Host alternation of chikungunya virus increases fitness while restricting population diversity and adaptability to novel selective pressures

The mechanisms by which RNA arboviruses, including chikungunya virus (CHIKV), evolve and maintain the ability to infect vertebrate and invertebrate hosts are poorly understood. To understand how host specificity shapes arbovirus populations, we studied CHIKV populations passaged alternately between invertebrate and vertebrate cells (invertebrate ↔ vertebrate) to simulate natural alternation and contrasted the results with those for populations that were artificially released from cycling by passage in single cell types. These CHIKV populations were characterized by measuring genetic diversity, changes in fitness, and adaptability to novel selective pressures. The greatest fitness increases were observed in alternately passaged CHIKV, without drastic changes in population diversity. The greatest increases in genetic diversity were observed after serial passage and correlated with greater adaptability. These results suggest an evolutionary trade-off between maintaining fitness for invertebrate ↔ vertebrate cell cycling, where maximum adaptability is possible only via enhanced population diversity and extensive exploration of sequence space.Emergence of pathogenic RNA viruses is associated with their genomic variability and environmental changes that lead to novel host contacts. Many new and reemerging RNA viruses have been introduced into humans (10). Arthropod-borne viruses (arboviruses), including dengue virus (DENV), have caused recent epidemics by changing their host ranges to increase infection of humans (54). Adaptation to the urban mosquito Aedes albopictus may have expanded a 2005-2006 outbreak of Chikungunya virus (CHIKV) in Reunion Island, France (46), that subsequently circulated among humans in the absence of other amplifying hosts.Despite these emergence events, the evolutionary processes that mediate arbovirus host range changes are poorly understood, partly since arbovirus evolution is understudied. Arboviruses are transmitted horizontally between arthropod vectors and vertebrate reservoir hosts. They replicate rapidly and achieve large population sizes. Polymerases of RNA viruses lack proofreading to repair errors, leading to one substitution per ∼10⁻⁴ nucleotides (nt) copied (11, 36), corresponding to one error per 10-kb genome. This polymerase infidelity leads to diversification that produces closely related but nonidentical RNAs that together form a spectrum of mutants. Although arbovirus mutant spectra have been observed in nature (1, 8, 20, 55, 57), the diversity and divergence within the spectrum are not well described, and the phenotypic roles of minority RNAs are unknown. Understanding the mutation distribution in a heterogeneous arbovirus population is important, given that any variant can be favored by selection and ultimately affect fitness (12, 13). However, the relationships between fitness and RNA virus population diversity are poorly understood.Studies with other RNA viruses, including human immunodeficiency virus (HIV) (3, 58, 60), hepatitis C virus (14, 15, 25), and poliovirus (30, 52), indicate that intrahost population diversity is important for virus evolution, fitness, and pathogenesis. Unlike these vertebrate-only RNA viruses, arboviruses obligately cycle between vertebrates and arthropods, a process that imposes additional selective constraints on evolution and adaptation. Sequence comparisons of RNA arbovirus isolates show that they are relatively stable (18, 19), and genetic studies reveal that evolution is dominated by purifying selection (20-22, 57). This constancy of consensus sequence may derive from the need to infect disparate hosts that present conflicting demands for adaptation where sequence changes that improve fitness in one host may not be maintained in the alternate organism.Experimental evolution studies have been performed to study fitness trade-offs and the unique ability of arboviruses to simultaneously evolve to vertebrate and invertebrate hosts. In vitro evolution studies reveal three general patterns of arbovirus evolution: (i) fitness gains after serial passage in vertebrate or invertebrate cells (except in certain cases [7]) and losses in bypassed host cell types (DENV, Eastern equine encephalitis virus [EEEV], Sindbis virus [SINV], and vesicular stomatitis virus [VSV]) (17, 28, 51, 56); (ii) reduced fitness in new cells (VSV) (28), and (iii) fitness increases after alternating (invertebrate ↔ vertebrate) passage (DENV, EEEV, SINV, VSV) (17, 28, 51, 56). Together, these studies suggest that constraints on fitness differ in insect and vertebrate cells and can be virus specific but that arbovirus fitness is not limited by alternating between vertebrate and invertebrate hosts.An in vivo study revealed a similar pattern of arbovirus evolution: vertebrate-passaged Venezuelan equine encephalitis virus (VEEV) was five times more fit than its unpassaged parent, and mosquito-passaged VEEV was more infectious for vectors (6). Consensus sequences revealed that, despite becoming more fit, mutations in passaged populations were slight or absent. This suggests that fitness increases were mediated by minority genomes in the mutant spectrum. However, a major limitation of the in vivo experiments and other arbovirus evolution studies is that sequencing of individual RNAs from the mutant spectrum in passaged intrahost populations was not performed (although notable exceptions for flaviviruses exist [5, 22]). The identity of minority variants within intrahost arbovirus populations and their influences on phenotype have not been extensively examined.The goal of this study, therefore, is to understand how obligate host cycling shapes an intrahost arbovirus population. RNA viruses can tolerate increased niche breadth when they evolve in heterogeneous environments (48), a trait which may promote easier emergence and therefore help explain why arboviruses frequently emerge or reemerge to cause human and veterinary disease. One limitation of increasing breadth may be restricted genetic diversity, where only genomes accepted in both hosts can be maintained. To explore this possibility, we described arbovirus populations after invertebrate ↔ vertebrate cell cycling and compared them to populations that were artificially released from alternating passage via serial vertebrate or invertebrate cell transfer. Since previous studies indicate that intrahost RNA virus population diversity can determine phenotype (3, 37, 52) and given that increases in arbovirus fitness are not always associated with consensus genome changes (6, 27), we determined how arbovirus population diversity (mutation frequency) and distance (number of mutations by which each RNA differs from the consensus) relate to fitness. We predicted that genetic diversity and distance between RNAs in the mutant spectrum and the corresponding consensus are correlated with fitness and that diverse populations better escape varied selective pressures than genetically homogeneous populations since, by chance, they are more likely to contain an advantageous mutation(s) when the pressure is applied.Chikungunya virus (family Togaviridae, genus Alphavirus) was selected primarily because of its medical relevance. CHIKV has caused outbreaks of human disease characterized by arthralgia and myalgia since the 18th century and since 2004 in Africa, Indian Ocean islands, Southeast Asia, and Italy (32). Furthermore, no adaptation studies have been performed for CHIKV, and no intrahost population studies have been conducted for an alphavirus. An infectious clone was generated from the strain used for passages and was genetically marked to differentiate its RNAs from passaged CHIKV, so that after direct competition, the fitnesses of passaged populations could be compared to those for progenitors.     

