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      Viral Interactions with PDZ Domain-Containing Proteins—An Oncogenic Trait?

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          Abstract

          Many of the human viruses with oncogenic capabilities, either in their natural host or in experimental systems (hepatitis B and C, human T cell leukaemia virus type 1, Kaposi sarcoma herpesvirus, human immunodeficiency virus, high-risk human papillomaviruses and adenovirus type 9), encode in their limited genome the ability to target cellular proteins containing PSD95/ DLG/ZO-1 (PDZ) interaction modules. In many cases (but not always), the viruses have evolved to bind the PDZ domains using the same short linear peptide motifs found in host protein-PDZ interactions, and in some cases regulate the interactions in a similar fashion by phosphorylation. What is striking is that the diverse viruses target a common subset of PDZ proteins that are intimately involved in controlling cell polarity and the structure and function of intercellular junctions, including tight junctions. Cell polarity is fundamental to the control of cell proliferation and cell survival and disruption of polarity and the signal transduction pathways involved is a key event in tumourigenesis. This review focuses on the oncogenic viruses and the role of targeting PDZ proteins in the virus life cycle and the contribution of virus-PDZ protein interactions to virus-mediated oncogenesis. We highlight how many of the viral associations with PDZ proteins lead to deregulation of PI3K/AKT signalling, benefitting virus replication but as a consequence also contributing to oncogenesis.

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          Severe Acute Respiratory Syndrome Coronavirus Envelope Protein Ion Channel Activity Promotes Virus Fitness and Pathogenesis

          Introduction Coronaviruses (CoVs) are vertebrate pathogens that cause severe diseases in a wide range of animals and infections in humans that until recently were limited to common colds [1]. Nevertheless, by the end of 2002, a novel coronavirus causing the severe acute respiratory syndrome (SARS-CoV) emerged in China and rapidly spread worldwide causing around 8000 infections leading to death in 10% of the cases [2], [3]. Since then, CoVs surveillance programs were intensified, and two additional human coronaviruses, already circulating in the human population, were identified as the causative agents of several cases of pneumonia and bronchiolitis (HCoV-HKU1 and HCoV-NL63) [4]. Furthermore, in 2012 a novel coronavirus infecting humans, the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) appeared in Saudi Arabia and disseminated to nine additional countries [5], [6]. To date, 182 cases of MERS-CoV have been reported, which has led to 79 fatalities (http://www.who.int). Clinical presentation of infected individuals involves acute pneumonia, sometimes accompanied by renal disease [7]. CoVs similar to SARS-CoV and MERS-CoV have also been isolated from bats widely distributed throughout the world [8]–[13], which represents a potential reservoir for outbreaks of novel zoonoses into humans. Therefore, understanding the virulence mechanisms of these pathogens, will allow the development of effective therapies in order to prevent and control future outbreaks. SARS-CoV is an enveloped virus containing a positive sense RNA genome of 29.7 kb, one of the largest viral RNA genomes known. The genome encodes a viral replicase involved in the synthesis of new genomes and in the generation of a nested set of subgenomic messenger RNAs, encoding both structural proteins present in all CoVs: Spike (S), Envelope (E), Membrane (M) and Nucleoprotein (N), and a group of proteins specific for SARS-CoV: 3a, 3b, 6, 7a, 7b, 8a, 8b, and 9b [14]. SARS-CoV E protein is a 76-amino acid transmembrane protein actively synthesized during viral infection, that mainly localizes at the ERGIC region of the cell, where virus budding and morphogenesis take place [15]–[18]. Different requirements of E protein during the virus cycle have been described among CoVs. Elimination of E gene in transmissible gastroenteritis coronavirus (TGEV) or MERS-CoV leads to a replication-competent propagation-deficient phenotype [19]–[21]. In contrast, deletion of E gene from mouse hepatitis virus (MHV) or SARS-CoV does not abolish virus production, although viral titers are significantly reduced by 1000 to 20-fold, respectively [16], [22]. Interestingly, E gene deleted SARS-CoV (SARS-CoV-ΔE) was attenuated in three animal models, and confers protection against challenge with parental virus in immunized hamsters, and in young or aged mice, representing a promising vaccine candidate [16], [23]–[27]. Cells infected with SARS-CoV-ΔE show increased stress and apoptotic markers compared to wild type virus, perhaps resulting in a decreased productivity of infection [28]. Additionally, elimination of the E gene diminishes inflammation induced by SARS-CoV through the NF-κB pathway [27]. Remarkably, SARS-CoV E protein was found to self-interact forming a pentameric structure that delimits an ion conductive pore, which may play a role in virus-host interaction [29]–[32]. E protein ion conductivity was also confirmed for a set of CoVs from different genera [33]. The ion channel (IC) activity of SARS-CoV E protein was mapped within the transmembrane domain of the protein by using synthetic peptides [31], [34], [35]. Recent studies determined that both ion conductance and selectivity of E protein ion channel were highly controlled by the charge of the lipid membranes in which the pores were assembled. This suggests that lipid head-groups are components of the channel structure facing the lumen of the pore, a novel concept for CoV E protein ion channel [34], [36]. Chemically synthesized SARS-CoV E protein showed slight preference for cations over anions when reconstituted in lipids that mimicked both charge and composition of ERGIC membranes, and displayed no specific selectivity for a particular cation [34], [36]. In addition, point mutations that suppressed SARS-CoV E protein IC activity (N15A and V25F) have been identified and confirmed [34], [35]. Several reports have analyzed the relevance of CoV E protein transmembrane domain, which contains ion-conduction properties, in virus maturation and production. Insertion of alanine residues within the transmembrane domain of MHV E protein rendered crippled viruses that evolutionary reverted to restore a proper structure of the alpha helix within the transmembrane domain [37]. Interchanging the genus β CoV MHV E protein transmembrane domain by those of CoVs from different genera revealed that only domains belonging to genus β, and γ, but not α, functionally replaced MHV E transmembrane domain in terms of viral production. It was speculated that this effect was a consequence of the possible different ion selectivity of these domains [38]. Replacement of genus γ CoV infectious bronchitis virus (IBV) E protein transmembrane domain, which displays IC activity, for vesicular stomatitis virus (VSV) G protein transmembrane domain lacking this function, interfered with an efficient trafficking and release of the viral progeny in the infected cells [39]. In contrast, mutation of threonine at position 16 to alanine, which is the amino acid change predicted to inhibit IC activity in IBV E protein did not affect virus-like particles formation, suggesting a multifunctional role of E protein [40]. Besides the E protein, SARS-CoV encodes two other ion-conducting proteins, 3a and 8a [41], [42]. In a related virus, human coronavirus 229E (HCoV-229E), novel IC activity has been described within the 4a protein [43]. The abundance and conservation of IC activity suggests an importance of influencing ion homeostasis within cells during the CoV infection cycle. Modulation of the cellular ion balance seems to be a common issue for viruses, as a growing list of viroporins are being identified, especially within RNA viruses [44]. Highly pathogenic human viruses such as influenza A virus, human immunodeficiency virus (HIV), hepatitis C virus (HCV) and several picornaviruses, among others, encode at least one viroporin [45]–[49]. Viroporins have been involved in virus entry, trafficking, morphogenesis, maturation and even virulence [50]–[53]. Influenza virus M2 is essential for viral RNA release from infections virions within the endosome into the cell cytoplasm [45] and also for raising the pH at the trans-Golgi network lumen, which prevents premature activation of hemaglutinin, which may render non-infectious virions [54]. Similarly, HCV p7 protein equilibrates the pH at the Golgi apparatus, protecting acid-sensitive intracellular virions [51]. Coxsackievirus 2B protein alters Golgi and endoplasmic reticulum (ER) Ca2+ and H+ concentrations, which in turn delay protein transport through the secretory pathway facilitating virus assembly and preventing major histocompatibility complex (MHC) molecules from reaching the cell surface [48], [55], [56]. A recent finding described that influenza M2 protein IC activity triggers NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome activation [52]. Furthermore, mutant versions of M2 protein that conduct Na+ and K+ ions apart from H+ ions more strongly elicited the inflammasome response [52]. This novel mechanism of immune system activation has also been proven for other viroporins [53], [57]–[59]. Viral proteins with IC activity impact different aspects of the virus life cycle, however, the involvement of their IC activity in pathogenesis remain to be further explored. Previous findings demonstrated that SARS-CoV E protein is a virulence determinant. In this manuscript we analyze the contribution of E protein IC activity in pathogenesis. Two recombinant viruses, each one containing a single point mutation suppressing IC activity, were generated by reverse genetics. Mutant viruses showed a tendency to evolve and restore E protein IC architecture and activity after serial infections, and viruses with deficient IC activity were outcompeted by those displaying this function after co-infections. This highlights the importance of IC activity in virus fitness. Interestingly, infection of mice with a set of viruses lacking or displaying E protein IC activity, revealed that the activation of inflammasome pathway, and the exacerbated inflammatory response induced by SARS-CoV was decreased in infections by on channel deficient viruses. In addition, less lung damage and proper localization of Na+/K+ ATPase within epithelia, which prevents edema accumulation, was detected for the mice infected with the viruses lacking E protein IC activity. As a consequence, increased survival of the infected animals was observed when E protein ion conductivity was absent. Therefore, E protein IC activity is required for inflammasome activation and a novel determinant for the virulence of highly pathogenic SARS-CoV. Results SARS-CoV E protein IC activity is not essential for virus production in cell culture Deletion of SARS-CoV E gene resulted in a virus that was attenuated in three animal models, as we have previously shown [16], [23], [24], [26], [27]. E gene codes for the small multifunctional E protein, which displays IC activity [31], [34]–[36]. To specifically test the relevance of IC activity in virus virulence, residues involved in E protein ion conductance were firstly identified. To this end a set of synthetic peptides representing the transmembrane domain of E protein were evaluated for their IC activity. These peptides contained point mutations that affect different conserved residues, or residues predicted to face the lumen of the channel pore [34]. Mutations N15A and V25F within the transmembrane domain of E protein completely disrupted IC activity [34], [35]. Accordingly, two recombinant viruses containing each of these two changes in the E gene, rSARS-CoV-E-N15A (N15A) and rSARS-CoV-E-V25F (V25F), were engineered ( Fig. 1 ). A SARS-CoV with a mouse adapted (MA15) genetic background [27], [60] was used to generate these viruses, as infection of mice with SARS-CoV MA15 accurately reproduces the symptoms of human disease [27], [60]. The mutant viruses were efficiently rescued, cloned by three rounds of plaque purification, and their sequence was confirmed (data not shown). To test whether the introduced mutations may alter E protein subcellular localization affecting other functions of the protein, Vero E6 cells were infected with the wt virus, the viruses lacking IC activity (N15A and V25F) or a virus missing E gene (ΔE) as a control. Immunofluorescence analysis showed similar colocalization patterns of E protein and ERGIC, the subcellular compartment where E protein mainly accumulates during infection, for both the wt and the mutant viruses ( Fig. 2A ), indicating that other functions of E protein associated with its localization are most likely not affected. 