Activation of Natural Killer Cells by Newcastle Disease Virus Hemagglutinin-Neuraminidase

The avian paramyxovirus Newcastle disease virus (NDV) selectively replicates in tumor cells and is known to stimulate T-cell-, macrophage-, and NK cell-mediated responses. The mechanisms of NK cell activation by NDV are poorly understood so far. We studied the expression of ligand structures for activating NK cell receptors on NDV-infected tumor cells. Upon infection with the nonlytic NDV strain Ulster and the lytic strain MTH-68/H, human carcinoma and melanoma cells showed enhanced expression of ligands for the natural cytotoxicity receptors NKp44 and NKp46, but not NKp30. Ligands for the activating receptor NKG2D were partially downregulated. Soluble NKp44-Fc and NKp46-Fc, but not NKp30-Fc, chimeric proteins bound specifically to NDV-infected tumor cells and to NDV particle-coated plates. Hemagglutinin-neuraminidase (HN) of the virus serves as a ligand structure for NKp44 and NKp46, as indicated by the blockade of binding to NDV-infected cells and viral particles in the presence of anti-HN antibodies and by binding to cells transfected with HN cDNA. Consistent with the recognition of sialic acid moieties by the viral lectin HN, the binding of NKp44-Fc and NKp46-Fc was lost after desialylation. NKp44- and NKp46-CD3ζ lacZ-inducible reporter cells were activated by NDV-infected cells. NDV-infected tumor cells stimulated NK cells to produce increased amounts of the effector lymphokines gamma interferon and tumor necrosis factor alpha. Primary NK cells and the NK line NK-92 lysed NDV-infected tumor cells with enhanced efficiency, an effect that was eliminated by the treatment of target cells with the neuraminidase inhibitor Neu5Ac2en. These results suggest that direct activation of NK cells contributes to the antitumor effects of NDV.Virulent strains of Newcastle disease virus (NDV) infect domestic poultry and other birds, causing a rapidly spreading viral disease that affects the alimentary and respiratory tracts as well as the central nervous system (55). In humans, however, NDV is well tolerated (17, 18). Other than mild fever for a day, only a few adverse effects have been reported. NDV, also known as avian paramyxovirus 1, is an enveloped virus containing a negative-sense, single-stranded RNA genome which codes for six proteins in the order (from 3′ to 5′) of nucleoprotein, phosphoprotein, matrix protein, fusion (F) protein, hemagglutinin-neuraminidase (HN), and large polymerase protein (19). There are many different strains of NDV, classified as either lytic or nonlytic for different types of cells. Lytic and nonlytic NDV strains both replicate much more efficiently in human cancer cells than they do in most normal human cells (43). Viruses of both strain types have been investigated as potential anticancer agents (30, 49, 52). The NDV strains that have been evaluated most widely for the treatment of cancer are 73-T, MTH-68, and Ulster (1, 7, 11, 17, 18, 53, 54, 56, 71).Initial binding of NDV to a host cell takes place through the interaction of HN molecules in the virus coat with sialic acid-containing molecules on the cell surface (31). NDV neuraminidase has strict specificity for the hydrolysis of the NeuAc-α2,3-Gal linkage, with no hydrolysis of the NeuAc-α2,6-Gal linkage (41).NDV infection of tumor cells not only improves T-cell responses (53, 58, 68), but has also been reported to vigorously stimulate innate immune responses. In the course of NDV infection, large amounts of alpha interferon (IFN-α) are released (68) and in turn activate dendritic cells and NK cells and polarize, in concert with interleukin-12 (IL-12), toward a Th1 T-cell response (33, 44, 47). In addition, NDV induces antitumor cytotoxicity in murine macrophages which produce increased amounts of tumor necrosis factor alpha (TNF-α) and nitric oxide (51, 60) and in human monocytes through the induction of TRAIL (64). Little is known about the NDV-mediated activation of NK cells. The coincubation of peripheral blood mononuclear cells with NDV was shown previously to stimulate NK-mediated cytotoxicity (70). Enhanced cytotoxicity correlates with the induction of IFN-α (70). It is not known, however, whether NDV-infected cells can directly activate NK cells and, if so, which molecular interactions are involved.The cytolytic activity of NK cells against virus-infected or tumor cells is regulated by the engagement of activating or inhibitory NK cell surface receptors, the actions of cytokines, and cross talk with other immune cells (32, 39). Most inhibitory receptors recognize particular major histocompatibility complex (MHC) class I alleles and thereby ensure the tolerance of NK cells against self antigens (38). Activating receptors on human NK cells include CD16; NKG2D; the natural cytotoxicity receptors (NCR) NKp30, NKp44, and NKp46; as well as NKp80; DNAM-1; and various stimulatory coreceptors (32).NCR are important activating receptors for the antitumor and antiviral activities of NK cells (5, 32, 37). Heparan sulfate has been discussed previously as a cellular ligand for NKp46, NKp44, and NKp30 (9, 26, 27), and nuclear factor BAT3, which can be released from tumor cells under stress conditions, has been described as a cellular ligand for NKp30 (42). Ligands for NKp30 and NKp44 can be detected on the surfaces and in the intracellular compartments of several kinds of tumor cells (10). Moreover, a number of pathogen-derived NCR ligands have been reported. The hemagglutinin protein of influenza virus and the HN of Sendai virus can bind to NKp46 and NKp44 and activate NK cells (3, 24, 34). The pp65 protein of human cytomegalovirus has been shown to bind NKp30 and inhibit its function (4). Human immunodeficiency virus, vaccinia virus, and herpes simplex virus have also been shown to upregulate the expression of cellular NCR ligands in infected cells (13, 14, 62). The Plasmodium falciparum erythrocyte membrane protein 1 is involved in the NCR-mediated NK cell attack against infected erythrocytes (36). Furthermore, NKp46 recognizes cells infected with mycobacteria (22, 61), and NKp44 was recently reported to directly bind to the surfaces of mycobacteria and other bacteria (21).In this study, we investigated the expression of ligand structures for NCR and NKG2D on NDV-infected cells. We demonstrate that NDV HN proteins which are strongly expressed on NDV-infected tumor cells function as activating ligand structures for NKp44 and NKp46 but that cellular ligands for NKG2D are partially downregulated during NDV infection.     