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.     

Reovirus Activates Transforming Growth Factor β and Bone Morphogenetic Protein Signaling Pathways in the Central Nervous System That Contribute to Neuronal Survival following Infection

Viral infections of the central nervous system (CNS) are important causes of worldwide morbidity and mortality, and understanding how viruses perturb host cell signaling pathways will facilitate identification of novel antiviral therapies. We now show that reovirus infection activates transforming growth factor β (TGF-β) and bone morphogenetic protein (BMP) signaling in a murine model of encephalitis in vivo. TGF-β receptor I (TGF-βRI) expression is increased and its downstream signaling factor, SMAD3, is activated in the brains of reovirus-infected mice. TGF-β signaling is neuroprotective, as inhibition with a TGF-βRI inhibitor increases death of infected neurons. Similarly, BMP receptor I expression is increased and its downstream signaling factor, SMAD1, is activated in reovirus-infected neurons in the brains of infected mice in vivo. Activated SMAD1 and SMAD3 were both detected in regions of brain infected by reovirus, but activated SMAD1 was found predominantly in uninfected neurons in close proximity to infected neurons. Treatment of reovirus-infected primary mouse cortical neurons with a BMP agonist reduced apoptosis. These data provide the first evidence for the activation of TGF-β and BMP signaling pathways following neurotropic viral infection and suggest that these signaling pathways normally function as part of the host''s protective innate immune response against CNS viral infection.The transforming growth factor β (TGF-β) superfamily of growth factors regulates multiple cellular functions including inflammation, cell growth, differentiation, migration, and apoptosis (33). In excess of 30 genes represent the TGF-β superfamily in mammals including three TGF-β genes, four activin β-chains (nodal), 10 bone morphogenetic proteins (BMPs), and 11 growth and differentiation factors. The receptors for the TGF-β superfamily of ligands form the only known transmembrane Ser-Thr kinases (33). The signaling pathways are similar for all ligands. Briefly, a TGF-β ligand binds to and brings into proximity a TGF-β receptor type I (TGF-βRI) and a TGF-β receptor type II (TGF-βRII), assembling a heterotetrameric complex (45). The constitutively active type II receptor kinase phosphorylates the type I receptor at several serine and threonine residues in a glycine- and serine-rich juxtamembrane domain, resulting in the recruitment and phosphorylation at two C-terminal serine residues in the MH2 domain of the receptor-regulated SMADs (R-SMAD): SMAD1, SMAD2, SMAD3, SMAD5, and SMAD8 (33). Phosphorylated R-SMAD proteins form complexes with the common mediator SMAD4, translocate to the nucleus, and alter gene expression. Each type I receptor typically binds a specific TGF-β superfamily ligand and activates a subset of R-SMADs. The TGF-β-activin-nodal ligands signal through specific type I receptors to activate SMAD2 or SMAD3, and the BMP-growth and differentiation factor ligands signal through specific type I receptors and activate SMAD1, SMAD5, or SMAD8 (33).Members of the TGF-β superfamily modulate innate immune responses to multiple infections by controlling inflammation and repair after injury (25). In addition, TGF-β signaling controls apoptosis and viral replication in several viral systems including polyomaviruses such as BK virus (1) and JC virus (16, 30), human immunodeficiency virus (16), Epstein-Barr virus reactivation (17), and hepatitis C virus (26). In the case of hepatitis C virus, the synergistic activation of BMP signaling and alpha interferon suppresses viral replication (35). In noninfectious models of disease, previous studies have shown that modulating TGF-β signaling is protective in a murine model of Alzheimer''s disease (36), and augmenting BMP signal activation can protect cells and neurons following oxidative stress (15), stroke (40), or other cellular injuries (3, 44). However, to our knowledge, the roles of TGF-β and BMP signaling have not been studied following acute viral infection in the central nervous system (CNS).Reovirus infection is a well-characterized experimental system utilized to study viral pathogenesis. Serotype 3 strains of reovirus (Abney [T3A] and Dearing [T3D]) induce apoptosis in vitro and in vivo by activating caspase-3-dependent cell death (4, 28). Reovirus-induced encephalitis in vivo is largely a result of virus-induced apoptosis with little associated infiltrate of inflammatory cells. Caspase 3 activation is initiated by reovirus-induced activation of death receptors and is augmented by mitochondrial apoptotic signaling (6, 24, 31). Previous studies have also demonstrated that virus-induced signaling events affect cell survival and cell death. Reovirus-induced selective activation of mitogen-activated protein kinases such as c-Jun N-terminal kinase (JNK) are vital to apoptosis in vitro and in a murine model of reovirus-induced encephalitis (2, 9). Similarly, the activation and subsequent inhibition of NF-κB signaling are important determinants of apoptosis (5, 7, 10). These pathways are likely to act in part by regulating critical components of either death receptor or mitochondrial apoptotic signaling. For example, reovirus-induced inhibition of NF-κB activation decreases cellular levels of c-FLIP, a caspase 8 inhibitor, and inhibition of JNK signaling decreases mitochondrial release of proapoptotic proteins cytochrome c and SMAC (5, 8). While many of these signaling pathways modulate apoptosis, the reovirus model of pathogenesis has been utilized to understand the interferon response to viral infection in cell culture, in myocardial cells, and in the CNS as well (18, 22, 34). Understanding the cellular response to viral infection will lead to the identification of new targets for antiviral therapy.Studies of neuroinvasive viral infections including those with Sindbis virus, West Nile virus, herpes simplex virus, and cytomegalovirus have shown that apoptosis is an important mechanism of neuronal cell death (11, 20, 27, 32). In many cases of neuroinvasive viral infection, exemplified by West Nile virus, viremia has ended by the time that the patient presents with acute symptoms; yet, ongoing virus-induced injury in the CNS results in significant morbidity and mortality (13, 21). There are currently no proven effective therapies for acute CNS viral infections other than acyclovir therapy for herpes simplex virus encephalitis, and even with optimal treatment of herpes simplex virus encephalitis, morbidity and mortality remain significant. The goal of our studies is to utilize the reovirus system to identify potential novel therapeutic targets that will enhance neuroprotection following CNS viral infection.We show here for the first time that TGF-β and BMP are activated in response to viral infection in a model of murine viral encephalitis in vivo. We extend these findings by showing that virus-activated BMP signaling protects mouse cortical neurons from cell death.     