10.1371/journal.ppat.1004077.g001 Figure 1 Engineering of rSARS-CoVs lacking E protein ion channel (IC) activity. SARS-CoV genome is represented at the top, and the region expanded shows wild type SARS-CoV E protein sequence (wt) and its different domains: amino terminal (N-terminal), transmembrane (TMD) and carboxy terminal (C-terminal). To generate viruses lacking E protein ion channel activity (rSARS-CoV-EIC−) the amino acid changes N15A or V25F were introduced within viral genome to generate two recombinant viruses. The positions of the mutated residues within the transmembrane domain of a simplified E protein oligomer inserted in a lipid membrane are shown at the bottom. 10.1371/journal.ppat.1004077.g002 Figure 2 Subcellular localization of rSARS-CoV-EIC− E proteins, growth kinetics and plaque size. (A) Vero E6 cells were infected either with the mutant viruses (N15A and V25F), the parental virus (wt) or a virus lacking E gene (ΔE) at an MOI of 0.3, fixed at 24 hpi and E protein (green) and ERGIC (red) were labeled with specific antibodies. Nuclei were stained with DAPI (blue). Original magnification was 126×. Right graphic on the panel represents the percentage of colocalization between E protein and ERGIC, calculated with Leica LAS AF v2.6.0 software. (B) Vero E6 and DBT-mACE2 cells were infected at an MOI of 0.001 with mutant viruses lacking IC activity (N15A and V25F), the parental virus (wt) or a virus lacking E gene (ΔE), and viral progeny was titrated at the indicated times post-infection. Error bars represent the standard deviation of three independent experiments. (C) Plaque morphology of the parental, the mutant viruses N15A and V25F and a ΔE virus. Deletions or mutations within the E gene of several CoVs sometimes led to crippled viruses or to lower virus yields [16], [20]–[22], [37], [39]. To test whether inhibition of E protein IC activity affects virus production, growth kinetics were performed in the monkey Vero E6 and mouse DBT-mACE2 cells [61]. Minor differences in growth rates were observed between the parental virus (wt), that contains E protein IC activity, and the mutant viruses that lack E protein IC activity ( Fig. 2B ), indicating that this function was not essential for virus growth in cell culture. More striking differences in plaque phenotypes were observed. Mutant viruses lacking E protein IC activity, apparently formed smaller plaques than wt virus, and V25F virus plaques were smaller than N15A virus ( Fig. 2C ). A possible explanation for all these data could be that infection foci productivity and area may be quite similar regardless of E protein IC activity, as determined by viral titration, but higher cytopathic effect may be induced when E protein IC is present, rendering bigger plaques. Elimination of full-length E protein induced more severe growth defects ( Fig. 2B and Fig. 2C ), suggesting that other functions of the protein contributing to virus production, apart from IC activity, may be affected. SARS-CoV E protein IC activity improves viral fitness Inhibition of E protein IC activity slightly reduced virus production in cell culture in a relatively short period of time, but these differences were not significant. To further explore whether ion conductivity could improve viral growth and fitness, a long-term competition assay was performed between the wt virus and the N15A mutant lacking IC activity, that was relative stable through passages as will be described below. Vero E6 cells were co-infected with N15A mutant and the wt virus in a proportion 7∶3, and the supernatant was serially passaged for 20 times every 24 hours. The E gene was sequenced every 4 passages, revealing that the proportion of wt virus steadily increased over the passages, accompanied by a decrease in the abundance of the N15A mutant. From passage 8 on, the wt virus took and maintained majority over the N15A mutant ( Fig. 3 ). These results suggested that E protein IC activity for SARS-CoV confers a selective advantage improving virus production. 10.1371/journal.ppat.1004077.g003 Figure 3 Effect of SARS-CoV E protein IC activity on viral fitness. Competition assays between the parental virus (wt, black circles) displaying IC activity (EIC+) and a mutant virus (N15A, red squares) lacking IC activity (EIC−) were performed. Vero E6 cells were co-infected with mutant and parental viruses at a ratio 7∶3 and supernatants were serially passaged 20 times every 24 hours. Relative abundance of each virus was determined by sequencing E gene within viral progeny. Error bars represent the standard deviation from three independent experiments. SARS-CoV E protein IC activity confers virulence in vivo To specifically analyze the contribution of E protein IC activity to SARS-CoV virulence, BALB/c mice were intranasally inoculated with the wt virus displaying E protein IC activity, or three independently-isolated clones of the mutant viruses N15A and V25F lacking E protein IC activity, and mice were monitored daily for 10 days (n = 5/virus clone). All infected animals showed disease symptoms at 2 days post infection (dpi), reflected by slower movements and ruffled fur (data not shown). Mice infected with the wt virus started to lose weight by day 2, and by day 5 all of them died ( Fig. 4 ). Interestingly, although mice infected with the three clones of N15A mutant started to lose weight in a similar fashion, at day 4 almost all of them started to regain weight, recover from the disease, and 80–100% survived ( Fig. 4 ). In contrast to N15A, mice infected with V25F virus experimented similar weight losses and survival rates (from 0 to 20%) than the wt virus ( Fig. 4 ). A possible explanation for this apparent discrepancy was the reversion of the introduced mutation or the incorporation of compensatory mutations restoring E protein IC activity. To test whether this was the case, total RNA was collected from the lungs of infected mice at 2 and 4 dpi or from the lungs of mice that died after infection. The virus genome region containing E gene was sequenced, as it was the target of the point mutations inhibiting IC activity, and therefore a likely place to incorporate compensatory mutations. E genes from wt virus and N15A mutant virus remained stable during the course of the experiment, since no changes were found in viral RNA extracted either from lungs of several mice at 2 and 4 dpi or from dead mice ( Fig. 5A ). In contrast, V25F viruses incorporated mutations in the E gene that led to amino acid changes either in the same position of the mutation that abolished IC activity (F25C) or in relatively close positions within the E protein transmembrane domain: L19A, F20L, F26L, L27S, T30I and L37R ( Fig. 5A ). These evolved variants of the V25F virus appeared as early as 2 days after mice infection and, in some cases (T30I mutant), completely overgrew the original virus by day 2. The tentative compensatory mutations were also present in the viral population at 4 dpi and in dead mice ( Fig. 5A ). Overall, the data obtained with wt and N15A viruses, which were genetically stable throughout the experiment, suggest that E protein IC activity is required for a virulent phenotype. 10.1371/journal.ppat.1004077.g004 Figure 4 Pathogenesis caused by rSARS-CoV-EIC− in BALB/c mice. Groups of five 16 week-old BALB/c mice were mock infected (Mock, green circles) or infected with 100000 PFU of either the parental virus (wt, black circles) or several clones of the mutant viruses missing IC activity: N15A C1, N15A C2 and N15A C3 (red, orange and deep-red squares, respectively), and V25F C1, V25F C2 and V25F C3 (dark blue, blue and light blue triangles, respectively). Mean weight losses (left graph) and survival (right graph) during 10 days following infection are represented for each group. Error bars represent the standard deviation for mice weights per experimental condition. 10.1371/journal.ppat.1004077.g005 Figure 5 Stability of rSARS-CoV-EIC− after serial infections. (A) Groups of eleven 16 week-old BALB/c mice were infected with 100000 PFU of either the parental virus (wt) or three clones of the mutant viruses missing IC activity: N15A C1, N15A C2, N15A C3, V25F C1, V25F C2 and V25F C3. At 2 dpi and 4 dpi 3 mice of each group were sacrificed, lung RNA was extracted, and E gene was sequenced. The rest of the mice (5 per group) participated in the weight-loss and survival experiment. When any mouse died, from 4 to 10 dpi, lung RNA was extracted and E gene was sequenced. Bars represent different E protein sequences, either that of parental or the mutant viruses. The central colored part represents the transmembrane domain of the protein. Letters and numbers in red represent the amino acid changes detected after viral evolution and their relative position within transmembrane domain, respectively. Numbers accompanying bars indicate from how many mice (first number) out of the total of the animals analyzed (second number) arose the indicated sequence change. Dead mice are indicated by a †. (B) Vero E6 cells were infected with the wt virus or the mutant clones N15A C1 and N15A C2, V25F C1 and V25F C2 at an initial MOI of 0.5, and supernatants were serially passaged for 24 times every 24 hours. E gene in the viral population was sequenced at passages 0, 8, 16 and 24. Colored bars represent the transmembrane domain of different E protein sequences and letters and numbers in red represent the amino acid mutations identified and their relative position, respectively. Viruses missing E protein IC activity are prone to evolve and restore ion conductivity To further analyze the evolution of the mutant viruses lacking E protein IC activity, two clones of the mutants N15A and V25F were serially passaged in cell culture. Throughout the 24 serial passages, E gene was sequenced at passages 0, 8, 16 and 24 for the two mutant viruses and wt as control. As observed during in vivo infection, the wt virus remained stable during the passages ( Fig. 5B ). V25F viruses rapidly incorporated additional mutations within E gene (L19A, L27S and T30I), reproducing our in vivo observations. The viruses incorporating T30I mutation completely out-competed the original V25F mutant by passage 8 ( Fig. 5B ). In contrast, N15A viruses either remained stable or incorporated a mutation in the E gene (A15D) that appeared late, at passage 24, suggesting that this mutant was more stable, confirming our in vivo results ( Fig. 5B ). The data obtained in cell culture or after mice infection indicate that SARS-CoVs lacking E protein IC activity incorporated mutations at the E gene that directly reverted the original mutation that suppressed IC activity (A15D and F25C) or modified residues mapping to a close position of the E protein transmembrane domain. These modified residues face the original mutation inhibiting IC activity, when the ion channel is assembled ( Fig. 6 ). To analyze whether these mutations restored IC activity, synthetic peptides representing the E protein transmembrane domain containing the mutations obtained after viral evolution in vivo and in cell culture (N15D, V25L, V25F L19A, V25F F26C, V25F L27S, V25F T30I, V25F L37R), were synthesized. The IC activity of these peptides was evaluated in artificial lipid membranes as previously described [34]. Whereas peptides containing the original mutations N15A and V25F did not show any conductance, all the peptides containing the mutations obtained after viral evolution displayed similar conductance values than a wild type peptide ( Fig. 7 ), indicating that all these compensatory mutations restored E protein IC activity. 10.1371/journal.ppat.1004077.g006 Figure 6 Spatial distribution of the mutations obtained in rSARS-CoV-EIC− after serial infections. (A) Left diagram represents a top view of E protein transmembrane domain and the spatial distribution of the amino acids within the alpha helix. Blue and red circles correspond to amino acids N15 and V25, respectively, originally mutated to inhibit IC activity. Yellow circles surround the amino acids that changed after evolution of V25F mutant. Arrow at position 15 points the lumen of the ion channel pore. Right graphic depicts the pentamer conformation of E protein that forms the ion conductive pore and the positions of both the mutated residue at position 25 and the evolved mutations at positions 19, 25, 26, 27, 30 and 37. Evolved changes map close to the originally mutated residue in the monomer-monomer interface. (B) Pentameric model of SARS-CoV E protein from a lateral (left) or a top view (right). This model was first proposed from linear dichroism of isotopically labeled E protein transmembrane peptides in lipid bilayers [29], [32]. The residues involved in ion channel inhibition (N15 in blue and V25 in red) or mutated after viral evolution (L19, F26, L27, T30 and L37 in yellow) are highlighted. 