Xiaoyong Bao

Human Metapneumovirus Glycoprotein G Inhibits Innate Immune Responses

Human metapneumovirus (hMPV) is a leading cause of acute respiratory tract infection in infants, as well as in the elderly and immunocompromised patients. No effective treatment or vaccine for hMPV is currently available. A recombinant hMPV lacking the G protein (rhMPV-ΔG) was recently developed as a potential vaccine candidate and shown to be attenuated in the respiratory tract of a rodent model of infection. The mechanism of its attenuation, as well as the role of G protein in modulation of hMPV-induced cellular responses in vitro, as well as in vivo, is currently unknown. In this study, we found that rhMPV-ΔG-infected airway epithelial cells produced higher levels of chemokines and type I interferon (IFN) compared to cells infected with rhMPV-WT. Infection of airway epithelial cells with rhMPV-ΔG enhanced activation of transcription factors belonging to the nuclear factor (NF)-κB and interferon regulatory factor (IRF) families, as revealed by increased nuclear translocation and/or phosphorylation of these transcription factors. Compared to rhMPV-WT, rhMPV-ΔG also increased IRF- and NF-κB-dependent gene transcription, which was reversely inhibited by G protein expression. Since RNA helicases have been shown to play a fundamental role in initiating viral-induced cellular signaling, we investigated whether retinoic induced gene (RIG)-I was the target of G protein inhibitory activity. We found that indeed G protein associated with RIG-I and inhibited RIG-I-dependent gene transcription, identifying an important mechanism by which hMPV affects innate immune responses. This is the first study investigating the role of hMPV G protein in cellular signaling and identifies G as an important virulence factor, as it inhibits the production of important immune and antiviral mediators by targeting RIG-I, a major intracellular viral RNA sensor.

Change in permeant size selectivity by phosphorylation of connexin 43 gap-junctional hemichannels by PKC

Gap-junctional channels, permeable to large hydrophilic solutes of up to Mr ≈ 1,000, are responsible for cell-to-cell communication. Phosphorylation of connexin 43 (Cx43) by PKC abolishes the permeability of gap-junctional channels and hemichannels to large hydrophilic solutes, but not to small inorganic ions. Here, we report on a methodology to produce purified hemichannels of controlled subunit composition and apply it to the generation of hemichannels with variable number of PKC-phosphorylated subunits. The subunit composition was determined by luminescence resonance energy transfer. We show that all Cx43 subunits in the hemichannel hexamer have to be phosphorylated to abolish sucrose (Mr 342) permeability. We also show that the hemichannel pores with all subunits phosphorylated by PKC have a sizable diameter, allowing for permeation of the small hydrophilic solute ethyleneglycol (Mr 62). These results indicate that phosphorylation of Cx43 by PKC alters the hemichannel size selectivity and explain why PKC activity affects dye transfer between cells without consistent effects on electrical communication.

Exosomes and Their Role in the Life Cycle and Pathogenesis of RNA Viruses.

Exosomes are membrane-enclosed vesicles actively released into the extracellular space, whose content reflect the physiological/pathological state of the cells they originate from. These vesicles participate in cell-to-cell communication and transfer of biologically active proteins, lipids, and RNAs. Their role in viral infections is just beginning to be appreciated. RNA viruses are an important class of pathogens and affect millions of people worldwide. Recent studies on Human Immunodeficiency Virus (HIV), Hepatitis C Virus (HCV), human T-cell lymphotropic virus (HTLV), and Dengue Virus (DENV) have demonstrated that exosomes released from infected cells harbor and deliver many regulatory factors including viral RNA and proteins, viral and cellular miRNA, and other host functional genetic elements to neighboring cells, helping to establish productive infections and modulating cellular responses. Exosomes can either spread or limit an infection depending on the type of pathogen and target cells, and can be exploited as candidates for development of antiviral or vaccine treatments. This review summarizes recent progress made in understanding the role of exosomes in RNA virus infections with an emphasis on their potential contribution to pathogenesis.