Genome-Scale Phylogenetic Analyses of Chikungunya Virus Reveal Independent Emergences of Recent Epidemics and Various Evolutionary Rates

Chikungunya virus (CHIKV), a mosquito-borne alphavirus, has traditionally circulated in Africa and Asia, causing human febrile illness accompanied by severe, chronic joint pain. In Africa, epidemic emergence of CHIKV involves the transition from an enzootic, sylvatic cycle involving arboreal mosquito vectors and nonhuman primates, into an urban cycle where peridomestic mosquitoes transmit among humans. In Asia, however, CHIKV appears to circulate only in the endemic, urban cycle. Recently, CHIKV emerged into the Indian Ocean and the Indian subcontinent to cause major epidemics. To examine patterns of CHIKV evolution and the origins of these outbreaks, as well as to examine whether evolutionary rates that vary between enzootic and epidemic transmission, we sequenced the genomes of 40 CHIKV strains and performed a phylogenetic analysis representing the most comprehensive study of its kind to date. We inferred that extant CHIKV strains evolved from an ancestor that existed within the last 500 years and that some geographic overlap exists between two main enzootic lineages previously thought to be geographically separated within Africa. We estimated that CHIKV was introduced from Africa into Asia 70 to 90 years ago. The recent Indian Ocean and Indian subcontinent epidemics appear to have emerged independently from the mainland of East Africa. This finding underscores the importance of surveillance to rapidly detect and control African outbreaks before exportation can occur. Significantly higher rates of nucleotide substitution appear to occur during urban than during enzootic transmission. These results suggest fundamental differences in transmission modes and/or dynamics in these two transmission cycles.Chikungunya virus (CHIKV; Togaviridae: Alphavirus) is an arbovirus (arthropod-borne virus) vectored by Aedes mosquitoes to humans in tropical and subtropical regions of Africa and Asia (Fig. ​(Fig.1;1; reviewed in references 26 and 46). CHIKV has a single-stranded, positive-sense RNA genome of ∼12 kb and causes chikungunya fever (CHIK), a febrile illness associated with severe arthralgia and rash (2, 15, 31, 35); the name is derived from a Bantu language word describing the severe arthritic signs (32), which can persist for years. Thus, CHIK has enormous economic costs in addition to its public health impact (9). Because the signs and symptoms of CHIK overlap with those of dengue and because CHIKV is transmitted sympatrically in urban areas by the same mosquito vectors, it is grossly underreported in the absence of laboratory diagnostics (10, 37).Open in a separate windowFIG. 1.Distribution of the CHIKV strains used in this study. The map, based on a world map template from http://www.presentationmagazine.com, was edited with permission.CHIKV was first isolated during a 1953 outbreak in present-day Tanzania by Ross (48, 49). Since then, outbreaks have been documented in Africa and Asia, including the Indian subcontinent (Fig. ​(Fig.1)1) (1, 4). In 2005, CHIKV emerged from East Africa to cause an explosive urban epidemic in popular tourist island destinations in the Indian Ocean (Fig. ​(Fig.1;1; reviewed in reference 31). In late 2005, CHIKV spread into the Indian subcontinent, where millions of people have been affected (5). However, the geographic source of spread into India, from the mainland of Africa or from the Indian Ocean Islands, has not been delineated. India had seen large epidemics of CHIK in the past (reviewed in reference 30), but CHIKV apparently disappeared during the 1970s (5). Since 2006, CHIKV has been imported into Europe and the western hemisphere (including the United States) via many viremic travelers, and an epidemic was initiated in Italy by a traveler from India (4, 11, 47). The dramatic spread since 1980 of dengue viruses (DENV) throughout tropical America, via the same vectors, portends the severity of the public health problem if CHIKV becomes established in the western hemisphere.The first phylogenetic analysis of CHIKV (45) identified three geographically associated genotypes: the West African (WAf), East/Central/South African (ECSA), and Asian genotypes. More recent analyses indicate that the recent Indian Ocean and Indian strains form a monophyletic group within the ECSA lineage (5, 12, 14, 27, 40, 51, 52). However, most CHIKV phylogenetic studies (1, 14, 28, 29, 38, 40, 41, 47, 52) have utilized only partial sequences from the envelope glycoprotein E1 gene, preventing a robust assessment of some of the relationships among strains and of their evolutionary dynamics.The CHIKV strains represented in different geographic lineages apparently circulate in different ecological cycles. In Asia, CHIKV appears to circulate primarily in an urban transmission cycle involving the peridomestic mosquitoes Aedes aegypti and A. albopictus, as well as humans (25, 45). Asian epidemics typically infect thousands-to-millions of people over the course of several years (46). In contrast, African CHIKV circulates primarily in a sylvatic/enzootic cycle, transmitted by arboreal primatophilic Aedes mosquitoes (e.g., A. furcifer and A. africanus) and probably relies on nonhuman primates as reservoir hosts (reviewed in reference 16). Epidemics in rural Africa usually occur on a much smaller scale than in Asia, likely a result of the lower human population densities, and possibly more stable herd immunity. Although the assignments of “urban” and “sylvatic/enzootic” are based on the most common mode of transmission, CHIKV strains of African origin are capable of urban transmission by A. aegypti and A. albopictus, as evidenced by outbreaks in the Democratic Republic of the Congo (41), Nigeria (36), Kenya (27), and Gabon (42). The ecological differences between the sylvatic/enzootic (henceforth called enzootic) and urban/endemic/epidemic transmission cycles (henceforth called epidemic) such as seasonality of vector larval habitats, vertebrate host abundance and herd immunity, and vector host preferences, prompted us to hypothesize that the evolutionary dynamics of CHIKV may differ between the two transmission cycles. To test this hypothesis, to provide more robust estimates of the evolutionary relationships among the CHIKV strains including the sources of the recent epidemics, and to elucidate the temporal and spatial history of CHIKV evolution, we performed an extensive, genome-scale phylogenetic analysis, utilizing complete open reading frame (ORF) sequences of a large collection of 80 isolates with broad temporal, spatial, and host coverage.     

