Host Cell Restriction Factors of Paramyxoviruses and Pneumoviruses.

The paramyxo- and pneumovirus family includes a wide range of viruses that can cause respiratory and/or systemic infections in humans and animals. The significant disease burden of these viruses is further exacerbated by the limited therapeutics that are currently available. Host cellular proteins that can antagonize or limit virus replication are therefore a promising area of research to identify candidate molecules with the potential for host-targeted therapies. Host proteins known as host cell restriction factors are constitutively expressed and/or induced in response to virus infection and include proteins from interferon-stimulated genes (ISGs). Many ISG proteins have been identified but relatively few have been characterized in detail and most studies have focused on studying their antiviral activities against particular viruses, such as influenza A viruses and human immunodeficiency virus (HIV)-1. This review summarizes current literature regarding host cell restriction factors against paramyxo- and pneumoviruses, on which there is more limited data. Alongside discussion of known restriction factors, this review also considers viral countermeasures in overcoming host restriction, the strengths and limitations in different experimental approaches in studies reported to date, and the challenges in reconciling differences between in vitro and in vivo data. Furthermore, this review provides an outlook regarding the landscape of emerging technologies and tools available to study host cell restriction factors, as well as the suitability of these proteins as targets for broad-spectrum antiviral therapeutics.

A gammaherpesvirus licenses CD8 T cells to protect the host from pneumovirus-induced immunopathologies.

Human respiratory syncytial virus (RSV) is a pneumovirus that causes severe infections in infants worldwide. Despite intensive research, safe and effective vaccines against RSV have remained elusive. The main reason is that RSV infection of children previously immunized with formalin-inactivated-RSV vaccines has been associated with exacerbated pathology, a phenomenon called RSV vaccine-enhanced respiratory disease. In parallel, despite the high RSV prevalence, only a minor proportion of children develop severe diseases. Interestingly, variation in the immune responses against RSV or following RSV vaccination could be linked with differences of exposure to microbes during childhood. Gammaherpesviruses (γHVs), such as the Epstein-Barr virus, are persistent viruses that deeply influence the immune system of their host and could therefore affect the development of pneumovirus-induced immunopathologies for the long term. Here, we showed that a previous ɣHV infection protects against both pneumovirus vaccine-enhanced disease and pneumovirus primary infection and that CD8 T cells are essential for this protection. These observations shed a new light on the understanding of pneumovirus-induced diseases and open new perspectives for the development of vaccine strategies.

The Absence of Interferon-ß Promotor Stimulator-1 (IPS-1) Predisposes to Bronchiolitis and Asthma-like Pathology in Response to Pneumoviral Infection in Mice.

Respiratory syncytial virus (RSV)-bronchiolitis is a major cause of infant morbidity and mortality and a risk factor for subsequent asthma. We showed previously that toll-like receptor (TLR)7 in plasmacytoid dendritic cells (pDCs) is critical for protection against bronchiolitis and asthma in mice infected with pneumonia virus of mice (PVM), the mouse homolog of RSV. This lack of redundancy was unexpected as interferon-ß promotor stimulator-1 (IPS-1) signalling, downstream of RIG-I-like receptor (RLR) and not TLR7 activation, contributes to host defence in hRSV-inoculated adult mice. To further clarify the role of IPS-1 signalling, we inoculated IPS-1-/- and WT mice with PVM in early-life, and again in later-life, to model the association between bronchiolitis and asthma. IPS-1 deficiency predisposed to severe PVM bronchiolitis, characterised by neutrophilic inflammation and necroptotic airway epithelial cell death, high mobility group box 1 (HMGB1) and IL-33 release, and downstream type-2 inflammation. Secondary infection induced an eosinophilic asthma-like pathophysiology in IPS-1-/- but not WT mice. Mechanistically, we identified that IPS-1 is necessary for pDC recruitment, IFN-α production and viral control. Our findings suggest that TLR7 and RLR signalling work collaboratively to optimally control the host response to pneumovirus infection thereby protecting against viral bronchiolitis and subsequent asthma.

