scholarly journals Deoxyribonucleoside Triphosphate Pool Imbalances In Vivo Are Associated with an Increased Retroviral Mutation Rate

1998 ◽  
Vol 72 (10) ◽  
pp. 7941-7949 ◽  
Author(s):  
John G. Julias ◽  
Vinay K. Pathak
Keyword(s):  
Reverse Transcription ◽  
Leukemia Virus ◽  
Retroviral Vectors ◽  
Mutation Rates ◽  
Target Cells ◽  
Dntp Pool ◽  
Deoxyribonucleoside Triphosphate ◽  
Spleen Necrosis

ABSTRACT Deoxyribonucleoside triphosphate (dNTP) pool imbalances are associated with an increase in the rate of misincorporation and hypermutation during in vitro reverse transcription reactions. However, the effects of in vivo dNTP pool imbalances on the accuracy of reverse transcription are unknown. We sought to determine the effects of in vivo dNTP pool imbalances on retroviral mutation rates and to test our hypothesis that 3′-azido-3′-deoxythymidine (AZT) increases the retroviral mutation rates through induction of dNTP pool imbalances. D17 cells were treated with thymidine, hydroxyurea (HU), or AZT, and the effects on in vivo dNTP pools were measured. Thymidine and HU treatments induced significant dNTP pool imbalances. In contrast, AZT treatment had very little effect on the dNTP pools. The effects of in vivo dNTP pool imbalances induced by thymidine and HU treatments on the retroviral mutation rates were also determined. Spleen necrosis virus (SNV)-based and murine leukemia virus (MLV)-based retroviral vectors that expressed the lacZ mutant reporter gene were used. The frequencies of inactivating mutations introduced in thelacZ gene in a single replication cycle provided a measure of the retroviral mutation rates. Treatment of D17 target cells with 500 μM thymidine increased the SNV and MLV mutant frequencies 4.7- and 4-fold, respectively. Treatment of D17 target cells with 2 mM HU increased the SNV and MLV mutant frequencies 2.1- and 2.7-fold, respectively. These results demonstrate that dNTP pool imbalances are associated with an increase in the in vivo retroviral mutation rates, but AZT treatment results in an increase in the retroviral mutation rates by a mechanism not involving alterations in dNTP pools.

scholarly journals Adaptation of Chimeric Retroviruses In Vitro and In Vivo: Isolation of Avian Retroviral Vectors with Extended Host Range

2001 ◽  
Vol 75 (11) ◽  
pp. 4973-4983 ◽  
Author(s):  
Eugene V. Barsov ◽  
William S. Payne ◽  
Stephen H. Hughes
Keyword(s):  
Host Range ◽  
Mammalian Cells ◽  
Leukemia Virus ◽  
Retroviral Vectors ◽  
Retroviral Vector ◽  
General Strategy ◽  
Coding Region ◽  
Chimeric Viruses

ABSTRACT We have designed and characterized two new replication-competent avian sarcoma/leukosis virus-based retroviral vectors with amphotropic and ecotropic host ranges. The amphotropic vector RCASBP-M2C(797-8), was obtained by passaging the chimeric retroviral vector RCASBP-M2C(4070A) (6) in chicken embryos. The ecotropic vector, RCASBP(Eco), was created by replacing theenv-coding region in the retroviral vector RCASBP(A) with the env region from an ecotropic murine leukemia virus. It replicates efficiently in avian DFJ8 cells that express murine ecotropic receptor. For both vectors, permanent cell lines that produce viral stocks with titers of about 5 × 106 CFU/ml on mammalian cells can be easily established by passaging transfected avian cells. Some chimeric viruses, for example, RCASBP(Eco), replicate efficiently without modifications. For those chimeric viruses that do require modification, adaptation by passage in vitro or in vivo is a general strategy. This strategy has been used to prepare vectors with altered host range and could potentially be used to develop vectors that would be useful for targeted gene delivery.

scholarly journals Role of Murine Leukemia Virus Reverse Transcriptase Deoxyribonucleoside Triphosphate-Binding Site in Retroviral Replication and In Vivo Fidelity