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.     

Andes Virus Recognition of Human and Syrian Hamster β3 Integrins Is Determined by an L33P Substitution in the PSI Domain

Andes virus (ANDV) causes a fatal hantavirus pulmonary syndrome (HPS) in humans and Syrian hamsters. Human α_vβ₃ integrins are receptors for several pathogenic hantaviruses, and the function of α_vβ₃ integrins on endothelial cells suggests a role for α_vβ₃ in hantavirus directed vascular permeability. We determined here that ANDV infection of human endothelial cells or Syrian hamster-derived BHK-21 cells was selectively inhibited by the high-affinity α_vβ₃ integrin ligand vitronectin and by antibodies to α_vβ₃ integrins. Further, antibodies to the β₃ integrin PSI domain, as well as PSI domain polypeptides derived from human and Syrian hamster β₃ subunits, but not murine or bovine β₃, inhibited ANDV infection of both BHK-21 and human endothelial cells. These findings suggest that ANDV interacts with β₃ subunits through PSI domain residues conserved in both Syrian hamster and human β₃ integrins. Sequencing the Syrian hamster β₃ integrin PSI domain revealed eight differences between Syrian hamster and human β₃ integrins. Analysis of residues within the PSI domains of human, Syrian hamster, murine, and bovine β₃ integrins identified unique proline substitutions at residues 32 and 33 of murine and bovine PSI domains that could determine ANDV recognition. Mutagenizing the human β₃ PSI domain to contain the L33P substitution present in bovine β₃ integrin abolished the ability of the PSI domain to inhibit ANDV infectivity. Conversely, mutagenizing either the bovine PSI domain, P33L, or the murine PSI domain, S32P, to the residue present human β₃ permitted PSI mutants to inhibit ANDV infection. Similarly, CHO cells transfected with the full-length bovine β₃ integrin containing the P33L mutation permitted infection by ANDV. These findings indicate that human and Syrian hamster α_vβ₃ integrins are key receptors for ANDV and that specific residues within the β₃ integrin PSI domain are required for ANDV infection. Since L33P is a naturally occurring human β₃ polymorphism, these findings further suggest the importance of specific β₃ integrin residues in hantavirus infection. These findings rationalize determining the role of β₃ integrins in hantavirus pathogenesis in the Syrian hamster model.Hantaviruses persistently infect specific small mammal hosts and are spread to humans by the inhalation of aerosolized excreted virus (41, 42). Hantaviruses predominantly infect endothelial cells and cause one of two vascular leak-based diseases: hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS) (41). Hantavirus diseases are characterized by increased vascular permeability and acute thrombocytopenia in the absence of endothelial cell lysis (36, 41, 42, 54). In general, hantaviruses are not spread from person to person; however, the Andes hantavirus (ANDV) is an exception, since there are several reports of person-to-person transmission of ANDV infection (11, 37, 47, 52). ANDV is also unique in its ability to cause an HPS-like disease in Syrian hamsters and serves as the best-characterized hantavirus disease model with a long onset, symptoms, and pathogenesis nearly identical to that of HPS patients (20, 21, 50).Hantavirus infection of the endothelium alters endothelial cell barrier functions through direct and immunological responses (8, 14). Although the means by which hantaviruses cause pulmonary edema or hemorrhagic disease has been widely conjectured, the mechanisms by which hantaviruses elicit pathogenic human responses have yet to be defined. Hantaviruses coat the surface of infected VeroE6 cells days after infection (17), and this further suggests that dynamic hantavirus interactions with immune and endothelial cells are likely to contribute to viral pathogenesis. Hantavirus pathogenesis has been suggested to involve CD8⁺ T cells, tumor necrosis factor alpha or other cytokines, viremia, and the dysregulation of β₃ integrins (7, 8, 13-16, 25-28, 32, 34, 38, 44-46). However, these responses have not been demonstrated to contribute to hantavirus pathogenesis, and in some cases there are conflicting data on their involvement (18, 25-28, 34, 35, 44, 45, 48). Immune complex deposition clearly contributes to HFRS patient disease and renal sequelae (4, 7), but it is unclear what triggers vascular permeability in HPS and HFRS diseases or why hemorrhage occurs in HFRS patients but not in HPS patients (8, 36, 54). Acute thrombocytopenia is common to both diseases, and platelet dysfunction resulting from defective platelet aggregation is reported in HFRS patients (7, 8).Pathogenic hantaviruses have in common their ability to interact with α_IIbβ₃ and α_vβ₃ integrins present on platelets and endothelial cells (13, 16), and β₃ integrins have primary roles in regulating vascular integrity (1, 2, 6, 19, 22, 39, 40). Consistent with the presence of cell surface displayed virus (17), pathogenic hantaviruses uniquely block α_vβ₃ directed endothelial cell migration and enhance endothelial cell permeability for 3 to 5 days postinfection (14, 15). Pathogenic hantaviruses dysregulate β₃ integrin functions by binding domains present at the apex of inactive β₃ integrin conformers (38). α_vβ₃ forms a complex with vascular endothelial cell growth factor receptor 2 (VEGFR2) and normally regulates VEGF-directed endothelial cell permeability (2, 3, 10, 39, 40). However, both β₃ integrin knockouts and hantavirus-infected endothelial cells result in increased VEGF-induced permeability, presumably by disrupting VEGFR2-β₃ integrin complex formation (2, 14, 19, 39, 40). This suggests that at least one means for hantaviruses to increase vascular permeability occurs through interactions with β₃ integrins that are required for normal platelet and endothelial cell functions.α_vβ₃ and α_IIbβ₃ integrins exist in two conformations: an active extended conformation where the ligand binding head domain is present at the apex of the heterodimer and a basal, inactive bent conformation where the globular head of the integrin is folded toward the cell membrane (30, 53, 55). Pathogenic HTN and NY-1 hantaviruses bind to the N-terminal plexin-semaphorin-integrin (PSI) domain of β₃ integrin subunits and are selective for bent, inactive α_vβ₃ integrin conformers (38). Pathogenic hantavirus binding to inactive α_vβ₃ integrins is consistent with the selective inhibitory effect of hantaviruses on α_vβ₃ function and endothelial cell permeability (14, 15, 38). Although the mechanism of hantavirus induced vascular permeability has yet to be defined, there is a clear role for β₃ integrin dysfunction in vascular permeability deficits (5, 6, 22, 29, 39, 40, 51) which make an understanding of hantavirus interactions with β₃ subunits important for both entry and disease processes.The similarity between HPS disease in humans and Syrian hamsters (20, 21) suggests that pathogenic mechanisms of ANDV disease are likely to be coincident. Curiously, other hantaviruses (Sin Nombre virus [SNV] and Hantaan virus [HTNV]) are restricted in Syrian hamsters and fail to cause disease in this animal, even though they are prominent causes of human disease (50). Although the host range restriction for SNV and HTNV in Syrian hamsters has not been defined (33), the pathogenesis of ANDV in Syrian hamsters suggests that both human and Syrian hamster β₃ integrins may similarly be used by ANDV and contribute to pathogenesis.We demonstrate here that ANDV infection of the Syrian hamster BHK-21 cell line and human endothelial cells is dependent on α_vβ₃ and inhibited by α_vβ₃ specific ligands and antibodies. Further, polypeptides expressing the N-terminal 53 residues of human and Syrian hamster β₃ subunits block ANDV infection. This further indicates that ANDV interaction with the N-terminal 53 residues of both human and Syrian hamster β₃ integrins is required for viral entry. We also demonstrate that ANDV recognition of human and Syrian hamster β₃ integrins is determined by proline substitutions at residues 32/33 within the β₃ integrin PSI domain. These results define unique ANDV interactions with human and Syrian hamster β₃ integrins.     

