1,720,962 research outputs found
Chk2 is required for HSV-1 ICP0-mediated G2/M arrest and enhancement of virus growth
No full textCP0 is a multi-functional herpes simplex virus type 1 (HSV-1) immediate-early (IE) gene product that contributes to efficient virus growth and reactivation from latency. Here we show that HSV-1-induced cell-cycle arrest at the G2/M border requires ICP0 and Chk2 kinase and that ICP0 expression by transfection or infection induces ATM-dependent phosphorylation of Chk2 and Cdc25C. Infection of cells with a replication-defective mutant virus deleted for all the regulatory IE genes except ICP0 (TOZ22R) induced G2/M arrest whereas a mutant virus deleted in addition for ICP0 (QOZ22R) failed to do so. Chk2-deficient cells and cells expressing a kinase-deficient Chk2 did not undergo cell-cycle arrest in response to TOZ22R infection. Chk2 deficiency diminished the growth of wild-type HSV-1, but not the growth of an ICP0-deleted recombinant virus. Together, these results are consistent with the interpretation that ICP0 activates a DNA damage response pathway to arrest cells in G2/M phase and promote virus growt
Rapid method for construction of recombinant HSV gene transfer vectors
No full textHerpes simplex virus type 1 (HSV-1) is a neurotrophic human pathogen that naturally persists in neurons in a latent state and carries a large number of viral functions which can be replaced by foreign genes to create a vector for gene therapy applications. In this report we describe a two-step method for insertion/deletion mutagenesis of HSV genes and the efficient insertion of transgenes into these locations in the viral genome. The first step is the insertion of a receptor gene (lacZ) cassette flanked by Pacl restriction enzyme sites not otherwise found in the viral genome, using standard marker transfer procedures to interrupt a portion of the target HSV gene. The second step is substitution of the reporter gene with foreign cDNAs by digestion of the vector DNA with Pacl to remove the lacZ gene and subsequent repair of the vector genome by homologous recombination with a transgene expression plasmid. Potential recombinants indentified by a 'clear plaque' phenotype after X-gal staining arose at high frequency (80-100%). Of these, recombinants containing the transgene in place of the lacZ gene ranged from 19-65%. Insertion of the transgene expression construct into the viral genome eliminates the Pacl sites, allowing this method to be used repeatedly for the sequential deletion of multiple HSV genes while inserting multiple transgenes. This procedure was repeated in succession to produce a vector carrying two independent expression cassettes at distinct viral loci
Engineering herpes simplex virus vectors for CNS applications
No full textWe have engineered highly defective HSV genomic vectors (i) deleted for multiple IE gene functions which fail to initiate lytic viral replication, (ii) that express few viral functions, (iii) that display reduced toxicity even for primary neurons in culture, (iv) that are able to incorporate multiple transgenes or single large genes, (v) that are able to efficiently establish latency in neurons and serve as a platform for long-term gene expression using the latency promoter system, (vi) that cannot reactivate from latency or spread to other nerves of tissues, (vii) that can enhance or regulate transgene expression, and (viii) that can be targeted to specific cell types using their normal receptor-recognition ligands modified to contain novel attachment functions. These vectors may prove useful in applications in which short-term gene expression and multiple gene products are required to achieve a therapeutic outcome such as tumor-cell killing and vaccination. Expression of these therapeutic genes may be coordinately regulated or controlled by drug-responsive transactivators that will enable regulation of the level and duration of therapeutic gene expression. Applications that require long-term expression of transgene in PNS neurons, such as diabetic neuropathy, may employ the LAP system for expression of therapeutic genes, such as NGF. Although we have been able to redirect virus binding using novel ligands introduced into the viral glycoproteins, significant improvements will be required to more efficiently bind the virus to the target cell without perturbing normal cellular pathways and still enable the virus to enter the target cell by a mechanism similar to that naturally employed. Future experiments will be designed using vectors that are capable of regulation of therapeutic gene expression using their own native promoters, thus responding to the natural stimuli involved in their production. Further work will be necessary to modify the vector for non- nervous-system approaches to treat diseases of other tissues such as arthritis, muscular dystrophy, or autoimmune disorders
Connexin 43-enhanced suicide gene therapy using herpesviral vectors
No full textTumor cell transduction with the herpes simplex virus (HSV) thymidine kinase (tk) gene and treatment with ganciclovir (GCV) is a widely studied cancer gene therapy. Connexin (Cx)-dependent gap junctions between cells facilitate the intercellular spread of TK-activated GCV, thereby creating a bystander effect that improves tumor cell killing. However, tumor cells often have reduced connexin expression, thus thwarting bystander killing and the effectiveness of TK/GCV gene therapy. To improve the effectiveness of this therapy, we compared an HSV vector (TOCX) expressing Cx43 in addition to TK with an isogenic tk vector (TOZ.1) for their abilities to induce bystander killing of Cx-positive U-87 MG human glioblastoma cells and Cx-negative L929 fibrosarcoma cells in vitro and in vivo. The results showed that low-multiplicity infection of U-87 MG cells with TOCX only minimally increased GCV-mediated cell death compared with infection by TOZ.1, consistent with the endogenous level of Cx in these cells. In contrast, bystander killing of L929 cells was markedly enhanced by vector-mediated expression of Cx. In vivo experiments in which U-87 MG cells were preinfected at low multiplicity and injected into the flanks of nude mice showed complete cures of all animals in the TOCX group following GCV treatment, whereas untreated animals uniformly formed fatal tumors. TOCX injection into U-87 MG intradermal and intracranial tumors resulted in prolonged survival of the host animals in a GCV-dependent manner. Together, these results suggest that the combination of TK and Cx may be beneficial for the treatment of human glioblastoma. © 2000 American Society for Gene Therapy