Signaling via pattern recognition receptors NOD2 and TLR2 contributes to immunomodulatory control of lethal pneumovirus infection.

Pattern recognition receptors (PRRs) engage microbial components in the lung, although their role in providing primary host defense against respiratory virus infection is not fully understood. We have previously shown that Gram-positive Lactobacillus plantarum (Lp) administered to the respiratory tract promotes full and sustained protection in response to an otherwise lethal mouse pneumovirus (PVM) infection, a robust example of heterologous immunity. While Lp engages PRRs TLR2 and NOD2 in ex vivo signaling assays, we found that Lp-mediated protection was unimpaired in single gene-deleted TLR2(-/-) and NOD2(-/-) mice. Here we demonstrate substantial loss of Lp-mediated protection in a double gene-deleted NOD2(-/-)TLR2(-/-) strain. Furthermore, we demonstrate protection against PVM infection by administration of the bi-functional NOD2-TLR2 agonist, CL-429. The bi-functional NOD2-TLR2 ligand CL-429 not only suppresses virus-induced inflammation, it is significantly more effective at preventing lethal infection than equivalent amounts of mono-molecular TLR2 and NOD2 agonists. Interestingly, and in contrast to biochemical NOD2 and/or TLR2 agonists, Lp remained capable of eliciting primary proinflammatory responses from NOD2(-/-)TLR2(-/-) mice in vivo and from alveolar macrophages challenged ex vivo. Taken together, we conclude that coordinate engagement of NOD2 and TLR2 constitutes a key step in the genesis of Lp-mediated protection from a lethal respiratory virus infection, and represents a critical target for modulation of virus-induced inflammatory pathology.

The response of aged mice to primary infection and re-infection with pneumonia virus of mice depends on their genetic background.

The pneumonia virus of mice (PVM) model is used to study respiratory syncytial virus (RSV) pathogenesis. The outcome of PVM infection varies in different inbred mouse strains, BALB/c being highly susceptible and C57BL/6 more resistant. As the disease symptoms induced by RSV infection can become more severe as people age, we examined the primary and secondary immune responses to infection with PVM in aged BALB/c and C57BL/6 mice. Based on clinical parameters, aged C57BL/6 mice displayed less severe disease than young adult mice when infected with 3000pfu of PVM-15, while BALB/c mice were equally susceptible at both ages showing significant weight loss and high levels of virus replication. Furthermore, after primary infection the CD4(+) T cell numbers in the lungs were higher in young adult mice, while the CD8(+) T cell numbers were comparable in both age groups and strains. When either C57BL/6 or BALB/c mice were infected with PVM as young adults and then re-infected as aged mice, they were protected from clinical disease, while virus replication was reduced. In contrast to mice with a primary PVM-infection, re-infected mice did not have infiltration of neutrophils or inflammatory mediators in the lung. BALB/c mice had higher virus neutralizing antibody levels in the serum and lung than C57BL/6 mice upon re-infection. Re-infection with PVM led to significant influx of effector CD4(+) T cells into the lungs when compared to aged mice with a primary infection, while this cell population was decreased in the lung draining lymph nodes in both mouse strains. After re-infection the effector CD8(+) T cell population was also decreased in the lung draining lymph nodes in both mouse strain when compared to aged mice after primary infection. However, the central memory CD4(+) and CD8(+) T cells were significantly enhanced in numbers in the lungs and draining lymph nodes of both mouse strains after re-infection, and these numbers were higher for C57BL/6 mice.

Blunted inflammatory and mucosal IgA responses to pneumonia virus of mice in C57BL/6 neonates are correlated to reduced protective immunity upon re-infection as elderly mice.

