediated by a heterodimer of Tap and NXT1, and the function of NXT1 on this pathway is to regulate the affinity of the Tap-RNA complex for nucleoporins within the nuclear pore complex. We propose that NXT1-dependent binding of the Tap-RNA complex to the nucleoporin p62, which we have reconstituted in vitro using recombinant proteins, represents a single step of the translocation reaction.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2001

Human immunodeficiency virus type 1 particles pseudotyped with envelope proteins that fuse at low pH no longer require Nef for optimal infectivity.

We have investigated the effects of Nef on infectivity in the context of various viral envelope proteins. These experiments were performed with a minimal vector system where Nef is the only accessory protein present. Our results support the hypothesis that the route of entry influences the ability of Nef to enhance human immunodeficiency virus (HIV) infectivity. We show that HIV particles pseudotyped with Ebola virus glycoprotein or vesicular stomatitis virus glycoprotein (VSV-G), which fuse at low pH, do not require Nef for optimal infectivity. In contrast, Nef significantly enhances the infectivity of virus particles that contain envelope proteins that fuse at neutral pH (CCR5-dependent HIV Env, CXCR4-dependent HIV Env, or amphotropic murine leukemia virus Env). In addition, our results demonstrate that virus particles containing mixed CXCR4-dependent HIV and VSV-G envelope proteins show a conditional requirement for Nef for optimal infectivity, depending on which protein is allowed to facilitate entry.

Animals↗

PubMed2001

RNA trafficking signals in human immunodeficiency virus type 1.

Intracellular trafficking of retroviral RNAs is a potential mechanism to target viral gene expression to specific regions of infected cells. Here we show that the human immunodeficiency virus type 1 (HIV-1) genome contains two sequences similar to the hnRNP A2 response element (A2RE), a cis-acting RNA trafficking sequence that binds to the trans-acting trafficking factor, hnRNP A2, and mediates a specific RNA trafficking pathway characterized extensively in oligodendrocytes. The two HIV-1 sequences, designated A2RE-1, within the major homology region of the gag gene, and A2RE-2, in a region of overlap between the vpr and tat genes, both bind to hnRNP A2 in vitro and are necessary and sufficient for RNA transport in oligodendrocytes in vivo. A single base change (A8G) in either sequence reduces hnRNP A2 binding and, in the case of A2RE-2, inhibits RNA transport. A2RE-mediated RNA transport is microtubule and hnRNP A2 dependent. Differentially labelled gag and vpr RNAs, containing A2RE-1 and A2RE-2, respectively, coassemble into the same RNA trafficking granules and are cotransported to the periphery of the cell. tat RNA, although it contains A2RE-2, is not transported as efficiently as vpr RNA. An A2RE/hnRNP A2-mediated trafficking pathway for HIV RNA is proposed, and the role of RNA trafficking in targeting HIV gene expression is discussed.

Animals↗

PubMed2001

NXT1 (p15) is a crucial cellular cofactor in TAP-dependent export of intron-containing RNA in mammalian cells.

TAP, the human homologue of the yeast protein Mex67p, has been proposed to serve a role in mRNA export in mammalian cells. We have examined the ability of TAP to mediate export of Rev response element (RRE)-containing human immunodeficiency virus (HIV) RNA, a well-characterized export substrate in mammalian cells. To do this, the TAP gene was fused in frame to either RevM10 or RevDelta78-79. These proteins are nonfunctional Rev mutant proteins that can bind to HIV RNA containing the RRE in vivo but are unable to mediate the export of this RNA to the cytoplasm. However, the fusion of TAP to either of these mutant proteins gave rise to chimeric proteins that were able to complement Rev function. Significantly, cotransfection with a vector expressing NXT1 (p15), an NTF2-related cellular factor that binds to TAP, led to dramatic enhancement of the ability of the chimeric proteins to mediate RNA export. Mutant-protein analysis demonstrated that the domain necessary for nuclear export mapped to the C-terminal region of TAP and required the domain that interacts with NXT1, as well as the region that has been shown to interact with nucleoporins. RevM10-TAP function was leptomycin B insensitive. In contrast, the function of this protein was inhibited by DeltaCAN, a protein consisting of part of the FG repeat domain of CAN/Nup214. These results show that TAP can complement Rev nuclear export signal function and redirect the export of intron-containing RNA to a CRM1-independent pathway. These experiments support the role of TAP as an RNA export factor in mammalian cells. In addition, they indicate that NXT1 serves as a crucial cellular cofactor in this process.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2000

Intravirion targeting of a functional anti-human immunodeficiency virus ribozyme directed to pol.

