Type I Interferon-Sensitive Recombinant Newcastle Disease Virus for Oncolytic Virotherapy

Newcastle disease virus (NDV), an avian paramyxovirus, is tumor selective and intrinsically oncolytic because of its potent ability to induce apoptosis. Several studies have demonstrated that NDV is selectively cytotoxic to tumor cells but not normal cells due to defects in the interferon (IFN) antiviral responses of tumor cells. Many naturally occurring strains of NDV have an intact IFN-antagonistic function and can still replicate in normal human cells. To avoid potential toxicity issues with NDV, especially in cancer patients with immunosuppression, safe NDV-oncolytic vectors are needed. We compared the cell killing abilities of (i) a recombinant NDV (rNDV) strain, Beaudette C, containing an IFN-antagonistic, wild-type V protein (rBC), (ii) an isogenic recombinant virus with a mutant V protein (rBC-Edit virus) that induces increased IFN in infected cells and whose replication is restricted in normal human cells, and (iii) a recombinant LaSota virus with a virulent F protein cleavage site that is as interferon sensitive as rBC-Edit virus (LaSota V.F. virus). Our results indicated that the tumor-selective replication of rNDV is determined by the differential regulation of IFN-α and downstream antiviral genes induced by IFN-α, especially through the IRF-7 pathway. In a nude mouse model of human fibrosarcoma, we show that the IFN-sensitive NDV variants are as effective as IFN-resistant rBC virus in clearing the tumor burden. In addition, mice treated with rNDV exhibited no signs of toxicity to the viruses. These findings indicate that augmentation of innate immune responses by NDV results in selective oncolysis and offer a novel and safe virotherapy platform.Several naturally occurring or engineered oncolytic viruses are emerging as novel tools for selective growth in and killing of a variety of tumor cells (1, 21, 34, 41). It has been consistently reported that during tumor evolution, diminished interferon (IFN) responsiveness coevolves as a frequent genetic defect (4, 31, 32, 41). Any defects in responsiveness to interferon will afford permissiveness of tumors for replication of oncolytic viruses by blunting the antiviral innate immune system. Thus, it was suggested that oncolytic viruses could be engineered to induce strong IFN response and/or to be defective in antagonizing the IFN signaling. This would result in virus replication in tumor cells with IFN defects but in reduced or crippled virus replication in normal cells, with the absence of toxicity (42). A variety of oncolytic viruses have been engineered to exploit tumor-specific genetic defects (3, 12, 24, 42, 46) and shown to be potent oncolytic agents.Newcastle disease virus (NDV), an avian paramyxovirus, is a promising broad-spectrum oncolytic agent (27, 29, 30, 37). Nonengineered, naturally occurring strains of NDV such as 73-T (6), MTH68 (7), PV701 (28, 35), and NDV-HUJ (11) have been successfully employed in several clinical studies for tumor regression. NDV is inherently oncolytic and tumor selective, sparing normal cells (9, 15, 37). The tumor selectivity of NDV is considered to be due to a defective IFN response in tumor cells (10, 23, 37). NDV is a strong inducer of type I IFN in many types of cells (18). In normal cells, a robust IFN-mediated antiviral response limits the replication of NDV (9, 23). This known sensitivity of NDV to cellular antiviral mechanisms affords a wide safety margin for its use in humans.Recent studies have indicated that improved therapeutic vectors of NDV could be engineered through reverse genetics for enhanced oncolytic efficacy from an increased anti-tumor response and interleukin 2 (IL-2) receptor-mediated targeting (5, 9, 44, 46). Therefore, we reasoned that recombinant NDVs (rNDVs) that are susceptible to cellular innate immune responses would be safer and more effective oncolytic agents. Even though NDV is an avian virus and induces a strong IFN response in normal human cells, it still expresses IFN-antagonizing activity. Ablation of the expression of V protein, which is responsible for this anti-IFN activity, may further reduce the ability of NDV to infect and kill normal human cells without affecting tumor cell infection and lysis. Here, we describe the relative oncolytic efficacies of three rNDV strains differing in IFN antagonism. The rNDV variants with an IFN-sensitive phenotype had parallel therapeutic efficacies in xenotransplanted human fibrosarcoma cells in a nude mouse model and offer great potential as recombinant vectors in therapy of human malignancies.     

