Retroviral Vector Infection Research Articles

IDO1, FAT10, IFI6, and GILT Are Involved in the Antiretroviral Activity of γ-Interferon and IDO1 Restricts Retrovirus Infection by Autophagy Enhancement.

Gamma-interferon (γ-IFN) significantly inhibits infection by replication-defective viral vectors derived from the human immunodeficiency virus type 1 (HIV-1) or murine leukemia virus (MLV) but the underlying mechanism remains unclear. Previously we reported that knockdown of γ-IFN-inducible lysosomal thiolreductase (GILT) abrogates the antiviral activity of γ-IFN in TE671 cells but not in HeLa cells, suggesting that other γ-IFN-inducible host factors are involved in its antiviral activity in HeLa cells. We identified cellular factors, the expression of which are induced by γ-IFN in HeLa cells, using a microarray, and analyzed the effects of 11 γ-IFN-induced factors on retroviral vector infection. Our results showed that the exogenous expression of FAT10, IFI6, or IDO1 significantly inhibits both HIV-1- and MLV-based vector infections. The antiviral activity of γ-IFN was decreased in HeLa cells, in which the function of IDO1, IFI6, FAT10, and GILT were simultaneously inhibited. IDO1 is an enzyme that metabolizes an essential amino acid, tryptophan. However, IDO1 did not restrict retroviral vector infection in Atg3-silencing HeLa cells, in which autophagy did not occur. This study found that IDO1, IFI6, FAT10, and GILT are involved in the antiviral activity of γ-IFN, and IDO1 inhibits retroviral infection by inducing autophagy.

Molecular Biology Techniques Q&A

Molecular Biology Techniques Q&A

Detailed characterization of the mouse glioma 261 tumor model for experimental glioblastoma therapy

Mouse glioma 261 (Gl261) cells are used frequently in experimental glioblastoma therapy; however, no detailed description of the Gl261 tumor model is available. Here we present that Gl261 cells carry point mutations in the K-ras and p53 genes. Basal major histocompatibility complex (MHC)I, but not MHCII, expression was detected in Gl261 cells. The introduction of interferon-gamma-encoding genes increased expression of both MHCI and MHCII. A low amount of B7-1 and B7-2 RNA was detected in wild-type cells, but cytokine production did not change expression levels. Gl261 cells were transduced efficiently by adenoviral vectors; the infectivity of retroviral vectors was limited. Low numbers of transplanted Gl261 cells formed both subcutaneous and intracranial tumors in C57BL/6 mice. The cells were moderately immunogenic: prevaccination of mice with irradiated tumor cells 7 days before intracranial tumor challenge prevented tumor formation in approximately 90% of mice. When vaccination was carried out on the day or 3 days after tumor challenge, no surviving animals could be found. In vitro-growing cells were radiosensitive: less than 2 Gy was required to achieve 50% cell mortality. Local tumor irradiation with 4 Gy X-rays in brain tumor-bearing mice slowed down tumor progression, but none of the mice were cured off the tumor. In conclusion, the Gl261 brain tumor model might be efficiently used to study the antitumor effects of various therapeutic modalities, but the moderate immunogenicity of the cells should be considered.

Activities of wildtype and mutant p53 in suppression of homologous recombination as measured by a retroviral vector system

DNA repair of double strand breaks, interstrand DNA cross-links, and other types of DNA damage utilizes the processes of homologous recombination and non-homologous end joining to repair the damage. Aberrant homologous recombination is likely to be responsible for a significant fraction of chromosomal deletions, duplications, and translocations that are observed in cancer cells. To facilitate measurement of homologous recombination frequencies in normal cells, mutant cells, and cancer cells, we have developed a high titer retroviral vector containing tandem repeats of mutant versions of a GFP-Zeocin resistance fusion gene and an intact neomycin resistance marker. Recombination between the tandem repeats regenerates a functional GFP-Zeo R marker that can be easily scored. This retroviral vector was used to assess homologous recombination frequencies in human cancer cells and rodent fibroblasts with differing dosages of wild type or mutant p53. Absence of wild type p53 stimulated spontaneous and ionizing radiation-induced homologous recombination, confirming previous studies. Moreover, p53 +/− mouse fibroblasts show elevated levels of homologous recombination compared to their p53 +/+ counterparts following retroviral vector infection, indicating that p53 is haploinsufficient for suppression of homologous recombination. Transfection of vector-containing p53 null Saos-2 cells with various human cancer-associated p53 mutants revealed that these altered p53 proteins retain some recombination suppression function despite being totally inactive for transcriptional transactivation. The retroviral vector utilized in these studies may be useful in performing recombination assays on a wide array of cell types, including those not readily transfected by normal vectors.

