Cell Cycle Arrest by Transforming Growth Factor β1 Enhances Replication of Respiratory Syncytial Virus in Lung Epithelial Cells

Respiratory syncytial virus (RSV) is a common respiratory viral infection in children which is associated with immune dysregulation and subsequent induction and exacerbations of asthma. We recently reported that treatment of primary human epithelial cells (PHBE cells) with transforming growth factor β (TGF-β) enhanced RSV replication. Here, we report that the enhancement of RSV replication is mediated by induction of cell cycle arrest. These data were confirmed by using pharmacologic inhibitors of cell cycle progression, which significantly enhanced RSV replication. Our data also showed that RSV infection alone resulted in cell cycle arrest in A549 and PHBE cells. Interestingly, our data showed that RSV infection induced the expression of TGF-β in epithelial cells. Blocking of TGF-β with anti-TGF-β antibody or use of a specific TGF-β receptor signaling inhibitor resulted in rescue of the RSV-induced cell cycle arrest, suggesting an autocrine mechanism. Collectively, our data demonstrate that RSV regulates the cell cycle through TGF-β in order to enhance its replication. These findings identify a novel pathway for upregulation of virus replication and suggest a plausible mechanism for association of RSV with immune dysregulation and asthma.Respiratory syncytial virus (RSV) is a single-stranded RNA virus and is a common cause of severe respiratory infections in children. RSV predominantly infects lung epithelial cells, inducing bronchiolitis, and in high-risk individuals it can cause lung fibrosis, airway hyperresponsiveness, mucus secretion, and edema. Interestingly, there is substantial evidence to show that RSV infection induces a dysregulation of the immune response (13, 14, 24, 28, 49). However, the molecular underpinnings of this immune dysregulation are not yet completely understood.It has been established that through its interaction with the immune system, RSV is associated with development and exacerbations of asthma, which is a chronic inflammatory respiratory disease (17, 18, 36, 41). In comparison to healthy individuals, those with asthma have an exaggerated inflammatory response during respiratory virus infections. Despite many studies reporting the involvement of RSV with asthma development and exacerbations, the underlining mechanisms are not yet fully delineated.Previously, we reported that transforming growth factor β (TGF-β) treatment enhanced RSV replication (30). TGF-β is a pleiotropic cytokine with diverse effects on T-cell differentiation and immune regulation and potent anti-inflammatory functions (21, 27, 33, 45). In the lung microenvironment TGF-β inhibits cell proliferation, induces mucus secretion, and regulates airway fibrosis and remodeling (2, 5, 6, 20, 23, 34, 39, 46), all of which are hallmarks of chronic asthma. Specifically, it has been reported that TGF-β expression is elevated in bronchoalveolar lavage fluids and lung tissue of asthmatic patients (9, 32, 48).In addition, genetic studies have found an association between asthma phenotype and TGF-β (19, 26, 38, 43). These studies have identified several single-nucleotide polymorphisms (C509T, T869C, and G915C) in the promoter and coding region of TGF-β that contributed to the increase in gene expression and are significantly associated with childhood wheezing, asthma diagnosis, and asthma severity. Despite this correlation between TGF-β and asthma, the interaction between this key cytokine and respiratory viral infection is poorly understood.A well-known function of TGF-β is the regulation of cell cycle progression. Activation of TGF-β-induced signaling pathways promotes cell cycle arrest in both the G₀/G₁ and G₂/M phases of the cell cycle (7, 8, 25, 29, 40, 42, 44). In the current study, our data showed that TGF-β induction of cell cycle arrest was beneficial to RSV replication. The association of cell cycle arrest with RSV replication was determined by using three different pharmacological inhibitors of cell cycle progression, which enhanced RSV replication. Interestingly, RSV infection alone resulted in secretion of active TGF-β. Treatment of epithelial cells with anti-TGF-β or a specific inhibitor of TGF-β receptor (TGF-βR) signaling resulted in a reduction in RSV replication.In the current study, our data uncover a new pathway for virus regulation of the cell cycle. These findings support our hypothesis that RSV regulates and utilizes TGF-β in lung epithelium to enhance its replication, which may contribute to the physiological changes in the lung leading to immune dysregulation, asthma development, and exacerbations.     

Prophylactic Treatment with a G Glycoprotein Monoclonal Antibody Reduces Pulmonary Inflammation in Respiratory Syncytial Virus (RSV)-Challenged Na?ve and Formalin-Inactivated RSV-Immunized BALB/c Mice

We examined whether prophylactically administered anti-respiratory syncytial virus (anti-RSV) G monoclonal antibody (MAb) would decrease the pulmonary inflammation associated with primary RSV infection and formalin-inactivated RSV (FI-RSV)-enhanced disease in mice. MAb 131-2G administration 1 day prior to primary infection reduced the pulmonary inflammatory response and the level of RSV replication. Further, intact or F(ab′)₂ forms of MAb 131-2G administered 1 day prior to infection in FI-RSV-vaccinated mice reduced enhanced inflammation and disease. This study shows that an anti-RSV G protein MAb might provide prophylaxis against both primary infection and FI-RSV-associated enhanced disease. It is possible that antibodies with similar reactivities might prevent enhanced disease and improve the safety of nonlive virus vaccines.Respiratory syncytial virus (RSV) infection in infants and young children causes substantial bronchiolitis and pneumonia (11, 27, 28, 40) resulting in 40,000 to 125,000 hospitalizations in the United States each year (27). RSV is also a prominent cause of respiratory illness in older children; those of any age with compromised cardiac, pulmonary, or immune systems; and the elderly (6, 7, 11, 17, 18, 39). Despite extensive efforts toward vaccine development (3, 5, 8, 20, 30, 38), none is yet available. Currently, only preventive measures are available that focus on infection control to decrease transmission and prophylactic administration of a humanized IgG monoclonal antibody (MAb) directed against the F protein of RSV (palivizumab) that is recommended for high-risk infants and young children (4, 7, 17). To date, no treatment has been highly effective for active RSV infection (17, 21).The first candidate vaccine, a formalin-inactivated RSV (FI-RSV) vaccine developed in the 1960s, not only failed to protect against disease but led to severe RSV-associated lower respiratory tract infection in young vaccine recipients upon subsequent natural infection (8, 16). The experience with FI-RSV has limited nonlive RSV vaccine development for the RSV-naïve infant and young child. Understanding the factors contributing to disease pathogenesis and FI-RSV vaccine-enhanced disease may identify ways to prevent such a response and to help achieve a safe and effective vaccine.The RSV G, or attachment, protein has been implicated in the pathogenesis of disease after primary infection and FI-RSV-enhanced disease (2, 26, 31). The central conserved region of the G protein contains four evolutionarily conserved cysteines in a cysteine noose structure, within which lies a CX3C chemokine motif (9, 29, 34). The G protein CX3C motif is also immunoactive, as suggested by studies with the mouse model that show that G protein CX3C motif interaction with CX3CR1 alters pulmonary inflammation (41), RSV-specific T-cell responses (12), FI-RSV vaccine-enhanced disease, and expression of the neurokinin substance P (14) and also depresses respiratory rates (32). Recent studies demonstrated that therapeutic treatment with a murine anti-RSV G protein monoclonal antibody (MAb 131-2G) which blocks binding to CX3CR1 can reduce pulmonary inflammation associated with primary infection (13, 23). These findings led us to hypothesize that prophylactic administration of this anti-RSV G monoclonal antibody may also diminish pulmonary inflammation associated with RSV infection in naïve and in FI-RSV-vaccinated mice. In this study, we evaluate the impact of prophylactic administration of MAb 131-2G on the pulmonary inflammatory response to primary infection and to RSV challenge following FI-RSV immunization in mice.     