Respiratory syncytial virus is a major cause of bronchiolitis in infants and pneumonia virus of mice (PVM) causes similar disease in mice. The impact of PVM infection in BALB/c and C57BL/6 neonates, and upon re-infection as elderly mice, was compared. As previously shown for adult mice, PVM caused more disease in BALB/c than in C57BL/6 neonates. After PVM-15 infection BALB/c neonates showed higher production of inflammatory mediators, more influx of plasmacytoid dendritic cells and higher IFN-α expression, and more IgA in the lungs than C57BL/6 neonates. After re-infection as elderly, BALB/c mice developed virus neutralizing antibodies in serum and lung, and were protected from clinical disease, whereas C57BL/6 mice did not develop an anamnestic response and were not protected. These results suggest that an effective local innate response, as well as priming of mucosal adaptive responses in neonates after PVM-15 infection is correlated to decreased susceptibility and protection upon re-infection.

Sustained inflammation and differential expression of interferons type I and III in PVM-infected interferon-gamma (IFNγ) gene-deleted mice.

Interferon gamma (IFNγ) has complex immunomodulatory and antiviral properties. While IFNγ is detected in the airways in response to infection with the pneumovirus pathogen, pneumonia virus of mice (PVM; Family Paramyxoviridae), its role in promoting disease has not been fully explored. Here, we evaluate PVM infection in IFNγ(-/-) mice. Although the IFNγ gene-deletion has no impact on weight loss, survival or virus kinetics, expression of IFNß, IFNλ2/3 and IFN-stimulated 2-5' oligoadenylate synthetases was significantly diminished compared to wild-type counterparts. Furthermore, PVM infection in IFNγ(-/-) mice promoted prominent inflammation, including eosinophil and neutrophil infiltration into the airways and lung parenchyma, observed several days after peak virus titer. Potential mechanisms include over-production of chemoattractant and eosinophil-active cytokines (CXCL1, CCL11, CCL3 and IL5) in PVM-infected IFNγ(-/-) mice; likewise, IFNγ actively antagonized IL5-dependent eosinophil survival ex vivo. Our results may have clinical implications for pneumovirus infection in individuals with IFNγ signaling defects.

Protective role of P2Y2 receptor against lung infection induced by pneumonia virus of mice.

ATP released in the early inflammatory processes acts as a danger signal by binding to purinergic receptors expressed on immune cells. A major contribution of the P2Y(2) receptor of ATP/UTP to dendritic cell function and Th2 lymphocyte recruitment during asthmatic airway inflammation was previously reported. We investigated here the involvement of P2Y(2) receptor in lung inflammation initiated by pneumonia virus of mice infection. We demonstrated that P2Y(2) (-/-) mice display a severe increase in morbidity and mortality rate in response to the virus. Lower survival of P2Y(2) (-/-) mice was not significantly correlated with excessive inflammation despite the higher level of neutrophil recruiters in their bronchoalveolar fluids. Interestingly, we observed reduced ATP level and lower numbers of dendritic cells, CD4(+) T cells and CD8(+) T cells in P2Y(2) (-/-) compared to P2Y(2) (+/+) infected lungs. Lower level of IL-12 and higher level of IL-6 in bronchoalveolar fluid support an inhibition of Th1 response in P2Y(2) (-/-) infected mice. Quantification of DC recruiter expression revealed comparable IP-10 and MIP-3α levels but a reduced BRAK level in P2Y(2) (-/-) compared to P2Y(2) (+/+) bronchoalveolar fluids. The increased morbidity and mortality of P2Y(2) (-/-) mice could be the consequence of a lower viral clearance leading to a more persistent viral load correlated with the observed higher viral titer. The decreased viral clearance could result from the defective Th1 response to PVM with a lack of DC and T cell infiltration. In conclusion, P2Y(2) receptor, previously described as a target in cystic fibrosis therapy and as a mediator of Th2 response in asthma, may also regulate Th1 response protecting mice against lung viral infection.

Depletion of alveolar macrophages prolongs survival in response to acute pneumovirus infection.