Targeting retroviral vector infection to cells that express heregulin receptors using a TVA-heregulin bridge protein.

Previously we have shown that it is possible to target retroviral vectors to cells using avian sarcoma and leukosis virus (ASLV) receptor-ligand and receptor-single-chain antibody bridge proteins (now designated as GATEs for guided adaptors for targeted entry). In this report we were interested in determining whether this approach can be used to deliver retroviral vectors specifically to cells that express heregulin receptors. Heregulin receptors are attractive targets for retroviral vector-based gene delivery protocols since they are often overexpressed on the surfaces of cancer cells. To explore this possibility, the TVA-herbeta1 protein was generated, consisting of the extracellular domain of the TVA receptor for ASLV-A fused to the EGF-like region of heregulin beta1. TVA-herbeta1 bound specifically to cells that express heregulin receptors, rendering them susceptible to efficient and specific infection by subgroup A ASLV vectors. In addition, these activities of TVA-herbeta1 were abrogated specifically in the presence of another bridge protein that contained the same ligand domain. These data confirm that the GATE protein TVA-herbeta1 mediates targeted retroviral infection via cell surface heregulin receptors.

Relationship between cell proliferation, cell-cycle phase, and retroviral vector production in FLYRD18 human packaging cells.

The relatively low concentrations of retroviral vectors produced by most packaging cells requires the optimization and intensification of their production to make a commercially viable product for gene therapy. While a number of reports exist concerning target cell-cycle effects on retroviral vector infection efficiency, no studies have been reported on the effects of packaging cell cycle on vector production. We have studied the effect of proliferation of the human packaging cell line, FLYRD18, on vector production. In addition, the titer levels of vector produced by cells in each phase of the cell cycle were compared. Numerous studies suggested progression of the cells through the cell cycle to be essential for vector production. However, vector release was found not to be predominant in any particular phase of the cell cycle. These findings indicate that packaging cell proliferation is important for optimal virus production and that arrest of the cells in any particular phase of the cell cycle affords no benefits in retroviral vector production. In contrast to previous reports (using other cell lines), we observed no temporary inhibition of cell cycle progression after detachment of cells from their substratum and that virus production occurred immediately after re-plating of the cells. The findings in this report are important for determining the optimal culture conditions for vector production by packaging cells in vitro.

Transgene Expression in Zebrafish: A Comparison of Retroviral-Vector and DNA-Injection Approaches

To assess alternative methods for introducing expressing transgenes into the germ line of zebrafish, transgenic fish that express a nuclear-targeted, enhanced, green fluorescent protein (eGFP) gene were produced using both pseudotyped retroviral vector infection and DNA microinjection of embryos. Germ-line transgenic founders were identified and the embryonic progeny of these founders were evaluated for the extent and pattern of eGFP expression. To compare the two modes of transgenesis, both vectors used the Xenopus translational elongation factor 1-α enhancer/promoter regulatory cassette. Several transgenic founder fish which transferred eGFP expression to their progeny were identified. The gene expression patterns are described and compared for the two modes of gene transfer. Transient expression of eGFP was detected 1 day after introducing the transgenes via either DNA microinjection or retroviral vector infection. In both cases of gene transfer, transgenic females produced eGFP-positive progeny even before the zygotic genome was turned on. Therefore, GFP was being provided by the oocyte before fertilization. A transgenic female revealed eGFP expression in her ovarian follicles. The qualitative patterns of gene expression in the transgenic progeny embryos after zygotic induction of gene expression were similar and independent of the mode of transgenesis. The appearance of newly synthesized GFP is detectable within 5–7 h after fertilization. The variability of the extent of eGFP expression from transgenic founder to transgenic founder was wider for the DNA-injection transgenics than for the retroviral vector-produced transgenics. The ability to provide expressing germ-line transgenic progeny via retroviral vector infection provides both an alternative mode of transgenesis for zebrafish work and a possible means of easily assessing the insertional mutagenesis frequency of retroviral vector infection of zebrafish embryos. However, because of the transfer of GFP from oocyte to embryo, the stability of GFP may create problems of analysis in embryos which develop as quickly as those of zebrafish.

