The Experts below are selected from a list of 1971 Experts worldwide ranked by ideXlab platform
Ralph Dornburg - One of the best experts on this subject based on the ideXlab platform.
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Spleen Necrosis Virus based vector delivery of anti hiv 1 genes potently protects human hematopoietic cells from hiv 1 infection
Virology, 2005Co-Authors: Elena Marusich, Ralph Dornburg, Zahida Parveen, Muhammad Mukhtar, David S Strayer, Roger J PomerantzAbstract:Abstract In this study, we report on the efficacy of using a Spleen Necrosis Virus (SNV)-based vector delivery system to block human immunodeficiency Virus type I (HIV-1) replication in human hematopoietic cells. These efforts were directed towards the development of human immune system cell resistance to HIV-1 infection, based on the strategy of “intracellular immunization” via generation of a series of anti-HIV-1 therapeutic constructs carrying scFvs, single-chain variable fragments, against HIV-1 integrase and reverse transcriptase in combination with the trans-dominant mutant of HIV-1 Rev, RevM10. The efficiency of the anti-HIV-1 constructs were tested in viral challenge assays with different doses of HIV-1 NL4-3, Bal, 89.6 and R7-GFP strains. These experiments demonstrated the reduction of HIV-1 replication by these retroviral vector constructs in a range of 4- to 10-fold in CD4+ T-lymphocytes, human peripheral blood mononuclear cells (PBMCs), and primary human macrophages. We observed selective efficiency of SNV-based therapeutics in H9, C8166 and Jurkat T-lymphocytic cell lines, demonstrating the most efficient inhibition of HIV-1 replication in Jurkat T-cells. Thus, these data are the first demonstration of the ability of SNV-based retroviral vectors with select transgenes, which may have certain molecular advantages over other retroviral vector systems, to combat HIV-1 replication in human hematopoietic cells and support the potential for using SNV-expressed constructs in anti-HIV-1 molecular therapeutics.
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cross packaging of human immunodeficiency Virus type 1 vector rna by Spleen Necrosis Virus proteins construction of a new generation of Spleen Necrosis Virus derived retroviral vectors
Journal of Virology, 2004Co-Authors: Zahida Parveen, Ralph Dornburg, Muhammad Mukhtar, Adrienne Goodrich, Edward Acheampong, Roger J PomerantzAbstract:The ability of the nonlentiviral retroVirus Spleen Necrosis Virus (SNV) to cross-package the genomic RNA of the distantly related human immunodeficiency Virus type 1 (HIV-1) and vice versa was analyzed. Such a model may allow us to further study HIV-1 replication and pathogenesis, as well as to develop safe gene therapy vectors. Our results suggest that SNV can cross-package HIV-1 genomic RNA but with lower efficiency than HIV-1 proteins. However, HIV-1-specific proteins were unable to cross-package SNV RNA. We also constructed SNV-based gag-pol chimeric variants by replacing the SNV integrase with the HIV-1 integrase, based on multiple sequence alignments and domain analyses. These analyses revealed that there are conserved domains in all retroviral integrase open reading frames (orf), despite the divergence in the primary sequences. The transcomplementation assays suggested that SNV proteins recognized one of the chimeric variants. This demonstrated that HIV-1 integrase is functional in the SNV gag-pol orf with a lower transduction efficiency, utilizing homologous (SNV) RNA, as well as the heterologous vector RNA of HIV-1. These findings suggest that homology in the conserved sequences of the integrase protein may not be fully competent in the replacement of protein(s) from one retroVirus to another, and there are likely several other factors involved in each of the steps related to replication, integration, and infection. However, further studies to dissect the gag-pol region will be critical for understanding the mechanisms involved in the cleavage of reverse transcriptase, RNase H, and integrase. These studies should provide further insight into the design and development of novel molecular approaches to block HIV-1 replication and to construct a new generation of SNV-based vectors.