Killing of Avian and Swine Influenza Virus by Natural Killer Cells

Today, global attention is focused on two influenza virus strains: the current pandemic strain, swine origin influenza virus (H1N1-2009), and the highly pathogenic avian influenza virus, H5N1. At present, the infection caused by the H1N1-2009 is moderate, with mortality rates of less <1%. In contrast, infection with the H5N1 virus resulted in high mortality rates, and ca. 60% of the infected patients succumb to the infection. Thus, one of the world greatest concerns is that the H5N1 virus will evolve to allow an efficient human infection and human-to-human transmission. Natural killer (NK) cells are one of the innate immune components playing an important role in fighting against influenza viruses. One of the major NK activating receptors involved in NK cell cytotoxicity is NKp46. We previously demonstrated that NKp46 recognizes the hemagglutinin proteins of B and A influenza virus strains. Whether NKp46 could also interact with H1N1-2009 virus or with the avian influenza virus is still unknown. We analyzed the immunological properties of both the avian and the H1N1-2009 influenza viruses. We show that NKp46 recognizes the hemagglutinins of H1N1-2009 and H5 and that this recognition leads to virus killing both in vitro and in vivo. However, importantly, while the swine H1-NKp46 interactions lead to the direct killing of the infected cells, the H5-NKp46 interactions were unable to elicit direct killing, probably because the NKp46 binding sites for these two viruses are different.Natural killer (NK) cells, which comprise 5 to 15% of peripheral blood lymphocytes, are a key frontline defense against a number of pathogens, including intracellular bacteria, parasites, and most importantly with respect to the present study, viruses (6, 40). The antiviral mechanisms by which NK cells operate include both cytotoxic activity and cytokine/chemokine secretion (21). The NK killing activity is executed by numerous receptors, including NKG2D, NKp80, CD16, and the natural cytotoxic receptors (NCRs): NKp30, NKp44, and NKp46 (7, 10, 25).Although the cellular ligands for NKG2D were identified (31, 38), the identity of several of the cellular ligands for the human NCRs is still unknown, except for BAT3 and B7-H6, which are ligands for NKp30 (8, 30). In contrast, viral ligands were identified for the NCRs, and we demonstrated that pp65 of HCMV interacts with NKp30 (3) and that various influenza virus hemagglutinins (HAs) are ligands for the NKp44 and NKp46 receptors (5, 22). Supporting these observations, it was recently shown that the HA-neuraminidase of Newcastle disease virus could also interact with NKp46 and NKp44 but not with NKp30 (17). Furthermore, we have shown in vivo that in the absence of NCR1 (the mouse homologue of NKp46), A/PR8 influenza virus infection is lethal (14).Human influenza virus (H1 and H3 subtype) infections pose a major threat to the entire population, as exemplified by the three major influenza pandemics that occurred during the 20th century. The Asian (A/H2N2) in 1957 to 1958 and the Hong Kong (A/H3N2) pandemics in 1968 to 1969 resulted in the deaths of 1 to 2 million people and the 1918 “Spanish flu” (A/H1N1) pandemic killed around 50 million people (18). At present, the worldwide concern regarding influenza pandemics concentrates mainly on two viruses: the A/H1N1 swine origin influenza virus (H1N1-2009), which currently causes only a moderate pandemic (the mortality rates are ca. 1%) but is more pathogenic than a regular seasonal influenza virus (19, 26, 27), and the avian influenza virus carrying the unique H5 HA (20). The avian influenza virus is quite deadly and, although it remains a zoonotic infection, ca. 60% of infected humans died due to the infection (28).The unique properties of the H5 protein of the avian influenza virus are one of the main reasons for the virulence of the virus. The H5 of the avian influenza virus binds to cell surface glycoproteins or glycolipids containing terminal sialyl-galactosyl residues linked by 2-3-linkage [Neu5Ac(α2-3)Gal] that are found in the human conjunctiva and ciliated portion of the respiratory columnar epithelium (33). In contrast, human viruses (including all three strains that caused the pandemics described above and the H1N1-2009) bind to receptors that mostly contain terminal 2-6-linked sialyl-galactosyl moieties [Neu5Ac(α2-6)Gal]. Such glycosylations are predominant on epithelial cells in the nasal mucosa, paranasal sinuses, pharynx, trachea, and bronchi (33, 37). It has been suggested that the lack of human-to-human transmission of avian influenza viruses is due to their α2,3-SA receptor binding preference, and the concern is that genetic changes in H5 might alter its preference from α2,3-SA to α2,6-SA, allowing human-to-human transmission.In our previous studies (4, 22) we showed that the interaction between NKp46 and influenza virus HAs depends on the sialylation of the NKp46 receptor. We further demonstrated that the sialic acid residues, which are linked via α2,6 to the threonine 225 residue of NKp46, are crucial for the NKp46 interactions with the various influenza virus HAs (4).We show that, both in vitro and in vivo, the killing of H1N1-2009-infected cells is correlated with the degree of NKp46 binding. Surprisingly, we observed that although NKp46 efficiently recognized the avian H5 HA, such interactions were unable to elicit the direct killing of the infected cells. By using mutagenesis analysis experiments and killing assays we demonstrate that NKp46 interacts with H1 and H5 at distinct sites, since we show that the sugar carrying residue at position 225 is crucial for the NKp46-H1N1-2009 interactions, whereas the interaction of H5 with NKp46 depends on both residues 216 and 225.     

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.     

Protein Tyrosine Kinase 6 Directly Phosphorylates AKT and Promotes AKT Activation in Response to Epidermal Growth Factor

Protein tyrosine kinase 6 (PTK6) is a nonmyristoylated Src-related intracellular tyrosine kinase. Although not expressed in the normal mammary gland, PTK6 is expressed in a majority of human breast tumors examined, and it has been linked to ErbB receptor signaling and AKT activation. Here we demonstrate that AKT is a direct substrate of PTK6 and that AKT tyrosine residues 315 and 326 are phosphorylated by PTK6. Association of PTK6 with AKT occurs through the SH3 domain of PTK6 and is enhanced through SH2 domain-mediated interactions following tyrosine phosphorylation of AKT. Using Src, Yes, and Fyn null mouse embryonic fibroblasts (SYF cells), we show that PTK6 phosphorylates AKT in a Src family kinase-independent manner. Introduction of PTK6 into SYF cells sensitized these cells to physiological levels of epidermal growth factor (EGF) and increased AKT activation. Stable introduction of active PTK6 into SYF cells also resulted in increased proliferation. Knockdown of PTK6 in the BPH-1 human prostate epithelial cell line led to decreased AKT activation in response to EGF. Our data indicate that in addition to promoting growth factor receptor-mediated activation of AKT, PTK6 can directly activate AKT to promote oncogenic signaling.Protein tyrosine kinase 6 (PTK6; also known as the breast tumor kinase BRK) is an intracellular Src-related tyrosine kinase (9, 48). Human PTK6 was identified in cultured human melanocytes (32) and breast tumor cells (39), while its mouse orthologue was cloned from normal small intestinal epithelial cell RNA (50). Although PTK6 shares overall structural similarity with Src family tyrosine kinases, it lacks an N-terminal myristoylation consensus sequence for membrane targeting (39, 51). As a consequence, PTK6 is localized to different cellular compartments, including the nucleus (14, 15). PTK6 is expressed in normal differentiated epithelial cells of the gastrointestinal tract (34, 42, 51), prostate (14), and skin (51-53). Expression of PTK6 is upregulated in different types of cancers, including breast carcinomas (6, 39, 54), colon cancer (34), ovarian cancer (47), head and neck cancers (33), and metastatic melanoma cells (16). The significance of apparent opposing signaling roles for PTK6 in normal differentiation and cancer is still poorly understood.In human breast tumor cells, PTK6 enhances signaling from members of the ErbB receptor family (10, 29, 30, 36, 40, 49, 54). In the HB4a immortalized human mammary gland luminal epithelial cell line, PTK6 promoted epidermal growth factor (EGF)-induced ErbB3 tyrosine phosphorylation and AKT activation (29). In response to EGF stimulation, PTK6 promoted phosphorylation of the focal adhesion protein paxillin and Rac1-mediated cell migration (10). PTK6 can be activated by the ErbB3 ligand heregulin and promotes activation of extracellular signal-regulated kinase 5 (ERK5) and p38 mitogen-activated protein kinase (MAPK) in breast cancer cells (40). PTK6 can also phosphorylate p190RhoGAP-A and stimulate its activity, leading to RhoA inactivation and Ras activation and thereby promoting EGF-dependent breast cancer cell migration and proliferation (49). Expression of PTK6 has been correlated with ErbB2 expression in human breast cancers (4, 5, 54).AKT (also called protein kinase B) is a serine-threonine kinase that is activated downstream of growth factor receptors (38). It is a key player in signaling pathways that regulate energy metabolism, proliferation, and cell survival (7, 45). Aberrant activation of AKT through diverse mechanisms has been discovered in different cancers (2). AKT activation requires phosphorylation of AKT on threonine residue 308 and serine residue 473. The significance of phosphorylation of AKT on tyrosine residues is less well understood. Src has been shown to phosphorylate AKT on conserved tyrosine residues 315 and 326 near the activation loop (11). Substitution of these two tyrosine residues with phenylalanine abolished AKT kinase activity stimulated by EGF (11). Use of the Src family inhibitor PP2 impaired AKT activation following IGF-1 stimulation of oligodendrocytes (13). The RET/PTC receptor tyrosine kinase that responds to glial cell-line-derived neurotrophic factor also phosphorylated AKT tyrosine residue 315 promoting activation of AKT (28). AKT tyrosine residue 474 was phosphorylated when cells were treated with the tyrosine phosphatase inhibitor pervanadate, and phosphorylation of tyrosine 474 contributed to full activation of AKT (12). Recently, the nonreceptor tyrosine kinase Ack1 was shown to regulate AKT tyrosine phosphorylation and activation (37).Here we show that AKT is a cytoplasmic substrate of the intracellular tyrosine kinase PTK6. We identify the tyrosine residues on AKT that are targeted by PTK6, and we demonstrate that tyrosine phosphorylation plays a role in regulating association between PTK6 and AKT. In addition, we show that PTK6 promotes AKT activation and cell proliferation in a Src-independent manner.     

