ABSTRACT
Pathophysiological hypoxia, which fosters the glioma stem-like cell (GSC) phenotype, is present in high-grade gliomas and has been linked to tumor development, invasiveness and resistance to chemotherapy and radiation. Oncolytic virotherapy with engineered herpes simplex virus-1 (HSV-1) is a promising therapy for glioblastoma; however, the efficacy of γ(1)34.5-deleted HSVs, which have been used in clinical trials, was diminished in hypoxia. We investigated the ability of a chimeric human cytolomegalovirus (HCMV)/HSV-1 virus, which expresses the human CMV protein kinase R evasion gene IRS1 and is in preparation for clinical trials, to infect and kill adult and pediatric patient-derived glioblastoma xenografts in hypoxia and normoxia. Infectivity, cytotoxicity and viral recovery were significantly greater with the chimeric virus compared with the γ(1)34.5-deleted virus, regardless of oxygen tension. The chimeric virus infected and killed CD133+ GSCs similarly to wild-type HSV-1. Increased activation of mitogen-activated protein kinase p38 and its substrate heat-shock protein 27 (Hsp27) was seen after viral infection in normoxia compared with hypoxia. Hsp27 knockdown or p38 inhibition reduced virus recovery, indicating that the p38 pathway has a role in the reduced efficacy of the γ(1)34.5-deleted virus in hypoxia. Taken together, these findings demonstrate that chimeric HCMV/HSV-1 efficiently targets both CD133+ GSCs and glioma cells in hypoxia.
Subject(s)
Cytomegalovirus/metabolism , Glioblastoma/therapy , Herpesvirus 1, Human/genetics , Oncolytic Virotherapy , Protein Kinases/metabolism , Viral Proteins/metabolism , Animals , Cell Hypoxia , Cell Line, Tumor , Cytomegalovirus/genetics , Glioblastoma/metabolism , HSP27 Heat-Shock Proteins/metabolism , Humans , Mice, Nude , Organisms, Genetically Modified , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
Limited expression and distribution of nectin-1, the major herpes simplex virus (HSV) type-1 entry-receptor, within tumors has been proposed as an impediment to oncolytic HSV (oHSV) therapy. To determine whether resistance to oHSVs in malignant peripheral nerve sheath tumors (MPNSTs) was explained by this hypothesis, nectin-1 expression and oHSV viral yields were assessed in a panel of MPNST cell lines using γ134.5-attenuated (Δγ134.5) oHSVs and a γ134.5 wild-type (wt) virus for comparison. Although there was a correlation between nectin-1 levels and viral yields with the wt virus (R=0.75, P =0.03), there was no correlation for Δγ134.5 viruses (G207, R7020 or C101) and a modest trend for the second-generation oHSV C134 (R=0.62, P=0.10). Nectin-1 overexpression in resistant MPNST cell lines did not improve Δγ134.5 oHSV output. While multistep replication assays showed that nectin-1 overexpression improved Δγ134.5 oHSV cell-to-cell spread, it did not confer a sensitive phenotype to resistant cells. Finally, oHSV yields were not improved with increased nectin-1 in vivo. We conclude that nectin-1 expression is not the primary obstacle of productive infection for Δγ134.5 oHSVs in MPNST cell lines. In contrast, viruses that are competent in their ability to counter the antiviral response may derive benefit with higher nectin-1 expression.
