Biomedical subjects

B K Felber

Publications and source records attributed to B K Felber.

At least 19 recordsLinked to original sources

PubMed2002

Design and in vivo immunogenicity of a polyvalent vaccine based on SIVmac regulatory genes.

Most vaccine modalities for human immunodeficiency virus type 1 (HIV-1) tested for immunogenicity and efficacy in the SIVmac (simian immunodeficiency virus) macaque model do not include the viral regulatory proteins. Because viral regulatory proteins are expressed early during the virus life cycle and represent an additional source of antigens, their inclusion as a vaccine component may increase the overall virus-specific immune response in vaccinees. However, at least two of the early proteins, Tat and Nef, may be immunosuppressive, limiting their usefulness as components of an SIV vaccine. We have constructed a polyvalent chimeric protein in which the open reading frames for Tat and Nef have been reassorted and the nuclear localization sequence for Tat and Rev and the myristoylation site for Nef have been removed. The resulting DNA plasmid (pDNA-SIV-Retanef) (pDNA-SIV-RTN) encodes a protein of 55 kDa (Retanef) that localizes at the steady state in the cytoplasma of transfected cells. Both the DNA-SIV-RTN and the highly attenuated recombinant poxvirus vector NYVAC-SIV-RTN were demonstrated to be immunogenic in SIVmac251-infected macaques treated with ART as well as in naive macaques. An equivalent strategy may be used for the generation of polyvalent antigens encoding the regulatory proteins in a HIV-1 vaccine candidate.

Animals↗

PubMed2001

Potentiation of simian immunodeficiency virus (SIV)-specific CD4(+) and CD8(+) T cell responses by a DNA-SIV and NYVAC-SIV prime/boost regimen.

T cell-mediated immune responses play an important role in the containment of HIV-1 replication. Therefore, an effective vaccine against HIV-1 should be able to elicit high frequencies of virus-specific CD8(+) and CD4(+) T cells. The highly attenuated poxvirus-based vaccine candidate, NYVAC-SIV-gag-pol-env (NYVAC-SIV-gpe), has been shown to induce and/or expand SIV-specific CD4(+) and CD8(+) T cell responses in both naive and infected macaques. In this study, the immunogenicity of NYVAC-SIV-gpe alone was compared with a combination regimen where priming with an optimized DNA-SIV-gag-env vaccine candidate was followed by a NYVAC-SIV-gpe boost. In macaques immunized with the prime-boost regimen, the extent and durability of CD8(+) T cell response to an immunodominant SIV gag epitope was increased and these animals recognized a broader array of subdominant SIV epitopes in the cytolytic assay. In addition, the prime-boost regimen significantly enhanced the proliferative responses to both SIV gag and env proteins. Thus, the combination of these vaccine modalities may represent a valuable strategy in the development of a vaccine for HIV.

Animals↗

PubMed2001

Identification of a novel posttranscriptional regulatory element by using a rev- and RRE-mutated human immunodeficiency virus type 1 DNA proviral clone as a molecular trap.

Human immunodeficiency virus (HIV) and all other lentiviruses utilize the essential viral protein Rev, which binds to RRE RNA, to export their unspliced and partially spliced mRNAs from the nucleus. We used a rev- and RRE-defective HIV type 1 (HIV-1) molecular clone in complementation experiments to establish a method for the rapid isolation of posttranscriptional regulatory elements from the mammalian genome by selecting for rescue of virus replication. Viruses rescued by this method contained a novel element with homology to rodent intracisternal A-particle (IAP) retroelements. A functional element was contained within a 247-nucleotide fragment named RNA transport element (RTE), which was able to promote replication of the Rev- and RRE-defective HIV-1 in both human lymphoid cell lines and primary lymphocytes, demonstrating its potent posttranscriptional function. RTE was functional in many cell types, indicating that the cellular factors that recognize RTE are widely expressed and evolutionarily conserved. RTE also promoted RNA export from Xenopus oocyte nuclei. RTE-mediated RNA transport was CRM1 independent, and RTE did not show high affinity for binding to mRNA export factor TAP/NXF1. Since CRM1 and TAP/NXF1 are critical export receptors associated with the two recognized mRNA export pathways, these results suggest that RTE functions via a distinct export mechanism. Taken together, our results identify a novel posttranscriptional control element that uses a conserved cellular export mechanism.