Correction of fanconi anemia Type C phenotypic abnormalities using a clinically suitable retroviral vector infection protocol

Fanconi anemia (FA) is a complex autosomal recessive disease with hematologic manifestations characterized by a progressive hypoplastic anemia, hypersensitivity to clastogenic agents, and an increased incidence of acute myelogenous leukemia. The cDNA that corrects one of four FA complementation subtypes, named Fanconi anemia Type C (FAC) has recently been identified. We constructed a a simplified recombinant retrovirus (vMFGFAC) encoding only the FAC cDNA, and tested its ability to correct the FAC defect in a lymphocytic cell line and primary mobilized blood progenitor cells. In addition, the gene transfer efficiency using a clinically applicable gene transfer protocol into normal primitive hematopoietic progenitor cells, high proliferating potential colony forming cells (HPP-CFC), derived from CD34+ purified cord blood cells was examined. The gene transfer efficiency was significantly enhanced when cells were transduced with supernatant while adherent to a 30/35 KD fragment of fibronectin, FN30/35, and was similar to efficiency obtained by coculture with retrovirus packaging cells. Transduction of an FAC deficient lymphoid cell line with vMFGFAC supernatant resulted in an enhanced cell viability, and G-CSF mobilized peripheral blood cells from an FAC-deficient patient transduced with the vMFGFAC virus demonstrated enhanced progenitor cell colony formation. These data indicate that the vMFGFAC virus allows functional complementation of FAC in lymphoblasts and primary hematopoietic progenitors, and that primitive cord blood hematopoietic stem/progenitor cells can be transduced at an efficiency comparable to protocols using cocultivation if adherent to FN 30/35 fragment.

Correction of Fanconi anemia type C phenotypic abnormalities using a clinically suitable retroviral vector infection protocol.

Fanconi anemia (FA) is a complex autosomal recessive disease with hematologic manifestations characterized by a progressive hypoplastic anemia, hypersensitivity to clastogenic agents, and an increased incidence of acute myelogenous leukemia. The cDNA that corrects one of four FA complementation subtypes, named Fanconi anemia Type C (FAC) has recently been identified. We constructed a a simplified recombinant retrovirus (vMFGFAC) encoding only the FAC cDNA, and tested its ability to correct the FAC defect in a lymphocytic cell line and primary mobilized blood progenitor cells. In addition, the gene transfer efficiency using a clinically applicable gene transfer protocol into normal primitive hematopoietic progenitor cells, high proliferating potential colony forming cells (HPP-CFC), derived from CD34+ purified cord blood cells was examined. The gene transfer efficiency was significantly enhanced when cells were transduced with supernatant while adherent to a 30/35 KD fragment of fibronectin, FN30/35, and was similar to efficiency obtained by coculture with retrovirus packaging cells. Transduction of an FAC deficient lymphoid cell line with vMFGFAC supernatant resulted in an enhanced cell viability, and G-CSF mobilized peripheral blood cells from an FAC-deficient patient transduced with the vMFGFAC virus demonstrated enhanced progenitor cell colony formation. These data indicate that the vMFGFAC virus allows functional complementation of FAC in lymphoblasts and primary hematopoietic progenitors, and that primitive cord blood hematopoietic stem/progenitor cells can be transduced at an efficiency comparable to protocols using cocultivation if adherent to FN 30/35 fragment.

