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Sample records for amphotropic pseudotyped retroviral

  1. Generation of high-titer pseudotyped retroviral vectors with very broad host range.

    PubMed

    Yee, J K; Friedmann, T; Burns, J C

    1994-01-01

    Encapsidation of the VSV G protein into the virions of MoMLV-derived retroviral vectors in the absence of other VSV-encoded proteins is shown to be an efficient process, although the exact mechanism for this process is currently unclear. Unlike the conventional retroviral vectors bearing the amphotropic envelope protein, the pseudotyped virus has the ability to withstand the shearing forces encountered during ultracentrifugation. This property of the pseudotyped virus enables the generation of high-titer retroviral vector stocks and has potential application for in vivo gene therapy studies. We have found as many as four copies of a pseudotyped vector to integrate into the genome of a single cell when a high multiplicity of infection was used to infect the cells. Multiple integration events were not observed with amphotropic retroviral vectors, probably because of their low virus titers. In addition, when retroviral vectors are pseudotyped with the VSV G protein, they acquire the host range of VSV and are able to infect nonmammalian cells derived from fish, Xenopus, mosquito, and Lepidoptera. Since techniques for efficient gene transfer in some of these nonmammalian systems are not currently available, retrovirus-mediated gene transfer described here should be useful for transgenic and other genetic studies in lower vertebrate species. The inability to establish a stable cell line expressing the VSV G protein, however, limits large-scale production of the pseudotyped retroviral vectors. Generation of stable packaging cell lines for the pseudotyped retroviral vectors is a major challenge for the future. PMID:7823872

  2. Safety testing for replication-competent retrovirus associated with gibbon ape leukemia virus-pseudotyped retroviral vectors.

    PubMed

    Chen, J; Reeves, L; Cornetta, K

    2001-01-01

    The potential pathogenicity of replication-competent retroviruses (RCR) requires vigilant testing to exclude inadvertent contamination of clinical gene therapy vector products with RCR. Pseudotyped vectors using the gibbon ape leukemia virus (GALV) envelope have entered into clinical trials but specific recommendations regarding methods for screening of vector product and analysis of clinical samples have not been set forth. Unfortunately, current screening assays used for detecting amphotropic RCR are not suitable for GALV-pseudotyped RCR. We modified the extended S+/L- assay for RCR detection by using human 293 cells for virus amplification. Of five cell lines tested, 293 cells were selected because they combined a high transduction efficiency and an ability to generate RCR at high titer. After optimizing the amplification assay, a dilution of GALV virus could consistently be detected at a dilution of 10(-6). In coculture experiments, one GALV-infected cell could be consistently detected in 10(6) uninfected cells. A PCR-based assay was developed that was capable of detecting 100 copies of a GALV envelope containing plasmid diluted in 1 microg of DNA obtained from uninfected cells. PCR was also able to detect one GALV-infected cell in 10(6) uninfected cells. These assays will be suitable for testing of vector preparations and for monitoring of clinical samples from patients treated in clinical gene therapy protocols. The assays developed are similar in methodology and sensitivity to those currently used for certification of amphotropic retroviral vectors. PMID:11177543

  3. High-efficiency gene transfer into CD34+ cells with a human immunodeficiency virus type 1-based retroviral vector pseudotyped with vesicular stomatitis virus envelope glycoprotein G.

    PubMed Central

    Akkina, R K; Walton, R M; Chen, M L; Li, Q X; Planelles, V; Chen, I S

    1996-01-01

    Currently, amphotropic retroviral vectors are widely used for gene transfer into CD34+ hematopoietic progenitor cells. The relatively low levels of transduction efficiency associated with these vectors in human cells is due to low viral titers and limitations in concentrating the virus because of the inherent fragility of retroviral envelopes. Here we show that a human immunodeficiency virus type 1 (HIV-1)-based retroviral vector containing the firefly luciferase reporter gene can be pseudotyped with a broad-host-range vesicular stomatitis virus envelope glycoprotein G (VSV-G). Higher-efficiency gene transfer into CD34+ cells was achieved with a VSV-G-pseudotyped HIV-1 vector than with a vector packaged in an amphotropic envelope. Concentration of virus without loss of viral infectivity permitted a higher multiplicity of infection, with a consequent higher efficiency of gene transfer, reaching 2.8 copies per cell. These vectors also showed remarkable stability during storage at 4 degrees C for a week. In addition, there was no significant loss of titer after freezing and thawing of the stock virus. The ability of VSV-G-pseudotyped retroviral vectors to achieve a severalfold increase in levels of transduction into CD34+ cells will allow high-efficiency gene transfer into hematopoietic progenitor cells for gene therapy purposes. Furthermore, since it has now become possible to infect CD34+ cells with pseudotyped HIV-1 with a high level of efficiency in vitro, many important questions regarding the effect of HIV-1 on lineage-specific differentiation of hematopoietic progenitors can now be addressed. PMID:8642689

  4. A sensitive retroviral pseudotype assay for influenza H5N1‐neutralizing antibodies

    PubMed Central

    Temperton, Nigel J.; Hoschler, Katja; Major, Diane; Nicolson, Carolyn; Manvell, Ruth; Hien, Vo Minh; Ha, Do Quang; De Jong, Menno; Zambon, Maria; Takeuchi, Yasuhiro; Weiss, Robin A.

    2007-01-01

    Background  The World Health Organisation (WHO) recommended the development of simple, safe, sensitive and specific neutralization assays for avian influenza antibodies. We have used retroviral pseudotypes bearing influenza H5 hemagglutinin (HA) as safe, surrogate viruses for influenza neutralization assays which can be carried out at Biosafety Level 2. Results  Using our assay, sera from patients who had recovered from infection with influenza H5N1, and sera from animals experimentally immunized or infected with H5 tested positive for the presence of neutralizing antibodies to H5N1. Pseudotype neutralizing antibody titers were compared with titers obtained by hemagglutinin inhibition (HI) assays and microneutralization (MN) assays using live virus, and showed a high degree of correlation, sensitivity and specificity. Conclusions  The pseudotype neutralization assay is as sensitive as horse erythrocyte HI and MN for the detection of antibodies to H5N1. It is safer, and can be applied in a high‐throughput format for human and animal surveillance and for the evaluation of vaccines. PMID:19453415

  5. Variable regions A and B in the envelope glycoproteins of feline leukemia virus subgroup B and amphotropic murine leukemia virus interact with discrete receptor domains.

    PubMed Central

    Tailor, C S; Kabat, D

    1997-01-01

    The surface (SU) envelope glycoproteins of feline leukemia virus subgroup B (FeLV-B) and amphotropic murine leukemia virus (A-MLV) are highly related, even in the variable regions VRA and VRB that have been shown to be required for receptor recognition. However, FeLV-B and A-MLV use different sodium-dependent phosphate symporters, Pit1 and Pit2, respectively, as receptors for infection. Pit1 and Pit2 are predicted to have 10 membrane-spanning domains and five extracellular loops. The close relationship of the retroviral envelopes enabled us to generate pseudotype virions carrying chimeric FeLV-B/A-MLV envelope glycoproteins. We found that some of the pseudotype viruses could not use Pit1 or Pit2 proteins but could efficiently utilize specific chimeric Pit1/Pit2 proteins as receptors. By studying Mus dunni tail fibroblasts expressing chimeric Pit1/Pit2 proteins and pseudotype virions carrying chimeric FeLV-B/A-MLV envelopes, we show that FeLV-B and A-MLV VRA and VRB interact in a modular manner with specific receptor domains. Our results suggest that FeLV-B VRA interacts with Pit1 extracellular loops 4 and 5 and that residues Phe-60 and Pro-61 of FeLV-B VRA are essential for receptor choice. However, this interaction is insufficient for infection, and an additional interaction between FeLV-B VRB and Pit1 loop 2 is essential. Similarly, A-MLV infection requires interaction of A-MLV VRA with Pit2 loops 4 and 5 and VRB with Pit2 loop 2, with residues Tyr-60 and Val-61 of A-MLV VRA being critical for receptor recognition. Together, our results suggest that FeLV-B and A-MLV infections require two major discrete interactions between the viral SU envelope glycoproteins and their respective receptors. We propose a common two-step mechanism for interaction between retroviral envelope glycoproteins and cell surface receptors. PMID:9371598

  6. Receptor choice determinants in the envelope glycoproteins of amphotropic, xenotropic, and polytropic murine leukemia viruses.

    PubMed Central

    Battini, J L; Heard, J M; Danos, O

    1992-01-01

    The envelope glycoproteins (SU) of mammalian type C retroviruses possess an amino-terminal domain of about 200 residues, which is involved in binding a cell surface receptor. In this domain, highly conserved amino acid sequences are interrupted by two segments of variable length and sequence, VRA and VRB. We have studied the role of these variable regions in receptor recognition and binding by constructing chimeric molecules in which portions of the amino-terminal domains from amphotropic (4070A), xenotropic (NZB), and polytropic (MCF 247) murine leukemia virus SU proteins were permuted. These chimeras, which exchanged either one or two variable regions, were expressed at the surface of replication-defective viral particles by a pseudotyping assay. Wild-type or recombinant env genes were transfected into a cell line producing Moloney murine leukemia virus particles devoid of envelope glycoproteins in which a retrovirus vector genome carrying an Escherichia coli lacZ gene was packaged. The host range and sensitivity to interference of pseudotyped virions were assayed, and we observed which permutations resulted in receptor switch or loss of function. Our results indicate that the determinants of receptor choice are found within the just 120 amino acids of SU proteins. Downstream sequences contribute to the stabilization of the receptor-specific structure. PMID:1310758

  7. A Proline-Rich Motif Downstream of the Receptor Binding Domain Modulates Conformation and Fusogenicity of Murine Retroviral Envelopes

    PubMed Central

    Lavillette, Dimitri; Maurice, Marielle; Roche, Catherine; Russell, Stephen J.; Sitbon, Marc; Cosset, François-Loïc

    1998-01-01

    The entry of retroviruses into cells depends on receptor recognition by the viral envelope surface subunit SU followed by membrane fusion, which is thought to be mediated by a fusion peptide located at the amino terminus of the envelope transmembrane subunit TM. Several fusion determinants have been previously identified in murine leukemia virus (MLV) envelopes, but their functional interrelationships as well as the processes involved in fusion activation upon retroviral receptor recognition remain unelucidated. Despite both structural and functional similarities of their envelope glycoproteins, ecotropic and amphotropic MLVs display two different postbinding properties: (i) while amphotropic MLVs fuse the cells at neutral pH, penetration of ecotropic MLVs is relatively acid pH dependent and (ii) ecotropic envelopes are more efficient than amphotropic envelopes in inducing cell-to-cell fusion and syncytium formation. By exploiting the latter characteristic in the analysis of chimeras of ecotropic and amphotropic MLV envelopes, we show here that substitution of the ecotropic MLV proline-rich region (PRR), located in the SU between the amino-terminal receptor binding domain and the TM-interacting SU carboxy-terminal domains, is sufficient to revert the amphotropic low-fusogenic phenotype into a high-fusogenic one. Furthermore, we have identified potential β-turns in the PRR that control the stability of SU-TM associations as well as the thresholds required to trigger either cell-to-cell or virus-to-cell fusion. These data, demonstrating that the PRR functions as a signal which induces envelope conformational changes leading to fusion, have enabled us to derive envelopes which can infect cells harboring low levels of available amphotropic receptors. PMID:9811733

  8. Introduction of new genetic material into human myeloid leukemic blast stem cells by retroviral infection

    SciTech Connect

    Smith, L.J.; Benchimol, S.

    1988-02-01

    An amphotropic retroviral vector containing the bacterial neomycin phosphotransferase gene (neo) was used to infect blast cells from patients with acute myeloblastic leukemia. The infected cells acquired a G418-resistant phenotype that was stable as measured in a clonogenic assay and in long-term suspension culture. Thus, gene transfer into stem cells was accomplished by this procedure. This approach for manipulating gene expression in blast stem cells provides a means to assess the roles of a variety of genes in self-renewal, differentiation, and leukemogenesis.

  9. Effects of stoichiometry of retroviral components on virus production.

    PubMed

    Yap, M W; Kingsman, S M; Kingsman, A J

    2000-09-01

    A study was conducted to investigate the effects of increasing the amount of each retroviral component on vector production. It was found that, while the components of both amphotropic and ecotropic vectors were expressed independently of each other in a transient transfection system, increasing the amount of the gag/gag-pol component resulted in a decrease in virus titres for the amphotropic particles but not ecotropic particles. Analyses of the virus stocks produced indicated that the negative effect on titres was closely linked to the availability of envelope proteins for virion incorporation. The negative effect was not observed for ecotropic particle production in 293T cells, where the ecotropic receptor was absent, but was manifested when production was conducted in 293/12 cells expressing the ecotropic receptor. This suggested that the premature interaction between envelope and receptor in producer cells could limit the amount of envelope available for virion incorporation. In designing optimal vector production systems it is essential, therefore, to balance the concentration of the vector components and to ensure that there is never an excess of Gag/Gag-Pol. PMID:10950977

  10. Green Fluorescent Protein-Tagged Retroviral Envelope Protein for Analysis of Virus-Cell Interactions

    PubMed Central

    Spitzer, Dirk; Dittmar, Kurt E. J.; Rohde, Manfred; Hauser, Hansjörg; Wirth, Dagmar

    2003-01-01

    Fluorescent retroviral envelope (Env) proteins were developed for direct visualization of viral particles. By fusing the enhanced green fluorescent protein (eGFP) to the N terminus of the amphotropic 4070A envelope protein, extracellular presentation of eGFP was achieved. Viruses incorporated the modified Env protein and efficiently infected cells. We used the GFP-tagged viruses for staining retrovirus receptor-positive cells, thereby circumventing indirect labeling techniques. By generating cells which conditionally expressed the GFP-tagged Env protein, we could confirm an inverse correlation between retroviral Env expression and infectivity (superinfection). eGFP-tagged virus particles are suitable for monitoring the dynamics of virus-cell interactions. PMID:12719600

  11. Infectious Entry by Amphotropic as well as Ecotropic Murine Leukemia Viruses Occurs through an Endocytic Pathway

    PubMed Central

    Katen, Louis J.; Januszeski, Michael M.; Anderson, W. French; Hasenkrug, Kim J.; Evans, Leonard H.

    2001-01-01

    Infectious entry of enveloped viruses is thought to proceed by one of two mechanisms. pH-dependent viruses enter the cells by receptor-mediated endocytosis and are inhibited by transient treatment with agents that prevent acidification of vesicles in the endocytic pathway, while pH-independent viruses are not inhibited by such agents and are thought to enter the cell by direct fusion with the plasma membrane. Nearly all retroviruses, including amphotropic murine leukemia virus (MuLV) and human immunodeficiency virus type 1, are classified as pH independent. However, ecotropic MuLV is considered to be a pH-dependent virus. We have examined the infectious entry of ecotropic and amphotropic MuLVs and found that they were equally inhibited by NH4Cl and bafilomycin A. These agents inhibited both viruses only partially over the course of the experiments. Agents that block the acidification of endocytic vesicles also arrest vesicular trafficking. Thus, partial inhibition of the MuLVs could be the result of virus inactivation during arrest in this pathway. In support of this contention, we found that that the loss of infectivity of the MuLVs during treatment of target cells with the drugs closely corresponded to the loss of activity due to spontaneous inactivation at 37°C in the same period of time. Furthermore, the drugs had no effect on the efficiency of infection under conditions in which the duration of infection was held to a very short period to minimize the effects of spontaneous inactivation. These results indicate that the infectious processes of both ecotropic and amphotropic MuLVs were arrested rather than aborted by transient treatment of the cells with the drugs. We also found that infectious viruses were efficiently internalized during treatment. This indicated that the arrest occurred in an intracellular compartment and that the infectious process of both the amphotropic and ecotropic MuLVs very likely involved endocytosis. An important aspect of this study

  12. Pseudotyping of G-Gene-Deficient Rabies Virus.

    PubMed

    Hagendorf, Nadin; Conzelmann, Karl-Klaus

    2015-12-01

    G-deleted fluorescent rabies virus (RV) pseudotyped with RV G proteins, SAD ΔG eGFP (RV CVS-G), can be used as single-round vectors for efficient retrograde labeling of neurons. For these experiments, as well as for monosynaptic tracing, which involves pseudotyping in situ, the use of the CVS strain G is recommended because of its high tropism for neurons. Pseudotype virus stocks generated by transfection of pCAGGS-G (or in MG139-on cells) contain the G protein of the vaccine strain SAD L16, which is broader in its tropism, and infects astrocytes, glia, and oligodendrocytes. We also describe a procedure for pseudotyping with ASLV Env A, which uses a cell-line expressing a version of the EnvA protein that is incorporated efficiently into the RV envelope (EnvARG(RGct)). PMID:26631128

  13. Transduction patterns of pseudotyped lentiviral vectors in the nervous system.

    PubMed

    Wong, Liang-Fong; Azzouz, Mimoun; Walmsley, Lucy E; Askham, Zoe; Wilkes, Fraser J; Mitrophanous, Kyriacos A; Kingsman, Susan M; Mazarakis, Nicholas D

    2004-01-01

    We have developed a non-primate-based lentiviral vector based on the equine infectious anemia virus (EIAV) for efficient gene transfer to the central and peripheral nervous systems. Previously we have demonstrated that pseudotyping lentiviral vectors with the rabies virus glycoprotein confers retrograde axonal transport to these vectors. In the present study we have successfully produced high-titer EIAV vectors pseudotyped with envelope glycoproteins from Rhabdovirus vesicular stomatitis virus (VSV) serotypes (Indiana and Chandipura strains); rabies virus [various Evelyn-Rokitnicki-Abelseth ERA strains and challenge virus standard (CVS)]; Lyssavirus Mokola virus, a rabies-related virus; and Arenavirus lymphocytic choriomeningitis virus (LCMV). These vectors were delivered to the striatum or spinal cord of adult rats or muscle of neonatal mice by direct injection. We report that the lentiviral vectors pseudotyped with envelopes from the VSV Indiana strain, wild-type ERA, and CVS strains resulted in strong transduction in the striatum, while Mokola- and LCMV-pseudotyped vectors exhibited moderate and weak transduction, respectively. Furthermore ERA- and CVS-pseudotyped lentiviral vectors demonstrated retrograde transport and expression in distal neurons after injection in brain, spinal cord, and muscle. The differences in transduction efficiencies and retrograde transport conferred by these envelope glycoproteins present novel opportunities in designing therapeutic strategies for different neurological diseases. PMID:14741783

  14. Broad host range of human T-cell leukemia virus type 1 demonstrated with an improved pseudotyping system.

    PubMed Central

    Sutton, R E; Littman, D R

    1996-01-01

    Studies of human T-cell leukemia virus type 1 (HTLV-1) have been hampered by the difficulty of achieving high cell-free and cell-associated infectious titers. Current retroviral pseudotyping systems using the HTLV-1 envelope generate titers of less than 200 infectious particles per ml. We describe here an improved system for pseudotyping using a defective human immunodeficiency virus (HIV) type 1 genome in combination with HTLV-1 env in 293T producer cells. Introduction of additional copies of rev and treatment of cells with sodium butyrate resulted in a cell-associated titer of 10(5)/ml and cell-free titers of greater than 10(4)/ml . By using this system, we found that the host range of HTLV-1 is even greater than previously suspected. Earlier studies which assigned a chromosomal location for the HTLV-1 receptor may therefore reflect cell-to-cell variation in receptor number rather than the absolute presence or absence of a receptor. The generation of higher-titer HIV(HTLV-1) may facilitate identification of the cellular receptor and investigations of the pathophysiology of HTLV-1 infection. PMID:8794391

  15. Human Immunodeficiency Virus Type 1 Particles Pseudotyped with Envelope Proteins That Fuse at Low pH No Longer Require Nef for Optimal Infectivity

    PubMed Central

    Chazal, Nathalie; Singer, Gregory; Aiken, Christopher; Hammarskjöld, Marie-Louise; Rekosh, David

    2001-01-01

    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. PMID:11264394

  16. Induction of methotrexate resistance by retroviral-mediated transfer of a mutant dihydrofolate reductase gene

    SciTech Connect

    Ricciardone, M.D.

    1986-01-01

    Methotrexate (MTX), a folate analog which inhibits the enzyme dihydrofolate reductase (DHFR), is an effective antineoplastic drug. However, MTX-induced myelosuppression limits the effectiveness of this agent. Selective induction of MTX resistance in bone marrow stem cells, prior to treatment with MTX, might prevent this toxicity and improve the therapeutic index of the drug. In these studies drug resistance was transferred to mouse and human bone marrow stem cells by retroviral expression vectors containing coding sequences of a mutant DHFR with a decreased affinity for MTX. Three retroviral expression vectors were analyzed. The CIS DR vector contained the mutant DHFR gene inserted into the replication-defective amphotropic 4070 virus, Cistor. The other vectors contained the mutant DHFR inserted into either the env region (SDHT1) or gag-pol region (SDHT2) of a replication-defective spleen focus-forming virus. All three constructs induced approximately a 200-fold resistance to MTX when transfected into NIH3T3 cells. Amphotropic infectious retroviruses were obtained by transfecting the mutant DHFR vectors into a packaging cell line, which supplied the gag, pol, and env proteins for virus production. Virus titers of 4.5 x 10/sup 3/ colony-forming units (CFU)/ml (CIS DR), 1.5 x 10/sup 4/ CFU/ml (SDHT2), and 5 x 10/sup 5/ CFU/ml (SDHT1) were measured by the transfer of MTX resistance to NIH3T3 cells. The amphotropic SDHT1 virus efficiently induced MTX resistance in cells of several species, including mouse NIH3T3 cells (5 x 10/sup 5/ CFU/ml), monkey CV1 cells (4 x 10/sup 3/ CFU/ml), and human MCF-7 cells (6 x 10/sup 4/ CFU/ml). When cocultured with SDHT1 virus-producing cells, both mouse and human bone marrow cells could be infected and rendered resistant to MTX. Mouse cytotoxic T lymphocytes and mouse helper T lymphocytes can also be made resistant to MTX.

  17. Analysis of 4070A envelope levels in retroviral preparations and effect on target cell transduction efficiency.

    PubMed

    Slingsby, J H; Baban, D; Sutton, J; Esapa, M; Price, T; Kingsman, S M; Kingsman, A J; Slade, A

    2000-07-01

    A number of stable producer cell lines for high-titer Mo-MuLV vectors have been constructed. Development has previously centered on increasing end-point titers by producing maximal levels of Mo-MuLV Gag/Pol, envelope glycoproteins, and retroviral RNA genomes. We describe the production yields and transduction efficiency characteristics of two Mo-MuLV packaging cell lines, FLYA13 and TEFLYA. Although they both produce 4070A-pseudotyped retroviral vectors reproducibly at >1 x 10(6) LFU ml(-1), the transduction efficiency of unconcentrated and concentrated virus from FLYA13 lines is poor compared with vector preparations from TEFLYA lines. A powerful inhibitor of retroviral transduction is secreted by FLYA13 packaging cells. We show that the inhibitory factor does not affect transduction of target cells by RD114-pseudotyped vectors. This suggests that the inhibitory factor functions at the level of envelope-receptor interactions. Phosphate starvation of target cells shows a two-fold increase in Pit2 receptor mRNA and causes some improvement in FLYA13 virus transduction efficiency. Western blots show that FLYA13 viral samples contain an eight-fold higher ratio of 4070A envelope to p30gag than that of virus produced by TEFLYA producer cell lines. This study correlates overexpression of 4070A envelope glycoprotein in retroviral preparations with a reduction of transduction efficiency at high multiplicities of infection. We suggest that TEFLYA packaging cells express preferable levels of 4070A compared with FLYA13, which not only enables high-titer stocks to be generated, but also facilitates a high efficiency of transduction of target cells. PMID:10910141

  18. Structural modeling of carbonaceous mesophase amphotropic mixtures under uniaxial extensional flow.

    PubMed

    Golmohammadi, Mojdeh; Rey, Alejandro D

    2010-07-21

    The extended Maier-Saupe model for binary mixtures of model carbonaceous mesophases (uniaxial discotic nematogens) under externally imposed flow, formulated in previous studies [M. Golmohammadi and A. D. Rey, Liquid Crystals 36, 75 (2009); M. Golmohammadi and A. D. Rey, Entropy 10, 183 (2008)], is used to characterize the effect of uniaxial extensional flow and concentration on phase behavior and structure of these mesogenic blends. The generic thermorheological phase diagram of the single-phase binary mixture, given in terms of temperature (T) and Deborah (De) number, shows the existence of four T-De transition lines that define regions that correspond to the following quadrupolar tensor order parameter structures: (i) oblate (perpendicular, parallel), (ii) prolate (perpendicular, parallel), (iii) scalene O(perpendicular, parallel), and (iv) scalene P(perpendicular, parallel), where the symbols (perpendicular, parallel) indicate alignment of the tensor order ellipsoid with respect to the extension axis. It is found that with increasing T the dominant component of the mixture exhibits weak deviations from the well-known pure species response to uniaxial extensional flow (uniaxial perpendicular nematic-->biaxial nematic-->uniaxial parallel paranematic). In contrast, the slaved component shows a strong deviation from the pure species response. This deviation is dictated by the asymmetric viscoelastic coupling effects emanating from the dominant component. Changes in conformation (oblate <==> prolate) and orientation (perpendicular <==> parallel) are effected through changes in pairs of eigenvalues of the quadrupolar tensor order parameter. The complexity of the structural sensitivity to temperature and extensional flow is a reflection of the dual lyotropic/thermotropic nature (amphotropic nature) of the mixture and their cooperation/competition. The analysis demonstrates that the simple structures (biaxial nematic and uniaxial paranematic) observed in pure discotic

  19. Oncogenicity of human N-ras oncogene and proto-oncogene introduced into retroviral vectors

    SciTech Connect

    Souyri, M.; Vigon, I.; Charon, M.; Tambourin, P. )

    1989-09-01

    The N-ras gene is the only member of the ras family which has never been naturally transduced into a retrovirus. In order to study the in vitro and in vivo oncogenicity of N-ras and to compare its pathogenicity to that of H-ras, the authors have inserted an activated or a normal form of human N-ras cDNA into a slightly modified Harvey murine sarcoma virus-derived vector in which the H-ras p21 coding region had been deleted. The resulting constructions were transfected into NIH 3T3 cells. The activated N-ras-containing construct (HSN) induced 10{sup 4} foci per {mu}g of DNA and was found to be as transforming as H-ras was. After infection of the transfected cells by either the ecotropic Moloney murine leukemia virus or the amphotropic 4070A helper viruses, rescued transforming viruses were injected into newborn mice. Both pseudotypes of HSN virus containing activated N-ras induced the typical Harvey disease with similar latency. However, they found that the virus which contained normal N-ras p21 (HSn) was also pathogenic and induced splenomegaly, lymphadenopathies, and sarcoma in mice after a latency of 3 to 7 weeks. In addition, Moloney murine leukemia virus pseudotypes of N-ras caused neurological disorders in 30% of the infected animals. These results differed markedly from those of previous experiments in which the authors had inserted the activated form of N-ras in the pSV(X) vector: the resulting SVN-ras virus was transforming on NIH 3T3 cells but was poorly oncogenic in vivo. Altogether, these data demonstrated unequivocally that N-ras is potentially as oncogenic as H-ras and that such oncogenic effect could depend on the vector environment.

  20. Pantropic retroviral vectors integrate and express in cells of the malaria mosquito, Anopheles gambiae.

    PubMed Central

    Matsubara, T; Beeman, R W; Shike, H; Besansky, N J; Mukabayire, O; Higgs, S; James, A A; Burns, J C

    1996-01-01

    The lack of efficient mechanisms for stable genetic transformation of medically important insects, such as anopheline mosquitoes, is the single most important impediment to progress in identifying novel control strategies. Currently available techniques for foreign gene expression in insect cells in culture lack the benefit of stable inheritance conferred by integration. To overcome this problem, a new class of pantropic retroviral vectors has been developed in which the amphotropic envelope is completely replaced by the G glycoprotein of vesicular stomatitis virus. The broadened host cell range of these particles allowed successful entry, integration, and expression of heterologous genes in cultured cells of Anopheles gambiae, the principle mosquito vector responsible for the transmission of over 100 million cases of malaria each year. Mosquito cells in culture infected with a pantropic vector expressing hygromycin phosphotransferase from the Drosophila hsp70 promoter were resistant to the antibiotic hygromycin B. Integrated provirus was detected in infected mosquito cell clones grown in selective media. Thus, pantropic retroviral vectors hold promise as a transformation system for mosquitoes in vivo. Images Fig. 2 Fig. 4 Fig. 5 PMID:8650240

  1. The use of pseudotypes to study viruses, virus sero-epidemiology and vaccination.

    PubMed

    Bentley, Emma M; Mather, Stuart T; Temperton, Nigel J

    2015-06-12

    The globalization of the world's economies, accompanied by increasing international travel, changing climates, altered human behaviour and demographics is leading to the emergence of different viral diseases, many of which are highly pathogenic and hence are considered of great public and animal health importance. To undertake basic research and therapeutic development, many of these viruses require handling by highly trained staff in BSL-3/4 facilities not readily available to the majority of the global R&D community. In order to circumvent the enhanced biosafety requirement, the development of non-pathogenic, replication-defective pseudotyped viruses is an effective and established solution to permit the study of many aspects of virus biology in a low containment biosafety level (BSL)-1/2 laboratory. Under the spectre of the unfolding Ebola crisis, this timely conference (the second to be organised by the Viral Pseudotype Unit, www.viralpseudotypeunit.info*) discusses the recent advances in pseudotype technology and how it is revolutionizing the study of important human and animal pathogens (human and avian influenza viruses, rabies/lyssaviruses, HIV, Marburg and Ebola viruses). Key topics addressed in this conference include the exploitation of pseudotypes for serology and serosurveillance, immunogenicity testing of current and next-generation vaccines and new pseudotype assay formats (multiplexing, kit development). The first pseudotype-focused Euroscicon conference organised by the Viral Pseudotype Unit was recently reviewed [1]. PMID:25936665

  2. Production and neurotropism of lentivirus vectors pseudotyped with lyssavirus envelope glycoproteins.

    PubMed

    Desmaris, N; Bosch, A; Salaün, C; Petit, C; Prévost, M C; Tordo, N; Perrin, P; Schwartz, O; de Rocquigny, H; Heard, J M

    2001-08-01

    We investigated the production efficiency and the gene transfer capacity in the central nervous system of HIV-1-based vectors pseudotyped with either the G protein of the Mokola lyssaviruses (MK-G), a neurotropic virus causing rabies disease, or the vesiculo-stomatitis G protein (VSV-G). Both envelopes induced syncitia in cell cultures. They were incorporated into vector particles and mature virions were observed by electron microscopy. Vector production was two- to sixfold more efficient with VSV-G than with MK-G. For equivalent amounts of physical particles, vector titration was 5- to 25-fold higher with VSV-G than with MK-G pseudotypes on cultured cells, and in vivo gene expression in mouse brain was more intense. Thus, VSV-G pseudotypes were produced more efficiently and were more infectious than MK-G pseudotypes. Tropism for brain cells was analyzed by intrastriatal injections in rats. Both pseudotypes preferentially transduced neurons (70-90% of transduced cells). Retrograde axonal transport was investigated by instilling vector suspensions in the rat nasal cavity. Both pseudotypes were efficiently transported to olfactive neuron bodies. Thus, although coating HIV-1 particles with rabdhovirus envelope glycoproteins enables them to enter neuronal cells efficiently, pseudotyping is not sufficient to confer the powerful neurotropism of lyssaviruses to lentivirus vectors. PMID:11482987

  3. Gene targeting with retroviral vectors

    SciTech Connect

    Ellis, J.; Bernstein, A. )

    1989-04-01

    The authors have designed and constructed integration-defective retroviral vectors to explore their potential for gene targeting in mammalian cells. Two nonoverlapping deletion mutants of the bacterial neomycin resistance (neo) gene were used to detect homologous recombination events between viral and chromosomal sequences. Stable neo gene correction events were selected at a frequency of approximately 1 G418/sup r/ cell per 3 x 10/sup 6/ infected cells. Analysis of the functional neo gene in independent targeted cell clones indicated that unintegrated retroviral linear DNA recombined with the target by gene conversion for variable distances into regions of nonhomology. In addition, transient neo gene correction events which were associated with the complete loss of the chromosomal target sequences were observed. These results demonstrated that retroviral vectors can recombine with homologous chromosomal sequences in rodent and human cells.

  4. Pseudotype-Based Neutralization Assays for Influenza: A Systematic Analysis

    PubMed Central

    Carnell, George William; Ferrara, Francesca; Grehan, Keith; Thompson, Craig Peter; Temperton, Nigel James

    2015-01-01

    The use of vaccination against the influenza virus remains the most effective method of mitigating the significant morbidity and mortality caused by this virus. Antibodies elicited by currently licensed influenza vaccines are predominantly hemagglutination-inhibition (HI)-competent antibodies that target the globular head of hemagglutinin (HA) thus inhibiting influenza virus entry into target cells. These antibodies predominantly confer homosubtypic/strain specific protection and only rarely confer heterosubtypic protection. However, recent academia or pharma-led R&D toward the production of a “universal vaccine” has centered on the elicitation of antibodies directed against the stalk of the influenza HA that has been shown to confer broad protection across a range of different subtypes (H1–H16). The accurate and sensitive measurement of antibody responses elicited by these “next-generation” influenza vaccines is, however, hampered by the lack of sensitivity of the traditional influenza serological assays HI, single radial hemolysis, and microneutralization. Assays utilizing pseudotypes, chimeric viruses bearing influenza glycoproteins, have been shown to be highly efficient for the measurement of homosubtypic and heterosubtypic broadly neutralizing antibodies, making them ideal serological tools for the study of cross-protective responses against multiple influenza subtypes with pandemic potential. In this review, we will analyze and compare literature involving the production of influenza pseudotypes with particular emphasis on their use in serum antibody neutralization assays. This will enable us to establish the parameters required for optimization and propose a consensus protocol to be employed for the further deployment of these assays in influenza vaccine immunogenicity studies. PMID:25972865

  5. RETROVIRAL INTEGRASE: THEN AND NOW

    PubMed Central

    Andrake, Mark D.; Skalka, Anna Marie

    2016-01-01

    The retroviral integrases are virally encoded, specialized recombinases that catalyze the insertion of viral DNA into the host cell’s DNA, a process that is essential for virus propagation. We have learned a great deal since the existence of an integrated form of retroviral DNA (the provirus) was first proposed by Howard Temin in 1964. Initial studies focused on the genetics and biochemistry of avian and murine virus DNA integration, but the pace of discovery increased substantially with advances in technology, and an influx of investigators focused on the human immunodeficiency virus (HIV). We begin with a brief account of the scientific landscape in which some of the earliest discoveries were made, and summarize research that led to our current understanding of the biochemistry of integration. A more detailed account of recent analyses of integrase structure follows, as they have provided valuable insights into enzyme function and raised important new questions. PMID:26958915

  6. Use of recombinant lentivirus pseudotyped with vesicular stomatitis virus glycoprotein G for efficient generation of human anti-cancer chimeric T cells by transduction of human peripheral blood lymphocytes in vitro

    PubMed Central

    Simmons, Anthony; Whitehead, Robert P; Kolokoltsov, Andrey A; Davey, Robert A

    2006-01-01

    Background Genetic redirection of lymphocytes that have been genetically engineered to recognize antigens other than those originally programmed in their germlines is a potentially powerful tool for immunotherapy of cancers and potentially also of persistent viral infections. The basis for this procedure is that both cancers and some viruses have developed strikingly similar mechanisms of evading attacks by host immune mechanisms. To redirect human peripheral blood lymphocytes (PBLs) with a chimeric T cell receptor (chTCR) so that they recognize a new target requires a high degree of transfection efficiency, a process that is regarded as technically demanding. Results Infection with a retroviral vector carrying a chTCR cassette was shown to transduce 100% of rapidly dividing murine T cells but typically, only ~10% of PBLs could be infected with the same vector. In contrast with other retroviruses, lentiviruses integrate their genomes into non-dividing cells. To increase host cell range, vesicular stomatitis virus G protein was pseudotyped with a lentivirus vector, which resulted in ~100% PBL transduction efficiency. Signaling of PBLs bearing chimeric receptors was shown by specific proliferation on exposure to cells expressing cognate ligand. Further, T-bodies against CEA showed a startling abilty to cause regression of maligant colon tumors in a nude mouse model of human cancer. Conclusion A lentivirus/VSV pseudotyped virus, which does not require replicating cells for integration of its genome, efficiently transduced a high proportion of human PBLs with chTCRs against CEA. PBLs transduced by infection with a lentivirus/VSV pseudotyped vector were able to proliferate specifically in vitro on exposure to CEA-expressing cells and further they had a startling therapeutic effect in a mouse model of human colon cancer. PMID:16507098

  7. Pseudotypes of vesicular stomatitis virus with the envelope properties of mammalian and primate retroviruses.

    PubMed

    Schnitzer, T J; Weiss, R A; Zavada, J

    1977-09-01

    By employing improved techniques it has been possible to produce and characterize a representative spectrum of mammalian and primate retrovirus pseudotypes of vesicular stomatitis virus (VSV). Selection of appropriate cell lines for both the production and subsequent detection of the VSV pseudotypes has been the most important factor in permitting their demonstration. The host range for penetration of these retrovirus pseudotypes of VSV has been defined and found to differ from that reported for the replication of the corresponding retroviruses. Additionally, retroviruses having an identical host range for replication were distinguishable by differences in their host range for penetration, implying that restriction of replication may be occurring by different mechanisms. Studies of the plaque-forming efficiency of retrovirus pseudotypes of VSV in cell lines nonpermissive for replication of the corresponding retroviruses permitted a distinction to be made between the restriction of replication occurring as a consequence of postpenetration events and that occurring as a consequence of a block of penetration itself. The demonstration of primate retrovirus pseudotypes of VSV permits the use of VSV as a probe for the detection of this group of viruses. PMID:197255

  8. Continuum Theory of Retroviral Capsids

    NASA Astrophysics Data System (ADS)

    Nguyen, T. T.; Bruinsma, R. F.; Gelbart, W. M.

    2006-02-01

    We present a self-assembly phase diagram for the shape of retroviral capsids, based on continuum elasticity theory. The spontaneous curvature of the capsid proteins drives a weakly first-order transition from spherical to spherocylindrical shapes. The conical capsid shape which characterizes the HIV-1 retrovirus is never stable under unconstrained energy minimization. Only under conditions of fixed volume and/or fixed spanning length can the conical shape be a minimum energy structure. Our results indicate that, unlike the capsids of small viruses, retrovirus capsids are not uniquely determined by the molecular structure of the constituent proteins but depend in an essential way on physical constraints present during assembly.

  9. Design and selection of Toca 511 for clinical use: modified retroviral replicating vector with improved stability and gene expression.

    PubMed

    Perez, Omar D; Logg, Christopher R; Hiraoka, Kei; Diago, Oscar; Burnett, Ryan; Inagaki, Akihito; Jolson, Dawn; Amundson, Karin; Buckley, Taylor; Lohse, Dan; Lin, Amy; Burrascano, Cindy; Ibanez, Carlos; Kasahara, Noriyuki; Gruber, Harry E; Jolly, Douglas J

    2012-09-01

    Retroviral replicating vectors (RRVs) are a nonlytic alternative to oncolytic replicating viruses as anticancer agents, being selective both for dividing cells and for cells that have defects in innate immunity and interferon responsiveness. Tumor cells fit both these descriptions. Previous publications have described a prototype based on an amphotropic murine leukemia virus (MLV), encoding yeast cytosine deaminase (CD) that converts the prodrug 5-fluorocytosine (5-FC) to the potent anticancer drug, 5-fluorouracil (5-FU) in an infected tumor. We report here the selection of one lead clinical candidate based on a general design goal to optimize the genetic stability of the virus and the CD activity produced by the delivered transgene. Vectors were tested for titer, genetic stability, CD protein and enzyme activity, ability to confer susceptibility to 5-FC, and preliminary in vivo antitumor activity and stability. One vector, Toca 511, (aka T5.0002) encoding an optimized CD, shows a threefold increased specific activity in infected cells over infection with the prototype RRV and shows markedly higher genetic stability. Animal testing demonstrated that Toca 511 replicates stably in human tumor xenografts and, after 5-FC administration, causes complete regression of such xenografts. Toca 511 (vocimagene amiretrorepvec) has been taken forward to preclinical and clinical trials. PMID:22547150

  10. Selection of Novel Vesicular Stomatitis Virus Glycoprotein Variants from a Peptide Insertion Library for Enhanced Purification of Retroviral and Lentiviral Vectors

    PubMed Central

    Yu, Julie H.; Schaffer, David V.

    2006-01-01

    The introduction of new features or functions that are not present in an original protein is a significant challenge in protein engineering. For example, modifications to vesicular stomatitis virus glycoprotein (VSV-G), which is commonly used to pseudotype retroviral and lentiviral vectors for gene delivery, have been hindered by a lack of structural knowledge of the protein. We have developed a transposon-based approach that randomly incorporates designed polypeptides throughout a protein to generate saturated insertion libraries and a subsequent high-throughput selection process in mammalian cells that enables the identification of optimal insertion sites for a novel designed functionality. This method was applied to VSV-G in order to construct a comprehensive library of mutants whose combined members have a His6 tag inserted at likely every site in the original protein sequence. Selecting the library via iterative retroviral infections of mammalian cells led to the identification of several VSV-G-His6 variants that were able to package high-titer viral vectors and could be purified by Ni-nitrilotriacetic acid affinity chromatography. Column purification of vectors reduced protein and DNA impurities more than 5,000-fold and 14,000-fold, respectively, from the viral supernatant. This substantially improved purity elicited a weaker immune response in the brain, without altering the infectivity or tropism from wild-type VSV-G-pseudotyped vectors. This work applies a powerful new tool for protein engineering to construct novel viral envelope variants that can greatly improve the safety and use of retroviral and lentiviral vectors for clinical gene therapy. Furthermore, this approach of library generation and selection can readily be extended to other challenges in protein engineering. PMID:16537595

  11. Investigating antibody neutralization of lyssaviruses using lentiviral pseudotypes: a cross-species comparison.

    PubMed

    Wright, Edward; Temperton, Nigel J; Marston, Denise A; McElhinney, Lorraine M; Fooks, Anthony R; Weiss, Robin A

    2008-09-01

    Cross-neutralization between rabies virus (RABV) and two European bat lyssaviruses (EBLV-1 and -2) was analysed using lentiviral pseudotypes as antigen vectors. Glycoprotein (G-protein) cDNA from RABV challenge virus standard-11 (CVS-11) and EBLV-1 and -2 were cloned and co-expressed with human immunodeficiency virus (HIV) or murine leukemia virus (MLV) gag-pol and packageable green fluorescent protein (GFP) or luciferase reporter genes in human cells. The harvested lentiviral (HIV) vector infected over 40% of baby hamster kidney (BHK) target cells, providing high-titre pseudotype stocks. Tests on blinded antibody-positive (n=15) and -negative (n=45) sera, predetermined by the fluorescent antibody virus neutralization (FAVN) test approved by the World Health Organization (WHO) and Office International des Epizooties (OIE), revealed that the CVS-11 pseudotype assay had 100% concordance with FAVN and strongly correlated with neutralization titres (r2=0.89). Cross-neutralization tests using sera from RABV-vaccinated humans and animals on pseudotypes with CVS-11, EBLV-1 and EBLV-2 envelopes showed that the relative neutralization titres correlated broadly with the degree of G-protein diversity. Pseudotypes have three major advantages over live-virus neutralization tests: (i) they can be handled in low-biohazard-level laboratories; (ii) the use of reporter genes such as GFP or beta-galactosidase will allow the assay to be undertaken at low cost in laboratories worldwide; (iii) each assay requires <10 microl serum. This robust microassay will improve our understanding of the protective humoral immunity that current rabies vaccines confer against emerging lyssaviruses, and will be applicable to surveillance studies, thus helping to control the spread of rabies. PMID:18753230

  12. Rabies-virus-glycoprotein-pseudotyped recombinant baculovirus vaccine confers complete protection against lethal rabies virus challenge in a mouse model.