10.1371/journal.ppat.1004077.g007 Figure 7 E protein IC activity of the rSARS-CoV-EIC− evolved variants. Synthetic peptides representing E protein transmembrane domain of the parental virus (wt) the mutant viruses (MUT) lacking IC activity (N15A and V25F) and their evolved revertants (REV) obtained after infections of mice or cell culture (N15D, V25L, V25F L19A, V25F F26C, V25F L27S, V25F T30I and V25F L37R) were reconstituted in artificial lipid bilayers, and their IC activity was analyzed as mean conductance values. Negative controls (C−) indicate conductance values obtained in the absence of any peptide. Error bars represent the variations obtained in 100 independent experiments. Genetically engineered revertant viruses restoring E protein IC activity show a virulent phenotype in mice A correlation between IC activity and virulence was found in vivo, where N15A viruses lacking IC activity were attenuated compared to wt virus competent in IC activity. Mutant virus V25F, originally lacking ion conductivity, rapidly incorporated compensatory mutations upon infection in vivo that restored IC activity and thus caused pathogenicity. To test whether the recovery of IC activity was the unique determinant of virulence, and to rule out effects of other mutations arising outside of the E gene, recombinant viruses containing a set of the compensatory mutations that restored IC activity (rSARS-CoV-EICrev): rSARS-CoV-E-V25F L27S (V25F L27S), rSARS-CoV-E-V25F T30I (V25F T30I), rSARS-CoV-E-V25F L37R (V25F L37R) were engineered, rescued and tested in mice. These viruses were virulent in mice in terms of weight loss and survival rates, causing similar disease as that caused by the wt virus ( Fig. 8 ). We sought to confirm this data on another genetic background, so a recombinant SARS-CoV containing the mutation that restored IC activity in N15A mutant after cell culture passage was engineered rSARS-CoV-E-N15D (N15D) and evaluated. In agreement with the V25F revertants, the mutant N15D induced similar morbidity and mortality as wt ( Fig. 8 ), confirming that E protein IC activity is a determinant of virus pathogenesis. 10.1371/journal.ppat.1004077.g008 Figure 8 Pathogenesis caused by rSARS-CoV-EICrev in BALB/c mice. Groups of five 16 week-old BALB/c mice were mock infected (Mock, green circles) or infected with 100000 PFU of either the parental virus (wt, black circles) or the genetically engineered revertant viruses recovering IC activity: N15D (deep-red diamonds), V25F L27S (fuchsia triangles), V25F T30I (pink triangles) and V25F L37R (green triangles). Mean weight losses (left graph) and survival (right graph) during 10 days are represented for each group. Error bars represent the standard deviation for mice weights per experimental condition. SARS-CoV E protein IC activity is dispensable for efficient growth in vivo Although E protein IC activity is not essential for virus growth in cell culture ( Fig. 2B ), it is possible that production of virus in vivo further depends on ion conductivity. To test if the attenuation observed in vivo with IC inactive viruses is due to lower virus production, 16 week-old BALB/c mice were intranasally inoculated with the wt virus, the genetically engineered revertant viruses N15D and V25F T30I displaying IC activity, or the N15A mutant lacking IC activity. Mice lungs were collected at 2 and 4 days post infection, homogenized, and viral titers were determined. Interestingly, the virus lacking IC activity (N15A) grew to the same extent or even better than the wt and the revertant viruses, respectively, reaching titers higher than 108 and 107 PFU/gr of lung tissue at 2 and 4 dpi, respectively ( Fig. 9 ). These data indicate that E protein IC activity does not significantly affect virus production in vivo, under these experimental conditions. Therefore the attenuation of the virus lacking IC activity is likely due to a host-specific effect mediated by the ion channel in the mouse, and not to a reduction in virus yields. 10.1371/journal.ppat.1004077.g009 Figure 9 Effects of SARS-CoV E protein IC activity on virus growth in BALB/c mice lungs. Groups of six 16 week-old BALB/c mice were infected with 100000 PFU of viruses displaying E protein IC activity (EIC+), either the parental virus (wt, black columns) or the genetically engineered revertant viruses V25F T30I (purple columns) and N15D (deep-red columns) or with the mutant lacking IC activity (EIC−) N15A (red columns). At 2 and 4 days post infection (dpi) 3 mice from each group were sacrificed to determine virus titers. Viruses with E protein IC activity induced edema accumulation after SARS-CoV infection To analyze the mechanisms by which IC inactivity confers less virulence, lung sections of mock-infected mice, or of those infected with the wt virus, IC revertants and N15A mutant were collected at 2 and 4 dpi, stained with hematoxylin and eosin and examined for histopathological changes. Mock-infected animals showed wide free alveolar and bronchiolar airways and no evidence of leukocyte infiltrates ( Fig. 10A ). Animals infected with the viruses displaying IC activity, presented swollen alveoli walls and leukocyte infiltrates in the infected areas at both time points ( Fig. 10A ). The histopathology caused by IC proficient viruses was even more dramatic at 4 dpi, where cell infiltrates were more abundant, and air spaces were collapsed by a profuse lung edema, which is the ultimate cause of acute respiratory distress syndrome (ARDS) that leads to lung failure and death ( Fig. 10A ). Edema accumulation at 4 dpi was also reflected by a marked increase (>1.5 fold) in the weight of lungs in animals infected with viruses competent in E protein ion conductivity ( Fig. 10B ). In contrast, mice infected with the virus lacking IC activity (N15A) showed moderate swollen lung epithelia and lung infiltrates that reflected a productive viral infection. However, at 4dpi, lung airways remained free from pulmonary edema, reflected by both the lung sections and in the minimal change of lung weight ( Fig. 10A and 10B ). Such moderate changes in the lung may retain efficient oxygen exchange. These data suggested that E protein IC activity contributes to SARS-CoV induced lung edema. 10.1371/journal.ppat.1004077.g010 Figure 10 SARS-CoV E protein IC activity and lung pathology. Groups of six 16 week-old BALB/c mice were mock infected (Mock) or infected with 100000 PFU of viruses displaying E protein IC activity (EIC+), either the parental virus (wt) or the genetically engineered revertant viruses V25F T30I and N15D or with the mutant lacking IC activity (EIC−) N15A. At 2 and 4 dpi 3 mice from each group were sacrificed and their lungs were collected. (A) Lungs were fixed in formalin, paraffin embedded, sectioned and processed for hematoxylin and eosin staining. Asterisks indicate edema accumulation in both bronchiolar and alveolar airways. Original magnification was 20×. (B) When collected and prior to fixation lungs were weighted. Error bars indicate the standard deviation from 3 mice lungs per each condition. Statistically significant data are indicated with an asterisk (Student's t-test p-value 24) of passages in cell culture, it is possible that after serial passages in vivo this mutant could also revert, as ion conductivity confers better fitness for the virus. Although both N15A and V25F mutations equally disrupted IC activity, the mechanisms by which this is achieved could be different. Replacement of N at position 15 to A, an amino acid predicted to be located facing the channel lumen, is not likely to affect the channel architecture. In fact, the rotational orientation in lipid bilayers of a labeled synthetic transmembrane peptide bearing this mutation was entirely consistent with that of a pentameric model [29]. In contrast, mutation at V25 implies the introduction of a larger side chain (replacement of V to F) at the monomer-monomer interface, which is likely to affect the overall structure of the homo-oligomer and therefore inhibit ion conductivity by causing larger structural changes. This may also explain the higher number of compensatory mutations found in V25F with respect to N15A virus, as the ways to recover a stable oligomer are more varied than those needed to recover channel activity. The compensatory mutations incorporated by the V25F mutant mapped to the opposite face of the transmembrane helix, although they are adjacent when the E protein pentamer is formed. Therefore, the compensatory mutations most likely restored the interaction and assembly between the mutated monomers, reinforcing our hypothesis ( Fig. 6 ). IC activity restoration through virus passage suggests that this function is important for the virus. Several of the mutations restoring ion channel activity appeared both in mice and in cell culture. Therefore, it seems that reverting E protein ion conductivity, and not adaptation to mice, was its main goal. Nevertheless, the possibility that these mutations could also improve mouse adaptation through an ion channel dependent or independent mechanism cannot be fully excluded. E protein IC activity was also involved in SARS-CoV pathogenesis as tested in the mouse model. Viruses lacking IC activity that were stable during multiple passages (N15A mutants) caused reduced mortality, whereas the wt and the mutant viruses restoring IC activity during mice infection (V25F background-evolved variants) caused high mortality rates. Furthermore, genetically engineered viruses containing the point mutations necessary to recover E protein IC activity induced similar mortality as wt virus, reinforcing that E protein IC activity contributes to SARS-CoV pathogenicity. The relevance of viroporins in virus virulence has also been shown in other viruses, such as respiratory syncytial virus SH protein, influenza A virus M2 protein and classical swine fever virus p7 protein [67]–[70], by deleting a large fraction of or the entire protein. Viroporins may play other critical functions apart from ion conduction. Therefore, a direct correlation between IC activity and virulence could not be formally established. To our knowledge, this is the first time in which the IC activity of a viroporin is directly linked to the virulence of the virus. The infection with highly pathogenic respiratory viruses, including SARS-CoV, is one of the causative agents of acute lung injury (ALI) and its most severe form, ARDS [71]. Just in the United States, 200,000 ARDS cases are reported annually with a 40% mortality rate [72]. Late stages of ARDS are characterized by development of pulmonary edema that leads to an impaired gas exchange, hypoxemia and eventually death. Infection of mice with rSARS-CoV-MA15 resulted in an abundant edema accumulation both in alveolar and bronchiolar spaces at late times post infection (4 dpi), which correlated with mortality. This phenotype was reproduced upon mice infection with other highly-virulent SARS-CoVs displaying IC activity based on alternative E protein sequences (revertant viruses). On the other side, infection of mice with the attenuated mutant lacking E protein IC activity (N15A) caused significantly reduced edema accumulation, likely contributing to a majority of the animals surviving. Collectively, these data indicate that E protein IC activity in vivo promotes lung pathology through edema accumulation. The pulmonary epithelia regulate water levels present within air spaces, a critical parameter for gas exchange, and play a critical role in edema clearance [72], [73]. Epithelial cells create an osmotic gradient mainly through a coordinated Na+ transport first from the airways to the cell cytoplasm through epithelial sodium channels (ENaC), located at the apical part of the plasma membrane, and then to the interstitium by Na+/K+ ATPase, present at the basolateral region of the plasma membrane. This vectorial transport of Na+ is accompanied by a water removal from the airspace and edema resolution [72], [73]. The integrity of alveolar and bronchiolar epithelia was analyzed by labeling of Na+/K+ ATPase in the lungs of mice infected with the virus containing or lacking IC activity. Interestingly, animals infected with the wt virus presented a strong disassembly of bronchiolar epithelia and mislocalization of Na+/K+ ATPase from its basolateral distribution within cells at late times, coincident with edema accumulation. In contrast, epithelia integrity was clearly preserved in the lungs of animals infected with the virus missing E protein IC activity. Intact lung epithelia may be required for proper function of the main components involved in edema resolution (Na+/K+ ATPase and ENaC), which may explain the lack of edema and therefore the attenuation observed for this virus. As previously described for SARS-CoV, differences in viral tropism within lung cells, without affecting viral production, can induce different pathologies [74]. Nevertheless, we have observed no significant differences in the infection patterns in the presence or absence of E protein ion channel activity, suggesting that