Ribozymes are catalytic RNAs that offer several advantages as specific therapeutic genes against human immunodeficiency virus type 1 (HIV-1). Significant challenges in antiviral uses of ribozymes include (1) how best to express and to deliver this agent and (2) what is the best locale to target ribozymes against HIV-1 RNA. To explore the former, we have previously characterized several vector systems for efficient expression/delivery of anti-HIV-1 ribozymes (Dropulic et al., 1992; Dropulic and Jeang, 1994a; Smith et al., 1997). Here, to investigate an optimal locale for ribozyme-targeting, we asked whether it might be advantageous to direct ribozymes into HIV-1 virions as opposed to the more conventional approach of targeting ribozymes into infected cells. Two series of experiments were performed. First, we demonstrated that anti-HIV-1 ribozymes could indeed be packaged specifically and efficiently into virions. Second, we compared the virus suppressing activity of a packageable ribozyme with its counterpart, which cannot be packaged into HIV-1 virions. Our results showed that although both ribozymes cleaved HIV-1 genomic RNA in vitro with equivalent efficiencies, the former ribozyme demonstrated significantly higher virus-suppressing activity than the latter. These findings provide proof-of-principle that to combat productive HIV-1 replication, intravirion targeting is more effective than intracellular targeting of ribozymes.

Base Sequence↗

PubMed1999

An intact TAR element and cytoplasmic localization are necessary for efficient packaging of human immunodeficiency virus type 1 genomic RNA.

Although most reports defining the human immunodeficiency virus type 1 (HIV-1) genomic RNA packaging signal have focused on the region downstream of the major 5' splice site, others have suggested that sequences upstream of the splice site may also play an important role. In this study we have directly examined the role played by the HIV-1 TAR region in RNA packaging. For these experiments we used a proviral expression system that is largely independent of Tat for transcriptional activation. This allowed us to create constructs that efficiently expressed RNAs carrying mutations in TAR and to determine the ability of these RNAs to be packaged. Our results indicate that loss of sequences in TAR significantly reduce the ability of a viral RNA to be packaged. The requirement for TAR sequences in RNA packaging was further examined by using a series of missense mutations positioned throughout the entire TAR structure. TAR mutations previously shown to influence Tat transactivation, such as G31U in the upper loop region or UCU to AAG in the bulge (nucleotides [nt] 22 to 24), failed to have any effect on RNA packaging. Mutations which disrupted the portion of the TAR stem immediately below the bulge also had little effect. In contrast, dramatic effects on RNA packaging were observed with constructs containing mutations in the lower portion of the TAR stem. Point mutations which altered nt 5 to 9, 10 to 15, 44 to 49, or 50 to 54 all reduced RNA packaging 11- to 25-fold. However, compensatory double mutations which restored the stem structure were able to restore packaging. These results indicate that an intact lower stem structure, rather than a specific sequence, is required for RNA packaging. Our results also showed that RNA molecules retained within the nucleus cannot be packaged, unless they are transported to the cytoplasm by either Rev/Rev response element or the Mason-Pfizer monkey virus constitutive transport element.

5' Untranslated Regions↗

PubMed1999

Selection and characterization of human immunodeficiency virus type 1 mutants that are resistant to inhibition by the transdominant negative RevM10 protein.