    PubMed

    Wu, Qunfeng; Yu, Fulai; Xu, Jinfang; Li, Yang; Chen, Huanchun; Xiao, Shaobo; Fu, Zhen F; Fang, Liurong

    2014-06-25

    Rabies virus has been an ongoing threat to humans and animals. Here, we developed a new strategy to generate a rabies virus vaccine based on a pseudotyped baculovirus. The recombinant baculovirus (BV-RVG/RVG) was pseudotyped with the rabies virus glycoprotein (RVG) and also simultaneously expressed another RVG under the control of the immediate early CMV promoter. In vitro, this RVG-pseudotyped baculovirus vector induced syncytium formation in insect cells and displayed more efficient gene delivery into mammalian cells. Mice immunized with BV-RVG/RVG developed higher levels of virus-neutralizing antibodies, and conferred 100% protection against rabies viral challenge. These data indicate that the RVG-pseudotyped baculovirus BV-RVG/RVG can be used as an alternative strategy to develop a safe and efficacious vaccine against the rabies virus. PMID:24793501

  13. Package of NDV-Pseudotyped HIV-Luc Virus and Its Application in the Neutralization Assay for NDV Infection

    PubMed Central

    Liu, Peixin; Li, Tao; Si, Wei; Xiu, Jinsheng; Liu, Henggui

    2014-01-01

    Newcastle disease virus (NDV) is a member of the Paramyxovirinae subfamily and can infect most species of birds. It has been a great threat for the poultry industry all around the world. In this report, we successfully produced infectious pseudotyped pNL4-3-Luc-R−E− (HIV-Luc) viruses with the HN and F envelope proteins of NDV. Further investigation revealed the cytoplasmic domains of HN and F, especially HN, plays a significant role in the infection efficiency of these pseudotyped HIV-Luc viruses. Replacement of, or direct fusion to the cytoplasmic domain of the HN protein by that of vesicular stomatitis virus G (VSV-G) could greatly enhance or destroy the infective potential of HN and F-pseudotyped (NDV-pseudotyped) HIV-Luc virus. We further established a novel neutralization assay to evaluate neutralizing antibodies against NDV with the NDV-pseudotyped HIV-Luc viruses. Comparative neutralization data indicate that the results determined by using the NDV-pseudotyped HIV-Luc viruses are as reliable as those by the conventional virus-neutralization assay (VN test) with native NDV. Moreover, the results show that the novel neutralization assay is more sensitive than the VN test. PMID:24937158

  14. Identification of entry inhibitors of Ebola virus pseudotyped vectors from a myxobacterial compound library.

    PubMed

    Beck, Simon; Henß, Lisa; Weidner, Tatjana; Herrmann, Jennifer; Müller, Rolf; Chao, Yu-Kai; Grimm, Christian; Weber, Christopher; Sliva, Katja; Schnierle, Barbara S

    2016-08-01

    Myxobacteria produce secondary metabolites many of which were described to have various biological effects including anti-fungal, anti-bacterial and anti-viral activity. The majority of these metabolites are novel scaffolds with unique modes-of-action and hence might be potential leads for drug discovery. Here, we tested a myxobacterial natural product library for compounds with inhibitory activity against Ebola virus (EBOV). The assay was performed with a surrogate system using Ebola envelope glycoprotein (GP) pseudotyped lentiviral vectors. EBOV specificity was proven by counter-screening with vesicular stomatitis virus G protein pseudotyped vectors. Two compounds were identified that preferentially inhibited EBOV GP mediated cell entry: Chondramides that act on the actin skeleton but might be too toxic and noricumazole A, a potassium channel inhibitor, which might constitute a novel pathway to inhibit Ebola virus cell entry. PMID:27241689

  15. Altering Entry Site Preference of Lentiviral Vectors into Neuronal Cells by Pseudotyping with Envelope Glycoproteins.

    PubMed

    Kobayashi, Kenta; Kato, Shigeki; Inoue, Ken-Ichi; Takada, Masahiko; Kobayashi, Kazuto

    2016-01-01

    A lentiviral vector system provides a powerful strategy for gene therapy trials against a variety of neurological and neurodegenerative disorders. Pseudotyping of lentiviral vectors with different envelope glycoproteins not only confers the neurotropism to the vectors, but also alters the preference of sites of vector entry into neuronal cells. One major group of lentiviral vectors is a pseudotype with vesicular stomatitis virus glycoprotein (VSV-G) that enters preferentially cell body areas (somata/dendrites) of neurons and transduces them. Another group contains lentiviral vectors pseudotyped with fusion envelope glycoproteins composed of different sets of rabies virus glycoprotein and VSV-G segments that enter predominantly axon terminals of neurons and are transported through axons retrogradely to their cell bodies, resulting in enhanced retrograde gene transfer. This retrograde gene transfer takes a considerable advantage of delivering the transgene into neuronal cell bodies situated in regions distant from the injection site of the vectors. The rational use of these two vector groups characterized by different entry mechanisms will further extend the strategy for gene therapy of neurological and neurodegenerative disorders. PMID:26611586

  16. Pseudotypes of human T-cell leukemia virus types 1 and 2: neutralization by patients' sera.

    PubMed Central

    Clapham, P; Nagy, K; Weiss, R A

    1984-01-01

    Pseudotypes of vesicular stomatitis virus (VSV) bearing envelope antigens of human T-cell leukemia virus (HTLV) types 1 and 2 were prepared by propagating VSV in cells lines productively infected with HTLV. Plaque assays of VSV (HTLV) pseudotypes were employed to determine the presence of (i) HTLV receptors on cells and (ii) neutralizing antibodies in the serum of patients with adult T-cell leukemia-lymphoma (ATLL). Cell surface receptors for HTLV-1 and HTLV-2 were found on nonlymphoid cells of human and mammalian origin. Neutralizing antibodies specific to VSV(HTLV-1) were found in sera of ATLL patients in titers varying from 1:50 to 1:30,000 and did not correlate closely with antibody titers for internal viral antigens. Sera from ATLL patients in the United Kingdom (Caribbean immigrants), United States, and Japan completely neutralized VSV (HTLV-1), indicating that the HTLV isolates from these distinct geographic regions represent a single envelope serotype. Neutralization of VSV (HTLV-1) was more specific and more sensitive than assays of syncytium inhibition. No cross-neutralization was observed between bovine leukosis virus and HTLV, and only limited cross-reaction was found for envelope antigens of HTLV-1 and HTLV-2. These studies show that VSV (HTLV) pseudotypes can be readily used to screen for neutralizing antibodies in patients' sera and to distinguish HTLV envelope serotypes. PMID:6326149

  17. A recombinant pseudotyped lentivirus expressing the envelope glycoprotein of Hantaan virus induced protective immunity in mice

    PubMed Central

    2013-01-01

    Background Hantaviruses cause acute hemorrhagic fever with renal syndrome (HFRS). Currently, several types of inactivated HFRS vaccines are widely used, however the limited ability of these immunogen to elicit neutralizing antibodies restricts vaccine efficacy. Development of an effective vaccine to overcome this weakness is must. Methods In the present study, a recombinant pseudotyped lentivirus bearing the hantaan virus (HTNV) envelope glycoproteins (GP), rLV-M, was constructed. C57BL/6 mice were immunized with the rLV-M and a series of immunological assays were conducted to determine the immunogenicity of the recombinant pseudotyped lentivirus. The humoral and cell-mediated immune responses induced by rLV-M were compared with those of the inactivated HFRS vaccine. Results Indirect immunofluorescence assay (IFA) showed the rLV-M expressed target proteins in HEK-293cells. In mice, the rLV-M efficiently induced GP-specific humoral responses and protection against HTNV infection. Furthermore, the rLV-M induced higher neutralizing antibody titers than the inactivated HFRS vaccine control. Conclusions The results indicated the potential of using a pseudotyped lentivirus as a delivery vector for a hantavirus vaccine immunogen. PMID:24093752

  18. Effect of cell lysates on retroviral transduction efficiency of cells in suspension culture.

    PubMed

    Beauchesne, Pascal R; Bruce, Katherine J; Bowen, Bruce D; Piret, James M

    2010-04-15

    Recombinant retroviruses are effective vectors able to integrate transgenes into the target cell's genome to achieve longer-term expression. This study investigates the effect of cell lysis products, a common cell culture by-product, on the transduction of suspension cells by gammaretroviral vectors. Cell lysates derived from human and murine suspension cell lines significantly increased the transduction of human TF-1 and K-562 cell lines by gibbon ape leukemia virus-pseudotyped retroviral vectors without altering tropism. The transduction efficiency of TF-1 cells increased as a function of lysate concentration and decreased with increasing target cell concentrations. This was adequately predicted using a saturation equation based on the lysed-to-target cell concentration ratio, R, where: Fold increase = 1+Fold_(Max) (R/(K_(L)+R)). Lysate completely masked the effects of fibronectin when the two were added in combination. With protamine sulfate, the transduction efficiency was increased by lysate to 58% from 20% for protamine sulfate alone. Overall, the presence of cell lysate significantly influenced the outcome of the transduction process, either alone or in the presence of protamine sulfate or fibronectin. PMID:20014140

  19. Studies of retroviral infection in humanized mice

    PubMed Central

    Marsden, Matthew D.; Zack, Jerome A.

    2015-01-01

    Many important aspects of human retroviral infections cannot be fully evaluated using only in vitro systems or unmodified animal models. An alternative approach involves the use of humanized mice, which consist of immunodeficient mice that have been transplanted with human cells and/or tissues. Certain humanized mouse models can support robust infection with human retroviruses including different strains of human immunodeficiency virus (HIV) and human T cell leukemia virus (HTLV). These models have provided wide-ranging insights into retroviral biology, including detailed information on primary infection, in vivo replication and pathogenesis, latent/persistent reservoir formation, and novel therapeutic interventions. Here we describe the humanized mouse models that are most commonly utilized to study retroviral infections, and outline some of the important discoveries that these models have produced during several decades of intensive research. PMID:25680625

  20. Bats and Rodents Shape Mammalian Retroviral Phylogeny.

    PubMed

    Cui, Jie; Tachedjian, Gilda; Wang, Lin-Fa

    2015-01-01

    Endogenous retroviruses (ERVs) represent past retroviral infections and accordingly can provide an ideal framework to infer virus-host interaction over their evolutionary history. In this study, we target high quality Pol sequences from 7,994 Class I and 8,119 Class II ERVs from 69 mammalian genomes and surprisingly find that retroviruses harbored by bats and rodents combined occupy the major phylogenetic diversity of both classes. By analyzing transmission patterns of 30 well-defined ERV clades, we corroborate the previously published observation that rodents are more competent as originators of mammalian retroviruses and reveal that bats are more capable of receiving retroviruses from non-bat mammalian origins. The powerful retroviral hosting ability of bats is further supported by a detailed analysis revealing that the novel bat gammaretrovirus, Rhinolophus ferrumequinum retrovirus, likely originated from tree shrews. Taken together, this study advances our understanding of host-shaped mammalian retroviral evolution in general. PMID:26548564

  1. Bats and Rodents Shape Mammalian Retroviral Phylogeny

    PubMed Central

    Cui, Jie; Tachedjian, Gilda; Wang, Lin-Fa

    2015-01-01

    Endogenous retroviruses (ERVs) represent past retroviral infections and accordingly can provide an ideal framework to infer virus-host interaction over their evolutionary history. In this study, we target high quality Pol sequences from 7,994 Class I and 8,119 Class II ERVs from 69 mammalian genomes and surprisingly find that retroviruses harbored by bats and rodents combined occupy the major phylogenetic diversity of both classes. By analyzing transmission patterns of 30 well-defined ERV clades, we corroborate the previously published observation that rodents are more competent as originators of mammalian retroviruses and reveal that bats are more capable of receiving retroviruses from non-bat mammalian origins. The powerful retroviral hosting ability of bats is further supported by a detailed analysis revealing that the novel bat gammaretrovirus, Rhinolophus ferrumequinum retrovirus, likely originated from tree shrews. Taken together, this study advances our understanding of host-shaped mammalian retroviral evolution in general. PMID:26548564

  2. A replication-competent retrovirus arising from a split-function packaging cell line was generated by recombination events between the vector, one of the packaging constructs, and endogenous retroviral sequences.

    PubMed

    Chong, H; Starkey, W; Vile, R G

    1998-04-01

    Previously we reported the presence of a replication-competent retrovirus in supernatant from a vector-producing line derived from a widely used split-function amphotropic packaging cell line. Rigorous routine screening of all retroviral stocks produced in our laboratory has not, previously or since, indicated the presence of such a virus. Replication-competent retroviruses have never previously been used in our laboratory, and stringent screening of all routinely used cell lines has not revealed the presence of any helper viruses. Therefore, it is highly unlikely that this virus represents an adventitious cross-contaminant or had been imported unknowingly with our cell line stocks. PCR studies with DNA from infected cell lines and Northern blot analysis and reverse transcriptase PCR with RNA from infected cells suggest that the helper virus arose by recombination events, at sites of partial homology, between sequences in the vector, one of the packaging constructs, and endogenous retroviral elements. These recombinations were not present in stocks of the packaging cell line or in an initial stock of the vector-producing line, indicating that these events occurred while the vector-producing line was being passaged for harvest of supernatant stocks. PMID:9525583

  3. Ultrasound-Targeted Retroviral Gene Delivery

    NASA Astrophysics Data System (ADS)

    Taylor, Sarah L.; Rahim, Ahad A.; Bush, Nigel L.; Bamber, Jeffrey C.; Porter, Colin D.

    2007-05-01

    This study demonstrates the ability of focused ultrasound to target retroviral gene delivery. Key to our experiments was the use of non-infectious virus particles lacking the envelope protein required for receptor-mediated entry. The novelty of our approach is that spatial control at a distance is exerted upon viral delivery by subsequent exposure to ultrasound, leading to stable gene delivery. The technology is ideally suited to controlling gene delivery in vivo following systemic vector administration. Our data provide a solution to the critical issue of obtaining tissue specificity with retroviral vectors and impart stability of expression to ultrasound-mediated gene delivery.

  4. A robust lentiviral pseudotype neutralisation assay for in-field serosurveillance of rabies and lyssaviruses in Africa.

    PubMed

    Wright, Edward; McNabb, Suzanne; Goddard, Trudy; Horton, Daniel L; Lembo, Tiziana; Nel, Louis H; Weiss, Robin A; Cleaveland, Sarah; Fooks, Anthony R

    2009-11-27

    The inflexibility of existing serological techniques for detection of rabies in surveillance constrains the benefit to be gained from many current control strategies. We analysed 304 serum samples from Tanzanian dogs for the detection of rabies antibodies in a pseudotype assay using lentiviral vectors bearing the CVS-11 envelope glycoprotein. Compared with the widely used gold standard fluorescent antibody virus neutralisation assay, a specificity of 100% and sensitivity of 94.4% with a strong correlation of antibody titres (r=0.915) were observed with the pseudotype assay. To increase the assay's surveillance specificity in Africa we incorporated the envelope glycoprotein of local viruses, Lagos bat virus, Duvenhage virus or Mokola virus and also cloned the lacZ gene to provide a reporter element. Neutralisation assays using pseudotypes bearing these glycoproteins reveal that they provide a greater sensitivity compared to similar live virus assays and will therefore allow a more accurate determination of the distribution of these highly pathogenic infections and the threat they pose to human health. Importantly, the CVS-11 pseudotypes were highly stable during freeze-thaw cycles and storage at room temperature. These results suggest the proposed pseudotype assay is a suitable option for undertaking lyssavirus serosurveillance in areas most affected by these infections. PMID:19925950

  5. Immunogenicity and efficacy of immunodeficiency virus-like particles pseudotyped with the G protein of vesicular stomatitis virus

    SciTech Connect

    Kuate, Seraphin; Stahl-Hennig, Christiane; Stoiber, Heribert; Nchinda, Godwin; Floto, Anja; Franz, Monika; Sauermann, Ulrike; Bredl, Simon; Deml, Ludwig; Ignatius, Ralf; Norley, Steve; Racz, Paul; Tenner-Racz, Klara; Steinman, Ralph M.; Wagner, Ralf; Uberla, Klaus . E-mail: klaus.ueberla@ruhr-uni-bochum.de

    2006-07-20

    Vaccination with exogenous antigens such as recombinant viral proteins, immunodeficiency virus-derived whole inactivated virus particles, or virus-like particles (VLP) has generally failed to provide sufficient protection in animal models for AIDS. Pseudotyping VLPs with the vesicular stomatitis virus G protein (VSV-G), which is known to mediate entry into dendritic cells, might allow more efficient stimulation of immune responses. Therefore, we pseudotyped noninfectious immunodeficiency virus-like particles with VSV-G and carried out a preliminary screen of their immunogenicity and vaccination efficacy. Incorporation of VSV-G into HIV-1 VLPs led to hundred-fold higher antibody titers to HIV-1 Gag and enhancement of T cell responses in mice. Repeated vaccination of rhesus monkeys for 65 weeks with VSV-G pseudotyped simian immunodeficiency virus (SIV)-like particles (VLP[G]) provided initial evidence for efficient suppression of viral load after mucosal challenge with the SIVmac239 virus. Challenge of monkeys after a 28 week vaccination regimen with VLP[G] led to a reduction in peak viremia, but persistent suppression of viral load was not achieved. Due to limitations in the number of animals available for this study, improved efficacy of VSV-G pseudotyped VLPs in nonhuman primates could not be demonstrated. However, mouse experiments revealed that pseudotyping of VLPs with fusion-competent VSV-G clearly improves their immunogenicity. Additional strategies, particularly adjuvants, should be considered to provide greater protection against a challenge with pathogenic immunodeficiency virus.

  6. Cleavage of Hemagglutinin-Bearing Lentiviral Pseudotypes and Their Use in the Study of Influenza Virus Persistence

    PubMed Central

    Sawoo, Olivier; Dublineau, Amélie; Batéjat, Christophe; Zhou, Paul; Manuguerra, Jean-Claude; Leclercq, India

    2014-01-01

    Influenza A viruses (IAVs) are a major cause of infectious respiratory human diseases and their transmission is dependent upon the environment. However, the role of environmental factors on IAV survival outside the host still raises many questions. In this study, we used lentiviral pseudotypes to study the influence of the hemagglutinin protein in IAV survival. High-titered and cleaved influenza-based lentiviral pseudoparticles, through the use of a combination of two proteases (HAT and TMPRSS2) were produced. Pseudoparticles bearing hemagglutinin proteins derived from different H1N1, H3N2 and H5N1 IAV strains were subjected to various environmental parameters over time and tested for viability through single-cycle infectivity assays. We showed that pseudotypes with different HAs have different persistence profiles in water as previously shown with IAVs. Our results also showed that pseudotypes derived from H1N1 pandemic virus survived longer than those derived from seasonal H1N1 virus from 1999, at high temperature and salinity, as previously shown with their viral counterparts. Similarly, increasing temperature and salinity had a negative effect on the survival of the H3N2 and H5N1 pseudotypes. These results showed that pseudotypes with the same lentiviral core, but which differ in their surface glycoproteins, survived differently outside the host, suggesting a role for the HA in virus stability. PMID:25166303

  7. PEGylation of a Vesicular Stomatitis Virus G Pseudotyped Lentivirus Vector Prevents Inactivation in Serum

    PubMed Central

    Croyle, Maria A.; Callahan, Shellie M.; Auricchio, Alberto; Schumer, Gregg; Linse, Klause D.; Wilson, James M.; Brunner, Lane J.; Kobinger, Gary P.

    2004-01-01

    One disadvantage of vesicular stomatitis virus G (VSV-G) pseudotyped lentivirus vectors for clinical application is inactivation of the vector by human serum complement. To prevent this, monomethoxypoly(ethylene) glycol was conjugated to a VSV-G-human immunodeficiency virus vector expressing Escherichia coli beta-galactosidase. The modification did not affect transduction efficiency in vitro and protected the vector from inactivation in complement-active human and mouse sera. Blood from mice dosed intravenously with either the unmodified or the PEGylated virus particles was assayed for active vector by a limiting-dilution assay to evaluate transduction efficiency and for p24, an indicator of the total number of virus particles present. PEGylation extended the circulation half-life of active vector by a factor of 5 and reduced the rate of vector inactivation in the serum by a factor of 1,000. Pharmacokinetic profiles for the total number of virus particles present in the circulation were unaffected by PEGylation. Modification of the vector with poly(ethylene) glycol significantly enhanced transduction efficiency in the bone marrow and in the spleen 14 days after systemic administration of the virus. These results, in concert with the pharmacokinetic profiles, indicate that PEGylation does protect the virus from inactivation in the serum and, as a result, improves the transduction efficiency of VSV-G pseudotyped lentivirus vectors in susceptible organs in vivo. PMID:14694122

  8. Molecular Architecture of the Retroviral Capsid.

    PubMed

    Perilla, Juan R; Gronenborn, Angela M

    2016-05-01

    Retroviral capsid cores are proteinaceous containers that self-assemble to encase the viral genome and a handful of proteins that promote infection. Their function is to protect and aid in the delivery of viral genes to the nucleus of the host, and, in many cases, infection pathways are influenced by capsid-cellular interactions. From a mathematical perspective, capsid cores are polyhedral cages and, as such, follow well-defined geometric rules. However, marked morphological differences in shapes exist, depending on virus type. Given the specific roles of capsid in the viral life cycle, the availability of detailed molecular structures, particularly at assembly interfaces, opens novel avenues for targeted drug development against these pathogens. Here, we summarize recent advances in the structure and understanding of retroviral capsid, with particular emphasis on assemblies and the capsid cores. PMID:27039020

  9. Retroviral Transduction of Murine Primary T Lymphocytes

    PubMed Central

    Lee, James; Sadelain, Michel; Brentjens, Renier

    2016-01-01

    Summary In comparison to human T cells, efficient retroviral gene transfer and subsequent expansion of murine primary T cells is more difficult to achieve. Herein, we describe an optimized gene transfer protocol utilizing an ecotropic viral vector to transduce primary murine T cells activated with magnetic beads coated with agonistic anti-CD3 and CD28 antibodies. Activated T cells are subsequently centrifuged (spinoculated) on RetroNectin-coated tissue culture plates in the context of retroviral supernatant. Variables found to be critical to high gene transfer and subsequent efficient T cell expansion included CD3/CD28 magnetic bead to cell ratio, time from T cell activation to initial spinoculation, frequency of T cell spinoculation, interleukin-2 concentration in the medium, and the initial purity of the T cell preparation. PMID:19110621

  10. Retroviral DNA Transposition: Themes and Variations

    PubMed Central

    Skalka, Anna Marie

    2015-01-01

    SUMMARY Retroviruses and LTR retrotransposons are transposable elements that encapsidate the RNAs that are intermediates in the transposition of DNA copies of their genomes (proviruses), from one cell (or one locus) to another. Mechanistic similarities in DNA transposase enzymes and retroviral/retrotransposon integrases underscore the close evolutionary relationship among these elements. The retroviruses are very ancient infectious agents, presumed to have evolved from Ty3/Gypsy LTR retrotransposons (1), and DNA copies of their sequences can be found embedded in the genomes of most, if not all, members of the tree of life. All retroviruses share a specific gene arrangement and similar replication strategies. However, given their ancestries and occupation of diverse evolutionary niches, it should not be surprising that unique sequences have been acquired in some retroviral genomes and that the details of the mechanism by which their transposition is accomplished can vary. While every step in the retrovirus lifecycle is, in some sense, relevant to transposition, this Chapter focuses mainly on the early phase of retroviral replication, during which viral DNA is synthesized and integrated into its host genome. Some of the initial studies that set the stage for current understanding are highlighted, as well as more recent findings obtained through use of an ever-expanding technological toolbox including genomics, proteomics, and siRNA screening. Persistence in the area of structural biology has provided new insight into conserved mechanisms as well as variations in detail among retroviruses, which can also be instructive. PMID:25844274

  11. The design of artificial retroviral restriction factors

    SciTech Connect

    Yap, Melvyn W.; Mortuza, Gulnahar B.; Taylor, Ian A.; Stoye, Jonathan P.

    2007-09-01

    In addition to the ability to bind the retroviral capsid protein, the retroviral restriction factors Fv1, Trim5{alpha} and Trim5-CypA share the common property of containing sequences that promote self-association. Otherwise Fv1 and Trim5{alpha} appear unrelated. Mutational analyses showed that restriction was invariably lost when changes designed to disrupt the sequences responsible for multimerization were introduced. A novel restriction protein could be obtained by substituting sequences from the self-associating domain of Fv1 for the Trim5 sequences in Trim5-CypA. Similarly, a fusion protein containing cyclophilin A joined to arfaptin2, a protein known to form extended dimers, was also shown to restrict HIV-1. Hence, multimerization of a capsid-binding domain could be the common minimum design feature for capsid-dependent retroviral restriction factors. However, not all domains that promote multimerization can substitute for the N-terminal domains of Fv1 and Trim5{alpha}. Moreover, only CypA can provide a capsid-binding site with different N-terminal domains. It is suggested that the spatial relationship between the multiple target binding sites may be important for restriction.

  12. Targeted gene transfer to lymphocytes using murine leukaemia virus vectors pseudotyped with spleen necrosis virus envelope proteins.

    PubMed

    Engelstädter, M; Buchholz, C J; Bobkova, M; Steidl, S; Merget-Millitzer, H; Willemsen, R A; Stitz, J; Cichutek, K

    2001-08-01

    In contrast to murine leukaemia virus (MLV)-derived vector systems, vector particles derived from the avian spleen necrosis virus (SNV) have been successfully targeted to subsets of human cells by envelope modification with antibody fragments (scFv). However, an in vivo application of the SNV vector system in gene transfer protocols is hampered by its lack of resistance against human complement. To overcome this limitation we established pseudotyping of MLV vector particles produced in human packaging cell lines with the SNV envelope (Env) protein. Three variants of SNV Env proteins differing in the length of their cytoplasmic domains were all efficiently incorporated into MLV core particles. These pseudotype particles infected the SNV permissive cell line D17 at titers of up to 10(5) IU/ml. A stable packaging cell line (MS4) of human origin released MLV(SNV) pseudotype vectors that were resistant against human complement inactivation. To redirect their tropism to human T cells, MS4 cells were transfected with the expression gene encoding the scFv 7A5 in fusion with the transmembrane domain (TM) of the SNV Env protein, previously shown to retarget SNV vector particles to human lymphocytes. MLV(SNV-7A5)-vector particles released from these cells were selectively infectious for human T cell lines. The data provide a proof of principle for targeting MLV-derived vectors to subpopulations of human cells through pseudotyping with SNV targeting envelopes. PMID:11509952

  13. Factors that influence VSV-G pseudotyping and transduction efficiency of lentiviral vectors-in vitro and in vivo implications.

    PubMed

    Farley, Daniel C; Iqball, Sharifah; Smith, Joanne C; Miskin, James E; Kingsman, Susan M; Mitrophanous, Kyriacos A

    2007-05-01

    Pseudotyping viral vectors with vesicular stomatitis virus glycoprotein (VSV-G) enables the transduction of an extensive range of cell types from different species. We have discovered two important parameters of the VSV-G-pseudotyping phenomenon that relate directly to the transduction potential of lentiviral vectors: (1) the glycosylation status of VSV-G, and (2) the quantity of glycoprotein associated with virions. We measured production-cell and virion-associated quantities of two isoform variants of VSV-G, which differ in their glycosylation status, VSV-G1 and VSV-G2, and assessed the impact of this difference on the efficiency of mammalian cell transduction by lentiviral vectors. The glycosylation of VSV-G at N336 allowed greater maximal expression of VSV-G in HEK293T cells, thus facilitating vector pseudotyping. The transduction of primate cell lines was substantially affected (up to 50-fold) by the degree of VSV-G1 or VSV-G2 incorporation, whereas other cell lines, such as D17 (canine), were less sensitive to virion-associated VSV-G1/2 quantities. These data indicate that the minimum required concentration of virion-associated VSV-G differs substantially between cell species/types. The implications of these data with regard to VSV-G-pseudotyped vector production, titration, and use in host-cell restriction studies, are discussed. PMID:17366519

  14. Rabies virus glycoprotein pseudotyping of lentiviral vectors enables retrograde axonal transport and access to the nervous system after peripheral delivery.

    PubMed

    Mazarakis, N D; Azzouz, M; Rohll, J B; Ellard, F M; Wilkes, F J; Olsen, A L; Carter, E E; Barber, R D; Baban, D F; Kingsman, S M; Kingsman, A J; O'Malley, K; Mitrophanous, K A

    2001-09-15

    In this report it is demonstrated for the first time that rabies-G envelope of the rabies virus is sufficient to confer retrograde axonal transport to a heterologous virus/vector. After delivery of rabies-G pseudotyped equine infectious anaemia virus (EIAV) based vectors encoding a marker gene to the rat striatum, neurons in regions distal from but projecting to the injection site, such as the dopaminergic neurons of the substantia nigra pars compacta, become transduced. This retrograde transport to appropriate distal neurons was also demonstrated after delivery to substantia nigra, hippocampus and spinal cord and did not occur when vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped vectors were delivered to these sites. In addition, peripheral administration of rabies-G pseudotyped vectors to the rat gastrocnemius muscle leads to gene transfer in motoneurons of lumbar spinal cord. In contrast the same vector pseudotyped with VSV-G transduced muscle cells surrounding the injection site, but did not result in expression in any cells in the spinal cord. Long-term expression was observed after gene transfer in the nervous system and a minimal immune response which, together with the possibility of non-invasive administration, greatly extends the utility of lentiviral vectors for gene therapy of human neurological disease. PMID:11590128

  15. Analysis of the entry mechanism of Crimean-Congo hemorrhagic fever virus, using a vesicular stomatitis virus pseudotyping system.

    PubMed

    Suda, Yuto; Fukushi, Shuetsu; Tani, Hideki; Murakami, Shin; Saijo, Masayuki; Horimoto, Taisuke; Shimojima, Masayuki

    2016-06-01

    Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne disease causing severe hemorrhagic symptoms with a nearly 30 % case-fatality rate in humans. The experimental use of CCHF virus (CCHFV), which causes CCHF, requires high-biosafety-level (BSL) containment. In contrast, pseudotyping of various viral glycoproteins (GPs) onto vesicular stomatitis virus (VSV) can be used in facilities with lower BSL containment, and this has facilitated studies on the viral entry mechanism and the measurement of neutralizing activity, especially for highly pathogenic viruses. In the present study, we generated high titers of pseudotyped VSV bearing the CCHFV envelope GP and analyzed the mechanisms involved in CCHFV infection. A partial deletion of the CCHFV GP cytoplasmic domain increased the titer of the pseudotyped VSV, the entry mechanism of which was dependent on the CCHFV envelope GP. Using the pseudotype virus, DC-SIGN (a calcium-dependent [C-type] lectin cell-surface molecule) was revealed to enhance viral infection and act as an entry factor for CCHFV. PMID:26935918

  16. Effect of gamma radiation on retroviral recombination.

    PubMed

    Hu, W S; Temin, H M

    1992-07-01

    To elucidate the mechanism(s) of retroviral recombination, we exposed virions to gamma radiation prior to infecting target cells. By using previously described spleen necrosis virus-based vectors containing multiple markers, recombinant proviruses were studied after a single round of retrovirus replication. The current models of retroviral recombination predict that breaking virion RNA should promote minus-strand recombination (forced copy-choice model), decrease or not affect plus-strand recombination (strand displacement/assimilation model), and shift plus-strand recombination towards the 3' end of the genome. However, we found that while gamma irradiation of virions reduced the amount of recoverable viral RNA, it did not primarily cause breaks. Thus, the frequency of selected recombinants was not significantly altered with greater doses of radiation. In spite of this, the irradiation did decrease the number of recombinants with only one internal template switch. As a result, the average number of additional internal template switches in the recombinant proviruses increased from 0.7 to 1.4 as infectivity decreased to 6%. The unselected internal template switches tended to be 5' of the selected crossover even in the recombinants from irradiated viruses, inconsistent with a plus-strand recombination mechanism. PMID:1602553

  17. Retroviral Integrations in Gene Therapy Trials

    PubMed Central

    Biasco, Luca; Baricordi, Cristina; Aiuti, Alessandro

    2012-01-01

    γ-Retroviral and lentiviral vectors allow the permanent integration of a therapeutic transgene in target cells and have provided in the last decade a delivery platform for several successful gene therapy (GT) clinical approaches. However, the occurrence of adverse events due to insertional mutagenesis in GT treated patients poses a strong challenge to the scientific community to identify the mechanisms at the basis of vector-driven genotoxicity. Along the last decade, the study of retroviral integration sites became a fundamental tool to monitor vector–host interaction in patients overtime. This review is aimed at critically revising the data derived from insertional profiling, with a particular focus on the evidences collected from GT clinical trials. We discuss the controversies and open issues associated to the interpretation of integration site analysis during patient's follow up, with an update on the latest results derived from the use of high-throughput technologies. Finally, we provide a perspective on the future technical development and on the application of these studies to address broader biological questions, from basic virology to human hematopoiesis. PMID:22252453

  18. The Use of Recombinant Pseudotype Virus-Like Particles Harbouring Inserted Target Antigen to Generate Antibodies against Cellular Marker p16INK4A

    PubMed Central

    Lasickienė, Rita; Gedvilaite, Alma; Norkiene, Milda; Simanaviciene, Vaida; Sezaite, Indre; Dekaminaviciute, Dovile; Shikova, Evelina; Zvirbliene, Aurelija

    2012-01-01

    Protein engineering provides an opportunity to generate new immunogens with desired features. Previously, we have demonstrated that hamster polyomavirus major capsid protein VP1-derived virus-like particles (VLPs) are highly immunogenic and can be employed for the insertion of foreign epitopes at certain surface-exposed positions. In the current study, we have designed pseudotype VLPs consisting of an intact VP1 protein and VP2 protein fused with the target antigen—cellular marker p16INK4A—at its N terminus. Both proteins coexpressed in yeast were self-assembled to pseudotype VLPs harbouring the inserted antigen on the surface. The pseudotype VLPs were used for generation of antibodies against p16INK4A that represents a potential biomarker for cells transformed by high-risk human papillomavirus (HPV). The pseudotype VLPs induced in immunized mice a strong immune response against the target antigen. The antisera raised against pseudotype VLPs showed specific immunostaining of p16INK4A protein in malignant cervical tissue. Spleen cells of the immunized mice were used to generate monoclonal antibodies against p16INK4A protein. The specificity of antibodies was proven by the immunostaining of HPV-transformed cells. In conclusion, the current study demonstrates the potential of pseudotype VLPs with inserted target antigen as a new type of immunogens to generate antibodies of high diagnostic value. PMID:22629125

  19. Hepatitis C virus quasispecies and pseudotype analysis from acute infection to chronicity in HIV-1 co-infected individuals.

    PubMed

    Ferns, R Bridget; Tarr, Alexander W; Hue, Stephane; Urbanowicz, Richard A; McClure, C Patrick; Gilson, Richard; Ball, Jonathan K; Nastouli, Eleni; Garson, Jeremy A; Pillay, Deenan

    2016-05-01

    HIV-1 infected patients who acquire HCV infection have higher rates of chronicity and liver disease progression than patients with HCV mono-infection. Understanding early events in this pathogenic process is important. We applied single genome sequencing of the E1 to NS3 regions and viral pseudotype neutralization assays to explore the consequences of viral quasispecies evolution from pre-seroconversion to chronicity in four co-infected individuals (mean follow up 566 days). We observed that one to three founder viruses were transmitted. Relatively low viral sequence diversity, possibly related to an impaired immune response, due to HIV infection was observed in three patients. However, the fourth patient, after an early purifying selection displayed increasing E2 sequence evolution, possibly related to being on suppressive antiretroviral therapy. Viral pseudotypes generated from HCV variants showed relative resistance to neutralization by autologous plasma but not to plasma collected from later time points, confirming ongoing virus escape from antibody neutralization. PMID:26971243

  20. Host Range of Small-Ruminant Lentivirus Cytopathic Variants Determined with a Selectable Caprine Arthritis- Encephalitis Virus Pseudotype System

    PubMed Central

    Hötzel, Isidro; Cheevers, William P.

    2001-01-01

    The small-ruminant lentiviruses ovine maedi-visna virus (MVV) and caprine arthritis-encephalitis virus (CAEV) cause encephalitis, progressive pneumonia, arthritis, and mastitis in sheep and goats. Icelandic MVV strains, which are lytic in tissue culture, have a wide species distribution of functional receptors, which includes human cells. In contrast, functional receptors for the nonlytic CAEV CO are absent from human cells. To determine if the wide species distribution of functional receptors is a common property of MVV strains or related to cytopathic phenotype, we tested the infectivity of viruses pseudotyped with the envelope glycoproteins of MVV K1514, CAEV CO, and lytic and nonlytic North American MVV strains to cells of different species. Replication-defective CAEV proviral constructs lacking the env, tat, and vif genes and carrying the neomycin phosphotransferase gene in the vif-tat region were developed for the infectivity assays. Cotransfection of human 293T cells with these proviral constructs and plasmids expressing CAEV, MVV, or vesicular stomatitis virus envelope glycoproteins produced infectious pseudotyped virus which induced resistance of infected cells to G418. Using these pseudotypes, we confirmed the wide species distribution of Icelandic MVV receptors and the narrow host range of CAEV. However, functional receptors for the two North American MVV strains tested, unlike the Icelandic MVV and similar to CAEV, were limited to cells of ruminant species, regardless of cytopathic phenotype. The results indicate a differential receptor recognition by MVV strains which is unrelated to cytopathic phenotype. PMID:11462010

  1. Retroviruses Pseudotyped with the Severe Acute Respiratory Syndrome Coronavirus Spike Protein Efficiently Infect Cells Expressing Angiotensin-Converting Enzyme 2

    PubMed Central

    Moore, Michael J.; Dorfman, Tatyana; Li, Wenhui; Wong, Swee Kee; Li, Yanhan; Kuhn, Jens H.; Coderre, James; Vasilieva, Natalya; Han, Zhongchao; Greenough, Thomas C.; Farzan, Michael; Choe, Hyeryun

    2004-01-01

    Infection of receptor-bearing cells by coronaviruses is mediated by their spike (S) proteins. The coronavirus (SARS-CoV) that causes severe acute respiratory syndrome (SARS) infects cells expressing the receptor angiotensin-converting enzyme 2 (ACE2). Here we show that codon optimization of the SARS-CoV S-protein gene substantially enhanced S-protein expression. We also found that two retroviruses, simian immunodeficiency virus (SIV) and murine leukemia virus, both expressing green fluorescent protein and pseudotyped with SARS-CoV S protein or S-protein variants, efficiently infected HEK293T cells stably expressing ACE2. Infection mediated by an S-protein variant whose cytoplasmic domain had been truncated and altered to include a fragment of the cytoplasmic tail of the human immunodeficiency virus type 1 envelope glycoprotein was, in both cases, substantially more efficient than that mediated by wild-type S protein. Using S-protein-pseudotyped SIV, we found that the enzymatic activity of ACE2 made no contribution to S-protein-mediated infection. Finally, we show that a soluble and catalytically inactive form of ACE2 potently blocked infection by S-protein-pseudotyped retrovirus and by SARS-CoV. These results permit studies of SARS-CoV entry inhibitors without the use of live virus and suggest a candidate therapy for SARS. PMID:15367630

  2. Membrane interaction of retroviral Gag proteins

    PubMed Central

    Dick, Robert A.; Vogt, Volker M.

    2014-01-01

    Assembly of an infectious retroviral particle relies on multimerization of the Gag polyprotein at the inner leaflet of the plasma membrane. The three domains of Gag common to all retroviruses – MA, CA, and NC – provide the signals for membrane binding, assembly, and viral RNA packaging, respectively. These signals do not function independently of one another. For example, Gag multimerization enhances membrane binding and is more efficient when NC is interacting with RNA. MA binding to the plasma membrane is governed by several principles, including electrostatics, recognition of specific lipid head groups, hydrophobic interactions, and membrane order. HIV-1 uses many of these principles while Rous sarcoma virus (RSV) appears to use fewer. This review describes the principles that govern Gag interactions with membranes, focusing on RSV and HIV-1 Gag. The review also defines lipid and membrane behavior, and discusses the complexities in determining how lipid and membrane behavior impact Gag membrane binding. PMID:24808894

  3. Retroviral infections transmitted by blood transfusion.

    PubMed Central

    Sandler, S. G.; Fang, C.; Williams, A.

    1990-01-01

    Modifications in donor screening and the introduction of laboratory testing of donated blood for anti-HIV-1 and anti-HTLV-I have resulted in a significant reduction in the risks of retroviral infections from blood transfusion. Presently, the American Red Cross detects an average of eight carriers of human immunodeficiency virus, type 1 (HIV-1) per 100,000 otherwise acceptable blood donors (0.008 percent), compared with an average of 35 per 100,000 (0.035 percent) when testing for HIV-1 antibodies began in 1985. Surveillance studies in the United States indicate a small likelihood that HIV-2 carriers will pass current screening procedures and be accepted as blood donors. Even if an HIV-2-infected person were to be accepted as a blood donor, there is a 42-92 percent likelihood that this person's blood would be detected as infective for HIV-2 and excluded because of serological cross-reactions that occur in the EIA for HIV-1 antibodies. During 1989, which was the first year that donated blood was routinely tested for antibodies to human T-lymphotropic virus, type I (HTLV-I) in the United States, approximately nine in 100,000 donors (0.009 percent) were confirmed positive for antibodies to HTLV-I, and their donated blood was excluded. Subsequent testing has revealed that a significant number of these persons whose sera was reactive by the HTLV-I EIA were, in fact, infected by HTLV-II. Epidemiological studies of human retroviral infections (HIV-1, HIV-2, HTLV-I, and HTLV-II) continue to provide important data and direction for improving criteria for qualifying blood donors. PMID:1981409

  4. Extensive Replication of a Retroviral Replicating Vector Can Expand the A Bulge in the Encephalomyocarditis Virus Internal Ribosome Entry Site and Change Translation Efficiency of the Downstream Transgene.