Gap-junctional Hemichannels Are Activated by ATP Depletion in Human Renal Proximal Tubule Cells

We present evidence suggesting that gap-junctional hemichannels (GJH) may be involved in acute ischemic injury of human renal proximal tubule cells (hPT cells). Two GJH, from neighboring cells, join to form an intercellular gap junction channel (GJC). Undocked GJH are permeable to hydrophilic molecules up to 1 kDa, and their opening can significantly alter cell homeostasis. Both GJC and GJH formed by connexin 43 (Cx43) are activated by dephosphorylation. Hence, we tested whether GJH activation during ATP depletion contributes to cell damage in renal ischemia. We found that hPT cells in primary culture express Cx43 (RT-PCR and Western-blot analysis) at the plasma membrane region (immunofluorescence). Divalent-cation removal or pharmacological ATP depletion increased cell loading with the hydrophilic dye 5/6 carboxy-fluorescein (CF, 376 Da) but not with fluorescein-labeled dextran (>1500 Da). Endocytosis and activation of P2X channels were experimentally ruled out. Several GJC blockers inhibited the loading elicited by PKC inhibition. Double labeling (CF and propidium iodide) showed that both Ca2+ removal and ATP depletion increase the percentage of necrotic cells. Gadolinium reduced both the loading and the degree of necrosis during divalent-cation removal or ATP depletion. In conclusion, GJH activation may play an important role in the damage of human renal proximal tubule cells during ATP depletion. These studies are the first to provide evidence supporting a role of GJH in causing injury in epithelial cells in general and in renal-tubule cells in particular.

A novel mechanism for the inhibition of interferon regulatory factor-3-dependent gene expression by human respiratory syncytial virus NS1 protein

Human respiratory syncytial virus (RSV), a leading cause of respiratory tract infections in infants, inhibits type I interferon (IFN)-dependent signalling, as well as IFN synthesis. RSV non-structural protein NS1 plays a significant role in this inhibition; however, the mechanism(s) responsible is not fully known. The transcription factor interferon regulatory factor (IRF)-3 is essential for viral-induced IFN-β synthesis. In this study, we found that NS1 protein inhibits IRF-3-dependent gene transcription in constitutively active IRF-3 overexpressing cells, demonstrating that NS1 directly targets IRF-3. Our data also demonstrate that NS1 associates with IRF-3 and its transcriptional coactivator CBP, leading to disrupted association of IRF-3 to CBP and subsequent reduced binding of IRF-3 to the IFN-β promoter without blocking viral-induced IRF-3 phosphorylation, nuclear translocation and dimerization, thereby identifying a novel molecular mechanism by which RSV inhibits IFN-β synthesis.

Human Metapneumovirus Small Hydrophobic Protein Inhibits NF-κB Transcriptional Activity

ABSTRACT Human metapneumovirus, a leading cause of respiratory tract infections in infants, encodes a small hydrophobic (SH) protein of unknown function. In this study, we showed that infection of airway epithelial cells or mice with recombinant human metapneumovirus lacking SH expression (rhMPV-ΔSH) enhanced secretion of proinflammatory mediators, including interleukin 6 (IL-6) and IL-8, encoded by two NF-kB-dependent genes, compared to infection with wild-type rhMPV. RhMPV-ΔSH infection resulted in enhanced NF-kB-dependent gene transcription and in increased levels of phosphorylated and acetylated NF-kB without affecting its nuclear translocation, identifying a possible novel mechanism by which paramyxovirus SH proteins modulate NF-kB activation.

Mechanism of the defect in gap-junctional communication by expression of a connexin 26 mutant associated with dominant deafness

Gap‐junctional channels (connexin oligomers) are large‐diameter aqueous pores formed by head‐to‐head association of two gap‐junctional hemichannels, one from each of the adjacent cells. Profound hearing loss of genetic origin is common, and mutations of connexin 26 (Cx26) are the most frequent cause of this disorder. The Cx26 R75W mutant has been associated with disruption of cell‐to‐cell communication and profound hearing loss, but the mechanism of the gap‐junctional defect is unknown. Here, we show that Cx26 R75W forms gap‐junctional hemichannels that display altered voltage dependency and reduced permeability, and which cannot form functional gap‐junctional channels between neighboring cells. The R75W phenotype is dominant at the gap‐junction channel but not at the hemichannel level. Therefore, the absence of gap‐junctional communication caused by R75W expression is due to defective gap‐junction formation by functional hemichannels.