Active Transport, Cell Nucleus↗

PubMed2001

Retroviral constitutive transport element evolved from cellular TAP(NXF1)-binding sequences.

The constitutive transport element (CTE) of type D retroviruses serves as a signal of nuclear export of unspliced viral RNAs. The human TAP(NXF1) protein, a cellular mRNA export factor, directly binds to CTE and mediates nuclear export of CTE-containing RNAs. Here, we use genomic SELEX (systematic evolution of ligands by exponential enrichment) to show that the human genome encodes a family of high-affinity TAP ligands. These TAP-binding elements (TBE) are 15-bp minisatellite repeats that are homologous to the core TAP-binding sites in CTE. The repeats are positioned similarly in the RNA secondary structures of CTE and TBE. Like CTE, TBE is an active nuclear export signal. CTE elements of different species share sequence similarities to TBE in the regions that are neutral for CTE function. This conservation points to a possible common ancestry of the two elements, and in fact, TBE has properties expected from a primordial CTE. Additionally, a molecular fossil of a TBE-like minisatellite is found in the genome of a modern retroelement. These findings constitute direct evidence of an evolutionary link between TBE-related minisatellites and CTE.

Active Transport, Cell Nucleus↗

PubMed2000

Unusual CD4+CD8+ phenotype in a greek patient diagnosed with adult T-cell leukemia positive for human T-cell leukemia virus type I (HTLV-I).

We describe the first Greek patient diagnosed with Adult T cell leukemia (ATL) characterized by an expansion of CD4+CD8+ double positive lymphocytes. Low levels of plasma antibodies against HTLV-I Env and Gag proteins were detected. Analysis of the the patient's DNA revealed that she was infected by a cosmopolitan strain of HTLV-I. Since HTLV-I usually leads to the expansion of CD4+ cells, this patient illustrates a rare immunophenotype, which suggests that the HTLV-I-induced proliferative response may occur in a pre-T cell stage.

Base Sequence↗

PubMed2000

The mRNA export in Caenorhabditis elegans is mediated by Ce-NXF-1, an ortholog of human TAP/NXF and Saccharomyces cerevisiae Mex67p.

Human TAP and Saccharomyces cerevisiae Mex67p belong to a family of proteins that mediate mRNA export. Computer searches identified previously two Caenorhabditis elegans genes, C15H11.3 and C115H11.6, that encode putative homologs of hTAP and Mex67p (Segref et al., EMBO J, 1997, 16:3256-3271). Using RNA interference experiments in C. elegans, we found that functional knockout of C15H11.3 resulted in nuclear accumulation of poly(A)-containing RNAs and was lethal for both embryos and adult nematodes. No embryonic or progeny abnormality was observed in functional knockout of C15H11.6. Taken together, these data established that the C15H11.3 gene product is an ortholog of hTAP and Mex67p; thus, it was named Ce-NXF-1. Ce-NXF-1 binds RNA directly and is a nucleocytoplasmic shuttle protein accumulating in the nucleoplasm and at the nuclear rim. The rim association is mediated via unique signals present in the C-terminal portion of all TAP/NXF and Mex67p proteins. This region was shown to interact with the FG-repeat domains of nucleoporins Nup98, Nup153, and Nup214, indicating that the rim association occurs through components of the nuclear pore complex. In summary, Ce-NXF-1 belongs together with hTAP and Mex67p to a family of proteins that participate in mRNA export and can provide a direct molecular link between mRNAs and components of the nuclear pore complex. Therefore, despite differences in mRNA metabolism between these species, they utilize a conserved mRNA transport mechanism.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1999

Nucleoporins nup98 and nup214 participate in nuclear export of human immunodeficiency virus type 1 Rev.

Human immunodeficiency virus type 1 (HIV-1) Rev contains a leucine-rich nuclear export signal that is essential for its nucleocytoplasmic export mediated by hCRM1. We examined the role of selected nucleoporins, which are located in peripheral structures of the nuclear pore complex and are thought to be involved in export, in Rev function in human cells. First, we found that upon actinomycin D treatment, Nup98, but not Nup214 or Nup153, is able to translocate to the cytoplasm of HeLa cells, demonstrating that Nup98 may act as a soluble factor. We further showed that Rev can recruit Nup98 and Nup214, but not Nup153, to the nucleolus. We also found that the isolated FG-containing repeat domains of Nup98 and Nup214, but not those of Nup153, competitively inhibit the Rev/RRE-mediated expression of HIV. Taken together, the recruitment of Nup98 and Nup214 by Rev and the competitive inhibition exhibited by their NP domains demonstrate direct participation of Nup98 and Nup214 in the Rev-hCRM1-mediated export.

