Development of a novel, high-efficacy oncolytic herpes simplex virus type 1 platform equipped with two distinct retargeting modalities.

To retarget oncolytic herpes simplex virus (oHSV) to cancer-specific antigens, we designed a novel, double-retargeted oHSV platform that uses single-chain antibodies (scFvs) incorporated into both glycoprotein H and a bispecific adapter expressed from the viral genome to mediate infection predominantly via tumor-associated antigens. Successful retargeting was achieved using a nectin-1-detargeted HSV that remains capable of interacting with herpesvirus entry mediator (HVEM), the second canonical HSV entry receptor, and is, therefore, recognized by the adapter consisting of the virus-binding N-terminal 82 residues of HVEM fused to the target-specific scFv. We tested both an epithelial cell adhesion molecule (EpCAM)- and a human epidermal growth factor receptor 2-specific scFv separately and together to target cells expressing one, the other, or both receptors. Our results show not only dose-dependent, target receptor-specific infection in vitro, but also enhanced virus spread compared with single-retargeted virus. In addition, we observed effective infection and spreading of the EpCAM double-retargeted virus in vivo. Remarkably, a single intravenous dose of the EpCAM-specific virus eliminated all detectable tumors in a subcutaneous xenograft model, and the same intravenous dose seemed to be harmless in immunocompetent FVB/N mice. Our findings suggest that our double-retargeted oHSV platform can provide a potent, versatile, and systemically deliverable class of anti-cancer therapeutics that specifically target cancer cells while ensuring safety.

Three-Dimensional Culture of Salivary Gland Stem Cell in Orthotropic Decellularized Extracellular Matrix Hydrogels.

Radiotherapy in patients with cancer can kill cancer cells but also damage normal cells or tissues. During the treatment of patients with head and neck cancer or thyroid cancer, hyposalivation is a representative chronic side effect of radio-damaged salivary glands (SGs). The major symptom of hyposalivation is mouth dryness, resulting in several subsequent long-term complications. No effective therapeutic approaches have been developed to manage this symptom. In this study, we developed the first rat SG tissue-derived decellularized extracellular matrix hydrogel (DSGM-hydrogel) as a functional orthotropic bioscaffold for future efficient SG stem cell therapy. DSGM-hydrogels were characterized by rheological or biochemical analyses, and rat SG stem/progenitor cells (rSGSCs) were then subjected to three-dimensional culture in the DSGM-hydrogels. Interestingly, DSGM-hydrogel-embedded rSGSCs survived and expressed SG functional differentiation marker of amylase IA and increased enzyme activity of α-amylase in protein level, whereas they showed reduced levels of adult ductal stem/progenitor markers, including c-Kit, c-Met, and CD44. Furthermore, the expression levels of basic epithelial tight junction markers were recovered to levels similar to those naked SG tissues after culture in DSGM-hydrogels in transcription level. Therefore, our findings suggested that the DSGM-hydrogels could provide an appropriate microenvironment for stem/progenitor cell survival and a source of SG cytodifferentiation. This approach could be an applicable method to SG stem cell research as a potential source for an organoid and for clinical regenerative reagents to manage radio-damaged SGs in vivo. Impact Statement In this study, we established the first rat salivary gland (SG) tissue-derived decellularized extracellular matrix hydrogel (DSGM-hydrogel) and assessed the role of this hydrogel as a functional orthotropic bioscaffold. Our findings provide important insights into the applications of the DSGM-hydrogel as a biocompatible matrix for regenerative therapy of radio-damaged SGs.

Detection of Methylated SEPT9 in Korean Colorectal Cancer Patients: Comparison with Previous Studies.