Newcastle Disease Virus-Like Particles Containing Respiratory Syncytial Virus G Protein Induced Protection in BALB/c Mice,with No Evidence of Immunopathology

Respiratory syncytial virus (RSV) is the leading cause of serious respiratory infections in children as well as a serious cause of disease in elderly and immunosuppressed populations. There are no licensed vaccines available to prevent RSV disease. We have developed a virus-like particle (VLP) vaccine candidate for protection from RSV. The VLP is composed of the NP and M proteins of Newcastle disease virus (NDV) and a chimeric protein containing the cytoplasmic and transmembrane domains of the NDV HN protein and the ectodomain of the human RSV G protein (H/G). Immunization of mice with 10 or 40 μg total VLP-H/G protein by intraperitoneal or intramuscular inoculation stimulated antibody responses to G protein which were as good as or better than those stimulated by comparable amounts of UV-inactivated RSV. Immunization of mice with two doses or even a single dose of these particles resulted in the complete protection of mice from RSV replication in the lungs. Immunization with these particles induced neutralizing antibodies with modest titers. Upon RSV challenge of VLP-H/G-immunized mice, no enhanced pathology in the lungs was observed, although lungs of mice immunized in parallel with formalin-inactivated RSV (FI-RSV) showed the significant pathology that has previously been documented after immunization with FI-RSV. Thus, the VLP-H/G candidate vaccine was immunogenic in BALB/c mice and prevented replication of RSV in murine lungs, with no evidence of immunopathology. These data support further development of virus-like particle vaccine candidates for protection against RSV.Human respiratory syncytial virus (RSV), a member of the Paramyxoviridae family, is the primary cause of serious lower respiratory tract infections in infants and young children and is an important pathogen in elderly and immunocompromised populations worldwide (15, 16, 23, 42). RSV infections can induce a wide spectrum of respiratory diseases, ranging from common cold-like symptoms to more serious disease, such as bronchiolitis or pneumonia (16, 57). Despite the significance of this pathogen, no vaccine is available. Strategies utilizing traditional subunit vaccines or attenuated virus preparations as well as live virus vectors and DNA vaccines have not resulted in a licensed vaccine (reviewed in reference 42). Complicating RSV vaccine development are previous vaccine trials of a formalin-inactivated vaccine (FI-RSV), which predisposed infants to more severe disease upon natural exposure to live virus. These studies have raised concerns about the safety of all subsequently developed RSV vaccines (reviewed in references 15 and 42).Both soluble and cell-mediated immune responses have been proposed to be important for protection from RSV infections (3, 13-15, 29, 42, 67). The RSV F protein, one of the two major antigens expressed on virion surfaces (15), is thought to be the most important target of neutralizing and protective antibodies (15, 25, 72). Indeed, monoclonal antibodies specific for the RSV F protein are used clinically for RSV disease prophylaxis in high-risk infants (4, 61). The F protein is also a major target of CD8 T cells in mice (12), but the association between cell-mediated immunity and protection from RSV disease has not been established (62). The role of the G protein, the other major antigen on virion surfaces, in stimulating protective immune responses is less clear, although it is thought that antibodies to this molecule do have a role in protection (54, 68). No CD8 T-cell epitopes have been reported for this protein. The G protein is unlike other paramyxovirus glycoproteins. Its ectodomain is heavily glycosylated by N-linked and, primarily, O-linked carbohydrates (77). The estimated 24 or 25 O-linked carbohydrate side chains and 4 N-linked side chains increase the molecular mass of the protein, as synthesized in Vero cells, from 32.5 kDa to approximately 90 kDa (15, 16). This extensive glycosylation may help to mask the underlying polypeptide backbone from immune recognition (15).A previous RSV vaccine, FI-RSV, resulted not in protection but in disease enhancement upon subsequent live virus infection (37, 38). Many subsequent studies have attempted to define the reasons for this response. These studies have consistently shown that enhanced disease is characterized by unbalanced Th2-biased cytokine responses, weak CD8 T-cell responses, pronounced eosinophilia, and induction of low-affinity and nonneutralizing antibodies (20, 21, 63, 64, 75). It is less clear which precise properties of the FI-RSV vaccine led to these results (reviewed in reference 42). The absence of these characteristics of enhanced disease is now one of the benchmarks for development of a successful RSV vaccine. Thus far, no vaccine approach reported has resulted in both the absence of enhanced disease upon RSV challenge and adequate, long-lasting protective responses in animal models (42).A virus-like particle (VLP) vaccine strategy has not been reported for RSV. VLPs are large particles, the size of viruses, composed of repeating structural arrays on their surfaces and in their cores, and these structures mimic those of infectious viruses (reviewed in references 36 and 56). VLPs are formed by the assembly of the structural proteins and lipids into particles, but without the incorporation of the viral genome. Thus, VLPs are incapable of the multiple rounds of infection typical of an infectious virus, yet they retain the superb antigenicity of virus particles. Native viral antigens arrayed on VLP surfaces and in their cores likely contribute to potent humoral responses, CD4 T-cell proliferation, and expansion of cytotoxic CD8 T cells, unlike less immunogenic subunit vaccines, which are often comprised of individual purified viral proteins (9-11, 27, 41, 43, 66, 70). The potential of VLPs as safe, effective vaccines for viral disease is increasingly being recognized. Indeed, two VLP vaccines are now licensed for use in humans, namely, the papillomavirus vaccine and the hepatitis B virus vaccine, and a number of other VLP vaccines are being evaluated in preclinical and clinical trials (reviewed in reference 36). Therefore, VLPs expressing one or both RSV glycoproteins may be an attractive strategy for designing an effective RSV vaccine.There is only one report of VLPs formed with RSV proteins (73). These particles have not been well characterized, nor is their efficiency of release known. Furthermore, their detection requires incorporation of a minigenome. However, we have previously reported that the expression of the four major structural proteins of Newcastle disease virus (NDV), an avian paramyxovirus, results in the very efficient release of particles that structurally and functionally resemble virus particles (60; L. W. McGinnes et al., unpublished data). Furthermore, we have found that these particles (ND VLPs) stimulate potent anti-NDV immune responses in mice, including neutralizing antibody responses (McGinnes et al., unpublished data). These results led us to test the hypothesis that ND VLPs could serve as a platform for the expression of antigens from human viruses, including RSV G and F proteins, and that these particles could serve as an effective RSV vaccine.In this study, we report that the ectodomain of the RSV G protein, fused to the cytoplasmic tail (CT) and the transmembrane (TM) domain of the NDV hemagglutinin-neuraminidase (HN) protein, can be incorporated efficiently into VLPs containing the NDV NP and M proteins and that these particles can be prepared quantitatively and used as an immunogen. We demonstrate that immunization with these particles stimulated robust soluble immune responses. Furthermore, these particles conferred protection in BALB/c mice, characterized by increased viral clearance in lung tissue, after live RSV challenge. Importantly, infectious RSV challenge of mice following VLP-H/G immunization did not result in the enhanced lung pathology typified by FI-RSV immunization (17, 18, 55).     

Protective Efficacy and Immunogenicity of an Adenoviral Vector Vaccine Encoding the Codon-Optimized F Protein of Respiratory Syncytial Virus