Animals↗

PubMed1999

Evaluation of novel human immunodeficiency virus type 1 Gag DNA vaccines for protein expression in mammalian cells and induction of immune responses.

Human immunodeficiency virus (HIV)-specific cytotoxic T lymphocytes (CTL) are an important parameter of host defenses that limit viral replication after infection. Induction of effective CTL against conserved viral proteins such as Gag may be essential to the development of a safe and effective HIV type 1 (HIV-1) vaccine. DNA vaccination represents a novel strategy for inducing potent CD8(+) CTL responses in vivo. However, expression of HIV-1 structural proteins by DNA vectors has been hampered by a stringent requirement for coexpression with other viral components, such as Rev and RRE. Furthermore, even with Rev and RRE present, the level of expression of HIV-1 Gag, Pol, or Env is very low in murine cells. These problems have limited our ability to address the key issue of how to generate effective CTL responses to Gag in a mouse model. To overcome this problem, we compared several novel DNA expression vectors for HIV-1 Gag protein expression in primate and mouse cells and for generating immune responses in mice after DNA vaccination. A DNA vector containing wild type HIV-1 gag coding sequences did not induce detectable Gag expression in any of the cells tested. Attempts to increase nuclear export of Gag expression RNA by adding the constitutive transport element yielded only a moderate increase in Gag expression in monkey-derived COS cells and an even lower increase in Gag expression in HeLa cells or several mouse cell lines. In contrast, silent-site mutations in the HIV-1 gag coding sequences significantly increased Gag expression levels in all cells tested. Furthermore, this construct induced both Gag-specific antibody and CTL responses in mice after DNA vaccination. Using this construct, we achieved stable expression of HIV-1 Gag in the mouse cell line p815, which can now be used as a target cell for measuring HIV-1 Gag-specific CTL responses in immunized mice. The DNA vectors described in this study should make it possible to systematically evaluate the approaches for maximizing the induction of CTL responses against HIV-1 Gag in mouse and other animal systems.

AIDS Vaccines↗

PubMed1999

Persistent infection of rhesus macaques by the rev-independent Nef(-) simian immunodeficiency virus SIVmac239: replication kinetics and genomic stability.

We generated previously a Nef(-), replication-competent clone of SIVmac239 in which the Rev protein and the Rev-responsive element were replaced by the constitutive transport element (CTE) of simian retrovirus type 1 (A. S. von Gegerfelt and B. K. Felber, Virology 232:291-299, 1997). In the present report, we show that this virus was able to infect and replicate in rhesus macaques. The Rev-independent Nef(-) simian immunodeficiency virus induced a persistent humoral immune response in all monkeys, although viral loads were very low. Upon propagation in the monkeys, the genotype remained stable and the virus retained its in vitro growth characteristics. The infected monkeys showed normal hematological values and no signs of disease at more than 18 months post-virus exposure. Therefore, replacement of the essential Rev regulation by the CTE generated a virus variant that retained its replicative capacity both in vitro and in vivo, albeit at low levels.

Animals↗

PubMed1999

Identification of novel import and export signals of human TAP, the protein that binds to the constitutive transport element of the type D retrovirus mRNAs.

The nuclear export of the unspliced type D retrovirus mRNA depends on the cis-acting constitutive transport RNA element (CTE) that has been shown to interact with the human TAP (hTAP) protein promoting the export of the CTE-containing mRNAs. We report here that hTAP is a 619-amino-acid protein extending the previously identified protein by another 60 residues at the N terminus and that hTAP shares high homology with the predicted rat and mouse TAP proteins. We found that hTAP is a nuclear protein that accumulates in the nuclear rim and the nucleoplasm. We further demonstrated that hTAP is able to shuttle between the nucleus and the cytoplasm. Identification of the signals responsible for nuclear import (NLS) and export (NES) revealed that they are distinct but partially overlapping. NLS and NES of hTAP are active transferable signals that do not share similarities with known elements. The C-terminal portion contributes further to hTAP's nuclear retention and contains a signal(s) for nuclear rim association. Taken together, our data show that hTAP is a dynamic protein capable of bidirectional trafficking across the nuclear envelope. These data further support hTAP's role as an export factor of the CTE-containing mRNAs.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1998

TAP, the human homolog of Mex67p, mediates CTE-dependent RNA export from the nucleus.