BACKGROUND: This study aimed to investigate the detection of methylated Septin 9 (mSEPT9) in Korean patients with colorectal cancer (CRC) and compare the results with those of previous studies. METHODS: A total of 127 plasma samples (111 patients with untreated CRC, 5 patients with adenomas, and 11 CRC patients treated with concurrent chemoradiotherapy before surgery) were collected. mSEPT9 was measured qualitatively with the Abbott RealTime ms9 Colorectal Cancer Assay. RESULTS: mSEPT9 was detected in 44 of 111 (39.6%) cases of untreated CRC but was not detected in the adenoma cases. The difference in the sensitivity of mSEPT9 among patients with adenomas and those with each stage of untreated CRC was statistically significant (Dukes' staging, p = 0.002 and TNM staging, p = 0.008). The sensitivity of mSEPT9 for each of the stages (I - IV) of untreated CRC patients were 20.7%, 54.1%, 36.6%, and 75.0%, respectively. The positive mSEPT9 results in untreated CRC patients reverted to negative in 19 of 21 patients (90.5%) after treatment. CONCLUSIONS: Compared to previous studies, the overall sensitivity of mSEPT9 was lower, but similar patterns were found in the sensitivities for each stage. Additionally, mSEPT9 appeared to have potential as a monitoring tool for CRC.

Fbxw7ß is an inducing mediator of dexamethasone-induced skeletal muscle atrophy in vivo with the axis of Fbxw7ß-myogenin-atrogenes.

Muscle atrophy is induced by several pathways, e.g., it can be attributed to inherited cachectic symptoms, genetic disorders, sarcopenia, or chronic side effects of treatments. However, the underlying regulatory mechanisms that contribute to muscle atrophy have not been fully elucidated. In this study, we evaluated the role of Fbxw7ß, an ubiquitin E3 ligase, in a dexamethasone-induced muscle atrophy model. In this model, endogenous Fbxw7ß was up-regulated; furthermore, the Fbxw7ß-myogenin-atrogene axis was upregulated, supporting our previous results linking Fbxw7ß to muscle atrophy in vitro. Also, muscle atrophy was associated with the Fbxw7ß-myogenin-atrogene axis and the down-regulation of Dach2, a repressor of myogenin. Taken together, these results suggest that the ubiquitin E3 ligase Fbxw7ß and the Fbxw7ß-myogenin-atrogene axis have important roles in a dexamethasone-induced muscle atrophy model in vivo and in vitro. Additionally, the Fbxw7ß-Dach2-myogenin-atrogene axis is a potential mechanism underlying muscle atrophy in cases of abnormal Fbxw7ß expression-induced muscle atrophy or myogenic degenerative disease.

MicroRNA miR-4779 suppresses tumor growth by inducing apoptosis and cell cycle arrest through direct targeting of PAK2 and CCND3.

Depending on the function of their target genes, microRNAs (miRNAs) act as either tumor suppressors or oncogenes. Therefore, miRNAs represent a novel therapeutic strategy for prevention and management of cancer by targeting of onco-miRNAs or mimicking of tumor suppressor miRNAs. Herein, we identified novel tumor suppressor miRNAs and investigated their molecular mechanisms. To identify novel tumor suppressor miRNAs, we used 532 human miRNA mimic libraries and measured cell viability using MTS assays. The function of miR-4779 was then analyzed using cell cycle analyses and apoptosis, colony forming, and soft agar assays. Target genes of miR-4779 were predicted using TargetScan and miRDB databases and were confirmed using luciferase assays. Levels of miR-4779 and target genes in colon cancer tissue samples from patients were evaluated using qRT-PCR and western blotting analyses. Finally, in vivo tumor suppressive effects of miR-4779 were evaluated in HCT116 xenografts. In this study, miR-4779 inhibited cancer cell growth by inducing apoptosis and cell cycle arrest, and the putative survival factors PAK2 and CCND3 were identified as direct targets of miR-4779. In subsequent experiments, PAK2 knockdown induced cell cycle arrest and CCND3 knockdown induced cell cycle arrest and apoptosis. In addition, miR-4779 suppressed tumor growth and tumorigenesis in an in vivo HCT116 xenograft model. Finally, miR-4779 expression was low in 9 of 10 colon cancer tissues, whereas PAK2 and CCND3 expressions were significantly high in colon cancer tissues. The novel tumor suppressor miR-4779 inhibits cancer cell growth via cell cycle arrest and apoptosis by directly targeting PAK2 and CCND3. The present data indicate the potential of miR-4779 as a therapeutic target for miRNA-based cancer therapy.

