Suppression of Pulmonary Innate Immunity by Pneumoviruses

Pneumonia Virus of Mice (PVM) and Respiratory Syncytial Virus (RSV) are negative sense, single-stranded, enveloped RNA viruses from Pneumovirus genus, Paramyxoviridae family. RSV is the leading cause of respiratory diseases in infants. PVM causes similar respiratory illness in mice. PVM is used as an animal model to study RSV pathogenesis because of its similarity with RSV infection. Viral infection induces type I interferon (IFN) response as an antiviral strategy. PVM and RSV both have two non-structural (NS) proteins that are known to be IFN antagonists. While RSV can target different signaling components of IFN pathway, the mechanism of IFN suppression for PVM was unknown. We have identified that PVM can also target different signaling components of IFN pathway to circumvent the host immune system. Our observations showed that PVM NS proteins facilitate proteasome-mediated degradation of RIG-I, IRF3, STAT2 in IFN pathway by direct interactions with them. Production of several Interferon Stimulated Genes (ISGs) is the distal part of the IFN pathway. We have identified that NS proteins of PVM can also target a few of them such as TRAFD1, IFITM1, ISG20, and IDO for complete suppression of the host immune system. RSV NS proteins play a similar role to suppress IFN pathway by targeting TBK1, RIG-I, IRF3, IRF7, and STAT2. Our study has identified one ISG, OASL, that has antiviral properties against RSV and documented that to counteract this antiviral property of OASL, RSV NS proteins can degrade OASL in a proteasome-dependent way. These above observations help us to delineate the complete suppression mechanism for the whole Pneumovirus genus, both for PVM and RSV by providing the first experimental evidence of signaling components from the IFN pathway targeted by PVM to suppress the IFN response. PVM is a clinically relevant animal model that will help us to find new therapeutic strategies against Pneumovirus infection. RSV study with one of those important ISGs, OASL, is also important to uncover the target substrates of the entire IFN pathway. Together these findings help us to delineate new immune modulatory strategies for the whole Pneumovirus genus

Suppression of Pulmonary Innate Immunity by Pneumoviruses

Pneumonia Virus of Mice (PVM) and Respiratory Syncytial Virus (RSV) are negative sense, single-stranded, enveloped RNA viruses from Pneumovirus genus, Paramyxoviridae family. RSV is the leading cause of respiratory diseases in infants. PVM causes similar respiratory illness in mice. PVM is used as an animal model to study RSV pathogenesis because of its similarity with RSV infection. Viral infection induces type I interferon (IFN) response as an antiviral strategy. PVM and RSV both have two non-structural (NS) proteins that are known to be IFN antagonists. While RSV can target different signaling components of IFN pathway, the mechanism of IFN suppression for PVM was unknown. We have identified that PVM can also target different signaling components of IFN pathway to circumvent the host immune system. Our observations showed that PVM NS proteins facilitate proteasome-mediated degradation of RIG-I, IRF3, STAT2 in IFN pathway by direct interactions with them. Production of several Interferon Stimulated Genes (ISGs) is the distal part of the IFN pathway. We have identified that NS proteins of PVM can also target a few of them such as TRAFD1, IFITM1, ISG20, and IDO for complete suppression of the host immune system. RSV NS proteins play a similar role to suppress IFN pathway by targeting TBK1, RIG-I, IRF3, IRF7, and STAT2. Our study has identified one ISG, OASL, that has antiviral properties against RSV and documented that to counteract this antiviral property of OASL, RSV NS proteins can degrade OASL in a proteasome-dependent way. These above observations help us to delineate the complete suppression mechanism for the whole Pneumovirus genus, both for PVM and RSV by providing the first experimental evidence of signaling components from the IFN pathway targeted by PVM to suppress the IFN response. PVM is a clinically relevant animal model that will help us to find new therapeutic strategies against Pneumovirus infection. RSV study with one of those important ISGs, OASL, is also important to uncover the target substrates of the entire IFN pathway. Together these findings help us to delineate new immune modulatory strategies for the whole Pneumovirus genus

Domain truncation studies reveal that the streptokinase-plasmin activator complex utilizes long range protein-protein interactions with macromolecular substrate to maximize catalytic turnover