A general method for the generation of high-titer, pantropic retroviral vectors: highly efficient infection of primary hepatocytes.

Retroviral vectors have been central components in many studies leading to human gene therapy. However, the generally low titers and inefficient infectivity of retroviral vectors in human cells have limited their use. We previously reported that the G protein of vesicular stomatitis virus can serve as the exclusive envelope protein component for one specific retroviral vector, LGRNL, that expresses vesicular stomatitis virus G. We now report a more useful general transient transfection scheme for producing very high-titer vesicular stomatitis virus G-enveloped pseudotypes from any Moloney murine leukemia-based retroviral vector without having to rely on the expression of the cytotoxic G protein from the retroviral vector itself. We also demonstrate very high efficiency of infection with a pseudotyped lacZ vector in primary mouse hepatocytes. We suggest that pseudotyped retroviral vectors carrying reporter genes will permit genetic studies in many previously inaccessible vertebrate and invertebrate systems. Furthermore, because these vectors represent retroviral vectors of sufficiently high titer to allow efficient direct retroviral-mediated in vivo gene transfer, we also suggest that pseudotyped vectors carrying potentially therapeutic genes will become useful to test the potential for in vivo gene therapy.

Granulocyte colony‐stimulating factor treatment of mice modulates differently the sensitivity of blood and bone marrow hematopoietic progenitors to retroviral vector infection

The effect of recombinant human granulocyte colony-stimulating factor (G-CSF) administration to mice on the retroviral-mediated transfer of the selectable marker gene neo to hematopoietic cells was studied. After G-CSF treatment, blood mononuclear cells and bone marrow cells were infected with the retrovirus, and the efficiency of gene transfer into myeloid progenitors was increased in peripheral blood and decreased in bone marrow cells. Bone marrow four to six months after transplantation from G-CSF treated mice revealed the presence of the transferred neo gene in 4 out of 104 mice, which is an eightfold lower proportion than in the control group. These data suggest that G-CSF in vivo treatment decreases bone marrow sensitivity and increases the sensitivity of peripheral blood cells to retroviral infection.

8-Cl-cAMP induces truncation and down-regulation of the RI alpha subunit and up-regulation of the RII beta subunit of cAMP-dependent protein kinase leading to type II holoenzyme-dependent growth inhibition and differentiation of HL-60 leukemia cells.

8-Cl-cAMP, a site-selective cAMP analog, induces growth inhibition in a variety of cell types of human cancer cell lines. This inhibitory effect of 8-Cl-cAMP was related to its ability to differentially regulate type I versus type II cAMP-dependent protein kinase. In the present study we demonstrated a unique mechanism of action of 8-Cl-cAMP in the regulation of these kinase isozymes in HL-60 human promyelocytic leukemia cells. High-performance liquid chromatography (HPLC) resolved various isoforms of protein kinase present in HL-60 cells. In control cells, type I protein kinase (PKI) comprised more than 90% and type II protein kinase (PKII) less than 10% of the total cAMP-stimulated kinase activity. Treatment with 8-Cl-cAMP (5 microM, 72 h) decreased PKI to a level below 30% of that in untreated control cells and markedly increased PKII composed of three peaks. Photoaffinity labeling/SDS-polyacrylamide gel electrophoresis of column fractions identified the molecular species of regulatory (R) subunits present in protein kinases. Control cells contained high levels of the 48-kDa protein (RI) that composed PKI and low levels of the 50-kDa RII associated with PKII. 8-Cl-cAMP treatment brought about a decrease in the 48-kDa RI along with an increased formation of the truncated 34-kDa RI associated with PKI and an increase in the 50-54-kDa species of RII associated with PKII. A similar protein kinase profile as that shown by 8-Cl-cAMP treatment was observed in cells infected with the human RII beta retroviral vector: the 48-kDa RI of PKI decreased and the 52- and 54-kDa RII associated with PKII increased as compared with uninfected control cells. However, unlike 8-Cl-cAMP treatment, RII beta retroviral vector infection brought about no increase in the 34-kDa-truncated RI but exhibited an increase in the free 48-kDa RI subunit. As the 48-kDa RI and the 50-kDa RII were present in control cells, the enhanced expression of the 52- and 54-kDa RII proteins was due to overexpression of the RII beta gene. We identified the 48-kDa RI as RI alpha, the 50-kDa RII as RII alpha, the 52-kDa RII as RII beta, and the 54-kDa RII as the phosphorylated form of either the RII alpha or RII beta subunit. In vivo labeling experiments using [3H]8-Cl-cAMP demonstrated that 8-Cl-cAMP enters cells and binds to both PKI and PKII.(ABSTRACT TRUNCATED AT 400 WORDS)

