1,721,011 research outputs found
Deletion of the S component inverted repeat sequence c’ and nonessential genes US1 through US5 from the herpes simplex virus type 1 genome substantially impairs productive viral infection in cell culture and pathogenesis in the rat central nervous system
No full textNovel mutations in gB and gH circumvent the requirement for known gD receptors in herpes simplex virus 1 entry and cell-to-cell spread
No full textBoth entry and cell-to-cell spread of herpes simplex virus (HSV) involve a cascade of cooperative interactions among the essential glycoproteins D, B, and H/L (gD, gB, and gH/gL, respectively) initiated by the binding of gD to a cognate HSV entry receptor. We previously reported that a variant (D285N/A549T) of glycoprotein B (gB:NT) enabled primary virus entry into cells that were devoid of typical HSV entry receptors. Here, we compared the activities of the gB:NT variant with those of a newly selected variant of glycoprotein H (gH:KV) and a frequently coselected gB variant (gB:S668N). In combination, gH:KV and gB:S668N enabled primary virus entry into cells that lacked established HSV entry receptors as efficiently as did gB:NT, but separately, each variant enabled only limited entry. Remarkably, gH:KV uniquely facilitated secondary virus spread between cells that lacked canonical entry receptors. Transient expression of the four essential entry glycoproteins revealed that gH:KV, but not gB:NT, induced fusion between cells lacking the standard receptors. Because the involvement of gD remained essential for virus spread and cell fusion, we propose that gH:KV mimics a transition state of gH that responds efficiently to weak signals from gD to reach the active state. Computational modeling of the structures of wild-type gH and gH:KV revealed relatively subtle differences that may have accounted for our experimental findings. Our study shows that (i) the dependence of HSV-1 entry and spread on specific gD receptors can be reduced by sequence changes in the downstream effectors gB and gH, and (ii) the relative roles of gB and gH are different in entry and spread
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
Detection of HSV-1 latency associated transcript expression in trigeminal ganglion by in situ reverse transcriptase PCR
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Heparan sulfate proteoglycan binding by herpes simplex virus type 1 glycoproteins B and C, which differ in their contributions to virus attachment, penetration, and cell-to-cell spread
No full textHerpes simplex virus type 1 (HSV-1) mutants defective for envelope glycoprotein C (gC) and gB are highly impaired in the ability to attach to cell surface heparan sulfate (HS) moieties of proteoglycans, the initial virus receptor. Here we report studies aimed at defining the HS binding element of HSV-1 (strain KOS) gB and determining whether this structure is functionally independent of gB's role in extracellular virus penetration or intercellular virus spread. A mutant form of gB deleted for a putative HS binding lysine-rich (pK) sequence (residues 68 to 76) was transiently expressed in Vero cells and shown to be processed normally, leading to exposure on the cell surface. Solubilized gBpK- also had substantially lower affinity for heparin-acrylic beads than did wild-type gB, confirming that the HS binding domain had been inactivated. The gBpK- gene was used to rescue a KOS gB null mutant virus to produce the replication-competent mutant KgBpK- . Compared with wild-type virus, KgBpK- showed reduced binding to mouse L cells (ca. 20%), while a gC null mutant virus in which the gC coding sequence was replaced by the lacZ gene (KCZ) was substantially more impaired (ca. 65%- reduced binding), indicating that the contribution of gC to HS binding was greater than that of gB. The effect of combining both mutations into a single virus (KgBpK-gC-) was additive (ca. 80%-reduced binding to HS) and displayed a binding activity similar to that observed for KOS virus attachment to sog9 cells, a glycosaminoglycan-deficient L-cell line. Cell-adsorbed individual and double HS mutant viruses exhibited a lower rate of virus entry following attachment, suggesting that HS binding plays a role in the process of virus penetration. Moreover, the KgBpK- mutant virus produced small plaques on Vero cells in the presence of neutralizing antibody where plaque formation depended on cell-to-cell virus spread. These studies permitted the following conclusions: (i) the pK sequence is not essential for gB processing or function in virus infection, (ii) the lysine-rich sequence of gB is responsible for HS binding, and (iii) binding to HS is cooperatively linked to the process of efficient virus entry and lateral spread but is not absolutely required for virus infectivity
Ectopic matrix metalloproteinase-9 expression in human brain tumor cells enhances oncolytic HSV vector infection.
No full textOncolytic herpes simplex virus (oHSV) vectors have shown promise in the treatment of patients with recurrent brain tumors although few complete responses have accrued. Impediments to effective therapy include limited vector distribution on delivery, a consequence of injected virion particle trapping in the tumor extracellular matrix (ECM). To enhance virus delivery and spread, we investigated the use of the matrix metalloproteinase-9 (MMP-9) as a means to degrade collagen type IV, a major component of the ECM and basement membranes of gliomas that is absent in normal brain tissue. SK-N-AS neuroblastoma cells were transduced for constitutive, elevated expression of MMP-9, which did not enhance tumor cell migration in vitro or tumor progression in a murine xenograft brain tumor model. MMP-9 expression improved the distribution and infection of oHSV vectors in spheroid model in vitro. Furthermore, MMP9 induced a vector infection over larger areas of brain tumors in vivo. These results suggest that vector delivery and distribution in vivo can be improved by compromising the ECM, potentially enhancing oncolytic efficacy
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
Cytokine therapy in immune-mediated demyelinating diseases of the central nervous system: a novel gene therapy approach
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