Intracellular immunization with RevM10, a transdominant negative form of the Rev protein, efficiently inhibits human immunodeficiency virus (HIV) replication in vitro and gene therapy protocols that use this modality are currently being evaluated in human clinical trials. Development of resistance to this kind of therapy has not been previously reported. Here we show that RevM10-resistant HIV type 1 (HIV-1) variants can be selected by in vitro passage of HIV-1 in a T-lymphoblastoid cell line constitutively expressing RevM10. Unexpectedly, the selected variants showed changes in the Rev response element (RRE) but no changes in Rev. Replacement of the wild-type RRE with a mutated RRE resulted in a virus that showed increased resistance to RevM10. After repeated passages of the resistant variant in cells expressing RevM10, a virus with an additional mutation in the viral vpu gene was selected. Surprisingly, a virus containing only this vpu mutation also showed some resistance to inhibition by RevM10.

Amino Acid Sequence↗

PubMed1997

The constitutive transport element (CTE) of Mason-Pfizer monkey virus (MPMV) accesses a cellular mRNA export pathway.

The constitutive transport elements (CTEs) of type D retroviruses are cis-acting elements that promote nuclear export of incompletely spliced mRNAs. Unlike the Rev response element (RRE) of human immunodeficiency virus type 1 (HIV-1), CTEs depend entirely on factors encoded by the host cell genome. We show that an RNA comprised almost entirely of the CTE of Mason-Pfizer monkey virus (CTE RNA) is exported efficiently from Xenopus oocyte nuclei. The CTE RNA and an RNA containing the RRE of HIV-1 (plus Rev) have little effect on export of one another, demonstrating differences in host cell requirements of these two viral mRNA export pathways. Surprisingly, even very low amounts of CTE RNA block export of normal mRNAs, apparently through the sequestration of cellular mRNA export factors. Export of a CTE-containing lariat occurs when wild-type CTE, but not a mutant form, is inserted into the pre-mRNA. The CTE has two symmetric structures, either of which supports export and the titration of mRNA export factors, but both of which are required for maximal inhibition of mRNA export. Two host proteins bind specifically to the CTE but not to non-functional variants, making these proteins candidates for the sequestered mRNA export factors.

Animals↗

PubMed1997

A class of nonribosomal nucleolar components is located in chromosome periphery and in nucleolus-derived foci during anaphase and telophase.

. The subcellular location of several nonribosomal nucleolar proteins was examined at various stages of mitosis in synchronized mammalian cell lines including HeLa, 3T3, COS-7 and HIV-1 Rev-expressing CMT3 cells. Nucleolar proteins B23, fibrillarin, nucleolin and p52 as well as U3 snoRNA were located partially in the peripheral regions of chromosomes from prometaphase to early telophase. However, these proteins were also found in large cytoplasmic particles, 1-2 microm in diameter, termed nucleolus-derived foci (NDF). The NDF reached maximum numbers (as many as 100 per cell) during mid- to late anaphase, after which their number declined to a few or none during late telophase. The decline in the number of NDF approximately coincided with the appearance of prenucleolar bodies and reforming nucleoli. The HIV-1 Rev protein and a mutant Rev protein defective in its nuclear export signal were also found in the NDF. The mutant Rev protein precisely followed the pattern of localization of the above nucleolar proteins, whereas the wild-type Rev did not enter nuclei until G1 phase. The nucleolar shuttling phosphoprotein Nopp140 did not follow the above pattern of localization during mitosis: it dispersed in the cytoplasm from prometaphase through early telophase and was not found in the NDF. Although the NDF and mitotic coiled bodies disappeared from the cytoplasm at approximately the same time during mitosis, protein B23 was not found in mitotic coiled bodies, nor was p80 coilin present in the NDF. These results suggest that a class of proteins involved in preribosomal RNA processing associate with chromosome periphery and with NDF as part of a system to conserve and deliver preexisting components to reforming nucleoli during mitosis.

Anaphase↗

PubMed1997

The effect of viral regulatory protein expression on gene delivery by human immunodeficiency virus type 1 vectors produced in stable packaging cell lines.