    PubMed

    Lin, Amy H; Liu, Yanzheng; Burrascano, Cynthia; Cunanan, Kathrina; Logg, Christopher R; Robbins, Joan M; Kasahara, Noriyuki; Gruber, Harry; Ibañez, Carlos; Jolly, Douglas J

    2016-04-01

    We have developed retroviral replicating vectors (RRV) derived from Moloney murine gammaretrovirus with an amphotropic envelope and an encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES)-transgene cassette downstream of the env gene. During long-term (180 days) replication of the vector in animals, a bulge of 7 adenosine residues (A's) in the J-K bifurcation domain sometimes serially added A's. Therefore, vectors with 4-12 A's in the A bulge in the J-K bifurcation domain were generated, and the impact of the variants on transgene protein expression, vector stability, and IRES sequence upon multiple infection cycles was assessed in RRV encoding yeast-derived cytosine deaminase and green fluorescent protein in vitro. For transgene protein expression, after multiple infection cycles, RRV-IRES with 5-7 A's gave roughly comparable levels, 4 and 8 A's were within about 4-5-fold of the 6 A's, whereas 10 and 12 A's were marked lower. In terms of stability, after 10 infection cycles, expansion of A's appeared to be a more frequent event affecting transgene protein expression than viral genome deletions or rearrangement: 4 and 5 A's appeared completely stable; 6, 7, and particularly 8 A's showed some level of expansion in the A bulge; 10 and 12 A's underwent both expansion and transgene deletion. The strong relative translational activity of the 5 A's in the EMCV IRES has not been reported previously. The 5A RRV-IRES may have utility for preclinical and clinical applications where extended replication is required. PMID:26918465

  5. Adenovirus hexon modifications influence in vitro properties of pseudotyped human adenovirus type 5 vectors.

    PubMed

    Solanki, Manish; Zhang, Wenli; Jing, Liu; Ehrhardt, Anja

    2016-01-01

    Commonly used human adenovirus (HAdV)-5-based vectors are restricted by their tropism and pre-existing immunity. Here, we characterized novel HAdV-5 vectors pseudotyped with hypervariable regions (HVRs) and surface domains (SDs) of other HAdV types. Hexon-modified HAdV-5 vectors (HV-HVR5, HV-HVR12, HV-SD12 and HV-SD4) could be reconstituted and amplified in human embryonic kidney cells. After infection of various cell lines, we measured transgene expression levels by performing luciferase reporter assays or coagulation factor IX (FIX) ELISA. Dose-dependent studies revealed that luciferase expression levels were comparable for HV-HVR5, HV-SD12 and HV-SD4, whereas HV-HVR12 expression levels were significantly lower. Vector genome copy numbers (VCNs) from genomic DNA and nuclear extracts were then determined by quantitative real-time PCR. Surprisingly, determination of cell- and nuclear fraction-associated VCNs revealed increased VCNs for HV-HVR12 compared with HV-SD12 and HV-HVR5. Increased nuclear fraction-associated HV-HVR12 DNA molecules and decreased transgene expression levels were independent of the cell line used, and we observed the same effect for a hexon-modified high-capacity adenoviral vector encoding canine FIX. In conclusion, studying hexon-modified adenoviruses in vitro demonstrated that HVRs but also flanking hexon regions influence uptake and transgene expression of adenoviral vectors. PMID:26519158

  6. Efficacy of an adeno-associated virus 8-pseudotyped vector in glycogen storage disease type II.

    PubMed

    Sun, Baodong; Zhang, Haoyue; Franco, Luis M; Young, Sarah P; Schneider, Ayn; Bird, Andrew; Amalfitano, Andrea; Chen, Y-T; Koeberl, Dwight D

    2005-01-01

    Glycogen storage disease type II (GSD-II; Pompe disease) causes death in infancy from cardiorespiratory failure. The underlying deficiency of acid alpha-glucosidase (GAA; acid maltase) can be corrected by liver-targeted gene therapy in GSD-II, if secretion of GAA is accompanied by receptor-mediated uptake in cardiac and skeletal muscle. An adeno-associated virus (AAV) vector encoding human (h) GAA was pseudotyped as AAV8 (AAV2/8) and injected intravenously into immunodeficient GSD-II mice. High levels of hGAA were maintained in plasma for 24 weeks following AAV2/8 vector administration. A marked increase in vector copy number in the liver was demonstrated for the AAV2/8 vector compared to the analogous AAV2/2 vector. GAA deficiency in the heart and skeletal muscle was corrected with the AAV2/8 vector in male GSD-II mice, consistent with receptor-mediated uptake of hGAA. Male GSD-II mice demonstrated complete correction of glycogen storage in heart and diaphragm with the AAV2/8 vector, while female GSD-II mice had correction only in the heart. A biomarker for GSD-II was reduced in both sexes following AAV2/8 vector administration. Therefore, GAA production with an AAV2/8 vector in a depot organ, the liver, generated evidence for efficacious gene therapy in a mouse model for GSD-II. PMID:15585406

  7. Pseudotyping of lentiviral vector with novel vesiculovirus envelope glycoproteins derived from Chandipura and Piry viruses.

    PubMed

    Hu, Shuang; Mohan Kumar, Dipu; Sax, Chelsea; Schuler, Clayton; Akkina, Ramesh

    2016-01-15

    While the envelope glycoprotein of vesicular stomatitis virus (VSV-G) is widely used for pseudotyping of lentiviral vectors, sub-optimal gene transfer into certain cell types and its sensitivity to inactivation by human complement hinders its broader applications. To find alternative candidates, here we evaluated two serologically distinct novel viral envelopes derived from Chandipura (CNV-G) and Piry (PRV-G) vesiculoviruses. Both permitted generation of high titer psuedotyped lentiviral vectors with a capacity for high efficiency gene transfer into various cell types from different species. In human lymphoid and hematopoietic stem cells, their transduction efficiency was significantly lower than that of VSV-G. However, both novel envelopes were found to be more resistant to inactivation by human serum complement compared to VSV-G. Thus CNV-G and PRV-G envelopes can be harnessed for multiple uses in the future based on the cell type that needs to be gene transduced and possibly for in vivo gene transfer. PMID:26650691

  8. Capture of syncytin-Mar1, a fusogenic endogenous retroviral envelope gene involved in placentation in the Rodentia squirrel-related clade.

    PubMed

    Redelsperger, François; Cornelis, Guillaume; Vernochet, Cécile; Tennant, Bud C; Catzeflis, François; Mulot, Baptiste; Heidmann, Odile; Heidmann, Thierry; Dupressoir, Anne

    2014-07-01

    Syncytin genes are fusogenic envelope protein (env) genes of retroviral origin that have been captured for a function in placentation. Within rodents, two such genes have previously been identified in the mouse-related clade, allowing a demonstration of their essential role via knockout mice. Here, we searched for similar genes in a second major clade of the Rodentia order, the squirrel-related clade, taking advantage of the complete sequencing of the ground squirrel Ictidomys tridecemlineatus genome. In silico search for env genes with full coding capacity identified several candidate genes with one displaying placenta-specific expression, as revealed by quantitative reverse transcription-PCR analysis of a large panel of tissues. This gene belongs to a degenerate endogenous retroviral element, with recognizable hallmarks of an integrated provirus. Cloning of the gene in an expression vector for ex vivo cell-cell fusion and pseudotype assays demonstrated fusogenicity on a large panel of mammalian cells. In situ hybridization on placenta sections showed specific expression in domains where trophoblast cells fuse into a syncytiotrophoblast at the fetomaternal interface, consistent with a role in syncytium formation. Finally, we show that the gene is conserved among the tribe Marmotini, thus dating its capture back to about at least 25 million years ago, with evidence for purifying selection and conservation of fusogenic activity. This gene that we named syncytin-Mar1 is distinct from all seven Syncytin genes identified to date in eutherian mammals and is likely to be a major effector of placentation in its related clade. Importance: Syncytin genes are fusogenic envelope genes of retroviral origin, ancestrally captured for a function in placentation. Within rodents, two such genes had been previously identified in the mouse-related clade. Here, in the squirrel-related rodent clade, we identified the envelope gene of an endogenous retrovirus with all the features of a

  9. Capture of syncytin-Mar1, a Fusogenic Endogenous Retroviral Envelope Gene Involved in Placentation in the Rodentia Squirrel-Related Clade

    PubMed Central

    Redelsperger, François; Cornelis, Guillaume; Vernochet, Cécile; Tennant, Bud C.; Catzeflis, François; Mulot, Baptiste; Heidmann, Odile; Dupressoir, Anne

    2014-01-01

    ABSTRACT Syncytin genes are fusogenic envelope protein (env) genes of retroviral origin that have been captured for a function in placentation. Within rodents, two such genes have previously been identified in the mouse-related clade, allowing a demonstration of their essential role via knockout mice. Here, we searched for similar genes in a second major clade of the Rodentia order, the squirrel-related clade, taking advantage of the complete sequencing of the ground squirrel Ictidomys tridecemlineatus genome. In silico search for env genes with full coding capacity identified several candidate genes with one displaying placenta-specific expression, as revealed by quantitative reverse transcription-PCR analysis of a large panel of tissues. This gene belongs to a degenerate endogenous retroviral element, with recognizable hallmarks of an integrated provirus. Cloning of the gene in an expression vector for ex vivo cell-cell fusion and pseudotype assays demonstrated fusogenicity on a large panel of mammalian cells. In situ hybridization on placenta sections showed specific expression in domains where trophoblast cells fuse into a syncytiotrophoblast at the fetomaternal interface, consistent with a role in syncytium formation. Finally, we show that the gene is conserved among the tribe Marmotini, thus dating its capture back to about at least 25 million years ago, with evidence for purifying selection and conservation of fusogenic activity. This gene that we named syncytin-Mar1 is distinct from all seven Syncytin genes identified to date in eutherian mammals and is likely to be a major effector of placentation in its related clade. IMPORTANCE Syncytin genes are fusogenic envelope genes of retroviral origin, ancestrally captured for a function in placentation. Within rodents, two such genes had been previously identified in the mouse-related clade. Here, in the squirrel-related rodent clade, we identified the envelope gene of an endogenous retrovirus with all the

  10. Membrane-mediated interaction between retroviral capsids

    NASA Astrophysics Data System (ADS)

    Zhang, Rui; Nguyen, Toan

    2012-02-01

    A retrovirus is an RNA virus that is replicated through a unique strategy of reverse transcription. Unlike regular enveloped viruses which are assembled inside the host cells, the assembly of retroviral capsids happens right on the cell membrane. During the assembly process, the partially formed capsids deform the membrane, giving rise to an elastic energy. When two such partial capsids approach each other, this elastic energy changes. Or in other words, the two partial capsids interact with each other via the membrane. This membrane mediated interaction between partial capsids plays an important role in the kinetics of the assembly process. In this work, this membrane mediated interaction is calculated both analytically and numerically. It is worth noting that the diferential equation determining the membrane shape in general nonlinear and cannot be solved analytically,except in the linear region of small deformations. And it is exactly the nonlinear regime that is important for the assembly kinetics of retroviruses as it provides a large energy barrier. The theory developed here is applicable to more generic cases of membrane mediated interactions between two membrane-embedded proteins.

  11. Structural basis for retroviral integration into nucleosomes

    PubMed Central

    Maskell, Daniel P.; Renault, Ludovic; Serrao, Erik; Lesbats, Paul; Matadeen, Rishi; Hare, Stephen; Lindemann, Dirk; Engelman, Alan N.; Costa, Alessandro; Cherepanov, Peter

    2015-01-01

    Retroviral integration is catalyzed by a tetramer of integrase (IN) assembled on viral DNA ends in a stable complex, known as the intasome1,2. How the intasome interfaces with chromosomal DNA, which exists in the form of nucleosomal arrays, is currently unknown. Here we show that the prototype foamy virus (PFV) intasome is proficient at stable capture of nucleosomes as targets for integration. Single-particle cryo-electron microscopy (EM) reveals a multivalent intasome-nucleosome interface involving both gyres of nucleosomal DNA and one H2A-H2B heterodimer. While the histone octamer remains intact, the DNA is lifted from the surface of the H2A-H2B heterodimer to allow integration at strongly preferred superhelix location (SHL) ±3.5 positions. Amino acid substitutions disrupting these contacts impinge on the ability of the intasome to engage nucleosomes in vitro and redistribute viral integration sites on the genomic scale. Our findings elucidate the molecular basis for nucleosome capture by the viral DNA recombination machinery and the underlying nucleosome plasticity that allows integration. PMID:26061770

  12. An Automated HIV-1 Env-Pseudotyped Virus Production for Global HIV Vaccine Trials

    PubMed Central

    Fuss, Martina; Mazzotta, Angela S.; Sarzotti-Kelsoe, Marcella; Ozaki, Daniel A.; Montefiori, David C.; von Briesen, Hagen; Zimmermann, Heiko; Meyerhans, Andreas

    2012-01-01

    Background Infections with HIV still represent a major human health problem worldwide and a vaccine is the only long-term option to fight efficiently against this virus. Standardized assessments of HIV-specific immune responses in vaccine trials are essential for prioritizing vaccine candidates in preclinical and clinical stages of development. With respect to neutralizing antibodies, assays with HIV-1 Env-pseudotyped viruses are a high priority. To cover the increasing demands of HIV pseudoviruses, a complete cell culture and transfection automation system has been developed. Methodology/Principal Findings The automation system for HIV pseudovirus production comprises a modified Tecan-based Cellerity system. It covers an area of 5×3 meters and includes a robot platform, a cell counting machine, a CO2 incubator for cell cultivation and a media refrigerator. The processes for cell handling, transfection and pseudovirus production have been implemented according to manual standard operating procedures and are controlled and scheduled autonomously by the system. The system is housed in a biosafety level II cabinet that guarantees protection of personnel, environment and the product. HIV pseudovirus stocks in a scale from 140 ml to 1000 ml have been produced on the automated system. Parallel manual production of HIV pseudoviruses and comparisons (bridging assays) confirmed that the automated produced pseudoviruses were of equivalent quality as those produced manually. In addition, the automated method was fully validated according to Good Clinical Laboratory Practice (GCLP) guidelines, including the validation parameters accuracy, precision, robustness and specificity. Conclusions An automated HIV pseudovirus production system has been successfully established. It allows the high quality production of HIV pseudoviruses under GCLP conditions. In its present form, the installed module enables the production of 1000 ml of virus-containing cell culture supernatant per

  13. Transduction of rat pancreatic islets with pseudotyped adeno-associated virus vectors

    PubMed Central

    Craig, Anthony T; Gavrilova, Oksana; Dwyer, Nancy K; Jou, William; Pack, Stephanie; Liu, Eric; Pechhold, Klaus; Schmidt, Michael; McAlister, Victor J; Chiorini, John A; Blanchette-Mackie, E Joan; Harlan, David M; Owens, Roland A

    2009-01-01

    Background Pancreatic islet transplantation is a promising treatment for type I diabetes mellitus, but current immunosuppressive strategies do not consistently provide long-term survival of transplanted islets. We are therefore investigating the use of adeno-associated viruses (AAVs) as gene therapy vectors to transduce rat islets with immunosuppressive genes prior to transplantation into diabetic mice. Results We compared the transduction efficiency of AAV2 vectors with an AAV2 capsid (AAV2/2) to AAV2 vectors pseudotyped with AAV5 (AAV2/5), AAV8 (AAV2/8) or bovine adeno-associated virus (BAAV) capsids, or an AAV2 capsid with an insertion of the low density lipoprotein receptor ligand from apolipoprotein E (AAV2apoE), on cultured islets, in the presence of helper adenovirus infection to speed expression of a GFP transgene. Confocal microscopy and flow cytometry were used. The AAV2/5 vector was superior to AAV2/2 and AAV2/8 in rat islets. Flow cytometry indicated AAV2/5-mediated gene expression in approximately 9% of rat islet cells and almost 12% of insulin-positive cells. The AAV2/8 vector had a higher dependence on the helper virus multiplicity of infection than the AAV 2/5 vector. In addition, the BAAV and AAV2apoE vectors were superior to AAV2/2 for transducing rat islets. Rat islets (300 per mouse) transduced with an AAV2/5 vector harboring the immunosuppressive transgene, tgfβ1, retain the ability to correct hyperglycemia when transplanted into immune-deficient diabetic mice. Conclusion AAV2/5 vectors may therefore be useful for pre-treating donor islets prior to transplantation. PMID:19450275

  14. Construction and immunogenicity of recombinant pseudotype baculovirus expressing the capsid protein of porcine circovirus type 2 in mice.

    PubMed

    Fan, Huiying; Pan, Yongfei; Fang, Liurong; Wang, Dang; Wang, Shengping; Jiang, Yunbo; Chen, Huanchun; Xiao, Shaobo

    2008-06-01

    Baculovirus has emerged recently as a novel and attractive gene delivery vehicle for mammalian cells. Porcine circovirus type 2 (PCV2) is known to be associated with post-weaning multisystemic wasting syndrome (PMWS), an emerging swine disease which results in tremendous economic losses. In this study, baculovirus pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G) was used as a vector to express capsid (Cap) protein, the most important immunogen of PCV2, under the transcriptional control of cytomegalovirus immediate early (CMV-IE) enhancer/promoter. The resultant recombinant baculovirus (BV-G-ORF2) efficiently transduced and expressed the Cap protein in mammalian cells, as demonstrated by Western blot and flow cytometric analyses. After direct vaccination with 1x10(8) or 1x10(9)plaque forming units (PFU)/mouse of BV-G-ORF2, significant PCV2-specific ELISA antibodies, neutralizing antibodies, as well as cellular immune responses could be induced in mice. BV-G-ORF2 exhibited better immunogenicity than a DNA vaccine encoding the Cap protein, even at a dose of 1x10(8)PFU/mouse. Taken together, the improved immunogenicity of BV-G-ORF2, together with the unique advantages of pseudotype baculovirus, including easy manipulation, simple scale-up, lack of toxicity, and no pre-existing antibody against baculovirus in the hosts, indicate that pseudotype baculovirus-mediated gene delivery can be utilized as an alternative strategy to develop a new generation of vaccines against PCV2 infection. PMID:18394722

  15. Deciphering the impact of parameters influencing transgene expression kinetics after repeated cell transduction with integration-deficient retroviral vectors.

    PubMed

    Schott, Juliane W; Jaeschke, Nico M; Hoffmann, Dirk; Maetzig, Tobias; Ballmaier, Matthias; Godinho, Tamaryin; Cathomen, Toni; Schambach, Axel

    2015-05-01

    Lentiviral and gammaretroviral vectors are state-of-the-art tools for transgene expression within target cells. The integration of these vectors can be deliberately suppressed to derive a transient gene expression system based on extrachromosomal circular episomes with intact coding regions. These episomes can be used to deliver DNA templates and to express RNA or protein. Importantly, transient gene transfer avoids the genotoxic side effects of integrating vectors. Restricting their applicability, episomes are rapidly lost upon dilution in dividing target cells. Addressing this limitation, we could establish comparably stable percentages of transgene-positive cells over prolonged time periods in proliferating cells by repeated transductions. Flow cytometry was applied for kinetic analyses to decipher the impact of individual parameters on the kinetics of fluoroprotein expression after episomal retransduction and to visualize sequential and simultaneous transfer of heterologous fluoroproteins. Expression windows could be exactly timed by the number of transduction steps. The kinetics of signal loss was affected by the cell proliferation rate. The transfer of genes encoding fluoroproteins with different half-lives revealed a major impact of protein stability on temporal signal distribution and accumulation, determining optimal retransduction intervals. In addition, sequential transductions proved broad applicability in different cell types and using different envelope pseudotypes without receptor overload. Stable percentages of cells coexpressing multiple transgenes could be generated upon repeated coadministration of different episomal vectors. Alternatively, defined patterns of transgene expression could be recapitulated by sequential transductions. Altogether, we established a methodology to control and adjust a temporally defined window of transgene expression using retroviral episomal vectors. Combined with the highly efficient cell entry of these vectors while

  16. Enhanced Central Nervous System Transduction with Lentiviral Vectors Pseudotyped with RVG/HIV-1gp41 Chimeric Envelope Glycoproteins

    PubMed Central

    Trabalza, Antonio; Eleftheriadou, Ioanna; Sgourou, Argyro; Liao, Ting-Yi; Patsali, Petros; Lee, Heyne

    2014-01-01

    ABSTRACT To investigate the potential benefits which may arise from pseudotyping the HIV-1 lentiviral vector with its homologous gp41 envelope glycoprotein (GP) cytoplasmic tail (CT), we created chimeric RVG/HIV-1gp41 GPs composed of the extracellular and transmembrane sequences of RVG and either the full-length gp41 CT or C terminus gp41 truncations sequentially removing existing conserved motifs. Lentiviruses (LVs) pseudotyped with the chimeric GPs were evaluated in terms of particle release (physical titer), biological titers, infectivity, and in vivo central nervous system (CNS) transduction. We report here that LVs carrying shorter CTs expressed higher levels of envelope GP and showed a higher average infectivity than those bearing full-length GPs. Interestingly, complete removal of GP CT led to vectors with the highest transduction efficiency. Removal of all C-terminal gp41 CT conserved motifs, leaving just 17 amino acids (aa), appeared to preserve infectivity and resulted in a significantly increased physical titer. Furthermore, incorporation of these 17 aa in the RVG CT notably enhanced the physical titer. In vivo stereotaxic delivery of LV vectors exhibiting the best in vitro titers into rodent striatum facilitated efficient transduction of the CNS at the site of injection. A particular observation was the improved retrograde transduction of neurons in connected distal sites that resulted from the chimeric envelope R5 which included the “Kennedy” sequence (Ken) and lentivirus lytic peptide 2 (LLP2) conserved motifs in the CT, and although it did not exhibit a comparable high titer upon pseudotyping, it led to a significant increase in distal retrograde transduction of neurons. IMPORTANCE In this study, we have produced novel chimeric envelopes bearing the extracellular domain of rabies fused to the cytoplasmic tail (CT) of gp41 and pseudotyped lentiviral vectors with them. Here we report novel effects on the transduction efficiency and physical titer

  17. The Host RNAs in Retroviral Particles.

    PubMed

    Telesnitsky, Alice; Wolin, Sandra L

    2016-01-01

    As they assemble, retroviruses encapsidate both their genomic RNAs and several types of host RNA. Whereas limited amounts of messenger RNA (mRNA) are detectable within virion populations, the predominant classes of encapsidated host RNAs do not encode proteins, but instead include endogenous retroelements and several classes of non-coding RNA (ncRNA), some of which are packaged in significant molar excess to the viral genome. Surprisingly, although the most abundant host RNAs in retroviruses are also abundant in cells, unusual forms of these RNAs are packaged preferentially, suggesting that these RNAs are recruited early in their biogenesis: before associating with their cognate protein partners, and/or from transient or rare RNA populations. These RNAs' packaging determinants differ from the viral genome's, and several of the abundantly packaged host ncRNAs serve cells as the scaffolds of ribonucleoprotein particles. Because virion assembly is equally efficient whether or not genomic RNA is available, yet RNA appears critical to the structural integrity of retroviral particles, it seems possible that the selectively encapsidated host ncRNAs might play roles in assembly. Indeed, some host ncRNAs appear to act during replication, as some transfer RNA (tRNA) species may contribute to nuclear import of human immunodeficiency virus 1 (HIV-1) reverse transcription complexes, and other tRNA interactions with the viral Gag protein aid correct trafficking to plasma membrane assembly sites. However, despite high conservation of packaging for certain host RNAs, replication roles for most of these selectively encapsidated RNAs-if any-have remained elusive. PMID:27548206

  18. The Host RNAs in Retroviral Particles

    PubMed Central

    Telesnitsky, Alice; Wolin, Sandra L.

    2016-01-01

    As they assemble, retroviruses encapsidate both their genomic RNAs and several types of host RNA. Whereas limited amounts of messenger RNA (mRNA) are detectable within virion populations, the predominant classes of encapsidated host RNAs do not encode proteins, but instead include endogenous retroelements and several classes of non-coding RNA (ncRNA), some of which are packaged in significant molar excess to the viral genome. Surprisingly, although the most abundant host RNAs in retroviruses are also abundant in cells, unusual forms of these RNAs are packaged preferentially, suggesting that these RNAs are recruited early in their biogenesis: before associating with their cognate protein partners, and/or from transient or rare RNA populations. These RNAs’ packaging determinants differ from the viral genome’s, and several of the abundantly packaged host ncRNAs serve cells as the scaffolds of ribonucleoprotein particles. Because virion assembly is equally efficient whether or not genomic RNA is available, yet RNA appears critical to the structural integrity of retroviral particles, it seems possible that the selectively encapsidated host ncRNAs might play roles in assembly. Indeed, some host ncRNAs appear to act during replication, as some transfer RNA (tRNA) species may contribute to nuclear import of human immunodeficiency virus 1 (HIV-1) reverse transcription complexes, and other tRNA interactions with the viral Gag protein aid correct trafficking to plasma membrane assembly sites. However, despite high conservation of packaging for certain host RNAs, replication roles for most of these selectively encapsidated RNAs—if any—have remained elusive. PMID:27548206

  19. Retroviral retargeting by envelopes expressing an N-terminal binding domain.

    PubMed Central

    Cosset, F L; Morling, F J; Takeuchi, Y; Weiss, R A; Collins, M K; Russell, S J

    1995-01-01

    We have engineered ecotropic Moloney murine leukemia virus-derived envelopes targeted to cell surface molecules expressed on human cells by the N-terminal insertion of polypeptides able to bind either Ram-1 phosphate transporter (the first 208 amino acids of amphotropic murine leukemia virus surface protein) or epidermal growth factor receptor (EGFR) (the 53 amino acids of EGF). Both envelopes were correctly processed and incorporated into viral particles. Virions carrying these envelopes could specifically bind the new cell surface receptors. Virions targeted to Ram-1 could infect human cells, although the efficiency was reduced compared with that of virions carrying wild-type amphotropic murine leukemia virus envelopes. The infectivity of virions targeted to EGFR was blocked at a postbinding step, and our results suggest that EGFR-bound virions were rapidly trafficked to lysosomes. These data suggest that retroviruses require specific properties of cell surface molecules to allow the release of viral cores into the correct cell compartment. PMID:7666532

  20. Efficient generation of vesicular stomatitis virus (VSV)-pseudotypes bearing morbilliviral glycoproteins and their use in quantifying virus neutralising antibodies

    PubMed Central

    Logan, Nicola; McMonagle, Elizabeth; Drew, Angharad A.; Takahashi, Emi; McDonald, Michael; Baron, Michael D.; Gilbert, Martin; Cleaveland, Sarah; Haydon, Daniel T.; Hosie, Margaret J.; Willett, Brian J.

    2016-01-01

    Morbillivirus neutralising antibodies are traditionally measured using either plaque reduction neutralisation tests (PRNTs) or live virus microneutralisation tests (micro-NTs). While both test formats provide a reliable assessment of the strength and specificity of the humoral response, they are restricted by the limited number of viral strains that can be studied and often present significant biological safety concerns to the operator. In this study, we describe the adaptation of a replication-defective vesicular stomatitis virus (VSVΔG) based pseudotyping system for the measurement of morbillivirus neutralising antibodies. By expressing the haemagglutinin (H) and fusion (F) proteins of canine distemper virus (CDV) on VSVΔG pseudotypes bearing a luciferase marker gene, neutralising antibody titres could be measured rapidly and with high sensitivity. Further, by exchanging the glycoprotein expression construct, responses against distinct viral strains or species may be measured. Using this technique, we demonstrate cross neutralisation between CDV and peste des petits ruminants virus (PPRV). As an example of the value of the technique, we demonstrate that UK dogs vary in the breadth of immunity induced by CDV vaccination; in some dogs the neutralising response is CDV-specific while, in others, the neutralising response extends to the ruminant morbillivirus PPRV. This technique will facilitate a comprehensive comparison of cross-neutralisation to be conducted across the morbilliviruses. PMID:26706278

  1. Efficient generation of vesicular stomatitis virus (VSV)-pseudotypes bearing morbilliviral glycoproteins and their use in quantifying virus neutralising antibodies.

    PubMed

    Logan, Nicola; McMonagle, Elizabeth; Drew, Angharad A; Takahashi, Emi; McDonald, Michael; Baron, Michael D; Gilbert, Martin; Cleaveland, Sarah; Haydon, Daniel T; Hosie, Margaret J; Willett, Brian J

    2016-02-01

    Morbillivirus neutralising antibodies are traditionally measured using either plaque reduction neutralisation tests (PRNTs) or live virus microneutralisation tests (micro-NTs). While both test formats provide a reliable assessment of the strength and specificity of the humoral response, they are restricted by the limited number of viral strains that can be studied and often present significant biological safety concerns to the operator. In this study, we describe the adaptation of a replication-defective vesicular stomatitis virus (VSVΔG) based pseudotyping system for the measurement of morbillivirus neutralising antibodies. By expressing the haemagglutinin (H) and fusion (F) proteins of canine distemper virus (CDV) on VSVΔG pseudotypes bearing a luciferase marker gene, neutralising antibody titres could be measured rapidly and with high sensitivity. Further, by exchanging the glycoprotein expression construct, responses against distinct viral strains or species may be measured. Using this technique, we demonstrate cross neutralisation between CDV and peste des petits ruminants virus (PPRV). As an example of the value of the technique, we demonstrate that UK dogs vary in the breadth of immunity induced by CDV vaccination; in some dogs the neutralising response is CDV-specific while, in others, the neutralising response extends to the ruminant morbillivirus PPRV. This technique will facilitate a comprehensive comparison of cross-neutralisation to be conducted across the morbilliviruses. PMID:26706278

  2. Sporadic ALS/MND: a global neurodegeneration with retroviral involvement?

    PubMed

    Westarp, M E; Ferrante, P; Perron, H; Bartmann, P; Kornhuber, H H

    1995-05-01

    Sporadic amyotrophic lateral sclerosis may be an aetiologically heterogenous disease. We confirmed elevated circulating IgG immune complexes, and altered IgG seroreactivities against human retroviral antigens (HIV-2 and HTLV immunoblots) in overlapping subgroups of patients. Together with preliminary findings of a positive polymerase chain reactivity for human T-lymphotropic virus (HTLV.tax/rex) in blood leukocytes of 5 out of 14 sALS patients, we interpret this as evidence for a retroviral involvement in this relentlessly progressive, often asymmetrically spreading neurodegeneration. The possibility of a secondary phenomenon seems unlikely, yet cannot be completely ruled out. PMID:7595609

  3. Transduction of motor neurons and muscle fibers by intramuscular injection of HIV-1-based vectors pseudotyped with select rabies virus glycoproteins.

    PubMed

    Mentis, George Z; Gravell, Maneth; Hamilton, Rebecca; Shneider, Neil A; O'Donovan, Michael J; Schubert, Manfred

    2006-10-30

    For studies of motor neuron function or for therapeutic purposes, novel pseudotype HIV-1-based vectors were developed that are capable of expressing transgenes in motor neurons following injection into mouse hind limb muscles. To specifically target motor neurons, glycoproteins from two rabies virus (RV) isolates, the mouse-brain adapted challenge virus 24 (CVS-24) variants, CVS-N2c and CVS-B2c were evaluated for pseudotype formation with an HIV-1-based vector. Both RV glycoproteins incorporated into vector envelopes, and both pseudotypes yielded high titers with Hek293T and cortical plate neuron cultures. Increased neuronotropism by the CVS-N2c pseudotype was not observed, suggesting that vector tropism is not solely determined by the fusogenic viral glycoprotein. Vector injection into hind limb muscles resulted in EYFP reporter gene expression in the injected muscle fibers and in spinal cord motor neurons innervating the same muscle, indicating retrograde vector transport. Intramuscular vector injections into the soleus and tibialis anterior muscles transduced 26% and 16% of all motor neurons in each motor nucleus, respectively. These transduction efficiencies may allow novel approaches to functional studies of the motor system and the treatment of neuromuscular disease. PMID:16725205

  4. Characterization of retrovirus-based reporter viruses pseudotyped with the precursor membrane and envelope glycoproteins of four serotypes of dengue viruses

    SciTech Connect

    Hu, H.-P.; Hsieh, S.-C.; King, C.-C.; Wang, W.-K.

    2007-11-25

    In this study, we successfully established retrovirus-based reporter viruses pseudotyped with the precursor membrane and envelope (PrM/E) proteins of each of the four serotypes of dengue viruses, which caused the most important arboviral diseases in this century. Co-sedimentation of the dengue E protein and HIV-1 core proteins by sucrose gradient analysis of the pseudotype reporter virus of dengue virus type 2, D2(HIVluc), and detection of HIV-1 core proteins by immunoprecipitation with anti-E monoclonal antibody suggested that dengue viral proteins were incorporated into the pseudotype viral particles. The infectivity in target cells, as assessed by the luciferase activity, can be inhibited by the lysosomotropic agents, suggesting a pH-dependent mechanism of entry. Amino acid substitutions of the leucine at position 107, a critical residue at the fusion loop of E protein, with lysine resulted in severe impairment in infectivity, suggesting that entry of the pseudotype reporter virus is mediated through the fusogenic properties of E protein. With more and more dengue viral sequences available from different outbreaks worldwide, this sensitive and convenient tool has the potential to facilitate molecular characterization of the PrM/E proteins of dengue field isolates.

  5. TRIM5 acts as more than a retroviral restriction factor.

    PubMed

    de Silva, Suresh; Wu, Li

    2011-07-01

    The retrovirus restriction factor TRIM5α blocks post-entry infection of retroviruses in a species-specific manner. As a cellular E3 ubiquitin ligase, TRIM5α binds to the retroviral capsid lattice in the cytoplasm of an infected cell and accelerates the uncoating process of retroviral capsid, thus providing a potent restriction to HIV-1 and other retrovirus infections. The precise mechanism by which this restriction is imposed remains under scrutiny, and evidence is lacking to link the E3 ubiquitin ligase activity of TRIM5α to its ability to restrict retrovirus infection. In a recent study, Pertel and colleagues have uncovered the link between the two, providing compelling evidence to suggest that following the interaction with the retroviral capsid, TRIM5 triggers an antiviral innate immune response by functioning as a pattern recognition receptor. This unique function of TRIM5 is dependent on its association with the E2 ubiquitin-conjugating enzyme complex UBC13-UEV1A and subsequent activation of the TAK1 kinase complex and downstream genes involved in innate immune responses. These findings have defined a novel function for TRIM5 as a pattern recognition receptor in innate immune recognition and provided valuable mechanistic insight into its role as a retroviral restriction factor. Here we discuss the significance of these new findings in understanding TRIM5-mediated HIV restriction. PMID:21866272

  6. PEGylated Cationic Serum Albumin for Boosting Retroviral Gene Transfer.

    PubMed

    Palesch, David; Boldt, Felix; Müller, Janis A; Eisele, Klaus; Stürzel, Christina M; Wu, Yuzhou; Münch, Jan; Weil, Tanja

    2016-08-17

    Retroviral vectors are common tools for introducing genes into the genome of a cell. However, low transduction rates are a major limitation in retroviral gene transfer, especially in clinical applications. We generated cationic human serum albumin (cHSA) protected by a shell of poly(ethylene glycol) (PEG); this significantly enhanced retroviral gene transduction with potentially attractive pharmacokinetics and low immunogenicity. By screening a panel of chemically optimized HSA compounds, we identified a very potent enhancer that boosted the transduction rates of viral vectors. Confocal microscopy revealed a drastically increased number of viral particles attached to the surfaces of target cells. In accordance with the positive net charge of cationic and PEGylated HSA, this suggests a mechanism of action in which the repulsion of the negatively charged cellular and viral vector membranes is neutralized, thereby promoting attachment and ultimately transduction. Importantly, the transduction-enhancing PEGylated HSA derivative evaded recognition by HSA-specific antibodies and macrophage activation. Our findings hold great promise for facilitating improved retroviral gene transfer. PMID:27239020

  7. Production in yeast of pseudotype virus-like particles harboring functionally active antibody fragments neutralizing the cytolytic activity of vaginolysin

    PubMed Central

    2011-01-01

    Background Recombinant antibodies can be produced in different formats and different expression systems. Single chain variable fragments (scFvs) represent an attractive alternative to full-length antibodies and they can be easily produced in bacteria or yeast. However, the scFvs exhibit monovalent antigen-binding properties and short serum half-lives. The stability and avidity of the scFvs can be improved by their multimerization or fusion with IgG Fc domain. The aim of the current study was to investigate the possibilities to produce in yeast high-affinity scFv-Fc proteins neutralizing the cytolytic activity of vaginolysin (VLY), the main virulence factor of Gardnerella vaginalis. Results The scFv protein derived from hybridoma cell line producing high-affinity neutralizing antibodies against VLY was fused with human IgG1 Fc domain. Four different variants of anti-VLY scFv-Fc fusion proteins were constructed and produced in yeast Saccharomyces cerevisiae. The non-tagged scFv-Fc and hexahistidine-tagged scFv-Fc proteins were found predominantly as insoluble aggregates and therefore were not suitable for further purification and activity testing. The addition of yeast α-factor signal sequence did not support secretion of anti-VLY scFv-Fc but increased the amount of its intracellular soluble form. However, the purified protein showed a weak VLY-neutralizing capability. In contrast, the fusion of anti-VLY scFv-Fc molecules with hamster polyomavirus-derived VP2 protein and its co-expression with VP1 protein resulted in an effective production of pseudotype virus-like particles (VLPs) that exhibited strong VLY-binding activity. Recombinant scFv-Fc molecules displayed on the surface of VLPs neutralized VLY-mediated lysis of human erythrocytes and HeLa cells with high potency comparable to that of full-length antibody. Conclusions Recombinant scFv-Fc proteins were expressed in yeast with low efficiency. New approach to display the scFv-Fc molecules on the surface of

  8. Convergent capture of retroviral superantigens by mammalian herpesviruses.

    PubMed

    Aswad, Amr; Katzourakis, Aris

    2015-01-01

    Horizontal gene transfer from retroviruses to mammals is well documented and extensive, but is rare between unrelated viruses with distinct genome types. Three herpesviruses encode a gene with similarity to a retroviral superantigen gene (sag) of the unrelated mouse mammary tumour virus (MMTV). We uncover ancient retroviral sags in over 20 mammals to reconstruct their shared history with herpesviral sags, revealing that the acquisition is a convergent evolutionary event. A retrovirus circulating in South American primates over 10 million years ago was the source of sag in two monkey herpesviruses, and a different retrovirus was the source of sag in a Peruvian rodent herpesvirus. We further show through a timescaled phylogenetic analysis that a cross-species transmission of monkey herpesviruses occurred after the acquisition of sag. These results reveal that a diverse range of ancient sag-containing retroviruses independently donated sag twice from two separate lineages that are distinct from MMTV. PMID:26400439

  9. Convergent capture of retroviral superantigens by mammalian herpesviruses

    PubMed Central

    Aswad, Amr; Katzourakis, Aris

    2015-01-01

    Horizontal gene transfer from retroviruses to mammals is well documented and extensive, but is rare between unrelated viruses with distinct genome types. Three herpesviruses encode a gene with similarity to a retroviral superantigen gene (sag) of the unrelated mouse mammary tumour virus (MMTV). We uncover ancient retroviral sags in over 20 mammals to reconstruct their shared history with herpesviral sags, revealing that the acquisition is a convergent evolutionary event. A retrovirus circulating in South American primates over 10 million years ago was the source of sag in two monkey herpesviruses, and a different retrovirus was the source of sag in a Peruvian rodent herpesvirus. We further show through a timescaled phylogenetic analysis that a cross-species transmission of monkey herpesviruses occurred after the acquisition of sag. These results reveal that a diverse range of ancient sag-containing retroviruses independently donated sag twice from two separate lineages that are distinct from MMTV. PMID:26400439

  10. Retroviral Transduction of T Cells and T Cell Precursors.

    PubMed

    Simmons, Amie; Alberola-Ila, José

    2016-01-01

    Transduction of lymphoid progenitors with retroviral or lentiviral vectors is a powerful experimental strategy to tease out the role of a gene or pathway in T cell development via gain-of-function or loss-of-function strategies. Here we discuss different approaches to use this powerful technology, and present some protocols that we use to transduce murine HSCs, thymocytes, and lymphoid cell lines with these viral vectors. PMID:26294401

  11. Intrinsic retroviral reactivation in human preimplantation embryos and pluripotent cells.

    PubMed

    Grow, Edward J; Flynn, Ryan A; Chavez, Shawn L; Bayless, Nicholas L; Wossidlo, Mark; Wesche, Daniel J; Martin, Lance; Ware, Carol B; Blish, Catherine A; Chang, Howard Y; Pera, Renee A Reijo; Wysocka, Joanna

    2015-06-11

    Endogenous retroviruses (ERVs) are remnants of ancient retroviral infections, and comprise nearly 8% of the human genome. The most recently acquired human ERV is HERVK(HML-2), which repeatedly infected the primate lineage both before and after the divergence of the human and chimpanzee common ancestor. Unlike most other human ERVs, HERVK retained multiple copies of intact open reading frames encoding retroviral proteins. However, HERVK is transcriptionally silenced by the host, with the exception of in certain pathological contexts such as germ-cell tumours, melanoma or human immunodeficiency virus (HIV) infection. Here we demonstrate that DNA hypomethylation at long terminal repeat elements representing the most recent genomic integrations, together with transactivation by OCT4 (also known as POU5F1), synergistically facilitate HERVK expression. Consequently, HERVK is transcribed during normal human embryogenesis, beginning with embryonic genome activation at the eight-cell stage, continuing through the emergence of epiblast cells in preimplantation blastocysts, and ceasing during human embryonic stem cell derivation from blastocyst outgrowths. Remarkably, we detected HERVK viral-like particles and Gag proteins in human blastocysts, indicating that early human development proceeds in the presence of retroviral products. We further show that overexpression of one such product, the HERVK accessory protein Rec, in a pluripotent cell line is sufficient to increase IFITM1 levels on the cell surface and inhibit viral infection, suggesting at least one mechanism through which HERVK can induce viral restriction pathways in early embryonic cells. Moreover, Rec directly binds a subset of cellular RNAs and modulates their ribosome occupancy, indicating that complex interactions between retroviral proteins and host factors can fine-tune pathways of early human development. PMID:25896322

  12. Retroviral vectors for the transduction of autoregulated, bidirectional expression cassettes.

    PubMed

    Unsinger, J; Kröger, A; Hauser, H; Wirth, D

    2001-11-01

    Regulated transgene expression is increasingly used in research but is also needed for certain therapies. Regulatory systems are usually composed of two expression units, one bearing the gene of interest under control of a regulatable promoter and the other, a constitutively expressed transactivator that modulates the activity of the regulatable promoter. Because the cotransfer of two independent elements is not efficient in primary cells, single transduction step vectors conferring regulatable gene expression cassettes would be helpful. We have developed retroviral vectors containing an autoregulatory bidirectional expression cassette that encodes all components necessary for regulated expression of a gene of interest. The influence of the orientation of the reporter gene with respect to the viral long terminal repeat (LTR) and the effect of transcriptionally inactive LTRs were investigated using mouse leukemia virus (MLV) and self-inactivating (SIN)-based retroviral vectors. Strict regulation was observed when the reporter was inserted in antisense orientation with respect to the LTR, whereas a sense arrangement of the reporter resulted in a loss of regulation capacity. Expression and regulation of the antisense-orientated reporter gene were homogenous in infected cell pools and investigated cell clones. Long-term observations of infected cells over a period of 30 passages revealed stable expression and regulation. These autoregulated, bidirectional retroviral vectors combine the advantages of single-step transduction with strict regulation of the gene of interest in the infected target cells. PMID:11708885

  13. Repair of gaps in retroviral DNA integration intermediates.

    PubMed

    Yoder, K E; Bushman, F D

    2000-12-01

    Diverse mobile DNA elements are believed to pirate host cell enzymes to complete DNA transfer. Prominent examples are provided by retroviral cDNA integration and transposon insertion. These reactions initially involve the attachment of each element 3' DNA end to staggered sites in the host DNA by element-encoded integrase or transposase enzymes. Unfolding of such intermediates yields DNA gaps at each junction. It has been widely assumed that host DNA repair enzymes complete attachment of the remaining DNA ends, but the enzymes involved have not been identified for any system. We have synthesized DNA substrates containing the expected gap and 5' two-base flap structure present in retroviral integration intermediates and tested candidate enzymes for the ability to support repair in vitro. We find three required activities, two of which can be satisfied by multiple enzymes. These are a polymerase (polymerase beta, polymerase delta and its cofactor PCNA, or reverse transcriptase), a nuclease (flap endonuclease), and a ligase (ligase I, III, or IV and its cofactor XRCC4). A proposed pathway involving retroviral integrase and reverse transcriptase did not carry out repair under the conditions tested. In addition, prebinding of integrase protein to gapped DNA inhibited repair reactions, indicating that gap repair in vivo may require active disassembly of the integrase complex. PMID:11070016

  14. Efficient conditional gene expression following transplantation of retrovirally transduced bone marrow stem cells.

    PubMed

    Chung, Jie-Yu; Mackay, Fabienne; Alderuccio, Frank

    2015-01-01

    Retroviral gene therapy combined with bone marrow stem cell transplantation can be used to generate mice with ectopic gene expression in the bone marrow compartment in a quick and cost effective manner when compared to generating and maintaining transgenic mouse lines. However a limitation of this procedure is the lack of cell specificity in gene expression that is associated with the use of endogenous retroviral promoters. Restricting gene expression to specific cell subsets utilising tissue-specific promoter driven retroviral vectors is a challenge. Here we describe the generation of conditional expression of retrovirally encoded genes in specific bone marrow derived cell lineages utilising a Cre-dependent retroviral vector. By utilising Lck and CD19 restricted Cre transgenic bone marrow stem cells, we generate chimeric animals with T or B lymphocyte restricted gene expression respectively. The design of the Cre-dependent retroviral vector enables expression of encoded MOG and GFP genes only in association with Cre mediated DNA inversion. Importantly this strategy does not significantly increase the size of the retroviral vector; as such we are able to generate bone marrow chimeric animals with significantly higher chimerism levels than previous studies utilising Cre-dependent retroviral vectors and Cre transgenic bone marrow stem cells. This demonstrates that the use of Cre-dependent retroviral vectors is able to yield high chimerism levels for experimental use and represent a viable alternative to generating transgenic animals. PMID:25445328

