RESUMEN

MV-NIS is an Edmonston lineage oncolytic measles virus expressing the human sodium iodide symporter-a means for monitoring by non-invasive imaging of radioiodine. Patients with relapsed, refractory myeloma who had explored all other treatment options were eligible for this Phase I trial. Cohort 1 was treated with intravenous MV-NIS, and Cohort 2 received cyclophosphamide 2 days prior to MV-NIS. Thirty-two patients were treated. Cohort 1 initially enrolled to four dose levels without reaching maximum tolerated dose (MTD) and subsequently to two higher dose levels when improved virus manufacture technology made it possible. MTD was not reached in Cohort 1, and TCID50 1011 is the dose being used in a Phase II trial of single agent MV-NIS. Grade 3-4 adverse events in both cohorts at all dose levels were: neutropenia (n=9); leukocyte count decreased (n=5); thrombocytopenia (n=2); and CD4 lymphocytes decreased, anemia and lymphopenia (each n=1). MV-N RNA sequences were amplified from gargle specimens, blood and urine. 123I scans were positive in eight patients. One patient achieved a complete response; transient drops in serum free light chains were seen in other patients. MV-NIS is capable of replicating before being cleared by the immune system. Oncolytic viruses offer a promising new modality for the targeted infection and destruction of disseminated myeloma.

Asunto(s)

Terapia Genética , Vectores Genéticos/genética , Virus del Sarampión/genética , Mieloma Múltiple/genética , Mieloma Múltiple/terapia , Viroterapia Oncolítica , Virus Oncolíticos/genética , Simportadores/genética , Adulto , Anciano , Anciano de 80 o más Años , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos , Femenino , Ingeniería Genética , Terapia Genética/efectos adversos , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Humanos , Masculino , Ratones , Persona de Mediana Edad , Mieloma Múltiple/diagnóstico , Viroterapia Oncolítica/efectos adversos , Viroterapia Oncolítica/métodos , Recurrencia , Tomografía Computarizada por Tomografía Computarizada de Emisión de Fotón Único , Distribución Tisular , Resultado del Tratamiento , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Oncolytic measles virus (MV) strains have demonstrated broad spectrum preclinical anti-tumor efficacy, including breast cancer. Aurora A kinase controls mitotic spindle formation and has a critical role in malignant transformation. We hypothesized that the Aurora A kinase inhibitor MLN8237 (alisertib) can increase MV oncolytic effect and efficacy by causing mitotic arrest. Alisertib enhanced MV oncolysis in vitro and significantly improved outcome in vivo against breast cancer xenografts. In a disseminated MDA-231-lu-P4 lung metastatic model, the MV/alisertib combination treatment markedly increased median survival to 82.5 days with 20% of the animals being long-term survivors versus 48 days median survival for the control animals. Similarly, in a pleural effusion model of advanced breast cancer, the MV/alisertib combination significantly improved outcome with a 74.5 day median survival versus the single agent groups (57 and 40 days, respectively). Increased viral gene expression and IL-24 upregulation were demonstrated, representing possible mechanisms for the observed increase in anti-tumor effect. Inhibiting Aurora A kinase with alisertib represents a novel approach to enhance MV-mediated oncolysis and antitumor effect. Both oncolytic MV strains and alisertib are currently tested in clinical trials, this study therefore provides the basis for translational applications of this combinatorial strategy in the treatment of patients with advanced breast cancer.

Asunto(s)