Differential Activation of NK Cells by Influenza A Pseudotype H5N1 and 1918 and 2009 Pandemic H1N1 Viruses

Natural killer (NK) cells are the effectors of innate immunity and are recruited into the lung 48 h after influenza virus infection. Functional NK cell activation can be triggered by the interaction between viral hemagglutinin (HA) and natural cytotoxicity receptors NKp46 and NKp44 on the cell surface. Recently, novel subtypes of influenza viruses, such as H5N1 and 2009 pandemic H1N1, transmitted directly to the human population, with unusual mortality and morbidity rates. Here, the human NK cell responses to these viruses were studied. Differential activation of heterogeneous NK cells (upregulation of CD69 and CD107a and gamma interferon [IFN-γ] production as well as downregulation of NKp46) was observed following interactions with H5N1, 1918 H1N1, and 2009 H1N1 pseudotyped particles (pps), respectively, and the responses of the CD56^dim subset predominated. Much stronger NK activation was triggered by H5N1 and 1918 H1N1 pps than by 2009 H1N1 pps. The interaction of pps with NK cells and subsequent internalization were mediated by NKp46 partially. The NK cell activation by pps showed a dosage-dependent manner, while an increasing viral HA titer attenuated NK activation phenotypes, cytotoxicity, and IFN-γ production. The various host innate immune responses to different influenza virus subtypes or HA titers may be associated with disease severity.Influenza is a contagious, acute respiratory disease caused by influenza viruses and has caused substantial human morbidity and mortality over the past century (24, 27). The 1918-1919 pandemic caused by influenza virus type A H1N1 was responsible for an estimated 50 million deaths (21). In recent years, novel subtype influenza viruses, such as H5N1 and the 2009 pandemic H1N1, have been transmitted directly from animals to the human population. These infections were characterized by unusually high rates of severe respiratory disease and mortality among young patients (8, 18). Various genetic shifts have occurred in these viruses, allowing them to evade the host protective effects of specific antihemagglutinin (HA) or antineuraminidase (NA) antibodies (27). Therefore, host innate immunity in the early phase of infection, which includes a variety of pattern recognition molecules, inflammatory cytokines, and immune cells, such as macrophages and natural killer (NK) cells, plays a critical role in host defense.NK cells are bone marrow-derived, large, granular lymphocytes and are key effector cells in innate immunity for host defense against invading infectious pathogens and malignant transformation through cytolytic activity and production of cytokines, such as gamma interferon (IFN-γ) (10, 28, 43, 51). In humans, NK cells account for approximately 10% of all blood lymphocytes and are identified by their expression of the CD56 surface antigen and their lack of CD3. Two distinct subsets of human NK cells have been defined according to the cell surface density of CD56 expression (10). The majority (∼90% in blood) of human NK cells are CD56^dim, and a minor population (∼10% in blood) is CD56^bright. These NK subsets are functionally distinct, with the immunoregulatory CD56^bright cells producing abundant cytokines and the cytotoxic CD56^dim cells probably functioning as efficient effectors of natural and antibody-dependent target cell lysis (11).Many lines of evidence suggest that NK cells can be functionally activated by the interaction between natural cytotoxicity receptors (NCRs) on the cell surface and influenza virus HA protein or stress-induced proteins from infected cells (2, 13, 33, 44, 46). On the other hand, influenza virus is able to evade host immunity by infecting NK cells and triggering cell apoptosis or by attenuating NK cell lysis of H3N2-infected cells, owing to alterations in HA binding properties (35, 39). The infiltration of macrophages and lymphocytes into the lung and strong inflammatory responses were detected in H5N1 and the 1918 and 2009 pandemic H1N1 infections. Nevertheless, little is known about the precise roles of NK cells in these infections.In this study, the responses of NK cells to 1918 H1N1, 2009 H1N1, and H5N1 influenza A viruses were evaluated using three strains of influenza A virus pseudotyped particles (pps). Our findings may aid in understanding the pathogenicity of influenza viruses and its correlation with clinical severity.     