Oncolytic Herpes Simplex Virus Vectors Fully Retargeted to Tumor- Associated Antigens.

Oncolytic virotherapy is a novel therapeutic modality for malignant diseases that exploits selective viral replication in cancer cells. Herpes simplex virus (HSV) is a promising agent for oncolytic virotherapy due to its broad cell tropism and the identification of mutations that favor its replication in tumor over normal cells. However, these attenuating mutations also tend to limit the potency of current oncolytic HSV vectors that have entered clinical studies. As an alternative, vector retargeting to novel entry receptors has the potential to achieve tumor specificity at the stage of virus entry, eliminating the need for replication-attenuating mutations. Here, we summarize the molecular mechanism of HSV entry and recent advances in the development of fully retargeted HSV vectors for oncolytic virotherapy. Retargeted HSV vectors offer an attractive platform for the creation of a new generation of oncolytic HSV with improved efficacy and specificity.

Skeletal muscle atrophy is induced by Fbxw7ß via atrogene upregulation.

Muscle atrophy decreases skeletal muscle mass and is induced by inherited cachectic symptoms, genetic disorders, and sarcopenia. However, the molecular pathways associated with the onset of muscle atrophy are still unclear. In this study, we evaluated Fbxw7ß, a gene associated with the development of muscle atrophy in vitro and in vivo. Among the three Fbxw7 isoforms, ectopically overexpressed Fbxw7ß induced the expression of myogenin and major atrogene markers (atrogin-1 and MuRF-1) and reduced myoblast differentiation. In addition, endogenous expression of Fbxw7ß was also upregulated by dexamethasone, which mimics muscle atrophy in vitro, accompanied by induction of myogenin and atrogene expression in primary myoblasts. Functional analysis of Fbxw7ß using short hairpin RNA (shRNA) and a dominant-negative mutant (ΔFbox) suggested that Fbxw7ß regulated muscle atrophy in vitro and in vivo. In particular, ΔFbox did not reduce the sizes of muscle fibers and did not induce myogenin and atrogene expression in vivo. Therefore, our findings demonstrated, for the first time, that Fbxw7ß induced muscle atrophic phenotypes via atrogenes in adult muscle precursor cells and myofibers; this mechanism could be a potential therapeutic target for skeletal muscle atrophy.

Fbxw7ß, E3 ubiquitin ligase, negative regulation of primary myoblast differentiation, proliferation and migration.

Satellite cells attached to skeletal muscle fibers play a crucial role in skeletal muscle regeneration. During regeneration, the satellite cells proliferate, migrate to the damaged region, and fuse to each other. Although it is important to determine the cellular mechanisms controlling myoblast behavior, their regulators are not well understood. In this study, we evaluated the roles of Fbxw7 in primary myoblasts and determined its potential as a therapeutic target for muscle disease. We originally found that Fbxw7ß, one of the E3 ubiquitin ligase Fbxw7 subtypes, negatively regulates differentiation, proliferation and migration of myoblasts and satellite cells on muscle fiber. However, these phenomena were not observed in myoblasts expressing a dominant-negative, F-box deleted Fbxw7ß, mutant. Our results suggest that myoblast differentiation potential and muscle regeneration can be regulated by Fbxw7ß.

Patient-tailored application for Duchene muscular dystrophy on mdx mice based induced mesenchymal stem cells.

Mesenchymal stem cells (MSCs) may be used as powerful tools for the repair and regeneration of damaged tissues. However, isolating tissue specific-derived MSCs may cause pain and increased infection rates in patients, and repetitive isolations may be required. To overcome these difficulties, we have examined alternative methods for MSC production. Here, we show that induced pluripotent stem cells (iPSCs) may be differentiated into mesenchymal stem cells (iMSCs) following exposure to SB431542. Purified iMSCs were administered to mdx mice to study skeletal muscle regeneration in a murine model of muscular dystrophy. Purified iMSCs displayed fibroblast-like morphology, formed three-dimensional spheroid structures, and expressed characteristic mesenchymal stem cell surface markers such as CD29, CD33, CD73, CD90, and CD105. Moreover, iMSCs were capable of differentiating into adipogenic, osteogenic, and chondrogenic lineages. Transplanting iMSC cells to tibialis anterior skeletal muscle tissue in mdx mice lowered oxidative damage as evidenced by a reduction in nitrotyrosine levels, and normal dystrophin expression levels were restored. This study demonstrates the therapeutic potential of purified iMSCs in skeletal muscle regeneration in mdx mice, and suggests that iPSCs are a viable alternate source for deriving MSCs as needed.

