The PDZ1 and PDZ3 Domains of MAGI-1 Regulate the Eight-Exon Isoform of the Coxsackievirus and Adenovirus Receptor

Epithelial integrity is essential for homeostasis and poses a formidable barrier to pathogen entry. Major factors for viral entry into epithelial cells are the localization and abundance of the primary receptor. The coxsackievirus and adenovirus receptor (CAR) is a primary receptor for these two pathogenic groups of viruses. In polarized epithelia, a low-abundance, alternatively spliced eight-exon isoform of CAR, CAR(Ex8), is localized apically where it can support viral infection from the air-exposed surface. Using biochemical, cell biology, genetic, and spectroscopic approaches, we show that the levels of apical CAR(Ex8) are negatively regulated by the PDZ domain-containing protein MAGI-1 (membrane-associated guanylate kinase with inverted orientation protein-1) and that two MAGI-1 PDZ domains, PDZ1 and PDZ3, regulate CAR(Ex8) levels in opposing ways. Similar to full-length MAGI-1, expression of the isolated PDZ3 domain significantly reduces cell surface CAR(Ex8) abundance and adenovirus infection. In contrast, the PDZ1 domain is able to rescue CAR(Ex8) and adenovirus infection from MAGI-1-mediated suppression. These data suggest a novel cell-based strategy to either suppress viral infection or augment adenovirus-based gene therapy.     

Sulfatide Regulates Caspase-3-Independent Apoptosis of Influenza A Virus through Viral PB1-F2 Protein

Influenza A virus (IAV) generally causes caspase-dependent apoptosis based on caspase-3 activation, resulting in nuclear export of newly synthesized viral nucleoprotein (NP) and elevated virus replication. Sulfatide, a sulfated galactosylsphingolipid, enhances IAV replication through promoting newly synthesized viral NP export induced by association of sulfatide with hemagglutinin delivered to the cell surface. Here, we demonstrated that sulfatide is involved in caspase-3-independent apoptosis initiated by the PB1-F2 protein of IAV by using genetically sulfatide-produced cells and PB1-F2-deficient IAVs. Sulfatide-deficient COS7 cells showed no virus-induced apoptosis, whereas SulCOS1 cells, sulfatide-enriched COS7 cells that genetically expressed the two transferases required for sulfatide synthesis from ceramide, showed an increase in IAV replication and were susceptible to caspase-3-independent apoptosis. Additionally, PB1-F2-deficient IAVs, which were generated by using a plasmid-based reverse genetics system from a genetic background of A/WSN/33 (H1N1), demonstrated that PB1-F2 contributed to caspase-3-independent apoptosis in IAV-infected SulCOS1 cells. Our results show that sulfatide plays a critical role in efficient IAV propagation via caspase-3-independent apoptosis initiated by the PB1-F2 protein.     

Most Influenza A Virions Fail To Express at Least One Essential Viral Protein

Segmentation of the influenza A virus (IAV) genome enables rapid gene reassortment at the cost of complicating the task of assembling the full viral genome. By simultaneously probing for the expression of multiple viral proteins in MDCK cells infected at a low multiplicity with IAV, we observe that the majority of infected cells lack detectable expression of one or more essential viral proteins. Consistent with this observation, up to 90% of IAV-infected cells fail to release infectious progeny, indicating that many IAV virions scored as noninfectious by traditional infectivity assays are capable of single-round infection. This fraction was not significantly affected by target or producer cell type but varied widely between different IAV strains. These data indicate that IAV exists primarily as a swarm of complementation-dependent semi-infectious virions, and thus traditional, propagation-dependent assays of infectivity may drastically misrepresent the true infectious potential of a virus population.     