Cell-to-Cell Spread of the RNA Interference Response Suppresses Semliki Forest Virus (SFV) Infection of Mosquito Cell Cultures and Cannot Be Antagonized by SFV

In their vertebrate hosts, arboviruses such as Semliki Forest virus (SFV) (Togaviridae) generally counteract innate defenses and trigger cell death. In contrast, in mosquito cells, following an early phase of efficient virus production, a persistent infection with low levels of virus production is established. Whether arboviruses counteract RNA interference (RNAi), which provides an important antiviral defense system in mosquitoes, is an important question. Here we show that in Aedes albopictus-derived mosquito cells, SFV cannot prevent the establishment of an antiviral RNAi response or prevent the spread of protective antiviral double-stranded RNA/small interfering RNA (siRNA) from cell to cell, which can inhibit the replication of incoming virus. The expression of tombusvirus siRNA-binding protein p19 by SFV strongly enhanced virus spread between cultured cells rather than virus replication in initially infected cells. Our results indicate that the spread of the RNAi signal contributes to limiting virus dissemination.In animals, RNA interference (RNAi) was first described for Caenorhabditis elegans (27). The production or introduction of double-stranded RNA (dsRNA) in cells leads to the degradation of mRNAs containing homologous sequences by sequence-specific cleavage of mRNAs. Central to RNAi is the production of 21- to 26-nucleotide small interfering RNAs (siRNAs) from dsRNA and the assembly of an RNA-induced silencing complex (RISC), followed by the degradation of the target mRNA (23, 84). RNAi is a known antiviral strategy of plants (3, 53) and insects (21, 39, 51). Study of Drosophila melanogaster in particular has given important insights into RNAi responses against pathogenic viruses and viral RNAi inhibitors (31, 54, 83, 86, 91). RNAi is well characterized for Drosophila, and orthologs of antiviral RNAi genes have been found in Aedes and Culex spp. (13, 63).Arboviruses, or arthropod-borne viruses, are RNA viruses mainly of the families Bunyaviridae, Flaviviridae, and Togaviridae. The genus Alphavirus within the family Togaviridae contains several mosquito-borne pathogens: arboviruses such as Chikungunya virus (16) and equine encephalitis viruses (88). Replication of the prototype Sindbis virus and Semliki Forest virus (SFV) is well understood (44, 71, 74, 79). Their genome consists of a positive-stranded RNA with a 5′ cap and a 3′ poly(A) tail. The 5′ two-thirds encodes the nonstructural polyprotein P1234, which is cleaved into four replicase proteins, nsP1 to nsP4 (47, 58, 60). The structural polyprotein is encoded in the 3′ one-third of the genome and cleaved into capsid and glycoproteins after translation from a subgenomic mRNA (79). Cytoplasmic replication complexes are associated with cellular membranes (71). Viruses mature by budding at the plasma membrane (35).In nature, arboviruses are spread by arthropod vectors (predominantly mosquitoes, ticks, flies, and midges) to vertebrate hosts (87). Little is known about how arthropod cells react to arbovirus infection. In mosquito cell cultures, an acute phase with efficient virus production is generally followed by the establishment of a persistent infection with low levels of virus production (9). This is fundamentally different from the cytolytic events following arbovirus interactions with mammalian cells and pathogenic insect viruses with insect cells. Alphaviruses encode host response antagonists for mammalian cells (2, 7, 34, 38).RNAi has been described for mosquitoes (56) and, when induced before infection, antagonizes arboviruses and their replicons (1, 4, 14, 15, 29, 30, 32, 42, 64, 65). RNAi is also functional in various mosquito cell lines (1, 8, 43, 49, 52). In the absence of RNAi, alphavirus and flavivirus replication and/or dissemination is enhanced in both mosquitoes and Drosophila (14, 17, 31, 45, 72). RNAi inhibitors weakly enhance SFV replicon replication in tick and mosquito cells (5, 33), posing the questions of how, when, and where RNAi interferes with alphavirus infection in mosquito cells.Here we use an A. albopictus-derived mosquito cell line to study RNAi responses to SFV. Using reporter-based assays, we demonstrate that SFV cannot avoid or efficiently inhibit the establishment of an RNAi response. We also demonstrate that the RNAi signal can spread between mosquito cells. SFV cannot inhibit cell-to-cell spread of the RNAi signal, and spread of the virus-induced RNAi signal (dsRNA/siRNA) can inhibit the replication of incoming SFV in neighboring cells. Furthermore, we show that SFV expression of a siRNA-binding protein increases levels of virus replication mainly by enhancing virus spread between cells rather than replication in initially infected cells. Taken together, these findings suggest a novel mechanism, cell-to-cell spread of antiviral dsRNA/siRNA, by which RNAi limits SFV dissemination in mosquito cells.     

Influenza Virus Directly Infects Human Natural Killer Cells and Induces Cell Apoptosis