Antineoplásicos/uso terapéutico , Aurora Quinasa B/antagonistas & inhibidores , Azepinas/uso terapéutico , Neoplasias de la Mama/terapia , Virus del Sarampión , Viroterapia Oncolítica , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirimidinas/uso terapéutico , Adenocarcinoma/secundario , Adenocarcinoma/terapia , Animales , Aurora Quinasa B/fisiología , Azepinas/farmacología , Proteínas Bacterianas/genética , Neoplasias de la Mama/patología , Chlorocebus aethiops , Terapia Combinada , Femenino , Regulación de la Expresión Génica , Humanos , Cadenas lambda de Inmunoglobulina/genética , Interleucinas/biosíntesis , Neoplasias Pulmonares/secundario , Neoplasias Pulmonares/terapia , Ratones , Ratones Desnudos , Pirimidinas/farmacología , Transgenes , Células Vero , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Vesicular stomatitis virus (VSV) is neuropathogenic in rodents but can be attenuated 50-fold by engineering the mouse interferon-beta (IFN-ß) gene into its genome. Intravenously administered VSVs encoding IFN-ß have potent activity against subcutaneous tumors in the 5TGM1 mouse myeloma model, without attendant neurotoxicity. However, when 5TGM1 tumor cells were seeded intravenously, virus-treated mice with advanced myeloma developed clinical signs suggestive of meningoencephalitis. Co-administration of a known active antimyeloma agent did not prolong survival, further suggesting that deaths were due to viral toxicity, not tumor burden. Histological analysis revealed that systemically administered 5TGM1 cells seed to the CNS, forming meningeal tumor deposits, and that VSV infects and destroys these tumors. Death is presumably a consequence of meningeal damage and/or direct transmission of virus to adjacent neural tissue. In light of these studies, extreme caution is warranted in clinical testing of attenuated VSVs, particularly in patients with CNS tumor deposits.

Asunto(s)

Neoplasias Meníngeas/terapia , Neoplasias Meníngeas/virología , Mieloma Múltiple/terapia , Mieloma Múltiple/virología , Viroterapia Oncolítica/métodos , Virus de la Estomatitis Vesicular Indiana/genética , Vesiculovirus/genética , Animales , Modelos Animales de Enfermedad , Femenino , Interferón beta/genética , Neoplasias Meníngeas/patología , Ratones , Ratones Endogámicos C57BL , Mieloma Múltiple/patología

RESUMEN

Glioblastoma (GBM) is the most common primary brain tumor in adults and has a dismal prognosis despite multimodality treatment. Given the resistance of glioma stem cells (GSC) to chemotherapy and radiation therapy, their eradication could prevent tumor recurrence. We sought to evaluate the antitumor activity of measles virus (MV) derivatives against GSC. We generated neurosphere cultures from patient-derived primary tumor GBM xenografts, and we characterized them for the GSC markers CD133, SOX2, Nestin, ATF5 and OLIG2. Using the MV-strains MV-GFP, MV-CEA and MV-NIS we demonstrated infection, viral replication and significant cytopathic effect in vitro against GSC lines. In tumorigenicity experiments, GBM44 GSC were infected with MV in vitro and subsequently implanted into the right caudate nucleus of nude mice: significant prolongation of survival in mice implanted with infected GSC was observed, compared with mock-infected controls (P=0.0483). In therapy experiments in GBM6 and GBM12 GSC xenograft models, there was significant prolongation of survival in MV-GFP-treated animals compared with inactivated virus-treated controls (GBM6 P=0.0021, GBM12 P=0.0416). Abundant syncytia and viral replication was demonstrated in tumors of MV-treated mice. Measles virus derivatives have significant antitumor activity against glioma-derived stem cells in vitro and in vivo.

Asunto(s)

Neoplasias Encefálicas/terapia , Glioblastoma/terapia , Virus del Sarampión/genética , Células Madre Neoplásicas/virología , Viroterapia Oncolítica , Virus Oncolíticos/genética , Animales , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Células Gigantes/patología , Células Gigantes/virología , Humanos , Virus del Sarampión/fisiología , Ratones , Ratones Desnudos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Virus Oncolíticos/fisiología , Células Tumorales Cultivadas , Replicación Viral , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Oncolytic viruses can be neutralized in the bloodstream by antiviral antibodies whose titers increase progressively with each exposure, resulting in faster virus inactivation and further reductions in efficacy with each successive dose. A single dose of cyclophosphamide (CPA) at 370 mg m(-2) was not sufficient to control the primary antiviral immune responses in mice, squirrel monkeys and humans. We therefore tested clinically approved multidose CPA regimens, which are known to kill proliferating lymphocytes, to determine if more intensive CPA therapy can more effectively suppress antiviral antibody responses during virotherapy. In virus-susceptible mice, primary antibody responses to intravenously (i.v.) administered oncolytic measles virus (MV) or vesicular stomatitis virus (VSV) were partially or completely suppressed, respectively, by oral (1 mg × 8 days) or systemic (3 mg × 4 days) CPA regimens initiated 1 day before virus. When MV- or VSV-immune mice were re-challenged with the respective viruses and concurrently treated with four daily systemic doses of CPA, their anamnestic antibody responses were completely suppressed and antiviral antibody titers fell significantly below pre-booster levels. We conclude that the CPA regimen of four daily doses at 370 mg m(-2) should be evaluated clinically with i.v. virotherapy to control the antiviral antibody response and facilitate effective repeat dosing.