  15. A pseudotype baculovirus expressing the capsid protein of foot-and-mouth disease virus and a T-Cell immunogen shows enhanced immunogenicity in mice

    PubMed Central

    2011-01-01

    Background Foot-and-mouth disease (FMD) is a highly contagious disease of livestock which causes severe economic loss in cloven-hoofed animals. Vaccination is still a major strategy in developing countries to control FMD. Currently, inactivated vaccine of FMDV has been used in many countries with limited success and safety concerns. Development of a novel effective vaccine is must. Methods In the present study, two recombinant pseudotype baculoviruses, one expressing the capsid of foot-and-mouth disease virus (FMDV) under the control of a cytomegalovirus immediate early enhancer/promoter (CMV-IE), and the other the caspid plus a T-cell immunogen coding region under a CAG promoter were constructed, and their expression was characterized in mammalian cells. In addition, their immunogenicity in a mouse model was investigated. The humoral and cell-mediated immune responses induced by pseudotype baculovirus were compared with those of inactivated vaccine. Results Indirect immunofluorescence assay (IFA) and indirect sandwich-ELISA (IS-ELISA) showed both recombinant baculoviruses (with or without T-cell epitopes) were transduced efficiently and expressed target proteins in BHK-21 cells. In mice, intramuscular inoculation of recombinants with 1 × 109 or 1 × 1010 PFU/mouse induced the production of FMDV-specific neutralizing antibodies and gamma interferon (IFN-γ). Furthermore, recombinant baculovirus with T-cell epitopes had better immunogenicity than the recombinant without T-cell epitopes as demonstrated by significantly enhanced IFN-γ production (P < 0.01) and higher neutralizing antibody titer (P < 0.05). Although the inactivated vaccine produced the highest titer of neutralizing antibodies, a lower IFN-γ expression was observed compared to the two recombinant pseudotype baculoviruses. Conclusions These results indicate that pseudotype baculovirus-mediated gene delivery could be a alternative strategy to develop a new generation of vaccines against FMDV infection

  16. Critical Variables affecting clinical-grade production of the self-inactivating gamma-retroviral vector for the treatment of X-linked severe combined immunodeficiency

    PubMed Central

    van der Loo, JCM; Swaney, WP; Grassman, E; Terwilliger, A; Higashimoto, T; Schambach, A; Hacein-Bey-Abina, S; Nordling, DL; Cavazzana-Calvo, M; Thrasher, AJ; Williams, DA; Reeves, L; Malik, P

    2014-01-01

    Patients with X-linked severe combined immunodeficiency (SCID-X1) were successfully cured following gene therapy with a gamma-retroviral vector (gRV) expressing the common gamma chain of the interleukin-2 receptor (IL2RG). However, 5 of 20 patients developed leukemia from activation of cellular proto-oncogenes by viral enhancers in the long-terminal repeats (LTR) of the integrated vector. These events prompted the design of a gRV vector with self-inactivating (SIN) LTRs to enhance vector safety. Herein we report on the production of a clinical-grade SIN IL2RG gRV pseudotyped with the Gibbon Ape Leukemia Virus envelope for a new gene therapy trial for SCID-X1, and highlight variables that were found to be critical for transfection-based large-scale SIN gRV production. Successful clinical production required careful selection of culture medium without pre-added glutamine, reduced exposure of packaging cells to cell-dissociation enzyme, and presence of cations in wash buffer. The clinical vector was high titer; transduced 68–70% normal human CD34 + cells, as determined by colony-forming unit assays and by xenotransplantation in immunodeficient NOD.CB17-Prkdcscid/J (nonobese diabetic/severe combined immunodeficiency (NOD/SCID)) and NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NOD/SCID gamma (NSG))) mice; and resulted in the production of T cells in vitro from human SCID-X1 CD34 + cells. The vector was certified and released for the treatment of SCID-X1 in a multi-center international phase I/II trial. PMID:22551777

  17. Split-Intron Retroviral Vectors: Enhanced Expression with Improved Safety

    PubMed Central

    Ismail, Said I.; Kingsman, Susan M.; Kingsman, Alan J.; Uden, Mark

    2000-01-01

    The inclusion of retrovirus-derived introns within retrovirus-based expression vectors leads to a fraction of the resulting transcripts being spliced. Such splicing has been shown to markedly improve expression (W. J. Krall et al., Gene Ther. 3:37–48, 1996). One way to improve upon this still further might involve the use of more efficient introns instead of those from the provirus. Currently, however, incorporation of such introns remains self-defeating since they are removed in the nucleus of the producer cell. In the past, elaborate ways to overcome this problem have included the use of alphaviruses to make the vector transcripts within the cytoplasm, thus avoiding the nuclear splicing machinery during vector production (K. J. Li and H. Garoff, Proc. Natl. Acad. Sci. USA 95:3650–3654, 1998). We now present a novel design for the inclusion of introns within a retroviral vector. In essence, this is achieved by exploiting the retroviral replication process to copy not only the U3 promoter but also a synthetic splice donor to the 5′-long-terminal-repeat position during reverse transcription. Once copied, synthesized transcripts then contain a splice donor at their 5′ end capable of interacting with a consensus splice acceptor engineered downstream of the packaging signal. Upon transduction, we demonstrate these vectors to produce enhanced expression from near fully spliced (and thus packaging signal minus) transcripts. The unique design of these high titer and high-expression retroviral vectors may be of use in a number of gene therapy applications. PMID:10666267

  18. Split-intron retroviral vectors: enhanced expression with improved safety.

    PubMed

    Ismail, S I; Kingsman, S M; Kingsman, A J; Uden, M

    2000-03-01

    The inclusion of retrovirus-derived introns within retrovirus-based expression vectors leads to a fraction of the resulting transcripts being spliced. Such splicing has been shown to markedly improve expression (W. J. Krall et al., Gene Ther. 3:37-48, 1996). One way to improve upon this still further might involve the use of more efficient introns instead of those from the provirus. Currently, however, incorporation of such introns remains self-defeating since they are removed in the nucleus of the producer cell. In the past, elaborate ways to overcome this problem have included the use of alphaviruses to make the vector transcripts within the cytoplasm, thus avoiding the nuclear splicing machinery during vector production (K. J. Li and H. Garoff, Proc. Natl. Acad. Sci. USA 95:3650-3654, 1998). We now present a novel design for the inclusion of introns within a retroviral vector. In essence, this is achieved by exploiting the retroviral replication process to copy not only the U3 promoter but also a synthetic splice donor to the 5'-long-terminal-repeat position during reverse transcription. Once copied, synthesized transcripts then contain a splice donor at their 5' end capable of interacting with a consensus splice acceptor engineered downstream of the packaging signal. Upon transduction, we demonstrate these vectors to produce enhanced expression from near fully spliced (and thus packaging signal minus) transcripts. The unique design of these high titer and high-expression retroviral vectors may be of use in a number of gene therapy applications. PMID:10666267

  19. Retroviral Transcriptional Regulation and Embryonic Stem Cells: War and Peace

    PubMed Central

    Schlesinger, Sharon

    2014-01-01

    Retroviruses have evolved complex transcriptional enhancers and promoters that allow their replication in a wide range of tissue and cell types. Embryonic stem (ES) cells, however, characteristically suppress transcription of proviruses formed after infection by exogenous retroviruses and also of most members of the vast array of endogenous retroviruses in the genome. These cells have unusual profiles of transcribed genes and are poised to make rapid changes in those profiles upon induction of differentiation. Many of the transcription factors in ES cells control both host and retroviral genes coordinately, such that retroviral expression patterns can serve as markers of ES cell pluripotency. This overlap is not coincidental; retrovirus-derived regulatory sequences are often used to control cellular genes important for pluripotency. These sequences specify the temporal control and perhaps “noisy” control of cellular genes that direct proper cell gene expression in primitive cells and their differentiating progeny. The evidence suggests that the viral elements have been domesticated for host needs, reflecting the wide-ranging exploitation of any and all available DNA sequences in assembling regulatory networks. PMID:25547290

  20. Retroviral vectors for homologous recombination provide efficient cloning and expression in mammalian cells.

    PubMed

    Kobayashi, Eiji; Kishi, Hiroyuki; Ozawa, Tatsuhiko; Horii, Masae; Hamana, Hiroshi; Nagai, Terumi; Muraguchi, Atsushi

    2014-02-14

    Homologous recombination technologies enable high-throughput cloning and the seamless insertion of any DNA fragment into expression vectors. Additionally, retroviral vectors offer a fast and efficient method for transducing and expressing genes in mammalian cells, including lymphocytes. However, homologous recombination cannot be used to insert DNA fragments into retroviral vectors; retroviral vectors contain two homologous regions, the 5'- and 3'-long terminal repeats, between which homologous recombination occurs preferentially. In this study, we have modified a retroviral vector to enable the cloning of DNA fragments through homologous recombination. To this end, we inserted a bacterial selection marker in a region adjacent to the gene insertion site. We used the modified retroviral vector and homologous recombination to clone T-cell receptors (TCRs) from single Epstein Barr virus-specific human T cells in a high-throughput and comprehensive manner and to efficiently evaluate their function by transducing the TCRs into a murine T-cell line through retroviral infection. In conclusion, the modified retroviral vectors, in combination with the homologous recombination method, are powerful tools for the high-throughput cloning of cDNAs and their efficient functional analysis. PMID:24462869

  1. Retroviral vector performance in defined chromosomal Loci of modular packaging cell lines.

    PubMed

    Gama-Norton, L; Herrmann, S; Schucht, R; Coroadinha, A S; Löw, R; Alves, P M; Bartholomae, C C; Schmidt, M; Baum, C; Schambach, A; Hauser, H; Wirth, D

    2010-08-01

    The improvement of safety and titer of retroviral vectors produced in standard retroviral packaging cell lines is hampered because production relies on uncontrollable vector integration events. The influences of chromosomal surroundings make it difficult to dissect the performance of a specific vector from the chromosomal surroundings of the respective integration site. Taking advantage of a technology that relies on the use of packaging cell lines with predefined integration sites, we have systematically evaluated the performance of several retroviral vectors. In two previously established modular packaging cell lines (Flp293A and 293 FLEX) with single, defined chromosomal integration sites, retroviral vectors were integrated by means of Flp-mediated site-specific recombination. Vectors that are distinguished by different long terminal repeat promoters were introduced in either the sense or reverse orientation. The results show that the promoter, viral vector orientation, and integration site are the main determinants of the titer. Furthermore, we exploited the viral production systems to evaluate read-through activity. Read-through is thought to be caused by inefficient termination of vector transcription and is inherent to the nature of retroviral vectors. We assessed the frequency of transduction of sequences flanking the retroviral vectors from both integration sites. The approach presented here provides a platform for systematic design and evaluation of the efficiency and safety of retroviral vectors optimized for a given producer cell line. PMID:20222806

  2. Detection of a human intracisternal A-type retroviral particle antigenically related to HIV

    NASA Technical Reports Server (NTRS)

    Garry, R. F.; Fermin, C. D.; Hart, D. J.; Alexander, S. S.; Donehower, L. A.; Luo-Zhang, H.

    1990-01-01

    Sjogren's syndrome is an autoimmune disease that is characterized by dryness of the mouth and eyes. The loss of salivary and lacrimal gland function is accompanied by lymphocytic infiltration. Because similar symptoms and glandular pathology are observed in certain persons infected with human immunodeficiency virus (HIV), a search was initiated for a possible retroviral etiology in this syndrome. A human intracisternal A-type retroviral particle that is antigenically related to HIV was detected in lymphoblastoid cells exposed to homogenates of salivary tissue from patients with Sjogren's syndrome. Comparison of this retroviral particle to HIV indicates that they are distinguishable by several ultrastructural, physical, and enzymatic criteria.

  3. Mechanisms and Factors that Influence High Frequency Retroviral Recombination

    PubMed Central

    Delviks-Frankenberry, Krista; Galli, Andrea; Nikolaitchik, Olga; Mens, Helene; Pathak, Vinay K.; Hu, Wei-Shau

    2011-01-01

    With constantly changing environmental selection pressures, retroviruses rely upon recombination to reassort polymorphisms in their genomes and increase genetic diversity, which improves the chances for the survival of their population. Recombination occurs during DNA synthesis, whereby reverse transcriptase undergoes template switching events between the two copackaged RNAs, resulting in a viral recombinant with portions of the genetic information from each parental RNA. This review summarizes our current understanding of the factors and mechanisms influencing retroviral recombination, fidelity of the recombination process, and evaluates the subsequent viral diversity and fitness of the progeny recombinant. Specifically, the high mutation rates and high recombination frequencies of HIV-1 will be analyzed for their roles in influencing HIV-1 global diversity, as well as HIV-1 diagnosis, drug treatment, and vaccine development. PMID:21994801

  4. Adherence to Anti Retroviral Therapy (ART) During Muslim Ramadan Fasting

    PubMed Central

    Habib, A. G.; Shepherd, J. C.; Eng, M. K. L.; Babashani, M.; Jumare, J.; Yakubu, U.; Gebi, U. I.; Saad, M.; Ibrahim, H.; Blattner, W. A.

    2010-01-01

    Annual fasting during the month of Ramadan is observed in Muslim countries, some of which have widespread HIV infection. We studied treatment adherence and customary practices among 142 fasting `FT' and 101 non-fasting `NFT' patients on anti-retroviral therapy (ART) in Nigeria. Adherence on ART among FT and NFT patients was similar during Ramadan, 96% and 98%, and ever since commencement of ART, 80% and 88%, respectively. FT patients altered their typical daily behaviors by advancing morning and delaying evening doses thereby prolonging dosing intervals, eating heavier meals pre-dawn and on breakfast at sunset (78%), and changing or reducing their sleeping and waking times (40%). This preliminary study suggests that adherence and drug taking frequency appear uncompromised in FT HIV infected patients on ARVs. PMID:18521736

  5. CCR5 Gene Editing of Resting CD4+ T Cells by Transient ZFN Expression From HIV Envelope Pseudotyped Nonintegrating Lentivirus Confers HIV-1 Resistance in Humanized Mice

    PubMed Central

    Yi, Guohua; Choi, Jang Gi; Bharaj, Preeti; Abraham, Sojan; Dang, Ying; Kafri, Tal; Alozie, Ogechika; Manjunath, Manjunath N; Shankar, Premlata

    2014-01-01

    CCR5 disruption by zinc finger nucleases (ZFNs) is a promising method for HIV-1 gene therapy. However, successful clinical translation of this strategy necessitates the development of a safe and effective method for delivery into relevant cells. We used non-integrating lentivirus (NILV) for transient expression of ZFNs and pseudotyped the virus with HIV-envelope for targeted delivery to CD4+ T cells. Both activated and resting primary CD4+ T cells transduced with CCR5-ZFNs NILV showed resistance to HIV-1 infection in vitro. Furthermore, NILV transduced resting CD4+ T cells from HIV-1 seronegative individuals were resistant to HIV-1 challenge when reconstituted into NOD-scid IL2rγc null (NSG) mice. Likewise, endogenous virus replication was suppressed in NSG mice reconstituted with CCR5-ZFN–transduced resting CD4+ T cells from treatment naïve as well as ART-treated HIV-1 seropositive patients. Taken together, NILV pseudotyped with HIV envelope provides a simple and clinically viable strategy for HIV-1 gene therapy. PMID:25268698

  6. Retroviral DNA Integration Directed by HIV Integration Protein in Vitro

    NASA Astrophysics Data System (ADS)

    Bushman, Frederic D.; Fujiwara, Tamio; Craigie, Robert

    1990-09-01

    Efficient retroviral growth requires integration of a DNA copy of the viral RNA genome into a chromosome of the host. As a first step in analyzing the mechanism of integration of human immunodeficiency virus (HIV) DNA, a cell-free system was established that models the integration reaction. The in vitro system depends on the HIV integration (IN) protein, which was partially purified from insect cells engineered to express IN protein in large quantities. Integration was detected in a biological assay that scores the insertion of a linear DNA containing HIV terminal sequences into a λ DNA target. Some integration products generated in this assay contained five-base pair duplications of the target DNA at the recombination junctions, a characteristic of HIV integration in vivo; the remaining products contained aberrant junctional sequences that may have been produced in a variation of the normal reaction. These results indicate that HIV IN protein is the only viral protein required to insert model HIV DNA sequences into a target DNA in vitro.

  7. Retroviral Vectors for Analysis of Viral Mutagenesis and Recombination

    PubMed Central

    Rawson, Jonathan M.O.; Mansky, Louis M.

    2014-01-01

    Retrovirus population diversity within infected hosts is commonly high due in part to elevated rates of replication, mutation, and recombination. This high genetic diversity often complicates the development of effective diagnostics, vaccines, and antiviral drugs. This review highlights the diverse vectors and approaches that have been used to examine mutation and recombination in retroviruses. Retroviral vectors for these purposes can broadly be divided into two categories: those that utilize reporter genes as mutation or recombination targets and those that utilize viral genes as targets of mutation or recombination. Reporter gene vectors greatly facilitate the detection, quantification, and characterization of mutants and/or recombinants, but may not fully recapitulate the patterns of mutagenesis or recombination observed in native viral gene sequences. In contrast, the detection of mutations or recombination events directly in viral genes is more biologically relevant but also typically more challenging and inefficient. We will highlight the advantages and disadvantages of the various vectors and approaches used as well as propose ways in which they could be improved. PMID:25254386

  8. Epigenetics, drugs of abuse, and the retroviral promoter

    PubMed Central

    Shirazi, Jasmine; Shah, Sonia; Sagar, Divya; Nonnemacher, Michael R.; Wigdahl, Brian; Khan, Zafar K.; Jain, Pooja

    2013-01-01

    Drug abuse alone has been shown to cause epigenetic changes in brain tissue that have been shown to play roles in addictive behaviors. In conjunction with HIV-1 infection, it can cause epigenetic changes at the viral promoter that can result in altered gene expression, and exacerbate disease progression overall. This review entails an in-depth look at research conducted on the epigenetic effects of three of the most widely abused drugs (cannabinoids, opioids, and cocaine), with a particular focus on the mechanisms through which these drugs interact with HIV-1 infection at the viral promoter. Here we discuss the impact of this interplay on disease progression from the point of view of the nature of gene regulation at the level of chromatin accessibility, chromatin remodeling, and nucleosome repositioning. Given the importance of chromatin remodeling and DNA methylation in controlling the retroviral promoter, and the high susceptibility of the drug abusing population of individuals to HIV infection, it would be beneficial to understand the way in which the host genome is modified and regulated by drugs of abuse. PMID:24218017

  9. The Effect of Life History on Retroviral Genome Invasions

    PubMed Central

    Kanda, Ravinder K.; Coulson, Tim

    2015-01-01

    Endogenous retroviruses (ERV), or the remnants of past retroviral infections that are no longer active, are found in the genomes of most vertebrates, typically constituting approximately 10% of the genome. In some vertebrates, particularly in shorter-lived species like rodents, it is not unusual to find active endogenous retroviruses. In longer-lived species, including humans where substantial effort has been invested in searching for active ERVs, it is unusual to find them; to date none have been found in humans. Presumably the chance of detecting an active ERV infection is a function of the length of an ERV epidemic. Intuitively, given that ERVs or signatures of past ERV infections are passed from parents to offspring, we might expect to detect more active ERVs in species with longer generation times, as it should take more years for an infection to run its course in longer than in shorter lived species. This means the observation of more active ERV infections in shorter compared to longer-lived species is paradoxical. We explore this paradox using a modeling approach to investigate factors that influence ERV epidemic length. Our simple epidemiological model may explain why we find evidence of active ERV infections in shorter rather than longer-lived species. PMID:25692467

  10. A revised nomenclature for transcribed human endogenous retroviral loci

    PubMed Central

    2011-01-01

    Background Endogenous retroviruses (ERVs) and ERV-like sequences comprise 8% of the human genome. A hitherto unknown proportion of ERV loci are transcribed and thus contribute to the human transcriptome. A small proportion of these loci encode functional proteins. As the role of ERVs in normal and diseased biological processes is not yet established, transcribed ERV loci are of particular interest. As more transcribed ERV loci are likely to be identified in the near future, the development of a systematic nomenclature is important to ensure that all information on each locus can be easily retrieved. Results Here we present a revised nomenclature of transcribed human endogenous retroviral loci that sorts loci into groups based on Repbase classifications. Each symbol is of the format ERV + group symbol + unique number. Group symbols are based on a mixture of Repbase designations and well-supported symbols used in the literature. The presented guidelines will allow newly identified loci to be easily incorporated into the scheme. Conclusions The naming system will be employed by the HUGO Gene Nomenclature Committee for naming transcribed human ERV loci. We hope that the system will contribute to clarifying a certain aspect of a sometimes confusing nomenclature for human endogenous retroviruses. The presented system may also be employed for naming transcribed loci of human non-ERV repeat loci. PMID:21542922

  11. Evaluation of retroviral vector design in defined chromosomal loci by Flp-mediated cassette replacement.

    PubMed

    Verhoeyen, E; Hauser, H; Wirth, D

    2001-05-20

    Successful retroviral vector construction is still empirical. Test systems for vector efficiency are based on statistical comparison of numerous infectants with single proviral integrates, since their expression depends on the chromosomal surroundings. More reliable data would be obtained if different vector constructs were studied in an identical chromosomal context. Here, we demonstrate the use of a new method, in which chromosomal sites are provirally tagged in such a way that they can be targeted with other expression cassettes. The original tagging integrate is replaced in one step by the targeting element. This permits a reliable comparison of different retroviral vector configurations, eliminating the influence of neighboring chromosomal elements. We compared different retroviral vector types for coexpression of two genes: a vector containing an internal promoter and a vector with an internal ribosome entry site (IRES) element. In contrast to bicistronic retroviral vectors, dual-promoter proviruses exhibited rapid inactivation of the long terminal repeat (LTR)-driven gene expression. Targeted exchange of the dual-promoter provirus with a bicistronic retroviral cassette resulted in gain of expression stability. The reverse experiment confirmed this promoter interaction phenomenon since initial expression stability from a single-promoter bicistronic provirus was lost by targeted exchange with a dual-promoter cassette. In addition, targeting exchange of the dual-promoter provirus, replacing the LTR with an artificial (Tet) promoter restored expression stability. These observations, valid for various integration sites, prove the strong interaction between the LTR and the internal promoter. Our results have implications for retroviral vector design and suggest that retroviral coexpression of two genes is more predictable in the bicistronic configuration. PMID:11387058

  12. Pseudotyping Serotype 5 Adenovirus with the Fiber from Other Serotypes Uncovers a Key Role of the Fiber Protein in Adenovirus 5-Induced Thrombocytopenia.

    PubMed

    Raddi, Najat; Vigant, Frédéric; Wagner-Ballon, Oriane; Giraudier, Stéphane; Custers, Jerome; Hemmi, Silvio; Benihoud, Karim

    2016-02-01

    Adenovirus (Ad) infection in humans is associated with inflammatory responses and thrombocytopenia. Although several studies were conducted in mice models to understand molecular and cellular mechanisms of Ad-induced inflammatory responses, only few of them turned their interest toward the mechanisms of Ad-induced thrombocytopenia. Using different depletion methods, the present study ruled out any significant role of spleen, macrophages, and vitamin K-dependent factor in Ad-induced thrombocytopenia. Interestingly, mice displaying thrombocytopenia expressed high levels of cytokines/chemokines after Ad administration. Most importantly, pseudotyping adenovirus with the fiber protein from other serotypes was associated with reduction of both cytokine/chemokine production and thrombocytopenia. Altogether, our results suggest that capsid fiber protein (and more precisely its shaft) of Ad serotype 5 triggers the cytokine production that leads to Ad-induced thrombocytopenia. PMID:26757054

  13. Peptide nanofibrils boost retroviral gene transfer and provide a rapid means for concentrating viruses

    NASA Astrophysics Data System (ADS)

    Yolamanova, Maral; Meier, Christoph; Shaytan, Alexey K.; Vas, Virag; Bertoncini, Carlos W.; Arnold, Franziska; Zirafi, Onofrio; Usmani, Shariq M.; Müller, Janis A.; Sauter, Daniel; Goffinet, Christine; Palesch, David; Walther, Paul; Roan, Nadia R.; Geiger, Hartmut; Lunov, Oleg; Simmet, Thomas; Bohne, Jens; Schrezenmeier, Hubert; Schwarz, Klaus; Ständker, Ludger; Forssmann, Wolf-Georg; Salvatella, Xavier; Khalatur, Pavel G.; Khokhlov, Alexei R.; Knowles, Tuomas P. J.; Weil, Tanja; Kirchhoff, Frank; Münch, Jan

    2013-02-01

    Inefficient gene transfer and low virion concentrations are common limitations of retroviral transduction. We and others have previously shown that peptides derived from human semen form amyloid fibrils that boost retroviral gene delivery by promoting virion attachment to the target cells. However, application of these natural fibril-forming peptides is limited by moderate efficiencies, the high costs of peptide synthesis, and variability in fibril size and formation kinetics. Here, we report the development of nanofibrils that self-assemble in aqueous solution from a 12-residue peptide, termed enhancing factor C (EF-C). These artificial nanofibrils enhance retroviral gene transfer substantially more efficiently than semen-derived fibrils or other transduction enhancers. Moreover, EF-C nanofibrils allow the concentration of retroviral vectors by conventional low-speed centrifugation, and are safe and effective, as assessed in an ex vivo gene transfer study. Our results show that EF-C fibrils comprise a highly versatile, convenient and broadly applicable nanomaterial that holds the potential to significantly facilitate retroviral gene transfer in basic research and clinical applications.

  14. Larger Mammalian Body Size Leads to Lower Retroviral Activity

    PubMed Central

    Katzourakis, Aris; Magiorkinis, Gkikas; Lim, Aaron G.; Gupta, Sunetra; Belshaw, Robert; Gifford, Robert

    2014-01-01

    Retroviruses have been infecting mammals for at least 100 million years, leaving descendants in host genomes known as endogenous retroviruses (ERVs). The abundance of ERVs is partly determined by their mode of replication, but it has also been suggested that host life history traits could enhance or suppress their activity. We show that larger bodied species have lower levels of ERV activity by reconstructing the rate of ERV integration across 38 mammalian species. Body size explains 37% of the variance in ERV integration rate over the last 10 million years, controlling for the effect of confounding due to other life history traits. Furthermore, 68% of the variance in the mean age of ERVs per genome can also be explained by body size. These results indicate that body size limits the number of recently replicating ERVs due to their detrimental effects on their host. To comprehend the possible mechanistic links between body size and ERV integration we built a mathematical model, which shows that ERV abundance is favored by lower body size and higher horizontal transmission rates. We argue that because retroviral integration is tumorigenic, the negative correlation between body size and ERV numbers results from the necessity to reduce the risk of cancer, under the assumption that this risk scales positively with body size. Our model also fits the empirical observation that the lifetime risk of cancer is relatively invariant among mammals regardless of their body size, known as Peto's paradox, and indicates that larger bodied mammals may have evolved mechanisms to limit ERV activity. PMID:25033295

  15. A transient three-plasmid expression system for the production of high titer retroviral vectors.

    PubMed

    Soneoka, Y; Cannon, P M; Ramsdale, E E; Griffiths, J C; Romano, G; Kingsman, S M; Kingsman, A J

    1995-02-25

    We have constructed a series of MLV-based retroviral vectors and packaging components expressed from the CMV promoter and carried on plasmids containing SV40 origins of replication. These two features greatly enhanced retroviral gene expression when introduced into cell lines carrying the SV40 large T antigen. The two packaging components, gag-pol and env, were placed on separate plasmids to reduce helper virus formation. Using a highly transfectable human cell line and sodium butyrate to further increase expression of each component, we achieved helper-free viral stocks of approximately 10(7) infectious units/ml by 48 h after transient co-transfection with the three plasmid components. This system can be used both for the generation of high titer retroviral stocks for transduction and for the rapid screening of a large number of MLV gag-pol or env mutants. PMID:7899083

  16. An XMRV Derived Retroviral Vector as a Tool for Gene Transfer

    PubMed Central

    2011-01-01

    Background Retroviral vectors are widely used tools for gene delivery and gene therapy. They are useful for gene expression studies and genetic manipulation in vitro and in vivo. Many retroviral vectors are derived from the mouse gammaretrovirus, murine leukemia virus (MLV). These vectors have been widely used in gene therapy clinical trials. XMRV, initially found in prostate cancer tissue, was the first human gammaretrovirus described. Findings We developed a new retroviral vector based on XMRV called pXC. It was developed for gene transfer to human cells and is produced by transient cotransfection of LNCaP cells with pXC and XMRV-packaging plasmids. Conclusions We demonstrated that pXC mediates expression of inserted transgenes in cell lines. This new vector will be a useful tool for gene transfer in human and non-human cell lines, including gene therapy studies. PMID:21651801

  17. Evidence for integration of retroviral vectors in a novel human repeat sequence

    SciTech Connect

    Kurdi-Haidar, B.; Friedmann, T.

    1994-09-01

    Retroviruses have become attractive vehicles for the introduction of foreign genes into mammalian cells not only for gene therapy but also to serve as anchor points for long-range mapping purposes. The information relating to retroviral integration in mammalian cells is derived mostly from studies of rodent genomes. The absence of information regarding integration sites of murine-based retroviral vectors in human cells has prompted us to investigate the characteristics of integration sites in the human genome. We have constructed a Moloney murine leukemia virus-based retroviral vector that carries the pUC8 origin of replication and the chloramphenicol resistance gene to allow the rescue of the flanking genomic sequences in plasmid form. We have infected human primary fibroblasts and myoblasts with this retroviral vector and isolated independently transduced clones. Genomic DNA was obtained from independent clones and the genomic fragment carrying the provirus-host sequence boundary was isolated after digestion of the genomic DNA, circularization, and transformation by electroporation of E. coli C cells to chloramphenicol resistance. Restriction map and nucleotide sequence analysis of the rescued plasmids showed that a number of the clones shared the same integration site within the human genome. We have used the nucleotide sequence information about the human DNA adjacent to the 3{prime}LTR to design a PCR-based assay diagnostic for this common integration site. Analysis revealed the presence of the same integration site in four out of twelve human primary fibroblast clones infected with this specific retroviral vector, and in one out of twelve human primary myoblast clones infected with a second retroviral vector. Further analysis revealed the common integration site to be a previously unreported primate repeat present in monkey and human genomes and absent from rodent, bovine and avian genomes.

  18. Retroviral RNA identified in the cerebrospinal fluids and brains of individuals with schizophrenia

    PubMed Central

    Karlsson, Håkan; Bachmann, Silke; Schröder, Johannes; McArthur, Justin; Torrey, E. Fuller; Yolken, Robert H.

    2001-01-01

    Schizophrenia is a serious brain disease of uncertain etiology. A role for retroviruses in the etiopathogenesis of some cases of schizophrenia has been postulated on the basis of clinical and epidemiological observations. We found sequences homologous to retroviral pol genes in the cell-free cerebrospinal fluids (CSFs) of 10 of 35 (29%) individuals with recent-onset schizophrenia or schizoaffective disorder. Retroviral sequences also were identified in the CSFs of 1 of 20 individuals with chronic schizophrenia. However, retroviral sequences were not identified in any of the CSFs obtained from 22 individuals with noninflammatory neurological diseases or from 30 individuals without evidence of neurological or psychiatric diseases (χ2 = 19.25, P < 0.001). The nucleotide sequences identified in the CSFs of the individuals with schizophrenia or schizoaffective disorder were related to those of the human endogenous retroviral (HERV)-W family of endogenous retroviruses and to other retroviruses in the murine leukemia virus genus. Transcription of RNA homologous to members of the HERV-W family of retroviruses also was found to be up-regulated differentially in the frontal cortex regions of brains obtained postmortem from individuals with schizophrenia, as compared with corresponding tissue from individuals without psychiatric diseases. The transcriptional activation of certain retroviral elements within the central nervous system may be associated with the development of schizophrenia in at least some individuals. The further characterization of retroviral elements within the central nervous system of individuals with schizophrenia might lead to improved methods for the diagnosis and management of this disorder. PMID:11296294

  19. Improved retroviral suicide gene transfer in colon cancer cell lines after cell synchronization with methotrexate

    PubMed Central

    2011-01-01

    Background Cancer gene therapy by retroviral vectors is mainly limited by the level of transduction. Retroviral gene transfer requires target cell division. Cell synchronization, obtained by drugs inducing a reversible inhibition of DNA synthesis, could therefore be proposed to precondition target cells to retroviral gene transfer. We tested whether drug-mediated cell synchronization could enhance the transfer efficiency of a retroviral-mediated gene encoding herpes simplex virus thymidine kinase (HSV-tk) in two colon cancer cell lines, DHDK12 and HT29. Methods Synchronization was induced by methotrexate (MTX), aracytin (ara-C) or aphidicolin. Gene transfer efficiency was assessed by the level of HSV-TK expression. Transduced cells were driven by ganciclovir (GCV) towards apoptosis that was assessed using annexin V labeling by quantitative flow cytometry. Results DHDK12 and HT29 cells were synchronized in S phase with MTX but not ara-C or aphidicolin. In synchronized DHDK12 and HT29 cells, the HSV-TK transduction rates were 2 and 1.5-fold higher than those obtained in control cells, respectively. Furthermore, the rate of apoptosis was increased two-fold in MTX-treated DHDK12 cells after treatment with GCV. Conclusions Our findings indicate that MTX-mediated synchronization of target cells allowed a significant improvement of retroviral HSV-tk gene transfer, resulting in an increased cell apoptosis in response to GCV. Pharmacological control of cell cycle may thus be a useful strategy to optimize the efficiency of retroviral-mediated cancer gene therapy. PMID:21970612

  20. Quantum chemical modelling of reactivity and selectivity of 1,2-dithiolanes towards retroviral and cellular zinc fingers

    NASA Astrophysics Data System (ADS)

    Topol, Igor A.; Nemukhin, Alexander V.; Burt, Stanley K.

    Interactions of 1,2-dithiolane species with zinc-containing sites, which mimic the zinc finger domains of retroviral and the cellular zinc finger proteins, have been investigated by quantum chemistry tools. According to the calculations, the immediate domains of zinc binding sites in the cellular and retroviral zinc fingers interact differently with such agents of the disulphide family. Thus, when approaching the model cellular-type domains, the molecules of 1,2-dithiolanes experience considerable potential barriers along the reaction path. However, these species react practically barrier-less with the model retroviral-type domains at the correlated DFT level. The results of the quantum chemical modelling provide firm support to the selectivity of 1,2-dithiolanes towards retroviral and cellular zinc fingers. This can be of great practical importance for the design of therapeutics that accomplish functional inactivation of the zinc fingers of the human immunodeficiency virus (HIV-1) retroviral type nucleocapsid protein NCp7.

  1. Pseudotyping human immunodeficiency virus type 1 (HIV-1) by the glycoprotein of vesicular stomatitis virus targets HIV-1 entry to an endocytic pathway and suppresses both the requirement for Nef and the sensitivity to cyclosporin A.

    PubMed Central

    Aiken, C

    1997-01-01

    Human immunodeficiency virus type 1 (HIV-1) normally enters cells by direct fusion with the plasma membrane. In this report, HIV-1 particles capable of infecting cells through an endocytic pathway are described. Chimeric viruses composed of the HIV-1 core and the envelope glycoprotein of vesicular stomatitis virus (VSV-G) were constructed and are herein termed HIV-1(VSV) pseudotypes. HIV-1(VSV) pseudotypes were 20- to 130-fold more infectious than nonpseudotyped HIV-1. Infection by HIV-1(VSV) pseudotypes was markedly diminished by ammonium chloride and concanamycin A, a selective inhibitor of vacuolar H+ ATPases, demonstrating that these viruses require endosomal acidification to achieve productive infection. HIV-1 is thus capable of performing all of the viral functions necessary for infection when entry is targeted to an endocytic route. Maximal HIV-1 infectivity requires the presence of the viral Nef protein and the cellular protein cyclophilin A (CyPA) during virus assembly. Pseudotyping by VSV-G markedly suppressed the requirement for Nef. HIV-1(VSV) particles were also resistant to inhibition by cyclosporin A; however, the deleterious effect of a gag mutation inhibiting CyPA incorporation was not relieved by VSV-G. These results suggest that Nef acts at a step of the HIV-1 life cycle that is either circumvented or facilitated by targeting virus entry to an endocytic pathway. The findings also support the hypothesis that Nef and CyPA enhance HIV-1 infectivity through independent processes and demonstrate a mechanistic difference between reduction of HIV-1 infectivity by cyclosporin A and gag mutations that decrease HIV-1 incorporation of CyPA. PMID:9223476

  2. Cancellers - Exploring the Possibility of Receptor Decoy Traps As a Superior Anti-Retroviral Strategy.

    PubMed

    Jeremiah, Sundararaj Stanley; Ohba, Kenji; Yamamoto, Naoki

    2016-01-01

    The global Human Immunodeficiency Virus (HIV) pandemic is still spreading due to the lack of ideal anti-retroviral measures and their availability. Till date, all attempts to produce an efficient vaccine have ended with unsatisfactory results. The highly active anti-retroviral therapy (HAART) is the only effective weapon currently available and is widely being used for curtailing the HIV pandemic. However, the HAART is also expected to fail in the near future due to the emergence and dissemination of antiviral resistance. This review sheds light on the reasons for the failure of the conventional anti-viral measures against HIV and the novel anti-retroviral strategies currently being developed. The various principles to be considered for the success of a novel anti-retroviral strategy are elaborately emphasized and an innovative concept is proposed on these lines. The proposed concept intends to use receptor decoy traps (RDT) called cancellers which are erythrocytes expressing the HIV entry receptors on their surface. If successfully developed, the cancellers would be capable of active targeting of the free HIV particles leading to the trapping of the viruses within the canceller, resulting in the neutralization of infectivity of the trapped virus. The possible ways of translating this concept into reality and the probable hurdles that can be encountered in the process are subsequently discussed. Also, the scope of cancellers in therapeutic and/or preventive strategies against HIV infection is envisaged upon their successful development. PMID:25882216

  3. Use of intron-disrupted polyadenylation sites to enhance expression and safety of retroviral vectors.

    PubMed

    Ismail, S I; Rohll, J B; Kingsman, S M; Kingsman, A J; Uden, M

    2001-01-01

    Normal mRNA polyadenylation signals are composed of an AAUAAA motif and G/U box spaced 20 to 30 bp apart. If this spacing is increased further, then polyadenylation is disrupted. Previously it has been demonstrated that insertion of an intron will similarly disrupt this signal even though such introns are removed during a nuclear splicing reaction (X. Liu and J. Mertz, Nucleic Acids Res. 21:5256-5263, 1993). This observation has led to the suggestion that polyadenylation site selection is undertaken prior to intron excision. We now present results that both support and extend these observations and in doing so create a novel class of retroviral expression vector with improved qualities. We found that when an intron-disrupted polyadenylation signal is inserted within a retroviral expression vector, such a signal, although reformed in the producer cell, remains benign until transduction, where it is then preferentially used. Thus, we demonstrate that upon transduction these vectors now produce a majority of shortened subgenomic species and as a consequence have a reduced tendency for subsequent mobilization from transduced cells. In addition, we demonstrate that the use of this internal signal leads to enhanced expression from such vectors and that this is achieved without any loss in titer. Therefore, split polyadenylation signals confer enhanced performance and improved safety upon retroviral expression vectors into which they are inserted. Such split signals may prove useful for the future optimization of retroviral vectors in gene therapy. PMID:11119589

  4. Use of Intron-Disrupted Polyadenylation Sites To Enhance Expression and Safety of Retroviral Vectors

    PubMed Central

    Ismail, Said I.; Rohll, Jonathan B.; Kingsman, Susan M.; Kingsman, Alan J.; Uden, Mark

    2001-01-01

    Normal mRNA polyadenylation signals are composed of an AAUAAA motif and G/U box spaced 20 to 30 bp apart. If this spacing is increased further, then polyadenylation is disrupted. Previously it has been demonstrated that insertion of an intron will similarly disrupt this signal even though such introns are removed during a nuclear splicing reaction (X. Liu and J. Mertz, Nucleic Acids Res. 21:5256–5263, 1993). This observation has led to the suggestion that polyadenylation site selection is undertaken prior to intron excision. We now present results that both support and extend these observations and in doing so create a novel class of retroviral expression vector with improved qualities. We found that when an intron-disrupted polyadenylation signal is inserted within a retroviral expression vector, such a signal, although reformed in the producer cell, remains benign until transduction, where it is then preferentially used. Thus, we demonstrate that upon transduction these vectors now produce a majority of shortened subgenomic species and as a consequence have a reduced tendency for subsequent mobilization from transduced cells. In addition, we demonstrate that the use of this internal signal leads to enhanced expression from such vectors and that this is achieved without any loss in titer. Therefore, split polyadenylation signals confer enhanced performance and improved safety upon retroviral expression vectors into which they are inserted. Such split signals may prove useful for the future optimization of retroviral vectors in gene therapy. PMID:11119589

  5. XPB mediated retroviral cDNA degradation coincides with entry to the nucleus

    SciTech Connect

    Yoder, Kristine E.; Roddick, William; Hoellerbauer, Pia; Fishel, Richard

    2011-02-20

    Retroviruses must integrate their cDNA to a host chromosome, but a significant fraction of retroviral cDNA is degraded before integration. XPB and XPD are part of the TFIIH complex which mediates basal transcription and DNA nucleotide excision repair. Retroviral infection increases when XPB or XPD are mutant. Here we show that inhibition of mRNA or protein synthesis does not affect HIV cDNA accumulation suggesting that TFIIH transcription activity is not required for degradation. Other host factors implicated in the stability of cDNA are not components of the XPB and XPD degradation pathway. Although an increase of retroviral cDNA in XPB or XPD mutant cells correlates with an increase of integrated provirus, the integration efficiency of pre-integration complexes is unaffected. Finally, HIV and MMLV cDNA degradation appears to coincide with nuclear import. These results suggest that TFIIH mediated cDNA degradation is a nuclear host defense against retroviral infection.

  6. Inhibition of Marek's disease virus replication by retroviral vector-based RNA interference

    Technology Transfer Automated Retrieval System (TEKTRAN)

    RNA interference (RNAi) is a promising antiviral methodology. We recently demonstrated that retroviral vectors expressing short hairpin RNAs (shRNA-mirs) in the context of a modified endogenous micro-RNA (miRNA) can be effective in reducing replication of other retroviruses in chicken cells. In thi...

  7. Identification, Phylogeny, and Evolution of Retroviral Elements Based on Their Envelope Genes

    PubMed Central

    Bénit, Laurence; Dessen, Philippe; Heidmann, Thierry

    2001-01-01

    Phylogenetic analyses of retroviral elements, including endogenous retroviruses, have relied essentially on the retroviral pol gene expressing the highly conserved reverse transcriptase. This enzyme is essential for the life cycle of all retroid elements, but other genes are also endowed with conserved essential functions. Among them, the transmembrane (TM) subunit of the envelope gene is involved in virus entry through membrane fusion. It has also been reported to contain a domain, named the immunosuppressive domain, that has immunosuppressive properties most probably essential for virus spread within the host. This domain is conserved among a large series of retroviral elements, and we have therefore attempted to generate phylogenetic links between retroviral elements identified from databases following tentative alignments of the immunosuppressive domain and adjacent sequences. This allowed us to unravel a conserved organization among TM domains, also found in the Ebola and Marburg filoviruses, and to identify a large number of human endogenous retroviruses (HERVs) from sequence databases. The latter elements are part of previously identified families of HERVs, and some of them define new families. A general phylogenetic analysis based on the TM proteins of retroelements, and including those with no clearly identified immunosuppressive domain, could then be derived and compared with pol-based phylogenetic trees, providing a comprehensive survey of retroelements and definitive evidence for recombination events in the generation of both the endogenous and the present-day infectious retroviruses. PMID:11689652

  8. Genome adaptations of a tripartite motif protein for retroviral defense in cattle and sheep

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tripartite motif (TRIM) genes encode proteins composed of RING, B-box, and coiled coil motif domains. Primate TRIM5' has been shown to be a primary determinant of retroviral host cell range restriction in primates. TRIM5 restriction was originally thought to be a primate-specific defense mechanism...