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spliced Spleen Necrosis Virus vector rna is not encapsidated implications for retroviral replication and vector design
Molecular Therapy, 2004Co-Authors: Adrienne Goodrich, Ralph Dornburg, Zahida Parveen, Matthias J Schnell, Roger J PomerantzAbstract:RNA splicing is a complex event in the retroviral life cycle and can involve multiple steps, as well as cis-acting sequences, to maintain a proper balance of spliced and unspliced viral RNA for translation and encapsidation. The retroviral RNA can be processed by cellular machinery and enables the removal of intronic sequences. We aimed to utilize the removal of a synthetic intron for targeted gene expression. To analyze intron removal and gene expression, we have constructed a novel self-inactivating gene-activating (SIGA) vector for potential universal gene therapy. New vectors for gene therapy are necessary for safe and effective gene delivery in humans. The SIGA vector is derived from Spleen Necrosis Virus (SNV), which is an avian reticuloendotheliosis Virus. The vector was designed so that expression of a therapeutic gene is blocked in helper cell lines due to an intervening sequence containing various blocks in transcription and translation. However, after one round of retroviral replication, the intervening sequence should be removed by the cellular machinery and the therapeutic gene will be selectively expressed in target cells. Our studies show that the intervening sequence in SIGA vector RNA is partially spliced. However, spliced vector RNA was not transduced to target cells. Previous studies showed that an infectious SNV vector enabled transduction of spliced RNA. However, yet-undefined differences in infectious and replication-deficient retroviral replication may have an effect on the transduction of spliced RNA. The results of this study present key information on spliced RNA and its encapsidation, as well as data for the construction of a new generation of SNV-derived retroviral vectors.
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Spleen Necrosis Virus derived c type retroviral vectors for gene transfer to quiescent cells
Nature Biotechnology, 2000Co-Authors: Zahida Parveen, Klaus Cichutek, Martin Engelstädter, Anna Krupetsky, Roger J Pomerantz, Ralph DornburgAbstract:Gene therapy applications of retroviral vectors derived from C-type retroViruses have been limited to introducing genes into dividing target cells. Here, we report genetically engineered C-type retroviral vectors derived from Spleen Necrosis Virus (SNV), which are capable of infecting nondividing cells. This has been achieved by introducing a nuclear localization signal (NLS) sequence into the matrix protein (MA) of SNV by site-directed mutagenesis. This increased the efficiency of infecting nondividing cells and was sufficient to endow the Virus with the capability to efficiently infect growth-arrested human T lymphocytes and quiescent primary monocyte-derived macrophages. We demonstrate that this vector actively penetrates the nucleus of a target cell, and has potential use as a gene therapy vector to transfer genes into nondividing cells.
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avian reticuloendotheliosis Virus strain a and Spleen Necrosis Virus do not infect human cells
Journal of Virology, 2000Co-Authors: Rodolphe Gautier, An Jiang, Ralph Dornburg, V Rousseau, Thierry JaffredoAbstract:Spleen Necrosis Virus (SNV) and Reticuloendotheliosis Virus strain A (REV-A) belong to the family of reticuloendotheliosis Viruses and are 90% sequence related. SNV-derived retroviral vectors produced by the REV-A-based D17.2G packaging cell line were shown to infect human cells (H.-M. Koo, A. M. C. Brown, Y. Ron, and J. P. Dougherty, J. Virol. 65:4769–4776, 1991), while similar vectors produced by another SNV-based packaging cell line, DSH134G, are not infectious in human cells (reviewed by R. Dornburg, Gene Ther. 2:301–310, 1995). Here we describe a careful reevaluation of the infectivity of vectors produced from the most commonly used REV-A- or SNV-based packaging cells obtained from various sources with, among them, one batch of D17.2G packaging cells obtained from the American Type Culture Collection. None of these packaging cells produced vectors able to infect human cells. Thus, contrary to previously published data, we conclude that REV-based vectors are not infectious in human cells.
Kathleen Borislawrie - One of the best experts on this subject based on the ideXlab platform.
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hiv 1 and two avian retroviral 5 untranslated regions bind orthologous human and chicken rna binding proteins
Virology, 2015Co-Authors: Matthew S Stake, Deepali Singh, Gatikrushna Singh, Marcela J Hernandez, Rebecca Kaddis J Maldonado, Leslie J Parent, Kathleen BorislawrieAbstract:Essential host cofactors in retroVirus replication bind cis-acting sequences in the 5'untranslated region (UTR). Although host RBPs are crucial to all aspects of Virus biology, elucidating their roles in replication remains a challenge to the field. Here RNA affinity-coupled-proteomics generated a comprehensive, unbiased inventory of human and avian RNA binding proteins (RBPs) co-isolating with 5'UTRs of HIV-1, Spleen Necrosis Virus and Rous sarcoma Virus. Applying stringent biochemical and statistical criteria, we identified 185 RBP; 122 were previously implicated in retroVirus biology and 63 are new to the 5'UTR proteome. RNA electrophoretic mobility assays investigated paralogs present in the common ancestor of vertebrates and one hnRNP was identified as a central node to the biological process-anchored networks of HIV-1, SNV, and RSV 5' UTR-proteomes. This comprehensive view of the host constituents of retroviral RNPs is broadly applicable to investigation of viral replication and antiviral response in both human and avian cell lineages.