Asunto(s)

Ciclofosfamida/farmacología , Inmunidad Humoral/efectos de los fármacos , Viroterapia Oncolítica/métodos , Virus Oncolíticos/fisiología , Animales , Anticuerpos Antivirales/inmunología , Cricetinae , Ciclofosfamida/administración & dosificación , Relación Dosis-Respuesta a Droga , Humanos , Inmunidad Humoral/inmunología , Inmunosupresores/administración & dosificación , Inmunosupresores/farmacología , Sarampión/inmunología , Sarampión/virología , Virus del Sarampión/genética , Virus del Sarampión/inmunología , Virus del Sarampión/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Virus Oncolíticos/genética , Virus Oncolíticos/inmunología , Factores de Tiempo , Virus de la Estomatitis Vesicular Indiana/genética , Virus de la Estomatitis Vesicular Indiana/inmunología , Virus de la Estomatitis Vesicular Indiana/fisiología

RESUMEN

Current therapy for multiple myeloma is complex and prolonged. Antimyeloma drugs are combined in induction, consolidation and/or maintenance protocols to destroy bulky disease, then suppress or eradicate residual disease. Oncolytic viruses have the potential to mediate both tumor debulking and residual disease elimination, but this curative paradigm remains unproven. Here, we engineered an oncolytic vesicular stomatitis virus to minimize its neurotoxicity, enhance induction of antimyeloma immunity and facilitate noninvasive monitoring of its intratumoral spread. Using high-resolution imaging, autoradiography and immunohistochemistry, we demonstrate that the intravenously administered virus extravasates from tumor blood vessels in immunocompetent myeloma-bearing mice, nucleating multiple intratumoral infectious centers that expand rapidly and necrose at their centers, ultimately coalescing to cause extensive tumor destruction. This oncolytic tumor debulking phase lasts only for 72 h after virus administration, and is completed before antiviral antibodies become detectable in the bloodstream. Antimyeloma T cells, cross-primed as the virus-infected cells provoke an antiviral immune response, then eliminate residual uninfected myeloma cells. The study establishes a curative oncolytic paradigm for multiple myeloma where direct tumor debulking and immune eradication of minimal disease are mediated by a single intravenous dose of a single therapeutic agent. Clinical translation is underway.

Asunto(s)

Vectores Genéticos/administración & dosificación , Mieloma Múltiple/terapia , Viroterapia Oncolítica , Animales , Línea Celular Tumoral , Cricetinae , Terapia Genética , Vectores Genéticos/genética , Interferón beta/genética , Interferón beta/metabolismo , Ratones , Ratones Endogámicos C57BL , Mieloma Múltiple/genética , Mieloma Múltiple/inmunología , Virus Oncolíticos/genética , Simportadores/genética , Simportadores/metabolismo , Trasplante Isogénico , Virus de la Estomatitis Vesicular Indiana/genética

RESUMEN

The purpose of our study was to validate the ability of pinhole micro-single-photon emission computed tomography/computed tomography (SPECT/CT) to: 1) accurately resolve the intratumoral dispersion pattern and 2) quantify the infection percentage in solid tumors of an oncolytic measles virus encoding the human sodium iodide symporter (MV-NIS). Sodium iodide symporter (NIS) RNA level and dispersion pattern were determined in control and MV-NIS-infected BxPC-3 pancreatic tumor cells and mouse xenografts using quantitative, real-time, reverse transcriptase, polymerase chain reaction, autoradiography and immunohistochemistry (IHC). Mice with BxPC-3 xenografts were imaged with (123)I or (99)TcO(4) micro-SPECT/CT. Tumor dimensions and radionuclide localization were determined with imaging software. Linear regression and correlation analyses were performed to determine the relationship between tumor infection percentage and radionuclide uptake (% injected dose per gram) above background and a highly significant correlation was observed (r(2)=0.947). A detection threshold of 1.5-fold above the control tumor uptake (background) yielded a sensitivity of 2.7% MV-NIS-infected tumor cells. We reliably resolved multiple distinct intratumoral zones of infection from non-infected regions. Pinhole micro-SPECT/CT imaging using the NIS reporter demonstrated precise localization and quantitation of oncolytic MV-NIS infection, and can replace more time-consuming and expensive analyses (for example, autoradiography and IHC) that require animal killing.