The Oncolytic Activity of Newcastle Disease Virus NDV-HUJ on Chemoresistant Primary Melanoma Cells Is Dependent on the Proapoptotic Activity of the Inhibitor of Apoptosis Protein Livin

Patients with advanced melanoma usually do not benefit from conventional chemotherapy treatment. There is therefore a true need for a new kind of therapy for melanoma. One factor responsible for the poor prognosis of melanoma is the inhibitor of apoptosis protein (IAP) family member Livin. In this study, we applied a novel approach for the treatment of melanoma, using a unique strain of the oncolytic Newcastle disease virus (NDV-HUJ). We found that, unlike chemotherapeutic drugs, NDV-HUJ, a one-cycle replicating virus, overcomes the resistance to apoptosis of melanoma primary cultures that over express the Livin protein. In contrast, melanoma tumor cells that do not express Livin are relatively resistant to NDV-HUJ treatment. Furthermore, we show that NDV-HUJ-induced oncolysis is attributed to the dual function of Livin: although Livin inhibits apoptosis through the inhibition of caspases, under the robust apoptotic stimulation of NDV-HUJ, caspases can cleave Livin to create a truncated protein with a paradoxical proapoptotic activity. Thus, NDV-HUJ is a potent inducer of apoptosis that can overcome the antiapoptotic effect of Livin and allow cleavage of Livin into the proapoptotic tLivin protein. Moreover, the results indicate that the interferon system, which is functional in melanoma, is not involved in NDV-induced oncolysis. Taken together, our data offer the possibility of a new viral oncolytic treatment for chemoresistant melanoma.Newcastle disease virus (NDV) is an avian paramyxovirus that has a potential selective oncolytic effect on human tumors (5, 7, 13, 21, 25, 26). NDV''s natural host is avian, and while mammalian cells bear the sialic acid receptor for NDV and may be infected by the virus, the virus has limited replication capacity in normal mammalian cells (21). We recently reported the development of an attenuated (lentogenic) isolate of NDV (HUJ) that undergoes only one cycle replication in infected mammalian cells (7, 25). NDV-HUJ is a single clone derived from the parental strain NDV Hitchner B1, which contains a mixed viral population. The new virus clone is attenuated due to multiple passages in specific-pathogen-free (SPF) eggs, and its intracerebral pathogenicity index (ICPI) value is low (an ICPI of 0.01 versus an ICPI of 0.93 for the parental NDV Hitchner B1). Sequence analysis of NDV-HUJ indicated 156 changes at the nucleotide sequence level and multiple amino acid changes from the parental B1 virus in all six viral genes (see Fig. S1 in the supplemental material). Although NDV-HUJ is an attenuated virus in chicken, it retains a selective cytotoxic potential for cancer cells, as determined in vitro and in vivo, using murine and human lung carcinomas (25). The oncolytic effect of the virus is apoptosis dependent (25). NDV-HUJ has been applied to treat glioblastoma patients in a phase I/II clinical trials and found to be safe and potentially active (7).The inhibitors of apoptosis proteins (IAPs) are receiving increased attention as key players in the initiation of tumors, their progression, and resistance to chemotherapy treatment (17). To date, eight human IAPs have been identified, including Livin. IAPs are characterized by one or more repeats of a highly conserved 70-amino-acid domain termed the baculovirus IAP repeat (BIR) that can bind and inhibit caspases, some IAPs also contain a conserved sequence termed the RING finger. RING finger proteins might function as E3 ubiquitin ligases; however, the exact nature of the E3 ligase activity of IAPs is still largely unclear.IAPs inhibit apoptosis induced by a variety of stimuli, mainly through their ability to bind and inhibit specific caspases (17). Intense study has shown that the role of IAP in apoptosis regulation is highly diverse, with a prominent role in tumorigenesis and resistance to therapy. Among the human IAPs, XIAP is the best characterized and the most potent caspase inhibitor. The most recently discovered member of this family is Livin, found by us and others (3, 9, 12, 24). Livin contains a single BIR domain and a RING finger (3, 12). We previously found that Livin is specifically cleaved by caspases at the Asp52 residue to produce a large C-terminal fragment, containing both the BIR and the RING domains. After cleavage, truncated Livin (tLivin) acts paradoxically as a proapoptotic factor (18, 19).In the present study we show that chemoresistant melanoma primary cultures that highly express the Livin protein are sensitive to oncolytic NDV-HUJ treatment. This appears to be a result of activation of caspases 8, 3, and 7 that in turn cleave Livin to produce the tLivin. This is a novel regulatory mechanism in which NDV-HUJ can overcome the antiapoptosis function of Livin and expose the “good side” of Livin by inducing the cleavage of Livin to produce the proapoptotic tLivin that subsequently leads to metastatic melanoma cell death.     

Effect of Preexisting Immunity on Oncolytic Adenovirus Vector INGN 007 Antitumor Efficacy in Immunocompetent and Immunosuppressed Syrian Hamsters