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coordinate enhancement of transgene transcription and translation in a lentiviral vector
Retrovirology, 2006Co-Authors: Alper Yilmaz, Soledad Fernandez, Michael Dale Lairmore, Kathleen BorislawrieAbstract:Coordinate enhancement of transgene transcription and translation would be a potent approach to significantly improve protein output in a broad array of viral vectors and nonviral expression systems. Many vector transgenes are complementary DNA (cDNA). The lack of splicing can significantly reduce the efficiency of their translation. Some retroViruses contain a 5' terminal post-transcriptional control element (PCE) that facilitates translation of unspliced mRNA. Here we evaluated the potential for Spleen Necrosis Virus PCE to stimulate protein production from HIV-1 based lentiviral vector by: 1) improving translation of the internal transgene transcript; and 2) functionally synergizing with a transcriptional enhancer to achieve coordinate increases in RNA synthesis and translation. Derivatives of HIV-1 SIN self-inactivating lentiviral vector were created that contain PCE and cytomegaloVirus immediate early enhancer (CMV IE). Results from transfected cells and four different transduced cell types indicate that: 1) PCE enhanced transgene protein synthesis; 2) transcription from the internal promoter is enhanced by CMV IE; 3) PCE and CMV IE functioned synergistically to significantly increase transgene protein yield; 4) the magnitude of translation enhancement by PCE was similar in transfected and transduced cells; 5) differences were observed in steady state level of PCE vector RNA in transfected and transduced cells; 6) the lower steady state was not attributable to reduced RNA stability, but to lower cytoplasmic accumulation in transduced cells. PCE is a useful tool to improve post-transcriptional expression of lentiviral vector transgene. Coordinate enhancement of transcription and translation is conferred by the combination of PCE with CMV IE transcriptional enhancer and increased protein yield up to 11 to 17-fold in transfected cells. The incorporation of the vector proVirus into chromatin correlated with reduced cytoplasmic accumulation of PCE transgene RNA. We speculate that epigenetic modulation of promoter activity altered cotranscriptional recruitment of RNA processing factors and reduced the availability of fully processed transcript or the efficiency of export from the nucleus. Our results provide an example of the dynamic interplay between the transcription and post-transcription steps of gene expression and document that introduction of heterologous gene expression signals can yield disparate effects in transfected versus transduced cells.
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primary sequence and secondary structure motifs in Spleen Necrosis Virus ru5 confer translational utilization of unspliced human immunodeficiency Virus type 1 reporter rna
Journal of Virology, 2003Co-Authors: Tiffiney M Roberts, Kathleen BorislawrieAbstract:The 5' long terminal repeat (LTR) of Spleen Necrosis Virus (SNV) contains a unique posttranscriptional control element that facilitates Rev/Rev-responsive element-independent expression of unspliced human immunodeficiency Virus type 1 (HIV-1) gag reporter RNA. HIV-1 Gag expression is eliminated when SNV LTR is repositioned to the 3' untranslated region or when the RU5 region is positioned in the antisense orientation. RU5 corresponds to the 5' RNA terminus, and results presented here indicate that Gag production is sustained upon introduction of transcribed spacers that reposition SNV RU5 35 to 200 nucleotides downstream. Concordant results of deletion and point mutagenesis identified two functionally redundant and synergistic motifs (designated A and C) that are necessary and sufficient for SNV RU5 activity. Enzymatic analysis of SNV RU5 RNA structure determined that A and C correspond to stem-loop structures. Quantitative RNA and protein analysis of A and C mutants revealed that the structural integrity of A and C is necessary for protein production, and loss of function correlates with little change in steady-state level, splicing efficiency, or cytoplasmic accumulation of HIV-1 gag reporter RNA. Instead, the structural mutations eliminate cytoplasmic utilization as an mRNA template for Gag protein production. Point mutations of unpaired loop-and-bulge nucleotides that maintain the structure of A eliminate activity. The results show that the unpaired UUGU loop and U-rich bulges function together and are candidate SNV RU5 binding sites for the host cell protein(s) that directs cytoplasmic utilization of unspliced HIV-1 reporter RNA.