2000 ◽  
Vol 74 (22) ◽  
pp. 10349-10358 ◽  
Author(s):  
Elias K. Halvas ◽  
Evguenia S. Svarovskaia ◽  
Vinay K. Pathak
Keyword(s):  
Amino Acids ◽  
Viral Replication ◽  
Reverse Transcriptase ◽  
Binding Site ◽  
Reverse Transcription ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Deoxyribonucleoside Triphosphate ◽  
Murine Leukemia

ABSTRACT Retroviral populations exhibit a high evolutionary potential, giving rise to extensive genetic variation. Error-prone DNA synthesis catalyzed by reverse transcriptase (RT) generates variation in retroviral populations. Structural features within RTs are likely to contribute to the high rate of errors that occur during reverse transcription. We sought to determine whether amino acids within murine leukemia virus (MLV) RT that contact the deoxyribonucleoside triphosphate (dNTP) substrate are important for in vivo fidelity of reverse transcription. We utilized the previously described ANGIE P encapsidating cell line, which expresses the amphotropic MLV envelope and a retroviral vector (pGA-1). pGA-1 expresses the bacterial β-galactosidase gene (lacZ), which serves as a reporter of mutations. Extensive mutagenesis was performed on residues likely to interact with the dNTP substrate, and the effects of these mutations on the fidelity of reverse transcription were determined. As expected, most substitution mutations of amino acids that directly interact with the dNTP substrate significantly reduced viral titers (>10,000-fold), indicating that these residues played a critical role in catalysis and viral replication. However, the D153A and A154S substitutions, which are predicted to affect the interactions with the triphosphate, resulted in statistically significant increases in the mutation rate. In addition, the conservative substitution F155W, which may affect interactions with the base and the ribose, increased the mutation rate 2.8-fold. Substitutions of residues in the vicinity of the dNTP-binding site also resulted in statistically significant decreases in fidelity (1.3- to 2.4-fold). These results suggest that mutations of residues that contact the substrate dNTP can affect viral replication as well as alter the fidelity of reverse transcription.

scholarly journals Bromo- and Extraterminal Domain Chromatin Regulators Serve as Cofactors for Murine Leukemia Virus Integration

2013 ◽  
Vol 87 (23) ◽  
pp. 12721-12736 ◽  
Author(s):  
Saumya Shree Gupta ◽  
Tobias Maetzig ◽  
Goedele N. Maertens ◽  
Azar Sharif ◽  
Michael Rothe ◽  
...  
Keyword(s):  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Retroviral Vectors ◽  
Transcription Start ◽  
Preintegration Complex ◽  
Transcription Start Sites ◽  
Bet Inhibitors ◽  
Murine Leukemia

Retroviral integrase (IN) proteins catalyze the permanent integration of proviral genomes into host DNA with the help of cellular cofactors. Lens epithelium-derived growth factor (LEDGF) is a cofactor for lentiviruses, including human immunodeficiency virus type 1 (HIV-1), and targets lentiviral integration toward active transcription units in the host genome. In contrast to lentiviruses, murine leukemia virus (MLV), a gammaretrovirus, tends to integrate near transcription start sites. Here, we show that the bromodomain and extraterminal domain (BET) proteins BRD2, BRD3, and BRD4 interact with gammaretroviral INs and stimulate the catalytic activity of MLV INin vitro. We mapped the interaction site to a characteristic structural feature within the BET protein extraterminal (ET) domain and to three amino acids in MLV IN. The ET domains of different BET proteins stimulate MLV integrationin vitroand, in the case of BRD2, alsoin vivo. Furthermore, two small-molecule BET inhibitors, JQ1 and I-BET, decrease MLV integration and shift it away from transcription start sites. Our data suggest that BET proteins might act as chromatin-bound acceptors for the MLV preintegration complex. These results could pave a way to redirecting MLV DNA integration as a basis for creating safer retroviral vectors.

scholarly journals Discordance between Bovine Leukemia Virus Tax Immortalization In Vitro and Oncogenicity In Vivo

2000 ◽  
Vol 74 (21) ◽  
pp. 9895-9902 ◽  
Author(s):  
Jean-Claude Twizere ◽  
Pierre Kerkhofs ◽  
Arsène Burny ◽  
Daniel Portetelle ◽  
Richard Kettmann ◽  
...  
Keyword(s):  
Viral Replication ◽  
Bovine Leukemia Virus ◽  
Leukemia Virus ◽  
Wild Type Virus ◽  
Target Cells ◽  
Phosphorylation Sites ◽  
Primary Cells ◽  
Wild Type