Adenoviral vectors (AdV) have received considerable attention for vaccine development because of their high immunogenicity and efficacy. In previous studies, it was shown that DNA immunization of mice with codon-optimized expression plasmids encoding the fusion protein of respiratory syncytial virus (RSV F) resulted in enhanced protection against RSV challenge compared to immunization with plasmids carrying the wild-type cDNA sequence of RSV F. In this study, we constructed AdV carrying the codon-optimized full-length RSV F gene (AdV-F) or the soluble form of the RSV F gene (AdV-Fsol). BALB/c mice were immunized twice with AdV-F or AdV-Fsol and challenged with RSV intranasally. Substantial levels of antibody to RSV F were induced by both AdV vaccines, with peak neutralizing-antibody titers of 1:900. Consistently, the viral loads in lung homogenates and bronchoalveolar lavage fluids were significantly reduced by a factor of more than 60,000. The protection against viral challenge could be measured even 8 months after the booster immunization. AdV-F and AdV-Fsol induced similar levels of immunogenicity and protective efficacy. Therefore, these results encourage further development of AdV vaccines against RSV infection in humans.Human respiratory syncytial virus (RSV) is a highly infectious member of the paramyxovirus family causing upper and lower respiratory tract infections in humans. Serious acute RSV infections, including fatal cases of bronchiolitis and pneumonia, occur particularly in premature infants, immunocompromised adults, and patients with pre-existing chronic lung diseases or underlying heart defects (11, 12, 14, 39, 46, 56). In young children, RSV is the most common respiratory tract pathogen, accounting for approximately 50% of hospitalizations due to lower respiratory tract infections (21). In population-based surveillance studies for hospitalization in Europe, RSV was identified in 42 to 45% of enrolled children younger than 2 years with lower respiratory tract infections, and the rate of hospitalization due to RSV-induced respiratory illnesses was estimated at 3 to 6% among industrialized nations (45). Children with severe RSV infections suffer from oxygen deficiency with cyanosis and require intensive medical care. Furthermore, RSV infection in childhood is suspected to be a risk factor for development of asthma (36, 41, 43, 59). The urgent need for an RSV vaccine is further demonstrated by a study showing that levels of disease burden, mortality, and morbidity caused by RSV infections in the elderly are comparable to those induced by nonpandemic influenza A infections (11). However, the immunization of children with a formalin-inactivated (FI) RSV vaccine in the 1960s resulted in a more severe clinical illness, with two fatal cases, than in nonvaccinated infants following RSV infection, pointing out the difficulties in developing a safe and efficacious RSV vaccine (7, 29). It was shown previously that the enhanced disease severity and the development of pulmonary eosinophilia are mainly attributable to an excessive Th2-polarized immune response (15, 35, 57). Furthermore, the lack of high-affinity antibodies after poor Toll-like receptor stimulation has been suggested to be a key factor of the enhanced disease induced by FI RSV vaccination and subsequent RSV infection shown recently (8). However, the enhanced disease induced by FI RSV could partially be reversed by the chemical reduction of the carbonyl groups produced by prior treatment with aldehyde (34).Passive transfer of a neutralizing monoclonal antibody directed against RSV F (palivizumab) results in significant reduction of hospitalization rate due to RSV infection in children and preterm infants (16, 25), making RSV F a promising vaccine candidate for active immunization. Besides being a target for neutralizing antibodies, RSV F additionally contains cytotoxic-T-cell epitopes (1, 37). Moreover, RSV F based DNA vaccines induced encouraging immune responses of a balanced Th1/Th2 type in mice, as pulmonary eosinophilia and disease-enhancing effects were not observed after viral challenge (4, 5, 19, 31, 52). Additionally, RSV F is highly conserved between the two antigenic subgroups of RSV, which allows generation of cross-reactive antibodies after immunization (26).We recently showed that vaccination with codon-optimized RSV F expression plasmids induced improved humoral immune responses in mice compared to vaccination with wild-type cDNA expression plasmids (52). Consequently, viral load was reduced 13-fold in mice immunized with full-length RSV F and 170-fold in mice immunized with the soluble form of RSV F following RSV challenge in comparison to nonimmunized mice. Based on these results, we inserted the codon-optimized open reading frame (ORF) of both full-length RSV F and its soluble form into a replication-deficient adenoviral serotype 5 vector (AdV), generating AdV-F and AdV-Fsol, respectively, to further enhance the immunogenicity and efficiency of the delivered RSV F transgenes. AdVs were chosen because these viral vectors have been extensively studied and have proven their potential as vaccine vectors in multiple successful preclinical studies (reviewed in references 47, 24, and 51). AdVs are also potent inducers of both humoral and cellular immune responses in animal models and in humans (48, 49, 55). Furthermore, convenience of vector design, ease of handling and a robust antigen expression make AdVs a promising vaccine delivery platform. Another main advantage is their natural tropism for mucosal surfaces, which makes adenoviral vaccines convenient for the purpose of immunization against respiratory pathogens that preferentially initiate infection at the mucosal site (40).However, AdV vaccines expressing the wild-type RSV F protein were tested in several animal models without achieving convincing protection against RSV challenge (13, 22, 23). This might be due to poor RSV F expression levels caused by premature polyadenylation, which could be overcome by codon optimization (53). Hence, here we used the codon-optimized RSV F based AdVs AdV-F and AdV-Fsol and evaluated their potential as RSV vaccines, showing greatly improved vaccine efficacy.     

Respiratory Syncytial Virus Grown in Vero Cells Contains a Truncated Attachment Protein That Alters Its Infectivity and Dependence on Glycosaminoglycans

Human respiratory syncytial virus (RSV) contains a heavily glycosylated 90-kDa attachment glycoprotein (G). Infection of HEp-2 and Vero cells in culture depends largely on virion G protein binding to cell surface glycosaminoglycans (GAGs). This GAG-dependent phenotype has been described for RSV grown in HEp-2 cells, but we have found that it is greatly reduced by a single passage in Vero cells. Virions produced from Vero cells primarily display a 55-kDa G glycoprotein. This smaller G protein represents a post-Golgi compartment form that is lacking its C terminus, indicating that the C terminus is required for GAG dependency. Vero cell-grown virus infected primary well-differentiated human airway epithelial (HAE) cell cultures 600-fold less efficiently than did HEp-2 cell-grown virus, indicating that the C terminus of the G protein is also required for virus attachment to this model of the in vivo target cells. This reduced infectivity for HAE cell cultures is not likely to be due to the loss of GAG attachment since heparan sulfate, the primary GAG used by RSV for attachment to HEp-2 cells, is not detectable at the apical surface of HAE cell cultures where RSV enters. Growing RSV stocks in Vero cells could dramatically reduce the initial infection of the respiratory tract in animal models or in volunteers receiving attenuated virus vaccines, thereby reducing the efficiency of infection or the efficacy of the vaccine.Human respiratory syncytial virus (RSV) is a negative-sense, single-stranded RNA virus in the family Paramyxoviridae, subfamily Pneumovirinae. RSV causes mild respiratory disease in all age groups, but the disease can be severe or fatal in infants and the elderly (4, 9, 11). Initial attempts to produce a killed vaccine were not successful, resulting instead in enhanced disease upon infection (26, 41). Efforts to produce a live attenuated vaccine are ongoing (6, 7, 51).RSV produces three glycoproteins which are important for infection. The largest glycoprotein (G) is involved in attachment to the host cell (35), the fusion (F) glycoprotein mediates virion membrane fusion with the target cell membrane (2), and the small hydrophobic (SH) glycoprotein may attenuate apoptosis (15). The F protein is the only glycoprotein that is absolutely required for infection of cultured immortalized cells (27, 45) and syncytium formation, the most obvious cytopathic effect of RSV in immortalized cell culture. Although the G protein is not absolutely required for infection, it enhances infection and syncytium formation (45). The G protein attaches to cultured, immortalized cell lines (35) primarily via glycosaminoglycans (GAGs) on the cell surface (13, 22, 23, 30). GAGs are repeating disaccharide units of hexuronic acid and hexosamine that form unbranched polysaccharide chains and are found on the surface of most mammalian cells. The GAG type that appears most important for RSV infection of HEp-2 cells is heparan sulfate (HS) (23, 30).The G protein is a type II integral membrane protein with its N terminus on the cytoplasmic side of the membrane and its C terminus as the extracellular ectodomain (49). An unglycosylated region in the center of the protein contains four cysteines held together by disulfide bonds in a cysteine noose (19, 24, 33), followed, to the C-terminal side, by a predicted heparin-binding domain (HBD) (12, 13). The 32-kDa G protein, while in the endoplasmic reticulum (ER), is modified by the addition of multiple N-linked carbohydrate chains, depending on the strain. These N-linked additions would increase the molecular mass of G to 45 to 60 kDa. Previous reports have found G protein forms of this size in cells and in virions at low levels (5, 20, 21, 50). All of these reports suggest that these smaller forms of the G protein are partially glycosylated processing intermediates.Maturation of the N-linked carbohydrates of the G protein occurs in the Golgi compartment, where a large number of O-linked carbohydrate chains are added, resulting in an 84- to 92-kDa mature protein (14, 32, 35, 49). This size variation of the G protein is probably due, in part, to the difficulty in sizing heavily glycosylated molecules and variations in molecular mass markers.The G protein shares no homology with the glycoproteins of paramyxoviruses outside the Pneumovirinae subfamily. The high serine and threonine content and the high O-linked glycosylation levels are similar to those found in mucins. The amount of O-linked glycosylation is partially dependent on the cell type used to produce the virus (18).In the present study, we examined virus produced in HEp-2 and Vero cells, which are both commonly used to grow RSV in the laboratory, for dependence on GAGs by the ability to infect cells expressing GAG or deficient in GAG expression. We also examined the ability of the viruses to infect primary, well-differentiated human airway epithelial (HAE) cell cultures. In both systems, infectivity was greatly dependent upon the cell line used to grow the virus. Biochemical characterization of purified virus grown in these two cell lines revealed a smaller form of the RSV G protein in virions from Vero cells. Using C terminus-specific antibodies and a six-His tag at the C terminus of the G protein, we determined that the smaller G protein form was lacking its C terminus. These results highlight the importance of the C-terminal portion of the G protein and suggest that the cell line used to produce a virus can alter its infectivity.     