the virulence conferred by E protein IC activity does not depend on alternative tropisms. Pulmonary epithelia damage leading to ALI and ARDS is a consequence of a cytokine burst initiated, in this case, by viral infection. One of the key early-response cytokines driving proinflammatory activity in bronchoalveolar spaces is IL-1β [75]. IL-1β is mainly produced by macrophages and dendritic cells through inflammasome activation. Ion imbalances within cells have been described as triggers of this pathway [52]. The levels of active IL-1β secreted to the airways were enhanced when E protein IC activity was conserved in SARS-CoV infection. Taking into account that the presence or absence of SARS-CoV E protein IC activity did not interfere with the production of IL-1β precursors (mRNA and protein levels of pro-IL-1β), these results suggest that E protein ion conduction may induce inflammasome triggering resulting in secretion of mature IL-1β. In agreement with this hypothesis, release of active IL-1β has recently been reported for viroporins of other viruses [52], [53], [57], [59]. IL-1β is implicated in the development of diverse pathologies, including obesity, atherosclerosis, diabetes and several pulmonary illnesses such as asthma, pulmonary obstructive chronic disease and ARDS progression through edema accumulation [75]–[78]. Here, we report the implications of this cytokine in SARS-CoV pathology and E protein ion channel activity as a trigger of its production. ARDS progression involves the production of TNF, another early response cytokine, and IL-6, which exerts its function in a more sustained manner accumulating during the disease [63]. We found that after SARS-CoV infection, these patterns of cytokine expression were clearly reproduced. TNF and IL-6 accumulated to higher levels in the lungs of animals infected with the wt virus displaying IC activity compared to the mutant lacking ion conductivity. IL-1β enhances the production of TNF, and IL-6 is stimulated by both cytokines providing an integrated amplified inflammatory response, detrimental for pulmonary function [63]. Elevated amounts of these cytokines have been reported in bronchoalveolar lavages of SARS-CoV patients [79]. Therefore, the enhanced amounts of active IL-1β found in the animals infected with the wt virus may explain the increased levels of TNF and IL-6, which leads to severe pathology. It is important to note that the increased damage found in pulmonary epithelia infected with the virus displaying E protein IC activity may not be explained by a higher virus production, as suppression of E protein IC activity rendered similar growth in mice lungs during the analyzed time points. SARS-CoV early replication may be a relevant issue in the induction of pathology. We cannot exclude early replicative defects for the N15A mutant in mice, delaying virus growth during the first hours post-infection. Nevertheless, alternative explanations are also possible, as it has been described that some mutations at SARS-CoV S gene conferred increased virulence without affecting growth within mice, even at early times. This increased pathogenesis was mainly dependent on an exacerbated host response to the viral infection [74]. Accordingly, the enhanced inflammatory response triggered by E protein ion channel proficient viruses may be a major pathology inducer. In this study, we have shown that SARS-CoV E protein IC activity is a virulence determinant, influencing inflammatory responses, including those inflammasome-derived, pulmonary damage and disease outcome. Although not essential for virus production, E protein IC activity confers a selective advantage, as the parental virus, competent for ion conductivity, was more fit. Nevertheless, the virulence associated to E protein ion conductivity could represent a non-selectable consequence. SARS-CoV crossed species barriers from zoonotic reservoirs such as bats, palm civets and raccoon dogs to humans, causing a severe disease [71]. Possibly, in SARS-CoV infection of its natural hosts, E protein ion channel activity may not have a relevant impact in SARS-CoV pathogenesis, and therefore it was positively selected before crossing species barrier. In conclusion, this work provides several findings that may have translational relevance for other coronaviruses, such as the highly pathogenic MERS-CoV, and even on other viruses encoding proteins with IC activity. Materials and Methods Ethics statement Animal experimental protocols were approved by the Ethical Committee of The Center for Animal Health Research (CISA-INIA) (permit numbers: 2011-009 and 2011-09) in strict accordance with Spanish national Royal Decree (RD 1201/2005) and international EU guidelines 2010/63/UE about protection of animals used for experimentation and other scientific purposes and Spanish national law 32/2007 about animal welfare in their exploitation, transport and sacrifice and also in accordance with the Royal Decree (RD 1201/2005). Infected mice were housed in a ventilated rack (Allentown, NJ). Cells The African green monkey kidney-derived Vero E6 cells were kindly provided by Eric Snijder (Medical Center, University of Leiden, The Netherlands). The mouse delayed brain tumor cells stably expressing the murine receptor for SARS-CoV (DBT-mACE2) were generated as previously described [61]. Baby hamster kidney cells (BHK-21) were obtained from American Type Culture Collection (ATCC; CCL-10). Cells were grown at 37°C with an atmosphere of 98% humidity, in Dulbecco's modified Eagle medium (DMEM, GIBCO) supplemented with 25 mM HEPES, 2 mM L-glutamine (SIGMA), 1% non essential amino acids (SIGMA) and 10% fetal bovine serum (FBS, Biowhittaker). Mice Specific-pathogen-free 8 week-old BALB/c OlaHsd female mice were purchased from Harlan. Mice were maintained for 8 additional weeks in the animal care facility at the National Center of Biotechnology (Madrid). All protocols were approved by the Ethical Review Committee at the center for animal health research (CISA-INIA). For infection experiments, 16-week-old mice females were anesthetized with isoflurane and intranasally inoculated with 100000 PFU of the indicated viruses. All work with infected animals was performed in a BSL3 laboratory (CISA, INIA) equipped with a ventilated rack (Animal transport unit-Bio containment unit, Harvard) to store the animals during the experiment. Construction of mutant rSARS-CoVs-MA15 An infectious cDNA clone encoding a mouse adapted (MA15) SARS-CoV assembled in a bacterial artificial chromosome (BAC) in our laboratory [26] was used as the background to introduce the mutations that inhibited or restored E protein IC activity. Briefly, DNA fragments representing the nucleotides 26044 to 26779 of SARS-CoV-MA15 genome, flanked by the restriction sites BamHI and MfeI, respectively, were chemically synthesized (Bio Basic Inc). These fragments contained different point mutations within the E gene, that generated amino acid changes inhibiting IC activity: N15A (AAT to GCC) and V25F (GTA to TTC) or restoring this activity: N15D (GCC to GAC), V25F L19A (GTA to TTC and CTT to GCA), V25F L27S (GTA to TTC and TTG to TCG), V25F T30I (GTA to TTC and ACA to ATA), V25F L37R (GTA to TTC and CTT to CGT). The fragments containing these mutations were digested and exchanged in the original BAC. The genetic integrity of the cloned DNA was verified by restriction analysis and sequencing. Recovery of recombinant viruses from the cDNAs clones BHK cells were grown to 95% confluency in 12.5 cm2 flasks and transfected with 6 µg of the infectious cDNA clones and 18 µl of Lipofectamine 2000 (Invitrogen), according to the manufacturer's specifications. At 6 hours post transfection, cells were trypsinized, added to Vero E6 cells confluent monolayers grown in 12.5 cm2 flasks and incubated at 37°C for 72 h. Cell supernatants were harvested, passaged once on fresh cells and the recovered viruses were cloned by three rounds of plaque purification following standard procedures. Growth kinetics and plaque assays Vero E6 or DBT-mACE2 cells were grown to confluency on 12.5 cm2 flasks and infected at a multiplicity of infection (MOI) of 0.001. Cells supernatants were collected at 0, 6, 24, 48 and 72 hpi and titrated on Vero E6 cells. For virus titration and plaque detection, supernatants of infected cells were added to confluent monolayers of Vero E6 cells and incubated for 45 min at 37°C. Media was removed and cells were overlaid with DMEM containing 0.6% of low melting agarose and 2% of fetal calf serum (FCS). At 72 hpi cells were fixed with 10% formaldehyde and stained with crystal violet. Confocal microscopy Vero E6 cells were grown to 90% confluency on glass coverslips and infected with rSARS-CoV-ΔE, rSARS-CoV wt, rSARS-CoV-E-N15A and rSARS-CoV-E-V25F at an MOI of 0.3. At the indicated hpi, media were removed and cells were washed twice with PBS and fixed with 4% paraformaldehyde in PBS for 30 min at room temperature. Then, cells were washed twice with PBS and permeabilized for 10 min with 0.2% saponin and 10% FBS in PBS. Primary antibody incubations were performed in PBS containing 10% FBS and 0.2% saponin for 1 h 30 min at room temperature. Immunofluorescence was performed using a mouse mAb specific for ERGIC53 (dilution 1∶200, Alexis Biochemicals), and a rabbit pAb specific for E protein [15] at 1∶2000 dilution. Coverslips were washed four times with PBS between primary and secondary antibody incubations. Alexa 488- or Alexa 546-conjugated antibodies specific for the different species (dilution 1∶500, Invitrogen) were incubated for 45 min at room temperature in PBS containing 10% FBS. Nuclei were stained using DAPI (dilution 1∶200, Sigma). Coverslips were mounted in ProLong Gold anti-fade reagent (Invitrogen) and examined on a Leica SP5 confocal microscope (Leica Microsystems). Colocalization studies were performed using Leica LAS AF v2.6.0 software. Virus genome sequencing The genomic region including nucleotides 26017 to 26447 that contains the E gene was sequenced after RT and PCR reactions. Briefly, total RNA from infected cells or homogenized mice lungs, was collected and purified using RNeasy kit (Qiagen) according to the manufacturer's specifications. For RT reaction, 1 µg of RNA was used as template, random oligonucleotides primers and Thermoscript reverse transcriptase (Invitrogen). The product was subsequently subjected to a PCR reaction using the oligonucleotides E-VS (CTCTTCAGGAGTTGCTAATCCAGCAATGG) and E-RS (TCCAGGAGTTGTTTAAGCTCCTCAACGGTA) and the Vent polymerase (New England Biolabs), following manufacturer's recommendations. Sequence assembly and comparison with the consensus sequence of SARS-CoV-MA15 strain were performed with the SeqMan program (Lasergene, Madison, WI). Genetic stability through serial infections of SARS-CoVs lacking IC activity Confluent monolayers of Vero E6 cells grown in 12.5 cm2 flasks were infected at an MOI of 0.5 with the viruses rSARS-CoV wt, rSARS-CoV-E-N15A and rSARS-CoV-E-V25F. At 24 hpi, supernatants were collected and passaged on fresh monolayers of Vero E6 cells, performed 24 times, serially. E gene sequence was analyzed at passages 0, 8, 16 and 24 as described. For in vivo experiments, mice were intranasally inoculates with 100000 PFU of the viruses described above. Lungs were collected at days 2 and 4 post infection and incubated in RNAlater (Ambion) at 4°C for 48 hpi prior to −80°C freezing. To extract total RNA, lungs were homogenized in 2 ml of RLT lysis buffer (QIAGEN) containing 1% β-mercaptoethanol using gentleMACS Dissociator (Miltenyi Biotec). Samples were centrifuged at 3000 rpm during 10 min, and RNA was purified from supernatants using RNeasy kit (QIAGEN) as previously described. Peptide synthesis and ion channel measurements in artificial lipid membranes Synthetic peptides representing the transmembrane domain of SARS-CoV E protein (amino acids 7 to 38) encoding the point mutations that appeared after serial infections of the mutant viruses were generated by standard phase synthesis, purified by HPLC and their IC activity was tested in artificial lipid membranes, as previously described [34]. Competition assays Total RNA from co-infected cells was isolated and E gene was sequenced as described above. Relative abundance of the rSARS-CoV wt and rSARS-CoV-E-N15A viruses within viral population was determined by quantifying the relative amounts of their respective E gene genetic markers in the sequence obtained from the population. Mice infection and evaluation of virus virulence 16 week-old BALB/c mice females were intranasally inoculated with 100000 PFU of the viruses rSARS-CoV wt, rSARS-CoV-E-N15A, rSARS-CoV-E-V25F, rSARS-CoV-E-N15D, rSARS-CoV-E-V25F L27S, rSARS-CoV-E-V25F T30I and rSARS-CoV-E-V25F L37R in 50 µl of DMEM containing 2% FCS. Weight loss and survival of the infected mice were monitored for 10 days. Animals reaching weight losses higher than 25% of the initial body weight were sacrificed