Autonomous isolation, long-term culture and differentiation potential of adult salivary gland-derived stem/progenitor cells.

Salivary gland stem/progenitor cells belong to the endodermal lineage and may serve as good candidates to replace their dysfunctional counterparts. The objective of this study was to isolate large numbers of salivary gland tissue-derived stem cells (SGSCs) from adult rats in order to develop a clinically applicable method that does not involve sorting or stem cell induction by duct ligation. We analysed SGSCs isolated from normal rat salivary glands to determine whether they retained the major characteristics of stem cells, self-renewal and multipotency, especially with respect to the various endodermal cell types. SGSCs expressed high levels of integrin α6ß1 and c-kit, which are surface markers of SGSCs. In particular, the integrin α6ß1(+) /c-kit(+) salivary gland cells maintained the morphology, proliferation activity and multipotency of stem cells for up to 92 passages in 12 months. Furthermore, we analysed the capacity of SGSCs to differentiate into endoderm lineage cell types, such as acinar-like and insulin-secreting cells. When cultured on growth factor reduced matrigel, the morphology of progenitor cells changed to acinar-like structures and these cells expressed the acinar cell-specific marker, α-amylase, and tight junction markers. Moreover, reverse transcription-polymerase chain reaction (RT-PCR) data showed increased expression of pancreatic cell markers, including insulin, Pdx1, pan polypeptide and neurogenin-3, when these cells formed pancreatic clusters in the presence of activin A, exendin-4 and retinoic acid. These data demonstrate that adult salivary stem/progenitor cells may serve as a potential source for cell therapy in salivary gland hypofunction and diabetes.

Human salivary gland stem cells ameliorate hyposalivation of radiation-damaged rat salivary glands.

Salivary function in mammals may be defective for various reasons, such as aging, Sjogren's syndrome or radiation therapy in head and neck cancer patients. Recently, tissue-specific stem cell therapy has attracted public attention as a next-generation therapeutic reagent. In the present study, we isolated tissue-specific stem cells from the human submandibular salivary gland (hSGSCs). To efficiently isolate and amplify hSGSCs in large amounts, we developed a culture system (lasting 4-5 weeks) without any selection. After five passages, we obtained adherent cells that expressed mesenchymal stem cell surface antigen markers, such as CD44, CD49f, CD90 and CD105, but not the hematopoietic stem cell markers, CD34 and CD45, and that were able to undergo adipogenic, osteogenic and chondrogenic differentiation. In addition, hSGSCs were differentiated into amylase-expressing cells by using a two-step differentiation method. Transplantation of hSGSCs to radiation-damaged rat salivary glands rescued hyposalivation and body weight loss, restored acinar and duct cell structure, and decreased the amount of apoptotic cells. These data suggest that the isolated hSGSCs, which may have characteristics of mesenchymal-like stem cells, could be used as a cell therapy agent for the damaged salivary gland.

Effective treatment of an orthotopic xenograft model of human glioblastoma using an EGFR-retargeted oncolytic herpes simplex virus.