Influenza A virus interactions with macrophages: Lessons from epithelial cells

Influenza viruses are an important cause of respiratory infection worldwide. In humans, infection with seasonal influenza A virus (IAV) is generally restricted to the respiratory tract where productive infection of airway epithelial cells promotes viral amplification, dissemination, and disease. Alveolar macrophages (MΦ) are also among the first cells to detect and respond to IAV, where they play a pivotal role in mounting effective innate immune responses. In contrast to epithelial cells, IAV infection of MΦ is a “dead end” for most seasonal strains, where replication is abortive and newly synthesised virions are not released. Although the key replicative stages leading to productive IAV infection in epithelial cells are defined, there is limited knowledge about the abortive IAV life cycle in MΦ. In this review, we will explore host factors and viral elements that support the early stages (entry) through to the late stages (viral egress) of IAV replication in epithelial cells. Similarities, differences, and unknowns for each key stage of the IAV replicative cycle in MΦ will then be highlighted. Herein, we provide mechanistic insights into MΦ‐specific control of seasonal IAV replication through abortive infection, which may in turn, contribute to effective host defence.     

Endogenous Murine BST-2/Tetherin Is Not a Major Restriction Factor of Influenza A Virus Infection

BST-2 (tetherin, CD317, HM1.24) restricts virus growth by tethering enveloped viruses to the cell surface. The role of BST-2 during influenza A virus infection (IAV) is controversial. Here, we assessed the capacity of endogenous BST-2 to restrict IAV in primary murine cells. IAV infection increased BST-2 surface expression by primary macrophages, but not alveolar epithelial cells (AEC). BST-2-deficient AEC and macrophages displayed no difference in susceptibility to IAV infection relative to wild type cells. Furthermore, BST-2 played little role in infectious IAV release from either AEC or macrophages. To examine BST-2 during IAV infection in vivo, we infected BST-2-deficient mice. No difference in weight loss or in viral loads in the lungs and/or nasal tissues were detected between BST-2-deficient and wild type animals. This study rules out a major role for endogenous BST-2 in modulating IAV in the mouse model of infection.     

Fibroblast growth factor-9 expression in airway epithelial cells amplifies the type I interferon response and alters influenza A virus pathogenesis

Influenza A virus (IAV) preferentially infects conducting airway and alveolar epithelial cells in the lung. The outcome of these infections is impacted by the host response, including the production of various cytokines, chemokines, and growth factors. Fibroblast growth factor-9 (FGF9) is required for lung development, can display antiviral activity in vitro, and is upregulated in asymptomatic patients during early IAV infection. We therefore hypothesized that FGF9 would protect the lungs from respiratory virus infection and evaluated IAV pathogenesis in mice that overexpress FGF9 in club cells in the conducting airway epithelium (FGF9-OE mice). However, we found that FGF9-OE mice were highly susceptible to IAV and Sendai virus infection compared to control mice. FGF9-OE mice displayed elevated and persistent viral loads, increased expression of cytokines and chemokines, and increased numbers of infiltrating immune cells as early as 1 day post-infection (dpi). Gene expression analysis showed an elevated type I interferon (IFN) signature in the conducting airway epithelium and analysis of IAV tropism uncovered a dramatic shift in infection from the conducting airway epithelium to the alveolar epithelium in FGF9-OE lungs. These results demonstrate that FGF9 signaling primes the conducting airway epithelium to rapidly induce a localized IFN and proinflammatory cytokine response during viral infection. Although this response protects the airway epithelial cells from IAV infection, it allows for early and enhanced infection of the alveolar epithelium, ultimately leading to increased morbidity and mortality. Our study illuminates a novel role for FGF9 in regulating respiratory virus infection and pathogenesis.     