To explore the interdomain co-operativity during human plasminogen (HPG) activation by streptokinase (SK), we expressed the cDNAs corresponding to each SK domain individually (α , β , and γ ), and also their two-domain combinations, viz. αβ and βγ in Escherichia coli. After purification, α and β showed activator activities of approximately 0.4 and 0.05%, respectively, as compared with that of native SK, measured in the presence of human plasmin, but the bi-domain constructs αβ and βγ showed much higher co-factor activities (3.5 and 0.7% of native SK, respectively). Resonant Mirror-based binding studies showed that the single-domain constructs had significantly lower affinities for "partner" HPG, whereas the affinities of the two-domain constructs were remarkably native-like with regards to both binary-mode as well as ternary mode ("substrate") binding with HPG, suggesting that the vast difference in co-factor activity between the two- and three-domain structures did not arise merely from affinity differences between activator species and HPG. Remarkably, when the co-factor activities of the various constructs were measured with microplasminogen, the nearly 50-fold difference in the co-factor activity between the two- and three-domain SK constructs observed with full-length HPG as substrate was found to be dramatically attenuated, with all three types of constructs now exhibiting a low activity of approximately 1-2% compared to that of SK·HPN and HPG. Thus, the docking of substrate through the catalytic domain at the active site of SK-plasmin(ogen) is capable of engendering, at best, only a minimal level of co-factor activity in SK·HPN. Therefore, apart from conferring additional substrate affinity through kringle-mediated interactions, reported earlier (Dhar et al., 2002; J. Biol. Chem. 277, 13257), selective interactions between all three domains of SK and the kringle domains of substrate vastly accelerate the plasminogen activation reaction to near native levels

Involvement of a nine-residue loop of streptokinase in the generation of macromolecular substrate specificity by the activator complex through interaction with substrate kringle domains

The selective deletion of a discrete surface-exposed epitope (residues 254-262; 250-loop) in the β domain of streptokinase (SK) significantly decreased the rates of substrate human plasminogen (HPG) activation by the mutant (SKdel254-262). A kinetic analysis of SKdel254-262 revealed that its low HPG activator activity arose from a 5-6-fold increase in Km for HPG as substrate, with little alteration in kcat rates. This increase in the Km for the macromolecular substrate was proportional to a similar decrease in the binding affinity for substrate HPG as observed in a new resonant mirror-based assay for the real-time kinetic analysis of the docking of substrate HPG onto preformed binary complex. In contrast, studies on the interaction of the two proteins with microplasminogen showed no difference between the rates of activation of microplasminogen under conditions where HPG was activated differentially by nSK and SKdel254-262. The involvement of kringles was further indicated by a hypersusceptibility of the SKdel254-262. plasmin activator complex to ε-aminocaproic acid-mediated inhibition of substrate HPG activation in comparison with that of the nSK.plasmin activator complex. Further, ternary binding experiments on the resonant mirror showed that the binding affinity of kringles 1-5 of HPG to SKdel254-262.HPG was reduced by about 3-fold in comparison with that of nSK.HPG. Overall, these observations identify the 250 loop in the β domain of SK as an important structural determinant of the inordinately stringent substrate specificity of the SK.HPG activator complex and demonstrate that it promotes the binding of substrate HPG to the activator via the kringle(s) during the HPG activation process

Induction of interferon-stimulated genes by IRF3 promotes replication of Toxoplasma gondii.

Innate immunity is the first line of defense against microbial insult. The transcription factor, IRF3, is needed by mammalian cells to mount innate immune responses against many microbes, especially viruses. IRF3 remains inactive in the cytoplasm of uninfected cells; upon virus infection, it gets phosphorylated and then translocates to the nucleus, where it binds to the promoters of antiviral genes and induces their expression. Such genes include type I interferons (IFNs) as well as Interferon Stimulated Genes (ISGs). IRF3-/- cells support enhanced replication of many viruses and therefore, the corresponding mice are highly susceptible to viral pathogenesis. Here, we provide evidence for an unexpected pro-microbial role of IRF3: the replication of the protozoan parasite, Toxoplasma gondii, was significantly impaired in IRF3-/- cells. In exploring whether the transcriptional activity of IRF3 was important for its pro-parasitic function, we found that ISGs induced by parasite-activated IRF3 were indeed essential, whereas type I interferons were not important. To delineate the signaling pathway that activates IRF3 in response to parasite infection, we used genetically modified human and mouse cells. The pro-parasitic signaling pathway, which we termed PISA (Parasite-IRF3 Signaling Activation), activated IRF3 without any involvement of the Toll-like receptor or RIG-I-like receptor pathways, thereby ruling out a role of parasite-derived RNA species in activating PISA. Instead, PISA needed the presence of cGAS, STING, TBK1 and IRF3, indicating the necessity of DNA-triggered signaling. To evaluate the physiological significance of our in vitro findings, IRF3-/- mice were challenged with parasite infection and their morbidity and mortality were measured. Unlike WT mice, the IRF3-/- mice did not support replication of the parasite and were resistant to pathogenesis caused by it. Our results revealed a new paradigm in which the antiviral host factor, IRF3, plays a cell-intrinsic pro-parasitic role