  9. Identification of candidate cancer-causing genes in mouse brain tumors by retroviral tagging

    PubMed Central

    Johansson, Fredrik K.; Brodd, Josefin; Eklöf, Charlotta; Ferletta, Maria; Hesselager, Göran; Tiger, Carl-Fredrik; Uhrbom, Lene; Westermark, Bengt

    2004-01-01

    Murine retroviruses may cause malignant tumors in mice by insertional mutagenesis of host genes. The use of retroviral tagging as a means of identifying cancer-causing genes has, however, almost entirely been restricted to hematopoietic tumors. The aim of this study was to develop a system allowing for the retroviral tagging of candidate genes in malignant brain tumors. Mouse gliomas were induced by a recombinant Moloney murine leukemia virus encoding platelet-derived growth factor (PDGF) B-chain. The underlying idea was that tumors evolve through a combination of PDGF-mediated autocrine growth stimulation and insertional mutagenesis of genes that cooperate with PDGF in gliomagenesis. Common insertion sites (loci that were tagged in more than one tumor) were identified by cloning and sequencing retroviral flanking segments, followed by blast searches of mouse genome databases. A number of candidate brain tumor loci (Btls) were identified. Several of these Btls correspond to known tumor-causing genes; these findings strongly support the underlying idea of our experimental approach. Other Btls harbor genes with a hitherto unproven role in transformation or oncogenesis. Our findings indicate that retroviral tagging with a growth factor-encoding virus may be a powerful means of identifying candidate tumor-causing genes in nonhematopoietic tumors. PMID:15273287

  10. Identification of candidate cancer-causing genes in mouse brain tumors by retroviral tagging.

    PubMed

    Johansson, Fredrik K; Brodd, Josefin; Eklöf, Charlotta; Ferletta, Maria; Hesselager, Göran; Tiger, Carl-Fredrik; Uhrbom, Lene; Westermark, Bengt

    2004-08-01

    Murine retroviruses may cause malignant tumors in mice by insertional mutagenesis of host genes. The use of retroviral tagging as a means of identifying cancer-causing genes has, however, almost entirely been restricted to hematopoietic tumors. The aim of this study was to develop a system allowing for the retroviral tagging of candidate genes in malignant brain tumors. Mouse gliomas were induced by a recombinant Moloney murine leukemia virus encoding platelet-derived growth factor (PDGF) B-chain. The underlying idea was that tumors evolve through a combination of PDGF-mediated autocrine growth stimulation and insertional mutagenesis of genes that cooperate with PDGF in gliomagenesis. Common insertion sites (loci that were tagged in more than one tumor) were identified by cloning and sequencing retroviral flanking segments, followed by blast searches of mouse genome databases. A number of candidate brain tumor loci (Btls) were identified. Several of these Btls correspond to known tumor-causing genes; these findings strongly support the underlying idea of our experimental approach. Other Btls harbor genes with a hitherto unproven role in transformation or oncogenesis. Our findings indicate that retroviral tagging with a growth factor-encoding virus may be a powerful means of identifying candidate tumor-causing genes in nonhematopoietic tumors. PMID:15273287

  11. Retroviral vectors encoding ADA regulatory locus control region provide enhanced T-cell-specific transgene expression

    PubMed Central

    2009-01-01

    Background Murine retroviral vectors have been used in several hundred gene therapy clinical trials, but have fallen out of favor for a number of reasons. One issue is that gene expression from viral or internal promoters is highly variable and essentially unregulated. Moreover, with retroviral vectors, gene expression is usually silenced over time. Mammalian genes, in contrast, are characterized by highly regulated, precise levels of expression in both a temporal and a cell-specific manner. To ascertain if recapitulation of endogenous adenosine deaminase (ADA) expression can be achieved in a vector construct we created a new series of Moloney murine leukemia virus (MuLV) based retroviral vector that carry human regulatory elements including combinations of the ADA promoter, the ADA locus control region (LCR), ADA introns and human polyadenylation sequences in a self-inactivating vector backbone. Methods A MuLV-based retroviral vector with a self-inactivating (SIN) backbone, the phosphoglycerate kinase promoter (PGK) and the enhanced green fluorescent protein (eGFP), as a reporter gene, was generated. Subsequent vectors were constructed from this basic vector by deletion or addition of certain elements. The added elements that were assessed are the human ADA promoter, human ADA locus control region (LCR), introns 7, 8, and 11 from the human ADA gene, and human growth hormone polyadenylation signal. Retroviral vector particles were produced by transient three-plasmid transfection of 293T cells. Retroviral vectors encoding eGFP were titered by transducing 293A cells, and then the proportion of GFP-positive cells was determined using fluorescence-activated cell sorting (FACS). Non T-cell and T-cell lines were transduced at a multiplicity of infection (MOI) of 0.1 and the yield of eGFP transgene expression was evaluated by FACS analysis using mean fluorescent intensity (MFI) detection. Results Vectors that contained the ADA LCR were preferentially expressed in T

  12. High-resolution structure of a retroviral protease folded as a monomer

    SciTech Connect

    Gilski, Miroslaw; Kazmierczyk, Maciej; Krzywda, Szymon; Zábranská, Helena; Cooper, Seth; Popović, Zoran; Khatib, Firas; DiMaio, Frank; Thompson, James; Baker, David; Pichová, Iva; Jaskolski, Mariusz

    2011-11-01

    The crystal structure of Mason–Pfizer monkey virus protease folded as a monomer has been solved by molecular replacement using a model generated by players of the online game Foldit. The structure shows at high resolution the details of a retroviral protease folded as a monomer which can guide rational design of protease dimerization inhibitors as retroviral drugs. Mason–Pfizer monkey virus (M-PMV), a D-type retrovirus assembling in the cytoplasm, causes simian acquired immunodeficiency syndrome (SAIDS) in rhesus monkeys. Its pepsin-like aspartic protease (retropepsin) is an integral part of the expressed retroviral polyproteins. As in all retroviral life cycles, release and dimerization of the protease (PR) is strictly required for polyprotein processing and virion maturation. Biophysical and NMR studies have indicated that in the absence of substrates or inhibitors M-PMV PR should fold into a stable monomer, but the crystal structure of this protein could not be solved by molecular replacement despite countless attempts. Ultimately, a solution was obtained in mr-rosetta using a model constructed by players of the online protein-folding game Foldit. The structure indeed shows a monomeric protein, with the N- and C-termini completely disordered. On the other hand, the flap loop, which normally gates access to the active site of homodimeric retropepsins, is clearly traceable in the electron density. The flap has an unusual curled shape and a different orientation from both the open and closed states known from dimeric retropepsins. The overall fold of the protein follows the retropepsin canon, but the C{sup α} deviations are large and the active-site ‘DTG’ loop (here NTG) deviates up to 2.7 Å from the standard conformation. This structure of a monomeric retropepsin determined at high resolution (1.6 Å) provides important extra information for the design of dimerization inhibitors that might be developed as drugs for the treatment of retroviral infections

  13. RD-MolPack technology for the constitutive production of self-inactivating lentiviral vectors pseudotyped with the nontoxic RD114-TR envelope.

    PubMed

    Marin, Virna; Stornaiuolo, Anna; Piovan, Claudia; Corna, Stefano; Bossi, Sergio; Pema, Monika; Giuliani, Erica; Scavullo, Cinzia; Zucchelli, Eleonora; Bordignon, Claudio; Rizzardi, Gian Paolo; Bovolenta, Chiara

    2016-01-01

    To date, gene therapy with transiently derived lentivectors has been very successful to cure rare infant genetic diseases. However, transient manufacturing is unfeasible to treat adult malignancies because large vector lots are required. By contrast, stable manufacturing is the best option for high-incidence diseases since it reduces the production cost, which is the major current limitation to scale up the transient methods. We have previously developed the proprietary RD2-MolPack technology for the stable production of second-generation lentivectors, based on the RD114-TR envelope. Of note, opposite to vesicular stomatitis virus glycoprotein (VSV-G) envelope, RD114-TR does not need inducible expression thanks to lack of toxicity. Here, we present the construction of RD2- and RD3-MolPack cells for the production of self-inactivating lentivectors expressing green fluorescent protein (GFP) as a proof-of-concept of the feasibility and safety of this technology before its later therapeutic exploitation. We report that human T lymphocytes transduced with self-inactivating lentivectors derived from RD3-MolPack cells or with self-inactivating VSV-G pseudotyped lentivectors derived from transient transfection show identical T-cell memory differentiation phenotype and comparable transduction efficiency in all T-cell subsets. RD-MolPack technology represents, therefore, a straightforward tool to simplify and standardize lentivector manufacturing to engineer T-cells for frontline immunotherapy applications. PMID:27222840

  14. A VSV-G Pseudotyped Last Generation Lentiviral Vector Mediates High Level and Persistent Gene Transfer in Models of Airway Epithelium In Vitro and In Vivo

    PubMed Central

    Copreni, Elena; Palmieri, Lucia; Castellani, Stefano; Conese, Massimo

    2010-01-01

    The aim of this work was to evaluate the efficiency and duration of gene expression mediated by a VSV-G pseudotyped last generation lentiviral (LV) vector. We studied LV efficiency in ex-vivo models of respiratory epithelial cells, obtained from bronchial biopsies and nasal polyps, by GFP epifluorescence and cytofluorimetry. In vivo efficiency and persistence of gene expression was investigated by GFP immunohistochemistry and luciferase activity in lung cryosections and homogenates, respectively, upon intranasal and intratracheal administration protocols in C57Bl/6 mice. Both primary bronchial and nasal epithelial cells were transduced up to 70–80% 72 hr after the LV infection. In vivo nasal luciferase expression was increased by lysophosphatidylcholine pre-treatment of the nose. Conversely, the bronchial epithelium was transduced in the absence of any pre-conditioning treatment and luciferase expression lasted for at least 6 months without any decline. We conclude that a last generation LV vector is a promising gene transfer agent in the target organ of genetic and acquired lung diseases, as in the case of cystic fibrosis. PMID:21994695

  15. RD-MolPack technology for the constitutive production of self-inactivating lentiviral vectors pseudotyped with the nontoxic RD114-TR envelope

    PubMed Central

    Marin, Virna; Stornaiuolo, Anna; Piovan, Claudia; Corna, Stefano; Bossi, Sergio; Pema, Monika; Giuliani, Erica; Scavullo, Cinzia; Zucchelli, Eleonora; Bordignon, Claudio; Rizzardi, Gian Paolo; Bovolenta, Chiara

    2016-01-01

    To date, gene therapy with transiently derived lentivectors has been very successful to cure rare infant genetic diseases. However, transient manufacturing is unfeasible to treat adult malignancies because large vector lots are required. By contrast, stable manufacturing is the best option for high-incidence diseases since it reduces the production cost, which is the major current limitation to scale up the transient methods. We have previously developed the proprietary RD2-MolPack technology for the stable production of second-generation lentivectors, based on the RD114-TR envelope. Of note, opposite to vesicular stomatitis virus glycoprotein (VSV-G) envelope, RD114-TR does not need inducible expression thanks to lack of toxicity. Here, we present the construction of RD2- and RD3-MolPack cells for the production of self-inactivating lentivectors expressing green fluorescent protein (GFP) as a proof-of-concept of the feasibility and safety of this technology before its later therapeutic exploitation. We report that human T lymphocytes transduced with self-inactivating lentivectors derived from RD3-MolPack cells or with self-inactivating VSV-G pseudotyped lentivectors derived from transient transfection show identical T-cell memory differentiation phenotype and comparable transduction efficiency in all T-cell subsets. RD-MolPack technology represents, therefore, a straightforward tool to simplify and standardize lentivector manufacturing to engineer T-cells for frontline immunotherapy applications. PMID:27222840

  16. [Highly Active AntiRetroviral Therapy and opportunistic protozoan infections].

    PubMed

    Pozio, E

    2004-06-01

    Opportunistic parasite infections (OPIs) are an important cause of morbidity and mortality in persons infected with HIV. In industrialised countries, the use of Highly Active AntiRetroviral Therapy (HAART) results to be effective in suppressing the HIV viral load, with a quantitative and qualitative improvement in the CD4+ T-cell count followed by a strong reduction of opportunistic infections including those caused by parasites. These successes have been mainly attributed to the reconstitution of the cell immunity, which play the most important role in controlling OPIs. However, there are many clinical reports and several laboratory results, which suggest that the control of OPIs in HIV-positive persons under HAART is also induced by the anti-HIV protease inhibitors (PIs), which inhibit the aspartyl proteases of the parasites. The non-conventional use of HIV-PIs seems to be an alternative way for the treatment of parasitic infections, which should be deeply investigated. Of five longitudinal studies carried out before and after the introduction of HAART, four studies showed a strong reduction of toxoplasmic encephalitis (TE) in HIV-positive persons under HAART, whereas in another study, no difference was observed in the incidence rate of TE before and after the introduction of HAART. The influence of HAART in reducing TE has been also confirmed in a randomised, controlled clinical trial, which showed that there is no increase in the risk of developing TE after beginning HAART, even though HIV-infected persons with TE had a discontinuing prophylaxis for Toxoplasma gondii. Four HIV protease inhibitors were tested against the T. gondii virulent RH strain in vitro, alone or in association with pyrimethamine or sulfadiazine. Ritonavir and nelfinavir were highly inhibitory for the parasite growth. Furthermore, none of the antiviral drugs negatively affected the anti-Toxoplasma activity of pyrimethamine or sulfadiazine. In HIV-Leishmania co-infections, a changing pattern

  17. Adeno-associated virus Rep-mediated targeting of integrase-defective retroviral vector DNA circles into human chromosome 19

    SciTech Connect

    Huang, Shuohao; Kawabe, Yoshinori; Ito, Akira; Kamihira, Masamichi

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Adeno-associated virus (AAV) is capable of targeted integration in human cells. Black-Right-Pointing-Pointer Integrase-defective retroviral vector (IDRV) enables a circular DNA delivery. Black-Right-Pointing-Pointer A targeted integration system of IDRV DNA using the AAV integration mechanism. Black-Right-Pointing-Pointer Targeted IDRV integration ameliorates the safety concerns for retroviral vectors. -- Abstract: Retroviral vectors have been employed in clinical trials for gene therapy owing to their relative large packaging capacity, alterable cell tropism, and chromosomal integration for stable transgene expression. However, uncontrollable integrations of transgenes are likely to cause safety issues, such as insertional mutagenesis. A targeted transgene integration system for retroviral vectors, therefore, is a straightforward way to address the insertional mutagenesis issue. Adeno-associated virus (AAV) is the only known virus capable of targeted integration in human cells. In the presence of AAV Rep proteins, plasmids possessing the p5 integration efficiency element (p5IEE) can be integrated into the AAV integration site (AAVS1) in the human genome. In this report, we describe a system that can target the circular DNA derived from non-integrating retroviral vectors to the AAVS1 site by utilizing the Rep/p5IEE integration mechanism. Our results showed that after G418 selection 30% of collected clones had retroviral DNA targeted at the AAVS1 site.

  18. STRUCTURAL VIROLOGY. Conformational plasticity of a native retroviral capsid revealed by x-ray crystallography.

    PubMed

    Obal, G; Trajtenberg, F; Carrión, F; Tomé, L; Larrieux, N; Zhang, X; Pritsch, O; Buschiazzo, A

    2015-07-01

    Retroviruses depend on self-assembly of their capsid proteins (core particle) to yield infectious mature virions. Despite the essential role of the retroviral core, its high polymorphism has hindered high-resolution structural analyses. Here, we report the x-ray structure of the native capsid (CA) protein from bovine leukemia virus. CA is organized as hexamers that deviate substantially from sixfold symmetry, yet adjust to make two-dimensional pseudohexagonal arrays that mimic mature retroviral cores. Intra- and interhexameric quasi-equivalent contacts are uncovered, with flexible trimeric lateral contacts among hexamers, yet preserving very similar dimeric interfaces making the lattice. The conformation of each capsid subunit in the hexamer is therefore dictated by long-range interactions, revealing how the hexamers can also assemble into closed core particles, a relevant feature of retrovirus biology. PMID:26044299

  19. Hypoxia- and radiation-inducible, breast cell-specific targeting of retroviral vectors

    SciTech Connect

    Lipnik, Karoline; Greco, Olga; Scott, Simon; Knapp, Elzbieta; Mayrhofer, Elisabeth; Rosenfellner, Doris; Guenzburg, Walter H.; Salmons, Brian; Hohenadl, Christine . E-mail: christine.hohenadl@vu-wien.ac.at

    2006-05-25

    To facilitate a more efficient radiation and chemotherapy of mammary tumours, synthetic enhancer elements responsive to hypoxia and ionizing radiation were coupled to the mammary-specific minimal promoter of the murine whey acidic protein (WAP) encoding gene. The modified WAP promoter was introduced into a retroviral promoter conversion (ProCon) vector. Expression of a transduced reporter gene in response to hypoxia and radiation was analysed in stably infected mammary cancer cell lines and an up to 9-fold increase in gene expression demonstrated in comparison to the respective basic vector. Expression analyses in vitro, moreover, demonstrated a widely preserved mammary cell-specific promoter activity. For in vivo analyses, xenograft tumours consisting of infected human mammary adenocarcinoma cells were established in SCID/beige mice. Immunohistochemical analyses demonstrated a hypoxia-specific, markedly increased WAP promoter-driven expression in these tumours. Thus, this retroviral vector will facilitate a targeted gene therapeutic approach exploiting the unique environmental condition in solid tumours.

  20. Expression of human. alpha. sub 1 -antitrypsin in dogs after autologous transplantation of retroviral transduced hepatocytes

    SciTech Connect

    Kay, M.A.; Baley, P.; Rothenberg, S.; Leland, F; Fleming, L.; Ponder, K.P.; Liu, Tajen; Finegold, M.; Darlington, G.; Pokorny, W.; Woo, S.L.C. )

    1992-01-01

    The liver represents an excellent organ for gene therapy since many genetic disorders result from the deficiency of liver-specific gene products. The authors have previously demonstrated that transgenic mouse hepatocytes can be heterologously transplanted into congenic recipients where they survived indefinitely and continued to function as hepatocytes. Here they demonstrate the autologous transplantation of retrovirally transduced canine hepatocytes. In two animals they have transplanted hepatocytes transduced with a retroviral vector containing the human {alpha}{sub 1}-antitrypsin cDNA under transcriptional control of the cytomegalovirus promotor. Both animals had significant human {alpha}{sub 1}-antitrypsin in the serum for 1 month. The results suggest that gene therapy of hepatic deficiencies may be achieved by hepatocellular transplantation after genetic reconstruction with the use of promoters of cellular genes that are active in the normal liver.

  1. Immune Protection of Retroviral Vectors Upon Molecular Painting with the Complement Regulatory Protein CD59.

    PubMed

    Heider, Susanne; Kleinberger, Sandra; Kochan, Feliks; Dangerfield, John A; Metzner, Christoph

    2016-07-01

    Glycosylphosphatidylinositol anchoring is a type of post-translational modification that allows proteins to be presented on the exterior side of the cell membrane. Purified glycosylphosphatidylinositol-anchored protein can spontaneously re-insert into lipid bilayer membranes in a process termed Molecular Painting. Here, we demonstrate the possibility of inserting purified, recombinant CD59 into virus particles produced from a murine retroviral producer cell line. CD59 is a regulator of the complement system that helps protect healthy cells from the lytic activity of the complement cascade. In this study, we could show that Molecular Painting confers protection from complement activity upon murine retroviral vector particles. Indeed, increased infectivity of CD59-modified virus particles was observed upon challenge with human serum, indicating that Molecular Painting is suitable for modulating the immune system in gene therapy or vaccination applications. PMID:27170144

  2. Domesticated retroviral GAG gene in Drosophila: new functions for an old gene.

    PubMed

    Nefedova, L N; Kuzmin, I V; Makhnovskii, P A; Kim, A I

    2014-02-01

    The domestication of foreign genes is a powerful mechanism for new gene formation and genome evolution. It is known that domesticated retroviral gag genes in mammals not only take part in protecting against viral infection but also control cell division, apoptosis, function of the placenta, and other biological processes. In this study, we focused on the domesticated retroviral gag gene homolog (Grp) in the Drosophila melanogaster genome. According to the results of a bioinformatic analysis, the Grp gene product is primarily under purifying selection in Drosophilidae family. The Grp protein has been shown to be transmembrane. The Grp gene is expressed at the adult stage of D. melanogaster in gender-specific and tissue-specific manner. Also the Grp gene expression is increased in response to the gypsy retrovirus. A function of the protein as a component of the endosomic membrane is considered. PMID:24503082

  3. Thyroid epithelial cell transformation by a retroviral vector expressing SV40 large T.

    PubMed Central

    Burns, J. S.; Lemoine, L.; Lemoine, N. R.; Williams, E. D.; Wynford-Thomas, D.

    1989-01-01

    A recombinant murine retroviral vector encoding the SV40 virus large T antigen was used to infect stably an immortal line of differentiated rat thyroid epithelial cells, FRTL-5. Expression of SV40 T transformed these cells to anchorage independence and tumorigenicity but did not alter morphology or abolish tissue-specific functions and growth factor requirements. The resulting phenotype provides a model of well-differentiated human thyroid cancer. Images Figure 1 Figure 3 PMID:2544221

  4. Retroviral envelope gene captures and syncytin exaptation for placentation in marsupials

    PubMed Central

    Cornelis, Guillaume; Vernochet, Cécile; Carradec, Quentin; Souquere, Sylvie; Mulot, Baptiste; Catzeflis, François; Nilsson, Maria A.; Menzies, Brandon R.; Renfree, Marilyn B.; Pierron, Gérard; Zeller, Ulrich; Heidmann, Odile; Dupressoir, Anne; Heidmann, Thierry

    2015-01-01

    Syncytins are genes of retroviral origin captured by eutherian mammals, with a role in placentation. Here we show that some marsupials—which are the closest living relatives to eutherian mammals, although they diverged from the latter ∼190 Mya—also possess a syncytin gene. The gene identified in the South American marsupial opossum and dubbed syncytin-Opo1 has all of the characteristic features of a bona fide syncytin gene: It is fusogenic in an ex vivo cell–cell fusion assay; it is specifically expressed in the short-lived placenta at the level of the syncytial feto–maternal interface; and it is conserved in a functional state in a series of Monodelphis species. We further identify a nonfusogenic retroviral envelope gene that has been conserved for >80 My of evolution among all marsupials (including the opossum and the Australian tammar wallaby), with evidence for purifying selection and conservation of a canonical immunosuppressive domain, but with only limited expression in the placenta. This unusual captured gene, together with a third class of envelope genes from recently endogenized retroviruses—displaying strong expression in the uterine glands where retroviral particles can be detected—plausibly correspond to the different evolutionary statuses of a captured retroviral envelope gene, with only syncytin-Opo1 being the present-day bona fide syncytin active in the opossum and related species. This study would accordingly recapitulate the natural history of syncytin exaptation and evolution in a single species, and definitely extends the presence of such genes to all major placental mammalian clades. PMID:25605903

  5. Retroviral envelope gene captures and syncytin exaptation for placentation in marsupials.

    PubMed

    Cornelis, Guillaume; Vernochet, Cécile; Carradec, Quentin; Souquere, Sylvie; Mulot, Baptiste; Catzeflis, François; Nilsson, Maria A; Menzies, Brandon R; Renfree, Marilyn B; Pierron, Gérard; Zeller, Ulrich; Heidmann, Odile; Dupressoir, Anne; Heidmann, Thierry

    2015-02-01

    Syncytins are genes of retroviral origin captured by eutherian mammals, with a role in placentation. Here we show that some marsupials-which are the closest living relatives to eutherian mammals, although they diverged from the latter ∼190 Mya-also possess a syncytin gene. The gene identified in the South American marsupial opossum and dubbed syncytin-Opo1 has all of the characteristic features of a bona fide syncytin gene: It is fusogenic in an ex vivo cell-cell fusion assay; it is specifically expressed in the short-lived placenta at the level of the syncytial feto-maternal interface; and it is conserved in a functional state in a series of Monodelphis species. We further identify a nonfusogenic retroviral envelope gene that has been conserved for >80 My of evolution among all marsupials (including the opossum and the Australian tammar wallaby), with evidence for purifying selection and conservation of a canonical immunosuppressive domain, but with only limited expression in the placenta. This unusual captured gene, together with a third class of envelope genes from recently endogenized retroviruses-displaying strong expression in the uterine glands where retroviral particles can be detected-plausibly correspond to the different evolutionary statuses of a captured retroviral envelope gene, with only syncytin-Opo1 being the present-day bona fide syncytin active in the opossum and related species. This study would accordingly recapitulate the natural history of syncytin exaptation and evolution in a single species, and definitely extends the presence of such genes to all major placental mammalian clades. PMID:25605903

  6. Crystal structure of a monomeric retroviral protease solved by protein folding game players.

    PubMed

    Khatib, Firas; DiMaio, Frank; Cooper, Seth; Kazmierczyk, Maciej; Gilski, Miroslaw; Krzywda, Szymon; Zabranska, Helena; Pichova, Iva; Thompson, James; Popović, Zoran; Jaskolski, Mariusz; Baker, David

    2011-10-01

    Following the failure of a wide range of attempts to solve the crystal structure of M-PMV retroviral protease by molecular replacement, we challenged players of the protein folding game Foldit to produce accurate models of the protein. Remarkably, Foldit players were able to generate models of sufficient quality for successful molecular replacement and subsequent structure determination. The refined structure provides new insights for the design of antiretroviral drugs. PMID:21926992

  7. PML/TRIM19-Dependent Inhibition of Retroviral Reverse-Transcription by Daxx

    PubMed Central

    Portilho, Débora M.; Arhel, Nathalie J.; Chelbi-Alix, Mounira K.; Nisole, Sébastien

    2015-01-01

    PML (Promyelocytic Leukemia protein), also known as TRIM19, belongs to the family of tripartite motif (TRIM) proteins. PML is mainly expressed in the nucleus, where it forms dynamic structures known as PML nuclear bodies that recruit many other proteins, such as Sp100 and Daxx. While the role of PML/TRIM19 in antiviral defense is well documented, its effect on HIV-1 infection remains unclear. Here we show that infection by HIV-1 and other retroviruses triggers the formation of PML cytoplasmic bodies, as early as 30 minutes post-infection. Quantification of the number and size of PML cytoplasmic bodies revealed that they last approximately 8 h, with a peak at 2 h post-infection. PML re-localization is blocked by reverse-transcription inhibitors and is not observed following infection with unrelated viruses, suggesting it is specifically triggered by retroviral reverse-transcription. Furthermore, we show that PML interferes with an early step of retroviral infection since PML knockdown dramatically increases reverse-transcription efficiency. We demonstrate that PML does not inhibit directly retroviral infection but acts through the stabilization of one of its well-characterized partners, Daxx. In the presence of PML, cytoplasmic Daxx is found in the vicinity of incoming HIV-1 capsids and inhibits reverse-transcription. Interestingly, Daxx not only interferes with exogenous retroviral infections but can also inhibit retrotransposition of endogenous retroviruses, thus identifying Daxx as a broad cellular inhibitor of reverse-transcription. Altogether, these findings unravel a novel antiviral function for PML and PML nuclear body-associated protein Daxx. PMID:26566030

  8. Murine Leukemias with Retroviral Insertions at Lmo2 Are Predictive of the Leukemias Induced in SCID-X1 Patients Following Retroviral Gene Therapy

    PubMed Central

    Davé, Utpal P.; Akagi, Keiko; Tripathi, Rati; Cleveland, Susan M.; Thompson, Mary A.; Yi, Ming; Stephens, Robert; Downing, James R.; Jenkins, Nancy A.; Copeland, Neal G.

    2009-01-01

    Five X-linked severe combined immunodeficiency patients (SCID-X1) successfully treated with autologous bone marrow stem cells infected ex vivo with an IL2RG-containing retrovirus subsequently developed T-cell leukemia and four contained insertional mutations at LMO2. Genetic evidence also suggests a role for IL2RG in tumor formation, although this remains controversial. Here, we show that the genes and signaling pathways deregulated in murine leukemias with retroviral insertions at Lmo2 are similar to those deregulated in human leukemias with high LMO2 expression and are highly predictive of the leukemias induced in SCID-X1 patients. We also provide additional evidence supporting the notion that IL2RG and LMO2 cooperate in leukemia induction but are not sufficient and require additional cooperating mutations. The highly concordant nature of the genetic events giving rise to mouse and human leukemias with mutations at Lmo2 are an encouraging sign to those wanting to use mice to model human cancer and may help in designing safer methods for retroviral gene therapy. PMID:19461887

  9. Cryo-EM reveals a novel octameric integrase structure for β-retroviral intasome function

    PubMed Central

    Ballandras-Colas, Allison; Brown, Monica; Cook, Nicola J.; Dewdney, Tamaria G.; Demeler, Borries; Cherepanov, Peter; Lyumkis, Dmitry; Engelman, Alan N.

    2016-01-01

    Retroviral integrase (IN) catalyzes the integration of viral DNA (vDNA) into host target (tDNA), which is an essential step in the lifecycle of all retroviruses1. Prior structural characterization of IN-vDNA complexes, or intasomes, from the spumavirus prototype foamy virus (PFV) revealed a functional IN tetramer2–5, and it is generally believed that intasomes derived from other retroviral genera will employ tetrameric IN6–9. However, the intasomes of orthoretroviruses, which include all known pathogenic species, have not been characterized structurally. Using single-particle cryo-electron microscopy (cryo-EM) and X-ray crystallography, we determine here an unexpected octameric IN architecture for the β-retrovirus mouse mammary tumor virus (MMTV) intasome. The structure is composed of two core IN dimers, which interact with the vDNA ends and structurally mimic the PFV IN tetramer, and two flanking IN dimers that engage the core structure via their IN C-terminal domains (CTDs). Contrary to the belief that tetrameric IN components are sufficient to catalyze integration, the flanking IN dimers were necessary for MMTV IN activity. The IN octamer solves a conundrum for the β- as well as α-retroviruses by providing critical CTDs to the intasome core that cannot be provided in cis due to evolutionarily restrictive catalytic core domain (CCD)-CTD linker regions. The octameric architecture of the MMTV intasome provides a new paradigm for the structural basis of retroviral DNA integration. PMID:26887496

  10. HSV-2- and HIV-1- permissive cell lines co-infected by HSV-2 and HIV-1 co-replicate HSV-2 and HIV-1 without production of HSV-2/HIV-1 pseudotype particles

    PubMed Central

    LeGoff, Jérôme; Bouhlal, Hicham; Lecerf, Maxime; Klein, Christophe; Hocini, Hakim; Si-Mohamed, Ali; Muggeridge, Martin; Bélec, Laurent

    2007-01-01

    Background Herpes simplex virus type 2 (HSV-2) is a major cofactor of human immunodeficiency virus type 1 (HIV-1) sexual acquisition and transmission. In the present study, we investigated whether HIV-1 and HSV-2 may interact at the cellular level by forming HIV-1 hybrid virions pseudotyped with HSV-2 envelope glycoproteins, as was previously reported for HSV type 1. Methods We evaluated in vitro the production of HSV-2/HIV-1 pseudotypes in mononuclear CEM cells and epithelial HT29 and P4P cells. We analyzed the incorporation into the HIV-1 membrane of HSV-2 gB and gD, two major HSV-2 glycoproteins required for HSV-2 fusion with the cell membrane, in co-infected cells and in HIV-1-infected P4P cells transfected by plasmids coding for gB or gD. Results We show that HSV-2 and HIV-1 co-replicated in dually infected cells, and gB and gD were co-localized with gp160. However, HIV-1 particles, produced in HIV-1-infected cells expressing gB or gD after transfection or HSV-2 superinfection, did not incorporate either gB or gD in the viral membrane, and did not have the capacity to infect cells normally non-permissive for HIV-1, such as epithelial cells. Conclusion Our results do not support the hypothesis of HSV-2/HIV-1 pseudotype formation and involvement in the synergistic genital interactions between HIV-1 and HSV-2. PMID:17207276

  11. TRIM5 Retroviral Restriction Activity Correlates with the Ability To Induce Innate Immune Signaling

    PubMed Central

    Lascano, Josefina; Uchil, Pradeep D.; Mothes, Walther

    2015-01-01

    ABSTRACT Host restriction factor TRIM5 inhibits retroviral transduction in a species-specific manner by binding to and destabilizing the retroviral capsid lattice before reverse transcription is completed. However, the restriction mechanism may not be that simple since TRIM5 E3 ubiquitin ligase activity, the proteasome, autophagy, and TAK1-dependent AP-1 signaling have been suggested to contribute to restriction. Here, we show that, among a panel of seven primate and Carnivora TRIM5 orthologues, each of which has potential for potent retroviral restriction activity, all activated AP-1 signaling. In contrast, TRIM family paralogues most closely related to TRIM5 did not. While each primate species has a single TRIM5 gene, mice have at least seven TRIM5 homologues that cluster into two groups, Trim12a, -b, and -c and Trim30a, -b, -c, and -d. The three Trim12 proteins activated innate immune signaling, while the Trim30 proteins did not, though none of the murine Trim5 homologues restricted any of a panel of cloned retroviruses. To determine if any mouse TRIM5 homologues had potential for restriction activity, each was fused to the human immunodeficiency virus type 1 (HIV-1) CA binding protein cyclophilin A (CypA). The three Trim12-CypA fusions all activated AP-1 and restricted HIV-1 transduction, whereas the Trim30-CypA fusions did neither. AP-1 activation and HIV-1 restriction by the Trim12-CypA fusions were inhibited by disruption of TAK1. Overall then, these experiments demonstrate that there is a strong correlation between TRIM5 retroviral restriction activity and the ability to activate TAK1-dependent innate immune signaling. IMPORTANCE The importance of retroviruses for the evolution of susceptible host organisms cannot be overestimated. Eight percent of the human genome is retrovirus sequence, fixed in the germ line during past infection. Understanding how metazoa protect their genomes from mutagenic retrovirus infection is therefore of fundamental importance to

  12. Positive selection of Iris, a retroviral envelope-derived host gene in Drosophila melanogaster.

    PubMed

    Malik, Harmit S; Henikoff, Steven

    2005-10-01

    Eukaryotic genomes can usurp enzymatic functions encoded by mobile elements for their own use. A particularly interesting kind of acquisition involves the domestication of retroviral envelope genes, which confer infectious membrane-fusion ability to retroviruses. So far, these examples have been limited to vertebrate genomes, including primates where the domesticated envelope is under purifying selection to assist placental function. Here, we show that in Drosophila genomes, a previously unannotated gene (CG4715, renamed Iris) was domesticated from a novel, active Kanga lineage of insect retroviruses at least 25 million years ago, and has since been maintained as a host gene that is expressed in all adult tissues. Iris and the envelope genes from Kanga retroviruses are homologous to those found in insect baculoviruses and gypsy and roo insect retroviruses. Two separate envelope domestications from the Kanga and roo retroviruses have taken place, in fruit fly and mosquito genomes, respectively. Whereas retroviral envelopes are proteolytically cleaved into the ligand-interaction and membrane-fusion domains, Iris appears to lack this cleavage site. In the takahashii/suzukii species groups of Drosophila, we find that Iris has tandemly duplicated to give rise to two genes (Iris-A and Iris-B). Iris-B has significantly diverged from the Iris-A lineage, primarily because of the "invention" of an intron de novo in what was previously exonic sequence. Unlike domesticated retroviral envelope genes in mammals, we find that Iris has been subject to strong positive selection between Drosophila species. The rapid, adaptive evolution of Iris is sufficient to unambiguously distinguish the phylogenies of three closely related sibling species of Drosophila (D. simulans, D. sechellia, and D. mauritiana), a discriminative power previously described only for a putative "speciation gene." Iris represents the first instance of a retroviral envelope-derived host gene outside vertebrates

  13. TRIM5α Degradation via Autophagy Is Not Required for Retroviral Restriction

    PubMed Central

    Imam, Sabrina; Talley, Sarah; Nelson, Rachel S.; Dharan, Adarsh; O'Connor, Christopher; Hope, Thomas J.

    2016-01-01

    ABSTRACT TRIM5α is an interferon-inducible retroviral restriction factor that prevents infection by inducing the abortive disassembly of capsid cores recognized by its C-terminal PRY/SPRY domain. The mechanism by which TRIM5α mediates the disassembly of viral cores is poorly understood. Previous studies demonstrated that proteasome inhibitors abrogate the ability of TRIM5α to induce premature core disassembly and prevent reverse transcription; however, viral infection is still inhibited, indicating that the proteasome is partially involved in the restriction process. Alternatively, we and others have observed that TRIM5α associates with proteins involved in autophagic degradation pathways, and one recent study found that autophagic degradation is required for the restriction of retroviruses by TRIM5α. Here, we show that TRIM5α is basally degraded via autophagy in the absence of restriction-sensitive virus. We observe that the autophagy markers LC3b and lysosome-associated membrane protein 2A (LAMP2A) localize to a subset of TRIM5α cytoplasmic bodies, and inhibition of lysosomal degradation with bafilomycin A1 increases this association. To test the requirement for macroautophagy in restriction, we examined the ability of TRIM5α to restrict retroviral infection in cells depleted of the autophagic mediators ATG5, Beclin1, and p62. In all cases, restriction of retroviruses by human TRIM5α, rhesus macaque TRIM5α, and owl monkey TRIM-Cyp remained potent in cells depleted of these autophagic effectors by small interfering RNA (siRNA) knockdown or clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 genome editing. Collectively, these results are consistent with observations that the turnover of TRIM5α proteins is sensitive to autophagy inhibition; however, the data presented here do not support observations that the inhibition of autophagy abrogates retroviral restriction by TRIM5 proteins. IMPORTANCE Restriction factors are a class of

  14. Large-scale clinical-grade retroviral vector production in a fixed-bed bioreactor.

    PubMed

    Wang, Xiuyan; Olszewska, Malgorzata; Qu, Jinrong; Wasielewska, Teresa; Bartido, Shirley; Hermetet, Gregory; Sadelain, Michel; Rivière, Isabelle

    2015-04-01

    The successful genetic engineering of patient T cells with γ-retroviral vectors expressing chimeric antigen receptors or T-cell receptors for phase II clinical trials and beyond requires the large-scale manufacture of high-titer vector stocks. The production of retroviral vectors from stable packaging cell lines using roller bottles or 10- to 40-layer cell factories is limited by a narrow harvest window, labor intensity, open-system operations, and the requirement for significant incubator space. To circumvent these shortcomings, we optimized the production of vector stocks in a disposable fixed-bed bioreactor using good manufacturing practice-grade packaging cell lines. High-titer vector stocks were harvested over 10 days, representing a much broader harvest window than the 3-day harvest afforded by cell factories. For PG13 and 293Vec packaging cells, the average vector titer and the vector stocks' yield in the bioreactor were higher by 3.2- to 7.3-fold, and 5.6- to 13.1-fold, respectively, than those obtained in cell factories. The vector production was 10.4 and 18.6 times more efficient than in cell factories for PG13 and 293Vec cells, respectively. Furthermore, the vectors produced from the fixed-bed bioreactors passed the release test assays for clinical applications. Therefore, a single vector lot derived from 293Vec is suitable to transduce up to 500 patients cell doses in the context of large clinical trials using chimeric antigen receptors or T-cell receptors. These findings demonstrate for the first time that a robust fixed-bed bioreactor process can be used to produce γ-retroviral vector stocks scalable up to the commercialization phase. PMID:25751502

  15. A phase I trial of retroviral BRCA1sv gene therapy in ovarian cancer.

    PubMed

    Tait, D L; Obermiller, P S; Redlin-Frazier, S; Jensen, R A; Welcsh, P; Dann, J; King, M C; Johnson, D H; Holt, J T

    1997-11-01

    Gene transfer of BRCA1sv (a normal splice variant of BRCA1) into ovarian cancer cells produces growth inhibition in vitro and tumor suppression in nude mouse xenografts. As an initial step toward gene replacement therapy for ovarian cancer, we conducted a Phase I trial to assess the pharmacokinetics and toxicity of i.p. BRCA1sv retroviral vector therapy. Following placement of an indwelling Port-a-Cath in patients, a dose escalation study was performed of four daily i.p. infusions spanning doses from 3 to 300 ml (i.e., 10(10) viral particles) at half-log intervals (23 cycles in 12 patients). Gene transfer and expression were documented by PCR, Southern blot, reverse transcription-PCR, and nuclease protection assays. Pharmacokinetics were assessed by PCR and Southern blots detecting vector DNA, and toxicity was evaluated by clinical exam and fluid analysis. Three of 12 patients developed an acute sterile peritonitis, which spontaneously resolved within 48 h. Plasma and peritoneal antibodies to the retroviral envelope protein were detected only in patients treated with the highest dose levels but not in others, despite repeat dosing for an interval of up to 4 months. Eight patients showed stable disease for 4-16 weeks, and three patients showed tumor reduction with diminished miliary tumor implants at reoperation (two patients) and radiographic shrinkage of measurable disease (one patient). The vector-related complication of peritonitis was observed in three patients but resolved quickly as in preclinical mouse studies. Ovarian cancer may provide an important model for retroviral gene therapy studies due to vector stability, minimal antibody response, and access to tumor by i.p. therapy. PMID:9815585

  16. VlincRNAs controlled by retroviral elements are a hallmark of pluripotency and cancer

    PubMed Central

    2013-01-01

    Background The function of the non-coding portion of the human genome remains one of the most important questions of our time. Its vast complexity is exemplified by the recent identification of an unusual and notable component of the transcriptome - very long intergenic non-coding RNAs, termed vlincRNAs. Results Here we identify 2,147 vlincRNAs covering 10 percent of our genome. We show they are present not only in cancerous cells, but also in primary cells and normal human tissues, and are controlled by canonical promoters. Furthermore, vlincRNA promoters frequently originate from within endogenous retroviral sequences. Strikingly, the number of vlincRNAs expressed from endogenous retroviral promoters strongly correlates with pluripotency or the degree of malignant transformation. These results suggest a previously unknown connection between the pluripotent state and cancer via retroviral repeat-driven expression of vlincRNAs. Finally, we show that vlincRNAs can be syntenically conserved in humans and mouse and their depletion using RNAi can cause apoptosis in cancerous cells. Conclusions These intriguing observations suggest that vlincRNAs could create a framework that combines many existing short ESTs and lincRNAs into a landscape of very long transcripts functioning in the regulation of gene expression in the nucleus. Certain types of vlincRNAs participate at specific stages of normal development and, based on analysis of a limited set of cancerous and primary cell lines, they appear to be co-opted by cancer-associated transcriptional programs. This provides additional understanding of transcriptome regulation during the malignant state, and could lead to additional targets and options for its reversal. PMID:23876380

  17. Large-scale Clinical-grade Retroviral Vector Production in a Fixed-Bed Bioreactor

    PubMed Central

    Wang, Xiuyan; Olszewska, Malgorzata; Qu, Jinrong; Wasielewska, Teresa; Bartido, Shirley; Hermetet, Gregory; Sadelain, Michel

    2015-01-01

    The successful genetic engineering of patient T cells with γ-retroviral vectors expressing chimeric antigen receptors or T-cell receptors for phase II clinical trials and beyond requires the large-scale manufacture of high-titer vector stocks. The production of retroviral vectors from stable packaging cell lines using roller bottles or 10- to 40-layer cell factories is limited by a narrow harvest window, labor intensity, open-system operations, and the requirement for significant incubator space. To circumvent these shortcomings, we optimized the production of vector stocks in a disposable fixed-bed bioreactor using good manufacturing practice–grade packaging cell lines. High-titer vector stocks were harvested over 10 days, representing a much broader harvest window than the 3-day harvest afforded by cell factories. For PG13 and 293Vec packaging cells, the average vector titer and the vector stocks’ yield in the bioreactor were higher by 3.2- to 7.3-fold, and 5.6- to 13.1-fold, respectively, than those obtained in cell factories. The vector production was 10.4 and 18.6 times more efficient than in cell factories for PG13 and 293Vec cells, respectively. Furthermore, the vectors produced from the fixed-bed bioreactors passed the release test assays for clinical applications. Therefore, a single vector lot derived from 293Vec is suitable to transduce up to 500 patients cell doses in the context of large clinical trials using chimeric antigen receptors or T-cell receptors. These findings demonstrate for the first time that a robust fixed-bed bioreactor process can be used to produce γ-retroviral vector stocks scalable up to the commercialization phase. PMID:25751502

  18. High-resolution structure of a retroviral protease folded as a monomer.

    PubMed

    Gilski, Miroslaw; Kazmierczyk, Maciej; Krzywda, Szymon; Zábranská, Helena; Cooper, Seth; Popović, Zoran; Khatib, Firas; DiMaio, Frank; Thompson, James; Baker, David; Pichová, Iva; Jaskolski, Mariusz

    2011-11-01

    Mason-Pfizer monkey virus (M-PMV), a D-type retrovirus assembling in the cytoplasm, causes simian acquired immunodeficiency syndrome (SAIDS) in rhesus monkeys. Its pepsin-like aspartic protease (retropepsin) is an integral part of the expressed retroviral polyproteins. As in all retroviral life cycles, release and dimerization of the protease (PR) is strictly required for polyprotein processing and virion maturation. Biophysical and NMR studies have indicated that in the absence of substrates or inhibitors M-PMV PR should fold into a stable monomer, but the crystal structure of this protein could not be solved by molecular replacement despite countless attempts. Ultimately, a solution was obtained in mr-rosetta using a model constructed by players of the online protein-folding game Foldit. The structure indeed shows a monomeric protein, with the N- and C-termini completely disordered. On the other hand, the flap loop, which normally gates access to the active site of homodimeric retropepsins, is clearly traceable in the electron density. The flap has an unusual curled shape and a different orientation from both the open and closed states known from dimeric retropepsins. The overall fold of the protein follows the retropepsin canon, but the C(α) deviations are large and the active-site 'DTG' loop (here NTG) deviates up to 2.7 Å from the standard conformation. This structure of a monomeric retropepsin determined at high resolution (1.6 Å) provides important extra information for the design of dimerization inhibitors that might be developed as drugs for the treatment of retroviral infections, including AIDS. PMID:22101816

  19. Production of retroviral constructs for effective transfer and expression of T-cell receptor genes using Golden Gate cloning.