Sulfatide is required for efficient replication of influenza A virus

Sulfatide is abundantly expressed in various mammalian organs, including the intestines and trachea, in which influenza A viruses (IAVs) replicate. However, the function of sulfatide in IAV infection remains unknown. Sulfatide is synthesized by two transferases, ceramide galactosyltransferase (CGT) and cerebroside sulfotransferase (CST), and is degraded by arylsulfatase A (ASA). In this study, we demonstrated that sulfatide enhanced IAV replication through efficient translocation of the newly synthesized IAV nucleoprotein (NP) from the nucleus to the cytoplasm, by using genetically produced cells in which sulfatide expression was down-regulated by RNA interference against CST mRNA or overexpression of the ASA gene and in which sulfatide expression was up-regulated by overexpression of both the CST and CGT genes. Treatment of IAV-infected cells with an antisulfatide monoclonal antibody (MAb) or an anti-hemagglutinin (HA) MAb, which blocks the binding of IAV and sulfatide, resulted in a significant reduction in IAV replication and accumulation of the viral NP in the nucleus. Furthermore, antisulfatide MAb protected mice against lethal challenge with pathogenic influenza A/WSN/33 (H1N1) virus. These results indicate that association of sulfatide with HA delivered to the cell surface induces translocation of the newly synthesized IAV ribonucleoprotein complexes from the nucleus to the cytoplasm. Our findings provide new insights into IAV replication and suggest new therapeutic strategies.     

Pulmonary collectins modulate strain-specific influenza a virus infection and host responses

Collectins are secreted collagen-like lectins that bind, agglutinate, and neutralize influenza A virus (IAV) in vitro. Surfactant proteins A and D (SP-A and SP-D) are collectins expressed in the airway and alveolar epithelium and could have a role in the regulation of IAV infection in vivo. Previous studies have shown that binding of SP-D to IAV is dependent on the glycosylation of specific sites on the HA1 domain of hemagglutinin on the surface of IAV, while the binding of SP-A to the HA1 domain is dependent on the glycosylation of the carbohydrate recognition domain of SP-A. Here, using SP-A and SP-D gene-targeted mice on a common C57BL6 background, we report that viral replication and the host response as measured by weight loss, neutrophil influx into the lung, and local cytokine release are regulated by SP-D but not SP-A when the IAV is glycosylated at a specific site (N165) on the HA1 domain. SP-D does not protect against IAV infection with a strain lacking glycosylation at N165. With the exception of a small difference on day 2 after infection with X-79, we did not find any significant difference in viral load in SP-A(-/-) mice with either IAV strain, although small differences in the cytokine responses to IAV were detected in SP-A(-/-) mice. Mice deficient in both SP-A and SP-D responded to IAV similarly to mice deficient in SP-D alone. Since most strains of IAV currently circulating are glycosylated at N165, SP-D may play a role in protection from IAV infection.     

Enhanced acetylation of alpha-tubulin in influenza A virus infected epithelial cells

Acetylated microtubules (AcMTs), a post-translationally modified form of microtubules, promote polarized protein transport. Here we report that influenza A virus (IAV) induces the acetylation of microtubules in epithelial cells. By employing specific inhibitors and siRNA we demonstrate Rho GTPase-mediated downregulation of tubulin deacetylase activity in IAV-infected cells, resulting in increased tubulin acetylation. Further, we demonstrate that depolymerization/deacetylation or enhanced acetylation of microtubules decreased or increased, respectively, the release of virions from infected cells. IAV assembly requires the polarized delivery of viral components to apical plasma membrane. Our findings suggest the potential involvement of AcMTs in polarized trafficking of IAV components.     

Influenza A M2 recruits M1 to the plasma membrane: A fluorescence fluctuation microscopy study

Influenza A virus (IAV) is a respiratory pathogen that causes seasonal epidemics with significant mortality. One of the most abundant proteins in IAV particles is the matrix protein 1 (M1), which is essential for the virus structural stability. M1 organizes virion assembly and budding at the plasma membrane (PM), where it interacts with other viral components. The recruitment of M1 to the PM as well as its interaction with the other viral envelope proteins (hemagglutinin [HA], neuraminidase, matrix protein 2 [M2]) is controversially discussed in previous studies. Therefore, we used fluorescence fluctuation microscopy techniques (i.e., scanning fluorescence cross-correlation spectroscopy and number and brightness) to quantify the oligomeric state of M1 and its interactions with other viral proteins in co-transfected as well as infected cells. Our results indicate that M1 is recruited to the PM by M2, as a consequence of the strong interaction between the two proteins. In contrast, only a weak interaction between M1 and HA was observed. M1-HA interaction occurred only in the event that M1 was already bound to the PM. We therefore conclude that M2 initiates the assembly of IAV by recruiting M1 to the PM, possibly allowing its further interaction with other viral proteins.     