Immune responses against adenovirus (Ad) vectors pose a possible concern for the outcome of treatment efficacy. To address the role of preexisting immunity in oncolytic Ad vector antitumor efficacy following intratumoral injection of vector as well as tumor-to-tissue spread of the vector, we employed the Syrian hamster model. These animals are immunocompetent, and their tumors and tissues are permissive for replication of Ad type 5 (Ad5). We used the adenovirus death protein-overexpressing Ad5-based vector INGN 007. Subcutaneous tumors were established in groups of hamsters that were or were not immunized with Ad5. Half of the hamsters in these groups were immunosuppressed with cyclophosphamide. For all groups, tumors injected with INGN 007 grew significantly more slowly than those injected with buffer. Under immunocompetent conditions, there was no significant effect of preexisting immunity on vector antitumor efficacy. Soon after the tumors in naïve animals were injected with vector, the hamsters developed neutralizing antibody (NAb) and the difference in NAb titers between the naïve and immunized groups diminished. Under immunosuppressed conditions, preexisting NAb did significantly reduce vector efficacy. Thus, NAb do reduce vector efficacy to some extent, but immunosuppression is required to observe the effect. Regarding vector toxicity, there was spillover of vector from the tumor to the liver and lungs in naïve immunocompetent hamsters, and this was nearly eliminated in the immunized hamsters. Thus, preexisting immunity to Ad5 does not affect INGN 007 antitumor efficacy following intratumoral injection, but immunity prevents vector spillover from the tumor to the liver and lungs.Oncolytic (replication-competent) viral vectors are being investigated as a treatment for cancer (2, 19, 25, 27). Recently, an oncolytic adenovirus serotype 5 (Ad5)-based vector was approved for cancer therapy in humans for the first time (14, 42). Oncolytic vectors based on Ad, reovirus, herpes simplex virus type 1 (HSV-1), poxvirus, poliovirus, Newcastle disease virus, measles virus, and vesicular stomatitis virus (VSV) are being studied extensively in both preclinical and clinical settings (16, 20, 24). Oncolytic Ad vectors are popular due to the Ad safety profile and ease of manipulation and handling (6, 13, 18, 23).Oncolytic Ad vectors infect and kill cancer cells as a result of the normal Ad life cycle by replicating in cells and releasing progeny viruses. These vectors rely on replication and spread through the tumor to achieve efficacy. A majority of the human population is seropositive for Ad5, which is acquired as a childhood infection (4, 15, 39). Elimination of the vector by preexisting immunity to Ad or vector elimination by the adaptive immune response generated after administration of the vector poses a possible concern with respect to achieving significant antitumor efficacy. A key question is whether the oncolytic Ad vector can efficiently eliminate tumor cells faster than its own clearance by the immune system. Several studies show that suppressing the immune system enhances the efficacy of oncolytic vectors (10, 12, 31).Alternatively, studies show that activation of the adaptive immune system by the vector might increase tumor cell killing, thereby increasing vector antitumor efficacy (11, 21, 27, 34). Studies with oncolytic HSV and VSV show that these vectors induce long-term antitumor immunity (11, 21, 27, 34). Therefore, apart from direct cell lysis, oncolytic vectors may be able to achieve antitumor efficacy by activating the antitumor immune response. Therefore, induced or preexisting immunity to the vector can be either a hurdle or beneficial for vector efficacy.Most efforts to address the effect of preexisting immunity were performed by gene transfer studies with replication-defective Ad vectors (28, 41). These studies showed that preexisting immunity significantly reduces gene transfer and expression in the target organ. In contrast, other studies showed that preexisting immunity does not prevent gene transfer (26) and does not affect vector antitumor efficacy (1). Little work has been done to address the role of induced or preexisting immunity on the efficacy and toxicity of oncolytic Ad vectors (3, 39). Studies with these vectors have been difficult because of a lack of immunocompetent and permissive animal models. Ad replication is generally species specific, and human Ads replicate poorly in cells from most nonhuman species. Consequently, Ad vectors are commonly evaluated in immunodeficient mice bearing human tumor xenografts. However, this model cannot adequately address the effect of the host immune system on the vector-infected tumor or the toxicity of the vector in normal tissues.We recently developed a novel Syrian hamster model for the study of oncolytic Ad5-based vectors (30). These animals are both replication permissive for Ad5 and immunocompetent. In the present study, we modeled the effect of preexisting immunity to Ad5 on the efficacy of an oncolytic Ad vector, INGN 007, and the spillover of the vector from the site of injection to the liver and lungs.     

Induction and Inhibition of Type I Interferon Responses by Distinct Components of Lymphocytic Choriomeningitis Virus

Type I interferons (IFNs) play a critical role in the host defense against viruses. Lymphocytic choriomeningitis virus (LCMV) infection induces robust type I IFN production in its natural host, the mouse. However, the mechanisms underlying the induction of type I IFNs in response to LCMV infection have not yet been clearly defined. In the present study, we demonstrate that IRF7 is required for both the early phase (day 1 postinfection) and the late phase (day 2 postinfection) of the type I IFN response to LCMV, and melanoma differentiation-associated gene 5 (MDA5)/mitochondrial antiviral signaling protein (MAVS) signaling is crucial for the late phase of the type I IFN response to LCMV. We further demonstrate that LCMV genomic RNA itself (without other LCMV components) is able to induce type I IFN responses in various cell types by activation of the RNA helicases retinoic acid-inducible gene I (RIG-I) and MDA5. We also show that expression of the LCMV nucleoprotein (NP) inhibits the type I IFN response induced by LCMV RNA and other RIG-I/MDA5 ligands. These virus-host interactions may play important roles in the pathogeneses of LCMV and other human arenavirus diseases.Type I interferons (IFNs), namely, alpha interferon (IFN-α) and IFN-β, are not only essential for host innate defense against viral pathogens but also critically modulate the development of virus-specific adaptive immune responses (6, 8, 28, 30, 36, 50, 61). The importance of type I IFNs in host defense has been demonstrated by studying mice deficient in the type I IFN receptor, which are highly susceptible to most viral pathogens (2, 47, 62).Recent studies have suggested that the production of type I IFNs is controlled by different innate pattern recognition receptors (PRRs) (19, 32, 55, 60). There are three major classes of PRRs, including Toll-like receptors (TLRs) (3, 40), retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) (25, 48, 51), and nucleotide oligomerization domain (NOD)-like receptors (9, 22). TLRs are a group of transmembrane proteins expressed on either cell surfaces or endosomal compartments. RLRs localize in the cytosol. Both TLRs and RLRs are involved in detecting viral pathogens and controlling the production of type I IFNs (52, 60). In particular, the endosome-localized TLRs (TLR3, TLR7/8, and TLR9) play important roles in detecting virus-derived double-stranded RNA (dsRNA), single-stranded RNA (ssRNA), and DNA-containing unmethylated CpG motifs, respectively. In contrast, RIG-I detects virus-derived ssRNA with 5′-triphosphates (5′-PPPs) or short dsRNA (<1 kb), whereas melanoma differentiation-associated gene 5 (MDA5) is responsible for recognizing virus-derived long dsRNA as well as a synthetic mimic of viral dsRNA poly(I):poly(C) [poly(I·C)] (24, 60). Recognition of viral pathogen-associated molecular patterns (PAMPs) ultimately leads to the activation and nuclear translocation of interferon regulatory factors (IRFs) and nuclear factor κB (NF-κB), which, in turn, switches on a cascade of genes controlling the production of both type I IFNs and other proinflammatory cytokines (10, 11, 60).Lymphocytic choriomeningitis virus (LCMV) infection in its natural host, the mouse, is an excellent system to study the impact of virus-host interactions on viral pathogenesis and to address important issues related to human viral diseases (1, 45, 49, 67). LCMV infection induces type I IFNs as well as other proinflammatory chemokines and cytokines (6, 41). Our previous studies have demonstrated that TLR2, TLR6, and CD14 are involved in LCMV-induced proinflammatory chemokines and cytokines (66). The mechanism by which LCMV induces type I IFN responses, however, has not been clearly defined (7, 8, 31, 44). The role of the helicase family members RIG-I and MDA5 in virus-induced type I IFN responses has been recently established. RIG-I has been found to be critical in controlling the production of type I IFN in response to a number of RNA viruses, including influenza virus, rabies virus, Hantaan virus, vesicular stomatitis virus (VSV), Sendai virus (SeV), etc. In contrast, MDA5 is required for responses to picornaviruses (15, 25, 63).In the present study, we demonstrated that LCMV genomic RNA strongly activates type I IFNs through a RIG-I/MDA5-dependent signaling pathway. Our present study further demonstrated that the LCMV nucleoprotein (NP) blocks LCMV RNA- and other viral ligand-induced type I IFN responses.     