Subject(s)
Cell Adhesion Molecules/metabolism , Nerve Sheath Neoplasms/metabolism , Oncolytic Viruses/physiology , Receptors, Virus/metabolism , Simplexvirus/physiology , Animals , Cell Line, Tumor , Chlorocebus aethiops , Cricetulus , Humans , Mice , Nectins , Nerve Sheath Neoplasms/virology , Oncolytic Virotherapy , Oncolytic Viruses/metabolismABSTRACT
Oncolytic herpes simplex virus (HSV)-1 gamma(1)34.5-deletion mutants (Deltagamma(1)34.5 HSV) are promising agents for tumor therapy. The attenuating mutation renders the virus aneurovirulent but also limits late viral protein synthesis and efficient replication in many tumors. We tested whether one function of gamma(1)34.5 gene, which mediates late viral protein synthesis through host protein kinase R (PKR) antiviral response evasion, could be restored, without restoring the neurovirulence. We have previously reported the construction of two chimeric Deltagamma(1)34.5 HSV vectors (chimeric HSV), C130 and C134, which express the human cytomegalovirus (HCMV) PKR-evasion genes TRS1 and IRS1, respectively. We now demonstrate the following. The HCMV/HSV-1 chimeric viruses (i) maintain late viral protein synthesis in the human malignant glioma cells tested (D54-MG, U87-MG and U251-MG); (ii) replicate to higher titers than Deltagamma(1)34.5 HSV in malignant glioma cells in vitro and in vivo; (iii) are aneurovirulent; and (iv) are superior to other Deltagamma(1)34.5 HSV with both improved reduction of tumor volumes in vivo, and improved survival in two experimental murine brain tumor models. These findings demonstrate that transfer of HCMV IRS1 or TRS1 gene into Deltagamma(1)34.5 HSV significantly improves replication in malignant gliomas without restoring wild-type neurovirulence, resulting in enhanced tumor reduction and prolonged survival.
Subject(s)
Brain Neoplasms/therapy , Cytomegalovirus/genetics , Genetic Therapy/methods , Glioblastoma/therapy , Herpesvirus 1, Human/genetics , Oncolytic Virotherapy/methods , Animals , Brain Neoplasms/pathology , Cell Line, Tumor , Chimera , Genetic Engineering , Glioblastoma/pathology , Glioma , Mice , Mice, SCID , Neoplasm Transplantation , Neuroblastoma , Neurons/pathology , Neurons/virology , Oncolytic Viruses/genetics , Oncolytic Viruses/physiology , Transplantation, Heterologous , Virus ReplicationABSTRACT
In herpes simplex virus-infected cells, viral gamma134.5 protein blocks the shutoff of protein synthesis by activated protein kinase R (PKR) by directing the protein phosphatase 1alpha to dephosphorylate the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2alpha). The amino acid sequence of the gamma134.5 protein which interacts with the phosphatase has high homology to a domain of the eukaryotic protein GADD34. A class of compensatory mutants characterized by a deletion which results in the juxtaposition of the alpha47 promoter next to US11, a gamma2 (late) gene in wild-type virus-infected cells, has been described. In cells infected with these mutants, protein synthesis continues even in the absence of the gamma134.5 gene. In these cells, PKR is activated but eIF-2alpha is not phosphorylated, and the phosphatase is not redirected to dephosphorylate eIF-2alpha. We report the following: (i) in cells infected with these mutants, US11 protein was made early in infection; (ii) US11 protein bound PKR and was phosphorylated; (iii) in in vitro assays, US11 blocked the phosphorylation of eIF-2alpha by PKR activated by poly(I-C); and (iv) US11 was more effective if present in the reaction mixture during the activation of PKR than if added after PKR had been activated by poly(I-C). We conclude the following: (i) in cells infected with the compensatory mutants, US11 made early in infection binds to PKR and precludes the phosphorylation of eIF-2alpha, whereas US11 driven by its natural promoter and expressed late in infection is ineffective; and (ii) activation of PKR by double-stranded RNA is a common impediment countered by most viruses by different mechanisms. The gamma134.5 gene is not highly conserved among herpesviruses. A likely scenario is that acquisition by a progenitor of herpes simplex virus of a portion of the cellular GADD34 gene resulted in a more potent and reliable means of curbing the effects of activated PKR. US11 was retained as a gamma2 gene because, like many viral proteins, it has multiple functions.