according to the established euthanasia protocols. Virus growth in mice lungs and lung histology Mice were inoculated with 100000 PFU of the virus rSARS-CoV wt, rSARS-CoV-E-N15A, rSARS-CoV-E-N15D and rSARS-CoV-E-V25F T30I, sacrificed at days 2 and 4 post infection, and lungs were collected. To analyze viral growth, right lungs were homogenized in 2 ml of Phosphate Buffered Saline (PBS) containing 100 UI/ml penicillin, 100 µg/ml streptomycin, 50 µg/ml gentamicin and 0.5 µg/ml fungizone using MACS homogenizer (Miltenyi Biotec) according to manufacturer's protocols, and titered as previously described. To examine lung histopathology, left lungs of infected mice were incubated with 10% zinc formalin for 24 h at 4°C, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Immunofluorescence in lung sections Five micron sections of zinc formalin fixed lungs were deparaffined at 60°C and rehydrated by successive incubations in 100% xylol, 100% ethanol and 96% ethanol. Antigen unmask was performed by boiling the samples in citrate buffer (8.2 mM sodium citrate; 1.8 mM citric acid, pH 6.5) for 5 min at 110°C in a decloaking chamber (Biocare medical). Samples were then permeabilized with 0.25% Triton X-100 in PBS for 15 min and blocked with 10% bovine serum albumin (BSA) and 0.25% Triton X-100 in PBS for 30 min. Samples were labeled with a mouse monoclonal antibody specific for SARS-CoV N protein (kindly provided by Ying Fang, South Dakota State University) diluted 1∶250 and a rabbit monoclonal antibody specific for Na+/K+ ATPase alpha subunit (Abcam) diluted 1∶100 in 0.25% Triton X-100 and 10% BSA in PBS for 1 h 30 min at room temperature. Goat anti-mouse and goat anti-rabbit secondary antibodies bound to Alexa 488 and Alexa 594 fluorophores were used respectively at a dilution 1∶250 in 0.25% Triton X-100 and 10% BSA in PBS for 45 min at room temperature. Cell nuclei were stained with DAPI (1∶200). Tissues were mounted in ProLong antifade reagent (Invitrogen) and analyzed in a Leica TCS SP5 confocal microscope. RT-qPCR analysis RNA extracted from lungs of infected mice was prepared as described above, and subjected to retro transcriptase reactions using a High-Capacity cDNA transcription kit (Applied Biosystems) to generate cDNAs. PCR using Taqman assays specific for IL-1β (Mm01336189-m1) and 18S ribosomal RNA as a control (Mm03928990-g1) [80], [81] (Applied Biosystems) were performed. Data were acquired with an ABI Prism 7000 sequence detection system (Applied Biosystems) and analyzed using ABI Prism 7000 SDS v1.0 software. Gene expression relative to mock-infected samples is shown. Lung protein extracts preparation and western blot assays Lungs from infected mice were collected at 2 dpi and the right lung was homogenized in 1.2 mL of protein lysis buffer containing Tris/HCl 10 mM, EDTA 1 mM, NaCl 150 mM, IGEPAL 1%, and complete protease inhibitor (Roche) pH8, using MACS homogenizer (Miltenyi Biotec). Samples were centrifuged for 1 h at 4°C and 13000× g and supernatants were collected. Pro-IL-1β levels were analyzed by Western blotting using a goat anti mouse IL-1β/IL-1F2 antibody (R&D systems). As a loading control, beta-actin was labeled using a mouse monoclonal antibody (Abcam). Bound antibodies were detected using a rabbit anti goat and a rabbit anti mouse HRP conjugated antibodies and the Immobilon Western chemiluminiscence substrate (Millipore), following manufacturer's specifications. Densitometric analysis was performed in non-saturated exposures of several experimental replicates using Quantity One, version 4.5.1 software (BioRad). Levels of pro-IL-1β were normalized to the levels of beta-actin. Bronchoalveolar lavages Following euthanasia by cervical dislocation, the trachea was exposed and cannulated through the animal mouth with a 19 gauge tube. Lungs were lavaged three times with 400 µl of cold phosphate buffered saline (PBS). Samples were centrifuged for 10 minutes at 3000× g at 4°C to separate cellular content, and supernatants were collected to analyze their cytokine levels. Cytokine multiplex analysis Bronchoalveolar lavages were treated with IGEPAL reaching a final concentration of 0.2%, to inactivate sample infectivity. The expression of IL-1β, TNF and IL-6 was measured using Luminex technology and a mouse cytokine antibody bead kit (Milliplex map kit, Millipore) according to the manufacturer's specifications. Supporting Information Figure S1 Infection efficiency and cellular tropism within mice lungs, in the presence or absence of SARS-CoV E protein IC activity. 16 week-old BALB/c mice were infected with 100000 PFU of the parental virus (wt, black columns) displaying E protein IC activity or the mutant virus lacking IC activity N15A (red columns). At 2 and 4 dpi mice were sacrificed and their lungs were fixed in formalin, paraffin embedded, sectioned and processed for immunofluorescence. SARS-CoV N protein and cell nuclei were labeled to discriminate both non-infected and infected cells. The number of alveolar (alveo), bronchiolar (bronch) and overall infected cells (total) were calculated in several representative images, and represented as percentages of their corresponding total cells (infected plus non-infected). Error bars indicate the standard deviation from the data collected from different images. (TIF) Click here for additional data file.
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            Large-scale sequence analysis of avian influenza isolates.

            The spread of H5N1 avian influenza viruses (AIVs) from China to Europe has raised global concern about their potential to infect humans and cause a pandemic. In spite of their substantial threat to human health, remarkably little AIV whole-genome information is available. We report here a preliminary analysis of the first large-scale sequencing of AIVs, including 2196 AIV genes and 169 complete genomes. We combine this new information with public AIV data to identify new gene alleles, persistent genotypes, compensatory mutations, and a potential virulence determinant.
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              Is Open Access

              Exposure to HIV-1 Directly Impairs Mucosal Epithelial Barrier Integrity Allowing Microbial Translocation

              Introduction The mucosa presents a primary barrier against a multitude of micro-organisms present on the mucosal surfaces of the human body [1]. The intestinal and upper reproductive tract are lined by a continuous monolayer of columnar epithelial cells that is responsible for maintaining the physical and functional barrier to harmful microorganisms, such as bacteria and their products, including bacterial toxins as well as commensal organisms [2]–[4]. The preservation of the barrier function is dependent on the intactness of apical plasma membrane on the epithelial cells as well as the intercellular tight junctions. The disruption of the tight junctions can cause increased permeability, leading to “leakiness” such that normally excluded molecules can cross the mucosal epithelium by paracellular permeation, and could lead to inflammatory conditions in the mucosa. Various pathogenic organisms have developed strategies to either infect or traverse through the epithelial cells at mucosal surfaces, as part of the strategy to establish infection in the host. In fact, mucosal transmission account for majority of infections in humans [5]. Viruses such as rotavirus and astrovirus as well as bacteria such as enteropathogenic E. Coli and C. difficile are known to increase intestinal permeability by disrupting tight junctions, as part of their pathogenesis [6]–[9]. Increased permeability is also related to a number of other disease conditions that may or may not be related to infection by a pathogen. Crohn's disease, a chronic inflammatory condition of the intestines is characterized by defective tight junction barrier functions, manifested by increased intestinal permeability, although the etiology of the disease is not clearly understood [10]. HIV-1 infection is initiated primarily on mucosal surfaces, through heterosexual or homosexual transmission [1],[11]. In fact, mucosal transmission accounts for greater than 90% of HIV infection [12],[13]. A number of clinical studies have reported intestinal barrier dysfunction, especially during chronic stage of HIV infection [14]–[19]. However, the pathophysiologic mechanism associated with compromised barrier function and whether HIV-1 plays a direct role in this is still unclear. Currently, epithelial barrier defect during HIV-1 infection is thought to be a consequence of mucosal T cell activation following infection, which could lead to increased production of inflammatory cytokines [19],[20]. The intestinal barrier dysfunction has also been implicated as the cause of systemic immune activation during chronic phase of HIV infection, although a recent study has raised the possibility that this may not be universal phenomenon [20],[21]. Studies that have demonstrated immune activation propose this to be the main driving force for progressive immune failure leading to the immunodeficiency stage [22]–[24]. In these studies, HIV disease progression was shown to correlate with increased circulating level of LPS, considered an indicator of microbial translocation, in chronic HIV-infected individuals [25]. Interestingly, immune activation was observed in both the chronic as well as acute phase of HIV infection [25]. The source of or mechanism whereby microbial products could cross the epithelial barrier leading to immune activation have not been elucidated thus far. In the present study, we investigated the direct effects of HIV-1 exposure on intestinal and genital mucosal epithelia, where primary HIV-1 infection is frequently initiated. We show that in fact the impairment of epithelial barrier function can be a direct result of exposure to HIV-1. Using ex-vivo cultures of pure primary genital epithelium as well as an intestinal epithelial cell line, we show significantly decreased barrier functions and enhanced permeability that is not unique to the intestinal epithelium; similar increase in permeability was seen in the genital epithelium as well. Small amounts of both bacterial and viral translocation were seen following HIV-1 exposure. The mechanism appears to be mediated by increased production of inflammatory cytokines directly from the epithelial cells following exposure to HIV-1, including TNF-alpha, known to disrupt barrier functions. Further, we show that HIV-1 envelope protein gp120 was able to impair barrier functions in epithelial cells on its own. Neutralization of gp120 or exposure to HIV-1 lacking gp160 surface envelope glycoprotein did not have any effect on epithelial cells. These results provide strong evidence that exposure to HIV-1 may lead to impairment in barrier function of mucosal epithelium which could result both in translocation of HIV-1 and/or luminal bacteria that could serve as the source of immune activation during HIV-1 infection. Results Genital and intestinal epithelial monolayer transepithelial resistances (TER) are decreased following exposure to different strains of HIV-1 In order to study HIV-1 induced barrier defect in epithelial monolayers, HIV-1 (106 infectious viral units/ml) was added apically to confluent monolayers of differentiated primary female genital epithelial cells (ECs) or T84 intestinal epithelial cells grown in transwells. Transepithelial resistance (TER), a measure of epithelial monolayer integrity, was measured before and 24h post-infection and calculated as a percentage of pretreatment TER. Transepithelial resistances of primary endometrial epithelial monolayers exposed to various strains of HIV-1 were significantly reduced (p 0.05), while the wild type HIV showed significant decrease in TERs (p<0.01) (Figure 7B). This was further confirmed by intact ZO-1 staining seen in epithelial monolayers exposed to Env− HIV, similar to mock-treated cells (Figure 7C). Combined with the results from gp120 neutralizing antibody described above, these results indicate that the HIV-1 surface glycoprotein is responsible for disruption of epithelial barrier leading to increased permeability. HIV-1 exposure induces inflammatory cytokines in genital and intestinal epithelial monolayers Epithelial cells are known to secrete a variety of cytokines at constitutive levels. Many of these are upregulated or induced de novo in response to pathogens such as Neisseria gonorrhea [31]. Additionally, inflammatory cytokines have been shown to mediate enhanced permeability of intestinal epithelial cells [32]. We therefore examined the cytokine secretion profile of epithelial cells following HIV-1 exposure. Apical and basolateral supernatants of genital and intestinal monolayers were collected 24h post HIV-1 exposure and examined for presence of six cytokines known to be secreted by epithelial cells (Figure 8A–F). The T84 intestinal cell line constitutively secreted low levels of IL-10 and IL-1β. In comparison, the primary endometrial epithelial monolayers showed constitutive production of a larger array of cytokines, some of them in high amounts (IL-6, IL-8, MCP-1). Following HIV-1 exposure, there was a significant increase in production of TNF-α, IL-6, IL-8 and MCP-1 in T84 intestinal epithelial monolayers (Figures 8A–D). In primary endometrial ECs there was a significant increase in production of TNF-α, IL-6, MCP-1, IL-10, and IL-1β secretion after 24 hours of HIV-1 exposure (Figures 8A, B, E, F). 