Antiangiogenic Arming of an Oncolytic Vaccinia Virus Enhances Antitumor Efficacy in Renal Cell Cancer Models

Oncolytic vaccinia viruses have shown compelling results in preclinical cancer models and promising preliminary safety and antitumor activity in early clinical trials. However, to facilitate systemic application it would be useful to improve tumor targeting and antitumor efficacy further. Here we report the generation of vvdd-VEGFR-1-Ig, a targeted and armed oncolytic vaccinia virus. Tumor targeting was achieved by deletion of genes for thymidine kinase and vaccinia virus growth factor, which are necessary for replication in normal but not in cancer cells. Given the high vascularization typical of kidney cancers, we armed the virus with the soluble vascular endothelial growth factor (VEGF) receptor 1 protein for an antiangiogenic effect. Systemic application of high doses of vvdd-VEGFR-1-Ig resulted in cytokine induction in an immunocompromised mouse model. Upon histopathological analysis, splenic extramedullary hematopoiesis was seen in all virus-injected mice and was more pronounced in the vvdd-VEGFR-1-Ig group. Analysis of the innate immune response after intravenous virus injection revealed high transient and dose-dependent cytokine elevations. When medium and low doses were used for intratumoral or intravenous injection, vvdd-VEGFR-1-Ig exhibited a stronger antitumor effect than the unarmed control. Furthermore, expression of VEGFR-1-Ig was confirmed, and a concurrent antiangiogenic effect was seen. In an immunocompetent model, systemic vvdd-VEGFR-1-Ig exhibited superior antitumor efficacy compared to the unarmed control virus. In conclusion, the targeted and armed vvdd-VEGFR-1-Ig has promising anticancer activity in renal cell cancer models. Extramedullary hematopoiesis may be a sensitive indicator of vaccinia virus effects in mice.In 2002 renal cell cancer accounted for more than 200,000 cases and 100,000 deaths worldwide (33). Unfortunately, chemotherapy, radiotherapy, and immunotherapy yield low response rates (9, 17) in this cancer type. Thus, prognosis for patients is poor, especially when the disease is metastatic, as median survival is only 8 months (19). Although recently approved drugs, such as sorafenib, sunitinib, temsirolimus, and bevacizumab, have provided additional tools for treatment of renal cell cancer (7), they are usually not curative, and thus new treatment approaches are needed.Oncolytic vaccinia viruses are promising agents for cancer treatment and have shown compelling results in preclinical tumor models (40, 42, 45). Moreover, good safety and preliminary evidence of antitumor efficacy were seen in phase 1 clinical trials (22, 26, 32). Vaccinia virus has a strong oncolytic effect due to its fast replication cycle (45) and a high innate tropism to cancer tissue (34). Tumor targeting can be further improved by deleting vaccinia virus genes that are necessary for replication in normal cells but not in cancer cells. For example, deletions of either thymidine kinase (TK) or vaccinia virus growth factor (VGF) or both have been shown to reduce pathogenicity compared to wild-type virus (3, 5, 27). To enhance antitumor potency, oncolytic vaccinia viruses can be armed with therapeutic transgenes, such as immunostimulatory factors (26) or suicide genes (14, 16, 35). With regard to kidney cancer, an arming approach with antiangiogenenic molecules seems logical, considering the high vascularization characteristic of renal tumors (20).Vascular endothelial growth factor (VEGF) is a major player in tumor angiogenesis and is highly expressed in renal cell cancers (29). VEGF binds to the fms-like-tyrosine kinase receptor (flt-1 or VEGFR-1) and kinase domain region receptor (KDR or VEGFR-2) with high affinity (13). The soluble vascular endothelial growth factor receptor 1-Ig fusion protein (VEGFR-1-Ig) used in this study is derived from the membrane-bound VEGFR-1 and binds human and murine VEGF without inducing vascular endothelial cell mitogenesis (31). Blocking VEGF with this or closely related molecules has been shown to inhibit tumor growth in several cancer models (18, 21, 25, 39).Although tumor cell selective replication can be enhanced by deletion of TK and/or VGF to reduce pathogenicity (3, 5, 27), high doses of attenuated vaccinia virus may increase serum cytokine concentrations which parallel the onset of toxic events, as seen with other viral vectors (2, 38). The potential “early” toxicity associated with oncolytic vaccinia viruses has not been completely elucidated heretofore (36, 46).Given the high vascularization of renal cell cancers and the pressing need to generate new antitumor agents with increased safety and efficacy, we hypothesized that an oncolytic vaccinia virus targeted by TK and VGF deletions and armed with VEGFR-1-Ig would exhibit enhanced antitumor efficacy due to its antiangiogenic properties in renal cell cancer models compared to a nonarmed control virus, allowing reduction of the treatment dose.     