Subject(s)
Eukaryotic Initiation Factor-2/metabolism , Genes, Viral , Herpesvirus 1, Human/genetics , Herpesvirus 1, Human/metabolism , RNA-Binding Proteins/metabolism , Viral Proteins/genetics , Viral Proteins/metabolism , eIF-2 Kinase/metabolism , Animals , Cell Line , Chlorocebus aethiops , Enzyme Activation/drug effects , HeLa Cells , Humans , Mutation , Phosphorylation , Poly I-C/pharmacology , RNA-Binding Proteins/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Vero CellsABSTRACT
In cells infected with the herpes simplex virus 1 (HSV-1) recombinant R3616 lacking both copies of the gamma134.5 gene, the double-stranded protein kinase R (PKR) is activated, eIF-2alpha is phosphorylated, and protein synthesis is shut off. Although PKR is also activated in cells infected with the wild-type virus, the product of the gamma134.5 gene, infected-cell protein 34.5 (ICP34.5), binds protein phosphatase 1alpha and redirects it to dephosphorylate eIF-2alpha, thus enabling sustained protein synthesis. Serial passage in human cells of a mutant lacking the gamma134.5 gene yields second-site, compensatory mutants lacking various domains of the alpha47 gene situated next to the US11 gene (I. Mohr and Y. Gluzman, EMBO J. 15:4759-4766, 1996). We report the construction of two recombinant viruses: R5103, lacking the gamma134. 5, US8, -9, -10, and -11, and alpha47 (US12) genes; and R5104, derived from R5103 and carrying a chimeric DNA fragment containing the US10 gene and the promoter of the alpha47 gene fused to the coding domain of the US11 gene. R5104 exhibited a protein synthesis profile similar to that of wild-type virus, whereas protein synthesis was shut off in cells infected with R5103 virus. Studies on the wild-type parent and mutant viruses showed the following: (i) PKR was activated in cells infected with parent or mutant virus but not in mock-infected cells, consistent with earlier studies; (ii) lysates of R3616, R5103, and R5104 virus-infected cells lacked the phosphatase activity specific for eIF-2alpha characteristic of wild-type virus-infected cells; and (iii) lysates of R3616 and R5103, which lacked the second-site compensatory mutation, contained an activity which phosphorylated eIF-2alpha in vitro, whereas lysates of mock-infected cells or cells infected with HSV-1(F) or R5104 did not phosphorylate eIF-2alpha. We conclude that in contrast to wild-type virus-infected cells, which preclude the shutoff of protein synthesis by causing rapid dephosphorylation of eIF-2alpha, in cells infected with gamma134.5(-) virus carrying the compensatory mutation, eIF-2alpha is not phosphorylated. The activity made apparent by the second-site mutation may represent a more ancient mechanism evolved to preclude the shutoff of protein synthesis.
Subject(s)
Capsid Proteins , Eukaryotic Initiation Factor-2/metabolism , Herpesvirus 1, Human/metabolism , Mutation , Viral Proteins/physiology , Animals , Capsid/biosynthesis , Capsid/genetics , Cell Differentiation , Chlorocebus aethiops , Gene Deletion , Genes, Viral , Genotype , HeLa Cells , Herpesvirus 1, Human/genetics , Humans , Mutagenesis, Insertional , Phosphoproteins/biosynthesis , Phosphoproteins/genetics , Phosphoric Monoester Hydrolases/metabolism , Phosphorylation , Protein Kinases/metabolism , RNA-Binding Proteins/biosynthesis , RNA-Binding Proteins/genetics , Recombination, Genetic , Vero Cells , Viral Proteins/biosynthesis , Viral Proteins/geneticsABSTRACT
The character of diseases caused by alphaherpesviruses has changed over the last decade. The severity of disease and the frequency of acyclovir resistance has increased with the increase in the number of immunocompromised patients. Compounding the trend towards more virulent herpes disease is the current emphasis towards outpatient management of many diseases. Much of the current antiviral research focuses on providing drugs with (i) improved oral bioavailability and pharmacokinetics which permit less frequent oral or topical dosing for suppressive treatment of herpes simplex virus (HSV) infections, (ii) different mechanisms of action for synergic effects in treating resistant HSV infections in the immunocompromised host and (iii) improved efficacy. Future antiviral agents will probably target enzymes or viral factors essential for infection or will inhibit other steps in the viral infection cycle, such as viral entry, protein synthesis or capsid assembly. Medications that augment the immune response constitute another pathway for combating herpes viral infections. Many of the newer experimental agents target essential processes unique to herpesvirus replication and, therefore, potentially have high selectivity.