Asunto(s)

Vectores Genéticos/metabolismo , Virus Oncolíticos/metabolismo , Tomografía Computarizada de Emisión de Fotón Único , Animales , Línea Celular , Femenino , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Humanos , Radioisótopos de Yodo/metabolismo , Ratones , Ratones Desnudos , Neoplasias/diagnóstico por imagen , Virus Oncolíticos/genética , Sensibilidad y Especificidad , Simportadores/genética , Simportadores/metabolismo , Trasplante Heterólogo

RESUMEN

Off-target binding or vector sequestration can significantly limit the efficiency of systemic virotherapy. We report here that systemically administered oncolytic measles virus (MV) was rapidly sequestered by the mononuclear phagocytic system (MPS) of the liver and spleen in measles receptor CD46-positive and CD46-negative mice. Since scavenger receptors on Kupffer cells are responsible for the elimination of blood-borne pathogens, we investigated here if MV uptake was mediated by scavenger receptors on Kupffer cells. Pretreatment of cells with poly(I), a scavenger receptor ligand, reduced MV expression by 99% in murine (J774A.1) macrophages and by 50% in human (THP-1) macrophages. Pre-dosing of mice with poly(I) reduced MPS sequestration of MV and increased circulating levels of MV by 4 to 15-folds at 2 min post virus administration. Circulating virus was still detectable 30 min post infusion in mice pre-dosed with poly(I) whereas no detectable MV was found at 5-10 min post infusion if mice did not receive poly(I). MPS blockade by poly(I) enhanced virus delivery to human ovarian SKOV3ip.1 and myeloma KAS6/1 xenografts in mice. Higher gene expression and improved control of tumor growth was noted early post therapy. Based on these results, incorporation of MPS blockade into MV treatment regimens is warranted.

Asunto(s)

Virus del Sarampión/fisiología , Mieloma Múltiple/terapia , Viroterapia Oncolítica/métodos , Neoplasias Ováricas/terapia , Poli I/farmacología , Animales , Línea Celular , Línea Celular Tumoral , Chlorocebus aethiops , Femenino , Humanos , Inmunohistoquímica , Hígado/virología , Macrófagos/efectos de los fármacos , Macrófagos/virología , Virus del Sarampión/genética , Virus del Sarampión/inmunología , Ratones , Ratones Desnudos , Ratones Transgénicos , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/virología , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/virología , Bazo/virología , Células Vero , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Because of their ability to replicate, the dose-response relationships of oncolytic viruses cannot easily be predicted. To better understand the pharmacokinetics of virotherapy in relation to viral dose and schedule, we administered MV-CEA intraperitoneally in an orthotopic mouse model of ovarian cancer. MV-CEA is an attenuated oncolytic measles virus engineered to express soluble human carcinoembryonic antigen (CEA), and the virus is currently undergoing phase I clinical testing in patients with ovarian cancer. Plasma CEA levels correlate with numbers of virus-infected tumor cells at a given time, and were used as a surrogate to monitor the profiles of viral gene expression over time. The antineoplastic activity of single- or multiple-dose MV-CEA was apparent over a wide range of virus doses (10(3)-10(8) TCID(50)), with little reduction in observed antitumor efficacy, even at the lowest tested dose. However, analysis of CEA profiles of treated mice was highly informative, illustrating the variability in virus kinetics at different dose levels. The highest doses of virus were associated with higher initial levels of tumor cell killing, but the final outcome of MV-CEA therapy at all dose levels was a partial equilibrium between virus and tumor, resulting in significant slowing of tumor growth and enhanced survival of the mice.