Pathogenic Hantaviruses Andes Virus and Hantaan Virus Induce Adherens Junction Disassembly by Directing Vascular Endothelial Cadherin Internalization in Human Endothelial Cells

Hantaviruses infect endothelial cells and cause 2 vascular permeability-based diseases. Pathogenic hantaviruses enhance the permeability of endothelial cells in response to vascular endothelial growth factor (VEGF). However, the mechanism by which hantaviruses hyperpermeabilize endothelial cells has not been defined. The paracellular permeability of endothelial cells is uniquely determined by the homophilic assembly of vascular endothelial cadherin (VE-cadherin) within adherens junctions, which is regulated by VEGF receptor-2 (VEGFR2) responses. Here, we investigated VEGFR2 phosphorylation and the internalization of VE-cadherin within endothelial cells infected by pathogenic Andes virus (ANDV) and Hantaan virus (HTNV) and nonpathogenic Tula virus (TULV) hantaviruses. We found that VEGF addition to ANDV- and HTNV-infected endothelial cells results in the hyperphosphorylation of VEGFR2, while TULV infection failed to increase VEGFR2 phosphorylation. Concomitant with the VEGFR2 hyperphosphorylation, VE-cadherin was internalized to intracellular vesicles within ANDV- or HTNV-, but not TULV-, infected endothelial cells. Addition of angiopoietin-1 (Ang-1) or sphingosine-1-phosphate (S1P) to ANDV- or HTNV-infected cells blocked VE-cadherin internalization in response to VEGF. These findings are consistent with the ability of Ang-1 and S1P to inhibit hantavirus-induced endothelial cell permeability. Our results suggest that pathogenic hantaviruses disrupt fluid barrier properties of endothelial cell adherens junctions by enhancing VEGFR2-VE-cadherin pathway responses which increase paracellular permeability. These results provide a pathway-specific mechanism for the enhanced permeability of hantavirus-infected endothelial cells and suggest that stabilizing VE-cadherin within adherens junctions is a primary target for regulating endothelial cell permeability during pathogenic hantavirus infection.Hantaviruses cause 2 human diseases: hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS) (50). HPS and HFRS are multifactorial in nature and cause thrombocytopenia, immune and endothelial cell responses, and hypoxia, which contribute to disease (7, 11, 31, 42, 62). Although these syndromes sound quite different, they share common components which involve the ability of hantaviruses to infect endothelial cells and induce capillary permeability. Edema, which results from capillary leakage of fluid into tissues and organs, is a common finding in both HPS and HFRS patients (4, 7, 11, 31, 42, 62). In fact, both diseases can present with renal or pulmonary sequelae, and the renal or pulmonary focus of hantavirus diseases is likely to result from hantavirus infection of endothelial cells within vast glomerular and pulmonary capillary beds (4, 7, 11, 31, 42, 62). All hantaviruses predominantly infect endothelial cells which line capillaries (31, 42, 44, 61, 62), and endothelial cells have a primary role in maintaining fluid barrier functions of the vasculature (1, 12, 55). Although hantaviruses do not lyse endothelial cells (44, 61), this primary cellular target underlies hantavirus-induced changes in capillary integrity. As a result, understanding altered endothelial cell responses following hantavirus infection is fundamental to defining the mechanism of permeability induced by pathogenic hantaviruses (1, 12, 55).Pathogenic, but not nonpathogenic, hantaviruses use β₃ integrins on the surface of endothelial cells and platelets for attachment (19, 21, 23, 39, 46), and β₃ integrins play prominent roles in regulating vascular integrity (3, 6, 8, 24, 48). Pathogenic hantaviruses bind to basal, inactive conformations of β₃ integrins (35, 46, 53) and days after infection inhibit β₃ integrin-directed endothelial cell migration (20, 46). This may be the result of cell-associated virus (19, 20, 22) which keeps β₃ in an inactive state but could also occur through additional regulatory processes that have yet to be defined. Interestingly, the nonpathogenic hantaviruses Prospect Hill virus (PHV) and Tula virus (TULV) fail to alter β₃ integrin functions, and their entry is consistent with the use of discrete α₅β₁ integrins (21, 23, 36).On endothelial cells, α_vβ₃ integrins normally regulate permeabilizing effects of vascular endothelial growth factor receptor-2 (VEGFR2) (3, 24, 48, 51). VEGF was initially identified as an edema-causing vascular permeability factor (VPF) that is 50,000 times more potent than histamine in directing fluid across capillaries (12, 14). VEGF is responsible for disassembling adherens junctions between endothelial cells to permit cellular movement, wound repair, and angiogenesis (8, 10, 12, 13, 17, 26, 57). Extracellular domains of β₃ integrins and VEGFR2 reportedly form a coprecipitable complex (3), and knocking out β₃ causes capillary permeability that is augmented by VEGF addition (24, 47, 48). Pathogenic hantaviruses inhibit β₃ integrin functions days after infection and similarly enhance the permeability of endothelial cells in response to VEGF (22).Adherens junctions form the primary fluid barrier of endothelial cells, and VEGFR2 responses control adherens junction disassembly (10, 17, 34, 57, 63). Vascular endothelial cadherin (VE-cadherin) is an endothelial cell-specific adherens junction protein and the primary determinant of paracellular permeability within the vascular endothelium (30, 33, 34). Activation of VEGFR2, another endothelial cell-specific protein, triggers signaling responses resulting in VE-cadherin disassembly and endocytosis, which increases the permeability of endothelial cell junctions (10, 12, 17, 34). VEGF is induced by hypoxic conditions and released by endothelial cells, platelets, and immune cells (2, 15, 38, 52). VEGF acts locally on endothelial cells through the autocrine or paracrine activation of VEGFR2, and the disassembly of endothelial cell adherens junctions increases the availability of nutrients to tissues and facilitates leukocyte trafficking and diapedesis (10, 12, 17, 55). The importance of endothelial cell barrier integrity is often in conflict with requirements for endothelial cells to move in order to permit angiogenesis and repair or cell and fluid egress, and as a result, VEGF-induced VE-cadherin responses are tightly controlled (10, 17, 18, 32, 33, 59). This limits capillary permeability while dynamically responding to a variety of endothelial cell-specific factors and conditions. However, if unregulated, this process can result in localized capillary permeability and edema (2, 9, 10, 12, 14, 17, 29, 60).Interestingly, tissue edema and hypoxia are common findings in both HPS and HFRS patients (11, 31, 62), and the ability of pathogenic hantaviruses to infect human endothelial cells provides a means for hantaviruses to directly alter normal VEGF-VE-cadherin regulation. In fact, the permeability of endothelial cells infected by pathogenic Andes virus (ANDV) or Hantaan virus (HTNV) is dramatically enhanced in response to VEGF addition (22). This response is absent from endothelial cells comparably infected with the nonpathogenic TULV and suggests that enhanced VEGF-induced endothelial cell permeability is a common underlying response of both HPS- and HFRS-causing hantaviruses (22). In these studies, we comparatively investigate responses of human endothelial cells infected with pathogenic ANDV and HTNV, as well as nonpathogenic TULV.     