The constitutive transport element (CTE) of the type D retroviruses promotes nuclear export of unspliced viral RNAs apparently by recruiting host factor(s) required for export of cellular messenger RNAs. Here, we report the identification of TAP as the cellular factor that specifically binds to wild-type CTE but not to export-deficient CTE mutants. Microinjection experiments performed in Xenopus oocytes demonstrate that TAP directly stimulates CTE-dependent export. Furthermore, TAP overcomes the mRNA export block caused by the presence of saturating amounts of CTE RNA. Thus, TAP, like its yeast homolog Mex67p, is a bona fide mRNA nuclear export mediator. TAP is the second cellular RNA binding protein shown to be directly involved in the export of its target RNA.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1997

Mutational analysis of human prothymosin alpha reveals a bipartite nuclear localization signal.

Mutants of human prothymosin alpha with impaired ability to inhibit yeast Saccharomyces cerevisiae. cerevisiae cell growth were characterized. Two types of prothymosin alpha-inactivating mutations were observed. Mutations that belong to the first type compromised the nuclear entry of prothymosin alpha by affecting its nuclear localization signal. Analysis of subcellular distribution of GFP-prothymosin alpha fusions revealed a bipartite nuclear localization signal that is both necessary and sufficient for nuclear import of the protein in human cells. Mutations of the second type abrogated the inhibitory action of prothymosin alpha through an unknown mechanism, without influencing the nuclear import of the protein.

Amino Acid Sequence↗

PubMed1997

Replacement of posttranscriptional regulation in SIVmac239 generated a Rev-independent infectious virus able to propagate in rhesus peripheral blood mononuclear cells.

Lentiviruses control virion production via posttranscriptional regulation mediated by the viral Rev protein. In this study, we demonstrate that the Rev regulation of SIVmac239 can be replaced by the presence of the cis-acting transport element (CTE) of the type D simian retroviruses 1 (SRV-1). To avoid the possibility of generating revertants, the Rev-Independent SIV clones have both rev and the Rev responsive element (RRE) destroyed by multiple point mutations that do not affect the overlapping tat and env open reading frames. Virus stocks generated from these Rev-independent SIV molecular clones can infect and can be propagated in rhesus peripheral blood mononuclear cells (PBMCs). Therefore, the Rev/RRE system of SIVmac239 can be replaced by the SRV-1 CTE as previously shown for HIV-1. In both rhesus and human primary cells, the replicative capacity of the Rev-independent SIV is 10- to 20-fold lower than that of the wild-type virus. Rhesus PBMC-derived virus stocks of the Rev-independent SIV have lower infectivity. Interestingly, in CEM x 174 cells, no difference in replicative capacity between wild-type and Rev-independent SIV has been observed. The Rev-independent SIV has a stable genotype after several passages in primary cells. The availability of such Rev-Independent viruses will allow the study of the role of Rev in pathogenesis and the potential generation of attenuated SIV strains.

Animals↗

PubMed1997

Mutations in the nuclear export signal of human ran-binding protein RanBP1 block the Rev-mediated posttranscriptional regulation of human immunodeficiency virus type 1.

We identified a region in the human Ran GTPase-binding protein RanBP1 that shares similarities to the nuclear export signal of the inhibitor of the cAMP-dependent protein kinase. Mutational analysis confirmed that this region is responsible for the cytoplasmic accumulation of RanBP1 and can functionally replace the nuclear export signal of Rev of human immunodeficiency virus type 1. We showed that RanBP1 interferes with Rev-mediated expression of human immunodeficiency virus type 1, whereas the RanBP1 with inactivated nuclear export signal abrogates Rev function. Expression of a Rev-independent molecular clone, which is regulated via the constitutive transport element (CTE) of the simian retrovirus type 1, is not affected. These findings indicate that Rev and RanBP1 compete for the same nuclear export pathway, whereas Rev- and the CTE-mediated pathways are distinct. The inhibition of Rev function is independent of the ability of RanBP1 to associate with Ran and therefore, it is not likely a result of interference with Ran function. These data suggest that RanBP1 interacts with Rev at the putative nuclear receptor and, hence, shares a step in posttranscriptional pathway with Rev.

Amino Acid Sequence↗

PubMed1997

Inflammatory polyarthropathy and bone remodeling in HTLV-I Tax-transgenic mice.