Vaccination To Induce Antibodies Blocking the CX3C-CX3CR1 Interaction of Respiratory Syncytial Virus G Protein Reduces Pulmonary Inflammation and Virus Replication in Mice

Respiratory syncytial virus (RSV) infection causes substantial morbidity and some deaths in the young and elderly worldwide. There is no safe and effective vaccine available, although it is possible to reduce the hospitalization rate for high-risk children by anti-RSV antibody prophylaxis. RSV has been shown to modify the immune response to infection, a feature linked in part to RSV G protein CX3C chemokine mimicry. This study determined if vaccination with G protein polypeptides or peptides spanning the central conserved region of the G protein could induce antibodies that blocked G protein CX3C-CX3CR1 interaction and disease pathogenesis mediated by RSV infection. The results show that mice vaccinated with G protein peptides or polypeptides containing the CX3C motif generate antibodies that inhibit G protein CX3C-CX3CR1 binding and chemotaxis, reduce lung virus titers, and prevent body weight loss and pulmonary inflammation. The results suggest that RSV vaccines that induce antibodies that block G protein CX3C-CX3CR1 interaction may offer a new, safe, and efficacious RSV vaccine strategy.Human respiratory syncytial virus (RSV) is an important and ubiquitous respiratory virus causing serious lower respiratory tract diseases in infants and young children and substantial morbidity and mortality in the elderly and immunocompromised (7, 11, 20, 21). Despite substantial efforts to develop safe and effective RSV vaccines, none have been successful. The first RSV candidate vaccine, a formalin-inactivated alum-precipitated RSV (FI-RSV) preparation, did not confer protection and was associated with a greater risk of serious disease with subsequent natural infection (9, 60). Live attenuated and inactivated whole virus vaccine candidates have also failed to protect, as they were either insufficiently attenuated or demonstrated the potential for enhanced pulmonary disease upon subsequent RSV infection (6, 37, 39, 41, 45). Similarly, subunit vaccine candidates, such as purified F protein and a prokaryotically expressed fusion protein comprising a fragment of the RSV G protein (residues 130 to 230) fused by its N terminus to the albumin binding domain of streptococcal protein G (designated BBG2Na), have been shown to be inadequate (8, 33, 37, 41). The specific reasons for RSV vaccine failure remain to be answered but could be related to RSV-mediated circumvention of immunity and, more broadly, to the lack of durable immunity elicited in response to natural RSV infection, as people of all ages may experience repeated infections and disease throughout life (3, 41, 45).Evidence indicates that the RSV F protein is important in inducing protective immunity (19, 38), but studies evaluating a BBG2Na vaccine candidate in combination with different adjuvants and by different routes of administration have shown a role for G protein in protection against RSV in rodents (4, 10, 17, 32, 43, 44, 49, 51). The structural elements of the G protein fragment in the BBG2Na vaccine candidate implicated in protective efficacy were mapped, and five different B-cell epitopes were determined, i.e., residues 145 to 159, 164 to 176, 171 to 187, 172 to 187, and 190 to 204 (44, 48). Interestingly, immunogenicity of peptides with residues 145 to 159 was dependent on the orientation of the covalent peptide coupling to the carrier proteins, as mice vaccinated with C-terminally coupled peptides developed protective antibody titers, whereas mice vaccinated with N-terminal peptides did not. The focus of the BBG2Na vaccine studies centered on development of protective neutralizing antibodies, and the studies showed that vaccination or priming with the G protein fragment in BBG2Na did not induce signs of enhanced pulmonary pathology (17, 42, 46, 50).Despite the strong evidence that G protein peptides and polypeptides can induce protective immunity, the G protein has also been implicated in disease pathogenesis (30, 40, 41, 54). One of the disease mechanisms linked to the G protein is CX3C chemokine mimicry (56). RSV G protein has marked similarities to fractalkine, the only known CX3C chemokine, including similarities in structural features (56). Both G protein and fractalkine exist as membrane-bound and secreted forms, and both contain a CX3C chemokine motif that can bind to the fractalkine receptor, CX3CR1 (15, 27). Fractalkine functions to recruit immune cells to sites of inflammation, in particular, CX3CR1⁺ leukocytes, which include subsets of NK cells and CD4⁺ and CD8⁺ T cells (23). RSV G protein has been shown to have fractalkine-like leukocyte chemotactic activity in vitro (56). In vivo, RSV G protein acts as a fractalkine antagonist, modulating the immune response to infection by inhibiting fractalkine-mediated responses by altering the trafficking of CX3CR1⁺ cells and modifying the magnitude and cadence of cytokine and chemokine expression (23, 55). Infection of mice with a mutant RSV lacking the CX3C motif leads to a substantial increase of pulmonary NK cells and CD4⁺ and CD8⁺ cells compared to infection with wild-type RSV (23). This suggests that G protein CX3C-CX3CR1 interaction contributes to immune evasion and may contribute to disease pathogenesis. Thus, G protein CX3C interaction with CX3CR1 is an important target for disease intervention strategies against RSV infection.In the present study, we investigated a new RSV vaccine strategy, using G protein polypeptide and peptide vaccination to generate antibodies reactive to the central conserved cysteine noose region of the G protein to block G protein CX3C motif interaction with CX3CR1. We hypothesize that vaccines inducing G protein-CX3CR1 blocking antibodies will prevent much of the RSV G protein-mediated immune modulation and disease pathogenesis. Our results show that antibodies induced by the central conserved noose region of the G protein block G protein binding to CX3CR1, prevent body weight loss indicative of disease pathogenesis, decrease pulmonary inflammation, and decrease lung virus titers compared to antibodies reactive to N- and C-terminal regions of the G protein. These results suggest that a vaccine strategy to induce G protein CX3C-CX3CR1 blocking antibodies may be useful to prevent G protein-mediated immune modulation and disease pathogenesis.     

A Systemic Neutrophil Response Precedes Robust CD8+ T-Cell Activation during Natural Respiratory Syncytial Virus Infection in Infants

