Since we did not use drug selection, we expected that 8. One week after plating of the infected cells, cell clusters were counted; 26 After 19 days in culture, 25 of the 26 GFP-positive cell clusters remained positive and showed growth properties and morphology indistinguishable from those of nontransduced GFP-negative cell clusters.
C and D Mouse neuronal cells. A and C Phase-contrast micrographs. B and D Fluorescence micrographs. Mouse neuronal cells were positive for reactivity to the following antibodies: mouse anti-neurofilament , rabbit anti-tau, mouse anti-MAP2, and rabbit anti-rat neuron-specific enolase. Two types of primary cells, mouse neurons from fetal brain tissue Fig.
Primary cells were infected at an MOI of 1. The MOI is calculated based on the infectivity of lentivirus vectors in HeLa cells as described above. This result is consistent with previous reports indicating that lentivirus vectors pseudotyped with VSV-G display broad tropism 7 , We routinely find, however, that most retrovirus vectors infect HeLa cells efficiently, but they infect primary cells less efficiently.
Vpr-induced cell cycle arrest is followed by apoptosis 57 and is therefore deleterious to the target cell. We predicted that an SIV-packaged lentivirus vector which contains Vpx but not Vpr would not cause cell cycle arrest. At 48 h postinfection, cells were simultaneously stained for cell surface expression of Thy-1 and DNA contents as described previously At 48 h, cells were stained with fluorescein isothiocyanate-conjugated anti-Thy-1 antibody, fixed, permeabilized, and stained for DNA contents with propidium iodide.
Cell cycle analysis was performed with Multicycle AV software. The G 1 , S, and G 2 peaks left, middle, and right shaded areas, respectively are shown below the DNA profile dotted line. We investigated the potential for the emergence of replication-competent viruses in the lentivirus system.
CEMX cells infected with the second vector passage were then cultured for 14 days. The presence of replication-competent viruses was evaluated by use of GFP fluorescence and a p27 capture ELISA at days 7 and 14 after exposure to the second vector passage. In addition, supernatants from indicator CEMX cells at the same times were used to infect MAGI cells for detection of any potential recombinant viruses which may have retained the expression of Tat.
No blue foci could be identified in these cells at 3 days postexposure. Visual examination of second-passage CEMX cells and MAGI cells failed to reveal any cytopathic effects which might have been expected in the presence of replication-competent viruses.
The generation of helper virus in preparations of retrovirus vectors has been documented in numerous instances involving oncoviruses 10 , 13 , 29 , 51 , In later generations of vectors in which viral protein-coding regions were split in the packaging cells, requiring multiple crossover events to generate replication-competent recombinant virus, the frequency of recombination leading to helper virus was decreased but not eliminated Helper virus has the potential for inducing pathogenesis, as demonstrated by studies in which monkeys were infused with transduced bone marrow cells after ablation of endogenous marrow with gamma irradiation 13 , 51 , In these studies, helper virus gave rise to lymphoma in the monkeys.
A heterologous envelope, VSV-G, is supplied in trans as previously described 1 , 7 , 37 to provide broad cellular tropism. Our studies demonstrate that an RNA and envelope pseudotype vector is functional in gene transduction.
The potential safety of this vector system is based on the existence of low nucleotide sequence homology between HIV-1 and SIVmac. A second property of this vector with important implications for safety is that the packaging construct is derived from a nonvirulent SIVmac isolate. Thus, the potential for pathogenicity with this vector system should be minimal. HIV-1 vpr was shown to induce arrest in the G 2 phase of the cell cycle 20 , 46 , 49 , Induction of G 2 arrest by vpr is thought to lead to apoptosis 57 and is thus a deleterious function.
An ideal lentivirus vector for nondividing cells would encode the former but not the latter function of vpr. The two functions of HIV-1 vpr are segregated such that SIVmac vpx participates in infection of nondividing cells via nuclear transport of preintegration complexes 15 and vpr induces cell cycle arrest We have developed a lentivirus vector based on SIVmac which expresses vpx but not vpr. We demonstrate that this vector is able to infect nondividing cells but is unable to cause cell cycle arrest in proliferating cells.