Asunto(s)

Virus del Sarampión , Viroterapia Oncolítica , Neoplasias Ováricas/terapia , Animales , Antígeno Carcinoembrionario/biosíntesis , Antígeno Carcinoembrionario/sangre , Relación Dosis-Respuesta Inmunológica , Femenino , Humanos , Ratones , Neoplasias Ováricas/inmunología , Neoplasias Ováricas/patología , Carga Tumoral , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

To better understand retroviral entry, we have characterized the interactions between subgroup A avian leukosis virus [ALV(A)] envelope glycoproteins and Tva, the receptor for ALV(A), that result in receptor interference. We have recently shown that soluble forms of the chicken and quail Tva receptor (sTva), expressed from genes delivered by retroviral vectors, block ALV(A) infection of cultured chicken cells ( approximately 200-fold antiviral effect) and chickens (>98% of the birds were not infected). We hypothesized that inhibition of viral replication by sTva would select virus variants with mutations in the surface glycoprotein (SU) that altered the binding affinity of the subgroup A SU for the sTva protein and/or altered the normal receptor usage of the virus. Virus propagation in the presence of quail sTva-mIgG, the quail Tva extracellular region fused to the constant region of the mouse immunoglobulin G (IgG) protein, identified viruses with three mutations in the subgroup A hr1 region of SU, E149K, Y142N, and Y142N/E149K. These mutations reduced the binding affinity of the subgroup A envelope glycoproteins for quail sTva-mIgG (32-, 324-, and 4,739-fold, respectively) but did not alter their binding affinity for chicken sTva-mIgG. The ALV(A) mutants efficiently infected cells expressing the chicken Tva receptor but were 2-fold (E149K), 10-fold (Y142N), and 600-fold (Y142N/E149K) less efficient at infecting cells expressing the quail Tva receptor. These mutations identify key determinants of the interaction between the ALV(A) glycoproteins and the Tva receptor. We also conclude from these results that, at least for the wild-type and variant ALV(A)s tested, the receptor binding affinity was directly related to infection efficiency.

Asunto(s)

Virus de la Leucosis Aviar/genética , Receptores Virales/metabolismo , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/metabolismo , Secuencia de Aminoácidos , Animales , Antivirales/farmacología , Leucosis Aviar/virología , Virus de la Leucosis Aviar/efectos de los fármacos , Virus de la Leucosis Aviar/metabolismo , Virus de la Leucosis Aviar/patogenicidad , Proteínas Aviares , Línea Celular , Células Cultivadas , Pollos , Clonación Molecular , Inmunoglobulina G/metabolismo , Ratones , Datos de Secuencia Molecular , Mutación , Codorniz , Receptores Virales/genética , Proteínas del Envoltorio Viral/genética

RESUMEN

The host developing resistance to retroviral infection is believed to be a major force in the evolution of multiple receptor usage by retroviruses. The avian leukosis-sarcoma virus (ALV) group of retroviruses provides a powerful system for studying the envelope-receptor interactions involved in retrovirus entry; different members of this group of closely related viruses use distinct cellular receptors. Analysis of the ALV envelope subgroups suggests that the different ALVs evolved from a common ancestor by mutations in the env gene. Cells and animals that express subgroup A ALV envelope glycoproteins are highly resistant to ALV(A) infection due to receptor interference. In this study, we tested whether expression of a soluble form of subgroup A surface glycoprotein (SU) would result in receptor interference and whether this interference would select for resistant viruses with altered receptor usage. Chicken cells expressing the secreted ALV(A) SU immunoadhesin SU(A)-rIgG, which contains the subgroup A SU domain fused to the constant region of a rabbit immunoglobulin (IgG) heavy chain, showed significant receptor interference. A variant virus resistant to SU(A)-rIgG receptor interference was obtained. This virus had a six-amino-acid deletion in the subgroup A hr1 that altered receptor usage. This approach may identify regions of SU that play a critical role in receptor specificity.