10.1371/journal.ppat.1000852.g008 Figure 8 Primary endometrial EC and T84 intestinal monolayers were exposed to HIV-1 (ADA, 106 infectious viral units/ml, p24 280ng/ml) and apical and basolateral supernatants were collected 24 hours post-exposure and assayed by Luminex multi-analyte kit for the following cytokines: (A) TNF-α (B) IL-6, (C) IL-8, (D) MCP-1, (E) IL-10, (F) IL-1β. *p<0.01, **p<0.001. Data shown is representative of three separate experiments from different tissues, each experiment had 3–5 replicate cultures for each experimental condition. HIV-1 mediated TER decrease is reversed by treatment with anti-TNF antibody Of the cytokines that showed increased production in genital and intestinal epithelial cells following HIV-1 exposure, TNF-α is well known to disrupt epithelial cell tight junction assembly and increase intestinal cell permeability [33]. Since TNF-α was significantly up-regulated following HIV-1 exposure in both genital and intestinal ECs, we neutralized TNF-α to see whether this would affect barrier function alterations. Confluent T84 intestinal epithelial monolayers were treated with TNF-α (20 ng/ml), TNF-α+anti-TNF antibody (mouse anti-human TNF-α , 25 µg/ml), TNF-α+mouse serum (control), HIV-1 alone, HIV-1+anti-TNF antibody (mouse anti-human TNF-α antibody, 25 µg/ml) and HIV-1+mouse serum (control). The TER measurements were taken prior to and 24 hours following the treatments. As expected both TNF-α and HIV-1 caused a significant drop in TER values compared to untreated control monolayers (Figure 9). When epithelial monolayers were pre-treated with anti-TNF-α antibody prior to treatment with TNF-α and HIV-1, the TER values did not decrease significantly over 24 hours of exposure. Incubation of monolayers with normal mouse serum did not show the same effect as the anti-TNF-α antibody. These results provide direct evidence that TNF-α secreted by epithelial cells in response to HIV-1 exposure contributed significantly to the disruption of barrier function in epithelial cells. 10.1371/journal.ppat.1000852.g009 Figure 9 Primary endometrial epithelial monolayers were exposed to TNF-α or HIV-1 alone; TNF-α or HIV-1 (ADA,106 infectious viral units/ml, p24 280ng/ml) in combination with anti-TNF-α neutralizing antibody; TNF-α or HIV-1 in combination with normal mouse serum for 24 hours. TER measurements were taken as a measure of change in permeability and presented as percentage of pre-treatment TER. Data shown is representative of two separate experiments, each experiment had 3–5 replicate cultures for each experimental condition. Increased permeability correlates with translocation of virus and bacteria across the epithelial monolayers To correlate barrier dysfunction with increased permeability to luminal antigens, we examined bacterial and viral translocation across the epithelial monolayers post-exposure to HIV-1. Intestinal epithelial monolayers grown to confluence were exposed to HIV-1. TNF-α was used as a positive control since it is known to disrupt tight junctions and increase permeability. Because direct exposure to TNF-α for prolonged period of time causes irreversible damage to epithelial cells, TNF-α treatment was limited to 6 hours prior to addition of non-pathogenic E. Coli to allow observation of bacterial translocation. Exposure time for HIV-1 was chosen at 6 hours (for comparison with TNF-α) and 24 hours (based on our results of maximum permeability with continued viability). Six hours after addition of E. Coli to the apical compartment, basolateral supernatants were collected and plated on LB agar and bacterial colonies were quantified. Transepithelial resistance measured prior to and 24 hours following treatments to determine if addition of E. Coli had any effect on TERs (Figure 10A). In HIV-1 exposed monolayers TER decreased significantly within 6h of exposure as expected; further reduction was seen at 24 hours. In comparison, HIV-1 unexposed monolayers only and those that were untreated except with E. Coli, TER values were maintained at 106% and 89% percent respectively, of pre-treatment TER values. Bacterial translocation was seen only in monolayers following 24hours of HIV-1 exposure and 6 hours of TNF-α treatment (Figure 10B). Bacterial translocation seen following 24 hours of HIV-1 treatment was about 50% of that seen following 6 hours of TNF treatment. No significant bacterial translocation was seen after 6h of HIV-1 exposure. 10.1371/journal.ppat.1000852.g010 Figure 10 Bacterial and viral translocation across mucosal epithelial monolayers following HIV-1 exposure. (A) Bacterial translocation was measured in T84 intestinal monolayers. Confluent monolayers were left untreated or treated for 6 hours with TNF-α (20ng/ml), E. coli (108 CFU/ml) , TNF-α (20ng/ml) +E. coli (108 CFU/ml), HIV-1 or HIV-1 (6 or 24 hours)+E. coli (108 CFU/ml). (A) TER measurements following various treatments in the presence or absence of E. Coli. * p<0.001. (B) Basolateral supernatants were collected and bacterial counts were done. (C) Viral translocation was determined in endometrial EC monolayers exposed to HIV-1 (ADA, 106 infectious viral units/ml, p24 280ng/ml) on the apical side. Basolateral supernatants were collected after different time intervals infectious and viral counts were done on TZM/b-l indicator cell line. Viral counts are depicted as percentage of inoculum added to the apical compartment of monolayers. Data shown is representative of two separate experiments, each experiment had 3–5 replicate cultures for each experimental condition. In a separate experiment, lipopolysaccaride (LPS) leakage in HIV-1 exposed endometrial monolayers was determined. LPS was added on apical side of HIV-1 exposed and control monolayers and one hour later basolateral supernatants were collected and LPS leakage was measured. The LPS levels in basolateral supernatants were increased by 47.3±0.922% in HIV-1 exposed monolayers in comparison with LPS leakage in mock-treated control monolayers. We also measured translocation of HIV-1 through the primary endometrial monolayers (Figure 10C). At various time points following HIV-1 exposure on the apical side, basolateral supernatant was collected and HIV-1 infectious viral counts were determined TZMb-1 indicator cell assay. The results are presented as percent of inoculum virus added on apical side. Infectious viral counts were seen starting at 6 hours following exposure to HIV-1 (0.03% of inoculum) and infectious virus continued to accumulate in the basolateral compartment (0.08% of inoculum) up to 48 hours time, which was the last time point examined. Discussion To summarize, we were able to demonstrate that exposure to HIV-1 directly decreased the transepithelial resistance across intestinal and genital epithelial monolayers. The reduction in TER correlated with significant decrease in tight junction protein expression and increased permeability, indicating functional impairment of the barrier. The effect was specific for HIV-1 and reached significant levels within 2–4 hours following HIV-1 exposure. Similar reduction in tight junction functioning was observed following treatment of ECs with HIV-1 envelope protein gp120 but not tat, a regulatory protein. Neutralization of gp120 and exposure to an Env− HIV significantly abrogated the impairment of epithelial barrier, indicating that the effect was mediated by HIV-1 envelope glycoprotein. We further determined that exposure of the epithelial monolayers to HIV-1 led to enhanced production of a number of inflammatory cytokines, including TNF-α, by both intestinal and genital epithelial cells. When epithelial cells were exposed to HIV-1 in presence of anti-TNF antibody, there was no significant decrease in TER, indicating that TNF played a major role in impairing the barrier functions. In experiments designed to determine whether the disruption of epithelial barrier function could be directly associated with microbial leakage across the mucosa, we found evidence for small but significant bacterial and viral translocation across epithelial monolayers following HIV-1 exposure. To the best of our knowledge, this is the first study to demonstrate that HIV-1 can directly disrupt mucosal epithelial barrier functions that can lead to enhanced microbial translocation. Previous clinical studies have documented that in HIV-1 infected patients intestinal permeability is altered, characterized by diarrhea-induction [15]–[17] . A recent study showed impairment of barrier function in intestinal biopsies of HAART-naïve patients compared to those on HAART treatment [14]. Increased production of cytokines IL-2, IL-4, IL-5 and TNF-α was found in supernatants of cultured intestinal biopsies in this study. Their conclusion was that following infection, HIV replication in target cells leads to local increase of inflammatory cytokines in the intestinal mucosa, which induce barrier impairment. This supports previous studies where PBMCs co-cultured with HIV-infected macrophages resulted in increased production of a number of cytokines, including TNF-α, IL-1β, IFN-α and IFN-γ which were shown to compromise epithelial barrier function [34]. The prevailing opinion from these studies is that the effect on epithelial barrier is likely mediated via immune cell activation due to viral replication [14],[25]. Of note are other studies that were unable to show that mononuclear cells isolated from colon of infected patients produce increased amount of cytokines [35],[36]. Thus far the cellular source of inflammatory cytokines that could lead to barrier disruption in HIV infected patients remains controversial [20]. Based on our results, we would like to propose that the primary sources of the inflammatory cytokines that disrupt the mucosal barrier are the epithelial cells themselves. Our studies demonstrate that epithelial cells respond directly and rapidly to HIV envelope glycoprotein by production of increased levels of cytokines which lead to loss of barrier functions, rather than an indirect effect mediated by immune cells following HIV replication. This provides an alternate and more direct explanation as to why decrease in viral load following HAART treatment restores intestinal barrier functions [14]. Our results that demonstrate that barrier dysfunction can allow bacterial translocation could also provide explanation for increased levels of immune activation during acute infection, an observation noted in a previous study which examined immune activation following HIV infection in North American cohorts [25]. The mechanism demonstrated in the present study does not exclude the possibility that cytokines released from immune cells in the HIV-infected intestines could also contribute to further disruption of the barrier, more likely in the chronic phase of the infection. That viral exposure could directly lead to compromised barrier function has been shown before [6],[8],[9]. Many other viruses and even bacteria have been shown to directly compromise both epithelial and endothelial barrier integrity. Astrovirus, a single stranded RNA virus and a causative organism of common diarrhea was recently shown to increase epithelial barrier permeability in Caco-2 intestinal cells, modulated by its capsid protein, independent of viral replication [6]. Coxsackievirus has also been shown to directly compromise endothelial tight junctions [8]. Previous studies have shown that HIV-1 infection can compromise the blood-brain barrier thereby leading to progression of HIV-1 encephalitis (reviewed in [37]). The functioning of the tight junctions between endothelial cells, that form the blood-brain barrier, is quite similar to those present between mucosal epithelial cells. However, the mechanism elucidated by these studies was not a direct effect of HIV, but facilitated by production of TNF-α during chronic infection that mediated opening of paracellular route in endothelial lining, for viral entry into the brain. Interestingly, a recent study elucidated that HIV-1 tat protein can directly compromise the retinal epithelial barrier function [38]. Despite this evidence, no studies have so far examined the direct effect of HIV-1 exposure on mucosal epithelium. Our results show that increased permeability is mediated directly by HIV viral envelope glycoprotein. Further, given that significant disruption of tight junction proteins and decreased TERs occurred following