Severe primary respiratory syncytial virus (RSV) infections are characterized by bronchiolitis accompanied by wheezing. Controversy exists as to whether infants suffer from virus-induced lung pathology or from excessive immune responses. Furthermore, detailed knowledge about the development of primary T-cell responses to viral infections in infants is lacking. We studied the dynamics of innate neutrophil and adaptive T-cell responses in peripheral blood in relation to theviral load and parameters of disease in infants admitted to the intensive care unit with severe RSV infection. Analysis of primary T-cell responses showed substantial CD8⁺ T-cell activation, which peaked during convalescence. A strong neutrophil response, characterized by mobilization of bone marrow-derived neutrophil precursors, preceded the peak in T-cell activation. The kinetics of this neutrophil response followed the peak of clinical symptoms and the viral load with a 2- to 3-day delay. From the sequence of events, we conclude that CD8⁺ T-cell responses, initiated during primary RSV infections, are unlikely to contribute to disease when it is most severe. The mobilization of precursor neutrophils might reflect the strong neutrophil influx into the airways, which is a characteristic feature during RSV infections and might be an integral pathogenic process in the disease.Viral infections are characterized by a dynamic interplay between the pathogen and defensive innate and adaptive immune responses of the host (35, 38). Upon infection, virus-specific structural components are recognized by pattern recognition receptors of the host, which triggers a mechanism aimed at the suppression of virus replication and eventually virus elimination. Each virus has a characteristic signature of triggering innate immune receptors and methods to counteract immune responses of the host, which ultimately results in an immune response tailored to the particular properties of the infecting virus (6).Most insights into the sequence of events occurring during viral infections have been obtained from animal experiments, where the immunological control of viral infections can be studied in detail. In many murine models, the crucial role of CD8⁺ T cells in complete elimination of the virus during acute infections has been well established (9, 20, 27). However, both virus-induced damage and immune pathology might contribute to the disease, depending on the type of viral infection and/or the intensity of the innate and adaptive immune responses triggered (10, 20, 37, 41, 49, 60).Primary infections with respiratory syncytial virus (RSV) can cause severe bronchiolitis and pneumonia in infants (24). For RSV, the mouse is not a good model to study primary disease because the virus replicates poorly in murine cells. Hence, to obtain insight into the mechanism of disease caused by RSV, infection studies in humans or nonhuman primate models are needed. We and others have shown that RSV infection causes a strong influx of neutrophils into the airways (15, 25, 48). In addition, we have recently shown that substantial virus-specific CD8⁺ T-cell responses can be elicited in infants with severe RSV infections (25). However, it is still a controversial issue whether the severe manifestations of lower respiratory tract disease are caused directly by the virus or by innate and/or adaptive immune responses triggered by RSV (8, 20, 31, 57). In our previous work, we found no relation between the severity of disease and the number of virus-specific CD8⁺ T cells in peripheral blood (25). Moreover, a direct role of the viral load or different viral strains in disease severity has not been established convincingly (11, 59).Data on the development of primary T-cell responses in infants (<6 months old) during acute viral infections and after vaccinations are sparse. It is generally accepted that the infant immune system is immature and less effective than that of older children or adults. This has been shown by lower activation and/or Th2-polarized adaptive immune responses (1, 2, 58). For RSV-induced disease, it has been suggested that a Th2-biased immune response might be correlated with disease (39, 45, 50), but this idea has been challenged by others (4, 7, 12).Currently, there is no RSV vaccine, and the only preventive treatment available is a humanized neutralizing antibody specific for the fusion protein of RSV that is administered to high-risk groups and is effective in about 60% of children (29). Immune-suppressive or antiviral treatments during severe RSV disease have marginal to no effect (3, 23, 55). Insights into the kinetics of the viral load and disease course in relation to activation of the innate and adaptive immune response will shed light on factors that are attributed to severe RSV-induced disease and will possibly provide leads for the development of curative treatment. We therefore monitored the dynamics of these parameters in infants admitted to the pediatric intensive care unit (ICU) with severe primary RSV infections. During primary RSV infection, the peak values of the viral load and disease severity were followed by the exhaustion of the peripheral blood neutrophil pool, indicating a strong innate immune response closely associated with the peak of disease. We further showed that this natural respiratory infection elicited a strong primary CD8⁺ T-cell response in the very young patients (<3 months). This T-cell response was undetectable at the moment of hospitalization, when the infants were severely ill, and peaked at convalescence. Therefore, severe primary RSV disease does not seem to be caused by inadequate or exaggerated T-cell responses but is most likely initiated by viral damage followed by intense innate immune processes.     

Characterization of Respiratory Syncytial Virus M- and M2-Specific CD4 T Cells in a Murine Model

CD4 T cells have been shown to play an important role in the immunity and immunopathogenesis of respiratory syncytial virus (RSV) infection. We identified two novel CD4 T-cell epitopes in the RSV M and M2 proteins with core sequences M_213-223 (FKYIKPQSQFI) and M2_27-37 (YFEWPPHALLV). Peptides containing the epitopes stimulated RSV-specific CD4 T cells to produce gamma interferon (IFN-γ), interleukin 2 (IL-2), and other Th1- and Th2-type cytokines in an I-A^b-restricted pattern. Construction of fluorochrome-conjugated peptide-I-A^b class II tetramers revealed RSV M- and M2-specific CD4 T-cell responses in RSV-infected mice in a hierarchical pattern. Peptide-activated CD4 T cells from lungs were more activated and differentiated, and had greater IFN-γ expression, than CD4 T cells from the spleen, which, in contrast, produced greater levels of IL-2. In addition, M_209-223 peptide-activated CD4 T cells reduced IFN-γ and IL-2 production in M- and M2-specific CD8 T-cell responses to D^b-M_187-195 and K^d-M2_82-90 peptides more than M2_25-39 peptide-stimulated CD4 T cells. This correlated with the fact that I-A^b-M_209-223 tetramer-positive cells responding to primary RSV infection had a much higher frequency of FoxP3 expression than I-A^b-M2_26-39 tetramer-positive CD4 T cells, suggesting that the M-specific CD4 T-cell response has greater regulatory function. Characterization of epitope-specific CD4 T cells by novel fluorochrome-conjugated peptide-I-A^b tetramers allows detailed analysis of their roles in RSV pathogenesis and immunity.CD4 T lymphocytes play an important role in the resolution of primary viral infections and the prevention of reinfection by regulating a variety of humoral and cellular immune responses. CD4 T cells provide cytokines and other molecules to support the differentiation and expansion of antigen-specific CD8 T cells, which are major effectors for both virus clearance and immunopathology during primary infection with respiratory syncytial virus (RSV) (3, 17, 42, 43). CD4 T-cell help is mandatory for an effective B-cell response (14), which is necessary for producing neutralizing antibodies that prevent secondary RSV infection (12, 18, 21). A concurrent CD4 T-cell response also promotes the maintenance of CD8 T-cell surveillance and effector capacity (9). Previous studies have shown that interleukin 2 (IL-2) from CD4 T cells can restore CD8 T-cell function in lungs (10) and that IL-2 supplementation can increase the production of gamma interferon (IFN-γ) by CD8 T cells upon peptide stimulation in vitro (45).While CD4 T cells are important for providing support to host immunity, they have also been associated with immunopathogenesis by playing a key role in the Th2-biased T-cell response (34, 46), which may be the common mechanism of enhanced lung pathology and other disease syndromes shown in murine studies (2, 16, 17, 19, 35). Earlier studies showed the positive association of formalin-inactivated RSV (FI-RSV) immunization-mediated enhanced illness upon subsequent natural RSV infection with a Th2-biased CD4 T-cell response (19, 44). Th2-orientated CD4 T cells elicit severe pneumonia with extensive eosinophilic infiltrates in the lungs of FI-RSV-immunized mice (13, 24, 48). Patients with severe RSV disease showed an elevated Th2/Th1 cytokine ratio in nasal secretions and peripheral blood mononuclear cells (27, 29, 31, 38). Increased disease severity has also been associated with polymorphisms in Th2-related cytokine genes, such as the IL-4, IL-4 receptor, and IL-13 genes (11, 23, 36). Th2 cytokines from CD4 T cells can also diminish the CD8 T-cell response and delay viral clearance (4, 8).The evaluation of CD4 T-cell responses in viral infection is particularly relevant in the RSV model because of the association of RSV and allergic inflammation, which is largely mediated by CD4 T cells. Understanding the influence of CD4 T cells on CD8 T-cell responses and other immunological effector mechanisms is central to understanding RSV pathogenesis and developing preventive vaccine strategies for RSV. Our lab and others have demonstrated that CD8 T cells target RSV M and M2 proteins with cytolytic effector activities (28, 30, 39). In this study, we found that both RSV M and M2 proteins also contain CD4 T-cell epitopes. These epitopes have 11-mer amino acid core sequences and are associated with the major histocompatibility complex (MHC) class II molecule I-A^b. Fluorochrome-conjugated peptide-I-A^b molecule tetrameric complexes can identify RSV M- and M2-specific CD4 T cells from CB6F1 mice following RSV infection in a hierarchical pattern. Peptides containing the epitopes can stimulate CD4 T cells from RSV M or M2 DNA-immunized and virus-challenged mice and can lead to the production of IFN-γ, IL-2, and other Th1- and Th2-type cytokines that can modulate the CD8 T-cell response to RSV M and M2. We also found that CD4 T cells from the lungs and spleens of immunized mice have different phenotype and cytokine profiles upon in vitro stimulation. These observations suggest a regulatory role for CD4 T cells in the host response to RSV infection. The development of novel MHC class II tetramer reagents allows the characterization of epitope-specific CD4 T-cell responses to RSV and will enable the investigation of basic mechanisms by which CD4 T cells affect pathogenesis and immunity to viral infections.     