Cross-Protective Effect of Antisense Oligonucleotide Developed Against the Common 3′ NCR of Influenza A Virus Genome

The influenza A virus (IAV) has eight segmented single-stranded RNA genome containing a common and evolutionarily conserved non-coding region (NCRs) at 5′ and 3′ ends that are important for the virus replication. In this study, we designed an antisense oligonucleotide against the 3′ NCR of vital segments of the IAV genome to inhibit its replication. The results demonstrated that the co-transfection of Madine Darby Canine Kidney (MDCK) cells with the antisense oligonucleotide and the plasmids encoding the viral genes led to the down-regulation of the viral gene expression. The designed antisense molecules reduced the cytopathic effect caused by A/PR/8/34 (H1N1), A/Udorn/307/72 (H3N2), and A/New Caledonia/20/99 (H1N1) strains of IAV for almost 48 h. Furthermore, the intra-venous delivery of this oligonucleotide significantly reduced the viral titers in the lungs of infected mice and protected the mice from lethal effects of all the strains of influenza virus. The study demonstrated that the antisense oligonucleotide designed against the NCR region inhibits the expression of the viral genome. The decrease of the cytopathic effect in the MDCK cells and increase in survival of mice confirmed the reduction of virus multiplication and pathogenesis in the presence of antisense oligonucleotide. Thus, we demonstrate that a single antisense oligonucleotide is capable of providing protection against more than one strains of the IAV.     

Coxsackie and adenovirus receptor (CAR) is a product of Sertoli and germ cells in rat testes which is localized at the Sertoli-Sertoli and Sertoli-germ cell interface

The coxsackie and adenovirus receptor (CAR), a putative cell-cell adhesion molecule, has attracted wide interest due to its importance in viral pathogenesis and in mediating adenoviral gene delivery. However, the distribution pattern and physiological function of CAR in the testis is still not clear. Here, we identified CAR in Sertoli cells and germ cells of rats. In vivo studies have shown that CAR resides at the blood-testis barrier as well as at the ectoplasmic specialization. The persistent expression of CAR in rat testes from neonatal period throughout adulthood implicates its role in spermatogenesis. Using primary Sertoli cell cultures, we observed a significant induction of CAR during the formation of Sertoli cell epithelium. Furthermore, CAR was seen to be concentrated at inter-Sertoli cell junctions, co-localizing with tight junction protein marker ZO-1 and adherens junction protein N-cadherin. CAR was also found to be associated with proteins of Src kinase family and its protein level declined after TNFα treatment in Sertoli cell cultures. Immunofluorescent staining of isolated germ cells has revealed the presence of CAR on spermatogonia, spermatocytes, round spermatids and elongate spermatids. Taken together, we propose that CAR functions as an adhesion molecule in maintaining the inter-Sertoli cell junctions at the basal compartment of the seminiferous epithelium. In addition, CAR may confer adhesion between Sertoli and germ cells at the Sertoli-germ cell interface. It is possible that the receptor utilized by viral pathogens to breakthrough the epithelial barrier was also employed by developing germ cells to migrate through the inter-Sertoli cell junctions.     

Robust expression of vault RNAs induced by influenza A virus plays a critical role in suppression of PKR-mediated innate immunity