    PubMed

    Coren, Lori V; Jain, Sumiti; Trivett, Matthew T; Ohlen, Claes; Ott, David E

    2015-03-01

    Here we present an improved strategy for producing T-cell receptor (TCR)-expressing retroviral vectors using a Golden Gate cloning strategy. This method takes advantage of the modular nature of TCR genes by directly amplifying TCR α and β variable regions from RNA or cDNA, then cloning and fusing them with their respective constant region genes resident in a retroviral TCR expression vector. Our one-step approach greatly streamlines the TCR vector production process in comparison to the traditional three-step procedure that typically involves cloning whole TCR genes, producing a TCR expression cassette, and constructing a retroviral construct. To date, we have generated TCR vectors that transferred seven functional human/rhesus macaque TCRs into primary T cells. The approach also holds promise for the assembly of other genes with defined variable regions, such as immunoglobulins. PMID:25757546

  20. Targeted transgene insertion into the CHO cell genome using Cre recombinase-incorporating integrase-defective retroviral vectors.

    PubMed

    Kawabe, Yoshinori; Shimomura, Takuya; Huang, Shuohao; Imanishi, Suguru; Ito, Akira; Kamihira, Masamichi

    2016-07-01

    Retroviral vectors have served as efficient gene delivery tools in various biotechnology fields. However, viral DNA is randomly inserted into the genome, which can cause problems, such as insertional mutagenesis and gene silencing. Previously, we reported a site-specific gene integration system, in which a transgene is integrated into a predetermined chromosomal locus of Chinese hamster ovary (CHO) cells using integrase-defective retroviral vectors (IDRVs) and Cre recombinase. In this system, a Cre expression plasmid is transfected into founder cells before retroviral transduction. In practical applications of site-specific gene modification such as for hard-to-transfect cells or for in vivo gene delivery, both the transgene and the Cre protein into retroviral virions should be encapsulate. Here, we generated novel hybrid IDRVs in which viral genome and enzymatically active Cre can be delivered (Cre-IDRVs). Cre-IDRVs encoding marker genes, neomycin resistance and enhanced green fluorescent protein (EGFP), flanked by wild-type and mutated loxP sites were produced using an expression plasmid for a chimeric protein of Cre and retroviral gag-pol. After analyzing the incorporation of the Cre protein into retroviral virions by Western blotting, the Cre-IDRV was infected into founder CHO cells, in which marker genes (hygromycin resistance and red fluorescent protein) flanked with corresponding loxP sites are introduced into the genome. G418-resistant colonies expressing GFP appeared and the site-specific integration of the transgene into the expected chromosomal site was confirmed by PCR and sequencing of amplicons. Moreover, when Cre-IDRV carried a gene expression unit for a recombinant antibody, the recombinant cells in which the antibody expression cassette was integrated in a site-specific manner were generated and the cells produced the recombinant antibody. This method may provide a promising tool to perform site-specific gene modification according to Cre

  1. Entry Kinetics and Cell-Cell Transmission of Surface-Bound Retroviral Vector Particles

    PubMed Central

    O’Neill, Lee S.; Skinner, Amy M.; Woodward, Josha A.; Kurre, Peter

    2010-01-01

    Background Transduction with recombinant Human Immunodeficiency Virus (HIV) -1 derived lentivirus vectors is a multi-step process initiated by surface attachment and subsequent receptor-directed uptake into the target cell. We previously reported the retention of vesicular stomatitis virus G protein (VSV-G) pseudotyped particles on murine progenitor cells and their delayed cell-cell transfer. Methods To examine the underlying mechanism in more detail we used a combination of approaches focused on investigating the role of receptor-independent factors in modulating attachment. Results Studies of synchronized transduction herein reveal cell-type specific rates of vector particle clearance with substantial delays during particle entry into murine hematopoietic progenitor cells. The observed uptake kinetics from the surface of the 1° cell correlate inversely with the magnitude of transfer to 2° targets, corresponding with our initial observation of preferential cell-cell transfer in the context of brief vector exposures. We further demonstrate that vector particle entry into cells is associated with the cell–type specific abundance of extracellular matrix fibronectin. Residual particle – ECM binding and 2° transfer can be competitively disrupted by heparin exposure without affecting murine progenitor homing and repopulation. Conclusions While cellular attachment factors, including fibronectin, aid gene transfer by colocalizing particles to cells and disfavoring early dissociation from targets, they also appear to stabilize particles on the cell surface. Our study highlights the inadvertent consequences for cell entry and cell-cell transfer. PMID:20440757

  2. 3'-end processing and kinetics of 5'-end joining during retroviral integration in vivo.

    PubMed Central

    Roe, T; Chow, S A; Brown, P O

    1997-01-01

    Retroviral replication depends on integration of viral DNA into a host cell chromosome. Integration proceeds in three steps: 3'-end processing, the endonucleolytic removal of the two terminal nucleotides from each 3' end of the viral DNA; strand transfer, the joining of the 3' ends of viral DNA to host DNA; and 5'-end joining (or gap repair), the joining of the 5' ends of viral DNA to host DNA. The 5'-end joining step has never been investigated, either for retroviral integration or for any other transposition process. We have developed an assay for 5'-end joining in vivo and have examined the kinetics of 5'-end joining for Moloney murine leukemia virus (MLV). The interval between 3'-end and 5'-end joining is estimated to be less than 1 h. This assay will be a useful tool for examining whether viral or host components mediate 5'-end joining. MLV integrates its DNA only after its host cell has completed mitosis. We show that the extent of 3'-end processing is the same in unsynchronized and aphidicolin-arrested cells. 3'-end processing therefore does not depend on mitosis. PMID:8995657

  3. Retroviral Retention Activates a Syk-Dependent HemITAM in Human Tetherin

    PubMed Central

    Galão, Rui Pedro; Pickering, Suzanne; Curnock, Rachel; Neil, Stuart J.D.

    2014-01-01

    Summary Tetherin (BST2/CD317) restricts the release of enveloped viral particles from infected cells. Coupled to this virion retention, hominid tetherins induce proinflammatory gene expression via activating NF-κB. We investigated the events initiating this tetherin-induced signaling and show that physical retention of retroviral particles induces the phosphorylation of conserved tyrosine residues in the cytoplasmic tails of tetherin dimers. This phosphorylation induces the recruitment of spleen tyrosine kinase (Syk), which is required for downstream NF-κB activation, indicating that the tetherin cytoplasmic tail resembles the hemi-immunoreceptor tyrosine-based activation motifs (hemITAMs) found in C-type lectin pattern recognition receptors. Retroviral-induced tetherin signaling is coupled to the cortical actin cytoskeleton via the Rac-GAP-containing protein RICH2 (ARHGAP44), and a naturally occurring tetherin polymorphism with reduced RICH2 binding exhibits decreased phosphorylation and NF-κB activation. Thus, upon virion retention, this linkage to the actin cytoskeleton likely triggers tetherin phosphorylation and subsequent signal transduction to induce an antiviral state. PMID:25211072

  4. High Expression of Endogenous Retroviral Envelope Gene in the Equine Fetal Part of the Placenta

    PubMed Central

    Stefanetti, Valentina; Marenzoni, Maria Luisa; Passamonti, Fabrizio; Cappelli, Katia; Garcia-Etxebarria, Koldo; Coletti, Mauro; Capomaccio, Stefano

    2016-01-01

    Endogenous retroviruses (ERVs) are proviral phases of exogenous retroviruses that have co-evolved with vertebrate genomes for millions of years. Previous studies have identified the envelope (env) protein genes of retroviral origin preferentially expressed in the placenta which suggests a role in placentation based on their membrane fusogenic capacity and therefore they have been named syncytins. Until now, all the characterized syncytins have been associated with three invasive placentation types: the endotheliochorial (Carnivora), the synepitheliochorial (Ruminantia), and the hemochorial placentation (human, mouse) where they play a role in the syncytiotrophoblast formation. The purpose of the present study was to evaluate whether EqERV env RNA is expressed in horse tissues as well and investigate if the horse, possessing an epitheliochorial placenta, has “captured” a common retroviral env gene with syncytin-like properties in placental tissues. Interestingly, although in the equine placenta there is no syncytiotrophoblast layer at the maternal-fetal interface, our results showed that EqERV env RNA is highly expressed at that level, as expected for a candidate syncytin-like gene but with reduced abundance in the other somatic tissues (nearly 30-fold lower) thus suggesting a possible role in the placental tissue. Although the horse is one of the few domestic animals with a sequenced genome, few studies have been conducted about the EqERV and their expression in placental tissue has never been investigated. PMID:27176223

  5. The Development of Metabolic Risk Factors After the Initiation of the Second Line Anti- Retroviral Therapy

    PubMed Central

    Mittal, Apoorva; Achappa, Basavaprabhu; Madi, Deepak; Chowta, Mukta N; Ramapuram, John T; Rao, Satish; Unnikrishnan, B; Mahalingam, Soundarya

    2013-01-01

    Background and objective: A Highly Active Anti-Retroviral Therapy (HAART) is accompanied with several metabolic effects like adipose redistribution and insulin resistance. In this study, we evaluated the association between a HAART and lipodystrophy. Methods: A cross sectional study, whose subjects were Human Immunodeficiency Virus (HIV) infected patients, was conducted at a tertiary care hospital in south India. Among these, 27 were on protease inhibitors for at-least 6 months and 13 were drug naive patients. The assessments of lipodystrophy, fasting blood sugar and the fasting lipid profile were done and these parameters were compared in the two groups. Results: The analysis of the data which was collected, showed an elevation in the total cholesterol levels in the individuals who were on the protease inhibitors versus the drug naive patients. There was a significant elevation in the Low Density Lipoprotein (LDL) cholesterol levels and a decrease in High Density Lipoprotein (HDL) cholesterol levels in the individuals who were on protease inhibitors. It was also observed that the HDL cholesterol levels decreased with an increase in the duration of the therapy. The LDL cholesterol levels increased with the duration of the therapy. Conclusion: The human immunodeficiency virus infection is itself related to the metabolic complications which are aggravated on the use of second line anti retroviral therapy. Therefore, after initiating the treatment with protease inhibitors, a periodic evaluation of the serum lipid levels and the blood sugar profile should be done as a standard care. PMID:23542168

  6. Long terminal repeat of murine retroviral DNAs: sequence analysis, host-proviral junctions, and preintegration site.

    PubMed Central

    Van Beveren, C; Rands, E; Chattopadhyay, S K; Lowy, D R; Verma, I M

    1982-01-01

    The nucleotide sequence of the long terminal repeat (LTR) of three murine retroviral DNAs has been determined. The data indicate that the U5 region (sequences originating from the 5' end of the genome) of various LTRs is more conserved than the U3 region (sequences from the 3' end of the genome). The location and sequence of the control elements such as the 5' cap, "TATA-like" sequences, "CCAAT-box," and presumptive polyadenylic acid addition signal AATAAA in the various LTRs are nearly identical. Some murine retroviral DNAs contain a duplication of sequences within the LTR ranging in size from 58 to 100 base pairs. A variant of molecularly cloned Moloney murine sarcoma virus DNA in which one of the two LTRs integrated into the viral DNA was also analyzed. A 4-base-pair duplication was generated at the site of integration of LTR in the viral DNA. The host-viral junction of two molecularly cloned AKR-murine leukemia virus DNAs (clones 623 and 614) was determined. In the case of AKR-623 DNA, a 3- or 4-base-pair direct repeat of cellular sequences flanking the viral DNA was observed. However, AKR-614 DNA contained a 5-base-pair repeat of cellular sequences. The nucleotide sequence of the preintegration site of AKR-623 DNA revealed that the cellular sequences duplicated during integration are present only once. Finally, a striking homology between the sequences flanking the preintegration site and viral LTRs was observed. Images PMID:6281466

  7. Retroviral intasomes search for a target DNA by 1D diffusion which rarely results in integration.

    PubMed

    Jones, Nathan D; Lopez, Miguel A; Hanne, Jeungphill; Peake, Mitchell B; Lee, Jong-Bong; Fishel, Richard; Yoder, Kristine E

    2016-01-01

    Retroviruses must integrate their linear viral cDNA into the host genome for a productive infection. Integration is catalysed by the retrovirus-encoded integrase (IN), which forms a tetramer or octamer complex with the viral cDNA long terminal repeat (LTR) ends termed an intasome. IN removes two 3'-nucleotides from both LTR ends and catalyses strand transfer of the recessed 3'-hydroxyls into the target DNA separated by 4-6 bp. Host DNA repair restores the resulting 5'-Flap and single-stranded DNA (ssDNA) gap. Here we have used multiple single molecule imaging tools to determine that the prototype foamy virus (PFV) retroviral intasome searches for an integration site by one-dimensional (1D) rotation-coupled diffusion along DNA. Once a target site is identified, the time between PFV strand transfer events is 470 ms. The majority of PFV intasome search events were non-productive. These observations identify new dynamic IN functions and suggest that target site-selection limits retroviral integration. PMID:27108531

  8. Passive Immunotherapy for Retroviral Disease: Influence of Major Histocompatibility Complex Type and T-Cell Responsiveness

    NASA Astrophysics Data System (ADS)

    Hasenkrug, Kim J.; Brooks, Diane M.; Chesebro, Bruce

    1995-11-01

    Administration of virus-specific antibodies is known to be an effective early treatment for some viral infections. Such immunotherapy probably acts by antibody-mediated neutralization of viral infectivity and is often thought to function independently of T-cell-mediated immune responses. In the present experiments, we studied passive antibody therapy using Friend murine leukemia virus complex as a model for an immunosuppressive retroviral disease in adult mice. The results showed that antibody therapy could induce recovery from a well-established retroviral infection. However, the success of therapy was dependent on the presence of both CD4^+ and CD8^+ T lymphocytes. Thus, cell-mediated responses were required for recovery from infection even in the presence of therapeutic levels of antibody. The major histocompatibility type of the mice was also an important factor determining the relative success of antibody therapy in this system, but it was less critical for low-dose than for high-dose infections. Our results imply that limited T-cell responsiveness as dictated by major histocompatibility genes and/or stage of disease may have contributed to previous immunotherapy failures in AIDS patients. Possible strategies to improve the efficacy of future therapies are discussed.

  9. Endogenous non-retroviral RNA virus elements evidence a novel type of antiviral immunity.

    PubMed

    Honda, Tomoyuki; Tomonaga, Keizo

    2016-01-01

    Vertebrate genomes contain many virus-related sequences derived from both retroviruses and non-retroviral RNA and DNA viruses. Such non-retroviral RNA sequences are possibly produced by reverse-transcription and integration of viral mRNAs of ancient RNA viruses using retrotransposon machineries. We refer to this process as transcript reversion. During an ancient bornavirus infection, transcript reversion may have left bornavirus-related sequences, known as endogenous bornavirus-like nucleoproteins (EBLNs), in the genome. We have recently demonstrated that all Homo sapiens EBLNs are expressed in at least one tissue. Because species with EBLNs appear relatively protected against infection by a current bornavirus, Borna disease virus, it is speculated that EBLNs play some roles in antiviral immunity, as seen with some endogenous retroviruses. EBLNs can function as dominant negative forms of viral proteins, small RNAs targeting viral sequences, or DNA or RNA elements modulating the gene expression. Growing evidence reveals that various RNA viruses are reverse-transcribed and integrated into the genome of infected cells, suggesting transcript reversion generally occurs during ongoing infection. Considering this, transcript reversion-mediated interference with related viruses may be a novel type of antiviral immunity in vertebrates. Understanding the biological significance of transcript reversion will provide novel insights into host defenses against viral infections. PMID:27510928

  10. Mechanism of Nucleic Acid Chaperone Function of Retroviral Nuceleocapsid (NC) Proteins

    NASA Astrophysics Data System (ADS)

    Rouzina, Ioulia; Vo, My-Nuong; Stewart, Kristen; Musier-Forsyth, Karin; Cruceanu, Margareta; Williams, Mark

    2006-03-01

    Recent studies have highlighted two main activities of HIV-1 NC protein contributing to its function as a universal nucleic acid chaperone. Firstly, it is the ability of NC to weakly destabilize all nucleic acid,(NA), secondary structures, thus resolving the kinetic traps for NA refolding, while leaving the annealed state stable. Secondly, it is the ability of NC to aggregate NA, facilitating the nucleation step of bi-molecular annealing by increasing the local NA concentration. In this work we use single molecule DNA stretching and gel-based annealing assays to characterize these two chaperone activities of NC by using various HIV-1 NC mutants and several other retroviral NC proteins. Our results suggest that two NC functions are associated with its zinc fingers and cationic residues, respectively. NC proteins from other retroviruses have similar activities, although expressed to a different degree. Thus, NA aggregating ability improves, and NA duplex destabilizing activity decreases in the sequence: MLV NC, HIV NC, RSV NC. In contrast, HTLV NC protein works very differently from other NC proteins, and similarly to typical single stranded NA binding proteins. These features of retroviral NCs co-evolved with the structure of their genomes.

  11. Detection of sequences homologous to human retroviral DNA in multiple sclerosis by gene amplification

    SciTech Connect

    Greenberg, S.J.; Ehrlich, G.D.; Abbott, M.A.; Hurwitz, B.J.; Waldmann, T.A.; Poiesz, B.J. )

    1989-04-01

    Twenty-one patients with multiple sclerosis, chronic progressive type, were examined for DNA sequences homologous to a human retrovirus. Genomic DNA from peripheral blood mononuclear cells was analyzed for the presence of homologous sequences to the human T-cell leukemia/lymphoma virus type I (HTLV-I) long terminal repeat, 3{prime} gag, pol, and env domains by the enzymatic in vitro gene amplification technique, polymerase chain reaction. Positive identification of homologous pol sequences was made in the amplified DNA from six of these patients (29%). Three of these six patients (14%) also tested positive for the env region, but not for the other regions tested. In contrast, none of the samples from 35 normal individuals studied was positive when amplified and tested with the same primers and probes. Comparison of patterns obtained from controls and from patients with adult T-cell leukemia or tropical spastic paraparesis suggests that the DNA sequences identified are exogenous to the human genome and may correspond to a human retroviral species. The data support the detection of a human retroviral agent in some patients with multiple sclerosis.

  12. Retroviral intasomes search for a target DNA by 1D diffusion which rarely results in integration

    PubMed Central

    Jones, Nathan D.; Lopez Jr, Miguel A.; Hanne, Jeungphill; Peake, Mitchell B.; Lee, Jong-Bong; Fishel, Richard; Yoder, Kristine E.

    2016-01-01

    Retroviruses must integrate their linear viral cDNA into the host genome for a productive infection. Integration is catalysed by the retrovirus-encoded integrase (IN), which forms a tetramer or octamer complex with the viral cDNA long terminal repeat (LTR) ends termed an intasome. IN removes two 3′-nucleotides from both LTR ends and catalyses strand transfer of the recessed 3′-hydroxyls into the target DNA separated by 4–6 bp. Host DNA repair restores the resulting 5′-Flap and single-stranded DNA (ssDNA) gap. Here we have used multiple single molecule imaging tools to determine that the prototype foamy virus (PFV) retroviral intasome searches for an integration site by one-dimensional (1D) rotation-coupled diffusion along DNA. Once a target site is identified, the time between PFV strand transfer events is 470 ms. The majority of PFV intasome search events were non-productive. These observations identify new dynamic IN functions and suggest that target site-selection limits retroviral integration. PMID:27108531

  13. New insights into retroviral Gag–Gag and Gag–membrane interactions

    PubMed Central

    Maldonado, José O.; Martin, Jessica L.; Mueller, Joachim D.; Zhang, Wei; Mansky, Louis M.

    2014-01-01

    A critical aspect of viral replication is the assembly of virus particles, which are subsequently released as progeny virus. While a great deal of attention has been focused on better understanding this phase of the viral life cycle, many aspects of the molecular details remain poorly understood. This is certainly true for retroviruses, including that of the human immunodeficiency virus type 1 (HIV-1; a lentivirus) as well as for human T-cell leukemia virus type 1 (HTLV-1; a deltaretrovirus). This review discusses the retroviral Gag protein and its interactions with itself, the plasma membrane and the role of lipids in targeting Gag to virus assembly sites. Recent progress using sophisticated biophysical approaches to investigate – in a comparative manner – retroviral Gag–Gag and Gag–membrane interactions are discussed. Differences among retroviruses in Gag–Gag and Gag–membrane interactions imply dissimilar molecular aspects of the viral assembly pathway, including the interactions of Gag with lipids at the membrane. PMID:25009535

  14. Murine hematopoietic reconstitution after tagging and selection of retrovirally transduced bone marrow cells

    PubMed Central

    García-Hernández, B.; Castellanos, A.; López, A.; Orfao, A.; Sánchez-García, I.

    1997-01-01

    A major problem facing the effective treatment of patients with cancer is how to get the specific antitumor agent into every tumor cell. In this report we describe the use of a strategy that, by using retroviral vectors encoding a truncated human CD5 cDNA, allows the selection of only the infected cells, and we show the ability to obtain, before bone marrow transplantation, a population of 5-fluouraci-treated murine bone marrow cells that are 100% marked. This marked population of bone marrow cells is able to reconstitute the hematopoietic system in lethally irradiated mice, indicating that the surface marker lacks deleterious effects on the functionality of bone marrow cells. No gross abnormalities in hematopoiesis were detected in mice repopulated with CD5-expressing cells. Nevertheless, a significant proportion of the hematopoietic cells no longer expresses the surface marker CD5 in the 9-month-old recipient mice. This transcriptional inactivity of the proviral long terminal repeat (LTR) was accompanied by de novo methylation of the proviral sequences. Our results show that the use of the CD5 as a retrovirally encoded marker enables the rapid, efficient, and nontoxic selection in vitro of infected primary cells, which can entirely reconstitute the hematopoietic system in mice. These results should now greatly enhance the power of studies aimed at addressing questions such as generation of cancer-negative hematopoiesis. PMID:9371830

  15. Improved self-inactivating retroviral vectors derived from spleen necrosis virus.

    PubMed Central

    Olson, P; Nelson, S; Dornburg, R

    1994-01-01

    Self-inactivating (SIN) retroviral vectors contain a deletion spanning most of the right long terminal repeat's (LTR's) U3 region. Reverse transcription copies this deletion to both LTRs. As a result, there is no transcription from the 5' LTR, preventing further replication. Many previously developed SIN vectors, however, had reduced titers or were genetically unstable. Earlier, we reported that certain SIN vectors derived from spleen necrosis virus (SNV) experienced reconstitution of the U3-deleted LTR at high frequencies. This reconstitution occurred on the DNA level and appeared to be dependent on defined vector sequences. To study this phenomenon in more detail, we developed an almost completely U3-free retroviral vector. The promoter and enhancer of the left LTR were replaced with those of the cytomegalovirus immediate-early genes. This promoter swap did not impair the level of transcription or alter its start site. Our data indicate that SNV contains a strong initiator which resembles that of human immunodeficiency virus. We show that the vectors replicate with efficiencies similar to those of vectors possessing two wild-type LTRs. U3-deleted vectors carrying the hygromycin B phosphotransferase gene did not observably undergo LTR reconstitution, even when replicated in helper cells containing SNV-LTR sequences. However, vectors carrying the neomycin resistance gene did undergo LTR reconstitution with the use of homologous helper cell LTR sequences as template. This supports our earlier finding that sequences within the neomycin resistance gene can trigger recombination. Images PMID:7933088

  16. High Expression of Endogenous Retroviral Envelope Gene in the Equine Fetal Part of the Placenta.

    PubMed

    Stefanetti, Valentina; Marenzoni, Maria Luisa; Passamonti, Fabrizio; Cappelli, Katia; Garcia-Etxebarria, Koldo; Coletti, Mauro; Capomaccio, Stefano

    2016-01-01

    Endogenous retroviruses (ERVs) are proviral phases of exogenous retroviruses that have co-evolved with vertebrate genomes for millions of years. Previous studies have identified the envelope (env) protein genes of retroviral origin preferentially expressed in the placenta which suggests a role in placentation based on their membrane fusogenic capacity and therefore they have been named syncytins. Until now, all the characterized syncytins have been associated with three invasive placentation types: the endotheliochorial (Carnivora), the synepitheliochorial (Ruminantia), and the hemochorial placentation (human, mouse) where they play a role in the syncytiotrophoblast formation. The purpose of the present study was to evaluate whether EqERV env RNA is expressed in horse tissues as well and investigate if the horse, possessing an epitheliochorial placenta, has "captured" a common retroviral env gene with syncytin-like properties in placental tissues. Interestingly, although in the equine placenta there is no syncytiotrophoblast layer at the maternal-fetal interface, our results showed that EqERV env RNA is highly expressed at that level, as expected for a candidate syncytin-like gene but with reduced abundance in the other somatic tissues (nearly 30-fold lower) thus suggesting a possible role in the placental tissue. Although the horse is one of the few domestic animals with a sequenced genome, few studies have been conducted about the EqERV and their expression in placental tissue has never been investigated. PMID:27176223

  17. IL-12-conditioning improves retrovirally-mediated transduction efficiency of CD8+ T cells

    PubMed Central

    Andrijauskaite, Kristina; Suriano, Samantha; Cloud, Colleen A.; Li, Mingli; Kesarwani, Pravin; Stefanik, Leah S.; Moxley, Kelly M.; Salem, Mohamed L; Garrett-Mayer, Elizabeth; Paulos, Chrystal M.; Mehrotra, Shikhar; Kochenderfer, James N.; Cole, David J.; Rubinstein, Mark P.

    2016-01-01

    The ability to genetically modify T cells is a critical component to many immunotherapeutic strategies and research studies. However, the success of these approaches is often limited by transduction efficiency. Since retroviral vectors require cell division for integration, transduction efficiency is dependent on the appropriate activation and culture conditions for T cells. Naïve CD8+ T cells which are quiescent must be first activated to induce cell division to allow genetic modification. To optimize this process, we activated mouse T cells with a panel of different cytokines, including IL-2, IL-4, IL-6, IL-7, IL-12, IL-15 and IL-23, known to act on T cells. After activation, cytokines were removed, and activated T cells were retrovirally transduced. We found that IL-12 pre-conditioning of mouse T cells greatly enhanced transduction efficiency while preserving function and expansion potential. We also observed a similar transduction enhancing effect of IL-12 pre-conditioning on human T cells. These findings provide a simple method to improve the transduction efficiencies of CD8+ T cells. PMID:26182912

  18. Endogenous retroviral long terminal repeats within the HLA-DQ locus.

    PubMed Central

    Kambhu, S; Falldorf, P; Lee, J S

    1990-01-01

    Two endogenous retroviral long terminal repeats (LTRs) were found in the human major histocompatibility complex locus HLA-DQ. The solo LTRs, unlinked to retrovirus structural genes, are located approximately 5 kilobases apart from each other and in the same transcriptional orientation, which is opposite to that for the HLA-DQB1 gene. These elements exhibit greater than 90% homology to the LTRs of the human endogenous retrovirus HERV-K10. The conservation of putative regulatory elements found within the LTRs and their position relative to the HLA-DQB1 gene suggest that these elements may confer distinct regulatory properties on genes in the HLA-DQ region. Polymorphic variation between different HLA haplotypes for the presence of the LTRs at this location and of the molecular architecture within this subregion is supported by polymerase chain reaction and Southern blot analysis. Comparisons of chromosomes with and without the LTRs in this region will provide a unique opportunity in the human genome to analyze transposition or integration of retroviral sequences. Images PMID:2114643

  19. Endogenous non-retroviral RNA virus elements evidence a novel type of antiviral immunity

    PubMed Central

    Honda, Tomoyuki; Tomonaga, Keizo

    2016-01-01

    ABSTRACT Vertebrate genomes contain many virus-related sequences derived from both retroviruses and non-retroviral RNA and DNA viruses. Such non-retroviral RNA sequences are possibly produced by reverse-transcription and integration of viral mRNAs of ancient RNA viruses using retrotransposon machineries. We refer to this process as transcript reversion. During an ancient bornavirus infection, transcript reversion may have left bornavirus-related sequences, known as endogenous bornavirus-like nucleoproteins (EBLNs), in the genome. We have recently demonstrated that all Homo sapiens EBLNs are expressed in at least one tissue. Because species with EBLNs appear relatively protected against infection by a current bornavirus, Borna disease virus, it is speculated that EBLNs play some roles in antiviral immunity, as seen with some endogenous retroviruses. EBLNs can function as dominant negative forms of viral proteins, small RNAs targeting viral sequences, or DNA or RNA elements modulating the gene expression. Growing evidence reveals that various RNA viruses are reverse-transcribed and integrated into the genome of infected cells, suggesting transcript reversion generally occurs during ongoing infection. Considering this, transcript reversion-mediated interference with related viruses may be a novel type of antiviral immunity in vertebrates. Understanding the biological significance of transcript reversion will provide novel insights into host defenses against viral infections. PMID:27510928

  20. Detection of retroviral super-infection from non-invasive samples.

    PubMed

    Goffe, Adeelia S; Blasse, Anja; Mundry, Roger; Leendertz, Fabian H; Calvignac-Spencer, Sébastien

    2012-01-01

    While much attention has been focused on the molecular epidemiology of retroviruses in wild primate populations, the correlated question of the frequency and nature of super-infection events, i.e., the simultaneous infection of the same individual host with several strains of the same virus, has remained largely neglected. In particular, methods possibly allowing the investigation of super-infection from samples collected non-invasively (such as faeces) have never been properly compared. Here, we fill in this gap by assessing the costs and benefits of end-point dilution PCR (EPD-PCR) and multiple bulk-PCR cloning, as applied to a case study focusing on simian foamy virus super-infection in wild chimpanzees (Pan troglodytes). We show that, although considered to be the gold standard, EPD-PCR can lead to massive consumption of biological material when only low copy numbers of the target are expected. This constitutes a serious drawback in a field in which rarity of biological material is a fundamental constraint. In addition, we demonstrate that EPD-PCR results (single/multiple infection; founder strains) can be well predicted from multiple bulk-PCR clone experiments, by applying simple statistical and network analyses to sequence alignments. We therefore recommend the implementation of the latter method when the focus is put on retroviral super-infection and only low retroviral loads are encountered. PMID:22590569

  1. Recombinant adeno-associated virus-mediated high-efficiency, transient expression of the murine cationic amino acid transporter (ecotropic retroviral receptor) permits stable transduction of human HeLa cells by ecotropic retroviral vectors.

    PubMed Central

    Bertran, J; Miller, J L; Yang, Y; Fenimore-Justman, A; Rueda, F; Vanin, E F; Nienhuis, A W

    1996-01-01

    Adeno-associated virus has a broad host range, is nonpathogenic, and integrates into a preferred location on chromosome 19, features that have fostered development of recombinant adeno-associated viruses (rAAV) as gene transfer vectors for therapeutic applications. We have used an rAAV to transfer and express the murine cationic amino acid transporter which functions as the ecotropic retroviral receptor, thereby rendering human cells conditionally susceptible to infection by an ecotropic retroviral vector. The proportion of human HeLa cells expressing the receptor at 60 h varied as a function of the multiplicity of infection (MOI) with the rAAV. Cells expressing the ecotropic receptor were efficiently transduced with an ecotropic retroviral vector encoding a nucleus-localized form of beta-galactosidase. Cells coexpressing the ecotropic receptor and nucleus-localized beta-galactosidase were isolated by fluorescence-activated cell sorting, and cell lines were recovered by cloning at limiting dilution. After growth in culture, all clones contained the retroviral vector genome, but fewer than 10% (3 of 47) contained the rAAV genome and continued to express the ecotropic receptor. The ecotropic receptor coding sequences in the rAAV genome were under the control of a tetracycline-modulated promoter. In the presence of tetracycline, receptor expression was low and the proportion of cells transduced by the ecotropic retroviral vector was decreased. Modulation of receptor expression was achieved with both an episomal and an integrated form of the rAAV genome. These data establish that functional gene expression from an rAAV genome can occur transiently without genome integration. PMID:8794313

  2. Association of murine lupus and thymic full-length endogenous retroviral expression maps to a bone marrow stem cell

    SciTech Connect

    Krieg, A.M.; Gourley, M.F.; Steinberg, A.D. )

    1991-05-01

    Recent studies of thymic gene expression in murine lupus have demonstrated 8.4-kb (full-length size) modified polytropic (Mpmv) endogenous retroviral RNA. In contrast, normal control mouse strains do not produce detectable amounts of such RNA in their thymuses. Prior studies have attributed a defect in experimental tolerance in murine lupus to a bone marrow stem cell rather than to the thymic epithelium; in contrast, infectious retroviral expression has been associated with the thymic epithelium, rather than with the bone marrow stem cell. The present study was designed to determine whether the abnormal Mpmv expression associated with murine lupus mapped to thymic epithelium or to a marrow precursor. Lethally irradiated control and lupus-prone mice were reconstituted with T cell depleted bone marrow; one month later their thymuses were studied for endogenous retroviral RNA and protein expression. Recipients of bone marrow from nonautoimmune donors expressed neither 8.4-kb Mpmv RNA nor surface MCF gp70 in their thymuses. In contrast, recipients of bone marrow from autoimmune NZB or BXSB donors expressed thymic 8.4-kb Mpmv RNA and mink cell focus-forming gp70. These studies demonstrate that lupus-associated 8.4-kb Mpmv endogenous retroviral expression is determined by bone marrow stem cells.

  3. Expression of HSV-TK suicide gene in primary T lymphocytes: the dog as a preclinical model.

    PubMed

    Weissinger, E M; Franz, M; Voss, C; Bonini, C; Kremmer, E; Kolb, H J

    2000-03-01

    Expression of suicide genes (e.g. herpes simplex virus thymidine kinase,HSV-TK) in T cells is an appealing approach to regulate graft-versus-host disease in adoptive immunotherapy. Here we report the optimization of retroviral infection of canine T cells. Canine T cells were stimulated either with phytohemagglutinin (PHA, 2 microg/ml) for 24-72 hours or with 100 U/ml interleukin-2 for seven days. Stimulated cells were co-cultivated with irradiated virus-producing cells. Transduction efficiencies ranged from 4% to 45% using PG13, a gibbon ape leukemia virus envelope (env) pseudotyped packaging cell line. Infection of cells with GPenvAM12, expressing the amphotropic Moloney murine leukemia virus env, did not yield a satisfactory percentage of transduced cells. Enrichment of transduced cells was performed using immunoselection, and gave a purity of up to 98%. Transfusion of 1 x 10(6) transduced cells per kilogram body weight showed that transduced cells could convert mixed chimerism to 100% and transfer immunity to a specific antigen. Transduced cells were repeatedly detected in peripheral blood and bone marrow by polymerase chain reaction with primers specific for the HSV-TK gene. We have demonstrated the feasibility of using the canine model to study gene therapy as a preclinical model. PMID:10976536

  4. Features, processing states, and heterologous protein interactions in the modulation of the retroviral nucleocapsid protein function.

    PubMed

    Mirambeau, Gilles; Lyonnais, Sébastien; Gorelick, Robert J

    2010-01-01

    Retroviral nucleocapsid (NC) is central to viral replication. Nucleic acid chaperoning is a key function for NC through the action of its conserved basic amino acids and zinc-finger structures. NC manipulates genomic RNA from its packaging in the producer cell to reverse transcription into the infected host cell. This chaperone function, in conjunction with NC's aggregating properties, is up-modulated by successive NC processing events, from the Gag precursor to the fully mature protein, resulting in the condensation of the nucleocapsid within the capsid shell. Reverse transcription also depends on NC processing, whereas this process provokes NC dissociation from double-stranded DNA, leading to a preintegration complex (PIC), competent for host chromosomal integration. In addition NC interacts with cellular proteins, some of which are involved in viral budding, and also with several viral proteins. All of these properties are reviewed here, focusing on HIV-1 as a paradigmatic reference and highlighting the plasticity of the nucleocapsid architecture. PMID:21045549

  5. [Hybrid nanocarriers for controlled delivery of antitumour and retroviral drugs delivery].

    PubMed

    Horcajada, Patricia; Serre, Christian; Férey, Gérard; Couvreur, Patrick; Gref, Ruxandra

    2010-01-01

    The efficient delivery of drugs in the body requires the use of non-toxic nanocarriers. Most of the existing materials show poor drug loading and/or rapid release of the proportion of the drug that is simply adsorbed (or anchored) at the external surface of the nanocarrier. The new porous hybrid solids, with the ability to tune their structures and porosities are well suited to serve as nanocarriers for delivery and imaging applications. Here we show that specific non-toxic porous iron(III) - based metal - organic frameworks with engineered cores and surfaces, as well as imaging properties, function as superior nanocarriers for efficient controlled delivery of antitumour and retroviral drugs against cancer and AIDS. They also potentially associate therapeutics and diagnostics, and open the way for theranostics, or -personalized patient treatments. double dagger. PMID:20819715

  6. Feline mediastinal lymphoma: a retrospective study of signalment, retroviral status, response to chemotherapy and prognostic indicators.

    PubMed

    Fabrizio, Francesca; Calam, Amy E; Dobson, Jane M; Middleton, Stephanie A; Murphy, Sue; Taylor, Samantha S; Schwartz, Anita; Stell, Anneliese J

    2014-08-01

    Historically, feline mediastinal lymphoma has been associated with young age, positive feline leukaemia virus (FeLV) status, Siamese breed and short survival times. Recent studies following widespread FeLV vaccination in the UK are lacking. The aim of this retrospective multi-institutional study was to re-evaluate the signalment, retroviral status, response to chemotherapy, survival and prognostic indicators in feline mediastinal lymphoma cases in the post-vaccination era. Records of cats with clinical signs associated with a mediastinal mass and cytologically/histologically confirmed lymphoma were reviewed from five UK referral centres (1998-2010). Treatment response, survival and prognostic indicators were assessed in treated cats with follow-up data. Fifty-five cases were reviewed. The median age was 3 years (range, 0.5-12 years); 12 cats (21.8%) were Siamese; and the male to female ratio was 3.2:1.0. Five cats were FeLV-positive and two were feline immunodeficiency-positive. Chemotherapy response and survival was evaluated in 38 cats. Overall response was 94.7%; complete (CR) and partial response (PR) rates did not differ significantly between protocols: COP (cyclophosphamide, vincristine, prednisone) (n = 26, CR 61.5%, PR 34.0%); Madison-Wisconsin (MW) (n = 12, CR 66.7%, PR 25.0%). Overall median survival was 373 days (range, 20-2015 days) (COP 484 days [range, 20-980 days]; MW 211 days [range, 24-2015 days] [P = 0.892]). Cats achieving CR survived longer (980 days vs 42 days for PR; P = 0.032). Age, breed, sex, location (mediastinal vs mediastinal plus other sites), retroviral status and glucocorticoid pretreatment did not affect response or survival. Feline mediastinal lymphoma cases frequently responded to chemotherapy with durable survival times, particularly in cats achieving CR. The prevalence of FeLV-antigenaemic cats was low; males and young Siamese cats appeared to be over-represented. PMID:24366846

  7. Size distribution of retrovirally marked lineages matches prediction from population measurements of cell cycle behavior

    NASA Technical Reports Server (NTRS)

    Cai, Li; Hayes, Nancy L.; Takahashi, Takao; Caviness, Verne S Jr; Nowakowski, Richard S.