Role of retinoic acid inducible gene-I in human metapneumovirus-induced cellular signalling

Human metapneumovirus (HMPV) is a recently discovered pathogen that causes a significant proportion of respiratory infections in young infants, the elderly and immunocompromised patients. Very little is known regarding the cellular signalling elicited by this virus in airway epithelial cells, the target of HMPV infection. In this study, we investigated the role of the RNA helicases retinoic acid inducible gene-I (RIG-I) and melanoma differentiation-associated gene-5 (MDA-5) as the main pattern recognition receptors (PRRs) involved in viral detection and subsequent expression of proinflammatory and antiviral genes. HMPV infection readily induced RIG-I and MDA-5 gene and protein expression in A549 cells, a type II-like alveolar epithelial cell line. Expression of dominant-negative (DN) RIG-I or downregulation of RIG-I gene expression using small interfering RNA (siRNA) significantly decreased HMPV-induced beta interferon (IFN-beta), interleukin (IL)-8 and RANTES gene transcription, by inhibiting viral-induced activation of nuclear factor (NF)-kappaB and interferon regulatory factor (IRF), leading to enhanced viral replication. On the other hand, MDA-5 did not seem to play a significant role in HMPV-induced cellular responses. Mitochondrial antiviral signalling protein (MAVS), an adaptor protein linking both RIG-I and MDA-5 to downstream activation of IRF-3 and NF-kappaB, was also necessary for HMPV-induced cellular signalling. Expression of a DN MAVS significantly reduced IFN-beta and chemokine gene transcription, by inhibiting NF-kappaB- and IRF-dependent gene transcription, in response to HMPV infection. Our results show that HMPV activates the RIG-I-MAVS signalling pathway in airway epithelial cells, leading to the expression of important proinflammatory and antiviral molecules involved in the innate immune response to viruses.

Human Metapneumovirus Glycoprotein G Inhibits TLR4-Dependent Signaling in Monocyte-Derived Dendritic Cells

Human metapneumovirus (hMPV) is a major cause of upper and lower respiratory infections in children and adults. Recent work from our group demonstrated that hMPV G glycoprotein is an important virulence factor, responsible for inhibiting innate immune responses in airway epithelial cells. Myeloid dendritic cells (DCs) are potent APCs and play a major role in initiating and modulating the innate and adaptive immune responses. In this study, we found that TLR4 plays a major role in hMPV-induced activation of monocyte-derived DCs (moDCs), as downregulation of its expression by small interfering RNA significantly blocked hMPV-induced chemokine and type I IFN expression. Similar results were found in bone marrow-derived DCs from TLR4-deficient mice. moDCs infected with a virus lacking G protein expression produced higher levels of cytokines and chemokines compared with cells infected with wild-type virus, suggesting that G protein plays an inhibitory role in viral-induced cellular responses. Specifically, G protein affects TLR4-dependent signaling, as infection of moDCs with recombinant hMPV lacking G protein inhibited LPS-induced production of cytokine and chemokines significantly less than did wild-type virus, and treatment of moDCs with purified G protein resulted in a similar inhibition of LPS-dependent signaling. Our results demonstrate that hMPV G protein plays an important role in inhibiting host innate immune responses, likely affecting adaptive responses too.

Identification of human metapneumovirus-induced gene networks in airway epithelial cells by microarray analysis.

Human metapneumovirus (hMPV) is a major cause of lower respiratory tract infections in infants, elderly and immunocompromised patients. Little is known about the response to hMPV infection of airway epithelial cells, which play a pivotal role in initiating and shaping innate and adaptive immune responses. In this study, we analyzed the transcriptional profiles of airway epithelial cells infected with hMPV using high-density oligonucleotide microarrays. Of the 47,400 transcripts and variants represented on the Affimetrix GeneChip Human Genome HG-U133 plus 2 array, 1601 genes were significantly altered following hMPV infection. Altered genes were then assigned to functional categories and mapped to signaling pathways. Many up-regulated genes are involved in the initiation of pro-inflammatory and antiviral immune responses, including chemokines, cytokines, type I interferon and interferon-inducible proteins. Other important functional classes up-regulated by hMPV infection include cellular signaling, gene transcription and apoptosis. Notably, genes associated with antioxidant and membrane transport activity, several metabolic pathways and cell proliferation were down-regulated in response to hMPV infection. Real-time PCR and Western blot assays were used to confirm the expression of genes related to several of these functional groups. The overall result of this study provides novel information on host gene expression upon infection with hMPV and also serves as a foundation for future investigations of genes and pathways involved in the pathogenesis of this important viral infection. Furthermore, it can facilitate a comparative analysis of other paramyxoviral infections to determine the transcriptional changes that are conserved versus the one that are specific to individual pathogens.