ABSTRACT Bovine leukemia virus (BLV) Tax protein, a transcriptional activator of viral expression, is essential for viral replication in vivo. Tax is believed to be involved in leukemogenesis because of its second function, immortalization of primary cells in vitro. These activities of Tax can be dissociated on the basis of point mutations within specific regions of the protein. For example, mutation of the phosphorylation sites at serines 106 and 293 abrogates immortalization potential in vitro but maintains transcriptional activity. This type of mutant is thus particularly useful for unraveling the role of Tax immortalization activity during leukemogenesis independently of viral replication. In this report, we describe the biological properties of BLV recombinant proviruses mutated in the Tax phosphorylation sites (BLVTax106+293). Titration of the proviral loads by semiquantitative PCR revealed that the BLV mutants propagated at wild-type levels in vivo. Furthermore, two animals (sheep 480 and 296) infected with BLVTax106+293 developed leukemia or lymphosarcoma after 16 and 36 months, respectively. These periods of time are within the normal range of latencies preceding the onset of pathogenesis induced by wild-type viruses. The phenotype of the mutant-infected cells was characteristic of a B lymphocyte (immunoglobulin M positive) expressing CD11b and CD5 (except at the final stage for the latter marker), a pattern that is typical of wild-type virus-infected target cells. Interestingly, the transformed B lymphocytes from sheep 480 also coexpressed the CD8 marker, a phenotype rarely observed in tumor biopsies from chronic lymphocytic leukemia patients. Finally, direct sequencing of the tax gene demonstrated that the leukemic cells did not harbor revertant proviruses. We conclude that viruses expressing a Tax mutant unable to transform primary cells in culture are still pathogenic in the sheep animal model. Our data thus provide a clear example of the discordant conclusions that can be drawn from in vitro immortalization assays and in vivo experiments. These observations could be of interest for other systems, such as the related human T-cell leukemia virus type 1, which currently lack animal models allowing the study of the leukemogenic process.

scholarly journals Pausing during Reverse Transcription Increases the Rate of Retroviral Recombination

2006 ◽  
Vol 80 (5) ◽  
pp. 2483-2494 ◽  
Author(s):  
Christian Lanciault ◽  
James J. Champoux
Keyword(s):  
Reverse Transcriptase ◽  
Dna Synthesis ◽  
Reverse Transcription ◽  
Fluorescent Protein ◽  
Leukemia Virus ◽  
Primer Binding Site ◽  
Infection Assay ◽  
Green Fluorescent

ABSTRACT Retroviruses package two copies of genomic RNA into viral particles. During the minus-sense DNA synthesis step of reverse transcription, the nascent DNA can transfer multiple times between the two copies of the genome, resulting in recombination. The mechanism for this process is similar to the process of obligate strand transfers mediated by the repeat and primer binding site sequences. The location at which the DNA 3′ terminus completely transfers to the second RNA strand defines the point of crossover. Previous work in vitro demonstrated that reverse transcriptase pausing has a significant impact on the location of the crossover, with a proportion of complete transfer events occurring very close to pause sites. The role of pausing in vivo, however, is not clearly understood. By employing a murine leukemia virus-based single-cycle infection assay, strong pausing was shown to increase the probability of recombination, as reflected in the reconstitution of green fluorescent protein expression. The infection assay results were directly correlated with the presence of strong pause sites in reverse transcriptase primer extension assays in vitro. Conversely, when pausing was diminished in vitro, without changing the sequence of the RNA template involved in recombination, there was a significant reduction in recombination in vivo. Together, these data demonstrate that reverse transcriptase pausing, as observed in vitro, directly correlates with recombination during minus-sense DNA synthesis in vivo.