Asunto(s)

Proteínas de la Cápside , Proteínas del Envoltorio Viral/metabolismo , Alpharetrovirus , Secuencia de Aminoácidos , Animales , Cápside , Separación Celular , Pollos , Clonación Molecular , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Datos de Secuencia Molecular , Conejos , Receptores de IgG/metabolismo , Proteínas de los Retroviridae , Proteínas del Núcleo Viral

RESUMEN

Capsid-targeted viral inactivation (CTVI), a promising gene-based antiviral strategy against retroviruses, was designed to disrupt the retroviral life cycle by incorporating a degradative enzyme (e.g., nuclease) into viral particles during assembly, thereby reducing or eliminating the production of infectious virus. The experimental system used to develop the CTVI strategy for retroviruses is designed to block the production of infectious Moloney murine leukemia virus (Mo-MLV). Two nucleases, Escherichia coli ribonulease HI and Staphylococcus nuclease, have been shown to be tolerated by the cell as Mo-MLV Gag-nuclease fusion polyproteins and still be active in the viral particles. The goal of this study was to determine what cellular and viral factors limit CTVI in cultured cells. The avian DF-1 cell line greatly expanded our ability to test the antiviral efficacy of CTVI in long-term assays and to determine the mechanism(s) of CTVI action. The CTVI antiviral effect is dependent on the level of Mo-MLV Gag-nuclease fusion polyprotein expressed. The Mo-MLV Gag-nuclease polyproteins produce a long-term prophylactic antiviral effect after a low- or high-dose Mo-MLV challenge. The Mo-MLV Gag-nuclease fusions have a significant therapeutic effect ( approximately 1000-fold) on the production of infectious Mo-MLV. The therapeutic CTVI effect can be improved by a second delivery of the CTVI fusion gene. Both the prophylactic and the therapeutic CTVI antiviral approaches can virtually eliminate the production of infectious Mo-MLV in vitro and are only limited by the number of cells in the population that do not express adequate levels of the CTVI fusion polyprotein.

Asunto(s)

Cápside/genética , Virus de la Leucemia Murina de Moloney/genética , Virus de la Leucemia Murina de Moloney/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , ADN Recombinante/genética , Escherichia coli/enzimología , Escherichia coli/genética , Genes gag , Vectores Genéticos , Ratones , Nucleasa Microcócica/genética , Virus de la Leucemia Murina de Moloney/crecimiento & desarrollo , Proteínas Recombinantes de Fusión/genética , Ribonucleasa H/genética , Replicación Viral

RESUMEN

We previously reported that mitogenic activation of porcine peripheral blood mononuclear cells resulted in production of porcine endogenous retrovirus(es) (PERV[s]) capable of productively infecting human cells (C. Wilson et al., J. Virol. 72:3082-3087, 1998). We now extend that analysis to show that additional passage of isolated virus, named here PERV-NIH, through a human cell line yielded a viral population with a higher titer of infectious virus on human cells than the initial isolate. We show that in a single additional passage on a human cell line, the increase in infectivity for human cells is accounted for by selection against variants carrying pig-tropic envelope sequences (PERV-C) as well as by enrichment for replication-competent genomes. Sequence analysis of the envelope cDNA present in virions demonstrated that the envelope sequence of PERV-NIH is related to but distinct from previously reported PERV envelopes. The in vitro host range of PERV was studied in human primary cells and cell lines, as well as in cell lines from nonhuman primate and other species. This analysis reveals three patterns of susceptibility to infection among these host cells: (i) cells are resistant to infection in our assay; (ii) cells are infected by virus, as viral RNA is detected in the supernatant by reverse transcription-PCR, but the cells are not permissive to productive replication and spread; and (iii) cells are permissive to low-level productive replication. Certain cell lines were permissive for efficient productive infection and spread. These results may prove useful in designing appropriate animal models to assess the in vivo infectivity properties of PERV.

Asunto(s)

Retrovirus Endógenos/fisiología , Tropismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Adhesión Celular , Línea Celular , ADN Complementario , Humanos , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Porcinos