We describe the generation of stable human immunodeficiency virus type 1 (HIV-1)-packaging lines that constitutively express high levels of HIV-1 structural proteins in either a Rev-dependent or a Rev-independent fashion. These cell lines were used to assess gene transfer by using an HIV-1 vector expressing the hygromycin B resistance gene and to study the effects of Rev, Tat, and Nef on the vector titer. The Rev-independent cell lines were created by using gag-pol and env expression vectors that contain the Mason-Pfizer monkey virus (MPMV) constitutive transport element (CTE). Vector titers approaching 10(4) CFU/ml were routinely obtained with these cell lines, as well as with the Rev-dependent cell lines, with HeLa-CD4 cells as targets. The presence of Nef and Tat in the producer cell each increased the vector titer 5- to 10-fold. Rev, on the other hand, was absolutely essential for gene transfer, unless the MPMV CTE was present in the vector. In that case, by using the Rev-independent cell lines for packaging, Rev could be completely eliminated from the system without a reduction in vector titer.

Base Sequence↗

PubMed1997

A structured retroviral RNA element that mediates nucleocytoplasmic export of intron-containing RNA.

A common feature of gene expression in all retroviruses is that unspliced, intron-containing RNA is exported to the cytoplasm despite the fact that cellular RNAs which contain introns are usually restricted to the nucleus. In complex retroviruses, the export of intron-containing RNA is mediated by specific viral regulatory proteins (e.g., human immunodeficiency virus type 1 [HIV-1] Rev) that bind to elements in the viral RNA. However, simpler retroviruses do not encode such regulatory proteins. Here we show that the genome of the simpler retrovirus Mason-Pfizer monkey virus (MPMV) contains an element that serves as an autonomous nuclear export signal for intron-containing RNA. This element is essential for MPMV replication; however, its function can be complemented by HIV-1 Rev and the Rev-responsive element. The element can also facilitate the export of cellular intron-containing RNA. These results suggest that the MPMV element mimics cellular RNA transport signals and mediates RNA export through interaction with endogenous cellular factors.

Animals↗

PubMed1997

Secondary structure and mutational analysis of the Mason-Pfizer monkey virus RNA constitutive transport element.

The Mason-Pfizer monkey virus (MPMV) genome contains a cis-acting element that serves to facilitate nucleocytoplasmic export of intron-containing RNA. This element, known as the constitutive transport element (CTE), has been mapped to a 154-nt region close to the 3' end of the MPMV genome. The CTE contains a degenerate direct repeat of approximately 70 nt. We have probed the secondary structure of the CTE using double-strand- and single-strand-specific ribonucleases and chemical modification agents. A mutational analysis was also performed to confirm critical features of the CTE structure, as well as to identify regions that contain sequence-specific information required for function. Our results indicate that the CTE forms a long stem structure that contains a 9-nt terminal hairpin loop as well as two identical 16-nt inner loops. The inner loop sequences are rotated 180 degrees relative to each other within the structure. The mutational analysis shows that primary sequences in the loop regions are important for function, suggesting that they may contain binding sites for cellular proteins involved in RNA export. Interestingly, sequences with significant homology to the inner loop regions are found in the genomes of spumaviruses and mouse intracisternal A particles.

Base Sequence↗

PubMed1996

Location of the HIV-1 Rev protein during mitosis: inactivation of the nuclear export signal alters the pathway for postmitotic reentry into nucleoli.

The HIV-1 Rev protein localizes predominantly to the nucleolus of HIV-1-infected or Rev-expressing cells. The subcellular location of Rev during mitotic nucleolar disintegration was examined at various stages of mitosis in synchronized Rev-expressing CMT3 cells. During early prophase Rev was predominantly located in disintegrating nucleoli and began to accumulate at the peripheral regions of chromosomes in late prophase, eventually distributing uniformly on all chromosomes in prometaphase. In anaphase Rev remained associated with the perichromosomal regions, but significant amounts of Rev were also seen in numerous nucleolus-derived foci. The movement of Rev from disintegrating nucleoli to perichromosomal regions and foci was similar to that of nonribosomal nucleolar proteins, including fibrillarin, nucleolin, protein B23 and p52 of the granular component. During telophase Rev remained associated with perichromosomal regions and mitotic foci until the nuclear envelope started to reform. When nuclear envelope formation was complete in late telophase, nonribosomal nucleolar proteins were present in prenucleolar bodies (PNBs) which were eventually incorporated into nucleoli; at the same time, Rev was excluded from nuclei. In contrast, a trans-dominant negative Rev protein containing an inactive nuclear export signal reentered nuclei by the nonribosomal nucleolar protein pathway in late telophase, associating with PNBs and reformed nucleoli. Rev protein reentry into postmitotic nuclei was delayed until early G1 phase, but before the arrival of ribosomal protein S6. Thus, Rev behaves like a nonribosomal nucleolar protein through mitosis until early telophase; however, its nuclear reentry seems to require reestablishment of both a nuclear import system and active nucleoli.