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nuclear interactions are necessary for translational enhancement by Spleen Necrosis Virus ru5
Journal of Virology, 2002Co-Authors: Andrew W Dangel, Tiffiney M Roberts, Stacey Hull, Kathleen BorislawrieAbstract:The 5' long terminal repeat of Spleen Necrosis Virus (SNV) facilitates Rev/Rev-responsive element (RRE)-independent expression of intron-containing human immunodeficiency Virus type 1 (HIV-1) gag. The SNV RU5 region, which corresponds to the 165-nucleotide 5' RNA terminus, functions in a position- and orientation-dependent manner to enhance polysome association of intron-containing HIV-1 gag RNA and also nonviral luc RNA. Evidence is mounting that association with nuclear factors during intron removal licenses mRNAs for nuclear export, efficient translation, and nonsense-mediated decay. This project addressed the relationship between the nuclear export pathway of SNV RU5-reporter RNA and translational enhancement. Results of RNA transfection experiments suggest that cytoplasmic proteins are insufficient for SNV RU5 translational enhancement of gag or luc RNA. Reporter gene assays, leptomycin B (LMB) sensitivity experiments, and RNase protection assays indicate that RU5 gag RNA accesses a nuclear export pathway that is distinct from the LMB-inhibited leucine-rich nuclear export sequence-dependent CRM1 pathway, which is used by the HIV-1 RRE. As a unique tool with which to investigate the relationship between different RNA trafficking routes and translational enhancement, SNV RU5 and Rev/RRE were combined on a single gag RNA. We observed a less-than-synergistic effect on cytoplasmic mRNA utilization. Instead, Rev/RRE diverts RU5 gag RNA to the CRM1-dependent, LMB-inhibited pathway and abrogates translational enhancement by SNV RU5. Our study is the first to show that a nuclear factor(s) directs SNV RU5-containing RNAs to a distinct export pathway that is not inhibited by LMB and programs the intron-containing transcript for translational enhancement.
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the 5 rna terminus of Spleen Necrosis Virus stimulates translation of nonviral mrna
Journal of Virology, 2000Co-Authors: Tiffiney M Roberts, Kathleen BorislawrieAbstract:The RU5 region at the 5′ RNA terminus of Spleen Necrosis Virus (SNV) has been shown to facilitate expression of human immunodeficiency Virus type 1 (HIV) unspliced RNA independently of the Rev-responsive element (RRE) and Rev. The SNV sequences act as a distinct posttranscriptional control element to stimulate gag RNA nuclear export and association with polyribosomes. Here we sought to determine whether RU5 functions to neutralize the cis-acting inhibitory sequences (INSs) in HIV RNA that confer RRE/Rev dependence or functions as an independent stimulatory sequence. Experiments with HIV gag reporter plasmids that contain inactivated INS-1 indicated that neutralization of INSs does not account for RU5 function. Results with luciferase reporter gene (luc) plasmids further indicated that RU5 stimulates expression of a nonretroviral RNA that lacks INSs. Northern blot and RT-PCR analyses indicated that RU5 does not increase the steady-state levels or nuclear export of the luc transcript but rather that the U5 region facilitates efficient polyribosomal association of the mRNA. RU5 does not function as an internal ribosome entry site in bicistronic reporter plasmids, and it requires the 5′-proximal position for efficient function. Our results indicate that RU5 contains stimulatory sequences that function in a 5′-proximal position to enhance initiation of translation of a nonretroviral reporter gene RNA. We speculate that RU5 evolved to overcome the translation-inhibitory effect of the highly structured encapsidation signal and other replication motifs in the 5′ untranslated region of the retroviral RNA.
Roger J Pomerantz - One of the best experts on this subject based on the ideXlab platform.