RESUMEN

The interactions between the subgroup A avian leukosis virus [ALV(A)] envelope glycoproteins and soluble forms of the ALV(A) receptor Tva were analyzed both in vitro and in vivo by quantitating the ability of the soluble Tva proteins to inhibit ALV(A) entry into susceptible cells. Two soluble Tva proteins were tested: the 83-amino-acid Tva extracellular region fused to two epitope tags (sTva) or fused to the constant region of the mouse immunoglobulin G heavy chain (sTva-mIgG). Replication-competent ALV-based retroviral vectors with subgroup B or C env were used to deliver and express the two soluble tv-a (stva) genes in avian cells. In vitro, chicken embryo fibroblasts or DF-1 cells expressing sTva or sTva-mIgG proteins were much more resistant to infection by ALV(A) ( approximately 200-fold) than were control cells infected by only the vector. The antiviral effect was specific for ALV(A), which is consistent with a receptor interference mechanism. The antiviral effect of sTva-mIgG was positively correlated with the amount of sTva-mIgG protein. In vivo, the stva genes were delivered and expressed in line 0 chicken embryos by the ALV(B)-based vector RCASBP(B). Viremic chickens expressed relatively high levels of stva and stva-mIgG RNA in a broad range of tissues. High levels of sTva-mIgG protein were detected in the sera of chickens infected with RCASBP(B)stva-mIgG. Viremic chickens infected with RCASBP(B) alone, RCASBP(B)stva, or RCASBP(B)stva-mIgG were challenged separately with ALV(A) and ALV(C). Both sTva and sTva-mIgG significantly inhibited infection by ALV(A) (95 and 100% respectively) but had no measurable effect on ALV(C) infection. The results of this study indicate that a soluble receptor can effectively block infection of at least some retroviruses and demonstrates the utility of the ALV experimental system in characterizing the mechanism(s) of viral entry.

Asunto(s)

Virus de la Leucosis Aviar/fisiología , Receptores Virales/metabolismo , Animales , Virus de la Leucosis Aviar/metabolismo , Proteínas Aviares , Embrión de Pollo , Expresión Génica , Cadenas Pesadas de Inmunoglobulina/biosíntesis , Cadenas Pesadas de Inmunoglobulina/genética , Ratones , Receptores Virales/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proyectos de Investigación , Solubilidad

RESUMEN

The lack of a well-behaved permanent, adherent, nontransformed chicken cell line has made some experiments with avian leukosis-sarcoma viruses (ASLV) and vectors considerably more difficult. The EV-O-derived line, DF-1, supports the efficient replication of subgroups (A), (B), and (C) ASLV, as well as amphotrophic murine leukemia virus and an ASLV-derived vector that has its env gene derived from the env gene from an amphotrophic murine leukemia virus. The cell line responds appropriately to the expression of a transforming oncogene (v-myc) to a growth suppressor gene [p21(waf1)] and can be sorted (using FACS) if infected by an ASLV vector that expresses GFP.

Asunto(s)

Alpharetrovirus/fisiología , Línea Celular/virología , Replicación Viral , Animales , Transformación Celular Neoplásica , Pollos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/fisiología , Genes myc/fisiología , Vectores Genéticos , Virus de la Leucemia Murina/fisiología , Oncogenes/fisiología , Proteínas del Envoltorio Viral/fisiología

RESUMEN

The antiviral strategy of capsid-targeted viral inactivation (CTVI) was designed to disable newly produced virions by fusing a Gag or Gag-Pol polyprotein to a degradative enzyme (e.g., a nuclease or protease) that would cause the degradative enzyme to be inserted into virions during assembly. Several new experimental approaches have been developed that increase the antiviral effect of the CTVI strategy on retroviral replication in vitro. A Moloney murine leukemia virus (Mo-MLV) Gag-Escherichia coli RNase HI fusion has a strong antiviral effect when used prophylactically, inhibiting the spread of Mo-MLV and reducing virus titers 1,500- to 2,500-fold. A significant (approximately 100-fold) overall improvement of the CTVI prophylactic antiviral effect was produced by a modification in the culture conditions which presumably increases the efficiency of delivery and expression of the Mo-MLV Gag fusion polyproteins. The therapeutic effect of Mo-MLV Gag-RNase HI polyproteins is to reduce the production of infectious Mo-MLV up to 18-fold. An Mo-MLV Gag-degradative enzyme fusion junction was designed that can be cleaved by the Mo-MLV protease to release the degradative enzyme.