    2002-01-01

    Mechanisms that regulate neuron production in the developing mouse neocortex were examined by using a retroviral lineage marking method to determine the sizes of the lineages remaining in the proliferating population of the ventricular zone during the period of neuron production. The distribution of clade sizes obtained experimentally in four different injection-survival paradigms (E11-E13, E11-E14, E11-E15, and E12-E15) from a total of over 500 labeled lineages was compared with that obtained from three models in which the average behavior of the proliferating population [i.e., the proportion of cells remaining in the proliferative population (P) vs. that exiting the proliferative population (Q)] was quantitatively related to lineage size distribution. In model 1, different proportions of asymmetric, symmetric terminal, and symmetric nonterminal cell divisions coexisted during the entire developmental period. In model 2, the developmental period was divided into two epochs: During the first, asymmetric and symmetric nonterminal cell divisions occurred, but, during the second, asymmetric and symmetric terminal cell divisions occurred. In model 3, the shifts in P and Q are accounted for by changes in the proportions of the two types of symmetric cell divisions without the inclusion of any asymmetric cell divisions. The results obtained from the retroviral experiments were well accounted for by model 1 but not by model 2 or 3. These findings demonstrate that: 1) asymmetric and both types of symmetric cell divisions coexist during the entire period of neurogenesis in the mouse, 2) neuron production is regulated in the proliferative population by the independent decisions of the two daughter cells to reenter S phase, and 3) neurons are produced by both asymmetric and symmetric terminal cell divisions. In addition, the findings mean that cell death and/or tangential movements of cells in the proliferative population occur at only a low rate and that there are no

  8. Development of retroviral vectors for insertional mutagenesis in medaka haploid cells.

    PubMed

    Lin, Fan; Liu, Qizhi; Yuan, Yongming; Hong, Yunhan

    2015-12-01

    Insertional mutagenesis (IM) by retrovirus (RV) is a high-throughput approach for interrogating gene functions in model species. Haploid cell provides a unique system for genetic screening by IM and prosperous progress has been achieved in mammal cells. However, little was known in lower vertebrate cells. Here, we report development of retroviral vectors (rvSAchCVgfp, rvSAchCVpf and rvSAchSTpf) and establishment of IM library in medaka haploid cells. Each vector contains a modified gene trapping (GT) cassette, which could extend the mutated cell population including GT insertions not in-frame or in weakly expressed genes. Virus titration determined by flow cytometry showed that rvSAchSTpf possessed the highest supernatant virus titer (1.5×10(5)TU/ml) in medaka haploid cell, while rvSAchCVpf produced the lowest titer (2.8×10(4)TU/ml). However, quantification of proviral DNAs in transduced cells by droplet digital PCR (ddPCR) demonstrated that the "real titer" may be similar among the three vectors. Furthermore, an IM library was established by FACS of haploid cells transduced with rvSAchCVgfp at a MOI of 0.1. A single copy RV integration in the majority of cells was confirmed by ddPCR in the library. Notably, there was a significant decrease of haploid cell percentage after FACS, suggesting potential trapping for survival/growth essential genes. Our results demonstrated successful development of retroviral vectors for IM in medaka haploid cells, serving for haploid genetic screening of host factors for virus infection and genes underlying certain cellular processes in fish model. PMID:26192464

  9. Murine retroviral vector producer cells survival and toxicity in the peritoneal cavity of dogs.

    PubMed

    Link, C J; Moorman, D W; Ackerman, M; Levy, J P; Seregina, T

    2000-01-01

    Retroviral vector producer cells (VPC) can effectively transfer genes in vivo. To develop a safe method to target gene delivery into intraperitoneal tumors, we have examined the toxicity of intraperitoneal (i.p.) infusion of retroviral VPC in a xenogeneic canine model. Mongrel dogs were injected intraperitoneally (i.p.) with 2 x 10(9) murine LTKOSN.2 VPC. The animals did not demonstrate acute toxicity and tolerated the i.p. infusion of the cells without difficulty. Starting 7 days after i.p. injection, the dogs received intravenous injections of ganciclovir (GCV) twice daily (5 mg/kg) for 7 days. The treatment dogs underwent peritoneal washings on days 3, 7 and 14 after their initial infusion of cells to study the persistence of the VPC. GCV treatment did not cause significant toxicities. Dogs underwent serial blood tests to evaluate bone marrow, renal, liver and immunological function. Complete blood counts, electrolytes and renal function remained normal throughout the study. Although, transient mild elevations occurred of serum alkaline phosphate, the remaining hepatic enzymes remained normal. Histologic examination of tissues from animals sacrificed after the i.p. administration of the VPC revealed no tissue destruction of the normal peritoneal lining. The dogs mounted an antibody response to the murine VPC that was first observed 7 days post injection. PCR analysis of selected tissues after GCV administration did not reveal persistent vector sequences. These results demonstrated that the injection of xenogeneic VPC is not accompanied by significant adverse effects over a 1 month period following administration into the canine peritoneal cavity. These data support the potential clinical application of the VPC in Phase I clinical trials in humans. PMID:11212841

  10. Sites of Retroviral DNA Integration: From Basic Research to Clinical Applications

    PubMed Central

    Serrao, Erik; Engelman, Alan N.

    2016-01-01

    One of the most crucial steps in the life cycle of a retrovirus is the integration of the viral DNA (vDNA) copy of the RNA genome into the genome of an infected host cell. Integration provides for efficient viral gene expression as well as for the segregation of the viral genomes to daughter cells upon cell division. Some integrated viruses are not well expressed, and cells latently infected with HIV-1 can resist the action of potent antiretroviral drugs and remain dormant for decades. Intensive research has been dedicated to understanding the catalytic mechanism of integration, as well as the viral and cellular determinants that influence integration site distribution throughout the host genome. In this review we summarize the evolution of techniques that have been used to recover and map retroviral integration sites, from the early days that first indicated that integration could occur in multiple cellular DNA locations, to current technologies that map upwards of millions of unique integration sites from single in vitro integration reactions or cell culture infections. We further review important insights gained from the use of such mapping techniques, including the monitoring of cell clonal expansion in patients treated with retrovirus-based gene therapy vectors, or AIDS patients on suppressive antiretroviral therapy (ART). These insights span from integrase (IN) enzyme sequence preferences within target DNA (tDNA) at the sites of integration, to the roles of host cellular proteins in mediating global integration distribution, to the potential relationship between genomic location of vDNA integration site and retroviral latency. PMID:26508664

  11. Multiple Groups of Novel Retroviral Genomes in Pigs and Related Species

    PubMed Central

    Patience, Clive; Switzer, William M.; Takeuchi, Yasuhiro; Griffiths, David J.; Goward, Melanie E.; Heneine, Walid; Stoye, Jonathan P.; Weiss, Robin A.

    2001-01-01

    In view of the concern over potential infection hazards in the use of porcine tissues and organs for xenotransplantation to humans, we investigated the diversity of porcine endogenous retrovirus (PERV) genomes in the DNA of domestic pigs and related species. In addition to the three known envelope subgroups of infectious gamma retroviruses (PERV-A, -B, and -C), classed together here as PERV group γ1, four novel groups of gamma retrovirus (γ2 to γ5) and four novel groups of beta retrovirus (β1 to β4) genomes were detected in pig DNA using generic and specific PCR primers. PCR quantification indicated that the retroviral genome copy number in the Landrace × Duroc F1 hybrid pig ranged from 2 (β2 and γ5) to approximately 50 (γ1). The γ1, γ2, and β4 genomes were transcribed into RNA in adult kidney tissue. Apart from γ1, the retroviral genomes are not known to be infectious, and sequencing of a small number of amplified genome fragments revealed stop codons in putative open reading frames in several cases. Analysis of DNA from wild boar and other species of Old World pigs (Suidae) and New World peccaries (Tayassuidae) showed that one retrovirus group, β2, was common to all species tested, while the others were present among all Old World species but absent from New World species. The PERV-C subgroup of γ1 genomes segregated among domestic pigs and were absent from two African species (red river hog and warthog). Thus domestic swine and their phylogenetic relatives harbor multiple groups of hitherto undescribed PERV genomes. PMID:11222700

  12. Interleukin-Encoding Adenoviral Vectors as Genetic Adjuvant for Vaccination against Retroviral Infection

    PubMed Central

    Ohs, Inga; Windmann, Sonja; Wildner, Oliver; Dittmer, Ulf; Bayer, Wibke

    2013-01-01

    Interleukins (IL) are cytokines with stimulatory and modulatory functions in the immune system. In this study, we have chosen interleukins which are involved in the enhancement of TH2 responses and B cell functions to analyze their potential to improve a prophylactic adenovirus-based anti-retroviral vaccine with regard to antibody and virus-specific CD4+ T cell responses. Mice were vaccinated with an adenoviral vector which encodes and displays the Friend Virus (FV) surface envelope protein gp70 (Ad.pIXgp70) in combination with adenoviral vectors encoding the interleukins IL4, IL5, IL6, IL7 or IL23. Co-application of Ad.pIXgp70 with Ad.IL5, Ad.IL6 or Ad.IL23 resulted in improved protection with high control over FV-induced splenomegaly and reduced viral loads. Mice co-immunized with adenoviral vectors encoding IL5 or IL23 showed increased neutralizing antibody responses while mice co-immunized with Ad.IL6 or Ad.IL23 showed improved FV-specific CD4+ T cell responses compared to mice immunized with Ad.pIXgp70 alone. We show that the co-application of adenoviral vectors encoding specific interleukins is suitable to improve the vaccination efficacy of an anti-retroviral vaccine. Improved protection correlated with improved CD4+ T cell responses and especially with higher neutralizing antibody titers. The co-application of selected interleukin-encoding adenoviral vectors is a valuable tool for vaccination with regard to enhancement of antibody mediated immunity. PMID:24349306

  13. Sites of retroviral DNA integration: From basic research to clinical applications.

    PubMed

    Serrao, Erik; Engelman, Alan N

    2016-01-01

    One of the most crucial steps in the life cycle of a retrovirus is the integration of the viral DNA (vDNA) copy of the RNA genome into the genome of an infected host cell. Integration provides for efficient viral gene expression as well as for the segregation of viral genomes to daughter cells upon cell division. Some integrated viruses are not well expressed, and cells latently infected with human immunodeficiency virus type 1 (HIV-1) can resist the action of potent antiretroviral drugs and remain dormant for decades. Intensive research has been dedicated to understanding the catalytic mechanism of integration, as well as the viral and cellular determinants that influence integration site distribution throughout the host genome. In this review, we summarize the evolution of techniques that have been used to recover and map retroviral integration sites, from the early days that first indicated that integration could occur in multiple cellular DNA locations, to current technologies that map upwards of millions of unique integration sites from single in vitro integration reactions or cell culture infections. We further review important insights gained from the use of such mapping techniques, including the monitoring of cell clonal expansion in patients treated with retrovirus-based gene therapy vectors, or patients with acquired immune deficiency syndrome (AIDS) on suppressive antiretroviral therapy (ART). These insights span from integrase (IN) enzyme sequence preferences within target DNA (tDNA) at the sites of integration, to the roles of host cellular proteins in mediating global integration distribution, to the potential relationship between genomic location of vDNA integration site and retroviral latency. PMID:26508664

  14. Regulation of human heme oxygenase in endothelial cells by using sense and antisense retroviral constructs.

    PubMed

    Quan, S; Yang, L; Abraham, N G; Kappas, A

    2001-10-01

    Our objective was to determine whether overexpression and underexpression of human heme oxygenase (HHO)-1 could be controlled on a long-term basis by introduction of the HO-1 gene in sense (S) and antisense (AS) orientation with an appropriate vector into endothelial cells. Retroviral vector (LXSN) containing viral long terminal repeat promoter-driven human HO-1 S (LSN-HHO-1) and LXSN vectors containing HHO-1 promoter (HOP)-controlled HHO-1 S and AS (LSN-HOP-HHO-1 and LSN-HOP-HHO-1-AS) sequences were constructed and used to transfect rat lung microvessel endothelial cells (RLMV cells) and human dermal microvessel endothelial cells (HMEC-1 cells). RLMV cells transduced with HHO-1 S expressed human HO-1 mRNA and HO-1 protein associated with elevation in total HO activity compared with nontransduced cells. Vector-mediated expression of HHO-1 S or AS under control of HOP resulted in effective production of HO-1 or blocked induction of endogenous human HO-1 in HMEC-1 cells, respectively. Overexpression of HO-1 AS was associated with a long-term decrease (45%) of endogenous HO-1 protein and an increase (167%) in unmetabolized exogenous heme in HMEC-1 cells. Carbon monoxide (CO) production in HO-1 S- or AS-transduced HMEC-1 cells after heme treatment was increased (159%) or decreased (50%), respectively, compared with nontransduced cells. HO-2 protein levels did not change. These findings demonstrate that HHO-1 S and AS retroviral constructs are functional in enhancing and reducing HO activity, respectively, and thus can be used to regulate cellular heme levels, the activity of heme-dependent enzymes, and the rate of heme catabolism to CO and bilirubin. PMID:11593038

  15. Anti-Retroviral Lectins Have Modest Effects on Adherence of Trichomonas vaginalis to Epithelial Cells In Vitro and on Recovery of Tritrichomonas foetus in a Mouse Vaginal Model

    PubMed Central

    Chatterjee, Aparajita; Ratner, Daniel M.; Ryan, Christopher M.; Johnson, Patricia J.; O’Keefe, Barry R.; Secor, W. Evan; Anderson, Deborah J.; Robbins, Phillips W.; Samuelson, John

    2015-01-01

    Trichomonas vaginalis causes vaginitis and increases the risk of HIV transmission by heterosexual sex, while Tritrichomonas foetus causes premature abortion in cattle. Our goals were to determine the effects, if any, of anti-retroviral lectins, which are designed to prevent heterosexual transmission of HIV, on adherence of Trichomonas to ectocervical cells and on Tritrichomonas infections in a mouse model. We show that Trichomonas Asn-linked glycans (N-glycans), like those of HIV, bind the mannose-binding lectin (MBL) that is part of the innate immune system. N-glycans of Trichomonas and Tritrichomonas bind anti-retroviral lectins (cyanovirin-N and griffithsin) and the 2G12 monoclonal antibody, each of which binds HIV N-glycans. Binding of cyanovirin-N appears to be independent of susceptibility to metronidazole, the major drug used to treat Trichomonas. Anti-retroviral lectins, MBL, and galectin-1 cause Trichomonas to self-aggregate and precipitate. The anti-retroviral lectins also increase adherence of ricin-resistant mutants, which are less adherent than parent cells, to ectocervical cell monolayers and to organotypic EpiVaginal tissue cells. Topical application of either anti-retroviral lectins or yeast N-glycans decreases by 40 to 70% the recovery of Tritrichomonas from the mouse vagina. These results, which are explained by a few simple models, suggest that the anti-retroviral lectins have a modest potential for preventing or treating human infections with Trichomonas. PMID:26252012

  16. Equivalent inhibition of half-site and full-site retroviral strand transfer reactions by structurally diverse compounds.

    PubMed Central

    Hazuda, D; Felock, P; Hastings, J; Pramanik, B; Wolfe, A; Goodarzi, G; Vora, A; Brackmann, K; Grandgenett, D

    1997-01-01

    In vitro assay systems which use recombinant retroviral integrase (IN) and short DNA oligonucleotides fail to recapitulate the full-site integration reaction as it is known to occur in vivo. The relevance of using such circumscribed in vitro assays to define inhibitors of retroviral integration has not been formerly demonstrated. Therefore, we analyzed a series of structurally diverse inhibitors with respect to inhibition of both half-site and full-site strand transfer reactions with either recombinant or virion-produced IN. Half-site and full-site reactions catalyzed by avian myeloblastosis virus and human immunodeficiency virus type 1 (HIV-1) IN from virions are shown to be equivalently sensitive to inhibition by compounds which inhibit half-site reactions catalyzed by the recombinant HIV-1 IN. These studies therefore support the utility of using in vitro assays employing either recombinant or virion-derived IN to identify inhibitors of integration. PMID:8985421

  17. Spectrum of imaging appearances of intracranial cryptococcal infection in HIV/AIDS patients in the anti-retroviral therapy era.

    PubMed

    Offiah, Curtis E; Naseer, Aisha

    2016-01-01

    Cryptococcus neoformans infection is the most common fungal infection of the central nervous system (CNS) in advanced human immunodeficiency virus (HIV) and acquired immunodeficiency syndrome (AIDS) patients, but remains a relatively uncommon CNS infection in both the immunocompromised and immunocompetent patient population, rendering it a somewhat elusive and frequently overlooked diagnosis. The morbidity and mortality associated with CNS cryptococcal infection can be significantly reduced by early recognition of the imaging appearances by the radiologist in order to focus and expedite clinical management and treatment. The emergence and evolution of anti-retroviral therapy have also impacted significantly on the imaging appearances, morbidity, and mortality of this neuro-infection. The constellation of varied imaging appearances associated with cryptococcal CNS infection in the HIV and AIDS population in the era of highly active anti-retroviral therapy (HAART) will be presented in this review. PMID:26564776

  18. Retroviral insertional mutagenesis identifies genes that collaborate with NUP98‐HOXD13 during leukemic transformation

    PubMed Central

    Slape, Christopher; Hartung, Helge; Lin, Ying‐Wei; Bies, Juraj; Wolff, Linda; Aplan, Peter D

    2007-01-01

    The t(2;11)(q31;p15) chromosomal translocation results in a fusion between the NUP98 and HOXD13genes and has been observed in patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). We previously demonstrated that expression of the NUP98‐HOXD13 (NHD13) fusion gene in transgenic mice results in an invariably fatal MDS; approximately one third of mice die due to complications of severe pancytopenia, and about two thirds progress to a fatal acute leukemia. In the present study, we used retroviral insertional mutagenesis to identify genes that might collaborate with NHD13 as the MDS transformed to an acute leukemia. Newborn NHD13 transgenic mice and littermate controls were infected with the MOL4070LTR retrovirus. The onset of leukemia was accelerated, suggesting a synergistic effect between the NHD13 transgene and the genes neighbouring retroviral insertion events. We identified numerous common insertion sites located near protein‐coding genes, and confirmed dysregulation of a subset of these by expression analyses. Among these genes were Meis1, a known collaborator of HOX and NUP98‐HOX fusion genes, and Mn1, a transcriptional coactivator involved in human leukemia through fusion with the TEL gene. Other putative collaborators included Gata2, Erg and Epor. Of note, we identified a common insertion site that was >100 kb from the nearest coding gene, but within 20 kb of the miR29a/miR29b1 microRNA locus. Both of these miRNA were upregulated, demonstrating that retroviral insertional mutagenesis can target miRNA loci as well as protein‐coding loci. Our data provides new insights into NHD13 mediated leukemogenesis as well as retroviral insertional mutagenesis mechanisms. PMID:17545593

  19. Structural and functional studies of murine Mbo I repeat LTR (MRL) retroviral genes on the Y chromosome

    SciTech Connect

    Ch'ang, L.Y.; Hoyt, P.R.; Wang, T.H.; Kanagala, R.; Henley, D.C.; Yang, D.M.; Yang, W.K. )

    1991-03-15

    The mouse genome harbors approximately 200 copies of MRL retroviral elements (or MuRRs) that are preferentially expressed in the reproductive system. The MRL retroviral gene family is wildly distributed in the genus Mus. About 10% of the elements are located on the Y chromosome and the abundance is probably due to gene amplification. Multiple copies of Y chromosome-specific MRL retroviral sequences are present only in the genome of M spretus and M. musculus. Structural and sequence analyses revealed a truncation of the male-specific MRL elements isolated from a BALB/c mouse DNA library. Consequently, two-thirds of an intact LTR was retained at the 5{prime} end and the 3{prime} structure was disrupted immediately downstream of the pol gene with a concomitant loss of the 3{prime} LTR. Southern analysis of male and female mouse DNA confirmed that sequences adjacent to the 3{prime} breakpoint were Y chromosome specific. These sequences are length polymorphic in nature and appear to be co-amplified with MRL retroviral genes on the Y chromosome. A collinear cDNA of 9.5 kb containing fused MRL and Y chromosome sequences was also isolated from a testis library. The LTR of male-specific MRL elements was unable to drive the expression of the bacterial CAT gene in cultured mouse NIH/3T3 and mink CCL64 cells. However, when its enhancer domain was linked to an SV40 promoter, the CAT gene was expressed at a significant level. Differential binding activities to male-specific MRL were found in nuclear extracts of the liver, kidney, and testis.

  20. Quantitative analysis of recombination between YFP and CFP genes of FRET biosensors introduced by lentiviral or retroviral gene transfer.

    PubMed

    Komatsubara, Akira T; Matsuda, Michiyuki; Aoki, Kazuhiro

    2015-01-01

    Biosensors based on the principle of Förster (or fluorescence) resonance energy transfer (FRET) have been developed to visualize spatio-temporal dynamics of signalling molecules in living cells. Many of them adopt a backbone of intramolecular FRET biosensor with a cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) as donor and acceptor, respectively. However, there remains the difficulty of establishing cells stably expressing FRET biosensors with a YFP and CFP pair by lentiviral or retroviral gene transfer, due to the high incidence of recombination between YFP and CFP genes. To address this, we examined the effects of codon-diversification of YFP on the recombination of FRET biosensors introduced by lentivirus or retrovirus. The YFP gene that was fully codon-optimized to E.coli evaded the recombination in lentiviral or retroviral gene transfer, but the partially codon-diversified YFP did not. Further, the length of spacer between YFP and CFP genes clearly affected recombination efficiency, suggesting that the intramolecular template switching occurred in the reverse-transcription process. The simple mathematical model reproduced the experimental data sufficiently, yielding a recombination rate of 0.002-0.005 per base. Together, these results show that the codon-diversified YFP is a useful tool for expressing FRET biosensors by lentiviral or retroviral gene transfer. PMID:26290434

  1. Identifying risk factors of immune reconstitution inflammatory syndrome in AIDS patients receiving highly active anti-retroviral therapy.

    PubMed

    He, Bo; Zheng, Yuhuang; Liu, Meng; Zhou, Guoqiang; Chen, Xia; Mamadou, Diallo; He, Yan; Zhou, Huaying; Chen, Zi

    2013-01-01

    Immune reconstitution inflammation syndrome typically occurs within days after patients undergo highly active anti-retroviral therapy and is a big hurdle for effective treatment of AIDS patients. In this study, we monitored immune reconstitution inflammation syndrome occurrence in 238 AIDS patients treated with highly active anti-retroviral therapy. Among them, immune reconstitution inflammation syndrome occurred in 47 cases (19.7%). Immune reconstitution inflammation syndrome patients had significantly higher rate of opportunistic infection (p<0.001) and persistently lower CD4(+) cell count (p<0.001) compared to the non-immune reconstitution inflammation syndrome patients. In contrast, no significant differences in HIV RNA loads were observed between the immune reconstitution inflammation syndrome group and non-immune reconstitution inflammation syndrome group. These data suggest that a history of opportunistic infection and CD4(+) cell counts at baseline may function as risk factors for immune reconstitution inflammation syndrome occurrence in AIDS patients as well as potential prognostic markers. These findings will improve the management of AIDS with highly active anti-retroviral therapy. PMID:23434049

  2. Inhibition of HMGI-C protein synthesis suppresses retrovirally induced neoplastic transformation of rat thyroid cells.

    PubMed Central

    Berlingieri, M T; Manfioletti, G; Santoro, M; Bandiera, A; Visconti, R; Giancotti, V; Fusco, A

    1995-01-01

    Elevated expression of the three high-mobility group I (HMGI) proteins (HMGI, HMGY, and HMGI-C) has previously been correlated with the presence of a highly malignant phenotype in epithelial and fibroblastic rat thyroid cells and in experimental thyroid, lung, mammary, and skin carcinomas. Northern (RNA) blot and run-on analyses demonstrated that the induction of HMGI genes in transformed thyroid cells occurs at the transcriptional level. An antisense methodology to block HMGI-C protein synthesis was then used to analyze the role of this protein in the process of thyroid cell transformation. Transfection of an antisense construct for the HMGI-C cDNA into normal thyroid cells, followed by infection with transforming myeloproliferative sarcoma virus or Kirsten murine sarcoma virus, generated cell lines that expressed significant levels of the retroviral transforming oncogenes v-mos or v-ras-Ki and removed the dependency on thyroid-stimulating hormones. However, in contrast with untransfected cells or cells transfected with the sense construct, those containing the antisense construct did not demonstrate the appearance of any malignant phenotypic markers (growth in soft agar and tumorigenicity in athymic mice). A great reduction of the HMGI-C protein levels and the absence of the HMGI(Y) proteins was observed in the HMGI-C antisense-transfected, virally infected cells. Therefore, the HMGI-C protein seems to play a key role in the transformation of these thyroid cells. PMID:7862147

  3. Inhibition Profiling of Retroviral Protease Inhibitors Using an HIV-2 Modular System.

    PubMed

    Mahdi, Mohamed; Szojka, Zsófia; Mótyán, János András; Tőzsér, József

    2015-12-01

    Retroviral protease inhibitors (PIs) are fundamental pillars in the treatment of HIV infection and acquired immunodeficiency syndrome (AIDS). Currently used PIs are designed against HIV-1, and their effect on HIV-2 is understudied. Using a modular HIV-2 protease cassette system, inhibition profiling assays were carried out for protease inhibitors both in enzymatic and cell culture assays. Moreover, the treatment-associated resistance mutations (I54M, L90M) were introduced into the modular system, and comparative inhibition assays were performed to determine their effect on the susceptibility of the protease. Our results indicate that darunavir, saquinavir, indinavir and lopinavir were very effective HIV-2 protease inhibitors, while tipranavir, nelfinavir and amprenavir showed a decreased efficacy. I54M, L90M double mutation resulted in a significant reduction in the susceptibility to most of the inhibitors with the exception of tipranavir. To our knowledge, this modular system constitutes a novel approach in the field of HIV-2 protease characterization and susceptibility testing. PMID:26633459

  4. Biomaterial-Mediated Retroviral Gene Transfer Using Self-Assembled Monolayers

    PubMed Central

    Gersbach, Charles A.; Coyer, Sean R.; Le Doux, Joseph M.; García, Andrés J.

    2007-01-01

    Biomaterial-mediated gene delivery has recently emerged as a promising alternative to conventional gene transfer technologies that focus on direct delivery of viral vectors or DNA-polymer/matrix complexes. However, biomaterial-based strategies have primarily targeted transient gene expression vehicles, including plasmid DNA and adenovirus particles. This study expands on this work by characterizing biomaterial properties conducive to the surface immobilization of retroviral particles and subsequent transduction of mammalian cells at the cell-material interface. Self-assembled monolayers (SAMs) of functionally-terminated alkanethiols on gold were used to establish biomaterial surfaces of defined chemical composition. Gene transfer was observed to be greater than 90% on NH2-terminated surfaces, approximately 50% on COOH-functionalized surfaces, and undetectable on CH3-terminated SAMs, similar to controls of tissue culture-treated polystyrene. Gene delivery via the NH2-SAM was further characterized as a function of coating time, virus concentration, and cell seeding density. Finally, SAM-mediated gene delivery was comparable to fibronectin- and poly-L-lysine-based methods for gene transfer. This work is significant to establishing safe and effective gene therapy strategies, developing efficient methods for gene delivery, and supporting recent progress in the field of biomaterial-mediated gene transfer. PMID:17698189

  5. Boosting of HIV-1 Neutralizing Antibody Responses by a Distally Related Retroviral Envelope Protein

    PubMed Central

    Uchtenhagen, Hannes; Schiffner, Torben; Bowles, Emma; Heyndrickx, Leo; LaBranche, Celia; Applequist, Steven E.; Jansson, Marianne; De Silva, Thushan; Back, Jaap Willem; Achour, Adnane; Scarlatti, Gabriella; Fomsgaard, Anders; Montefiori, David; Stewart-Jones, Guillaume; Spetz, Anna-Lena

    2014-01-01

    Our knowledge of the binding sites for neutralizing antibodies (NAbs) that recognize a broad range of HIV-1 strains (bNAb) has substantially increased in recent years. However, gaps remain in our understanding of how to focus B-cell responses to vulnerable conserved sites within the HIV-1 envelope glycoprotein (Env). Here we report an immunization strategy composed of a trivalent HIV-1 (clade B envs) DNA prime, followed by a SIVmac239 gp140 Env protein boost that aimed to focus the immune response to structurally conserved parts of the HIV-1 and SIV Envs. Heterologous NAb titres, primarily to tier 1 HIV-1 isolates, elicited during the trivalent HIV-1 env prime, were significantly increased by the SIVmac239 gp140 protein boost in rabbits. Epitope mapping of antibody binding reactivity revealed preferential recognition of the C1, C2, V2, V3 and V5 regions. These results provide a proof of concept that a distally related retroviral SIV Env protein boost can increase pre-existing NAb responses against HIV-1. PMID:24829409

  6. MuLV IN Mutants Responsive to HDAC Inhibitors Enhance Transcription from Unintegrated Retroviral DNA

    PubMed Central

    Schneider, William M.; Wu, Dai-tze; Amin, Vaibhav; Aiyer, Sriram; Roth, Monica J.

    2012-01-01

    For Moloney murine leukemia virus (M-MuLV), sustained viral infections require expression from an integrated provirus. For many applications, non-integrating retroviral vectors have been utilized to avoid the unwanted effects of integration, however, the level of expression from unintegrated DNA is significantly less than that of integrated provirus. We find that unintegrated DNA expression can be increased in the presence of HDAC inhibitors, such as TSA, when applied in combination with integrase (IN) mutations. These mutants include an active site mutation as well as catalytically active INs bearing mutations of K376 in the MuLV C-terminal domain of IN. MuLV IN K376 is homologous to K266 in HIV-1 IN, a known substrate for acetylation. The MuLV IN protein is acetylated by p300 in vitro, however, the effect of HDAC inhibitors on gene expression from unintegrated DNA is not dependent on the acetylation state of MuLV IN K376. PMID:22365328

  7. Outer domains of integrase within retroviral intasomes are dispensible for catalysis of DNA integration.

    PubMed

    Li, Min; Lin, Shiqiang; Craigie, Robert

    2016-02-01

    Retroviral DNA integration is mediated by nucleoprotein complexes (intasomes) comprising a pair of viral DNA ends synapsed by a tetramer of integrase. Current integrase inhibitors act on intasomes rather than free integrase protein. Structural and functional studies of intasomes are essential to understand their mechanism of action and how the virus can escape by mutation. To date, prototype foamy virus (PFV) is the only retrovirus for which high-resolution structures of intasomes have been determined. In the PFV intasome structure, only the core domains of the outer subunits are ordered; the N-terminal domain, C-terminal domain, and N-terminal extension domain are disordered. Are these "missing domains" required for function or are they dispensable? We have devised a strategy to assemble "hetero-intasomes" in which the outer domains are not present as a tool to assess the functional role of the missing domains for catalysis of integration. We find that the disordered domains of outer subunits are not required for intasome assembly or catalytic activity as catalytic core domains can substitute for the outer subunits in the case of both PFV and HIV-1 intasomes. PMID:26537415

  8. A Self-inactivating γ-Retroviral Vector Reduces Manifestations of Mucopolysaccharidosis I in Mice

    PubMed Central

    Metcalf, Jason A; Ma, Xiucui; Linders, Bruce; Wu, Susan; Schambach, Axel; Ohlemiller, Kevin K; Kovacs, Attila; Bigg, Mark; He, Li; Tollefsen, Douglas M; Ponder, Katherine P

    2009-01-01

    Mucopolysaccharidosis I (MPS I) is a lysosomal storage disease due to deficiency in α-L-iduronidase (IDUA) that results in accumulation of glycosaminoglycans (GAGs) throughout the body, causing numerous clinical defects. Intravenous administration of a γ-retroviral vector (γ-RV) with an intact long terminal repeat (LTR) reduced the clinical manifestations of MPS I, but could cause insertional mutagenesis. Although self-inactivating (SIN) γ-RVs in which the enhancer and promoter elements in the viral LTR are absent after transduction reduces this risk, such vectors could be less effective. This report demonstrates that intravenous (i.v.) injection of a SIN γ-RV expressing canine IDUA from the liver-specific human α1-antitrypsin promoter into adult or newborn MPS I mice completely prevents biochemical abnormalities in several organs, and improved bone disease, vision, hearing, and aorta to a similar extent as was seen with administration of the LTR-intact vector to adults. Improvements were less profound than when using an LTR-intact γ-RV in newborns, which likely reflects a lower level of transduction and expression for the SIN vector-transduced mice, and might be overcome by using a higher dose of SIN vector. A SIN γ-RV vector ameliorates clinical manifestations of MPS I in mice and should be safer than an LTR-intact γ-RV. PMID:19844196

  9. B′-protein phosphatase 2A is a functional binding partner of delta-retroviral integrase

    PubMed Central

    Maertens, Goedele N.

    2016-01-01

    To establish infection, a retrovirus must insert a DNA copy of its RNA genome into host chromatin. This reaction is catalysed by the virally encoded enzyme integrase (IN) and is facilitated by viral genus-specific host factors. Herein, cellular serine/threonine protein phosphatase 2A (PP2A) is identified as a functional IN binding partner exclusive to δ-retroviruses, including human T cell lymphotropic virus type 1 and 2 (HTLV-1 and HTLV-2) and bovine leukaemia virus (BLV). PP2A is a heterotrimer composed of a scaffold, catalytic and one of any of four families of regulatory subunits, and the interaction is specific to the B′ family of the regulatory subunits. B′-PP2A and HTLV-1 IN display nuclear co-localization, and the B′ subunit stimulates concerted strand transfer activity of δ-retroviral INs in vitro. The protein–protein interaction interface maps to a patch of highly conserved residues on B′, which when mutated render B′ incapable of binding to and stimulating HTLV-1 and -2 IN strand transfer activity. PMID:26657642

  10. Mitochondrial DNA Haplogroups influence lipoatrophy after Highly Active Anti-retroviral Therapy

    PubMed Central

    Hendrickson, Sher L.; Kingsley, Lawrence A.; Ruiz-Pesini, Eduardo; Poole, Jason C.; Jacobson, Lisa P.; Palella, Frank J.; Bream, Jay H.; Wallace, Douglas C.; O’Brien, Stephen J.

    2009-01-01

    Although highly active retroviral therapy (HAART) has been extremely effective in lowering AIDS incidence among patients infected with HIV, certain drugs included in HAART can cause serious mitochondrial toxicities. One of the most frequent adverse events is lipoatrophy, which is the loss of subcutaneous fat in the face, arms, buttocks and/or legs as an adverse reaction to nucleoside reverse transcriptase inhibitors (NRTIs). The clinical symptoms of lipoatrophy resemble those of inherited mitochondrial diseases, which suggests that host mitochondrial genotype may play a role in susceptibility. We analyzed the association between mitochondrial haplogroup and severity of lipoatrophy in HIV-infected European American patients on HAART in the Multicenter AIDS cohort Study (MACS) and found that mitochondrial haplogroup H was strongly associated with increased atrophy (arms: p = 0.007, OR = 1.77, 95% CI = 1.17–2.69 legs: p = 0.037, OR = 1.54 95% CI = 1.03–2.31, and buttocks: p = 0.10, OR = 1.41 95% CI = 0.94–2.12). We also saw borderline significance for haplogroup T as protective against lipoatrophy (p = 0.05, OR = 0.52, 95% CI = 0.20–1.00). These data suggest that mitochondrial DNA haplogroup may influence the propensity for lipoatrophy in patients receiving NRTIs. PMID:19339895

  11. Effects of Hormonal Contraception on Anti-Retroviral Drug Metabolism, Pharmacokinetics and Pharmacodynamics

    PubMed Central

    Thurman, Andrea Ries; Anderson, Sharon; Doncel, Gustavo F

    2014-01-01

    Among women, human immunodeficiency virus type 1 (HIV-1) infection is most prevalent in those of reproductive age. These women are also at risk of unintended or mistimed pregnancies. Hormonal contraceptives (HCs) are one of the most commonly used methods of family planning world-wide. Therefore concurrent use of HC among women on anti-retroviral medications (ARVs) is increasingly common. ARVs are being investigated and have been approved for pre-exposure prophylaxis (PrEP), and therefore drug-drug interactions must also be considered in HIV-1 negative women who want to prevent both unintended pregnancy and HIV-1 infection. This article will review four main interactions: (1) the effect of HCs on ARV pharmacokinetics (PK) and pharmacodynamics (PD) during therapy, (2) the effect of ARVs on HC PK and PD, (3) the role of drug transporters on drug-drug interactions and (4) ongoing research into the effect of HCs on pre-exposure prophylaxis PK and PD. PMID:24521428

  12. B'-protein phosphatase 2A is a functional binding partner of delta-retroviral integrase.

    PubMed

    Maertens, Goedele N

    2016-01-01

    To establish infection, a retrovirus must insert a DNA copy of its RNA genome into host chromatin. This reaction is catalysed by the virally encoded enzyme integrase (IN) and is facilitated by viral genus-specific host factors. Herein, cellular serine/threonine protein phosphatase 2A (PP2A) is identified as a functional IN binding partner exclusive to δ-retroviruses, including human T cell lymphotropic virus type 1 and 2 (HTLV-1 and HTLV-2) and bovine leukaemia virus (BLV). PP2A is a heterotrimer composed of a scaffold, catalytic and one of any of four families of regulatory subunits, and the interaction is specific to the B' family of the regulatory subunits. B'-PP2A and HTLV-1 IN display nuclear co-localization, and the B' subunit stimulates concerted strand transfer activity of δ-retroviral INs in vitro. The protein-protein interaction interface maps to a patch of highly conserved residues on B', which when mutated render B' incapable of binding to and stimulating HTLV-1 and -2 IN strand transfer activity. PMID:26657642

  13. Inhibition Profiling of Retroviral Protease Inhibitors Using an HIV-2 Modular System

    PubMed Central

    Mahdi, Mohamed; Szojka, Zsófia; Mótyán, János András; Tőzsér, József

    2015-01-01

    Retroviral protease inhibitors (PIs) are fundamental pillars in the treatment of HIV infection and acquired immunodeficiency syndrome (AIDS). Currently used PIs are designed against HIV-1, and their effect on HIV-2 is understudied. Using a modular HIV-2 protease cassette system, inhibition profiling assays were carried out for protease inhibitors both in enzymatic and cell culture assays. Moreover, the treatment-associated resistance mutations (I54M, L90M) were introduced into the modular system, and comparative inhibition assays were performed to determine their effect on the susceptibility of the protease. Our results indicate that darunavir, saquinavir, indinavir and lopinavir were very effective HIV-2 protease inhibitors, while tipranavir, nelfinavir and amprenavir showed a decreased efficacy. I54M, L90M double mutation resulted in a significant reduction in the susceptibility to most of the inhibitors with the exception of tipranavir. To our knowledge, this modular system constitutes a novel approach in the field of HIV-2 protease characterization and susceptibility testing. PMID:26633459

  14. Growth factor enhanced retroviral gene transfer to the adult central nervous system.

    PubMed

    King, L A; Mitrophanous, K A; Clark, L A; Kim, V N; Rohll, J B; Kingsman, A J; Colello, R J

    2000-07-01

    The use of viral vectors for gene delivery into mammalian cells provides a new approach in the treatment of many human diseases. The first viral vector approved for human clinical trials was murine leukemia virus (MLV), which remains the most commonly used vector in clinical trials to date. However, the application of MLV vectors is limited since MLV requires cells to be actively dividing in order for transduction and therefore gene delivery to occur. This limitation precludes the use of MLV for delivering genes to the adult CNS, where very little cell division is occurring. However, we speculated that this inherent limitation of ML V may be overcome by utilizing the known mitogenic effect of growth factors on cells of the CNS. Specifically, an in vivo application of growth factor to the adult brain, if able to induce cell division, could enhance MLV-based gene transfer to the adult brain. We now show that an exogenous application of basic fibroblast growth factor induces cell division in vivo. Under these conditions, where cells of the adult brain are stimulated to divide, MLV-based gene transfer is significantly enhanced. This novel approach precludes any vector modifications and provides a simple and effective way of delivering genes to cells of the adult brain utilizing MLV-based retroviral vectors. PMID:10918476

  15. Onco-exaptation of an endogenous retroviral LTR drives IRF5 expression in Hodgkin lymphoma.

    PubMed

    Babaian, A; Romanish, M T; Gagnier, L; Kuo, L Y; Karimi, M M; Steidl, C; Mager, D L

    2016-05-12

    The transcription factor interferon regulatory factor 5 (IRF5) is upregulated in Hodgkin lymphoma (HL) and is a key regulator of the aberrant transcriptome characteristic of this disease. Here we show that IRF5 upregulation in HL is driven by transcriptional activation of a normally dormant endogenous retroviral LOR1a long terminal repeat (LTR) upstream of IRF5. Specifically, through screening of RNA-sequencing libraries, we detected LTR-IRF5 chimeric transcripts in multiple HL cell lines but not in normal B-cell controls. In HL, the LTR was in an open and hypomethylated epigenetic state, and we further show the LTR is the site of transcriptional initiation. Among HL cell lines, usage of the LTR promoter strongly correlates with overall levels of IRF5 mRNA and protein, indicating that LTR transcriptional awakening is a major contributor to IRF5 upregulation in HL. Taken together, oncogenic IRF5 overexpression in HL is the result of a specific LTR transcriptional activation. We propose that such LTR derepression is a distinct mechanism of oncogene activation ('onco-exaptation'), and that such a mechanism warrants further investigation in molecular and cancer research. PMID:26279299

  16. Retroviral infection of non-dividing cells: Old and new perspectives

    SciTech Connect

    Yamashita, Masahiro; Emerman, Michael . E-mail: memerman@fhcrc.org

    2006-01-05

    The dependence of retroviral replication on cell proliferation was described as early as 1958, although different classes of retroviruses are able to infect non-dividing cells with different efficiencies. For example, the human immunodeficiency virus (HIV) and other lentiviruses infect most non-dividing cells nearly as well as dividing cells, while the gammaretroviruses such as the murine leukemia virus (MLV) cannot infect non-dividing cells, and other retroviruses have intermediate phenotypes. One exception to the ability of HIV to infect non-dividing cells involves resting CD4+ T cells in vitro where there are multiple restrictions. However, recent data show that there is massive infection of non-activated CD4+ T cell during acute infection which suggests that the situation is different in vivo. Finally, much work trying to explain the difference between HIV and MLV in non-dividing cells has focused on describing the ability of HIV to enter the nucleus during interphase. However, we suggest that events in the viral lifecycle other than nuclear import may be more important in determining the ability of a given retrovirus to infect non-dividing cells.

  17. Single molecule DNA interaction kinetics of retroviral nucleic acid chaperone proteins

    NASA Astrophysics Data System (ADS)

    Williams, Mark

    2010-03-01

    Retroviral nucleocapsid (NC) proteins are essential for several viral replication processes including specific genomic RNA packaging and reverse transcription. The nucleic acid chaperone activity of NC facilitates the latter process. In this study, we use single molecule biophysical methods to quantify the DNA interactions of wild type and mutant human immunodeficiency virus type 1 (HIV-1) NC and Gag and human T-cell leukemia virus type 1 (HTLV-1) NC. We find that the nucleic acid interaction properties of these proteins differ significantly, with HIV-1 NC showing rapid protein binding kinetics, significant duplex destabilization, and strong DNA aggregation, all properties that are critical components of nucleic acid chaperone activity. In contrast, HTLV-1 NC exhibits significant destabilization activity but extremely slow DNA interaction kinetics and poor aggregating capability, which explains why HTLV-1 NC is a poor nucleic acid chaperone. To understand these results, we developed a new single molecule method for quantifying protein dissociation kinetics, and applied this method to probe the DNA interactions of wild type and mutant HIV-1 and HTLV-1 NC. We find that mutations to aromatic and charged residues strongly alter the proteins' nucleic acid interaction kinetics. Finally, in contrast to HIV-1 NC, HIV-1 Gag, the nucleic acid packaging protein that contains NC as a domain, exhibits relatively slow binding kinetics, which may negatively impact its ability to act as a nucleic acid chaperone.

  18. Integration Site and Clonal Expansion in Human Chronic Retroviral Infection and Gene Therapy

    PubMed Central

    Niederer, Heather A.; Bangham, Charles R. M.

    2014-01-01

    Retroviral vectors have been successfully used therapeutically to restore expression of genes in a range of single-gene diseases, including several primary immunodeficiency disorders. Although clinical trials have shown remarkable results, there have also been a number of severe adverse events involving malignant outgrowth of a transformed clonal population. This clonal expansion is influenced by the integration site profile of the viral integrase, the transgene expressed, and the effect of the viral promoters on the neighbouring host genome. Infection with the pathogenic human retrovirus HTLV-1 also causes clonal expansion of cells containing an integrated HTLV-1 provirus. Although the majority of HTLV-1-infected people remain asymptomatic, up to 5% develop an aggressive T cell malignancy. In this review we discuss recent findings on the role of the genomic integration site in determining the clonality and the potential for malignant transformation of cells carrying integrated HTLV-1 or gene therapy vectors, and how these results have contributed to the understanding of HTLV-1 pathogenesis and to improvements in gene therapy vector safety. PMID:25365582

  19. Effects of retroviral envelope-protein cleavage upon trafficking, incorporation, and membrane fusion

    SciTech Connect

    Apte, Swapna; Sanders, David Avram

    2010-09-15

    Retroviral envelope glycoproteins undergo proteolytic processing by cellular subtilisin-like proprotein convertases at a polybasic amino-acid site in order to produce the two functional subunits, SU and TM. Most previous studies have indicated that envelope-protein cleavage is required for rendering the protein competent for promoting membrane fusion and for virus infectivity. We have investigated the role of proteolytic processing of the Moloney murine leukemia virus envelope-protein through site-directed mutagenesis of the residues near the SU-TM cleavage site and have established that uncleaved glycoprotein is unable either to be incorporated into virus particles efficiently or to induce membrane fusion. Additionally, the results suggest that cleavage of the envelope protein plays an important role in intracellular trafficking of protein via the cellular secretory pathway. Based on our results it was concluded that a positively charged residue located at either P2 or P4 along with the arginine at P1 is essential for cleavage.