Analysis of the Viral Elements Required in the Nuclear Import of HIV-1 DNA

HIV-1 possesses an exquisite ability to infect cells independently from their cycling status by undergoing an active phase of nuclear import through the nuclear pore. This property has been ascribed to the presence of karyophilic elements present in viral nucleoprotein complexes, such as the matrix protein (MA); Vpr; the integrase (IN); and a cis-acting structure present in the newly synthesized DNA, the DNA flap. However, their role in nuclear import remains controversial at best. In the present study, we carried out a comprehensive analysis of the role of these elements in nuclear import in a comparison between several primary cell types, including stimulated lymphocytes, macrophages, and dendritic cells. We show that despite the fact that none of these elements is absolutely required for nuclear import, disruption of the central polypurine tract-central termination sequence (cPPT-CTS) clearly affects the kinetics of viral DNA entry into the nucleus. This effect is independent of the cell cycle status of the target cells and is observed in cycling as well as in nondividing primary cells, suggesting that nuclear import of viral DNA may occur similarly under both conditions. Nonetheless, this study indicates that other components are utilized along with the cPPT-CTS for an efficient entry of viral DNA into the nucleus.Lentiviruses display an exquisite ability to infect dividing and nondividing cells alike that is unequalled among Retroviridae. This property is thought to be due to the particular behavior or composition of the viral nucleoprotein complexes (NPCs) that are liberated into the cytoplasm of target cells upon virus-to-cell membrane fusion and that allow lentiviruses to traverse an intact nuclear membrane (17, 28, 29, 39, 52, 55, 67, 79). In the case of the human immunodeficiency type I virus (HIV-1), several studies over the years identified viral components of such structures with intrinsic karyophilic properties and thus perfect candidates for mediation of the passage of viral DNA (vDNA) through the nuclear pore: the matrix protein (MA); Vpr; the integrase (IN); and a three-stranded DNA flap, a structure present in neo-synthesized viral DNA, specified by the central polypurine tract-central termination sequence (cPPT-CTS). It is clear that these elements may mediate nuclear import directly or via the recruitment of the host''s proteins, and indeed, several cellular proteins have been found to influence HIV-1 infection during nuclear import, like the karyopherin α2 Rch1 (38); importin 7 (3, 30, 93); the transportin SR-2 (13, 20); or the nucleoporins Nup98 (27), Nup358/RANBP2, and Nup153 (13, 56).More recently, the capsid protein (CA), the main structural component of viral nucleoprotein complexes at least upon their cytoplasmic entry, has also been suggested to be involved in nuclear import or in postnuclear entry steps (14, 25, 74, 90, 92). Whether this is due to a role for CA in the shaping of viral nucleoprotein complexes or to a direct interaction between CA and proteins involved in nuclear import remains at present unknown.Despite a large number of reports, no single viral or cellular element has been described as absolutely necessary or sufficient to mediate lentiviral nuclear import, and important controversies as to the experimental evidences linking these elements to this step exist. For example, MA was among the first viral protein of HIV-1 described to be involved in nuclear import, and 2 transferable nuclear localization signals (NLSs) have been described to occur at its N and C termini (40). However, despite the fact that early studies indicated that the mutation of these NLSs perturbed HIV-1 nuclear import and infection specifically in nondividing cells, such as macrophages (86), these findings failed to be confirmed in more-recent studies (23, 33, 34, 57, 65, 75).Similarly, Vpr has been implicated by several studies of the nuclear import of HIV-1 DNA (1, 10, 21, 43, 45, 47, 64, 69, 72, 73, 85). Vpr does not possess classical NLSs, yet it displays a transferable nucleophilic activity when fused to heterologous proteins (49-51, 53, 77, 81) and has been shown to line onto the nuclear envelope (32, 36, 47, 51, 58), where it can truly facilitate the passage of the viral genome into the nucleus. However, the role of Vpr in this step remains controversial, as in some instances Vpr is not even required for viral replication in nondividing cells (1, 59).Conflicting results concerning the role of IN during HIV-1 nuclear import also exist. Indeed, several transferable NLSs have been described to occur in the catalytic core and the C-terminal DNA binding domains of IN, but for some of these, initial reports of nuclear entry defects (2, 9, 22, 46, 71) were later shown to result from defects at steps other than nuclear import (60, 62, 70, 83). These reports do not exclude a role for the remaining NLSs in IN during nuclear import, and they do not exclude the possibility that IN may mediate this step by associating with components of the cellular nuclear import machinery, such as importin alpha and beta (41), importin 7 (3, 30, 93, 98), and, more recently, transportin-SR2 (20).The central DNA flap, a structure present in lentiviruses and in at least 1 yeast retroelement (44), but not in other orthoretroviruses, has also been involved in the nuclear import of viral DNA (4, 6, 7, 31, 78, 84, 95, 96), and more recently, it has been proposed to provide a signal for viral nucleoprotein complexes uncoating in the proximity of the nuclear pore, with the consequence of providing a signal for import (8). However, various studies showed an absence or weakness of nuclear entry defects in viruses devoid of the DNA flap (24, 26, 44, 61).Overall, the importance of viral factors in HIV-1 nuclear import is still unclear. The discrepancies concerning the role of MA, IN, Vpr, and cPPT-CTS in HIV-1 nuclear import could in part be explained by their possible redundancy. To date, only one comprehensive study analyzed the role of these four viral potentially karyophilic elements together (91). This study showed that an HIV-1 chimera where these elements were either deleted or replaced by their murine leukemia virus (MLV) counterparts was, in spite of an important infectivity defect, still able to infect cycling and cell cycle-arrested cell lines to similar efficiencies. If this result indicated that the examined viral elements of HIV-1 were dispensable for the cell cycle independence of HIV, as infections proceeded equally in cycling and arrested cells, they did not prove that they were not required in nuclear import, because chimeras displayed a severe infectivity defect that precluded their comparison with the wild type (WT).Nuclear import and cell cycle independence may not be as simply linked as previously thought. On the one hand, there has been no formal demonstration that the passage through the nuclear pore, and thus nuclear import, is restricted to nondividing cells, and for what we know, this passage may be an obligatory step in HIV infection in all cells, irrespective of their cycling status. In support of this possibility, certain mutations in viral elements of HIV affect nuclear import in dividing as well as in nondividing cells (4, 6, 7, 31, 84, 95). On the other hand, cell cycle-independent infection may be a complex phenomenon that is made possible not only by the ability of viral DNA to traverse the nuclear membrane but also by its ability to cope with pre- and postnuclear entry events, as suggested by the phenotypes of certain CA mutants (74, 92).Given that the cellular environment plays an important role during the early steps of viral infection, we chose to analyze the role of the four karyophilic viral elements of HIV-1 during infection either alone or combined in a wide comparison between cells highly susceptible to infection and more-restrictive primary cell targets of HIV-1 in vivo, such as primary blood lymphocytes (PBLs), monocyte-derived macrophages (MDM), and dendritic cells (DCs).In this study, we show that an HIV-1-derived virus in which the 2 NLSs of MA are mutated and the IN, Vpr, and cPPT-CTS elements are removed displays no detectable nuclear import defect in HeLa cells independently of their cycling status. However, this mutant virus is partially impaired for nuclear entry in primary cells and more specifically in DCs and PBLs. We found that this partial defect is specified by the cPPT-CTS, while the 3 remaining elements seem to play no role in nuclear import. Thus, our study indicates that the central DNA flap specifies the most important role among the viral elements involved thus far in nuclear import. However, it also clearly indicates that the role played by the central DNA flap is not absolute and that its importance varies depending on the cell type, independently from the dividing status of the cell.     