treatment with UV inactivated virus, this phenomenon is independent of viral replication. Whether HIV-1 entry is required for the epithelial cell response is currently being examined. In our study, both the intestinal cell line and primary genital epithelial cells showed similar response to different strains of HIV-1: disruption of tight junctions, and increased permeability. However, we found the profile of cytokines produced constitutively by intestinal and genital epithelial cells was quite distinct. While the intestinal cell line T84 did not constitutively produce TNF-α, IL-6, IL-8 and MCP-1, there was significant induction of these cytokines following HIV-1 exposure. Primary genital epithelial cultures, on the other hand, constitutively produced TNF-α, IL-6, IL-8 and MCP-1 and production of TNF-α and IL-6 was significantly upregulated following HIV-1 exposure. Both types of ECs secreted minimal levels of IL-10 and IL-1β which was upregulated following HIV-1 exposure only in primary genital epithelial cells. The differences in the constitutive cytokine profile between genital and intestinal epithelial cells could be due to distinct characteristics of primary cells compared to cell lines. Alternatively, intestinal epithelial cells are likely to be more quiescent in terms of baseline cytokine production given their microenvironment where a variety of commensal organisms are always present in the lumen [39]. In comparison, upper genital tract epithelium exist in a relatively sterile environment and are known to actively secrete an array of cytokines [2]. Nevertheless, following exposure to HIV-1 both types of ECs responded with enhanced induction of inflammatory cytokines that mediated disruption of tight junctions. This indicates that as long as the viral load and exposure times are sufficient, HIV can likely disrupt any mucosal barrier in the body, independent of infection and replication. Among the cytokines that were upregulated, the direct effect of TNF-α on disruption of intestinal epithelial tight junction and increased permeability has been extensively characterized [33]. TNF-induced increase in permeability of Caco-2 cells is known to be mediated by NF-kB activation that downregulates ZO-1 protein expression [40]. ZO-1 proteins are integral part of the tight junction assembly and function as a scaffolding protein critical in maintaining the integrity of the tight junctions. The results from ZO-1 quantification (Figure 4) indicate that the disruption of tight junctions following HIV-1 exposure likely happens in two stages: initially there may be a displacement of ZO-1 that leads to disruption of tight junction integrity followed by marked reduction in the amount of ZO-1 and other tight junction proteins due to decreased transcription. Thus, TNF-α produced by the ECs in response to HIV-1 envelope glycoprotein could induce NF-kB activation and subsequent downregulation of tight junction proteins, including ZO-1. Our ongoing studies show that NF-kB translocation occurs within 1 hour of HIV-1 exposure (Nazli and Kaushic, unpublished). Whether there are distinct steps in this process that have discrete mechanisms is currently under investigation. Regardless of the detailed mechanism, the outcome of tight junction disruption is decrease in TER and leakage across the epithelial barrier. The finding that disruption of barrier function can result in small but significant amount of both viral and bacterial translocation across ECs following exposure to HIV-1 has profound implications. Although previous studies have demonstrated presence of LPS in serum of HIV infected patients and correlated it with immune activation in North American cohorts, the inference that microbial flora in the intestines was the source of LPS was indirect [25]. Our studies provide direct evidence that both bacteria and virus present on the apical side of mucosal epithelial cells during HIV-1 exposure could leak through because of the impairment of epithelial tight junctions and increased permeability. This could allow HIV-1 access to target cells located in the lamina propria of the mucosa as well allow bacterial translocation that could cause local immune activation. While the viral-epithelial interactions described here are novel, further investigation is needed to determine what role increased barrier permeability plays in initiating HIV-1 infection. HIV-1 transmission across intestinal and genital mucosa occurs predominantly via infected semen; currently the role of seminal plasma in HIV-1 transmission is far from clear. Recent studies indicate that seminal plasma can lead to inflammatory responses and facilitate HIV-1 transmission [41]–[43]. However, seminal plasma components such as TGF-β and HGF also enhance epithelial barrier functions [44]. Further, given the low efficiency of viral translocation seen in the ex-vivo model described here, the ability of HIV-1 to cross over in significant numbers, in vivo, would be depend presence of high viral load in the semen, most likely in acute phase of infection. While plasma and semen loads show overall correlation, compartmentalization between genital and blood viral loads is well recognized and more recent studies show that seminal plasma viral load can persist following treatment with HAART [45]–[47]. While the results from the present study elucidate a new mechanism that could lead to viral translocation across the epithelial barrier, more information is needed to understand how other factors like seminal plasma, stage of the infection and viral load may influence any viral leakage across the mucosal barrier. If under physiological conditions, viral leakage does occur because of barrier disruption, it could play a critical role in initiation of infection, especially in the presence of existing inflammation from other viral or bacterial co-infections [48]. In conclusion, the current study provides evidence for the first time that HIV-1 exposure at the mucosal surface leads to direct response by the mucosal epithelium, seen by production of inflammatory cytokines. This response is rapid, independent of viral infection and likely plays a key role in initiation of mucosal damage. This information will be critical for strategies to target control of mucosal damage. Methods Primary genital epithelial and intestinal cell line cultures Reproductive tract tissues were obtained from women aged 30–59 years (mean age 42.9+7.2) undergoing hysterectomy for benign gynecological reasons at Hamilton Health Sciences Hospital. Written informed consent was obtained from all patients, with the approval of Hamilton Health Sciences Research Ethics Board. The most common reasons for surgery were uterine fibroids and heavy bleeding. Tissues were first examined by pathologists and if they were deemed free from any malignant or other clinically observed disease, coin-sized pieces were collected for further processing. Detailed protocol for isolation and culture of genital epithelial cells (GEC) has been described previously [49]. Briefly, endometrial and cervical tissues were obtained from women undergoing hysterectomy and minced into small pieces and digested in an enzyme mixture for 1 hour at 37°C. Epithelial cells (EC) were isolated by a series of separations through nylon mesh filters of different pore sizes. EC were grown onto Matrigel™ (Becton, Dickinson and Company) coated, 0.4-µm pore-size polycarbonate membrane tissue culture inserts (BD Falcon, Mississauga, Canada) with primary tissue culture medium (DMEM/F12; Invitrogen, Canada) supplemented with 10 µM HEPES (Invitrogen, Canada), 2 µM l-glutamine (Invitrogen, Canada), 100 units/ml penicillin/streptomycin (Sigma–Aldrich, Oakville, Canada), 2.5% Nu Serum culture supplement (Becton, Dickinson and company, Franklin Lakes, USA), and 2.5% Hyclone defined fetal bovine serum (Hyclone, Logan, USA). Polarized monolayers were formed within 5–7 days. The purity of GEC monolayers was between 95% and 98%. There was no trace of any hematopoietic cells in the confluent monolayers. The methodology used for monitoring the purity of the epithelial monolayers and absence of CD45 staining in confluent cultures has been described in detail before [49]. The human colon-derived crypt-like T84 epithelial cell line was maintained and cultured as described previously [50]. Briefly T84 intestinal cells were grown and maintained in a 1∶1 (vol/vol) mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium, supplemented with 10% fetal bovine serum, 1.5% HEPES, and 2% penicillin-streptomycin (Life Technologies, Grand Island, NY) at 37°C in 5% CO2. T84 cells were seeded onto filter supports (0.5×106 cells/well, 0.4-µm pore-size polycarbonate membrane tissue culture inserts, BD Falcon, Mississauga, Canada) and grown for approximately 5–6 days till they reached confluency. The confluency of EC cultures and T84 monolayers was monitored microscopically and by trans-epithelial resistance (TER) across monolayers grown on cell culture inserts, using a volt ohm meter (EVOM; World Precision Instruments, Sarasota, FL, USA). Epithelial monolayers showing TER values higher than 1000 Ω/cm2 were considered completely confluent and used for further experiments. Virus strains, propagation and infection HIV-1 R5 and X4 -tropic laboratory strains were prepared by one of two methods. R5-tropic ADA and X4-tropic laboratory strain IIIB viral stocks were prepared by infection of adherent monocytes from human PBMCs (ADA) or from chronically infected H9 cell line (IIIB), followed by virus concentration by Amicon Ultra-15 filtration system (Millipore, Billerica, US). Virus stock preparations were checked for possible contamination by cellular factors by multiplex bead-based sandwich immunoassay (Luminex Corporation, Austin, TX, USA). TNF-α, IL-6, IL-8, MCP-1, MIP-1α, MIP-1β, RANTES, IL-1α, IL-1β were not detected in any viral stock (standard range of detection limit for different factors: 0.1–4.5 pg/ml). Laboratory strains of HIV-1 virus were also prepared by ultracentrifugation method. HIV-1 R5 laboratory strains ADA, Bal, and X4 strains IIIB, MN and NL4-3 and four clinical strains 11242 (dual), 11249 (R5), 4648 (R5), 7681 (X4) (Dr. Donald R. Branch, University of Toronto) were prepared in human PBMC preparations and concentrated by ultracentrifugation over 20% sucrose for 1 hour at 19,000 rpm (33,000g). All HIV-1 stocks were titered for infectious viral count/ml by TZMb-1 indicator cell assay as described previously [51]. TZMb-1 assay is based on infection of Hela cell line that has been stably transfected with CD4, CXCR4 and CCR5 receptors for HIV-1 attachment. The cell line also carries two indicator systems (β-galactosidase and luciferase systems) under the influence of HIV-1 promoter. HIV-1 infection is detected by staining the cells for β-galactosidase activity resulting in cells turning blue, indicative of HIV-1 replication or alternately by detection of luciferase activity. Infectious viral units/ml  =  infectious viral counts indicated by number of blue cells per well X dilution factor/ml. For HIV-1 exposure, primary epithelial cells, isolated from human female genital tract tissues or intestinal T84 cells were grown to confluence. Epithelial cell cultures were exposed apically or basolaterally to HIV-1 virus (105 infectious viral units/well/100µl, final concentration 106 infectious viral units/ml), corresponding to MOI of 1.0 or other viral doses as mentioned in individual experiments. The p24 values corresponding to this standard concentration of virus (106 infectious viral units/ml) varied, depending on the viral strain, as determined by p24 ELISA (Zeptometrix Corp., Buffalo, NY, USA); it corresponded to p24 concentration between 0.7–1110 ng/ml for X4 tropic lab strains and between 0.3–790 ng/ml for R5 tropic viruses. For clinical strains p24 concentrations used for HIV exposures were between 49–773 ng/ml. Mock infection controls included exposure to same volume of media without HIV-1 (media control, mock) or exposure to same volume of virus (and gp120) free supernatant from PBMC (for R5 HIV-1) or H-9 (X4 HIV-1) cell line cultures (R5 and X4 controls). Env-defective mutant, Env− (kind gift of D. Johnson, NCI) was on an NL4-3 backbone (X4-tropic HIV-1 laboratory strain) and was compared to wildtype NL4-3 for its effect on epithelial cell permeability [30]. T84 intestinal epithelial monolayers were exposed to wildtype (106 infectious units/ml, p24 79ng/ml) or Env− NL4-3 (p24, 79ng/ml) and TER were measured prior to and 24 hours post-exposure. Monolayers were fixed for ZO-1 staining. UV inactivation of HIV HIV-1 R5-tropic strain ADA and X4-tropic strain IIIB were inactivated by UV exposure. 