Protein kinase R (PKR) is a vital component of host innate immunity against viral infection. However, the mechanism underlying inactivation of PKR by influenza A virus (IAV) remains elusive. Here, we found that vault RNAs (vtRNAs) were greatly induced in A549 cells and mouse lungs after infection with IAV. The viral NS1 protein was shown to be the inducer triggering the upregulation of vtRNAs. Importantly, silencing vtRNA in A549 cells significantly inhibited IAV replication, whereas overexpression of vtRNAs markedly promoted the viral replication. Furthermore, in vivo studies showed that disrupting vtRNA expression in mice significantly decreased IAV replication in infected lungs. The vtRNA knockdown animals exhibited significantly enhanced resistance to IAV infection, as evidenced by attenuated acute lung injury and spleen atrophy and consequently increased survival rates. Interestingly, vtRNAs promoted viral replication through repressing the activation of PKR and the subsequent antiviral interferon response. In addition, increased expression of vtRNAs was required for efficient suppression of PKR by NS1 during IAV infection. Moreover, vtRNAs were also significantly upregulated by infections of several other viruses and involved in the inactivation of PKR signaling by these viruses. These results reveal a novel mechanism by which some viruses circumvent PKR-mediated innate immunity.     

Influenza-Infected Neutrophils within the Infected Lungs Act as Antigen Presenting Cells for Anti-Viral CD8+ T Cells

Influenza A virus (IAV) is a leading cause of respiratory tract disease worldwide. Anti-viral CD8⁺ T lymphocytes responding to IAV infection are believed to eliminate virally infected cells by direct cytolysis but may also contribute to pulmonary inflammation and tissue damage via the release of pro-inflammatory mediators following recognition of viral antigen displaying cells. We have previously demonstrated that IAV antigen expressing inflammatory cells of hematopoietic origin within the infected lung interstitium serve as antigen presenting cells (APC) for infiltrating effector CD8⁺ T lymphocytes; however, the spectrum of inflammatory cell types capable of serving as APC was not determined. Here, we demonstrate that viral antigen displaying neutrophils infiltrating the IAV infected lungs are an important cell type capable of acting as APC for effector CD8⁺ T lymphocytes in the infected lungs and that neutrophils expressing viral antigen as a result of direct infection by IAV exhibit the most potent APC activity. Our findings suggest that in addition to their suggested role in induction of the innate immune responses to IAV, virus clearance, and the development of pulmonary injury, neutrophils can serve as APCs to anti-viral effector CD8⁺ T cells within the infected lung interstitium.     

Robust expression of p27Kip1 induced by viral infection is critical for antiviral innate immunity

Influenza A virus (IAV) infection regulates the expression of numerous host genes. However, the precise mechanism underlying implication of these genes in IAV pathogenesis remains largely unknown. Here, we employed isobaric tags for relative and absolute quantification (iTRAQ) to identify host proteins regulated by IAV infection. iTRAQ analysis of mouse lungs infected or uninfected with IAV showed a total of 167 differentially upregulated proteins in response to the viral infection. Interestingly, we observed that p27Kip1, a potent cyclin‐dependent kinase inhibitor, was markedly induced by IAV both at mRNA and protein levels through in vitro and in vivo studies. Furthermore, it was shown that innate immune signalling positively regulated p27Kip1 expression in response to IAV infection. Ectopic expression of p27Kip1 in A549 cells dramatically inhibited IAV replication, whereas, p27Kip1 knockdown significantly enhanced the virus replication. in vivo experiments demonstrated that p27Kip1 knockout (KO) mice were more susceptible to IAV than wild‐type (WT) mice: exhibiting higher viral load in lung tissue, faster body‐weight loss, reduced survival rate and more severe organ damage. Moreover, we found that p27Kip1 overexpression facilitated the degradation of viral NS1 protein, caused a dramatic STAT1 activation and promoted the expression of IFN‐β and several critical antiviral interferon‐stimulated genes (ISGs). Increased p27Kip1 expression also restricted infections of several other viruses. Conversely, IAV‐infected p27Kip1 KO mice exhibited a sharp increase in NS1 protein accumulation, reduced level of STAT1 activation and decreased expression of IFN‐β and the ISGs in the lung compared to WT animals. These findings reveal a key role of p27Kip1 in enhancing antiviral innate immunity.     