Influenza is an acute respiratory viral disease that is transmitted in the first few days of infection. Evasion of host innate immune defenses, including natural killer (NK) cells, is important for the virus''s success as a pathogen of humans and other animals. NK cells encounter influenza viruses within the microenvironment of infected cells and are important for host innate immunity during influenza virus infection. It is therefore important to investigate the direct effects of influenza virus on NK cells. In this study, we demonstrated for the first time that influenza virus directly infects and replicates in primary human NK cells. Viral entry into NK cells was mediated by both clathrin- and caveolin-dependent endocytosis rather than through macropinocytosis and was dependent on the sialic acids on cell surfaces. In addition, influenza virus infection induced a marked apoptosis of NK cells. Our findings suggest that influenza virus can directly target and kill NK cells, a potential novel strategy of influenza virus to evade the NK cell innate immune defense that is likely to facilitate viral transmission and may also contribute to virus pathogenesis.Influenza is an acute respiratory virus infection that continues to pose endemic, zoonotic, and pandemic threats to human health, with significant morbidity and mortality (17). At the early phase of viral infection, innate immunity plays important roles in host defense by limiting viral replication and helping to initiate an adaptive immune response. Natural killer (NK) cells are key effector cells in innate immunity and play a critical role in the first line of host defense against acute viral infections by directly destroying infected cells without the need for prior antigen stimulation (7, 20). As influenza illness and virus transmission usually occur in the first few days of infection, the virus has to devise strategies to evade host innate immune responses, including NK cell immunity (15, 21).NK cells can recognize and kill influenza virus-infected cells (2, 10, 23); to counteract this killing, however, influenza virus has developed an escape strategy that inhibits NK cell cytotoxicity by increasing the binding of two inhibitory receptors to the infected cells after infection (1). The individuals with complete NK cell deficiency developed life-threatening varicella zoster virus and cytomegalovirus infection, but no severe influenza virus infection occurred (30, 40). Indeed, the interaction between human NK cells and influenza virus remains poorly understood. After influenza virus infection, respiratory epithelial cells release inflammatory chemokines that recruit NK cells to the site of infection (12). As a lytic virus, numerous influenza virus particles are released from the infected epithelia and macrophages (5, 9, 33). In the infected microenvironment, NK cells undoubtedly encounter these infective virus particles. It is therefore important to investigate the direct interaction of NK cells with influenza virus. Patients with severe influenza virus infection were shown to have diminished NK cells in peripheral blood and an almost complete absence of pulmonary NK cells, together with marked apoptosis (13, 42). During influenza virus infection in mice, a transient increase of NK cytotoxicity is followed by a marked decrease in NK cell activity, with a virus dose-dependent effect (8, 28). These data suggest that influenza virus may directly target NK cells as part of its immunoevasion strategies. However, no reports of the direct effects of influenza virus on human NK cells have so far been available.In this study, we demonstrated that influenza virus infects and replicates in primary human NK cells. Viral infection was dependent on sialic acids on the cells. The entry was mediated by both clathrin- and caveolin-dependent endocytosis rather than macropinocytosis. Influenza virus infection induced a marked apoptosis of NK cells, which contributed to reduced NK cell cytotoxicity. This, to the best of our knowledge, is the first paper to demonstrate that influenza virus can directly infect NK cells and induce cell apoptosis. These findings suggest that influenza virus may have developed a novel strategy to evade NK cell innate immune defenses, which is likely to facilitate viral transmission and may also contribute to virus pathogenesis.     

NKp46 O-Glycan Sequences That Are Involved in the Interaction with Hemagglutinin Type 1 of Influenza Virus

Natural killer (NK) cells serve as a crucial first-line defense against tumors and virus-infected cells. We previously showed that lysis of influenza virus (IV)-infected cells is mediated by the interaction between the NK receptor, NKp46, and the IV hemagglutinin (HA) type 1 expressed by the infected cells. This interaction requires the presence of sialyl groups on the NKp46-T225 O-glycoforms. In the current study, we analyzed the O-glycan sequences that are imperative for the interaction between recombinant NKp46 (rNKp46) and IV H1N1 strains. We first showed that rNKp46 binding to IV H1N1 is not mediated by a glycoform unique to the Thr225 site. We then characterized the O-glycan sequences that mediate the interaction of rNKp46 and IV H1N1; we employed rNKp46s with dissimilar glycosylation patterns and IV H1N1 strains with different sialic acid α2,3 and α2,6 linkage preferences. The branched α2,3-sialylated O-glycoform Neu5NAcα2,3-Galβ1,4-GlcNAcβ1,6[Neu5NAcα2,3-Galβ1,3]GalNAc competently mediated the interaction of rNKp46 with IV H1N1, manifesting a preference for α2,3 linkage. In contrast, the linear α2,3-sialylated O-glycoform Neu5NAcα2,3-Galβ1,3-GalNAc was not correlated with enhanced interaction between rNKp46 and IV H1N1 or a preference for α2,3 linkage. The branched α2,3- and α2,6-sialylated O-glycoform Neu5NAcα2,3-Galβ1,3[Neu5NAcα2,6]GalNAc competently mediated the interaction of rNKp46 with IV H1N1, manifesting a preference for α2,6 linkage. Previous viral HA-binding-specificity studies were performed with glycopolymer conjugates, free synthetic sialyl oligosaccharides, and sialidase-treated cells. This study shed light on the O-glycan sequences involved in the interaction of glycoprotein and viral hemagglutinins and may help in the design of agents inhibitory to hemagglutinin for influenza treatment.Hemagglutinin (HA) is the receptor-binding and membrane fusion protein of influenza virus (IV), as well as the target for infectivity-neutralizing antibodies (27). Terminal sialic acids of glycoproteins and glycolipids are the cellular receptors for the IV HA (27). Two major linkages between sialic acid and the penultimate galactose residues of carbohydrate side chains are found in nature, Neu5NAcα(2,3)-Gal and Neu5NAcα(2,6)-Gal (27); different HAs have different recognition specificities for these linkages and the sugar backbone beneath (23, 26, 30). However, all of the HA-binding specificity studies were performed with glycopolymer conjugates, free synthetic sialyl oligosaccharides, and sialidase-treated cells (8, 10, 20, 25). This could be sufficient for the design of IV-inhibitory agents, and yet, it contributes only partially to the understanding of the interaction of IV HAs with glycoproteins and glycolipids. We aimed to further explore the exact glycoform sequences conjugated to a specific glycoprotein''s glycosylation site that is recognized by different IV strains.For this purpose, we took advantage of our findings on the interaction of natural cytotoxicity receptors (NCRs) and IV HAs (2, 3, 13, 18, 19, 22, 34). We showed that the NKp44 and NKp46 NCRs but not the NKp30 NCR interact with IV HAs. This interaction requires the sialylation of NKp44 and NKp46 oligosaccharides, and the binding of these NCRs to viral HA is required for the lysis of virus-infected cells by NK cells (3, 13, 18). NKp46 displays two putative O-linked glycosylation sites at Thr125 and Thr225 and one N-linked glycosylation site at Asn216. In order to determine the specific sugar-carrying residue that is important for the HA1 recognition, site-directed mutagenesis of the three residues was performed to carry the glycan modifications. Only when Thr225 was replaced was a sharp decrease in the enhanced binding to IV HA1 and IV H1N1-infected cells observed (2). Therefore, for the NKp46 receptor, the interaction with IV HA1 is restricted to Thr225, one of its three glycosylation sites (2).We already showed that producing recombinant NKp46 (rNKp46) in different cell lines resulted in dissimilar glycosylation patterns and had a strong effect on the binding to its ligands (11). Therefore, we analyzed the O-glycan patterns of rNKp46 produced from various cell lines and utilized the dissimilar glycosylation patterns to elucidate the NKp46 O-glycan sequences that mediate the interaction with IV H1N1 strains. To associate the results with the IV preference for sialic acid α2,3 and/or α2,6 linkages, we employed A/PR/8/34 (H1N1), A/NC/20/99 (H1N1), and A/Brisbane/59/2007 (H1N1) grown in either hen egg amnion or Madin-Darby canine kidney (MDCK) cells. Our results pointed to two branched O-glycan sequences that mediated the interaction of the NKp46 glycoprotein with IV H1N1 in correlation with the sialic acid linkage preference of the IV strain.     