  20. Isolation, characterization, and genetic complementation of a cellular mutant resistant to retroviral infection

    PubMed Central

    Agarwal, Sumit; Harada, Josephine; Schreifels, Jeffrey; Lech, Patrycja; Nikolai, Bryan; Yamaguchi, Tomoyuki; Chanda, Sumit K.; Somia, Nikunj V.

    2006-01-01

    By using a genetic screen, we have isolated a mammalian cell line that is resistant to infection by retroviruses that are derived from the murine leukemia virus, human immunodeficiency virus type 1, and feline immunodeficiency virus. We demonstrate that the cell line is genetically recessive for the resistance, and hence it is lacking a factor enabling infection by retroviruses. The block to infection is early in the life cycle, at the poorly understood uncoating stage. We implicate the proteasome at uncoating by completely rescuing the resistant phenotype with the proteasomal inhibitor MG-132. We further report on the complementation cloning of a gene (MRI, modulator of retrovirus infection) that can also act to reverse the inhibition of infection in the mutant cell line. These data implicate a role for the proteasome during uncoating, and they suggest that MRI is a regulator of this activity. Finally, we reconcile our findings and other published data to suggest a model for the involvement of the proteasome in the early phase of the retroviral life cycle. PMID:17043244

  1. Neonatal Gene Therapy With a Gamma Retroviral Vector in Mucopolysaccharidosis VI Cats

    PubMed Central

    Ponder, Katherine P; O'Malley, Thomas M; Wang, Ping; O'Donnell, Patricia A; Traas, Anne M; Knox, Van W; Aguirre, Gustavo A; Ellinwood, N Matthew; Metcalf, Jason A; Wang, Bin; Parkinson-Lawrence, Emma J; Sleeper, Meg M; Brooks, Doug A; Hopwood, John J; Haskins, Mark E

    2012-01-01

    Mucopolysaccharidosis (MPS) VI is due to a deficiency in the activity of N-acetylgalactosamine 4-sulfatase (4S), also known as arylsulfatase B. Previously, retroviral vector (RV)-mediated neonatal gene therapy reduced the clinical manifestations of MPS I and MPS VII in mice and dogs. However, sulfatases require post-translational modification by sulfatase-modifying factors. MPS VI cats were injected intravenously (i.v.) with a gamma RV-expressing feline 4S, resulting in 5 ± 3 copies of RV per 100 cells in liver. Liver and serum 4S activity were 1,450 ± 1,720 U/mg (26-fold normal) and 107 ± 60 U/ml (13-fold normal), respectively, and were directly proportional to the liver 4S protein levels for individual cats. This study suggests that sulfatase-modifying factor (SUMF) activity in liver was sufficient to result in active enzyme despite overexpression of 4S. RV-treated MPS VI cats achieved higher body weights and longer appendicular skeleton lengths, had reduced articular cartilage erosion, and reduced aortic valve thickening and aortic dilatation compared with untreated MPS VI cats, although cervical vertebral bone lengths were not improved. This demonstrates that therapeutic expression of a functional sulfatase protein can be achieved with neonatal gene therapy using a gamma RV, but some aspects of bone disease remain difficult to treat. PMID:22395531

  2. Serial bone marrow transplantation reveals in vivo expression of the pCLPG retroviral vector

    PubMed Central

    2010-01-01

    Background Gene therapy in the hematopoietic system remains promising, though certain aspects of vector design, such as transcriptional control elements, continue to be studied. Our group has developed a retroviral vector where transgene expression is controlled by p53 with the intention of harnessing the dynamic and inducible nature of this tumor suppressor and transcription factor. We present here a test of in vivo expression provided by the p53-responsive vector, pCLPG. For this, we used a model of serial transplantation of transduced bone marrow cells. Results We observed, by flow cytometry, that the eGFP transgene was expressed at higher levels when the pCLPG vector was used as compared to the parental pCL retrovirus, where expression is directed by the native MoMLV LTR. Expression from the pCLPG vector was longer lasting, but did decay along with each sequential transplant. The detection of eGFP-positive cells containing either vector was successful only in the bone marrow compartment and was not observed in peripheral blood, spleen or thymus. Conclusions These findings indicate that the p53-responsive pCLPG retrovirus did offer expression in vivo and at a level that surpassed the non-modified, parental pCL vector. Our results indicate that the pCLPG platform may provide some advantages when applied in the hematopoietic system. PMID:20096105

  3. Host factors in retroviral integration and the selection of integration target sites

    PubMed Central

    Craigie, Robert; Bushman, Frederic D.

    2015-01-01

    In order to replicate, a retrovirus must integrate a DNA copy of the viral RNA genome into a chromosome of the host cell. The study of retroviral integration has advanced considerably in the last few years. Here we focus on host factor interactions and the linked area of integration targeting. Genome-wide screens for cellular factors affecting HIV replication have identified a series of host cell proteins that may mediate subcellular trafficking of integration complexes, nuclear import, and integration target site selection. The cell transcriptional co-activator protein LEDGF/p75 has been identified as a tethering factor important for HIV integration, and recently, BET proteins (Brd2, 4, and 4) have been identified as tethering factors for the gammaretroviruses. A new class of HIV inhibitors has been developed targeting the HIV-1 IN-LEDGF binding site, though surprisingly these inhibitors appear to block assembly late during replication and do not act at the integration step. Going forward, genome-wide studies of HIV-host interactions offer many new starting points to investigate HIV replication and identify potential new inhibitor targets. PMID:26104434

  4. Probing Retroviral and Retrotransposon Genome Structures: The “SHAPE” of Things to Come

    PubMed Central

    Sztuba-Solinska, Joanna; Le Grice, Stuart F. J.

    2012-01-01

    Understanding the nuances of RNA structure as they pertain to biological function remains a formidable challenge for retrovirus research and development of RNA-based therapeutics, an area of particular importance with respect to combating HIV infection. Although a variety of chemical and enzymatic RNA probing techniques have been successfully employed for more than 30 years, they primarily interrogate small (100–500 nt) RNAs that have been removed from their biological context, potentially eliminating long-range tertiary interactions (such as kissing loops and pseudoknots) that may play a critical regulatory role. Selective 2′ hydroxyl acylation analyzed by primer extension (SHAPE), pioneered recently by Merino and colleagues, represents a facile, user-friendly technology capable of interrogating RNA structure with a single reagent and, combined with automated capillary electrophoresis, can analyze an entire 10,000-nucleotide RNA genome in a matter of weeks. Despite these obvious advantages, SHAPE essentially provides a nucleotide “connectivity map,” conversion of which into a 3-D structure requires a variety of complementary approaches. This paper summarizes contributions from SHAPE towards our understanding of the structure of retroviral genomes, modifications to which technology that have been developed to address some of its limitations, and future challenges. PMID:22685659

  5. A rev1-vpu polymorphism unique to HIV-1 subtype A and C strains impairs envelope glycoprotein expression from rev-vpu-env cassettes and reduces virion infectivity in pseudotyping assays

    SciTech Connect

    Kraus, Matthias H.; Parrish, Nicholas F.; Shaw, Katharina S.; Decker, Julie M.; Keele, Brandon F.; Salazar-Gonzalez, Jesus F.; Grayson, Truman; McPherson, David T.; Ping, Li-Hua; Anderson, Jeffrey A.; Swanstrom, Ronald; Williamson, Carolyn; Shaw, George M.; Hahn, Beatrice H.

    2010-02-20

    Functional studies of HIV-1 envelope glycoproteins (Envs) commonly include the generation of pseudoviruses, which are produced by co-transfection of rev-vpu-env cassettes with an env-deficient provirus. Here, we describe six Env constructs from transmitted/founder HIV-1 that were defective in the pseudotyping assay, although two produced infectious virions when expressed from their cognate proviruses. All of these constructs exhibited an unusual gene arrangement in which the first exon of rev (rev1) and vpu were in the same reading frame without an intervening stop codon. Disruption of the rev1-vpu fusion gene by frameshift mutation, stop codon, or abrogation of the rev initiation codon restored pseudovirion infectivity. Introduction of the fusion gene into wildtype Env cassettes severely compromised their function. The defect was not due to altered env and rev transcription or a dominant negative effect of the expressed fusion protein, but seemed to be caused by inefficient translation at the env initiation codon. Although the rev1-vpu polymorphism affects Env expression only in vitro, it can cause problems in studies requiring Env complementation, such as analyses of co-receptor usage and neutralization properties, since 3% of subtype A, 20% of subtype C and 5% of CRF01{sub A}/E viruses encode the fusion gene. A solution is to eliminate the rev initiation codon when amplifying rev-vpu-env cassettes since this increases Env expression irrespective of the presence of the polymorphism.

  6. Retroviral Cyclin Controls Cyclin-Dependent Kinase 8-Mediated Transcription Elongation and Reinitiation

    PubMed Central

    Birkenheuer, Claire H.; Brewster, Connie D.; Quackenbush, Sandra L.

    2015-01-01

    ABSTRACT Walleye dermal sarcoma virus (WDSV) infection is associated with the seasonal development and regression of walleye dermal sarcoma. Previous work showed that the retroviral cyclin (RV-cyclin), encoded by WDSV, has separable cyclin box and transcription activation domains. It binds to cyclin-dependent kinase 8 (CDK8) and enhances its kinase activity. CDK8 is evolutionarily conserved and is frequently overexpressed in human cancers. It is normally activated by cyclin C and is required for transcription elongation of the serum response genes (immediate early genes [IEGs]) FOS, EGR1, and cJUN. The IEGs drive cell proliferation, and their expression is brief and highly regulated. Here we show that constitutive expression of RV-cyclin in the HCT116 colon cancer cell line significantly increases the level of IEG expression in response to serum stimulation. Quantitative reverse transcription-PCR (RT-PCR) and nuclear run-on assays provide evidence that RV-cyclin does not alter the initiation of IEG transcription but does enhance the overall rate of transcription elongation and maintains transcription reinitiation. RV-cyclin does not increase activating phosphorylation events in the mitogen-activated protein kinase pathway and does not inhibit decay of IEG mRNAs. At the EGR1 gene locus, RV-cyclin increases and maintains RNA polymerase II (Pol II) occupancy after serum stimulation, in conjunction with increased and extended EGR1 gene expression. The RV-cyclin increases CDK8 occupancy at the EGR1 gene locus before and after serum stimulation. Both of RV-cyclin's functional domains, i.e., the cyclin box and the activation domain, are necessary for the overall enhancement of IEG expression. RV-cyclin presents a novel and ancient mechanism of retrovirus-induced oncogenesis. IMPORTANCE The data reported here are important to both virology and cancer biology. The novel mechanism pinpoints CDK8 in the development of walleye dermal sarcoma and sheds light on CDK8's role in

  7. Cost-effectiveness of anti-retroviral therapy at a district hospital in southern Ethiopia

    PubMed Central

    Bikilla, Asfaw Demissie; Jerene, Degu; Robberstad, Bjarne; Lindtjørn, Bernt

    2009-01-01

    Background As the resource implications of expanding anti-retroviral therapy (ART) are likely to be large, there is a need to explore its cost-effectiveness. So far, there is no such information available from Ethiopia. Objective To assess the cost-effectiveness of ART for routine clinical practice in a district hospital setting in Ethiopia. Methods We estimated the unit cost of HIV-related care from the 2004/5 fiscal year expenditure of Arba Minch Hospital in southern Ethiopia. We estimated outpatient and inpatient service use from HIV-infected patients who received care and treatment at the hospital between January 2003 and March 2006. We measured the health effect as life years gained (LYG) for patients receiving ART compared with those not receiving such treatment. The study adopted a health care provider perspective and included both direct and overhead costs. We used Markov model to estimate the lifetime costs, health benefits and cost-effectiveness of ART. Findings ART yielded an undiscounted 9.4 years expected survival, and resulted in 7.1 extra LYG compared to patients not receiving ART. The lifetime incremental cost is US$2,215 and the undiscounted incremental cost per LYG is US$314. When discounted at 3%, the additional LYG decreases to 5.5 years and the incremental cost per LYG increases to US$325. Conclusion The undiscounted and discounted incremental costs per LYG from introducing ART were less than the per capita GDP threshold at the base year. Thus, ART could be regarded as cost-effective in a district hospital setting in Ethiopia. PMID:19615069

  8. Murine retroviral vector producer cells survival and toxicity in the dog liver.

    PubMed

    Link, C J; Seregina, T; Levy, J P; Martin, M; Ackermann, M; Moorman, D W

    2000-01-01

    To develop a safe method to target gene delivery into intrahepatic tumors, we examined the toxicity of intrahepatic (IH) injection of retroviral vector producer cells (VPC) into the canine liver. VPC have been demonstrated to effectively transfer genes in vivo. To evaluate for adverse effects form xenogeneic cell transplantation, mongrel dogs were injected IH with 1 x 10(9) murine LTKOSN.2 VPC divided into three aliquots. The animals were then monitored for acute toxicity induced by the VPC. The intraoperative IH injections of the cells were tolerated without difficulty. Starting 7 days after IH injection, the dogs then received intravenous ganciclovir (GCV) twice daily (5 mg/kg) for 7 days. GCV treatment did not cause significant toxicities. Dogs underwent serial blood tests to evaluate bone marrow, renal, liver and immunological function. Complete blood counts, electrolytes, liver function and renal function tests remained normal except for mild elevations of alkaline phosphatase. Histologic examination of liver tissues from the IH injection site revealed no apparent normal tissue destruction induced by the VPC. Two of the four treated dogs underwent liver biopsy on day 3. These biopsy specimens were cultured and persistent, viable VPC were recovered. The dogs mounted an antibody response to the murine VPC that was first demonstrated 5 days post injection. PCR analysis demonstrated low level gene transfer into dog liver tissue. Overall, our results demonstrate that IH xenogeneic VPC injections are not accompanied by significant adverse effects over a 1 month period following administration into the canine liver. These data support the safety aspects of using murine VPC in Phase I clinical trials. PMID:11212842

  9. Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites.

    PubMed

    Serrao, Erik; Cherepanov, Peter; Engelman, Alan N

    2016-01-01

    Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients. PMID:27023428

  10. Retroviral insertions in the VISION database identify molecular pathways in mouse lymphoid leukemia and lymphoma

    PubMed Central

    Weiser, Keith C.; Liu, Bin; Hansen, Gwenn M.; Skapura, Darlene; Hentges, Kathryn E.; Yarlagadda, Sujatha; Morse III, Herbert C.

    2007-01-01

    AKXD recombinant inbred (RI) strains develop a variety of leukemias and lymphomas due to somatically acquired insertions of retroviral DNA into the genome of hematopoetic cells that can mutate cellular proto-oncogenes and tumor suppressor genes. We generated a new set of tumors from nine AKXD RI strains selected for their propensity to develop B-cell tumors, the most common type of human hematopoietic cancers. We employed a PCR technique called viral insertion site amplification (VISA) to rapidly isolate genomic sequence at the site of provirus insertion. Here we describe 550 VISA sequence tags (VSTs) that identify 74 common insertion sites (CISs), of which 21 have not been identified previously. Several suspected proto-oncogenes and tumor suppressor genes lie near CISs, providing supportive evidence for their roles in cancer. Furthermore, numerous previously uncharacterized genes lie near CISs, providing a pool of candidate disease genes for future research. Pathway analysis of candidate genes identified several signaling pathways as common and powerful routes to blood cancer, including Notch, E-protein, NFκB, and Ras signaling. Misregulation of several Notch signaling genes was confirmed by quantitative RT-PCR. Our data suggest that analyses of insertional mutagenesis on a single genetic background are biased toward the identification of cooperating mutations. This tumor collection represents the most comprehensive study of the genetics of B-cell leukemia and lymphoma development in mice. We have deposited the VST sequences, CISs in a genome viewer, histopathology, and molecular tumor typing data in a public web database called VISION (Viral Insertion Sites Identifying Oncogenes), which is located at http://www.mouse-genome.bcm.tmc.edu/vision. PMID:17926094

  11. Local Sequence Targeting in the AID/APOBEC Family Differentially Impacts Retroviral Restriction and Antibody Diversification*

    PubMed Central

    Kohli, Rahul M.; Maul, Robert W.; Guminski, Amy F.; McClure, Rhonda L.; Gajula, Kiran S.; Saribasak, Huseyin; McMahon, Moira A.; Siliciano, Robert F.; Gearhart, Patricia J.; Stivers, James T.

    2010-01-01

    Nucleic acid cytidine deaminases of the activation-induced deaminase (AID)/APOBEC family are critical players in active and innate immune responses, playing roles as target-directed, purposeful mutators. AID specifically deaminates the host immunoglobulin (Ig) locus to evolve antibody specificity, whereas its close relative, APOBEC3G (A3G), lethally mutates the genomes of retroviral pathogens such as HIV. Understanding the basis for the target-specific action of these enzymes is essential, as mistargeting poses significant risks, potentially promoting oncogenesis (AID) or fostering drug resistance (A3G). AID prefers to deaminate cytosine in WRC (W = A/T, R = A/G) motifs, whereas A3G favors deamination of CCC motifs. This specificity is largely dictated by a single, divergent protein loop in the enzyme family that recognizes the DNA sequence. Through grafting of this substrate-recognition loop, we have created enzyme variants of A3G and AID with altered local targeting to directly evaluate the role of sequence specificity on immune function. We find that grafted loops placed in the A3G scaffold all produced efficient restriction of HIV but that foreign loops in the AID scaffold compromised hypermutation and class switch recombination. Local targeting, therefore, appears alterable for innate defense against retroviruses by A3G but important for adaptive antibody maturation catalyzed by AID. Notably, AID targeting within the Ig locus is proportionally correlated to its in vitro ability to target WRC sequences rather than non-WRC sequences. Although other mechanisms may also contribute, our results suggest that local sequence targeting by AID/APOBEC3 enzymes represents an elegant example of co-evolution of enzyme specificity with its target DNA sequence. PMID:20929867

  12. Histone H3.3 is required for endogenous retroviral element silencing in embryonic stem cells.

    PubMed

    Elsässer, Simon J; Noh, Kyung-Min; Diaz, Nichole; Allis, C David; Banaszynski, Laura A

    2015-06-11

    Transposable elements comprise roughly 40% of mammalian genomes. They have an active role in genetic variation, adaptation and evolution through the duplication or deletion of genes or their regulatory elements, and transposable elements themselves can act as alternative promoters for nearby genes, resulting in non-canonical regulation of transcription. However, transposable element activity can lead to detrimental genome instability, and hosts have evolved mechanisms to silence transposable element mobility appropriately. Recent studies have demonstrated that a subset of transposable elements, endogenous retroviral elements (ERVs) containing long terminal repeats (LTRs), are silenced through trimethylation of histone H3 on lysine 9 (H3K9me3) by ESET (also known as SETDB1 or KMT1E) and a co-repressor complex containing KRAB-associated protein 1 (KAP1; also known as TRIM28) in mouse embryonic stem cells. Here we show that the replacement histone variant H3.3 is enriched at class I and class II ERVs, notably those of the early transposon (ETn)/MusD family and intracisternal A-type particles (IAPs). Deposition at a subset of these elements is dependent upon the H3.3 chaperone complex containing α-thalassaemia/mental retardation syndrome X-linked (ATRX) and death-domain-associated protein (DAXX). We demonstrate that recruitment of DAXX, H3.3 and KAP1 to ERVs is co-dependent and occurs upstream of ESET, linking H3.3 to ERV-associated H3K9me3. Importantly, H3K9me3 is reduced at ERVs upon H3.3 deletion, resulting in derepression and dysregulation of adjacent, endogenous genes, along with increased retrotransposition of IAPs. Our study identifies a unique heterochromatin state marked by the presence of both H3.3 and H3K9me3, and establishes an important role for H3.3 in control of ERV retrotransposition in embryonic stem cells. PMID:25938714

  13. Ophthalmic manifestations of HIV in the highly active anti-retroviral therapy era.

    PubMed

    Mowatt, L

    2013-01-01

    HIV-related eye disease can be classified as retinal HIV microangiopathy, opportunistic infections, neuro-ophthalmic manifestations and unusual malignancies. There is a 52-100% lifetime accumulative risk of HIV patients developing eye problems. Seventy-seven per cent of patients with ocular manifestations of HIV had CD4 counts < 200 cells/μL. Cytomegalovirus (CMV) is the most prevalent opportunistic infection, however, Africa has a low incidence of this, and more commonly squamous cell carcinoma, compared to the western hemisphere. Due to highly active antiretroviral therapy (HAART), the anti-CMV therapy may be discontinued if the CD4+ T cell count is > 100 cells/μL for a minimum of three months. Despite HAART, patients with a CD4 count < 50 cells/μL have a similar risk of developing CMV retinitis as compared to the pre-HAART era. Opportunistic infections include CMV, herpetic retinopathy (progressive outer retinal necrosis - PORN), less commonly toxoplasmosis, pneumocystis and cryptococcus. Malignancies associated with HIV include Kaposi's sarcoma and conjunctival squamous cell carcinoma. Cranial nerve palsies, optic disc swelling and atrophy are characteristic neuro-ophthalmic features. They usually occur secondary to meningitis/encephalitis (from cryptococcus and tuberculosis). With the advent of HAART, new complications have developed in CMV retinitis: immune recovery uveitis (IRU) and cystoid macula oedema (CMO). Immune recovery uveitis occurs in 71% of patients if HAART is started before the induction of the anti-CMV treatment. However, this is reduced to 31% if HAART is started after the induction treatment. Molluscum contagiosum and Kaposi's sarcoma can spontaneously resolve on HAART. Highly active anti-retroviral therapy has reduced the frequencies of opportunistic infections and improved the remission duration in HIV patients. PMID:24756590

  14. Widespread Endogenization of Genome Sequences of Non-Retroviral RNA Viruses into Plant Genomes

    PubMed Central

    Tani, Akio; Saisho, Daisuke; Sakamoto, Wataru; Kanematsu, Satoko; Suzuki, Nobuhiro

    2011-01-01

    Non-retroviral RNA virus sequences (NRVSs) have been found in the chromosomes of vertebrates and fungi, but not plants. Here we report similarly endogenized NRVSs derived from plus-, negative-, and double-stranded RNA viruses in plant chromosomes. These sequences were found by searching public genomic sequence databases, and, importantly, most NRVSs were subsequently detected by direct molecular analyses of plant DNAs. The most widespread NRVSs were related to the coat protein (CP) genes of the family Partitiviridae which have bisegmented dsRNA genomes, and included plant- and fungus-infecting members. The CP of a novel fungal virus (Rosellinia necatrix partitivirus 2, RnPV2) had the greatest sequence similarity to Arabidopsis thaliana ILR2, which is thought to regulate the activities of the phytohormone auxin, indole-3-acetic acid (IAA). Furthermore, partitivirus CP-like sequences much more closely related to plant partitiviruses than to RnPV2 were identified in a wide range of plant species. In addition, the nucleocapsid protein genes of cytorhabdoviruses and varicosaviruses were found in species of over 9 plant families, including Brassicaceae and Solanaceae. A replicase-like sequence of a betaflexivirus was identified in the cucumber genome. The pattern of occurrence of NRVSs and the phylogenetic analyses of NRVSs and related viruses indicate that multiple independent integrations into many plant lineages may have occurred. For example, one of the NRVSs was retained in Ar. thaliana but not in Ar. lyrata or other related Camelina species, whereas another NRVS displayed the reverse pattern. Our study has shown that single- and double-stranded RNA viral sequences are widespread in plant genomes, and shows the potential of genome integrated NRVSs to contribute to resolve unclear phylogenetic relationships of plant species. PMID:21779172

  15. p62/Sequestosome-1 Associates with and Sustains the Expression of Retroviral Restriction Factor TRIM5α▿ †

    PubMed Central

    O'Connor, Christopher; Pertel, Thomas; Gray, Seth; Robia, Seth L.; Bakowska, Joanna C.; Luban, Jeremy; Campbell, Edward M.

    2010-01-01

    TRIM5 proteins mediate a potent block to the cross-species transmission of retroviruses, the most well known being the TRIM5α protein from rhesus macaques, which potently inhibits human immunodeficiency virus type 1 (HIV-1) infection. This restriction occurs at an early stage in the replication cycle and is mediated by the binding of TRIM5 proteins to determinants present in the retroviral capsid. TRIM5α, as well as other TRIM family proteins, has been shown to be regulated by interferons (IFN). Here we show that TRIM5α associates with another IFN-induced gene, sequestosome-1/p62 (p62). p62 plays a role in several signal transduction cascades that are important for maintaining the antiviral state of cells. Here we demonstrate that p62 localizes to both human and rhesus macaque TRIM5α cytoplasmic bodies, and fluorescence resonance energy transfer (FRET) analysis demonstrates that these proteins closely associate in these structures. When p62 expression was knocked down via small interfering RNA (siRNA), the number of TRIM5α cytoplasmic bodies and the level of TRIM5α protein expression were reduced in cell lines stably expressing epitope-tagged versions of TRIM5α. In accordance with these data, p62 knockdown resulted in reduced TRIM5α-mediated retroviral restriction in cells expressing epitope-tagged TRIM5α or expressing endogenously expressed human TRIM5α. p62 may therefore operate to enhance TRIM5α-mediated retroviral restriction, contributing to the antiviral state of cells following IFN treatment. PMID:20357094

  16. Gamma-Retroviral Vector Design for the Co-Expression of Artificial microRNAs and Therapeutic Proteins

    PubMed Central

    Park, Tristen S.; Zhang, Ling; Zheng, Zhili; Morgan, Richard A.

    2014-01-01

    To generate γ-retroviral vectors for stable conjoint expression of artificial microRNAs (amiR) and therapeutic genes in primary human lymphocytes, and to identify the design parameters that are key for successful vector generation. Gamma-retroviral vectors were designed to co-express both amiRs and a linked reporter gene, truncated CD34 (tCD34). Artificial miRs based on microRNAs miR-16, miR-142, miR-146b, miR-150, miR155, and miR-223 were inserted into sites within the intron of the vector and tested for tCD34 expression by flow cytometry (FACS). Different constructs were assembled with amiRs targeted to knockdown expression of suppressor of cytokine signaling 1 (SOCS1) or programmed cell death 1 (PDCD1, PD-1). Three of the six amiRs maintained tCD34 expression. Expansion of primary human T cells transduced with these amiR vectors, as well as transgene expression, were equivalent to control engineered T cells over a 40-day period. Knockdown of SOCS1 RNA and PD-1 expression by FACS was shown to vary between constructs, dependent on either the specific short interfering RNA sequence used in the amiR, or the microRNA backbone and location in the vector intron. Gamma-retroviral vectors that both efficiently knockdown endogenous gene expression and maintain linked transgene production can be produced, but empirical vector evaluations were best suited for optimal construct analysis. PMID:25019196

  17. A new family of retroviral long terminal repeat elements in the human genome identified by their homologies to an element 5{prime} to the spider monkey haptoglobin gene

    SciTech Connect

    Erickson, L.M.; Maeda, N.

    1995-06-10

    A new family of retroviral long terminal repeats that we name Spm-LTR has been identified as a result of DNA sequence comparisons between the entire Gen-Bank databank and an element, SPHP, located 5{prime} to the haptoglobin gene of spider monkeys. The 18 human Spm-LTR sequences so identified fall into three subtypes. There is no sequence similarity between Spm-LTR elements and any endogenous retroviral LTR sequences previously reported except for general features that define LTRs. However, a previously described repeated sequence (MER-4) forms a portion of the Spm-LTR sequence. 13 refs., 1 fig., 1 tab.

  18. Factors determining the risk of inadvertent retroviral transduction of male germ cells after in utero gene transfer in sheep.

    PubMed

    Park, Paul J; Colletti, Evan; Ozturk, Ferhat; Wood, Josh A; Tellez, Joe; Almeida-Porada, Graça; Porada, Christopher

    2009-03-01

    The possibility of permanent genetic changes to the germline is central to the bioethics of in utero gene therapy (IUGT) because of the concern of inadvertent potentially deleterious alterations to the gene pool. Despite presumed protection of the male germline due to early germ cell (GC) compartmentalization, we reported that GCs within the developing ovine testes are transduced at low levels after retrovirus-mediated IUGT, thus underscoring the need for a thorough understanding of GC development in clinically predictive models to determine the optimal time to perform IUGT and avoid germline modification. In the present studies, we used the fetal sheep model to analyze GCs for phenotype, location, proliferation, and incidence of transduction after IUGT at various fetal ages to learn when during development the nascent germline is likely to be at greatest risk of retrovirus-mediated alteration. Our studies show that although GCs were transduced at all injection ages, the levels of transduction varied by nearly 700-fold as a function of the age at transfer. After remaining largely quiescent as they migrated to/settled within nascent sex cords, GCs began active cycling before cord closure was complete, suggesting this is likely the point at which they would be most susceptible to retroviral transduction.Furthermore, we observed that compartmentalization of GCs continued into early postnatal life, suggesting the male germline may be vulnerable to low-level inadvertent retroviral vector modification throughout fetal life, but that this risk can be minimized by performing IUGT later in gestation. PMID:19301473

  19. Comprehensive profiling of retroviral integration sites using target enrichment methods from historical koala samples without an assembled reference genome.

    PubMed

    Cui, Pin; Löber, Ulrike; Alquezar-Planas, David E; Ishida, Yasuko; Courtiol, Alexandre; Timms, Peter; Johnson, Rebecca N; Lenz, Dorina; Helgen, Kristofer M; Roca, Alfred L; Hartman, Stefanie; Greenwood, Alex D

    2016-01-01

    Background. Retroviral integration into the host germline results in permanent viral colonization of vertebrate genomes. The koala retrovirus (KoRV) is currently invading the germline of the koala (Phascolarctos cinereus) and provides a unique opportunity for studying retroviral endogenization. Previous analysis of KoRV integration patterns in modern koalas demonstrate that they share integration sites primarily if they are related, indicating that the process is currently driven by vertical transmission rather than infection. However, due to methodological challenges, KoRV integrations have not been comprehensively characterized. Results. To overcome these challenges, we applied and compared three target enrichment techniques coupled with next generation sequencing (NGS) and a newly customized sequence-clustering based computational pipeline to determine the integration sites for 10 museum Queensland and New South Wales (NSW) koala samples collected between the 1870s and late 1980s. A secondary aim of this study sought to identify common integration sites across modern and historical specimens by comparing our dataset to previously published studies. Several million sequences were processed, and the KoRV integration sites in each koala were characterized. Conclusions. Although the three enrichment methods each exhibited bias in integration site retrieval, a combination of two methods, Primer Extension Capture and hybridization capture is recommended for future studies on historical samples. Moreover, identification of integration sites shows that the proportion of integration sites shared between any two koalas is quite small. PMID:27069793

  20. Comprehensive profiling of retroviral integration sites using target enrichment methods from historical koala samples without an assembled reference genome

    PubMed Central

    Alquezar-Planas, David E.; Ishida, Yasuko; Courtiol, Alexandre; Timms, Peter; Johnson, Rebecca N.; Lenz, Dorina; Helgen, Kristofer M.; Roca, Alfred L.; Hartman, Stefanie

    2016-01-01

    Background. Retroviral integration into the host germline results in permanent viral colonization of vertebrate genomes. The koala retrovirus (KoRV) is currently invading the germline of the koala (Phascolarctos cinereus) and provides a unique opportunity for studying retroviral endogenization. Previous analysis of KoRV integration patterns in modern koalas demonstrate that they share integration sites primarily if they are related, indicating that the process is currently driven by vertical transmission rather than infection. However, due to methodological challenges, KoRV integrations have not been comprehensively characterized. Results. To overcome these challenges, we applied and compared three target enrichment techniques coupled with next generation sequencing (NGS) and a newly customized sequence-clustering based computational pipeline to determine the integration sites for 10 museum Queensland and New South Wales (NSW) koala samples collected between the 1870s and late 1980s. A secondary aim of this study sought to identify common integration sites across modern and historical specimens by comparing our dataset to previously published studies. Several million sequences were processed, and the KoRV integration sites in each koala were characterized. Conclusions. Although the three enrichment methods each exhibited bias in integration site retrieval, a combination of two methods, Primer Extension Capture and hybridization capture is recommended for future studies on historical samples. Moreover, identification of integration sites shows that the proportion of integration sites shared between any two koalas is quite small. PMID:27069793

  1. Genome-Wide Analysis of Transposon and Retroviral Insertions Reveals Preferential Integrations in Regions of DNA Flexibility.

    PubMed

    Vrljicak, Pavle; Tao, Shijie; Varshney, Gaurav K; Quach, Helen Ngoc Bao; Joshi, Adita; LaFave, Matthew C; Burgess, Shawn M; Sampath, Karuna

    2016-01-01

    DNA transposons and retroviruses are important transgenic tools for genome engineering. An important consideration affecting the choice of transgenic vector is their insertion site preferences. Previous large-scale analyses of Ds transposon integration sites in plants were done on the basis of reporter gene expression or germ-line transmission, making it difficult to discern vertebrate integration preferences. Here, we compare over 1300 Ds transposon integration sites in zebrafish with Tol2 transposon and retroviral integration sites. Genome-wide analysis shows that Ds integration sites in the presence or absence of marker selection are remarkably similar and distributed throughout the genome. No strict motif was found, but a preference for structural features in the target DNA associated with DNA flexibility (Twist, Tilt, Rise, Roll, Shift, and Slide) was observed. Remarkably, this feature is also found in transposon and retroviral integrations in maize and mouse cells. Our findings show that structural features influence the integration of heterologous DNA in genomes, and have implications for targeted genome engineering. PMID:26818075

  2. Development of human cells with RXFP1 knockdown using retroviral delivery of microRNA against human RXFP1.

    PubMed

    Yong, K L; Callander, G E; Bergin, R; Samuel, C S; Bathgate, R A D

    2013-01-01

    To study the specific actions of relaxin through RXFP1 in human cells, it would be advantageous to develop cell populations with permanent RXFP1 knockdown (KD). We have developed and assessed four microRNA against human RXFP1. One of the four designed microRNA displayed significant RXFP1 KD as assessed by reduced relaxin binding when co-transfected with human RXFP1 into HEK-293T cells. The selected microRNA sequence was subsequently retrovirally delivered into the human dermal fibroblast cell line BJ3 which natively expresses RXFP1. The RXFP1 KD BJ3 cells displayed diminished RXFP1 mRNA expression and complete loss of ability of relaxin treatment to reduce collagen deposition after TGF-beta1 stimulation. The retroviral expression of miRNA to successfully silence RXFP1 expression is an invaluable tool to investigate receptor specificity, signalling and possible off-target effects of newly developed relaxin analogs. PMID:24640558

  3. Direct retroviral delivery of human cytochrome P450 2B6 for gene-directed enzyme prodrug therapy of cancer.

    PubMed

    Kan, O; Griffiths, L; Baban, D; Iqball, S; Uden, M; Spearman, H; Slingsby, J; Price, T; Esapa, M; Kingsman, S; Kingsman, A; Slade, A; Naylor, S

    2001-07-01

    Human cytochrome P450 2B6 (CYP2B6) metabolizes the prodrug cyclophosphamide (CPA) to produce phosphoramide mustard that cross-links DNA leading to cell death. We have constructed a novel retroviral vector encoding CYP2B6 (designated "MetXia-P450") and used it to transduce the human tumor cell lines HT29 and T47D. MetXia-P450 transduction sensitised these cells to the cytotoxic effects of the prodrug CPA. Results from in vitro experiments demonstrated adverse effects on the clonogenic survival of cyclophosphamide-treated cells transduced with MetXia-P450. Cytotoxic activity accompanied by bystander effect was particularly evident in 3-D multicellular spheroid models suggesting that this in vitro system may be a more appropriate model for assessing the efficacy of gene directed-enzyme prodrug therapy (GDEPT). We have applied this approach in a clinically relevant gene therapy protocol on established subcutaneous tumor xenografts. These studies show for the first time the efficacy of a P450-based GDEPT strategy mediated by a direct retroviral gene transfer in vivo. PMID:11498768

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

    PubMed Central

    Porter, Colin D.; Lukacs, Katalin V.; Box, Gary; Takeuchi, Yasuhiro; Collins, Mary K. L.

    1998-01-01

    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. PMID:9573249

  5. Genome-Wide Analysis of Transposon and Retroviral Insertions Reveals Preferential Integrations in Regions of DNA Flexibility

    PubMed Central

    Vrljicak, Pavle; Tao, Shijie; Varshney, Gaurav K.; Quach, Helen Ngoc Bao; Joshi, Adita; LaFave, Matthew C.; Burgess, Shawn M.; Sampath, Karuna

    2016-01-01

    DNA transposons and retroviruses are important transgenic tools for genome engineering. An important consideration affecting the choice of transgenic vector is their insertion site preferences. Previous large-scale analyses of Ds transposon integration sites in plants were done on the basis of reporter gene expression or germ-line transmission, making it difficult to discern vertebrate integration preferences. Here, we compare over 1300 Ds transposon integration sites in zebrafish with Tol2 transposon and retroviral integration sites. Genome-wide analysis shows that Ds integration sites in the presence or absence of marker selection are remarkably similar and distributed throughout the genome. No strict motif was found, but a preference for structural features in the target DNA associated with DNA flexibility (Twist, Tilt, Rise, Roll, Shift, and Slide) was observed. Remarkably, this feature is also found in transposon and retroviral integrations in maize and mouse cells. Our findings show that structural features influence the integration of heterologous DNA in genomes, and have implications for targeted genome engineering. PMID:26818075

  6. Factors Determining the Risk of Inadvertent Retroviral Transduction of Male Germ Cells After In Utero Gene Transfer in Sheep

    PubMed Central

    Park, Paul J.; Colletti, Evan; Ozturk, Ferhat; Wood, Josh A.; Tellez, Joe; Almeida-Porada, Graça

    2009-01-01

    Abstract The possibility of permanent genetic changes to the germline is central to the bioethics of in utero gene therapy (IUGT) because of the concern of inadvertent potentially deleterious alterations to the gene pool. Despite presumed protection of the male germline due to early germ cell (GC) compartmentalization, we reported that GCs within the developing ovine testes are transduced at low levels after retrovirus-mediated IUGT, thus underscoring the need for a thorough understanding of GC development in clinically predictive models to determine the optimal time to perform IUGT and avoid germline modification. In the present studies, we used the fetal sheep model to analyze GCs for phenotype, location, proliferation, and incidence of transduction after IUGT at various fetal ages to learn when during development the nascent germline is likely to be at greatest risk of retrovirus-mediated alteration. Our studies show that although GCs were transduced at all injection ages, the levels of transduction varied by nearly 700-fold as a function of the age at transfer. After remaining largely quiescent as they migrated to/settled within nascent sex cords, GCs began active cycling before cord closure was complete, suggesting this is likely the point at which they would be most susceptible to retroviral transduction. Furthermore, we observed that compartmentalization of GCs continued into early postnatal life, suggesting the male germline may be vulnerable to low-level inadvertent retroviral vector modification throughout fetal life, but that this risk can be minimized by performing IUGT later in gestation. PMID:19301473

  7. Biological activities of a synthetic peptide composed of two unlinked domains from a retroviral transmembrane protein sequence.

    PubMed Central

    Wegemer, D E; Kabat, K G; Kloetzer, W S

    1990-01-01

    We report several biological activities of a synthetic peptide whose sequence contains the highly conserved region of feline leukemia virus transmembrane protein (TM) synthetically linked to another short TM-derived sequence particularly rich in polar positive residues. This 29-amino-acid peptide blocked [3H]thymidine uptake 30 to 50% by concanavalin A-stimulated CD4(+)--but not CD8(+)-enriched murine splenocytes. Maximal suppression was detected at 12.5 micrograms (3 microM) to 75 micrograms (19 microM) per ml of growth medium; stimulation of [3H]thymidine uptake was observed at higher peptide concentrations. The synthetic peptide inhibited but did not stimulate [3H]thymidine uptake by mitogen-activated thymocytes and antibody production by splenocytes as determined in a liquid hemolytic plaque assay. Similarities are reported between a consensus sequence of diverse retroviral TMs and a region of alpha interferons shown by others to be important for antiviral and cytostatic properties. The TM sequence-derived synthetic peptide blocked in a nontoxic and sequence-specific manner the release of murine leukemia virus from two chronically infected cell lines. We suggest that some of the biological effects of retroviral TM are mediated through a common pathway shared with alpha interferons. Images PMID:1969500

  8. Retroviral envelope syncytin capture in an ancestrally diverged mammalian clade for placentation in the primitive Afrotherian tenrecs

    PubMed Central

    Cornelis, Guillaume; Vernochet, Cécile; Malicorne, Sébastien; Souquere, Sylvie; Tzika, Athanasia C.; Goodman, Steven M.; Catzeflis, François; Robinson, Terence J.; Milinkovitch, Michel C.; Pierron, Gérard; Heidmann, Odile; Dupressoir, Anne; Heidmann, Thierry

    2014-01-01

    Syncytins are fusogenic envelope (env) genes of retroviral origin that have been captured for a function in placentation. Syncytins have been identified in Euarchontoglires (primates, rodents, Leporidae) and Laurasiatheria (Carnivora, ruminants) placental mammals. Here, we searched for similar genes in species that retained characteristic features of primitive mammals, namely the Malagasy and mainland African Tenrecidae. They belong to the superorder Afrotheria, an early lineage that diverged from Euarchotonglires and Laurasiatheria 100 Mya, during the Cretaceous terrestrial revolution. An in silico search for env genes with full coding capacity within a Tenrecidae genome identified several candidates, with one displaying placenta-specific expression as revealed by RT-PCR analysis of a large panel of Setifer setosus tissues. Cloning of this endogenous retroviral env gene demonstrated fusogenicity in an ex vivo cell–cell fusion assay on a panel of mammalian cells. Refined analysis of placental architecture and ultrastructure combined with in situ hybridization demonstrated specific expression of the gene in multinucleate cellular masses and layers at the materno–fetal interface, consistent with a role in syncytium formation. This gene, which we named “syncytin-Ten1,” is conserved among Tenrecidae, with evidence of purifying selection and conservation of fusogenic activity. To our knowledge, it is the first syncytin identified to date within the ancestrally diverged Afrotheria superorder. PMID:25267646

  9. Infection by retroviral vectors outside of their host range in the presence of replication-defective adenovirus.

    PubMed Central

    Adams, R M; Wang, M; Steffen, D; Ledley, F D

    1995-01-01

    Retrovirus infection is normally limited to cells within a specific host range which express a cognate receptor that is recognized by the product of the env gene. We describe retrovirus infection of cells outside of their normal host range when the infection is performed in the presence of a replication-defective adenovirus (dl312). In the presence of adenovirus, several different ecotropic vectors are shown to infect human cell lines (HeLa and PLC/PRF), and a xenotropic vector is shown to infect murine cells (NIH 3T3). Infectivity is demonstrated by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) staining, selection with G418 for neomycin resistance, and PCR identification of the provirus in infected cells. Infectivity is quantitatively dependent upon both the concentration of adenovirus (10(6) to 10(8) PFU/ml) and the concentration of retrovirus. Infection requires the simultaneous presence of adenovirus in the retrovirus infection medium and is not stimulated by preincubation and removal of adenovirus from the cells before retrovirus infection. The presence of adenovirus is shown to enhance the uptake of fluorescently labeled retrovirus particles into cells outside of their normal host range, demonstrating that the adenovirus enhances viral entry into cells in the absence of the recognized cognate receptor. This observation suggests new opportunities for developing safe retroviral vectors for gene therapy and new mechanisms for the pathogenesis of retroviral disease. PMID:7853530

  10. ISOLATION OF AN ACTIVE LV1 GENE FROM CATTLE INDICATES THAT TRIPARTITE MOTIF PROTEIN-MEDIATED INNATE IMMUNITY TO RETROVIRAL INFECTION IS WIDESPREAD AMONG MAMMALS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lv1/TRIM5alpha (tripartite motif 5alpha) has recently emerged as an important factor influencing species-specific permissivity to retroviral infection in a range of primates, including humans. Old World monkey TRIM5alpha blocks human immunodeficiency virus type 1 (HIV-1) infectivity, and the human a...