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1.  Production of Bioactive Soluble Interleukin-15 in Complex with Interleukin-15 Receptor Alpha from a Conditionally-Replicating Oncolytic HSV-1 
PLoS ONE  2013;8(11):e81768.
Oncolytic type-1 herpes simplex viruses (oHSVs) lacking the γ134.5 neurovirulence gene are being evaluated for treatment of a variety of malignancies. oHSVs replicate within and directly kill permissive cancer cells. To augment their anti-tumor activity, oHSVs have been engineered to express immunostimulatory molecules, including cytokines, to elicit tumor-specific immune responses. Interleukin-15 (IL-15) holds potential as an immunotherapeutic cytokine because it has been demonstrated to promote both natural killer (NK) cell-mediated and CD8+ T cell-mediated cytotoxicity against cancer cells. The purpose of these studies was to engineer an oHSV producing bioactive IL-15. Two oHSVs were constructed encoding murine (m)IL-15 alone (J100) or with the mIL-15 receptor α (mIL-15Rα, J100D) to determine whether co-expression of these proteins is required for production of bioactive mIL-15 from oHSV. The following were demonstrated: i) both oHSVs retain replication competence and cytotoxicity in permissive tumor cell lines. ii) Enhanced production of mIL-15 was detected in cell lysates of neuro-2a cells following J100D infection as compared to J100 infection, suggesting that mIL-15Rα improved mIL-15 production. iii) Soluble mIL-15 in complex with mIL-15Rα was detected in supernates from J100D-infected, but not J100-infected, neuro-2a, GL261, and CT-2A cells. These cell lines vary in permissiveness to oHSV replication and cytotoxicity, demonstrating soluble mIL-15/IL-15Rα complex production from J100D was independent of direct oHSV effects. iv) The soluble mIL-15/IL-15Rα complex produced by J100D was bioactive, stimulating NK cells to proliferate and reduce the viability of syngeneic GL261 and CT-2A cells. v) J100 and J100D were aneurovirulent inasmuch as no neuropathologic effects were documented following direct inoculation into brains of CBA/J mice at up to 1x107 plaque forming units. The production of mIL-15/mIL-15Rα from multiple tumor lines, as well as the lack of neurovirulence, renders J100D suitable for investigating the combined effects of oHSV and mIL-15/IL-15Rα in various cancer models.
PMCID: PMC3842420  PMID: 24312353
2.  Targeting Pediatric Cancer Stem Cells with Oncolytic Virotherapy 
Pediatric research  2012;71(4 Pt 2):500-510.
Cancer stem cells (CSC), also termed “cancer initiating cells” or “cancer progenitor cells”, which have the ability to self-renew, proliferate, and maintain the neoplastic clone, have recently been discovered in a wide variety of pediatric tumors. These CSC are thought to be responsible for tumorigenesis, tumor maintenance, aggressiveness and recurrence due to inherent resistance to current treatment modalities such as chemotherapy and radiation. Oncolytic virotherapy offers a novel, targeted approach for eradicating pediatric CSC by utilizing mechanisms of cell killing that differ from conventional therapies. Moreover, oncolytic viruses have the ability to target specific features of CSC such as cell surface proteins, transcription factors, and the CSC microenvironment. Through genetic engineering, a wide variety of foreign genes may be expressed by oncolytic viruses to augment the oncolytic effect. We review the current data regarding the ability of several types of oncolytic viruses (herpes simplex virus-1 (HSV-1), adenovirus, reovirus, Seneca Valley virus, vaccinia virus, Newcastle disease virus, myxoma virus, vesicular stomatitis virus) to target and kill both CSC and tumor cells in pediatric tumors. We highlight advantages and limitations of each virus and potential ways next-generation engineered viruses may target resilient CSC.