Asunto(s)

Escherichia coli/enzimología , Productos del Gen gag/genética , Virus de la Leucemia Murina de Moloney/crecimiento & desarrollo , Ribonucleasa H/genética , Células 3T3 , Secuencia de Aminoácidos , Animales , Antivirales/farmacología , Secuencia de Bases , Embrión de Pollo , Endopeptidasas/metabolismo , Productos del Gen gag/metabolismo , Productos del Gen gag/farmacología , Ratones , Datos de Secuencia Molecular , Virus de la Leucemia Murina de Moloney/efectos de los fármacos , Virus de la Leucemia Murina de Moloney/metabolismo , Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Ribonucleasa H/metabolismo , Ribonucleasa H/farmacología

Asunto(s)

Técnicas de Transferencia de Gen , Vectores Genéticos , Músculos/fisiología , Retroviridae , Animales , Aves , Ratones

RESUMEN

Transgenic mouse lines have been developed that express the tv-a receptor under the control of the chicken beta-actin promoter. These mice express the tv-a receptor in most or all tissues and in the early embryo. An avian leukosis virus (ALV)-based retroviral vector system was used for the efficient delivery of genes into preimplantation mouse embryos from these transgenic lines. Experimental animals could be generated quickly and easily by infecting susceptible blastocysts with ALV-based retroviral vectors. Expression of the delivered genes was controlled by either the constitutive viral promoter contained in the long terminal repeat or an internal nonviral tissue-specific promoter. Mating the infected founder chimeric animals produced animals that carry the ALV provirus as a transgene. A subset of the integrated proviruses expressed the chloramphenicol acetyltransferase reporter gene from either the promoter in the long terminal repeat or an internal promoter, which we believe indicates that many of the sites that are accessible to viral DNA insertion in preimplantation embryos are incompatible with expression in older animals. This approach should prove useful for studies on murine cell lineage and development, providing models for studying oncogenesis, and testing gene therapy strategies.

Asunto(s)

Ratones Transgénicos/genética , Transducción Genética , Actinas/genética , Animales , Virus de la Leucosis Aviar/genética , Secuencia de Bases , Blastocisto , Embrión de Pollo , Cartilla de ADN/genética , Expresión Génica , Técnicas Genéticas , Vectores Genéticos , Ratones , Ratones Transgénicos/embriología , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Receptores Virales/genética

RESUMEN

Capsid-targeted viral inactivation is an antiviral strategy in which toxic fusion proteins are targeted to virions, where they inhibit viral multiplication by destroying viral components. These fusion proteins consist of a virion structural protein moiety and an enzymatic moiety such as a nuclease. Such fusion proteins can severely inhibit transposition of yeast retrotransposon Ty1, an element whose transposition mechanistically resembles retroviral multiplication. We demonstrate that expression of a murine retrovirus capsid-staphylococcal nuclease fusion protein inhibits multiplication of the corresponding murine leukemia virus by 30- to 100-fold. Staphylococcal nuclease is apparently inactive intracellularly and hence nontoxic to the host cell, but it is active extracellularly because of its requirement for high concentrations of Ca2+ ions. Virions assembled in and shed from cells expressing the fusion protein contain very small amounts of intact viral RNA, as would be predicted for nuclease-mediated inhibition of viral multiplication.

Asunto(s)

Antivirales/metabolismo , Cápside/metabolismo , Productos del Gen gag/metabolismo , Virus de la Leucemia Murina/crecimiento & desarrollo , Nucleasa Microcócica/metabolismo , Células 3T3 , Animales , Productos del Gen gag/genética , Productos del Gen gag/farmacología , Ratones , Nucleasa Microcócica/genética , Nucleasa Microcócica/farmacología , ARN Viral/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Virión/crecimiento & desarrollo , Replicación Viral

RESUMEN

Avian leukosis viruses (ALVs) have been used extensively as genetic vectors in avian systems, but their utility in mammals or mammalian cell lines is compromised by inefficient viral entry. We have overcome this limitation by generating transgenic mice that express the receptor for the subgroup A ALV under the control of the chicken alpha sk-actin promoter. The skeletal muscles of these transgenic animals are susceptible to efficient infection by subgroup A ALV. Because infection is restricted to cell lineages that express the transgene, the method has utility for studies of development and oncogenesis and will provide models for tissue-specific gene therapy.

Asunto(s)

Virus de la Leucosis Aviar/genética , Marcación de Gen/métodos , Receptores Virales/genética , Actinas/genética , Animales , Virus de la Leucosis Aviar/crecimiento & desarrollo , Vectores Genéticos , Ratones , Ratones Transgénicos , Regiones Promotoras Genéticas , Replicación Viral