Alveolar macrophages are immunoregulatory effector cells that interact directly with respiratory virus pathogens in vivo. We examined the role of alveolar macrophages in acute infection with pneumonia virus of mice (PVM), a rodent pneumovirus that replicates the clinical sequelae of severe human respiratory syncytial virus disease. We show that PVM replicates in primary mouse macrophage culture, releasing infectious virions and proinflammatory cytokines. Alveolar macrophages isolated from PVM-infected mice express activation markers Clec43 and CD86, cytokines TNFα, IL-1, IL-6, and numerous CC and CXC chemokines. Alveolar macrophage depletion prior to PVM infection results in small but statistically significant increases in virus recovery but paradoxically prolonged survival. In parallel, macrophage depleted PVM-infected mice exhibit enhanced NK cell recruitment and increased production of IFNγ by NK, CD4(+) and CD8(+) T cells. These results suggest a protective, immunomodulatory role for IFNγ, as overproduction secondary to macrophage depletion may promote survival despite increased virus recovery.

ChemR23 dampens lung inflammation and enhances anti-viral immunity in a mouse model of acute viral pneumonia.

Viral diseases of the respiratory tract, which include influenza pandemic, children acute bronchiolitis, and viral pneumonia of the elderly, represent major health problems. Plasmacytoid dendritic cells play an important role in anti-viral immunity, and these cells were recently shown to express ChemR23, the receptor for the chemoattractant protein chemerin, which is expressed by epithelial cells in the lung. Our aim was to determine the role played by the chemerin/ChemR23 system in the physiopathology of viral pneumonia, using the pneumonia virus of mice (PVM) as a model. Wild-type and ChemR23 knock-out mice were infected by PVM and followed for functional and inflammatory parameters. ChemR23(-/-) mice displayed higher mortality/morbidity, alteration of lung function, delayed viral clearance and increased neutrophilic infiltration. We demonstrated in these mice a lower recruitment of plasmacytoid dendritic cells and a reduction in type I interferon production. The role of plasmacytoid dendritic cells was further addressed by performing depletion and adoptive transfer experiments as well as by the generation of chimeric mice, demonstrating two opposite effects of the chemerin/ChemR23 system. First, the ChemR23-dependent recruitment of plasmacytoid dendritic cells contributes to adaptive immune responses and viral clearance, but also enhances the inflammatory response. Second, increased morbidity/mortality in ChemR23(-/-) mice is not due to defective plasmacytoid dendritic cells recruitment, but rather to the loss of an anti-inflammatory pathway involving ChemR23 expressed by non-leukocytic cells. The chemerin/ChemR23 system plays important roles in the physiopathology of viral pneumonia, and might therefore be considered as a therapeutic target for anti-viral and anti-inflammatory therapies.

Quantitative proteomic analysis of A549 cells infected with human respiratory syncytial virus.

Human respiratory syncytial virus (HRSV) is a major cause of pediatric lower respiratory tract disease to which there is no vaccine or efficacious chemotherapeutic strategy. Although RNA synthesis and virus assembly occur in the cytoplasm, HRSV is known to induce nuclear responses in the host cell as replication alters global gene expression. Quantitative proteomics was used to take an unbiased overview of the protein changes in transformed human alveolar basal epithelial cells infected with HRSV. Underpinning this was the use of stable isotope labeling with amino acids in cell culture coupled to LC-MS/MS, which allowed the direct and simultaneous identification and quantification of both cellular and viral proteins. To reduce sample complexity and increase data return on potential protein localization, cells were fractionated into nuclear and cytoplasmic extracts. This resulted in the identification of 1,140 cellular proteins and six viral proteins. The proteomics data were analyzed using Ingenuity Pathways Analysis to identify defined canonical pathways and functional groupings. Selected data were validated using Western blot, direct and indirect immunofluorescence confocal microscopy, and functional assays. The study served to validate and expand upon known HRSV-host cell interactions, including those associated with the antiviral response and alterations in subnuclear structures such as the nucleolus and ND10 (promyelocytic leukemia bodies). In addition, novel changes were observed in mitochondrial proteins and functions, cell cycle regulatory molecules, nuclear pore complex proteins and nucleocytoplasmic trafficking proteins. These data shed light into how the cell is potentially altered to create conditions more favorable for infection. Additionally, the study highlights the application and advantage of stable isotope labeling with amino acids in cell culture coupled to LC-MS/MS for the analysis of virus-host interactions.