IRF3 facilitates intracellular <i>T</i>. <i>gondii</i> replication regardless of parasite virulence type.

<p><b>A,</b> Stunted parasite replication in IRF3 -/- cells. Isogenic wild type and IRF3 -/- MEF cells were infected by <i>T</i>. <i>gondii</i> RH at an m.o.i. of 0.2, and at the indicated times (4, 18, 24 h) stained for parasites (green), cytoplasmic actin (red) and nucleus (DAPI, blue) and visualized by confocal microscopy. For each sample, several individual PVs are shown; number of parasites in 12–14 PVs was counted and the mean ± SD values are shown. <b>B,</b> Flow cytometry measurement of parasite growth. DCs isolated from wild type and IRF3 -/- mice were infected with <i>T</i>. <i>gondii</i> RH at 0.3 m.o.i., and cells were permeabilized and dual-stained for CD11c and parasitic SAG1 at 18 h post-infection, with uninfected wild type cells as control. Gating was set to count infected cells with 2 or more parasites per PV, as marked by contour plot (left), and the percentage of such cells in the total population is shown. <b>C,</b> IRF3 is important for optimal growth of diverse <i>T</i>. <i>gondii</i> strains. The indicated <i>T</i>. <i>gondii</i> strains, represented the three major types: GT1 (type I), ME49 (type II), and VEG (type III). Infection was performed as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004779#ppat.1004779.g001" target="_blank">Fig. 1A</a>, and total DNA was isolated at the indicated times. The amount of <i>T</i>. <i>gondii</i> DNA was quantified by qPCR of the genomic DNA as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004779#ppat.1004779.g001" target="_blank">Fig. 1</a>.</p

Requirement of IRF3-induced host genes for <i>T</i>. <i>gondii</i> replication.

<p><b>A,</b> Infection and immunoblotting was performed with HDAC6 KO and wild type MEF cells. <b>B</b>, MEF cells (wild type or IFNAR KO) were infected with <i>T</i>. <i>gondii</i> and the indicated proteins were measured by immunoblotting. <b>C</b>, <i>T</i>. <i>gondii</i> growth measured in IRF3 KO MEF cells; where indicated, recombinant murine IFN-β (2,000 U/ml, R&D Systems) was added, and parasite was added 6 h later. Immunoblotting was performed as above. Where shown, parasite growth was also quantified by qPCR of genomic DNA, and ISG56 (Ifit1) mRNA was quantified by qRT-PCR as described before [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004779#ppat.1004779.ref066" target="_blank">66</a>].</p

Essential role of a TLR3/4-independent cGAS-STING signaling axis in PISA and optimal <i>T</i>. <i>gondii</i> replication.

<p>Parasite infection and growth analyses were performed as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004779#ppat.1004779.g001" target="_blank">Fig. 1A</a>. <b>A,</b> MEFs (WT, RIG-I-/-, TLR3-/-) and primary macrophages (MyD88-/-, TLR4-/-) were used for comparing PISA (<i>Tg</i>SAG1 expression or IRF3 phosphorylation) upon <i>T</i>. <i>gondii</i> infection. <b>B/C,</b> STING-/- MEF cells (B) or human 293T cells (C) were transiently transfected with wild type or mutant (mt) FLAG-cGAS and/or HA-STING plasmids, and infected as shown. <b>D,</b> Parasite growth in various conditions used in the other panels, quantified by qPCR; error bars are from three data sets.</p