Glioblastoma multiforme (GBM) remains an untreatable human brain malignancy. Despite promising preclinical studies using oncolytic herpes simplex virus (oHSV) vectors, efficacy in patients has been limited by inefficient virus replication in tumor cells. This disappointing outcome can be attributed in part to attenuating mutations engineered into these viruses to prevent replication in normal cells. Alternatively, retargeting of fully replication-competent HSV to tumor-associated receptors has the potential to achieve tumor specificity without impairment of oncolytic activity. Here, we report the establishment of an HSV retargeting system that relies on the combination of two engineered viral glycoproteins, gD and gB, to mediate highly efficient HSV infection exclusively through recognition of the abundantly expressed epidermal growth factor receptor (EGFR) on glioblastoma cells. We demonstrate efficacy in vitro and in a heterotopic tumor model in mice. Evidence for systemically administered virus homing to the tumor mass is presented. Treatment of orthotopic primary human GBM xenografts demonstrated prolonged survival with up to 73% of animals showing a complete response as confirmed by magnetic resonance imaging. Our study describes an approach to HSV retargeting that is effective in a glioma model and may be applicable to the treatment of a broad range of tumor types.

Establishment of a bioluminescent imaging-based in vivo leukemia model by intra-bone marrow injection.

In vivo leukemia mouse models are usually generated by intraperitoneal (IP) or intravenous (IV) injection of leukemia cells. However, the pattern of leukemia development observed can be inconsistent. This study investigated injection directly into bone marrow [intra-bone marrow transplantation (IBMT)], the natural microenvironment of leukemia. A bioluminescent imaging-based leukemia animal model has been established by direct injection of a bioluminescent leukemia cells (CCRF-CEM/fLuc) into NOD/SCID mouse tibia bone marrow and compared with models established by IP and IV routes. The comparison revealed that a bioluminescent in vivo leukemia model established via IBMT could recapitulate leukemia more faithfully and facilitate improved quantification of leukemia engraftment kinetics with a wider range of bioluminescent intensity than IP or IV. IBMT of bioluminescent leukemic cells allowed quantification of dose-dependent responses to anti-leukemic drugs, thus validating this model as a potential preclinical anti-leukemic drug screening system. IBMT-leukemia cells isolated from peripheral blood of the model mice and then injected into new recipients successfully established a second generation IBMT in vivo model and demonstrated the reproducibility of the model. Bioluminescent imaging-based analysis of this IBMT-leukemia model could provide a means for the comprehensive evaluation of treatment responses with enhanced sensitivity in preclinical studies.

Recovery from radiation-induced bone marrow damage by HSP25 through Tie2 signaling.

PURPOSE: Whole-body radiation therapy can cause severe injury to the hematopoietic system, and therefore it is necessary to identify a novel strategy for overcoming this injury. METHODS AND MATERIALS: Mice were irradiated with 4.5 Gy after heat shock protein 25 (HSP25) gene transfer using an adenoviral vector. Then, peripheral blood cell counts, histopathological analysis, and Western blotting on bone marrow (BM) cells were performed. The interaction of HSP25 with Tie2 was investigated with mouse OP9 and human BM-derived mesenchymal stem cells to determine the mechanism of HSP25 in the hematopoietic system. RESULTS: HSP25 transfer increased BM regeneration and reduced apoptosis following whole-body exposure to ionizing radiation (IR). The decrease in Tie2 protein expression that followed irradiation of the BM was blocked by HSP25 transfer, and Tie2-positive cells were more abundant among the BM cells of HSP25-transferred mice, even after IR exposure. Following systemic RNA interference of Tie2 before IR, HSP25-mediated radioprotective effects were partially blocked in both mice and cell line systems. Stability of Tie2 was increased by HSP25, a response mediated by the interaction of HSP25 with Tie2. IR-induced tyrosine phosphorylation of Tie2 was augmented by HSP25 overexpression; downstream events in the Tie2 signaling pathway, including phosphorylation of AKT and EKR1/2, were also activated. CONCLUSIONS: HSP25 protects against radiation-induced BM damage by interacting with and stabilizing Tie2. This may be a novel strategy for HSP25-mediated radioprotection in BM.

The soluble amino-terminal region of HVEM mediates efficient herpes simplex virus type 1 infection of gD receptor-negative cells.