Animals↗

PubMed1995

5' regions of HIV-1 RNAs are not sufficient for encapsidation: implications for the HIV-1 packaging signal.

The location and nature of the HIV-1 packaging signal are largely unknown, despite several genetic and biochemical mutational analyses. In this report we present our attempts to define a minimal HIV-1 packaging signal through the generation of test RNAs containing small blocks of HIV-1 sequences. We constructed RNAs differing in the position and identity of the HIV-1 sequence and the segments of heterologous sequences. However, none of the vectors were efficiently, encapsidated by wild-type HIV-1 virions. These results contrast those of Moloney murine leukemia virus and Rous sarcoma virus, where small viral segments mediate the efficient encapsidation of heterologous RNAs. The results suggest that the HIV-1 packaging signal may be extremely dispersed or heavily context-dependent.

Animals↗

PubMed1995

Schistosoma mansoni: cloning and characterization of a gene encoding cytosolic Cu/Zn superoxide dismutase.

Two forms of Cu/Zn superoxide dismutase (SOD) have been identified in Schistosoma mansoni, signal peptide-containing SOD (SP-SOD, Simurda et al., 1988, Exp. Parasitol. 67, 73-84) and cytosolic SOD (CT-SOD, Hong et al., 1992, Exp. Parasitol. 75, 308-322). The SP-SOD gene has been previously characterized. We report the isolation and characterization of the gene encoding CT-SOD. A cDNA clone of CT-SOD was radiolabeled and used to screen an EMBL3 genomic library. Of 100,000 plaques, 1 hybridizing phage contained an insert of 14.6 kb. The CT-SOD gene spans 8.5 kb of chromosomal DNA and possesses three exons (150, 285, and 234 bp, respectively) and two introns (4.9 and 2.7 kb, respectively) which interrupt the coding region. Separate copies of the gene as determined by restriction fragment patterns on Southern blots are found on two YAC clones. Using YAC clones that contain the CT-SOD gene, fluorescence in situ hybridization on chromosomes identified a copy of the gene located on the middle region of chromosome 1 and another copy localized to the middle region of the long arm of chromosome 3. Primer extension and nuclease protection assay performed on total RNA define the transcription initiation site and allow the identification of potential cis-acting sequences, such as a CAAT box and a hexanucleotide GCCCGG. Comparison of this gene with Cu/Zn SOD genes from Drosophila, humans, and mice identifies conserved exons that represent functional domains in the SOD protein.

Amino Acid Sequence↗

PubMed1995

Expression and purification of the HIV type 1 Rev protein produced in Escherichia coli and its use in the generation of monoclonal antibodies.

We have developed a simple and rapid procedure for the purification of large amounts of Rev protein overexpressed in E. coli. The purification method, which does not require denaturation of the protein, takes advantage of the positively charged nature of Rev and the ability of Rev to interact with nucleic acids. The purified protein was used to develop three novel murine monoclonal antibodies against Rev. Using fusion proteins between glutathione S-transferase (GST) and various fragments of the Rev protein, we mapped the specificity of these antibodies to different regions of the Rev protein. One antibody, 3H6, is directed against the nucleolar localization/RRE-binding domain of Rev between amino acids 38 and 44. Another antibody, 3G4, recognizes an epitope between amino acids 90 and 116 of Rev. A third antibody, 2G2, does not recognize any of the fusion proteins, and may be directed against a conformational epitope. All three antibodies are able to detect Rev on Western blots and to immunoprecipitate Rev under native conditions. However, only 3H6 and 3G4 immunoprecipitate Rev under denaturing conditions and are able to detect Rev expressed in transfected cells by indirect immunofluorescence. These antibodies should prove useful in further studies of Rev function.