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

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

Utilization of Immunoglobulin G Fc Receptors by Human Immunodeficiency Virus Type 1: a Specific Role for Antibodies against the Membrane-Proximal External Region of gp41

Receptors (FcγRs) for the constant region of immunoglobulin G (IgG) are an important link between humoral immunity and cellular immunity. To help define the role of FcγRs in determining the fate of human immunodeficiency virus type 1 (HIV-1) immune complexes, cDNAs for the four major human Fcγ receptors (FcγRI, FcγRIIa, FcγRIIb, and FcγRIIIa) were stably expressed by lentiviral transduction in a cell line (TZM-bl) commonly used for standardized assessments of HIV-1 neutralization. Individual cell lines, each expressing a different FcγR, bound human IgG, as evidence that the physical properties of the receptors were preserved. In assays with a HIV-1 multisubtype panel, the neutralizing activities of two monoclonal antibodies (2F5 and 4E10) that target the membrane-proximal external region (MPER) of gp41 were potentiated by FcγRI and, to a lesser extent, by FcγRIIb. Moreover, the neutralizing activity of an HIV-1-positive plasma sample known to contain gp41 MPER-specific antibodies was potentiated by FcγRI. The neutralizing activities of monoclonal antibodies b12 and 2G12 and other HIV-1-positive plasma samples were rarely affected by any of the four FcγRs. Effects with gp41 MPER-specific antibodies were moderately stronger for IgG1 than for IgG3 and were ineffective for Fab. We conclude that FcγRI and FcγRIIb facilitate antibody-mediated neutralization of HIV-1 by a mechanism that is dependent on the Fc region, IgG subclass, and epitope specificity of antibody. The FcγR effects seen here suggests that the MPER of gp41 could have greater value for vaccines than previously recognized.Fc receptors (FcRs) are differentially expressed on a variety of cells of hematopoietic lineage, where they bind the constant region of antibody (Ab) and provide a link between humoral and cellular immunity. Humans possess two classes of FcRs for the constant region of IgG (FcγRs) that, when cross-linked, are distinguished by their ability to either activate or inhibit cell signaling (69, 77, 79). The activating receptors FcγRI (CD64), FcγRIIa (CD32), and FcγRIII (CD16) signal through an immunoreceptor tyrosine-based activation motif (ITAM), whereas FcγRIIb (CD32) contains an inhibitory motif (ITIM) that counters ITAM signals and B-cell receptor signals. It has been suggested that a balance between activating and inhibitory FcγRs coexpressed on the same cells plays an important role in regulating adaptive immunity (23, 68). Moreover, the inhibitory FcγRIIb, being the sole FcγR on B cells, appears to play an important role in regulating self-tolerance (23, 68). The biologic role of FcγRs may be further influenced by differences in their affinity for immunoglobulin G (IgG); thus, FcγRI is a high-affinity receptor that binds monomeric IgG (mIgG) and IgG immune complexes (IC), whereas FcγRIIa, FcγRIIb, and FcγRIIIa are medium- to low-affinity receptors that preferentially bind IgG IC (10, 49, 78). FcγRs also exhibit differences in their relative affinity for the four IgG subclasses (10), which has been suggested to influence the balance between activating and inhibitory FcγRs (67).In addition to their participation in acquired immunity, FcγRs can mediate several innate immune functions, including phagocytosis of opsonized pathogens, Ab-dependent cell cytotoxicity (ADCC), antigen uptake by professional antigen-presenting cells, and the production of inflammatory cytokines and chemokines (26, 35, 41, 48, 69). In some cases, interaction of Ab-coated viruses with FcγRs may be exploited by viruses as a means to facilitate entry into FcγR-expressing cells (2, 33, 47, 84). Several groups have reported FcγR-mediated Ab-dependent enhancement (ADE) of HIV-1 infection in vitro (47, 51, 58, 63, 94, 96), whereas other reports have implicated FcγRs in efficient inhibition of the virus in vitro (19, 21, 29, 44-46, 62, 98) and possibly as having beneficial effects against HIV-1 in vivo (5, 27, 28, 42). These conflicting results are further complicated by the fact that HIV-1-susceptible cells, such as monocytes and macrophages, can coexpress more than one FcγR (66, 77, 79).HIV-1 entry requires sequential interactions between the viral surface glycoprotein, gp120, and its cellular receptor (CD4) and coreceptor (usually CCR5 or CXCR4), followed by membrane fusion that is mediated by the viral transmembrane glycoprotein gp41 (17, 106). Abs neutralize the virus by binding either gp120 or gp41 and blocking entry into cells. Several human monoclonal Abs that neutralize a broad spectrum of HIV-1 variants have attracted considerable interest for vaccine design. Epitopes for these monoclonal Abs include the receptor binding domain of gp120 in the case of b12 (71, 86), a glycan-specific epitope on gp120 in the case of 2G12 (13, 85, 86), and two adjacent epitopes in the membrane-proximal external region (MPER) of g41 in the cases of 2F5 and 4E10 (3, 11, 38, 93). At least three of these monoclonal Abs have been shown to interact with FcRs and to mediate ADCC (42, 43).A highly standardized and validated assay for neutralizing Abs against HIV-1 that quantifies reductions in luciferase (Luc) reporter gene expression after a single round of virus infection in TZM-bl cells has been developed (60, 104). TZM-bl (also called JC53BL-13) is a CXCR4-positive HeLa cell line that was engineered to express CD4 and CCR5 and to contain integrated reporter genes for firefly Luc and Escherichia coli β-galactosidase under the control of the HIV-1 Tat-regulated promoter in the long terminal repeat terminal repeat sequence (74, 103). TZM-bl cells are permissive to infection by a wide variety of HIV-1, simian immunodeficiency virus, and human-simian immunodeficiency virus strains, including molecularly cloned Env-pseudotyped viruses. Here we report the creation and characterization of four new TZM-bl cell lines, each expressing one of the major human FcγRs. These new cell lines were used to gain a better understanding of the individual roles that FcγRs play in determining the fate of HIV-1 IC. Two FcγRs that potentiated the neutralizing activity of gp41 MPER-specific Abs were identified.     

Critical Role of Airway Macrophages in Modulating Disease Severity during Influenza Virus Infection of Mice