BACKGROUND: Previous studies from our own and other labs reported the surprising finding that the soluble V domain of the herpes simplex virus type 1 (HSV-1) entry receptor nectin-1 can both block HSV infection of receptor-bearing cells and mediate infection of receptor-deficient cells. Here we show that this property is not unique to nectin-1. We generated a pair of truncated, soluble forms of the other major HSV-1 entry receptor, herpes virus entry mediator (HVEM or HveA), and examined its effects on HSV-1 infection of receptor-deficient cells. RESULTS: In cultures of CHO-K1 cells, sHveA102 comprising the two amino-terminal cysteine-rich pseudorepeats (CRPs) of HVEM enabled infection of greater than 80% of the cells at an MOI of 3, while sHveA162 comprising the complete ectodomain failed to mediate infection. Both sHveA102 and sHveA162 blocked infection of CHO-K1 cells stably expressing HVEM in a dose-dependent manner, indicating that both were capable of binding to viral gD. We found that sHveA102-mediated infection involves pH-independent endocytosis whereas HSV infection of HVEM-expressing CHO-K1 cells is known to be pH-dependent. CONCLUSIONS: Our results suggest that the C-terminal portion of the soluble HVEM ectodomain inhibits gD activation and that this effect is neutralized in the full-length form of HVEM in normal infection.

Direct and indirect contribution of human embryonic stem cell-derived hepatocyte-like cells to liver repair in mice.

BACKGROUND & AIMS: Many studies of embryonic stem cells have investigated direct cell replacement of damaged tissues, but little is known about how donor cell-derived signals affect host tissue regeneration. We investigated the direct and indirect roles of human embryonic stem cell-derived cells in liver repair in mice. METHODS: To promote the initial differentiation of human embryonic stem cells into mesendoderm, we activated the ß-catenin signaling pathway with lithium; cells were then further differentiated into hepatocyte-like cells. The differentiated cells were purified by indocyanine green staining and laser microdissection and characterized by immunostaining, polymerase chain reaction, biochemical function, electron microscopy, and transplantation analyses. To investigate indirect effects of these cells, secreted proteins (secretomes) were analyzed by a label-free quantitative mass spectrometry. Carbon tetrachloride was used to induce acute liver injury in mice; cells or secreted proteins were administered by intrasplenic or intraperitoneal injection, respectively. RESULTS: The differentiated hepatocyte-like cells had multiple features of normal hepatocytes, engrafted efficiently into mice, and continued to have hepatic features; they promoted proliferation of host hepatocytes and revascularization of injured host liver tissues. Proteomic analysis identified proteins secreted from these cells that might promote host tissue repair. Injection of the secreted proteins into injured livers of mice promoted significant amounts of tissue regeneration without cell grafts. CONCLUSIONS: Hepatocyte-like cells derived from human embryonic stem cells contribute to recovery of injured liver tissues in mice, not only by cell replacement but also by delivering trophic factors that support endogenous liver regeneration.

Bispecific adapter-mediated retargeting of a receptor-restricted HSV-1 vector to CEA-bearing tumor cells.

The safety and efficacy of viral therapies for solid tumors can be enhanced by redirecting the virus infection to tumor-specific cell-surface markers. Successful retargeting of herpes simplex virus type 1 (HSV-1) has been achieved using vectors that carry a modified envelope glycoprotein D (gD) engineered to interact directly with novel receptors. In addition, soluble bridging molecules (adapters) have been used to link gD indirectly to cell-specific receptors. Here, we describe the development of an adapter connecting gD to the common tumor antigen carcinoembryonic antigen (CEA). The adapter consisted of a CEA-specific single-chain antibody fused to the gD-binding region of the gD receptor, herpes virus entry mediator (HVEM). We used this adapter in combination with a vector that is detargeted for recognition of the widely expressed gD receptor nectin-1, but retains an intact binding region for the less common HVEM. We show that the adapter enabled infection of HSV-resistant Chinese hamster ovary (CHO) cells expressing ectopic CEA and nectin-1/CEA-bearing human gastric carcinoma cells that are resistant to the vector alone. We observed cell-to-cell spread following adapter-mediated infection in vitro and reduced tumor growth in vivo, indicating that this method of vector retargeting may provide a novel strategy for tumor-specific delivery of tumoricidal HSV.

Radioprotective effect of heat shock protein 25 on submandibular glands of rats.