Pneumoviruses infect eosinophils and elicit MyD88-dependent release of chemoattractant cytokines and interleukin-6.

Eosinophils are recruited to the lung in response to infection with pneumovirus pathogens and have been associated with both the pathophysiologic sequelae of infection and, more recently, with accelerated virus clearance. Here, we demonstrate that the pneumovirus pathogens, respiratory syncytial virus (RSV) and pneumonia virus of mice (PVM), can infect human and mouse eosinophils, respectively, and that virus infection of eosinophils elicits the release of disease-related proinflammatory mediators from eosinophils. RSV replication in human eosinophils results in the release of infectious virions and in the release of the proinflammatory mediator, interleukin-6 (IL-6). PVM replication in cultured bone marrow eosinophils (bmEos) likewise results in release of infectious virions and the proinflammatory mediators IL-6, IP-10, CCL2, and CCL3. In contrast to the findings reported in lung tissue of RSV-challenged mice, PVM replication is accelerated in MyD88 gene-deleted bmEos, whereas release of cytokines is diminished. Interestingly, exogenous IL-6 suppresses virus replication in MyD88 gene-deleted bmEos, suggesting a role for a MyD88-dependent cytokine-mediated feedback circuit in modulating this response. Taken together, our findings suggest that eosinophils are targets of virus infection and may have varied and complex contributions to the pathogenesis and resolution of pneumovirus disease.

Pneumonia virus of mice: severe respiratory infection in a natural host.

Pneumonia virus of mice (PVM; family Paramyxoviridae, genus Pneumovirus) is a natural mouse pathogen that is closely related to human and bovine respiratory syncytial viruses. Among the prominent features of this infection, robust replication of PVM takes place in bronchial epithelial cells in response to a minimal virus inoculum. Virus replication in situ results in local production of proinflammatory cytokines (MIP-1alpha, MIP-2, MCP-1 and IFNgamma) and granulocyte recruitment to the lung. If left unchecked, PVM infection and the ensuing inflammatory response ultimately lead to pulmonary edema, respiratory compromise and death. In this review, we consider the recent studies using the PVM model that have provided important insights into the role of the inflammatory response in the pathogenesis of severe respiratory virus infection. We also highlight several works that have elucidated acquired immune responses to this pathogen, including T cell responses and the development of humoral immunity. Finally, we consider several immunomodulatory strategies that have been used successfully to reduce morbidity and mortality when administered to PVM-infected, symptomatic mice, and thus hold promise as realistic therapeutic strategies for severe respiratory virus infections in human subjects.

Suppression of pattern-recognition receptor TLR4 sensing does not alter lung responses to pneumovirus infection.

Toll-like receptors (TLR) are an important component in the innate immune response to a wide variety of pathogens. Recently, a series of studies has addressed the hypothesis that TLR4 also participates in the host innate response against respiratory syncytial virus (RSV), the leading cause of lower respiratory tract infections in infants and young children. In most of the studies available, RSV, which is not a natural pathogen of mice, has been systematically used in mouse models of human bronchiolitis, with conflicting results. Pneumonia virus of mice (PVM), a member of the pneumovirus genus, shares many similarities with RSV. The serological and structural relationships that exist between them suggest that the immune response to these viruses may be similar in their respective natural hosts. To determine the role of TLR4 in host defense against PVM, TLR4-competent and TLR4-deficient mice were intranasally infected with PVM. Variation of body weight, pulmonary function values, histopathology, and pulmonary viral loads were analyzed. None of the investigated clinical, functional, histological and virological parameters was different between strains, which demonstrates that the sensitivity of the mouse to its natural pneumovirus infection is independent of the presence or absence of TLR4 sensing.