106 infectious units/ml of virus was subjected to 25–100mJ/cm2 UV with a UV cross-linker (Fisher Scientific, USA). UV inactivation of virus was confirmed by titration on TZMb-1 cells. Gp120 and Tat treatments HIV-1 proteins gp120 envelope protein and soluble Tat protein were obtained from NIH AIDS Research & Reference Reagent Program. Epithelial cell cultures were treated with HIV-1 viral proteins gp120 (0.8nM, 0.1 µg/ml) or Tat (100 nM, 1.4ug/ml). A range of Gp120 concentration (50ng–1µg/ml) was tried based on those used in previous studies [28]. Concentration of tat was consistent with that used in other studies for cultured brain endothelial cells and corneal epithelial cells [29],[38]. HIV-1 proteins were allowed to interact with the epithelial cells for 24 hours at 37°C. Gp120 neutralization assay To test the role of gp120, HIV-1 IIIB was incubated at 37C with a recombinant human monoclonal neutralizing antibody against HIV-1 gp120 (IgG1, clone 2G12, Polymun Scientific, Austria) at a concentration of 35µg/ml or an isotype control antibody (Southern Biotechnology, Birmingham, USA) at same concentration for 1 hour. TERs were measured prior to and post-exposure. Quantitative real-time reverse transcriptase polymerase chain reaction of tight junction proteins Quantitation of tight junction gene expression in epithelial cells post-HIV-1 exposure and comparison with unexposed control epithelial cells was done by real time quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) with Syber Green. The tight junction genes examined were Claudin 1, 2, 3, 4, 5, ZO-1, and Occludin. ECs were lysed by Trizol reagent, total RNA was extracted by RNeasy mini kit (Qiagen Inc., ON, Canada) and treated with DNase column (RNase-free DNase set, Qiagen Inc., ON, Canada) to remove DNA contamination. The cDNA was synthesized by qScript™ cDNA supermix (Quanta Bioscience Inc., Gaithersburg, MD, US) according to manufacturer's protocol. Real-time PCR was performed for each tight junction gene mRNA and GAPDH (internal control) in AB7700 SDS V1.7 (Applied Biosystems, Foster City, CA) with the program: 50°C 2 min, 94°C for 10 minutes and 40 cycles at 94°C for 15 s and 60°C for 1 minute. To validate the quantitative real-time RT-PCR protocol, melting curve analysis was performed to check for the absence of primer dimers. The sequence of primers targeting tight junction genes was taken from published studies (Table 1). The quantitative PCR data was analyzed using the comparative CT method [52]. Briefly the difference in cycle times, ΔCT, was determined as the difference between the tested gene and the reference house keeping gene GAPDH. ΔΔCT was obtained by finding the difference between exposed and mock-treatment groups for each gene. The fold change was calculated as FC = 2−ΔΔCT and results were expressed as fold decrease following HIV exposure compared to mock-treated control cultures. Immunofluorescent staining for tight junction proteins Following treatment, EC monolayers were fixed in 4% Paraformaldehyde, permeabilized with 0.1% Triton X-100 (Mallinckrodt Inc., Paris, KY), and blocked for 30 minutes in blocking solution (5% bovine serum albumin and 5% goat serum (Sigma-Aldrich, ON, Canada) in 0.1% Triton X-100]. Primary antibodies (rabbit anti-human claudin-2, rabbit anti-human Occludin, or rabbit anti-human ZO-1 from Zymed Laboratories, CA, USA) were diluted (2 µg/ml) in blocking solution and incubated with monolayers for 1 hour at room temperature. Normal rabbit serum was used as a negative control to check the specificity of primary antibodies. Following incubation with primary antibodies the monolayers were washed with PBS and secondary antibody, Alexa Fluor 488 goat anti-rabbit IgG (1.5 µg/ml, Molecular Probes, Eugene, OR) was added for 1 hour at room temperature. Nuclear counterstaining was done with Propidium Iodide (500nM, Molecular Probes, Eugene, OR). After extensive washing, filters were excised from the polystyrene inserts and mounted on glass slides in mounting medium (Vectashield mounting medium, Vector Lab, CA, USA). All samples were imaged on an inverted confocal laser-scanning microscope (LSM 510, Zeiss, Germany) using standard operating conditions (63× objective, optical laser thickness of 1µm, image dimension of 512×512, lasers: argon (450nm) and HeNe (543nm) for ZO-1 and nuclear staining, respectively. For each experiment, confocal microscope settings for image acquisition and processing were identical between control and treated monolayers and 3 separate, random images were acquired and analyzed for each experimental condition. Each experiment was repeated at least 3 times. Monolayers were scanned in an apical to basolateral sequence and sequential image sets were analyzed by image analysis software (Image J, NIH) to measure the areas of both fluorescently stained ZO-1 and cellular nuclei. Images are presented as either en face to illustrate the distribution of tight junction protein immunoreactivity or as a composite Z-stack reconstruction, which shows the monolayer in transverse profile with the basally located nuclei identified by propidium iodide staining (red) and tight junction proteins by fluorescein isothiocyanate labeled secondary antibodies (green). For Figure 4A–C, optical sections (XY planes) through the apical regions of monolayers were stacked to represent complete tight junction ZO-1 staining distribution in order to make direction comparison between control and experimental counterparts. MTT viability assay MTT assay was used to determine viability of HIV-1 exposed monolayers and compared to unexposed control monolayers. The assay was performed according to manufactures instructions (Biotium Inc., CA, USA). Briefly, human primary endometrial epithelial cells and T84 intestinal epithelial cells were seeded on 96-well plates at a density of 103 cells/well and allowed to attach to the plate and grown for 5 days. Triplicate wells were treated with media or exposed with laboratory strains of HIV-1 (104 infectious viral units/ml, MOI 1∶1) in 100 µl quantity. After 24 hours incubation, 10 µl of MTT solution was added and incubated for 4h at 37°C. After incubation, the medium was discarded and the purple blue sediment was dissolved in 200 µl DMSO. The relative optical density (OD)/well were determined at a test wavelength of 570 nm in a ELISA reader using a 630 nm reference wavelength. The MTT assay is based on the cleavage of the yellow tetrazolium salt (MTT) to purple formazan by metabolically active cells, based on their mitochondrial activity. Cell viability was expressed as a percentage of untreated cells, which served as a negative control group and was designated 100%; the results are expressed as % of negative control. All assays were performed in triplicate. Blue Dextran leakage assay Blue Dextran dye was dissolved in primary medium (2.3 mg/ml, [26]) and added to the apical surface of confluent epithelial cell monolayers grown on 0.4µm pore size culture inserts. At various time intervals, post-HIV-1 exposure, 50ml of basolateral medium was sampled and replaced by equal volume of primary growth media. Blue Dextran dye in basolateral samples was measured using a microplate reader (Safire, tecan, NC, USA) at 610nm and the optical density was expressed as a % of density of dye added to the apical medium at the beginning of experiment (Time “0”). Cytokine analysis Apical and basolateral supernatants were analyzed for multiple cytokines using the Luminex multianalyte technology (Luminex Corporation, Austin, TX, USA) as described before [53]. Multiplex bead-based sandwich immunoassay kits (Upstate Biotech, Millipore, MA, USA) were used to measure levels of IL-1β, IL-6, IL-8, IL-10, MCP-1 and TNF-α, as per the manufacturer's instructions. Primary endometrial EC and T84 monolayers were exposed to HIV-1 (ADA strain, 106 infectious viral units/ml) and apical and basolateral supernatants were collected after 24 hours. Minimum detection limit for the cytokines were 0.1 pg/ml for TNF-α, 0.2 pg/ml for IL-8, 0.3 pg/ml for IL-6 and IL-10, 0.4 pg/ml for IL-1β, 0.9 pg/ml for MCP-1. Levels detected at or below this limit were considered and reported as undetectable. TNF-α neutralization assay Epithelial cells were grown to confluence and treated with TNF-α (20ng/ml) or HIV-1 (ADA, 106 infectious viral units /ml ) for 24 hours. To test the role of TNF-α, mouse anti-human TNF-α neutralizing antibody (25 µg/ml) (R&D Systems, USA) or normal mouse serum (25 µg/ml) was added to confluent monolayers for 1 hour at 37C prior to treatment with TNF-α (20ng/ml) or HIV-1. Barrier function was determined by TER measurements before and after treatment. Bacterial and HIV-1 translocation For bacterial translocation experiments, non-pathogenic E. coli strain HB101 was grown and cultured in Luria-Bertani (LB) broth (Invitrogen, Canada). T84 cells were grown to confluence on 3.0-µm-pore-size filters (BD Falcon, Canada), transferred to antibiotic-free Hanks solution, and treated with TNF-α (20ng/ml), E.coli (108 CFU/ml), HIV-1 (106 infectious virus units/ml) for 6h, HIV-1 (106 infectious viral units/ml) for 24h, TNF-α+E.coli, HIV-1 + E.coli at the same time for 6h and HIV-1 for 24h + E.coli for 6h. Some wells were left untreated as negative controls. TER was measured before and after treatment and basolateral supernatants were collected 6 hours after the addition of E. Coli to detect bacterial translocation to the basolateral side. The supernatants were diluted and plated on LB agar and incubated for 24h followed by enumeration of bacterial colony counts. For viral translocation, HIV-1 was added to the apical surface of confluent EC monolayers at a concentration of 105 infectious viral units/well and basolateral supernatants were collected at different time intervals. Viral counts were determined using TZMb-1 indicator cell assay. For assessment of LPS leakage, LPS (100ng/ml; from E.coli O26:B6; Sigma-Aldrich, MO, USA) was added to the apical surface of confluent EC monolayers, 24h post-exposure to HIV and compared with unexposed controls. Basolateral supernatants were collected 1 hour after addition of LPS and LPS leakage was measured by measuring LPS levels in the basolateral supernatants by Pyrochrome LPS detection kit (Cape Cod incorporated, MA, USA) according to the manufacturer's instructions. Statistical analysis GraphPad Prism Version 4 (GraphPad Software, San Diego, CA) was used to compare three or more means by 2 way analysis of variance (ANOVA). When an overall statistically significant difference was seen, post-tests were performed to compare pairs of treatments, using the Bonferroni method to adjust the p-value for multiple comparisons. An alpha value of 0.05 was set for statistical significance. p-Values for each analysis are indicated in figure legends. Accession numbers of genes and proteins TNF-a (NCBI Accession number AAD18091), IL-8 (NCBI Accession number CAA77745), IL-6 (NCBI Accession number AAD13886), IL-10 (NCBI Accession number AAA63207), IL-1b (NCBI Accession number AAC03536), MCP-1 (NCBI Accession number AABB29926). ZO-1 (GeneBank Accession number NM_003257), Occludin (GeneBank Accession number NM_002538), Claudin-1 (Genebank Accession number NM_021101, Claudin-2 (Genebank Accession number NM_020384), Claudin-3 (Genebank Accession number NM_001306), Claudin-4 (Genebank Accession number NM_001305), Claudin-5 (Genebank Accession number NM_003277), GAPDH (Genebank Accession number NM_002046).
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                Author and article information

                Contributors
                Role: Academic Editor
                Journal
                Pathogens
                Pathogens
                pathogens
                Pathogens
                MDPI
                2076-0817
                18 January 2016
                March 2016
                : 5
                : 1
                : 8
                Affiliations
                [1 ]Institute of Cancer and Genomic Sciences, College of Medical and Dental Sciences, University of Birmingham, Vincent Drive, Birmingham B15 2TT, UK; cdjames@ 123456vcu.edu
                [2 ]Present address; Virginia Commonwealth University, School of Dentistry, W. Baxter Perkinson Jr. Building, 521 North 11th Street, P.O. Box 980566, Richmond, VA 23298-0566, USA
                Author notes
                [* ]Correspondence: s.roberts@ 123456bham.ac.uk ; Tel.: +44-121-414-7459; Fax: +44-121-414-4486
                Article
                pathogens-05-00008
                10.3390/pathogens5010008
                4810129
                26797638
                5b2e2d1b-f074-41bf-a102-0cc9beffed1b
                © 2016 by the authors; licensee MDPI, Basel, Switzerland.

                This article is an open access article distributed under the terms and conditions of the Creative Commons by Attribution (CC-BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 23 November 2015
                : 15 January 2016
                Categories
                Review

                oncogenic viruses,pdz proteins,cell polarity,pi3k/akt signalling,hpv e6,adenovirus e4orf1,dlg1,scrib,tight junctions

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