Isolation of Human Monoclonal Antibodies That Potently Neutralize Human Cytomegalovirus Infection by Targeting Different Epitopes on the gH/gL/UL128-131A Complex

Human cytomegalovirus (HCMV) is a widely circulating pathogen that causes severe disease in immunocompromised patients and infected fetuses. By immortalizing memory B cells from HCMV-immune donors, we isolated a panel of human monoclonal antibodies that neutralized at extremely low concentrations (90% inhibitory concentration [IC₉₀] values ranging from 5 to 200 pM) HCMV infection of endothelial, epithelial, and myeloid cells. With the single exception of an antibody that bound to a conserved epitope in the UL128 gene product, all other antibodies bound to conformational epitopes that required expression of two or more proteins of the gH/gL/UL128-131A complex. Antibodies against gB, gH, or gM/gN were also isolated and, albeit less potent, were able to neutralize infection of both endothelial-epithelial cells and fibroblasts. This study describes unusually potent neutralizing antibodies against HCMV that might be used for passive immunotherapy and identifies, through the use of such antibodies, novel antigenic targets in HCMV for the design of immunogens capable of eliciting previously unknown neutralizing antibody responses.Human cytomegalovirus (HCMV) is a member of the herpesvirus family which is widely distributed in the human population and can cause severe disease in immunocompromised patients and upon infection of the fetus. HCMV infection causes clinical disease in 75% of patients in the first year after transplantation (58), while primary maternal infection is a major cause of congenital birth defects including hearing loss and mental retardation (5, 33, 45). Because of the danger posed by this virus, development of an effective vaccine is considered of highest priority (51).HCMV infection requires initial interaction with the cell surface through binding to heparan sulfate proteoglycans (8) and possibly other surface receptors (12, 23, 64, 65). The virus displays a broad host cell range (24, 53), being able to infect several cell types such as endothelial cells, epithelial cells (including retinal cells), smooth muscle cells, fibroblasts, leukocytes, and dendritic cells (21, 37, 44, 54). Endothelial cell tropism has been regarded as a potential virulence factor that might influence the clinical course of infection (16, 55), whereas infection of leukocytes has been considered a mechanism of viral spread (17, 43, 44). Extensive propagation of HCMV laboratory strains in fibroblasts results in deletions or mutations of genes in the UL131A-128 locus (1, 18, 21, 36, 62, 63), which are associated with the loss of the ability to infect endothelial cells, epithelial cells, and leukocytes (15, 43, 55, 61). Consistent with this notion, mouse monoclonal antibodies (MAbs) to UL128 or UL130 block infection of epithelial and endothelial cells but not of fibroblasts (63). Recently, it has been shown that UL128, UL130, and UL131A assemble with gH and gL to form a five-protein complex (thereafter designated gH/gL/UL128-131A) that is an alternative to the previously described gCIII complex made of gH, gL, and gO (22, 28, 48, 63).In immunocompetent individuals T-cell and antibody responses efficiently control HCMV infection and reduce pathological consequences of maternal-fetal transmission (13, 67), although this is usually not sufficient to eradicate the virus. Albeit with controversial results, HCMV immunoglobulins (Igs) have been administered to transplant patients in association with immunosuppressive treatments for prophylaxis of HCMV disease (56, 57), and a recent report suggests that they may be effective in controlling congenital infection and preventing disease in newborns (32). These products are plasma derivatives with relatively low potency in vitro (46) and have to be administered by intravenous infusion at very high doses in order to deliver sufficient amounts of neutralizing antibodies (4, 9, 32, 56, 57, 66).The whole spectrum of antigens targeted by HCMV-neutralizing antibodies remains poorly characterized. Using specific immunoabsorption to recombinant antigens and neutralization assays using fibroblasts as model target cells, it was estimated that 40 to 70% of the serum neutralizing activity is directed against gB (6). Other studies described human neutralizing antibodies specific for gB, gH, or gM/gN viral glycoproteins (6, 14, 26, 29, 34, 41, 52, 60). Remarkably, we have recently shown that human sera exhibit a more-than-100-fold-higher potency in neutralizing infection of endothelial cells than infection of fibroblasts (20). Similarly, CMV hyperimmunoglobulins have on average 48-fold-higher neutralizing activities against epithelial cell entry than against fibroblast entry (10). However, epitopes that are targeted by the antibodies that comprise epithelial or endothelial cell-specific neutralizing activity of human immune sera remain unknown.In this study we report the isolation of a large panel of human monoclonal antibodies with extraordinarily high potency in neutralizing HCMV infection of endothelial and epithelial cells and myeloid cells. With the exception of a single antibody that recognized a conserved epitope of UL128, all other antibodies recognized conformational epitopes that required expression of two or more proteins of the gH/gL/UL128-131A complex.     