Airway macrophages provide a first line of host defense against a range of airborne pathogens, including influenza virus. In this study, we show that influenza viruses differ markedly in their abilities to infect murine macrophages in vitro and that infection of macrophages is nonproductive and no infectious virus is released. Virus strain BJx109 (H3N2) infected macrophages with high efficiency and was associated with mild disease following intranasal infection of mice. In contrast, virus strain PR8 (H1N1) was poor in its ability to infect macrophages and highly virulent for mice. Depletion of airway macrophages by clodronate-loaded liposomes led to the development of severe viral pneumonia in BJx109-infected mice but did not modulate disease severity in PR8-infected mice. The severe disease observed in macrophage-depleted mice infected with BJx109 was associated with exacerbated virus replication in the airways, leading to severe airway inflammation, pulmonary edema, and vascular leakage, indicative of lung injury. Thymic atrophy, lymphopenia, and dysregulated cytokine and chemokine production were additional systemic manifestations associated with severe disease. Thus, airway macrophages play a critical role in limiting lung injury and associated disease caused by BJx109. Furthermore, the inability of PR8 to infect airway macrophages may be a critical factor contributing to its virulence for mice.Airway macrophages (Mφ) (AM), located at the interphase between air and lung tissue, provide the first line of defense following inhalation of airborne pathogens, including influenza viruses. In addition to phagocytosis of virions and virus-infected cells (16, 24), infection of AM represents an early event in recognition of the virus by the innate immune system. Following intranasal (i.n.) infection of mice, influenza virus replicates productively in type II epithelial cells lining the respiratory tract. Murine Mφ are also susceptible to influenza virus infection and viral proteins are produced, but replication is abortive and infectious progeny are not released (52, 69), although recent studies suggest limited release from mouse Mφ exposed to highly pathogenic H1N1 and H5N1 viruses (44). Following exposure to influenza virus, Mφ synthesize and release proinflammatory cytokines and alpha/beta interferon (26, 27, 45, 55), which further limit viral replication and spread within the respiratory tract. Inflammatory responses in the airways must be tightly regulated to ensure rapid virus clearance while avoiding excessive or chronic inflammation that may damage the delicate tissue-air interface.Liposome-encapsulated dichloromethylene diphosphonate (clodronate or CL2-MDP) is taken up by phagocytic Mφ and accumulates in the cytosol, resulting in Mφ death and depletion (66). Administration of clodronate liposomes (CL-LIP) has been widely used to selectively deplete airway Mφ in mouse models without affecting circulating monocytes (for examples, see references 4, 6, 8, 36, 47, 64, and 71). While CL-LIP has been used predominantly in rodent models of infection, it is noteworthy that CL-LIP treatment of pigs, a natural host of influenza virus, resulted in enhanced morbidity and mortality following infection with a human H1N1 subtype virus (30). In murine studies, depletion of airway Mφ prior to influenza virus infection led to increased cytotoxic CD8⁺ T-cell responses in the lungs of virus-infected mice (71); however, treatment with CL-LIP 48 h after infection was associated with impaired CD8⁺ T-cell responses (41). Furthermore, CL-LIP treatment prior to intranasal infection of mice with a recombinant virus bearing the surface glycoproteins of the 1918 pandemic strain led to exacerbated disease and mortality (64). Treatment of mice with CL-LIP has been associated with enhanced virus replication (41, 64, 71); however, the mechanisms by which airway Mφ initiate and modulate inflammatory responses and disease after influenza virus infection have not been fully elucidated.We observed in a previous study that influenza A virus strains show marked differences in their abilities to infect murine Mφ in vitro and implicated the Mφ mannose receptor (MMR) (CD206), a C-type lectin (46), in infectious virus entry (48). Virus strain BJx109, a reassortant virus bearing the surface glycoproteins of A/Beijing/353/89 (H3N2) and internal components derived from A/PR/8/34 (H1N1) (PR8) bears a highly glycosylated hemagglutinin (HA) molecule and was shown to infect Mφ to high levels, while the HA of PR8 is poorly glycosylated and the virus infected Mφ very poorly. In the current study, we demonstrate that intranasal infection of mice with BJx109 leads to mild clinical disease, while infection with an equivalent dose of PR8 leads to severe disease and rapid death. Depletion of airway Mφ by intranasal administration of CL-LIP prior to and during infection with influenza viruses had little effect on the course of PR8 infection; however, BJx109 infection of Mφ-depleted animals led to severe disease and death. Severe disease was associated with enhanced virus replication, severe airway inflammation, and pulmonary edema and vascular leakage, indicative of lung injury. Together, these data demonstrate that airway Mφ play a critical role in moderating disease severity during BJx109 infection. Furthermore, they suggest that the ability of PR8 to evade infectious uptake by airway Mφ is likely to be an important factor contributing to its virulence for mice.     

