1,720,975 research outputs found
A double mutation in glycoprotein gB compensates for ineffective gD-dependent initiation of herpes simplex virus type 1 infection.
No full textHerpes simplex virus (HSV) entry into cells is triggered by the binding of envelope glycoprotein D (gD) to a specific receptor, such as nectin-1 or herpesvirus entry mediator (HVEM), resulting in activation of the fusion effectors gB and gH and virus penetration. Here we report the identification of a hyperactive gB allele, D285N/A549T, selected by repeat passage of a gD mutant virus defective for nectin-1 binding through cells that express a gD-binding-impaired mutant nectin-1. The gB allele in a wild-type virus background enabled the use of other nectins as virus entry receptors. In addition, combination of the mutant allele with an epidermal growth factor receptor (EGFR)-retargeted gD gene yielded dramatically increased EGFR-specific virus entry compared to retargeted virus carrying wild-type gB. Entry of the gB mutant virus into nectin-1-bearing cells was markedly accelerated compared to that of wild-type virus, suggesting that the gB mutations affect a rate-limiting step in entry. Our observations indicate that ineffective gD activation can be complemented by hypersensitization of a downstream component of the entry cascade to gD signaling
Advances in the development of herpes simplex virus-based gene transfer vectors for the nervous system
No full textHerpes simplex virus (HSV) is an attractive candidate vector for treatment of nervous system disease by gene therapy. Here we review molecular aspects of the natural biology of HSV as it relates to vector design and application. Although gene transfer and transient expression was readily achieved using first generation replication defective HSV vectors, these vectors did not provide for long-term transgene expression, a prerequisite for effective treatment of neurodegenerative disease. The principal impediments to effective use of HSV vectors are residual toxicity of non- replicating vectors and the silencing of transgene expression from persisting latent viral genomes in neurons. Recent advances suggest that vectors deleted for multiple immediate early vital genes provide a solution to both of these problems and thereby provide for the first time insight into methods for the effective design of useful gene vectors for central nervous system applications
Construction of replication-defective herpes simplex virus vectors.
No full textAdvances in identification and characterization of gene products responsible for specific diseases of the nervous system have opened opportunities for novel therapies using gene transfer vectors for gene replacement. Herpes simplex virus (HSV)-based vectors are particularly well suited for gene delivery to neurons of the central and peripheral nervous systems. The authors have developed methods to delete HSV-1 IE gene functions and to subsequently introduce foreign genes into the HSV-1 genome using homologous recombination. This unit describes methods for generating cell lines that complement multiple essential gene deletion mutants as well for generating such replication-defective virus recombinants and inserting foreign DNA sequences into replication-defective viral genomes, the last step in preparing a vector. Three support protocols describe methods for preparing virus stocks, titering virus, and preparing viral DNA
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
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
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
Equine herpesvirus type 1 (EHV-1) utilizes microtubules, dynein, and ROCK1 to productively infect cells.
No full textTo initiate infection, equine herpesvirus type 1 (EHV-1) attaches to heparan sulfate on cell surfaces and then interacts with a putative glycoprotein D receptor(s). After attachment, virus entry occurs either by direct fusion of the virus envelope with the plasma membrane or via endocytosis followed by fusion between the virus envelope and an endosomal membrane. Upon fusion, de-enveloped virus particles are deposited into the cytoplasm and travel to the nucleus for viral replication. In this report, we examined the mechanism of EHV-1 intracellular trafficking and investigated the ability of EHV-1 to utilize specific cellular components to efficiently travel to the nucleus post-entry. Using a panel of microtubule-depolymerizing drugs and inhibitors of microtubule motor proteins, we show that EHV-1 infection is dependent on both the integrity of the microtubule network and the minus-end microtubule motor protein, dynein. In addition, we show that EHV-1 actively induces the acetylation of tubulin, a marker of microtubule stabilization, as early as 15 min post-infection. Finally, our data support a role for the cellular kinase, ROCK1, in virus trafficking to the nucleus
Progress in development of herpes simplex virus gene vectors for treatment of rheumatoid arthritis
No full textArthritis is presently incurable and poorly treatable, but there are good grounds for expecting gene therapy to improve matters considerably. Although local ex vivo delivery of anti-arthritic genes to the synovial lining of joints has shown considerable promise, intraarticular gene delivery may be desirable. Herpes simplex virus (HSV) may be a viable vector for in vivo transfer of anti-arthritic genes to joints. HSV has the advantages of high infectivity, large carrying capacity and high titer. The large packaging capacity would permit the inclusion of multiple anti-arthritic genes and necessary regulatory elements. Recombinant vectors produced by this laboratory infect synovial cells efficiently, permitting prolonged expression of transgenes in vitro and in vivo without evidence of cytotoxicity. Further improvements to this vector system include taking advantage of an endogenous HSV `stealthing' gene, ICP47, which interferes with formation of antigen-class I complexes. Inclusion of inducible promoters to appropriately regulate expression of anti-arthritic genes should further improve this system
Generation of herpesvirus entry mediator (HVEM)-restricted herpes simplex virus type 1 mutant viruses: resistance of HVEM-expressing cells and identification of mutations that rescue nectin-1 recognition.
No full textBoth initial infection and cell-to-cell spread by herpes simplex virus type 1 (HSV-1) require the interaction of the viral glycoprotein D (gD) with an entry receptor on the cell surface. The two major HSV entry receptors, herpesvirus entry mediator (HVEM) and nectin-1, mediate infection independently but are coexpressed on a variety of cells. To determine if both receptors are active in these instances, we have established mutant viruses that are selectively impaired for recognition of one or the other receptor. In plaque assays, these viruses showed approximately 1,000-fold selectivity for the matched receptor over the mismatched receptor. Separate assays showed that each virus is impaired for both infection and spread through the mismatched receptor. We tested several human tumor cell lines for susceptibility to these viruses and observed that HT29 colon carcinoma cells are susceptible to infection by nectin-1-restricted virus but are highly resistant to HVEM-restricted virus infection, despite readily detectable HVEM expression on the cell surface. HVEM cDNA isolated from HT29 cells rendered HSV-resistant cells permissive for infection by the HVEM-restricted virus, suggesting that HT29 cells lack a cofactor for HVEM-mediated infection or express an HVEM-specific inhibitory factor. Passaging of HVEM-restricted virus on nectin-1-expressing cells yielded a set of gD missense mutations that each restored functional recognition of nectin-1. These mutations identify residues that likely play a role in shaping the nectin-1 binding site of gD. Our findings illustrate the utility of these receptor-restricted viruses in studying the early events in HSV infection
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