Autophagy is involved in regulating influenza A virus RNA and protein synthesis associated with both modulation of Hsp90 induction and mTOR/p70S6K signaling pathway

Influenza A virus (IAV) infection triggers autophagosome formation, but inhibits the fusion of autophagosomes with lysosomes. However, the role of autophagy in IAV replication is still largely unclarified. In this study, we aim to reveal the role of autophagy in IAV replication and the molecular mechanisms underlying the regulation. By using autophagy-deficient (Atg7^−/−) MEFs, we demonstrated that autophagy deficiency significantly reduced the levels of viral proteins, mRNA and genomic RNAs (vRNAs) without affecting viral entry. We further found that autophagy deficiency lead to a transient increase in phosphorylation of mTOR and its downstream targets including 4E-BP1 and S6 at a very early stage of IAV infection, and markedly suppressed p70S6K phosphorylation at the late stage of IAV infection. Furthermore, autophagy deficiency resulted in impairment of Hsp90 induction in response to IAV infection. These results indicate that IAV regulates autophagy to benefit the accumulation of viral elements (synthesis of viral proteins and genomic RNA) during IAV replication. This regulation is associated with modulation of Hsp90 induction and mTOR/p70S6K signaling pathway. Our results provide important evidence for the role of autophagy in IAV replication and the mechanisms underlying the regulation.     

Interleukin-22 is produced by invariant natural killer T lymphocytes during influenza A virus infection: potential role in protection against lung epithelial damages

Invariant natural killer T (iNKT) cells are non-conventional lipid-reactive αβ T lymphocytes that play a key role in host responses during viral infections, in particular through the swift production of cytokines. Their beneficial role during experimental influenza A virus (IAV) infection has recently been proposed, although the mechanisms involved remain elusive. Here we show that during in vivo IAV infection, mouse pulmonary iNKT cells produce IFN-γ and IL-22, a Th17-related cytokine critical in mucosal immunity. Although permissive to viral replication, IL-22 production by iNKT cells is not due to IAV infection per se of these cells but is indirectly mediated by IAV-infected dendritic cells (DCs). We show that activation of the viral RNA sensors TLR7 and RIG-I in DCs is important for triggering IL-22 secretion by iNKT cells, whereas the NOD-like receptors NOD2 and NLRP3 are dispensable. Invariant NKT cells respond to IL-1β and IL-23 provided by infected DCs independently of the CD1d molecule to release IL-22. In vitro, IL-22 protects IAV-infected airway epithelial cells against mortality but has no role on viral replication. Finally, during early IAV infection, IL-22 plays a positive role in the control of lung epithelial damages. Overall, IAV infection of DCs activates iNKT cells, providing a rapid source of IL-22 that might be beneficial to preserve the lung epithelium integrity.     

Human h-ficolin inhibits replication of seasonal and pandemic influenza a viruses

The collectins have been shown to have a role in host defense against influenza A virus (IAV) and other significant viral pathogens (e.g., HIV). The ficolins are a related group of innate immune proteins that are present at relatively high concentrations in serum, but also in respiratory secretions; however, there has been little study of the role of ficolins in viral infection. In this study, we demonstrate that purified recombinant human H-ficolin and H-ficolin in human serum and bronchoalveolar lavage fluid bind to IAV and inhibit viral infectivity and hemagglutination activity in vitro. Removal of ficolins from human serum or bronchoalveolar lavage fluid reduces their antiviral activity. Inhibition of IAV did not involve the calcium-dependent lectin activity of H-ficolin. We demonstrate that H-ficolin is sialylated and that removal of sialic acid abrogates IAV inhibition, while addition of the neuraminidase inhibitor oseltamivir potentiates neutralization, hemagglutinin inhibition, and viral aggregation caused by H-ficolin. Pandemic and mouse-adapted strains of IAV are generally not inhibited by the collectins surfactant protein D or mannose binding lectin because of a paucity of glycan attachments on the hemagglutinin of these strains. In contrast, H-ficolin inhibited both the mouse-adapted PR-8 H1N1 strain and a pandemic H1N1 strain from 2009. H-ficolin also fixed complement to a surface coated with IAV. These findings suggest that H-ficolin contributes to host defense against IAV.