Human Herpesvirus 7 U21 Downregulates Classical and Nonclassical Class I Major Histocompatibility Complex Molecules from the Cell Surface

Herpesviruses have evolved numerous strategies to evade detection by the immune system. Notably, most of the herpesviruses interfere with viral antigen presentation to cytotoxic T lymphocytes (CTLs) by removing class I major histocompatibility complex (MHC) molecules from the infected cell surface. Clearly, since the herpesviruses have evolved an extensive array of mechanisms to remove class I MHC molecules from the cell surface, this strategy serves them well. However, class I MHC molecules often serve as inhibitory ligands for NK cells, so viral downregulation of all class I MHC molecules should leave the infected cell open to NK cell attack. Some viruses solve this problem by selectively downregulating certain class I MHC products, leaving other class I products at the cell surface to serve as inhibitory NK cell ligands. Here, we show that human herpesvirus 7 (HHV-7) U21 binds to and downregulates all of the human class I MHC gene products, as well as the murine class I molecule H-2K^b. HHV-7-infected cells must therefore possess other means of escaping NK cell detection.Human herpesvirus-7 (HHV-7) is a betaherpesvirus that infects over 90% of the population by the age of 3 (for a review, see reference 58). Like all other herpesviruses, HHV-7 establishes a latent or persistent infection, lasting for the lifetime of its host. Primary infection is usually accompanied by febrile illness, but long-term infection with the virus is asymptomatic (3, 53). HHV-7 is T-lymphotrophic, but it has also been found in salivary epithelial cells (30, 62).As viruses that remain latent or persistent throughout the life of their hosts, the herpesviruses must interact continually with the host immune system. In so doing, all herpesviruses have evolved mechanisms to interfere with viral antigen presentation by class I major histocompatibility complex (MHC) molecules as a means to escape detection by cytotoxic T lymphocytes (CTLs). Some herpesvirus gene products interfere with proteolysis of antigens or peptide transport into the endoplasmic reticulum (ER) (1, 20, 56, 61). Others retain or destroy class I molecules (2, 26, 59, 64), enhance the internalization of class I molecules, or divert class I molecules to lysosomes for degradation (11, 23, 25, 44). Judging from the number and molecular diversity of these strategies, the removal of MHC class I-peptide complexes from the cell surface must be evolutionarily advantageous to these viruses as a means of escaping immune detection. We have described one such immunoevasin, U21, from HHV-7. HHV-7 U21 binds to class I MHC molecules in the ER and diverts them to a lysosomal compartment, where they are degraded, effectively removing them from the cell surface (23). The mechanism of U21-mediated diversion of class I molecules to lysosomes is not known, but the relocalization of class I MHC molecules is specific—U21 does not cause the rerouting of either the transferrin receptor or CD4 to lysosomes (22, 23).Since the herpesviruses have evolved such an extensive array of mechanisms to remove class I MHC molecules from the cell surface of infected cells, this strategy must serve them well. However, when natural killer (NK) cells detect an absence of class I MHC molecules on the surface of a cell (i.e., “missing self”), they become activated to kill that cell. NK cells detect the absence of class I MHC molecules through interaction of NK cell receptors with NK cell receptor ligands present on the surface of the target cell (for a review, see references 6 and 7). When an NK cell surveys a potential target, it integrates the number and strength of the activating and inhibitory signals it receives; after weighing the balance, it either remains indifferent to the target or becomes activated to kill it.Class I MHC molecules are ligands for inhibitory NK cell receptors. Thus, when a virus removes class I MHC molecules from the cell surface to escape detection by CTLs, it simultaneously renders the cell vulnerable to NK cell attack. Not surprisingly, viruses have evolved counterstrategies to protect their host cells from NK cell-mediated attack. The class I MHC locus contains three classical class I gene products, HLA-A, -B, and -C, as well as other “nonclassical” products, including HLA-E and HLA-G. As a strategy to avoid both CTL and NK cell attack, some viral immunoevasins selectively downregulate HLA-A and HLA-B locus products, while leaving HLA-C, -E, and other inhibitory class I-like molecules at the plasma membrane (10, 16, 35). It has therefore been speculated that HLA-A and -B may be more effective at antigen presentation to CTLs than HLA-C (15, 40). The nonclassical class I molecule HLA-E, on the other hand, functions primarily to inhibit NK cell activation and does not present foreign antigen to CTLs (33). As such, its expression at the cell surface is even promoted by at least one immunoevasin, UL40 from human cytomegalovirus (HCMV) (54, 57).We do not know how HHV-7 responds to the selective pressures exerted by NK cells. We have shown previously that U21 can associate with and downregulate HLA-A and -B, but we do not yet know the full extent of its promiscuity (23). For this reason, we now examine the ability of U21 to bind to and downregulate the various classical and nonclassical class I MHC gene products. We find that, unlike many other viral immunoevasins, HHV-7 U21 can associate with and downregulate HLA-C, -E, and -G and even murine class I MHC molecules. In an infection, this would shift the balance of inhibitory NK cell ligands on the cell surface to favor NK cell attack, suggesting that HHV-7 might compensate for such an imbalance through other means of NK cell evasion.U21 is 55-kDa type I membrane protein with a short (50-amino-acid [aa]) cytoplasmic tail. We have shown that its transmembrane domain and cytoplasmic tail are not involved in its association with the lumenal domain of the class I molecule (22). In addition to gaining information about U21''s potential influence on CTL and NK cell detection of HHV-7-infected cells, we also hoped that a survey of its ability to associate with various class I MHC gene products might help to illuminate regions of the class I molecule important for association with U21.     