Antibodies, Monoclonal↗

PubMed1995

Characterization of deletion mutations in the capsid region of human immunodeficiency virus type 1 that affect particle formation and Gag-Pol precursor incorporation.

The core of human immunodeficiency virus type 1 is derived from two precursor polyproteins, Pr55gag and Pr160gag-pol. The Gag precursor can assemble into immature virus-like particles when expressed by itself, while the Gag-Pol precursor lacks particle-forming ability. We have shown previously that the Gag precursor is able to "rescue" the Gag-Pol precursor into virus-like particles when the two polyproteins are expressed in the same cell by using separate simian virus 40-based plasmid expression vectors. To understand this interaction in greater detail, we have made deletion mutations in the capsid-coding regions of Gag- and Gag-Pol-expressing plasmids and assayed for the abilities of these precursors to assemble into virus-like particles. When we tested the abilities of Gag-Pol precursors to be incorporated into particles of Gag by coexpressing the precursors, we found that mutant Gag-Pol precursors lacking a conserved region in retroviral capsid proteins, the major homology region (MHR), were excluded from wild-type Gag particles. Mutant precursors lacking MHR were also less efficient in processing the Gag precursor in trans. These results suggest that the MHR is critical for interactions between Gag and Gag-Pol molecules. In contrast to these results, expression of mutated Gag precursors alone showed that deletions in the capsid region, including those which removed the MHR, reduced the efficiency of particle formation by only 40 to 50%. The mutant particles, however, were clearly lighter than the wild type in sucrose density gradients. These results indicate that the requirements for Gag particle formation differ from the ones essential for efficient incorporation of the Gag-Pol precursor into these particles.

Animals↗

PubMed1995

The roles of nucleolar structure and function in the subcellular location of the HIV-1 Rev protein.

The human immunodeficiency virus 1 (HIV-1) Rev transactivator protein plays a critical role in the regulation of expression of structural proteins by controlling the pathway of mRNA transport. The Rev protein is located predominantly in the nucleoli of HIV-1 infected or Rev-expressing cells. Previous studies demonstrated that the Rev protein forms a specific complex in vitro with protein B23 which is suggested to be a nucleolar receptor and/or carrier for the Rev protein. To study the role of the nucleolus and nucleolar proteins in Rev function, transfected COS-7 or transformed CMT3 cells expressing the Rev protein were examined for subcellular locations of Rev and other proteins using indirect immunofluorescence and immunoelectron microscopy. One day after transfection the Rev protein was found in most cells only in the nucleolar dense fibrillar and granular components where it colocalized with protein B23. These were designated class 1 cells. In a second class of cells Rev and B23 accumulated in the nucleoplasm as well as in nucleoli. Treatment of class 1 cells with actinomycin D (AMD) under conditions that blocked only RNA polymerase I transcription caused Rev to completely redistribute from nucleoli to the cytoplasm. Simultaneously, protein B23 was partially released from nucleoli, mostly into the nucleoplasm, with detectable amounts in the cytoplasm. In cells recovering from AMD treatment in the presence of cycloheximide Rev and B23 showed coincident relocation to nucleoli. Class 2 cells were resistant to AMD-induced Rev redistribution. Selective inhibition of RNA polymerase II transcription by alpha-amanitin or by DRB did not cause Rev to be released into the cytoplasm suggesting that active preribosomal RNA transcription is required for the nucleolar location of Rev. However, treatment with either of the latter two drugs at higher doses and for longer times caused partial disruption of nucleoli accompanied by translocation of the Rev protein to the cytoplasm. These results suggest that the nucleolar location of Rev depends on continuous preribosomal RNA transcription and a substantially intact nucleolar structure.

Animals↗