The Respiratory Syncytial Virus Matrix Protein Possesses a Crm1-Mediated Nuclear Export Mechanism

The respiratory syncytial virus (RSV) matrix (M) protein is localized in the nucleus of infected cells early in infection but is mostly cytoplasmic late in infection. We have previously shown that M localizes in the nucleus through the action of the importin β1 nuclear import receptor. Here, we establish for the first time that M''s ability to shuttle to the cytoplasm is due to the action of the nuclear export receptor Crm1, as shown in infected cells, and in cells transfected to express green fluorescent protein (GFP)-M fusion proteins. Specific inhibition of Crm1-mediated nuclear export by leptomycin B increased M nuclear accumulation. Analysis of truncated and point-mutated M derivatives indicated that Crm1-dependent nuclear export of M is attributable to a nuclear export signal (NES) within residues 194 to 206. Importantly, inhibition of M nuclear export resulted in reduced virus production, and a recombinant RSV carrying a mutated NES could not be rescued by reverse genetics. That this is likely to be due to the inability of a nuclear export deficient M to localize to regions of virus assembly is indicated by the fact that a nuclear-export-deficient GFP-M fails to localize to regions of virus assembly when expressed in cells infected with wild-type RSV. Together, our data suggest that Crm1-dependent nuclear export of M is central to RSV infection, representing the first report of such a mechanism for a paramyxovirus M protein and with important implications for related paramyxoviruses.The Pneumovirus respiratory syncytial virus (RSV) within the Paramyxoviridae family is the most common cause of lower-respiratory-tract disease in infants (7). The negative-sense single-strand RNA genome of RSV encodes two nonstructural and nine structural proteins, comprising the envelope glycoproteins (F, G, and SH), the nucleocapsid proteins (N, P, and L), the nucleocapsid-associated proteins (M2-1 and M2-2), and the matrix (M) protein (1, 7, 11). Previously, we have shown that M protein localizes in the nucleus at early stages of infection, but later in infection it is localized mainly in the cytoplasm, in association with nucleocapsid-containing cytoplasmic inclusions (13, 16). The M proteins of other negative-strand viruses, such as Sendai virus, Newcastle disease virus, and vesicular stomatitis virus (VSV), have also been observed in the nucleus at early stages of infection (32, 40, 48). Interestingly, the M proteins of all of these viruses, including RSV, play major roles in virus assembly, which take place in the cytoplasm and at the cell membrane (11, 12, 24, 34, 36, 39), but the mechanisms by which trafficking between the nucleus and cytoplasm occurs are unknown.The importin β family member Crm1 (exportin 1) is known to mediate nuclear export of proteins bearing leucine-rich nuclear export signals (NES) (8, 9, 18, 19, 37, 42, 43), such as the human immunodeficiency virus type 1 Rev protein (4). In the case of the influenza virus matrix (M1) protein, binding to the influenza virus nuclear export protein, which possesses a Crm1-recognized NES, appears to be responsible for its export from the nucleus, bound to the influenza virus RNA (3).We have recently shown that RSV M localizes in the nucleus through a conventional nuclear import pathway dependent on the nuclear import receptor importin β1 (IMPβ1) and the guanine nucleotide-binding protein Ran (14). In the present study, we show for the first time that RSV M possesses a Crm1-dependent nuclear export pathway, based on experiments using the specific inhibitor leptomycin B (LMB) (25), both in RSV-infected cells and in green fluorescent protein (GFP)-M fusion protein-expressing transfected cells. We use truncated and point-mutated M derivatives to map the Crm1-recognized NES within the M sequence and show that Crm1-dependent nuclear export is critical to the RSV infectious cycle, since LMB treatment early in infection, inhibiting M export from the nucleus, reduces RSV virion production and a recombinant RSV carrying a NES mutation in M was unable to replicate, probably because M deficient in nuclear export could not localize to areas of virus assembly, as shown in RSV-infected cells transfected to express GFP-M. This is the first report of a Crm1-mediated nuclear export pathway for a paramyxovirus M protein, with implications for the trafficking and function of other paramyxovirus M proteins.     

The Rab11 Pathway Is Required for Influenza A Virus Budding and Filament Formation

Influenza A virus buds through the apical plasma membrane, forming enveloped virus particles that can take the shape of pleomorphic spheres or vastly elongated filaments. For either type of virion, the factors responsible for separation of viral and cell membranes are not known. We find that cellular Rab11 (a small GTP-binding protein involved in endocytic recycling) and Rab11-family interacting protein 3 ([FIP3] which plays a role in membrane trafficking and regulation of actin dynamics) are both required to support the formation of filamentous virions, while Rab11 is additionally involved in the final budding step of spherical particles. Cells transfected with Rab11 GTP-cycling mutants or depleted of Rab11 or FIP3 content by small interfering RNA treatment lost the ability to form virus filaments. Depletion of Rab11 resulted in up to a 100-fold decrease in titer of spherical virus released from cells. Scanning electron microscopy of Rab11-depleted cells showed high densities of virus particles apparently stalled in the process of budding. Transmission electron microscopy of thin sections confirmed that Rab11 depletion resulted in significant numbers of abnormally formed virus particles that had failed to pinch off from the plasma membrane. Based on these findings, we see a clear role for a Rab11-mediated pathway in influenza virus morphogenesis and budding.Influenza A virus is a highly infectious respiratory pathogen, causing 3 to 5 million severe cases yearly while the recent H1N1 pandemic has spread to over 200 countries and resulted in over 15,000 WHO-confirmed deaths since its emergence in March 2009 (57). Influenza virus particles are enveloped structures that contain nine identified viral polypeptides. The lipid envelope is derived by budding from the apical plasma membrane and contains the viral integral membrane proteins hemagglutinin (HA) and neuraminidase (NA) as well as the M2 ion channel. Internally, virus particles contain a matrix protein (M1), small quantities of the NS2/NEP polypeptide, and eight genomic segments of negative-sense RNA that are separately encapsidated into ribonucleoprotein (RNP) particles by the viral nucleoprotein (NP) and tripartite polymerase complex (PB1, PB2, and PA). M1 is thought to form a link between the RNPs and the cytoplasmic tails of the viral membrane proteins though M2 may also play a role (39). The minimal viral protein requirements for budding are disputed; while initial studies suggested that M1 was the main driver of budding (21, 34), more recent work proposes that the glycoproteins HA and NA are responsible (8).Further complicating the analysis of influenza A virus budding is the observation that most strains of the virus form two distinct types of virions: spherical particles approximately 100 nm in diameter and much longer filamentous particles up to 30 μm in length (38). Of the viral proteins, M1 is the primary determinant of particle shape (3, 17) although other virus genes also play a role. It is also likely that host factors are involved in the process as cells with fully differentiated apical and basolateral membranes produce more filaments than nonpolarized cell types (42). While it is tempting to speculate that virus morphology and budding are regulated by the same cellular process, the fact that spherical budding occurs in the absence of an intact actin cytoskeleton while filament formation does not (42, 48) indicates some level of divergence in the mechanisms responsible for spherical and filamentous virion morphogenesis.The means by which viral and cellular membranes are separated are also unclear. Unlike many other enveloped viruses, including retroviruses (19, 36, 52) and herpes simplex virus (12), influenza A virus does not utilize the cellular endosomal sorting complex required for transport (ESCRT) pathway (5, 8). However, recent reports indicate that some viruses, including human cytomegalovirus (HCMV) (32), the hantavirus Andes virus (44), and respiratory syncytial virus (RSV) may employ a Rab11-mediated pathway during assembly and/or budding (4, 51). The Rab family of small GTPases is involved in targeting vesicle trafficking, mediating a wide range of downstream processes including endosomal trafficking and membrane fusion/fission events (reviewed in references 53 and 58). Rab11 is involved in trafficking proteins and vesicles between the trans-Golgi network (TGN), recycling endosome, and the plasma membrane (9, 49, 50) as well as playing a role in actin remodeling, cytokinesis, and abscission (27, 41, 55). Apical recycling endosome (ARE) trafficking is of particular interest in the context of viral infection as other negative-sense RNA viruses have been shown to assemble and/or traffic virion components through the ARE prior to final assembly and budding at the plasma membrane (4, 44, 51). Rab11 function is modulated and targeted through interactions with Rab11 family interacting proteins (Rab11-FIPs) that direct it to specific subcellular locations (23, 25, 26) by binding to actin or microtubule-based motor proteins (24, 26, 47). While Rab11-FIPs recognize both isoforms of Rab11 (a and b [Rab11a/b]) through a conserved amphipathic α-helical motif, they differ in their ability to bind either the GTP-bound form of Rab11 (FIP1, FIP3, FIP4, and Rip11) or both the GTP and GDP-bound forms (FIP2) (23, 30). FIP1 and FIP2 have been implicated in RSV budding (4, 51) while FIP4 is important for trafficking of HCMV components (32). FIP3 has not previously been linked with virus budding but plays an important role in both cell motility and cytokinesis, regulating actin dynamics and endosomal membrane trafficking (29, 55).In light of the normal cellular functions of Rab11 and its effectors and of their reported involvement in the budding of other viruses, we examined the role of this cellular pathway in influenza virus budding. We find that Rab11-FIP3 is essential for filamentous but not spherical virion formation while Rab11 is required for both forms of virus budding.     