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Spleen Necrosis Virus based vector delivery of anti hiv 1 genes potently protects human hematopoietic cells from hiv 1 infection
Virology, 2005Co-Authors: Elena Marusich, Ralph Dornburg, Zahida Parveen, Muhammad Mukhtar, David S Strayer, Roger J PomerantzAbstract:Abstract In this study, we report on the efficacy of using a Spleen Necrosis Virus (SNV)-based vector delivery system to block human immunodeficiency Virus type I (HIV-1) replication in human hematopoietic cells. These efforts were directed towards the development of human immune system cell resistance to HIV-1 infection, based on the strategy of “intracellular immunization” via generation of a series of anti-HIV-1 therapeutic constructs carrying scFvs, single-chain variable fragments, against HIV-1 integrase and reverse transcriptase in combination with the trans-dominant mutant of HIV-1 Rev, RevM10. The efficiency of the anti-HIV-1 constructs were tested in viral challenge assays with different doses of HIV-1 NL4-3, Bal, 89.6 and R7-GFP strains. These experiments demonstrated the reduction of HIV-1 replication by these retroviral vector constructs in a range of 4- to 10-fold in CD4+ T-lymphocytes, human peripheral blood mononuclear cells (PBMCs), and primary human macrophages. We observed selective efficiency of SNV-based therapeutics in H9, C8166 and Jurkat T-lymphocytic cell lines, demonstrating the most efficient inhibition of HIV-1 replication in Jurkat T-cells. Thus, these data are the first demonstration of the ability of SNV-based retroviral vectors with select transgenes, which may have certain molecular advantages over other retroviral vector systems, to combat HIV-1 replication in human hematopoietic cells and support the potential for using SNV-expressed constructs in anti-HIV-1 molecular therapeutics.
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cross packaging of human immunodeficiency Virus type 1 vector rna by Spleen Necrosis Virus proteins construction of a new generation of Spleen Necrosis Virus derived retroviral vectors
Journal of Virology, 2004Co-Authors: Zahida Parveen, Ralph Dornburg, Muhammad Mukhtar, Adrienne Goodrich, Edward Acheampong, Roger J PomerantzAbstract:The ability of the nonlentiviral retroVirus Spleen Necrosis Virus (SNV) to cross-package the genomic RNA of the distantly related human immunodeficiency Virus type 1 (HIV-1) and vice versa was analyzed. Such a model may allow us to further study HIV-1 replication and pathogenesis, as well as to develop safe gene therapy vectors. Our results suggest that SNV can cross-package HIV-1 genomic RNA but with lower efficiency than HIV-1 proteins. However, HIV-1-specific proteins were unable to cross-package SNV RNA. We also constructed SNV-based gag-pol chimeric variants by replacing the SNV integrase with the HIV-1 integrase, based on multiple sequence alignments and domain analyses. These analyses revealed that there are conserved domains in all retroviral integrase open reading frames (orf), despite the divergence in the primary sequences. The transcomplementation assays suggested that SNV proteins recognized one of the chimeric variants. This demonstrated that HIV-1 integrase is functional in the SNV gag-pol orf with a lower transduction efficiency, utilizing homologous (SNV) RNA, as well as the heterologous vector RNA of HIV-1. These findings suggest that homology in the conserved sequences of the integrase protein may not be fully competent in the replacement of protein(s) from one retroVirus to another, and there are likely several other factors involved in each of the steps related to replication, integration, and infection. However, further studies to dissect the gag-pol region will be critical for understanding the mechanisms involved in the cleavage of reverse transcriptase, RNase H, and integrase. These studies should provide further insight into the design and development of novel molecular approaches to block HIV-1 replication and to construct a new generation of SNV-based vectors.
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spliced Spleen Necrosis Virus vector rna is not encapsidated implications for retroviral replication and vector design
Molecular Therapy, 2004Co-Authors: Adrienne Goodrich, Ralph Dornburg, Zahida Parveen, Matthias J Schnell, Roger J PomerantzAbstract:RNA splicing is a complex event in the retroviral life cycle and can involve multiple steps, as well as cis-acting sequences, to maintain a proper balance of spliced and unspliced viral RNA for translation and encapsidation. The retroviral RNA can be processed by cellular machinery and enables the removal of intronic sequences. We aimed to utilize the removal of a synthetic intron for targeted gene expression. To analyze intron removal and gene expression, we have constructed a novel self-inactivating gene-activating (SIGA) vector for potential universal gene therapy. New vectors for gene therapy are necessary for safe and effective gene delivery in humans. The SIGA vector is derived from Spleen Necrosis Virus (SNV), which is an avian reticuloendotheliosis Virus. The vector was designed so that expression of a therapeutic gene is blocked in helper cell lines due to an intervening sequence containing various blocks in transcription and translation. However, after one round of retroviral replication, the intervening sequence should be removed by the cellular machinery and the therapeutic gene will be selectively expressed in target cells. Our studies show that the intervening sequence in SIGA vector RNA is partially spliced. However, spliced vector RNA was not transduced to target cells. Previous studies showed that an infectious SNV vector enabled transduction of spliced RNA. However, yet-undefined differences in infectious and replication-deficient retroviral replication may have an effect on the transduction of spliced RNA. The results of this study present key information on spliced RNA and its encapsidation, as well as data for the construction of a new generation of SNV-derived retroviral vectors.