Expression of neuropilin-1 by human glomerular epithelial cells in vitro and in vivo

2001 ◽  
Vol 101 (4) ◽  
pp. 439-446 ◽  
Author(s):  
Steven J. HARPER ◽  
Chang Ying XING ◽  
Cathy WHITTLE ◽  
Robin PARRY ◽  
David GILLATT ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Reverse Transcription ◽  
Target Cells ◽  
Normal Kidney ◽  
Autocrine Loop ◽  
Reverse Transcription Pcr ◽  
Neuropilin 1 ◽  
Glomerular Epithelial Cells

Vascular endothelial growth factor (VEGF) is a potent promoter of endothelial mitogenesis and of endothelial permeability. Within the kidney it is synthesized primarily in the visceral glomerular epithelial cells (vGECs); however, the role of VEGF in the glomerulus remains unknown, as does the target cell upon which it acts. Although the target cells may be those of the glomerular endothelium, there are micro-anatomical reasons why this might not be the case. This, therefore, led us to consider the possibility that glomerular VEGF may bind to the vGECs themselves. Since it has been shown that vGECs do not express the main tyrosine kinase VEGF receptors, we chose to study vGEC expression of the more recently described VEGF isoform-specific receptors, the neuropilins. The expression of mRNAs for neuropilin-1, neuropilin-2 and soluble neuropilin was studied in whole kidney, sieved glomeruli and cultured podocytes by reverse transcription-PCR, and neuropilin-1 mRNA expression in isolated single glomeruli was analysed by nested reverse transcription-PCR. The expression of neuropilin-1 protein was investigated in cultured vGECs by Western blotting and immunocytochemistry, and in normal kidney sections by immunohistochemistry. Neuropilin-1 mRNA was detected in whole kidney, single and sieved glomeruli and cultured vGECs. Neuropilin-1 protein was detected in cultured vGECs and in vGECs in normal kidney sections by immunohistochemistry. Thus the present study suggests that vGECs may have the potential to bind the VEGF that they secrete. Functional studies will be required to address the potential significance of this finding in terms of an autocrine loop or VEGF sequestration.

scholarly journals Cationic Liposomes Enhance the Rate of Transduction by a Recombinant Retroviral Vector In Vitro and In Vivo

1998 ◽  
Vol 72 (6) ◽  
pp. 4832-4840 ◽  
Author(s):  
Colin D. Porter ◽  
Katalin V. Lukacs ◽  
Gary Box ◽  
Yasuhiro Takeuchi ◽  
Mary K. L. Collins
Keyword(s):  
Fold Increase ◽  
Retroviral Vectors ◽  
Cationic Liposomes ◽  
Receptor Interaction ◽  
Target Cells ◽  
Virus Concentration ◽  
First Order ◽  
Optimal Efficiency

ABSTRACT Cationic liposomes enhanced the rate of transduction of target cells with retroviral vectors. The greatest effect was seen with the formulation DC-Chol/DOPE, which gave a 20-fold increase in initial transduction rate. This allowed an efficiency of transduction after brief exposure of target cells to virus plus liposome that could be achieved only after extensive exposure to virus alone. Enhancement with DC-Chol/DOPE was optimal when stable virion-liposome complexes were preformed. The transduction rate for complexed virus, as for virus used alone or with the polycation Polybrene, showed first-order dependence on virus concentration. Cationic liposomes, but not Polybrene, were able to mediate envelope-independent transduction, but optimal efficiency required envelope-receptor interaction. When virus complexed with DC-Chol/DOPE was used to transduce human mesothelioma xenografts, transduction was enhanced four- to fivefold compared to that for virus alone. Since the efficacy of gene therapy is dependent on the number of cells modified, which is in turn dependent upon the balance between transduction and biological clearance of the vector, the ability of cationic liposomes to form stable complexes with retroviral vectors and enhance their rate of infection is likely to be important for in vivo application.

scholarly journals Detection and Induction of Equine Infectious Anemia Virus-Specific Cytotoxic T-Lymphocyte Responses by Use of Recombinant Retroviral Vectors

1999 ◽  
Vol 73 (4) ◽  
pp. 2762-2769 ◽  
Author(s):  
S. M. Lonning ◽  
W. Zhang ◽  
S. R. Leib ◽  
T. C. McGuire
Keyword(s):  
Equine Infectious Anemia Virus ◽  
Leukemia Virus ◽  
Retroviral Vectors ◽  
Retroviral Vector ◽  
Simian Virus ◽  
Target Cells ◽  
Equine Infectious Anemia ◽  
Anemia Virus ◽  
Ctl Responses