Characterization of Lassa Virus Glycoprotein Oligomerization and Influence of Cholesterol on Virus Replication

Mature glycoprotein spikes are inserted in the Lassa virus envelope and consist of the distal subunit GP-1, the transmembrane-spanning subunit GP-2, and the signal peptide, which originate from the precursor glycoprotein pre-GP-C by proteolytic processing. In this study, we analyzed the oligomeric structure of the viral surface glycoprotein. Chemical cross-linking studies of mature glycoprotein spikes from purified virus revealed the formation of trimers. Interestingly, sucrose density gradient analysis of cellularly expressed glycoprotein showed that in contrast to trimeric mature glycoprotein complexes, the noncleaved glycoprotein forms monomers and oligomers spanning a wide size range, indicating that maturation cleavage of GP by the cellular subtilase SKI-1/S1P is critical for formation of the correct oligomeric state. To shed light on a potential relation between cholesterol and GP trimer stability, we performed cholesterol depletion experiments. Although depletion of cholesterol had no effect on trimerization of the glycoprotein spike complex, our studies revealed that the cholesterol content of the viral envelope is important for the infectivity of Lassa virus. Analyses of the distribution of viral proteins in cholesterol-rich detergent-resistant membrane areas showed that Lassa virus buds from membrane areas other than those responsible for impaired infectivity due to cholesterol depletion of lipid rafts. Thus, derivation of the viral envelope from cholesterol-rich membrane areas is not a prerequisite for the impact of cholesterol on virus infectivity.Lassa virus (LASV) is a member of the family Arenaviridae, of which Lymphocytic choriomeningitis virus (LCMV) is the prototype. Arenaviruses comprise more than 20 species, divided into the Old World and New World virus complexes (19). The Old World arenaviruses include the human pathogenic LASV strains, Lujo virus, which was first identified in late 2008 and is associated with an unprecedented high case fatality rate in humans, the nonhuman pathogenic Ippy, Mobala, and Mopeia viruses, and the recently described Kodoko virus (10, 30, 49). The New World virus complex contains, among others, the South American hemorrhagic fever-causing viruses Junín virus, Machupo virus, Guanarito virus, Sabiá virus, and the recently discovered Chapare virus (22).Arenaviruses contain a bisegmented single-stranded RNA genome encoding the polymerase L, matrix protein Z, nucleoprotein NP, and glycoprotein GP. The bipartite ribonucleoprotein of LASV is surrounded by a lipid envelope derived from the plasma membrane of the host cell. The matrix protein Z has been identified as a major budding factor, which lines the interior of the viral lipid membrane, in which GP spikes are inserted (61, 75). The glycoprotein is synthesized as precursor protein pre-GP-C and is cotranslationally cleaved by signal peptidase into GP-C and the signal peptide, which exhibits unusual length, stability, and topology (3, 27, 28, 33, 70, 87). Moreover, the arenaviral signal peptide functions as trans-acting maturation factor (2, 26, 33). After processing by signal peptidase, GP-C of both New World and Old World arenaviruses is cleaved by the cellular subtilase subtilisin kexin isozyme-1/site-1 protease (SKI-1/S1P) into the distal subunit GP-1 and the membrane-anchored subunit GP-2 within the secretory pathway (5, 52, 63). For LCMV, it has been shown that GP-1 subunits are linked to each other by disulfide bonds and are noncovalently connected to GP-2 subunits (14, 24, 31). GP-1 is responsible for binding to the host cell receptor, while GP-2 mediates fusion between the virus envelope and the endosomal membrane at low pH due to a bipartite fusion peptide near the amino terminus (24, 36, 44). Sequence analysis of the LCMV GP-2 ectodomain revealed two heptad repeats that most likely form amphipathic helices important for this process (34, 86).In general, viral class I fusion proteins have triplets of α-helical structures in common, which contain heptad repeats (47, 73). In contrast, class II fusion proteins are characterized by β-sheets that form dimers in the prefusion status and trimers in the postfusion status (43). The class III fusion proteins are trimers that, unlike class I fusion proteins, were not proteolytically processed N-terminally of the fusion peptide, resulting in a fusion-active membrane-anchored subunit (39, 62). Previous studies with LCMV described a tetrameric organization of the glycoprotein spikes (14), while more recent data using a bacterially expressed truncated ectodomain of the LCMV GP-2 subunit pointed toward a trimeric spike structure (31). Due to these conflicting data regarding the oligomerization status of LCMV GP, it remains unclear to which class of fusion proteins the arenaviral glycoproteins belong.The state of oligomerization and the correct conformation of viral glycoproteins are crucial for membrane fusion during virus entry. The early steps of infection have been shown for several viruses to be dependent on the cholesterol content of the participating membranes (i.e., either the virus envelope or the host cell membrane) (4, 9, 15, 20, 21, 23, 40, 42, 53, 56, 76, 78, 79). In fact, it has been shown previously that entry of both LASV and LCMV is susceptible to cholesterol depletion of the target host cell membrane using methyl-β-cyclodextrin (MβCD) treatment (64, 71). Moreover, cholesterol not only plays an important role in the early steps during entry in the viral life cycle but also is critical in the virus assembly and release process. Several viruses of various families, including influenza virus, human immunodeficiency virus type 1 (HIV-1), measles virus, and Ebola virus, use the ordered environment of lipid raft microdomains. Due to their high levels of glycosphingolipids and cholesterol, these domains are characterized by insolubility in nonionic detergents under cold conditions (60, 72). Recent observations have suggested that budding of the New World arenavirus Junin virus occurs from detergent-soluble membrane areas (1). Assembly and release from distinct membrane microdomains that are detergent soluble have also been described for vesicular stomatitis virus (VSV) (12, 38, 68). At present, however, it is not known whether LASV requires cholesterol in its viral envelope for successful virus entry or whether specific membrane microdomains are important for LASV assembly and release.In this study, we first investigated the oligomeric state of the premature and mature LASV glycoprotein complexes. Since it has been shown for several membrane proteins that the oligomerization and conformation are dependent on cholesterol (58, 59, 76, 78), we further analyzed the dependence of the cholesterol content of the virus envelope on glycoprotein oligomerization and virus infectivity. Finally, we characterized the lipid membrane areas from which LASV is released.     

Virus Entry via the Alternative Coreceptors CCR3 and FPRL1 Differs by Human Immunodeficiency Virus Type 1 Subtype

Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding to CD4 and a chemokine receptor, most commonly CCR5. CXCR4 is a frequent alternative coreceptor (CoR) in subtype B and D HIV-1 infection, but the importance of many other alternative CoRs remains elusive. We have analyzed HIV-1 envelope (Env) proteins from 66 individuals infected with the major subtypes of HIV-1 to determine if virus entry into highly permissive NP-2 cell lines expressing most known alternative CoRs differed by HIV-1 subtype. We also performed linear regression analysis to determine if virus entry via the major CoR CCR5 correlated with use of any alternative CoR and if this correlation differed by subtype. Virus pseudotyped with subtype B Env showed robust entry via CCR3 that was highly correlated with CCR5 entry efficiency. By contrast, viruses pseudotyped with subtype A and C Env proteins were able to use the recently described alternative CoR FPRL1 more efficiently than CCR3, and use of FPRL1 was correlated with CCR5 entry. Subtype D Env was unable to use either CCR3 or FPRL1 efficiently, a unique pattern of alternative CoR use. These results suggest that each subtype of circulating HIV-1 may be subject to somewhat different selective pressures for Env-mediated entry into target cells and suggest that CCR3 may be used as a surrogate CoR by subtype B while FPRL1 may be used as a surrogate CoR by subtypes A and C. These data may provide insight into development of resistance to CCR5-targeted entry inhibitors and alternative entry pathways for each HIV-1 subtype.Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding first to CD4 and then to a coreceptor (CoR), of which C-C chemokine receptor 5 (CCR5) is the most common (6, 53). CXCR4 is an additional CoR for up to 50% of subtype B and D HIV-1 isolates at very late stages of disease (4, 7, 28, 35). Many other seven-membrane-spanning G-protein-coupled receptors (GPCRs) have been identified as alternative CoRs when expressed on various target cell lines in vitro, including CCR1 (76, 79), CCR2b (24), CCR3 (3, 5, 17, 32, 60), CCR8 (18, 34, 38), GPR1 (27, 65), GPR15/BOB (22), CXCR5 (39), CXCR6/Bonzo/STRL33/TYMSTR (9, 22, 25, 45, 46), APJ (26), CMKLR1/ChemR23 (49, 62), FPLR1 (67, 68), RDC1 (66), and D6 (55). HIV-2 and simian immunodeficiency virus SIVmac isolates more frequently show expanded use of these alternative CoRs than HIV-1 isolates (12, 30, 51, 74), and evidence that alternative CoRs other than CXCR4 mediate infection of primary target cells by HIV-1 isolates is sparse (18, 30, 53, 81). Genetic deficiency in CCR5 expression is highly protective against HIV-1 transmission (21, 36), establishing CCR5 as the primary CoR. The importance of alternative CoRs other than CXCR4 has remained elusive despite many studies (1, 30, 70, 81). Expansion of CoR use from CCR5 to include CXCR4 is frequently associated with the ability to use additional alternative CoRs for viral entry (8, 16, 20, 63, 79) in most but not all studies (29, 33, 40, 77, 78). This finding suggests that the sequence changes in HIV-1 env required for use of CXCR4 as an additional or alternative CoR (14, 15, 31, 37, 41, 57) are likely to increase the potential to use other alternative CoRs.We have used the highly permissive NP-2/CD4 human glioma cell line developed by Soda et al. (69) to classify virus entry via the alternative CoRs CCR1, CCR3, CCR8, GPR1, CXCR6, APJ, CMKLR1/ChemR23, FPRL1, and CXCR4. Full-length molecular clones of 66 env genes from most prevalent HIV-1 subtypes were used to generate infectious virus pseudotypes expressing a luciferase reporter construct (19, 57). Two types of analysis were performed: the level of virus entry mediated by each alternative CoR and linear regression of entry mediated by CCR5 versus all other alternative CoRs. We thus were able to identify patterns of alternative CoR use that were subtype specific and to determine if use of any alternative CoR was correlated or independent of CCR5-mediated entry. The results obtained have implications for the evolution of env function, and the analyses revealed important differences between subtype B Env function and all other HIV-1 subtypes.     

Ectromelia Virus Inhibitor of Complement Enzymes Protects Intracellular Mature Virus and Infected Cells from Mouse Complement

Poxviruses produce complement regulatory proteins to subvert the host''s immune response. Similar to the human pathogen variola virus, ectromelia virus has a limited host range and provides a mouse model where the virus and the host''s immune response have coevolved. We previously demonstrated that multiple components (C3, C4, and factor B) of the classical and alternative pathways are required to survive ectromelia virus infection. Complement''s role in the innate and adaptive immune responses likely drove the evolution of a virus-encoded virulence factor that regulates complement activation. In this study, we characterized the ectromelia virus inhibitor of complement enzymes (EMICE). Recombinant EMICE regulated complement activation on the surface of CHO cells, and it protected complement-sensitive intracellular mature virions (IMV) from neutralization in vitro. It accomplished this by serving as a cofactor for the inactivation of C3b and C4b and by dissociating the catalytic domain of the classical pathway C3 convertase. Infected murine cells initiated synthesis of EMICE within 4 to 6 h postinoculation. The levels were sufficient in the supernatant to protect the IMV, upon release, from complement-mediated neutralization. EMICE on the surface of infected murine cells also reduced complement activation by the alternative pathway. In contrast, classical pathway activation by high-titer antibody overwhelmed EMICE''s regulatory capacity. These results suggest that EMICE''s role is early during infection when it counteracts the innate immune response. In summary, ectromelia virus produced EMICE within a few hours of an infection, and EMICE in turn decreased complement activation on IMV and infected cells.Poxviruses encode in their large double-stranded DNA genomes many factors that modify the immune system (30, 56). The analysis of these molecules has revealed a delicate balance between viral pathogenesis and the host''s immune response (2, 21, 31, 61). Variola, vaccinia, monkeypox, cowpox, and ectromelia (ECTV) viruses each produce an orthologous complement regulatory protein (poxviral inhibitor of complement enzymes [PICE]) that has structural and functional homology to host proteins (14, 29, 34, 38, 41, 45, 54). The loss of the regulatory protein resulted in smaller local lesions with vaccinia virus lacking the vaccinia virus complement control protein (VCP) (29) and in a greater local inflammatory response in the case of cowpox lacking the inflammation-modulatory protein (IMP; the cowpox virus PICE) (35, 45, 46). Additionally, the complete loss of the monkeypox virus inhibitor of complement enzymes (MOPICE) may account for part of the reduced mortality observed in the West African compared to Congo basin strains of monkeypox virus (12).The complement system consists of proteins on the cell surface and in blood that recognize and destroy invading pathogens and infected host cells (36, 52). Viruses protect themselves from the antiviral effects of complement activation in a variety of ways, including hijacking the host''s complement regulatory proteins or producing their own inhibitors (7, 8, 15, 20, 23). Another effective strategy is to incorporate the host''s complement regulators in the outermost viral membrane, which then protects the virus from complement attack (62). The extracellular enveloped virus (EEV) produced by poxviruses acquires a unique outer membrane derived from the Golgi complex or early endosomes that contain the protective host complement regulators (58, 62). Poxviruses have multiple infectious forms, and the most abundant, intracellular mature virions (IMV), are released when infected cells lyse (58). The IMV lacks the outermost membrane found on EEV and is sensitive to complement-mediated neutralization. The multiple strategies viruses have evolved to evade the complement system underscore its importance to innate and adaptive immunity (15, 36).The most well-characterized PICE is VCP (24-29, 34, 49, 50, 53, 55, 59, 60). Originally described as a secreted complement inhibitor (34), VCP also attaches to the surface of infected cells through an interaction with the viral membrane protein A56 that requires an unpaired N-terminal cysteine (26). This extra cysteine also adds to the potency of the inhibitor by forming function-enhancing dimers (41). VCP and the smallpox virus inhibitor of complement enzymes (SPICE) bind heparin in vitro, and this may facilitate cell surface interactions (24, 38, 50, 59). The coevolution of variola virus with its only natural host, humans, likely explains the enhanced activity against human complement observed with SPICE compared to the other PICEs (54, 64).Our recent work with ECTV, the causative agent of mousepox infection, demonstrated that the classical and alternative pathways of the complement system are required for host survival (48). The mouse-specific pathogen ECTV causes severe disease in most strains and has coevolved with its natural host, analogous to variola virus in humans (9). This close host-virus relationship is particularly important for evaluating the role of the complement system, given the species specificity of many complement proteins, receptors, and regulators (10, 47, 62). Additionally, the availability of complement-deficient mice permits dissection of the complement activation pathways involved. Naïve C57BL/6 mouse serum neutralizes the IMV of ECTV in vitro, predominately through opsonization (48). Maximal neutralization requires natural antibody, classical-pathway activation, and amplification by the alternative pathway. C3 deficiency in the normally resistant C57BL/6 strain results in acute mortality, similar to immunodeficiencies in important elements of the antiviral immune response, including CD8⁺ T cells (19, 32), natural killer cells (18, 51), and gamma interferon (33). During ECTV infection, the complement system acts in the first few hours and days to delay the spread of infection, resulting in lower levels of viremia and viral burden in tissues (48).This study characterized the PICE produced by ECTV, ectromelia virus inhibitor of complement enzymes (EMICE), and assessed its complement regulatory activity. Recombinant EMICE (rEMICE) decreased activation of both human and mouse complement. Murine cells produced EMICE at 4 to 6 h postinfection prior to the release of the majority of the complement-sensitive IMV from infected cells. rEMICE protected ECTV IMV from complement-mediated neutralization. Further, EMICE produced during natural infection inhibited complement deposition on infected cells by the alternative pathway. ECTV likely produces this abundance of EMICE to protect both the IMV and infected cells.