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Spleen Necrosis Virus derived c type retroviral vectors for gene transfer to quiescent cells
Nature Biotechnology, 2000Co-Authors: Zahida Parveen, Klaus Cichutek, Martin Engelstädter, Anna Krupetsky, Roger J Pomerantz, Ralph DornburgAbstract:Gene therapy applications of retroviral vectors derived from C-type retroViruses have been limited to introducing genes into dividing target cells. Here, we report genetically engineered C-type retroviral vectors derived from Spleen Necrosis Virus (SNV), which are capable of infecting nondividing cells. This has been achieved by introducing a nuclear localization signal (NLS) sequence into the matrix protein (MA) of SNV by site-directed mutagenesis. This increased the efficiency of infecting nondividing cells and was sufficient to endow the Virus with the capability to efficiently infect growth-arrested human T lymphocytes and quiescent primary monocyte-derived macrophages. We demonstrate that this vector actively penetrates the nucleus of a target cell, and has potential use as a gene therapy vector to transfer genes into nondividing cells.
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a genetically engineered Spleen Necrosis Virus derived retroviral vector that displays the hiv type 1 glycoprotein 120 envelope peptide
Human Gene Therapy, 1999Co-Authors: An Jiang, Roger J Pomerantz, Hugh Fisher, Ralph DornburgAbstract:We reported that SNV-derived retroviral vectors, which display single-chain antibodies on the viral surface, enable cell type-specific gene delivery into various human cells. In particular, the SNV cell type-specific gene delivery vector system appears to be well suited to transduce genes into cells of the human hematopoietic system (Jiang et al., J. Virol. 72:10148-10156, 1998). Here, we report the construction of SNV vector particles that display the complete gp120 surface unit of the envelope protein of human immunodeficiency Virus type 1 (HIV-1) on the viral surface. The complete gp120-coding region of a T cell-tropic HIV-1 strain (LAI/BRU) was fused to a short peptide spacer coding region [(Gly4Ser)3] linking it to the SNV TM-coding region. The corresponding protein was expressed as a single 145-kDa peptide as expected. This peptide was nontoxic and could be stably expressed in dog D17 SNV-derived packaging cells. Particles harvested from stable packaging lines infected CD4+ human hematopoietic cells with titers exceeding 10(5) CFU/ml supernatant tissue culture medium. Titers in other, CD4- cell lines expressing various coreceptors of HIV-1 were 100-fold lower than titers obtained in CD4+ cells. Specificity of infection was demonstrated by antibody inhibition assays or by preincubating cells with SDF-1alpha, the ligand, which binds to the CXCR4 coreceptor, to which this gp120 binds. Our data indicate that binding of the HIV-1 gp120 to either CD4 or CXCR4 is sufficient to enable infection of human cells with SNV vector particles. We constructed retroviral vector particles that display chimeric HIV-1-SU-SNV-TM proteins plus wild-type SNV envelope on the viral surface. Such particles allowed efficient infection of CD4-positive human T lymphocytes, and, at a lower efficiency, also cells expressing CXCR4 without CD4. These data coincide with our earlier hypothesis that the chimeric envelope is required only to bind the vector particle to a cell surface receptor of the target cell, while membrane fusion is mediated by wild-type Env, which alone is not sufficient to enable infection of human cells.
An Jiang - One of the best experts on this subject based on the ideXlab platform.