PMCID: PMC3607376  PMID: 22430386
3.  Pediatric glioma stem cells: biologic strategies for oncolytic HSV virotherapy 
While glioblastoma multiforme (GBM) is the most common adult malignant brain tumor, GBMs in childhood represent less than 10% of pediatric malignant brain tumors and are phenotypically and molecularly distinct from adult GBMs. Similar to adult patients, outcomes for children with high-grade gliomas (HGGs) remain poor. Furthermore, the significant morbidity and mortality yielded by pediatric GBM is compounded by neurotoxicity for the developing brain caused by current therapies. Poor outcomes have been attributed to a subpopulation of chemotherapy and radiotherapy resistant cells, termed “glioma stem cells” (GSCs), “glioma progenitor cells,” or “glioma-initiating cells,” which have the ability to initiate and maintain the tumor and to repopulate the recurring tumor after conventional therapy. Future innovative therapies for pediatric HGG must be able to eradicate these therapy-resistant GSCs. Oncolytic herpes simplex viruses (oHSV), genetically engineered to be safe for normal cells and to express diverse foreign anti-tumor therapeutic genes, have been demonstrated in preclinical studies to infect and kill GSCs and tumor cells equally while sparing normal brain cells. In this review, we discuss the unique aspects of pediatric GSCs, including markers to identify them, the microenvironment they reside in, signaling pathways that regulate them, mechanisms of cellular resistance, and approaches to target GSCs, with a focus on the promising therapeutic, genetically engineered oHSV.
PMCID: PMC3584319  PMID: 23450706
pediatric; glioblastoma; glioma stem cells; cancer stem cells; herpes simplex virus; HSV; oncolytic; virotherapy
4.  Human Herpesviridae Methods of Natural Killer Cell Evasion 
Advances in Virology  2012;2012:359869.
Human herpesviruses cause diseases of considerable morbidity and mortality, ranging from encephalitis to hematologic malignancies. As evidence emerges about the role of innate immunity and natural killer (NK) cells in the control of herpesvirus infection, evidence of viral methods of innate immune evasion grows as well. These methods include interference with the ligands on infected cell surfaces that bind NK cell activating or inhibitory receptors. This paper summarizes the most extensively studied NK cell receptor/ligand pairs and then describes the methods of NK cell evasion used by all eight herpesviruses through these receptors and ligands. Although great strides have been made in elucidating their mechanisms, there is still a disparity between viruses in the amount of knowledge regarding innate immune evasion. Further research of herpesvirus innate immune evasion can provide insight for circumventing viral mechanisms in future therapies.
PMCID: PMC3399383  PMID: 22829821
5.  Δγ134.5 Herpes Simplex Viruses Encoding Human Cytomegalovirus IRS1 or TRS1 Induce Interferon Regulatory Factor 3 Phosphorylation and an Interferon-Stimulated Gene Response 
Journal of Virology  2012;86(1):610-614.
The chimeric herpes simplex viruses (HSV) are Δγ134.5 vectors encoding the human cytomegalovirus (HCMV) IRS1 or TRS1 genes. They are capable of late viral protein synthesis and are superior to Δγ134.5 HSVs in oncolytic activity. The interferon (IFN) response limits efficient HSV gene expression and replication. HCMV TRS1 and IRS1 restore one γ134.5 gene function: evasion of IFN-inducible protein kinase R, allowing late viral protein synthesis. Here we show that, unlike wild-type HSV, the chimeric HSV do not restore another γ134.5 function, the suppression of early IFN signaling mediated by IFN regulatory factor 3 (IRF3).
PMCID: PMC3255867  PMID: 22072777
6.  Hypoxia Moderates γ134.5-Deleted Herpes Simplex Virus Oncolytic Activity in Human Glioma Xenoline Primary Cultures1 
Translational Oncology  2012;5(3):200-207.