Transgenic mice carrying the tax gene of human T-cell lymphotropic virus type I displayed a high prevalence of arthropathy. The percentage of affected animals increased with age, reaching a peak of 43% at 20 months. Southern analysis of deoxyribonucleic acid (DNA) from tissue samples indicated that disease development was related to tax copy number. Histopathologic evaluation of the ankle joints disclosed deep erosion of the synovial lining, fibrous tissue proliferation together with angiogenesis, mononuclear cell infiltration, and activation of osteoclasts. Radiologic examination confirmed joint involvement and revealed bone architecture modifications. The phenotype exhibited by the affected animals closely resembles that of seronegative arthritis in humans, and may indicate tax protein as a causal agent of arthropathy observed in HTLV-I infected individuals.

Age Factors↗

PubMed1997

Posttranscriptional regulation by Rev protein of human immunodeficiency virus type 1 results in nonrandom nuclear localization of gag mRNA.

The expression of the human immunodeficiency virus type 1 mRNAs containing the Rev-responsive element is regulated at the posttranscriptional level by the viral Rev protein. Rev increases the nucleocytoplasmic export of these mRNAs, leading to high expression. Using in situ hybridization and electron microscopy, we investigated the localization of a subgenomic gag mRNA in the absence and presence of Rev. In addition to the previously shown cytoplasmic accumulation of the Rev-dependent mRNA, we observed that in the presence of Rev the nuclear gag mRNA accumulates nonrandomly and forms specific localization patterns at the nuclear membrane and in the nucleoplasm. Cellular mRNAs for beta-actin and glyceraldehyde-3-phosphate dehydrogenase were not found to form such patterns. These data suggest that Rev leads the gag mRNA to specific subnuclear locations, which further supports the transport function of Rev.

Actins↗

PubMed1997

Identification of an RNA sequence within an intracisternal-A particle element able to replace Rev-mediated posttranscriptional regulation of human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 (HIV-1) replication depends on the posttranscriptional regulation by the viral Rev protein and can be replaced with the posttranscriptional RNA control element (CTE) of the type D simian retroviruses. We have identified a sequence which shares only nucleotide sequences of the internal loops and secondary structure with the CTE and which is part of a novel murine intracisternal-A particle (IAP) retroelement, inserted within the transcribed mouse osteocalcin-related gene. This sequence, named CTE(IAP), can replace the Rev-mediated regulation of HIV-1, hence it is a posttranscriptional regulatory element. Related elements have been identified in other IAPs. These results suggest that insertional mutagenesis can affect gene expression by providing a functional posttranscriptional control element. The CTE(IAP) and CTEs of the type D simian retroviruses represent a novel class of RNA elements characterized by unique sequences within the internal loops which are predicted to represent the interaction site with cellular factor(s). These findings suggest that such elements may be involved in posttranscriptional regulation of cellular mRNAs.

Amino Acid Sequence↗

PubMed1997

Reduced viral load and lack of CD4 depletion in SCID-hu mice infected with Rev-independent clones of human immunodeficiency virus type 1.

The posttranscriptional control element CTE of the simian type D retrovirus has been shown to support replication of Rev-Rev-responsive-element (RRE)-deficient molecular clones of human immunodeficiency virus type 1 (HIV-1). Upon infection of peripheral blood mononuclear cells in vitro, these CTE-containing Rev-independent viruses that are nef+ or nef-minus showed lower replicative capacity and infectivity than the wild-type HIV-1. We studied the effects of Rev-RRE replacement by the CTE on HIV-1 expression with SCID-hu mice. The nef+ and nef-minus Rev-independent viruses established infection with kinetics slower than that of the nef-minus NL4-3. Most importantly, no depletion of CD4-bearing thymocytes was observed after 6 weeks for mice infected with these Rev-independent viruses. This is in contrast to the infection with both wild-type and nef-minus viruses, which led to varying depletion of thymocytes. These data suggest an attenuated phenotype for growth and cytotoxicity of the Rev-independent HIV-1 clones in SCID-hu mice, independent of the presence of Nef. The mutant viruses, which have the essential Rev-RRE regulatory system eliminated, display a distinct phenotype not previously observed with HIV mutant viruses having deletions of accessory genes. Therefore, replacement of the Rev-RRE regulatory axis may generate viruses with altered biological properties in vivo.

Animals↗