ABSTRACT Cytotoxic T lymphocytes (CTL) appear to be critical in resolving or reducing the severity of lentivirus infections. Retroviral vectors expressing the Gag/Pr or SU protein of the lentivirus equine infectious anemia virus (EIAV) were constructed and used to evaluate EIAV-specific CTL responses in horses. Three promoters, cytomegalovirus, simian virus SV40, and Moloney murine sarcoma virus (MoMSV) long terminal repeat (LTR), were used, and there was considerable variation in their ability to direct expression of Gag/Pr and SU. Vectors expressing EIAV proteins under the direction of MoMSV LTR and using the gibbon ape leukemia virus (GALV) Env for internalization were efficient at transducing equine kidney (EK) target cells and were effective targets for EIAV-specific CTL lysis. CTL from EIAV-infected horses caused lysis of retroviral vector-transduced EK cells expressing either Gag/Pr or SU in an ELA-A-restricted manner. In contrast, lysis of recombinant vaccinia virus-infected EK cells expressing Gag/Pr and SU/TM was often non-LA-A restricted. Five horses were immunized by direct intramuscular injection with a mixture of retroviral vectors expressing Gag/Pr or SU, and one responded with EIAV-specific CTL. This result indicates that retroviral vector stimulation of CTL in horses needs to be optimized, perhaps by inclusion of appropriate cytokine genes in the constructs. However, the studies demonstrated that retroviral vector-transduced target cells were very effective for in vitro dissection of EIAV-specific CTL responses.

scholarly journals Characterization of gP85gag as an antigen recognized by Moloney leukemia virus-specific cytolytic T cell clones that function in vivo.

1985 ◽  
Vol 162 (1) ◽  
pp. 128-144 ◽  
Author(s):  
F A van der Hoorn ◽  
T Lahaye ◽  
V Müller ◽  
M A Ogle ◽  
H D Engers
Keyword(s):  
Membrane Protein ◽  
Leukemia Virus ◽  
Tumor Formation ◽  
Target Cells ◽  
Moloney Leukemia Virus ◽  
Murine Sarcoma Virus ◽  
Loss Variant ◽  
Antigen Loss

The gag membrane protein gP85gag, encoded by Moloney murine leukemia virus (M-MLV), was identified as a target molecule recognized by Moloney murine sarcoma virus--M-MLV (M-MSV--M-MLV)-specific cytolytic T lymphocyte (CTL) clones. Target cells infected with Ab-X-MLV, an M-MLV-derived mutant virus not encoding gP85gag, were not lysed by the CTL clones. The same CTL clones were shown previously to induce the destruction of M-MLV-induced tumor cells in the peritoneal cavity. We have now characterized CTL-resistant antigen-loss tumor cell variants that have lost the surface antigen, but which retain transcriptionally silent M-MLV genomes. A cloned antigen-loss variant that reverted in vitro to the CTL-susceptible phenotype reexpressed M-MLV genomes that had undergone an insertion event in the region of the viral DNA coding for the gag membrane protein. Intravenous injection of virus-specific CTL clones inhibited tumor formation in mice injected subcutaneously with M-MSV--M-MLV.

Non-Viral Vectors for Gene Delivery

2018 ◽  
Vol 9 (1) ◽  
pp. 4-11 ◽  
Author(s):  
Aparna Bansal ◽  
Himanshu
Keyword(s):  
Gene Therapy ◽  
Viral Vectors ◽  
Transfection Efficiency ◽  
Gene Transfection ◽  
Expression System ◽  
Target Cells ◽  
Specific Expression ◽  
Naked Dna

Introduction: Gene therapy has emerged out as a promising therapeutic pave for the treatment of genetic and acquired diseases. Gene transfection into target cells using naked DNA is a simple and safe approach which has been further improved by combining vectors or gene carriers. Both viral and non-viral approaches have achieved a milestone to establish this technique, but non-viral approaches have attained a significant attention because of their favourable properties like less immunotoxicity and biosafety, easy to produce with versatile surface modifications, etc. Literature is rich in evidences which revealed that undoubtedly, non–viral vectors have acquired a unique place in gene therapy but still there are number of challenges which are to be overcome to increase their effectiveness and prove them ideal gene vectors. Conclusion: To date, tissue specific expression, long lasting gene expression system, enhanced gene transfection efficiency has been achieved with improvement in delivery methods using non-viral vectors. This review mainly summarizes the various physical and chemical methods for gene transfer in vitro and in vivo.

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