Hypoxia plays a critical role in the tumor microenvironment of high-grade gliomas by promoting the glioma stem cell (GSC)-like phenotype, which displays resistance to standard therapies. We tested three glioblastoma multiforme xenograft lines (xenolines) against γ134.5-deleted recombinant oncolytic herpes simplex virus (oHSV) C101 under 1% (hypoxia) and 20.8% (normoxia) oxygen tension for effects on oHSV infectivity, replication, and cytotoxicity in all tumor cells and CD133+ GSCs. Expression levels of CD133, a putative GSC marker, and CD111 (nectin-1), an adhesion molecule that is the most efficient method for HSV entry, increased significantly under hypoxia in all three xenolines. Despite increased CD111 expression under hypoxic conditions, oHSV infectivity, cytotoxicity and viral recovery were not improved or were diminished in all three xenolines under hypoxia. In contrast, wild-type HSV-1 equally infected xenoline cells in normoxia and hypoxia, suggesting that the 34.5 mutation plays a role in the decreased C101 infectivity in hypoxia. Importantly, CD133+ cells were not more resistant to oHSV than CD133- tumor cells regardless of oxygen tension. Furthermore, CD133 expression decreased as viral dose increased in two of the xenolines suggesting that up-regulation of CD133 in hypoxia was not the cause of reduced viral efficacy. Our findings that oHSV infectivity and cytotoxicity were diminished under hypoxia in several GBM xenolines likely have important implications for clinical applications of oHSV therapies, especially considering the vital role of hypoxia in the microenvironment of GBM tumors.
PMCID: PMC3384274  PMID: 22741039
7.  Strategies For The Rapid Construction Of Conditionally-Replicating HSV-1 Vectors Expressing Foreign Genes As Anti-Cancer Therapeutic Agents 
Molecular pharmaceutics  2010;8(1):44-49.
Conditionally replication-competent Herpes Simplex Virus Type 1 (HSV-1) vectors expressing foreign genes have been developed as experimental therapeutic agents. Traditional methods of virus construction, including growth selection based on thymidine kinase gene expression, and color selection based on a reporter gene expression are often time-consuming and relatively inefficient. This review summarizes the various strategies developed in recent years for the rapid and efficient construction of novel conditionally replication-competent mutant HSV expressing multiple foreign genes. Additionally, two new modifications of existing strategies, which have not been previously reported, are discussed.
PMCID: PMC3034828  PMID: 21142023
HSV-1; conditionally-replicating; virus construction; Δγ134.5; UL39; co-infection; bridge plasmid; marker rescue
8.  Herpesvirus Vectors for Therapy of Brain Tumors 
The Open Virology Journal  2010;4:103-108.
Genetically modified, conditionally-replicating Herpes Simplex Virus Type 1 (HSV-1) vectors for the treatment of malignant glioma have provided encouraging results in the handful of Phase I and Phase II clinical trials conducted to date. In recent years, a number of new strategies have been developed to improve anti-tumor activity of these attenuated vectors, through either introduction of foreign gene inserts to enhance tumor killing through a variety of mechanisms, or through combination with existing treatment regimens, including radiation and/or chemotherapeutics. Another promising new approach has been the engineering of novel oncolytic HSV vectors that retain wildtype replication, but are targeted to tumor cells through a variety of mechanisms. This review summarizes the latest advances in herpesvirus-mediated oncolytic therapies from both preclinical results and clinical trials with oncolytic HSV vectors in patients, and their implication for design of future trials.
PMCID: PMC2930154  PMID: 20811578
Oncolytic HSV therapy; brain tumor; G207; HSV1716; M032; chimeric HSV; C134; R5141; rQNestin34.5; phase I trial; glioma; γ1(34.5).
9.  Spontaneous and Engineered Compensatory HSV Mutants that Counteract the Host Antiviral PKR Response 
Viruses  2009;1(3):510-522.
A virulent recombinant HSV lacking the diploid γ134.5 gene (Δγ134.5) have been investigated over the last two decades both for anti-tumor therapy and as vaccine vectors. The first generation vectors, while safe, are incapable of sustained replication in the majority of treated patients. An interferon inducible host antiviral kinase, protein kinase R (PKR), limits late viral protein synthesis and replication of Δγ134.5 viruses. This review describes the development of new Δγ134.5 vectors, through serial passage selection and direct viral genome engineering, which demonstrate selective PKR evasion in targeted cells and improved viral replication without restoring neurovirulence.
PMCID: PMC3185541  PMID: 21994558
PKR; oncolytic HSV; Δγ134.5
10.  Serial Passage through Human Glioma Xenografts Selects for a Δγ134.5 Herpes Simplex Virus Type 1 Mutant That Exhibits Decreased Neurotoxicity and Prolongs Survival of Mice with Experimental Brain Tumors 
Journal of Virology  2006;80(15):7308-7315.