The influence of lentivirus accessory genes involved in virulence vpr , vpu , nef , and vif on the ability to transduce nondividing and primary cells was recently addressed in the context of an HIVderived lentivirus vector The study by Zufferey et al. In the experiments presented here, the individual roles of the SIVmac accessory genes were not directly evaluated. In addition, vif and vpx are present in the SIVmac packaging system, although their individual contributions remain to be evaluated by comparing constructs which differ in the presence of single genes, as was done by Zufferey et al.
Thus, future experiments involving the SIVmac packaging system will address the potential contributions of SIVmac vpr , vpx , nef , and vif. The genetic engineering experiments described in this work cover a rather small spectrum of the possibilities for the development and improvement of lentivirus vectors.
An additional safety feature which may be incorporated into future lentivirus systems is the use of self-inactivating transfer vectors 33 , In these vectors, the LTR are engineered such that following proviral integration, a viral promoter is not regenerated. Expression is then limited to an internal promoter specific for the gene of interest.
The lentivirus vector that we propose here provides proof of the feasibility of heterologous packaging. However, additional changes will have to be made before such a vector can be considered useful in vivo. These changes include deletion of tat , rev , and vpu from the transfer vector, as was described earlier for an HIVderived system with homologous packaging Deletion of rev will require the inclusion of a constitutive transport element of Mason-Pfizer monkey virus This element works in cis to allow unspliced and singly spliced mRNAs to be expressed at high levels in a Rev-independent manner.
Lentivirus vectors offer potential for the treatment of a wide variety of syndromes, including genetic and metabolic deficiencies, viral infection, and cancer. Inherited genetic defects, such as adenosine deaminase deficiency, familial hypercholesterolemia, cystic fibrosis, mucopolysaccharidosis type VII, type I and II diabetes, classical phenylketonuria, and Gaucher disease, may be overcome by lentivirus vector-mediated gene therapy because they constitute single-gene deficiencies for which the involved genes are known.
Certain types of cancer may also benefit from the use of lentivirus vectors. Hypoxia and lack of vascularization lead to the generation of tumor cells which exhibit limited or no proliferation. Partly because of the lack of growth, these cells are highly resistant to genotoxic agents. Viral diseases may also constitute appropriate targets for lentivirus gene delivery. In particular, a number of gene therapy approaches have been proposed for the treatment of HIV-1 infection.
Preliminary studies have used defective murine oncoviruses for the delivery of antisense RNAs, ribozymes, and trans -dominant proteins against HIV-1 replication. The usefulness of an HIVderived vector for delivery of an anti-HIV-1 strategy would be limited by inhibition of the vector itself. We thank W. Sutton, N. Haigwood, and S. Mossman for the generous contribution of antibodies and technical assistance for the detection of SIV p We also thank M. Sacco for providing assistance with digital imaging.
We thank P. Challita-Eid, R. Bambara, and E. Schwarz for critical reading of the manuscript. This work was supported by NIH research grants to V. RAI and J. Read article at publisher's site DOI : Free to read at jvi. J Allergy Clin Immunol , 10 Dec Munis AM. Viruses , 12 10 , 29 Sep Front Cell Dev Biol , , 14 Aug Viruses , 11 11 , 14 Nov Front Med , 13 1 , 05 Feb Cited by: 1 article PMID: To arrive at the top five similar articles we use a word-weighted algorithm to compare words from the Title and Abstract of each citation.
Hum Gene Ther , 12 7 , 01 May Cited by: 15 articles PMID: Virology , 2 , 01 Dec Cited by: 16 articles PMID: J Virol , 77 17 , 01 Sep Review Free to read. Mautino MR. Curr Gene Ther , 2 1 , 01 Feb Cited by: 14 articles PMID: Contact us. Europe PMC requires Javascript to function effectively. Recent Activity. Search life-sciences literature Over 39 million articles, preprints and more Search Advanced search.