Leukemia inhibitory factor improves survival of retroviral vector-infected hematopoietic stem cells in vitro, allowing efficient long-term expression of vector-encoded human adenosine deaminase in vivo.

Low recovery and poor retroviral vector infection efficiency of hematopoietic stem cells has hindered application of gene therapy for disease affecting blood-forming tissues. Developmental restriction (or death) of stem cells during ex vivo infection has contributed to these difficulties. In these studies we report that the cytokine leukemia inhibitory factor (LIF) directly or indirectly supported the survival of hematopoietic stem cells during culture of bone marrow with vector-producing fibroblasts, resulting in efficient recovery of stem cells able to compete for engraftment in irradiated recipient animals. The infection efficiency of hematopoietic stem cells recovered from these cultures was approximately 80%; and all recipients (20/20) of the LIF-treated marrow were stably engrafted with the progeny of provirus-bearing stem cells. Expression of vector-encoded human adenosine deaminase (hADA) was detected in all recipients at levels averaging 15-50% of endogenous murine ADA in all their hematolymphoid tissues. Survival of stem cells in untreated cultures was approximately 10% of that observed from LIF-treated cultures, resulting in poor engraftment of recipient animals with transplanted cells. The infection efficiency of the few stem cells recovered from untreated cultures, however, was high (approximately 80%), suggesting that LIF did not have an effect on infection efficiency per se, but acted at the level of stem cell survival. Consistent with the poor engraftment observed in the control animals, expression of vector-encoded ADA was only approximately 4-20% of the endogenous levels. These results support the postulated role of LIF as a regulator of hematopoiesis and suggest that cytokine stimulation can positively affect inefficient retroviral vector transduction in hematopoietic stem cells.

Retroviral vector infection and transplantation in rats of primary fetal rat hepatocytes.

Retroviral vectors were used to transduce recombinant DNA encoding firefly luciferase, Escherichia coli beta-galactosidase or human factor IX into fetal rat hepatocytes in primary culture. Hepatocytes were transduced optimally during a restricted time interval, 2-4 days post-plating. Although efficient and stable expression of reporter gene products was observed in vitro, it was affected differentially by culture conditions (plating density, media constituents) and chemical modulators of hepatocyte growth and differentiation (gelatin, hydrocortisone, isobutylmethylxanthine). Cultured cells, mock-infected or infected with a luciferase-expressing vector, were harvested non-enzymatically and injected subcutaneously into the dorsal neck fascia of neonatal syngeneic rats. Tissue isolated from injection sites one week later contained hepatocyte foci. In animals transplanted with infected cells, the preliminary results suggest that luciferase activity was present at these sites in proportion to the numbers of injected cells. These findings and previous observations made with hepatocytes from neonatal and adult primary cultures, indicate that from day 19 in utero through maturity the transient temporal 'period of susceptibility' to infection in vitro is independent of the developmental state of starting tissue. Transplantability of cultured fetal hepatocytes infected with retroviral vectors and stably expressing reporter gene products suggests that such cells might provide promising models for liver gene therapy.