Previous studies have described in vitro serial passage of a Δγ134.5 herpes simplex virus type 1 (HSV-1) strain in SK-N-SH neuroblastoma cells and selection of mutants that have acquired the ability to infect and replicate in this previously nonpermissive cell line. Here we describe the selection of a mutant HSV-1 strain by in vivo serial passage, which prolongs survival in two separate experimental murine brain tumor models. Two conditionally replication-competent Δγ134.5 viruses, M002, which expresses murine interleukin-12, and its parent virus, R3659, were serially passaged within human malignant glioma D54-MG cell lines in vitro or flank tumor xenografts in vivo. The major findings are (i) viruses passaged in vivo demonstrate decreased neurovirulence, whereas those passaged in vitro demonstrate a partial recovery of the neurovirulence associated with HSV-1; and (ii) vvD54-M002, the virus selected after in vivo serial passage of M002 in D54-MG tumors, improves survival in two independent murine brain tumor models compared to the parent (unpassaged) M002. Additionally, in vitro-passaged, but not in vivo-passaged, M002 displayed changes in the protein synthesis profile in previously nonpermissive cell lines, as well as early US11 transcription. Thus, a mutant HSV-1 strain expressing a foreign gene can be selected for enhanced antitumor efficacy via in vivo serial passage within flank D54-MG tumor xenografts. The enhanced antitumor efficacy of vvD54-M002 is not due to restoration of protein synthesis or early US11 expression. This finding emphasizes the contribution of the in vivo tumor environment for selecting novel oncolytic HSV specifically adapted for tumor cell destruction in vivo.
PMCID: PMC1563698  PMID: 16840311
11.  Human Cytomegalovirus TRS1 and IRS1 Gene Products Block the Double-Stranded-RNA-Activated Host Protein Shutoff Response Induced by Herpes Simplex Virus Type 1 Infection 
Journal of Virology  2005;79(14):8707-8715.
Human cytomegalovirus (HCMV) attachment and entry stimulates the expression of cellular interferon-inducible genes, many of which target important cellular functions necessary for viral replication. Double-stranded RNA-dependent host protein kinase (PKR) is an interferon-inducible gene product that limits viral replication by inhibiting protein translation in the infected cell. It was anticipated that HCMV encodes gene products that facilitate the evasion of this PKR-mediated antiviral response. Using a Δγ134.5 herpes simplex virus type 1 (HSV-1) recombinant that triggers PKR-mediated protein synthesis shutoff, experiments identified an HCMV gene product expressed in the initial hours of infection that allows continued protein synthesis in the infected cell. Recombinant HSV-1 viruses expressing either the HCMV TRS1 or IRS1 protein demonstrate that either of these HCMV gene products allows the Δγ134.5 recombinant viruses to evade PKR-mediated protein shutoff and maintain late viral protein synthesis.
PMCID: PMC1168740  PMID: 15994764
12.  The Herpes Simplex Virus Type 1 US11 Protein Interacts with Protein Kinase R in Infected Cells and Requires a 30-Amino-Acid Sequence Adjacent to a Kinase Substrate Domain 
Journal of Virology  2002;76(5):2029-2035.
The herpes simplex virus type 1 γ134.5 gene product precludes the host-mediated protein shutoff response induced by activated protein kinase R (PKR). Earlier studies demonstrated that recombinant viruses lacking the γ134.5 gene (Δγ134.5) developed secondary mutations that allowed earlier US11 expression and enabled continued protein synthesis. Further, in vitro studies demonstrated that a recombinant expressed US11 protein binds PKR, blocks the phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF-2α) by activated PKR, and, if provided prior to PKR activation, precluded PKR autophosphorylation. The present study furthers the hypothesis that early US11 production precludes PKR-mediated host protein shutoff by demonstrating that (i) US11 and PKR interact in the context of viral infection, (ii) this interaction is RNA dependent and requires a 30-amino-acid domain (amino acids 91 to 121) in the carboxyl domain of the US11 protein, (iii) the proteins biochemically colocalize in the S100 ribosomal fraction, and (iv) there is a PKR substrate domain immediately adjacent to the binding domain. The results suggest that the US11 interaction with PKR at the ribosome is RNA dependent and that the US11 protein contains a substrate domain with homology to eIF-2α in close proximity to an essential binding domain.