Attenuation of Vesicular Stomatitis Virus Encephalitis through MicroRNA Targeting

Vesicular stomatitis virus (VSV) has long been regarded as a promising recombinant vaccine platform and oncolytic agent but has not yet been tested in humans because it causes encephalomyelitis in rodents and primates. Recent studies have shown that specific tropisms of several viruses could be eliminated by engineering microRNA target sequences into their genomes, thereby inhibiting spread in tissues expressing cognate microRNAs. We therefore sought to determine whether microRNA targets could be engineered into VSV to ameliorate its neuropathogenicity. Using a panel of recombinant VSVs incorporating microRNA target sequences corresponding to neuron-specific or control microRNAs (in forward and reverse orientations), we tested viral replication kinetics in cell lines treated with microRNA mimics, neurotoxicity after direct intracerebral inoculation in mice, and antitumor efficacy. Compared to picornaviruses and adenoviruses, the engineered VSVs were relatively resistant to microRNA-mediated inhibition, but neurotoxicity could nevertheless be ameliorated significantly using this approach, without compromise to antitumor efficacy. Neurotoxicity was most profoundly reduced in a virus carrying four tandem copies of a neuronal mir125 target sequence inserted in the 3′-untranslated region of the viral polymerase (L) gene.Vesicular stomatitis virus (VSV) is a nonsegmented, negative-strand rhabdovirus widely used as a vaccine platform as well as an anticancer therapeutic. While VSV is predominantly a pathogen of livestock (34), it has a very broad species tropism. The cellular tropism of VSV is determined predominantly at postentry steps, since the G glycoprotein of the virus mediates entry into most tissues in nearly all animal species (10).Though viral entry can take place in nearly all cell types, in vivo models of VSV infection have revealed that the virus is highly sensitive to the innate immune response, limiting its pathogenesis (4). VSV is intensively responsive to type I interferon (IFN), as the double-stranded RNA (dsRNA)-dependent PKR (2), the downstream effector of pattern recognition receptors MyD88 (32), and other molecules mediate shutdown of viral translation and allow the adaptive immune response to clear the virus. The vulnerability of the virus to the type I IFN response, typically defective in many cancers, has been exploited to generate tumor-selective replication (49), such that the virus is now poised to enter phase I trials. However, the virus remains potently neurotoxic, causing lethal encephalitis not only in rodent models (7, 22, 53) but also in nonhuman primates (25).VSV very often infiltrates the central nervous system (CNS) through infection of the olfactory nerves (41). When administered intranasally, the virus replicates rapidly in the nasal epithelium and is transmitted to olfactory neurons, from which it then moves retrograde axonally to the brain and replicates robustly, causing neuropathogenesis. While intranasal inoculation does cause neuropathy in mice, neurotoxicity following viral administration also occurs when the virus is delivered intravascularly (47), intraperitoneally (42), and (not surprisingly) intracranially (13). Previously, other groups have modified the VSV genome to be more sensitive to cellular IFNs (49) and have actually encoded IFN in the virus (36). However, the former can result in attenuation of the virus, such that it has reduced anticancer potential, while the latter still results in lethal encephalitis (unpublished results). In order to mitigate the effects of VSV infection on the brain without perturbing the potent oncolytic activity of the virus, we utilized a microRNA (miRNA) targeting paradigm, whereby viral replication is restricted in the brain without altering the tropism of the virus for other tissues.To redirect the tissue tropism of anticancer therapeutics, we (26) and others (11, 14, 55) have previously exploited the tissue-specific expression of cellular miRNAs. miRNAs are ∼22-nucleotide (nt) regulatory RNAs that regulate a diverse and expansive array of cellular activities. Through recognition of sequence-complementary target elements, miRNAs can either translationally suppress or catalytically degrade both cellular (6) and viral (50) RNAs. We have determined that cellular miRNAs can potentially regulate numerous steps of a virus life cycle and that this regulation of the virus by endogenous miRNAs can then abrogate toxicities of replication-competent viruses (27; E. J. Kelly et al., unpublished data).miRNAs are known to be highly upregulated in many different tissues, including (but not limited to) muscle (40), lung (44), liver (15, 44), spleen (44, 46), and kidney (51). In addition, the brain has a number of upregulated miRNAs, with each different subtype of cell having a unique miRNA profile. miR-125 is highly upregulated in all cells in the brain (neurons, astrocytes, and glia cells), while miR-124 is found predominantly in neuronal cells (48). Glial cells and glioblastomas are thought to have decreased expression of miR-128 compared to neurons (17), while miR-134 is particularly abundant in dendrites of neurons in the hippocampus (43). In addition to these miRNAs, the tumor suppressor miRNA let-7 and miRs 9, 26, and 29 (51) are also found to be enriched in the brain, with expression varying not only between different cell types and regions of the brain but also temporally (48).MicroRNAs have previously been exploited to modulate the tissue tropism of nonreplicating lentiviral vectors (8, 9), as well as curbing known toxicities of replication-competent picornaviruses (5, 26), adenoviruses (11), herpes simplex virus 1 (33), and influenza A virus (39). In addition, a recombinant VSV encoding a tumor suppressor target was found to be responsive to sequence-complementary miRNAs in vitro, possibly by affecting expression of the matrix (M) protein (14), and evidence from Dicer-deficient mice suggests that endogenously expressed microRNA targets within the P and L genes of VSV could restrict enhanced pathogenicity of the virus (37). However, in vivo protection from neuropathogenesis by this means has not been demonstrated for VSV.Here we evaluate the efficiencies of different brain-specific miRNAs for shutting down gene expression and extensively characterize the ability of miRNA targeting to attenuate the neurotoxicity of vesicular stomatitis virus in vivo. We constructed and evaluated recombinant VSVs with miRNA target (miRT) insertions at different regions of the viral genome, with special focus upon those affecting viral L expression. In addition, we looked at the regulatory efficiency of different brain-specific miRNAs and the impact of miRT orientation on VSV replication and determined the impact of the virus on oncolytic activity in vivo.