Stimulation of retroviral vector infection of murine hematopoietic progenitors

Conditioned media (CM) from a cloned murine marrow-derived stromal cell line, AC6.21 (ALC), was shown to stimulate retroviral vector infection of hematopoietic progenitors in culture. Inclusion of ALC CM during cocultivation of normal murine bone marrow (BM) with vector-producing fibroblasts improved infection efficiency of day 13 spleen colony-forming cells (CFU-s) from 63% (15 provirus-positive spleen colonies/24 total), without added growth factor, to 90% (36 provirus-positive colonies/40 total). In addition, stimulation of BM cells with ALC CM during cocultivation improved retroviral infection of stem cells capable of repopulating the hematopoietic system of irradiated recipient animals. Because ALC CM was found to have 50 to 100 U/ml of IL-6 activity, purified recombinant human IL-6 was tested for an effect in this system. Stimulation with IL-6 alone increased retroviral infection efficiency of CFU-s from 15% (17 colonies provirus-positive/111 total analyzed) without added growth factor to 66% (97 provirus-positive colonies/148 total analyzed). These experiments support and extend previous studies which have demonstrated the necessity for growth factor stimulation in optimizing retroviral vector transduction of hematopoietic precursors.

Murine leukemia inhibitory factor enhances retroviral-vector infection efficiency of hematopoietic progenitors

We have investigated the in vitro effects of the cytokine leukemia inhibitory factor (LIF) on normal murine hematopoietic progenitors by measuring recovery and retroviral vector infection efficiency of 13-day posttransplant, spleen-colony-forming cell (CFU-S 13) in short-term culture. Up to a twofold increase in CFU-S13 recovery was observed, from 9.7 x 10(-5) cells in untreated controls to 17.8 to 19.5 x 10(-5) cells, depending on the concentration of LIF. Histologic analysis of spleen colonies from control and LIF-treated marrows demonstrated that there was no detectable alteration in the differentiative potential of CFU-S13. The efficiency of CFU-S13 infection was increased from 15% in untreated controls to 84% to 91% in LIF-treated marrows. Analysis of proviral integration sites in spleen colonies indicated that some CFU- S13 precursors were infected in the LIF-treated marrows.

Murine Leukemia Inhibitory Factor Enhances Retroviral-Vector Infection Efficiency of Hematopoietic Progenitors

We have investigated the in vitro effects of the cytokine leukemia inhibitory factor (LIF) on normal murine hematopoietic progenitors by measuring recovery and retroviral vector infection efficiency of 13-day posttransplant, spleen-colony-forming cell (CFU-S 13) in short-term culture. Up to a twofold increase in CFU-S13 recovery was observed, from 9.7 x 10(-5) cells in untreated controls to 17.8 to 19.5 x 10(-5) cells, depending on the concentration of LIF. Histologic analysis of spleen colonies from control and LIF-treated marrows demonstrated that there was no detectable alteration in the differentiative potential of CFU-S13. The efficiency of CFU-S13 infection was increased from 15% in untreated controls to 84% to 91% in LIF-treated marrows. Analysis of proviral integration sites in spleen colonies indicated that some CFU-S13 precursors were infected in the LIF-treated marrows.

Epitope insertion into the human hypoxanthine phosphoribosyltransferase protein and detection of the mutant protein by an anti-peptide antibody

The translational stop codon TAA of the human hypoxanthine phosphoribosyltransferase (HPRT) cDNA has been changed to GAA by site-specific mutagenesis. This modification extends the open reading frame to a downstream stop codon and results in the addition of a unique negatively charged hexapeptide to the C terminus of human HPRT protein. The mutated cDNA was transferred into HPRT-deficient rodent cells by retroviral vector infection, and the expressed enzyme was found to be fully active. An antibody against a synthetic octapeptide corresponding to the mutated HPRT C terminus precipitated the HPRT protein specifically from cells infected with the mutant virus and not infected with the wild-type HPRT virus. The technique of inserting a novel epitope into a protein by site-directed mutagenesis should be generally applicable in studies of the regulation of gene expression in vitro and in vivo.