PMCID: PMC135940  PMID: 11836380
13.  Second-Site Mutation Outside of the US10-12 Domain of Δγ134.5 Herpes Simplex Virus 1 Recombinant Blocks the Shutoff of Protein Synthesis Induced by Activated Protein Kinase R and Partially Restores Neurovirulence 
Journal of Virology  2002;76(3):942-949.
Earlier studies have shown that herpes simplex virus type 1 (HSV-1) activated protein kinase R (PKR) but that the product of the product of the γ134.5 gene binds and redirects the host phosphatase 1 to dephosphorylate the α subunit of eukaryotic translation initiation factor 2 (eIF-2α). In consequence, the γ134.5 gene product averts the threatened shutoff of protein synthesis caused by activated PKR. Serial passages of Δγ134.5 mutants in human cells led to isolation of two classes of second-site, compensatory mutants. The first, reported earlier, resulted from the juxtaposition of the α promoter of the US12 gene to the coding sequence of the US11 gene. The mutant blocks the phosphorylation of eIF-2α but does not restore the virulence phenotype of the wild-type virus. We report another class of second-site, compensatory mutants that do not map to the US10-12 domain of the HSV-1 genome. All mutants in this series exhibit sustained late protein synthesis, higher yields in human cells, and reduced phosphorylation of PKR that appears to be phosphatase dependent. Specific dephosphorylation of eIF-2α was not demonstrable. At least one mutant in this series exhibited a partial restoration of the virulence phenotype characteristic of the wild-type virus phenotype. The results suggest that the second-site mutations reflect activation of fossilized functions designed to block the interferon response pathways in cells infected with the progenitor of present HSV.
PMCID: PMC135782  PMID: 11773369
14.  The Herpes Simplex Virus US11 Protein Effectively Compensates for the γ134.5 Gene if Present before Activation of Protein Kinase R by Precluding Its Phosphorylation and That of the α Subunit of Eukaryotic Translation Initiation Factor 2 
Journal of Virology  1998;72(11):8620-8626.
In herpes simplex virus-infected cells, viral γ134.5 protein blocks the shutoff of protein synthesis by activated protein kinase R (PKR) by directing the protein phosphatase 1α to dephosphorylate the α subunit of eukaryotic translation initiation factor 2 (eIF-2α). The amino acid sequence of the γ134.5 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 α47 promoter next to US11, a γ2 (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 γ134.5 gene. In these cells, PKR is activated but eIF-2α is not phosphorylated, and the phosphatase is not redirected to dephosphorylate eIF-2α. 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-2α 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-2α, 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 γ134.5 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 γ2 gene because, like many viral proteins, it has multiple functions.
PMCID: PMC110273  PMID: 9765401
15.  The Second-Site Mutation in the Herpes Simplex Virus Recombinants Lacking the γ134.5 Genes Precludes Shutoff of Protein Synthesis by Blocking the Phosphorylation of eIF-2α 
Journal of Virology  1998;72(9):7005-7011.
In cells infected with the herpes simplex virus 1 (HSV-1) recombinant R3616 lacking both copies of the γ134.5 gene, the double-stranded protein kinase R (PKR) is activated, eIF-2α 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 γ134.5 gene, infected-cell protein 34.5 (ICP34.5), binds protein phosphatase 1α and redirects it to dephosphorylate eIF-2α, thus enabling sustained protein synthesis. Serial passage in human cells of a mutant lacking the γ134.5 gene yields second-site, compensatory mutants lacking various domains of the α47 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 γ134.5, US8, -9, -10, and -11, and α47 (US12) genes; and R5104, derived from R5103 and carrying a chimeric DNA fragment containing the US10 gene and the promoter of the α47 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-2α 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-2α in vitro, whereas lysates of mock-infected cells or cells infected with HSV-1(F) or R5104 did not phosphorylate eIF-2α. We conclude that in contrast to wild-type virus-infected cells, which preclude the shutoff of protein synthesis by causing rapid dephosphorylation of eIF-2α, in cells infected with γ134.5− virus carrying the compensatory mutation, eIF-2α 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.
PMCID: PMC109920  PMID: 9696792

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