Irradiation (IR) is a fundamental treatment modality for head and neck malignancies. However, a significant drawback of IR treatment is irreversible damage of salivary gland in the IR field. In the present study, we investigated whether heat shock protein (HSP) 25 could be used as a radioprotective molecule for radiation-induced salivary gland damage in rats. HSP25 as well as inducible HSP70 (HSP70i) that were delivered to the salivary gland via an adenoviral vector significantly ameliorated radiation-induced salivary fluid loss. Radiation-induced apoptosis, caspase-3 activation, and poly(ADP-ribose) polymerase cleavage in acinar cells, granular convoluted cells, and intercalated ductal cells were also inhibited by HSP25 or HSP70i transfer. The alteration of salivary contents, including amylase, protein, Ca+, Cl-, and Na+, was also attenuated by HSP25 transfer. Histological analysis revealed almost no radiation-induced damage in salivary gland when HSP25 was transferred. Aquaporin 5 expression in salivary gland was inhibited by radiation; and HSP25 transfer to salivary gland prevented this alteration. The protective effect of HSP70i on radiation-induced salivary gland damage was less or delayed than that of HSP25. These results indicate that HSP25 is a good candidate molecule to protect salivary gland from the toxicity of IR.

Soluble V domain of Nectin-1/HveC enables entry of herpes simplex virus type 1 (HSV-1) into HSV-resistant cells by binding to viral glycoprotein D.

Interaction of herpes simplex virus (HSV) glycoprotein D (gD) with specific cellular receptors is essential for HSV infection of susceptible cells. Virus mutants that lack gD can bind to the cell surface (attachment) but do not enter, implying that interaction of gD with its receptor(s) initiates the postattachment (entry) phase of HSV infection. In this report, we have studied HSV entry in the presence of the gD-binding variable (V) domain of the common gD receptor nectin-1/HveC to determine whether cell association of the gD receptor is required for HSV infection. In the presence of increasing amounts of the soluble nectin-1 V domain (sNec1(123)), increasing viral entry into HSV-resistant CHO-K1 cells was observed. At a multiplicity of 3 in the presence of optimal amounts of sNec1(123), approximately 90% of the cells were infected. The soluble V domain of nectin-2, a strain-specific HSV entry receptor, promoted entry of the HSV type 1 (HSV-1) Rid-1 mutant strain, but not of wild-type HSV-1. Preincubation and immunofluorescence studies indicated that free or gD-bound sNec1(123) did not associate with the cell surface. sNec1(123)-mediated entry was highly impaired by interference with the cell-binding activities of viral glycoproteins B and C. While gD has at least two functions, virus attachment to the cell and initiation of the virus entry process, our results demonstrate that the attachment function of gD is dispensable for entry provided that other means of attachment are available, such as gB and gC binding to cell surface glycosaminoglycans.

Herpes simplex virus targeting to the EGF receptor by a gD-specific soluble bridging molecule.

Herpes simplex virus 1 (HSV-1) enters cells via initial binding of envelope glycoproteins (g) C and B to cell-surface glycosaminoglycans (GAGs) and subsequent membrane fusion involving envelope gD, gB, and gH/gL. Current insights suggest that the fusion process is initiated by interaction of gD with a cognate cellular receptor, such as the widely distributed cell adhesion molecule nectin-1. To redirect the tropism of HSV-1, we have generated a soluble adapter protein (P-V528LH) comprising the gD-binding variable domain of nectin-1 fused to a single-chain antibody (528LH) recognizing the EGF receptor. The adapter molecule enabled HSV-1 entry into naturally nonpermissive CHO cells expressing the human EGF receptor, but not into CHO cells lacking the receptor, and entry was not observed when the antibody portion of the adapter was replaced with an antibody of different specificity. Adapter-mediated entry increased with the viral dose and was nearly as efficient as direct viral entry into nectin-1-bearing CHO cells. Entry depended on viral gD and was diminished in the absence of cellular GAGs. These experiments represent the first demonstration that a soluble molecule can direct HSV infection via a new receptor, supporting the possible utility of this approach for HSV retargeting.