Borna Disease Virus Requires Cholesterol in both Cellular Membrane and Viral Envelope for Efficient Cell Entry

Borna disease virus (BDV), the prototypic member of the family Bornaviridae within the order Mononegavirales, provides an important model for the investigation of viral persistence within the central nervous system (CNS) and of associated brain disorders. BDV is highly neurotropic and enters its target cell via receptor-mediated endocytosis, a process mediated by the virus surface glycoprotein (G), but the cellular factors and pathways determining BDV cell tropism within the CNS remain mostly unknown. Cholesterol has been shown to influence viral infections via its effects on different viral processes, including replication, budding, and cell entry. In this work, we show that cell entry, but not replication and gene expression, of BDV was drastically inhibited by depletion of cellular cholesterol levels. BDV G-mediated attachment to BDV-susceptible cells was cholesterol independent, but G localized to lipid rafts (LR) at the plasma membrane. LR structure and function critically depend on cholesterol, and hence, compromised structural integrity and function of LR caused by cholesterol depletion likely inhibited the initial stages of BDV cell internalization. Furthermore, we also show that viral-envelope cholesterol is required for BDV infectivity.Borna disease virus (BDV) is an enveloped virus with a nonsegmented negative-strand RNA genome whose organization (3′-N-p10/P-M-G-L-5′) is characteristic of mononegaviruses (6, 28, 46, 48). However, based on its unique genetics and biological features, BDV is considered to be the prototypic member of a new virus family, Bornaviridae, within the order Mononegavirales (8, 28, 46, 49).BDV can infect a variety of cell types in cell culture but in vivo exhibits exquisite neurotropism and causes central nervous system (CNS) disease in different vertebrate species, which is frequently manifested in behavioral abnormalities (19, 33, 44, 53). Both host and viral factors contribute to a variable period of incubation and heterogeneity in the symptoms and pathology associated with BDV infection (14, 16, 29, 42, 44). BDV provides an important model for the investigation of both immune-mediated pathological events associated with virus-induced neurological disease and mechanisms whereby noncytolytic viruses induce neurodevelopmental and behavioral disturbances in the absence of inflammation (15, 18, 41). Moreover, serological data and molecular epidemiological studies suggest that BDV, or a BDV-like virus, can infect humans and that it might be associated with certain neuropsychiatric disorders (17, 24), which further underscores the interest in understanding the mechanisms underlying BDV persistence in the CNS and its effect on brain cell functions. The achievement of these goals will require the elucidation of the determinants of BDV cell tropism within the CNS.BDV enters its target cell via receptor-mediated endocytosis, a process in which the BDV G protein plays a central role (1, 5, 13, 14, 39). Cleavage of BDV G by the cellular protease furin generates two functional subunits: GP1 (GP_N), involved in virus interaction with a yet-unidentified cell surface receptor (1, 39), and GP2 (GP_C), which mediates a pH-dependent fusion event between viral and cellular membranes (13). However, a detailed characterization of cellular factors and pathways involved in BDV cell entry remains to be done.Besides cell surface molecules that serve as viral receptors, many other cell factors, including nonproteinaceous molecules, can influence cell entry by virus (52). In this regard, cholesterol, which plays a critical role in cellular homeostasis (55), has also been identified as a key factor required for productive infection by different viruses. Accordingly, cholesterol participates in a variety of processes in virus-infected cells, including fusion events between viral and cellular membranes (3), viral replication (23), and budding (35, 37), as well as maintenance of lipid rafts (LR) (12) as scaffold structures where the viral receptor and coreceptor associate (11, 26, 32, 36). LR are specialized microdomains within cellular membranes constituted principally of proteins, sphingolipids, and cholesterol. LR facilitate the close proximity and interaction of specific sets of proteins and contribute to different processes associated with virus multiplication (38). Cholesterol can also influence virus infection by contributing to the maintenance of the properties of the viral envelope required for virus particle infectivity (21, 54). Here, we show for the first time that cholesterol plays a critical role in BDV infection. Depletion of cellular cholesterol prior to, but not after, BDV cell entry prevented productive BDV infection, likely due to disruption of plasma membrane LR that appear to be the cell entry point for BDV. In addition, we document that cholesterol also plays an essential role in the properties of the BDV envelope required for virus particle infectivity.     