Search articles by 'S M White'. White SM 1 ,. M Renda Search articles by 'M Renda'. Renda M ,. Nam NY ,. E Klimatcheva Search articles by 'E Klimatcheva'. Klimatcheva E ,. Y Zhu Search articles by 'Y Zhu'. Zhu Y ,. J Fisk Search articles by 'J Fisk'. Fisk J ,. M Halterman Search articles by 'M Halterman'. Halterman M ,. Rimel BJ ,. H Federoff Search articles by 'H Federoff'. Federoff H ,. S Pandya Search articles by 'S Pandya'. Pandya S ,. Rosenblatt JD ,. V Planelles Search articles by 'V Planelles'.
Planelles V. Affiliations 1 author 1. Search life-sciences literature Over 39 million articles, preprints and more Search Advanced search.
This website requires cookies, and the limited processing of your personal data in order to function. By using the site you are agreeing to this as outlined in our privacy notice and cookie policy. Search articles by 'S M White'. White SM 1 ,. M Renda Search articles by 'M Renda'. Renda M ,. Nam NY ,. E Klimatcheva Search articles by 'E Klimatcheva'.
Klimatcheva E ,. Y Zhu Search articles by 'Y Zhu'. Zhu Y ,. J Fisk Search articles by 'J Fisk'. Fisk J ,. M Halterman Search articles by 'M Halterman'. Halterman M ,. Rimel BJ ,. H Federoff Search articles by 'H Federoff'. Federoff H ,. S Pandya Search articles by 'S Pandya'. Pandya S ,. Rosenblatt JD ,. V Planelles Search articles by 'V Planelles'. Planelles V.
Affiliations 1 author 1. Share this article Share with email Share with twitter Share with linkedin Share with facebook. Abstract Lentivirus vectors based on human immunodeficiency virus HIV type 1 HIV-1 constitute a recent development in the field of gene therapy.
Free full text. J Virol. PMID: Sarah M. Bobbie J. Joseph D. Author information Article notes Copyright and License information Disclaimer. Phone: Fax: E-mail: ude. Received Aug 28; Accepted Dec This article has been cited by other articles in PMC. Go to:. Plasmid construction. Vector production. Immunologic detection of viral antigens. Infections of dividing and growth-arrested HeLa cells. Isolation and infection of primary mouse neurons.
Isolation and infection of human peripheral blood macrophages. Fluorescence microscopy and photography. Flow cytometry. Construction and characterization of an SIV-based packaging system.
Open in a separate window. Vector titers were calculated as described in Materials and Methods and expressed in infectious units IU. NA, not applicable. At 48 or 72 h postinfection, fluorescent cells were visually counted under a microscope.
Mock infections of the same cell types were performed in parallel and resulted in no detectable fluorescent cells. ND, not determined. Lack of induction of cell cycle arrest. Detection of replication-competent viruses. In vitro macrophage tropism of pathogenic and nonpathogenic molecular clones of simian immunodeficiency virus SIVmac Virology.
Identification of viral determinants of macrophage tropism for simian immunodeficiency virus SIVmac. The activation domain of simian immunodeficiency virus SIVmac Rev protein is structurally and functionally analogous to the HIV-1 Rev activation domain. Optimized survival of hippocampal neurons in Bsupplemented Neurobasal, a new serum-free medium combination. J Neurosci Res. HIV-1 nuclear import: in search of a leader. Front Biosci. Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells.
Nonreciprocal pseudotyping: murine leukemia virus proteins cannot efficiently package spleen necrosis virus-based vector RNA. J Gen Virol. Chong H, Vile R G. Gene Ther. Function of the human immunodeficiency virus type 1 and 2 Rev proteins is dependent on their ability to interact with a structured region present in env gene mRNA. Helper virus induced T cell lymphoma in nonhuman primates after retroviral mediated gene transfer.