Heavily Isotype-Dependent Protective Activities of Human Antibodies against Vaccinia Virus Extracellular Virion Antigen B5

Antibodies against the extracellular virion (EV or EEV) form of vaccinia virus are an important component of protective immunity in animal models and likely contribute to the protection of immunized humans against poxviruses. Using fully human monoclonal antibodies (MAbs), we now have shown that the protective attributes of the human anti-B5 antibody response to the smallpox vaccine (vaccinia virus) are heavily dependent on effector functions. By switching Fc domains of a single MAb, we have definitively shown that neutralization in vitro—and protection in vivo in a mouse model—by the human anti-B5 immunoglobulin G MAbs is isotype dependent, thereby demonstrating that efficient protection by these antibodies is not simply dependent on binding an appropriate vaccinia virion antigen with high affinity but in fact requires antibody effector function. The complement components C3 and C1q, but not C5, were required for neutralization. We also have demonstrated that human MAbs against B5 can potently direct complement-dependent cytotoxicity of vaccinia virus-infected cells. Each of these results was then extended to the polyclonal human antibody response to the smallpox vaccine. A model is proposed to explain the mechanism of EV neutralization. Altogether these findings enhance our understanding of the central protective activities of smallpox vaccine-elicited antibodies in immunized humans.The smallpox vaccine, live vaccinia virus (VACV), is frequently considered the gold standard of human vaccines and has been enormously effective in preventing smallpox disease. The smallpox vaccine led to the worldwide eradication of the disease via massive vaccination campaigns in the 1960s and 1970s, one of the greatest successes of modern medicine (30). However, despite the efficacy of the smallpox vaccine, the mechanisms of protection remain unclear. Understanding those mechanisms is key for developing immunologically sound vaccinology principles that can be applied to the design of future vaccines for other infectious diseases (3, 101).Clinical studies of fatal human cases of smallpox disease (variola virus infection) have shown that neutralizing antibody titers were either low or absent in patient serum (24, 68). In contrast, neutralizing antibody titers for the VACV intracellular mature virion (MV or IMV) were correlated with protection of vaccinees against smallpox (68). VACV immune globulin (VIG) (human polyclonal antibodies) is a promising treatment against smallpox (47), since it was able to reduce the number of smallpox cases ∼80% among variola-exposed individuals in four case-controlled clinical studies (43, 47, 52, 53, 69). In animal studies, neutralizing antibodies are crucial for protecting primates and mice against pathogenic poxviruses (3, 7, 17, 21, 27, 35, 61, 66, 85).The specificities and the functions of protective antipoxvirus antibodies have been areas of intensive research, and the mechanics of poxvirus neutralization have been debated for years. There are several interesting features and problems associated with the antibody response to variola virus and related poxviruses, including the large size of the viral particles and the various abundances of many distinct surface proteins (18, 75, 91, 93). Furthermore, poxviruses have two distinct virion forms, intracellular MV and extracellular enveloped virions (EV or EEV), each with a unique biology. Most importantly, MV and EV virions share no surface proteins (18, 93), and therefore, there is no single neutralizing antibody that can neutralize both virion forms. As such, an understanding of virion structure is required to develop knowledge regarding the targets of protective antibodies.Neutralizing antibodies confer protection mainly through the recognition of antigens on the surface of a virus. A number of groups have discovered neutralizing antibody targets of poxviruses in animals and humans (3). The relative roles of antibodies against MV and EV in protective immunity still remain somewhat unclear. There are compelling data that antibodies against MV (21, 35, 39, 66, 85, 90, 91) or EV (7, 16, 17, 36, 66, 91) are sufficient for protection, and a combination of antibodies against both targets is most protective (66). It remains controversial whether antibodies to one virion form are more important than those to the other (3, 61, 66). The most abundant viral particles are MV, which accumulate in infected cells and are released as cells die (75). Neutralization of MV is relatively well characterized (3, 8, 21, 35). EV, while less abundant, are critical for viral spread and virulence in vivo (93, 108). Neutralization of EV has remained more enigmatic (3).B5R (also known as B5 or WR187), one of five known EV-specific proteins, is highly conserved among different strains of VACV and in other orthopoxviruses (28, 49). B5 was identified as a protective antigen by Galmiche et al., and the available evidence indicated that the protection was mediated by anti-B5 antibodies (36). Since then, a series of studies have examined B5 as a potential recombinant vaccine antigen or as a target of therapeutic monoclonal antibodies (MAbs) (1, 2, 7, 17, 40, 46, 66, 91, 110). It is known that humans immunized with the smallpox vaccine make antibodies against B5 (5, 22, 62, 82). It is also known that animals receiving the smallpox vaccine generate antibodies against B5 (7, 20, 27, 70). Furthermore, previous neutralization assays have indicated that antibodies generated against B5 are primarily responsible for neutralization of VACV EV (5, 83). Recently Chen at al. generated chimpanzee-human fusion MAbs against B5 and showed that the MAbs can protect mice from lethal challenge with virulent VACV (17). We recently reported, in connection with a study using murine monoclonal antibodies, that neutralization of EV is highly complement dependent and the ability of anti-B5 MAbs to protect in vivo correlated with their ability to neutralize EV in a complement-dependent manner (7).The focus of the study described here was to elucidate the mechanisms of EV neutralization, focusing on the human antibody response to B5. Our overall goal is to understand underlying immunobiological and virological parameters that determine the emergence of protective antiviral immune responses in humans.     

The Mitogen-Activated Protein Kinase Scaffold KSR1 Is Required for Recruitment of Extracellular Signal-Regulated Kinase to the Immunological Synapse