Lipid Phosphate Phosphatase 3 Stabilization of β-Catenin Induces Endothelial Cell Migration and Formation of Branching Point Structures

Endothelial cell (EC) migration, cell-cell adhesion, and the formation of branching point structures are considered hallmarks of angiogenesis; however, the underlying mechanisms of these processes are not well understood. Lipid phosphate phosphatase 3 (LPP3) is a recently described p120-catenin-associated integrin ligand localized in adherens junctions (AJs) of ECs. Here, we tested the hypothesis that LPP3 stimulates β-catenin/lymphoid enhancer binding factor 1 (β-catenin/LEF-1) to induce EC migration and formation of branching point structures. In subconfluent ECs, LPP3 induced expression of fibronectin via β-catenin/LEF-1 signaling in a phosphatase and tensin homologue (PTEN)-dependent manner. In confluent ECs, depletion of p120-catenin restored LPP3-mediated β-catenin/LEF-1 signaling. Depletion of LPP3 resulted in destabilization of β-catenin, which in turn reduced fibronectin synthesis and deposition, which resulted in inhibition of EC migration. Accordingly, reexpression of β-catenin but not p120-catenin in LPP3-depleted ECs restored de novo synthesis of fibronectin, which mediated EC migration and formation of branching point structures. In confluent ECs, however, a fraction of p120-catenin associated and colocalized with LPP3 at the plasma membrane, via the C-terminal cytoplasmic domain, thereby limiting the ability of LPP3 to stimulate β-catenin/LEF-1 signaling. Thus, our study identified a key role for LPP3 in orchestrating PTEN-mediated β-catenin/LEF-1 signaling in EC migration, cell-cell adhesion, and formation of branching point structures.Angiogenesis, the formation of new blood vessels, involves several well-coordinated cellular processes, including endothelial cell (EC) migration, synthesis and deposition of extracellular matrix proteins, such as fibronectin, cell-cell adhesion, and formation of branching point structures (1-3, 19, 33); however, less is known about the underlying mechanisms of these processes (6, 8, 12, 14, 16, 17). For example, adherens junctions (AJs), which mediate cell-cell adhesion between ECs, may be involved in limiting the extent of cell migration (2, 14, 38, 40). VE-cadherin, a protein found in AJs, is a single-pass transmembrane polypeptide responsible for calcium-dependent homophilic interactions through its extracellular domains (2, 38, 40). The VE-cadherin cytoplasmic domain interacts with the Armadillo domain-containing proteins, β-catenin, γ-catenin (plakoglobin), and p120-catenin (p120ctn) (2, 15, 38, 40, 43). Genetic and biochemical evidence documents a crucial role of β-catenin in regulating cell adhesion as well as proliferation secondary to the central position of β-catenin in the Wnt signaling pathway (13, 16, 25, 31, 44). In addition, the juxtamembrane protein p120ctn regulates AJ stability via binding to VE-cadherin (2, 7, 9, 15, 21, 28, 32, 43). The absence of regulation or inappropriate regulation of β-catenin and VE-cadherin functions is linked to cardiovascular disease and tumor progression (2, 6).We previously identified lipid phosphate phosphatase 3 (LPP3), also known as phosphatidic acid phosphatase 2b (PAP2b), in a functional assay of angiogenesis (18, 19, 41, 42). LPP3 not only exhibits lipid phosphatase activity but also functions as a cell-associated integrin ligand (18, 19, 35, 41, 42). The known LPPs (LPP1, LPP2, and LPP3) (20-23) are six transmembrane domain-containing plasma membrane-bound enzymes that dephosphorylate sphingosine-1-phosphate (S1P) and its structural homologues, and thus, these phosphatases generate lipid mediators (4, 5, 23, 35, 39). All LPPs, which contain a single N-glycosylation site and a putative lipid phosphatase motif, are situated such that their N and C termini are within the cell (4, 5, 22, 23, 35, 39). Only the LPP3 isoform contains an Arg-Gly-Asp (RGD) sequence in the second extracellular loop, and this RGD sequence enables LPP3 to bind integrins (18, 19, 22). Transfection experiments with green fluorescent protein (GFP)-tagged LPP1 and LPP3 showed that LPP1 is apically sorted, whereas LPP3 colocalized with E-cadherin at cell-cell contact sites with other Madin-Darby canine kidney (MDCK) cells (22). Mutagenesis and domain swapping experiments established that LPP1 contains an apical targeting signal sequence (FDKTRL) in its N-terminal segment. In contrast, LPP3 contains a dityrosine (109Y/110Y) basolateral sorting motif (22). Interestingly, conventional deletion of Lpp3 is embryonic lethal, since the Lpp3 gene plays a critical role in extraembryonic vasculogenesis independent of its lipid phosphatase activity (11). In addition, an LPP3-neutralizing antibody was shown to prevent cell-cell interactions (19, 42) and angiogenesis (42). Here, we addressed the hypothesis that LPP3 plays a key role in EC migration, cell-cell adhesion, and formation of branching point structures by stimulating β-catenin/lymphoid enhancer binding factor 1 (β-catenin/LEF-1) signaling.