HSV vector cytotoxicity is inversely correlated with effective TK/GCV suicide gene therapy of rat gliosarcoma
No full textHerpes simplex virus (HSV)-mediated delivery of the HSV thymidine kinase (tk) gene to tumor cells in combination with ganciclovir (GCV) administration may provide an effective suicide gene therapy for destruction of malignant glioblastomas. However, because HSV is a highly cytotoxic agent, gene expression from the virus is short-lived which may limit the effectiveness of HSVtk/GCV therapy. Using different replication-defective HSVtk gene vectors, we compared HSV vector backgrounds for their cytotoxic activity on infection of 9L gliosarcoma cells in culture and brain tumors in rats and evaluated the impact of vector toxicity on the effectiveness of tk/GCV-mediated suicide gene therapy. As reported previously for other cell lines, a vector deleted for both copies of the immediate-early (IE) gene ICP4 (SOZ.1) was highly toxic for 9L cells in culture while a vector deleted in addition for the ICP22 and ICP27 IE genes (T.1) reduced or arrested 9L cell proliferation with more limited cell killing. Nevertheless, both vectors supported widespread killing of uninfected cells in the presence of GCV following low multiplicity infections, indicating that vector cytotoxicity did not preempt the production of vector-encoded TK enzyme necessary for the killing of uninfected cells by the HSV-tk/GCV bystander effect. Although an SOZ.1-related vector (SHZ.2) caused tumor cell necrosis in vivo, injection of SHZ.2 at multiple coordinates thoughout the tumor followed by GCV administration failed to prolong markedly the survival of tumor-bearing rats. In contrast, a single injection of T.1 produced a life-extending response to GCV. These results indicate that vector cytotoxicity can limit the efficacy of HSV-tk/GCV treatment in vivo, which may be due to premature termination of tk gene expression with attendant abortion of the bystander effect
Development of herpes simplex virus replication-defective multigene vectors for combination gene therapy applications
No full textSome gene therapy applications will require simultaneous expression of multiple gene products to achieve a therapeutic effect. In this study we describe the generation and characterization of replication incompetent herpes simplex virus type 1 (HSV-1) vectors (HX86Z or HX86G) carrying distinct and independently regulated expression cassettes for five transgenes (hIL-2, hGM-CSF, hB7.1, HSV-tk and lacZ or hIFNγ). The transgenes, representing 12 kb of DNA sequence, were recombined into separate loci of a single mutant virus vector deleted for 11.6 kb of vector sequences representing portions of nine viral genes, ICP4, ICP22, ICP27, ICP47, U(L)24, U(L)41, U(L)44, U(S)10 and U(S)11. Deletion of the immediate-early genes ICP4, ICP22 and ICP27 substantially reduced vector cytotoxicity, prevented early and late viral gene expression and left intact MHC class I antigen expression. Simultaneous expression of multiple transgenes was obtained for up to 7 days in primary human melanoma cells with peak expression at 2-3 days after infection. The transgenes were chosen for their potential to function synergistically in tumor destruction and vaccine gene therapy applications, but the method and vector employed could be applied to other multigene therapy strategies. This study demonstrates the potential for engineering large transgene capacity DNA viruses such as HSV-1 for expression of multiple transgenes
Deletion of multiple immediate-early genes from herpes simplex virus reduces cytotoxicity and permits long-term gene expression in neurons
No full textHerpes simplex virus type 1 (HSV-1) has many attractive features that suggest its utility for gene transfer to neurons. However, viral cytotoxicity and transient transgene expression limit practical applications even in the absence of vital replication. Mutant viruses deleted for the immediate-early (IE) gene, ICP4, an essential transcriptional transactivator, are toxic to many cell types in culture in which only the remaining IE genes are expressed. In order to test directly the toxicity of other IE gene products in neurons and develop a mutant background capable of long-term transgene expression, we generated mutants deleted for multiple IE genes in various combinations and tested their relative cytotoxicity in 9L rat gliosarcoma cells, Vero monkey kidney cells, and primary rat cortical and dorsal roof neurons in culture. Viral mutants deleted simultaneously for the IE genes encoding ICP4, ICP22 and ICP27 showed substantially reduced cytotoxicity compared with viruses deleted for ICP4 alone or ICP4 in combination with either ICP22, ICP27 or ICP47. Infection of neurons in culture with these triple IE deletion mutants substantially enhanced cell survival and permitted transgene expression for over 21 days. Such mutants may prove useful for efficient gene transfer and extended transgene expression in neurons in vitro and in vivo
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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