J Exp Med. Lack of competition results in efficient packaging of heterologous murine retroviral RNAs and reticuloendotheliosis virus encapsidation-minus RNAs by the reticuloendotheliosis virus helper cell line. Inhibition of viral DNA synthesis in stationary chicken embryo fibroblasts infected with avian retroviruses.
Lentiviral vectors for gene therapy of cystic fibrosis. Hum Gene Ther. Human immunodeficiency virus type 1 viral protein R Vpr arrests cells in the G 2 phase of the cell cycle by inhibiting p34 cdc2 activity. Cell cycle-dependent activation of Rous sarcoma virus-infected stationary chicken cells: avian leukosis virus group-specific antigens and ribonucleic acid. Importance of the nef gene for maintenance of high virus load and for development of AIDS.
Kewalramani V N, Emerman M. Vpx association with mature core structures of HIV Kimpton J, Emerman M. Detection of replication-competent and pseudotyped human immunodeficiency virus with a sensitive cell line on the basis of activation of an integrated beta-galactosidase gene. Packaging system for rapid production of murine leukemia virus vectors with variable tropism.
A highly conserved RNA folding region coincident with the Rev response element of primate immunodeficiency viruses. Nucleic Acids Res. Phenotypic characterization of the human fibrous histiocytoma giant cell tumor GCT cell line and its cytokine repertoire. Exp Hematol. The Vif protein of human and simian immunodeficiency viruses is packaged into virions and associates with viral core structures.
Construction and use of a safe and efficient amphotropic packaging cell line. Rhesus macaques inoculated with molecularly cloned simian immunodeficiency virus. J Med Primatol. Viral determinants of simian immunodeficiency virus SIV virulence in rhesus macaques assessed by using attenuated and pathogenic molecular clones of SIVmac. Immunization with a live, attenuated simian immunodeficiency virus SIV prevents early disease but not infection in rhesus macaques challenged with pathogenic SIV.
Miller A D. Development and application of retroviral vectors. Cold Spring Harbor, N. Improved retroviral vectors for gene transfer and expression. Development of a self-inactivating lentivirus vector. Complex determinants of macrophage tropism in env of simian immunodeficiency virus. Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector.
In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. J Acquired Immune Defic Syndr. Characterization of a replication-competent retrovirus resulting from recombination of packaging and vector sequences.
Effects of vif mutations on cell-free infectivity and replication of simian immunodeficiency virus. J Acquired Immun Defic Syndr. Park I W, Sodroski J. Functional analysis of the vpx, vpr, and nef genes of simian immunodeficiency virus. EMBO J. Fate of the human immunodeficiency virus type 1 provirus in infected cells: a role for vpr. A new reporter system for detection of retroviral infection. Vpr-induced cell cycle arrest is conserved among primate lentiviruses. Identification of a human immunodeficiency virus type 2 HIV-2 encapsidation determinant and transduction of nondividing human cells by HIVbased lentivirus vectors.
Efficient transduction of nondividing human cells by feline immunodeficiency virus lentiviral vectors. Nat Med. Cell cycle arrest by Vpr in HIV-1 virions and insensitivity to antiretroviral agents. Human immunodeficiency virus type 1 vpr gene induces phenotypic effects similar to those of the DNA-alkylating agent nitrogen mustard. An array of murine leukemia virus-related elements is transmitted and expressed in a primate recipient of retroviral gene transfer.
Simian immunodeficiency virus RNA is efficiently encapsidated by human immunodeficiency virus type 1 particles. The human immunodeficiency virus type 1 vpr gene prevents cell proliferation during chronic infection. Access to Document Link to publication in Scopus. Fingerprint Dive into the research topics of 'Lentivirus vectors using human and simian immunodeficiency virus elements'.
Together they form a unique fingerprint. View full fingerprint. Journal of virology , 73 4 , In: Journal of virology , Vol.
Journal of virology.
0コメント