KSR1 is a mitogen-activated protein (MAP) kinase scaffold that enhances the activation of the MAP kinase extracellular signal-regulated kinase (ERK). The function of KSR1 in NK cell function is not known. Here we show that KSR1 is required for efficient NK-mediated cytolysis and polarization of cytolytic granules. Single-cell analysis showed that ERK is activated in an all-or-none fashion in both wild-type and KSR1-deficient cells. In the absence of KSR1, however, the efficiency of ERK activation is attenuated. Imaging studies showed that KSR1 is recruited to the immunological synapse during T-cell activation and that membrane recruitment of KSR1 is required for recruitment of active ERK to the synapse.Kinase suppressor of Ras was originally identified in Drosophila melanogaster (53) and Caenorhabditis elegans (19, 32, 52) as a positive regulator of the extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase signaling pathway. It is thought to function as a MAP kinase scaffold because it can bind to Raf, MEK, and ERK (18, 19, 27, 28, 44, 59). While the exact function of KSR is unknown, preassembling the three components of the ERK MAP kinase cascade could function to enhance the efficiency of ERK activation, potentially regulate the subcellular location of ERK activation, and promote access to specific subcellular substrates (16, 45, 46).While only one isoform of KSR is expressed in Drosophila (53), two KSR isoforms have been identified in C. elegans (19, 32, 52) and most higher organisms. They are referred to as KSR1 and KSR2 (32, 43). While KSR1 mRNA and protein are detectable in a wide variety of cells and tissues, including brain, thymus, and muscle (10, 11, 29), little is known about the expression pattern of KSR2.We previously reported the phenotype of KSR1-deficient mice (30). These mice are born at Mendelian ratios and develop without any obvious defects. Using gel filtration, we showed that KSR1 promotes the formation of large signaling complexes containing KSR1, Raf, MEK, and ERK (30). Using both primary T cells stimulated with antibodies to the T-cell receptor as well as fibroblasts stimulated with growth factors, we showed that KSR1-deficient cells exhibit an attenuation of ERK activation with defects in cell proliferation.Here we explored the role of KSR1 in NK cell-mediated cytolysis. The killing of a target cell by a cytolytic T cell or NK cell is a complicated process that involves cell polarization with microtubule-dependent movement of cytolytic granules to an area that is proximal to the contact surface or immunological synapse (7, 33, 34, 48-50, 54). A variety of different signaling molecules are also involved, including calcium (23), phosphatidylinositol-3,4,5-triphosphate (13, 17), and activation of the ERK MAP kinase (6, 42, 56). Recently, the recruitment of activated ERK to the immunological synapse (IS) has been shown to be a feature of successful killing of a target by cytotoxic T lymphocytes (58).How active ERK is recruited to the synapse is not known. Since KSR1 is known to be recruited to the plasma membrane by Ras activation (24), and since the immunological synapse is one of the major sites of Ras activation (26, 41), it seemed plausible to test the hypothesis that KSR1 recruitment to the plasma membrane functions to recruit ERK to the immunological synapse and facilitate its activation. We found that KSR1 was recruited to the immunological synapse and that KSR1 appeared to be required for the localization of active ERK at the contact site. As KSR1-deficient cells exhibit a defect in killing, this suggests that KSR1 recruitment to the synapse may be important in the cytolytic killing of target cells.     

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.     

Analysis of Human Immunodeficiency Virus Type 1 Viremia and Provirus in Resting CD4+ T Cells Reveals a Novel Source of Residual Viremia in Patients on Antiretroviral Therapy

Highly active antiretroviral therapy (HAART) can reduce human immunodeficiency virus type 1 (HIV-1) viremia to clinically undetectable levels. Despite this dramatic reduction, some virus is present in the blood. In addition, a long-lived latent reservoir for HIV-1 exists in resting memory CD4⁺ T cells. This reservoir is believed to be a source of the residual viremia and is the focus of eradication efforts. Here, we use two measures of population structure—analysis of molecular variance and the Slatkin-Maddison test—to demonstrate that the residual viremia is genetically distinct from proviruses in resting CD4⁺ T cells but that proviruses in resting and activated CD4⁺ T cells belong to a single population. Residual viremia is genetically distinct from proviruses in activated CD4⁺ T cells, monocytes, and unfractionated peripheral blood mononuclear cells. The finding that some of the residual viremia in patients on HAART stems from an unidentified cellular source other than CD4⁺ T cells has implications for eradication efforts.Successful treatment of human immunodeficiency virus type 1 (HIV-1) infection with highly active antiretroviral therapy (HAART) reduces free virus in the blood to levels undetectable by the most sensitive clinical assays (18, 36). However, HIV-1 persists as a latent provirus in resting, memory CD4⁺ T lymphocytes (6, 9, 12, 16, 48) and perhaps in other cell types (45, 52). The latent reservoir in resting CD4⁺ T cells represents a barrier to eradication because of its long half-life (15, 37, 40-42) and because specifically targeting and purging this reservoir is inherently difficult (8, 25, 27).In addition to the latent reservoir in resting CD4⁺ T cells, patients on HAART also have a low amount of free virus in the plasma, typically at levels below the limit of detection of current clinical assays (13, 19, 35, 37). Because free virus has a short half-life (20, 47), residual viremia is indicative of active virus production. The continued presence of free virus in the plasma of patients on HAART indicates either ongoing replication (10, 13, 17, 19), release of virus after reactivation of latently infected CD4⁺ T cells (22, 24, 31, 50), release from other cellular reservoirs (7, 45, 52), or some combination of these mechanisms. Finding the cellular source of residual viremia is important because it will identify the cells that are still capable of producing virus in patients on HAART, cells that must be targeted in any eradication effort.Detailed analysis of this residual viremia has been hindered by technical challenges involved in working with very low concentrations of virus (13, 19, 35). Recently, new insights into the nature of residual viremia have been obtained through intensive patient sampling and enhanced ultrasensitive sequencing methods (1). In a subset of patients, most of the residual viremia consisted of a small number of viral clones (1, 46) produced by a cell type severely underrepresented in the peripheral circulation (1). These unique viral clones, termed predominant plasma clones (PPCs), persist unchanged for extended periods of time (1). The persistence of PPCs indicates that in some patients there may be another major cellular source of residual viremia (1). However, PPCs were observed in a small group of patients who started HAART with very low CD4 counts, and it has been unclear whether the PPC phenomenon extends beyond this group of patients. More importantly, it has been unclear whether the residual viremia generally consists of distinct virus populations produced by different cell types.Since the HIV-1 infection in most patients is initially established by a single viral clone (23, 51), with subsequent diversification (29), the presence of genetically distinct populations of virus in a single individual can reflect entry of viruses into compartments where replication occurs with limited subsequent intercompartmental mixing (32). Sophisticated genetic tests can detect such population structure in a sample of viral sequences (4, 39, 49). Using two complementary tests of population structure (14, 43), we analyzed viral sequences from multiple sources within individual patients in order to determine whether a source other than circulating resting CD4⁺ T cells contributes to residual viremia and viral persistence. Our results have important clinical implications for understanding HIV-1 persistence and treatment failure and for improving eradication strategies, which are currently focusing only on the latent CD4⁺ T-cell reservoir.