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avian reticuloendotheliosis Virus strain a and Spleen Necrosis Virus do not infect human cells
Journal of Virology, 2000Co-Authors: Rodolphe Gautier, An Jiang, Ralph Dornburg, V Rousseau, Thierry JaffredoAbstract:Spleen Necrosis Virus (SNV) and Reticuloendotheliosis Virus strain A (REV-A) belong to the family of reticuloendotheliosis Viruses and are 90% sequence related. SNV-derived retroviral vectors produced by the REV-A-based D17.2G packaging cell line were shown to infect human cells (H.-M. Koo, A. M. C. Brown, Y. Ron, and J. P. Dougherty, J. Virol. 65:4769–4776, 1991), while similar vectors produced by another SNV-based packaging cell line, DSH134G, are not infectious in human cells (reviewed by R. Dornburg, Gene Ther. 2:301–310, 1995). Here we describe a careful reevaluation of the infectivity of vectors produced from the most commonly used REV-A- or SNV-based packaging cells obtained from various sources with, among them, one batch of D17.2G packaging cells obtained from the American Type Culture Collection. None of these packaging cells produced vectors able to infect human cells. Thus, contrary to previously published data, we conclude that REV-based vectors are not infectious in human cells.
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a genetically engineered Spleen Necrosis Virus derived retroviral vector that displays the hiv type 1 glycoprotein 120 envelope peptide
Human Gene Therapy, 1999Co-Authors: An Jiang, Roger J Pomerantz, Hugh Fisher, Ralph DornburgAbstract:We reported that SNV-derived retroviral vectors, which display single-chain antibodies on the viral surface, enable cell type-specific gene delivery into various human cells. In particular, the SNV cell type-specific gene delivery vector system appears to be well suited to transduce genes into cells of the human hematopoietic system (Jiang et al., J. Virol. 72:10148-10156, 1998). Here, we report the construction of SNV vector particles that display the complete gp120 surface unit of the envelope protein of human immunodeficiency Virus type 1 (HIV-1) on the viral surface. The complete gp120-coding region of a T cell-tropic HIV-1 strain (LAI/BRU) was fused to a short peptide spacer coding region [(Gly4Ser)3] linking it to the SNV TM-coding region. The corresponding protein was expressed as a single 145-kDa peptide as expected. This peptide was nontoxic and could be stably expressed in dog D17 SNV-derived packaging cells. Particles harvested from stable packaging lines infected CD4+ human hematopoietic cells with titers exceeding 10(5) CFU/ml supernatant tissue culture medium. Titers in other, CD4- cell lines expressing various coreceptors of HIV-1 were 100-fold lower than titers obtained in CD4+ cells. Specificity of infection was demonstrated by antibody inhibition assays or by preincubating cells with SDF-1alpha, the ligand, which binds to the CXCR4 coreceptor, to which this gp120 binds. Our data indicate that binding of the HIV-1 gp120 to either CD4 or CXCR4 is sufficient to enable infection of human cells with SNV vector particles. We constructed retroviral vector particles that display chimeric HIV-1-SU-SNV-TM proteins plus wild-type SNV envelope on the viral surface. Such particles allowed efficient infection of CD4-positive human T lymphocytes, and, at a lower efficiency, also cells expressing CXCR4 without CD4. These data coincide with our earlier hypothesis that the chimeric envelope is required only to bind the vector particle to a cell surface receptor of the target cell, while membrane fusion is mediated by wild-type Env, which alone is not sufficient to enable infection of human cells.
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cell type specific gene transfer into human cells with retroviral vectors that display single chain antibodies
Journal of Virology, 1998Co-Authors: An Jiang, Tehua T Chu, Frank Nocken, Klaus Cichutek, Ralph DornburgAbstract:The successful application of human gene therapy protocols on a broad clinical basis will depend on the availability of in vivo cell-type-specific gene delivery systems. We have developed retroviral vector particles, derived from Spleen Necrosis Virus (SNV), that display the antigen binding site of an antibody on the viral surface. Using retroviral vectors derived from SNV that displayed single-chain antibodies (scAs) directed against a carcinoembryonic antigen-cross-reacting cell surface protein, we have shown that an efficient, cell-type-specific gene delivery can be obtained. In this study, we tested whether other scAs displayed on SNV vector particles can also lead to cell-type-specific gene delivery. We displayed the following scAs on the retroviral surface: one directed against the human cell surface antigen Her2neu, which belongs to the epidermal growth factor receptor family; one directed against the stem cell-specific antigen CD34; and one directed against the transferrin receptor, which is expressed on liver cells and various other tissues. We show that retroviral vectors displaying these scAs are competent for infection in human cells which express the antigen recognized by the scA. Infectivity was cell type specific, and titers above 10(5) CFU per ml of tissue culture supernatant medium were obtained. The density of the antigen on the target cell surface does not influence Virus titers in vitro. Our data indicate that the SNV vector system is well suited for the development of a large variety of cell-type-specific targeting vectors.
Klaus Cichutek - One of the best experts on this subject based on the ideXlab platform.
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envelope proteins of Spleen Necrosis Virus form infectious human immunodeficiency Virus type 1 pseudotype vector particles but fail to incorporate upon substitution of the cytoplasmic domain with that of gibbon ape leukemia Virus
Journal of General Virology, 2006Co-Authors: Joern Stitz, Christian J. Buchholz, Nina Wolfrum, Klaus CichutekAbstract:The wild-type (wt) envelope (Env) proteins of Spleen Necrosis Virus (SNV), together with the transmembrane (TM) protein fused to antibody domains (scFv), have been used for the generation of stable packaging cell lines releasing pseudotyped cell targeting vectors derived from SNV and Murine leukemia Virus (MLV). As a first step towards assessing whether HIV-1(SNV/TM-scFv) packaging cells could be established for the production of lentiviral cell targeting vectors, it is reported here that infectious HIV-1-derived particles pseudotyped with wt SNV Env proteins could be generated. Using novel chimeric SNV-derived Env proteins encompassing wt and engineered cytoplasmic domains (C-tail) of the Gibbon ape leukemia Virus (GaLV) TM protein, it was further shown that the wt C-tail not only excludes the GaLV TM protein from incorporation into HIV-1 particles, but confers this phenotype to other retroviral envelopes upon C-terminal fusion.
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Targeted gene transfer to lymphocytes using murine leukaemia Virus vectors pseudotyped with Spleen Necrosis Virus envelope proteins
Gene therapy, 2001Co-Authors: Martin Engelstädter, Christian J. Buchholz, Maria Bobkova, Stefanie Steidl, Heike Merget-millitzer, Ra Willemsen, Joern Stitz, Klaus CichutekAbstract:Targeted gene transfer to lymphocytes using murine leukaemia Virus vectors pseudotyped with Spleen Necrosis Virus envelope proteins
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Spleen Necrosis Virus derived c type retroviral vectors for gene transfer to quiescent cells
Nature Biotechnology, 2000Co-Authors: Zahida Parveen, Klaus Cichutek, Martin Engelstädter, Anna Krupetsky, Roger J Pomerantz, Ralph DornburgAbstract:Gene therapy applications of retroviral vectors derived from C-type retroViruses have been limited to introducing genes into dividing target cells. Here, we report genetically engineered C-type retroviral vectors derived from Spleen Necrosis Virus (SNV), which are capable of infecting nondividing cells. This has been achieved by introducing a nuclear localization signal (NLS) sequence into the matrix protein (MA) of SNV by site-directed mutagenesis. This increased the efficiency of infecting nondividing cells and was sufficient to endow the Virus with the capability to efficiently infect growth-arrested human T lymphocytes and quiescent primary monocyte-derived macrophages. We demonstrate that this vector actively penetrates the nucleus of a target cell, and has potential use as a gene therapy vector to transfer genes into nondividing cells.
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cell type specific gene transfer into human cells with retroviral vectors that display single chain antibodies
Journal of Virology, 1998Co-Authors: An Jiang, Tehua T Chu, Frank Nocken, Klaus Cichutek, Ralph DornburgAbstract:The successful application of human gene therapy protocols on a broad clinical basis will depend on the availability of in vivo cell-type-specific gene delivery systems. We have developed retroviral vector particles, derived from Spleen Necrosis Virus (SNV), that display the antigen binding site of an antibody on the viral surface. Using retroviral vectors derived from SNV that displayed single-chain antibodies (scAs) directed against a carcinoembryonic antigen-cross-reacting cell surface protein, we have shown that an efficient, cell-type-specific gene delivery can be obtained. In this study, we tested whether other scAs displayed on SNV vector particles can also lead to cell-type-specific gene delivery. We displayed the following scAs on the retroviral surface: one directed against the human cell surface antigen Her2neu, which belongs to the epidermal growth factor receptor family; one directed against the stem cell-specific antigen CD34; and one directed against the transferrin receptor, which is expressed on liver cells and various other tissues. We show that retroviral vectors displaying these scAs are competent for infection in human cells which express the antigen recognized by the scA. Infectivity was cell type specific, and titers above 10(5) CFU per ml of tissue culture supernatant medium were obtained. The density of the antigen on the target cell surface does not influence Virus titers in vitro. Our data indicate that the SNV vector system is well suited for the development of a large variety of cell-type-specific targeting vectors.