Activation of Innate Immune-Response Genes in Little Brown Bats (Myotis lucifugus) Infected with the Fungus Pseudogymnoascus destructans

Recently bats have been associated with the emergence of diseases, both as reservoirs for several new viral diseases in humans and other animals and, in the northern Americas, as hosts for a devastating fungal disease that threatens to drive several bat species to regional extinction. However, despite these catastrophic events little Information is available on bat defences or how they interact with their pathogens. Even less is known about the response of bats to infection during torpor or long-term hibernation. Using tissue samples collected at the termination of an experiment to explore the pathogenesis of White Nose Syndrome in Little Brown Bats, we determined if hibernating bats infected with the fungus Pseudogymnoascus destructans could respond to infection by activating genes responsible for innate immune and stress responses. Lesions due to fungal infection and, in some cases, secondary bacterial infections, were restricted to the skin. However, we were unable to obtain sufficient amounts of RNA from these sites. We therefore examined lungs for response at an epithelial surface not linked to the primary site of infection. We found that bats responded to infection with a significant increase in lungs of transcripts for Cathelicidin (an anti-microbial peptide) as well as the immune modulators tumor necrosis factor alpha and interleukins 10 and 23. In conclusion, hibernating bats can respond to experimental P. destructans infection by activating expression of innate immune response genes.Funding for this study was provided by a Fish and Wildlife Service grant to CRKW, TB and VM and by a Natural Sciences and Engineering Research Council (Discovery) grant to VM and a fellowship within the Postdoc Programme of the DAAD, German Academic Exchange Service (to LW). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.https://journals.plos.org/plosone/article?id=10.1371/journal.pone.011228

Immune System Modulation and Viral Persistence in Bats: Understanding Viral Spillover

Bats harbor a myriad of viruses and some of these viruses may have spilled over to other species including humans. Spillover events are rare and several factors must align to create the “perfect storm„ that would ultimately lead to a spillover. One of these factors is the increased shedding of virus by bats. Several studies have indicated that bats have unique defense mechanisms that allow them to be persistently or latently infected with viruses. Factors leading to an increase in the viral load of persistently infected bats would facilitate shedding of virus. This article reviews the unique nature of bat immune defenses that regulate virus replication and the various molecular mechanisms that play a role in altering the balanced bat⁻virus relationship

Interferon Regulatory Factor 3-Mediated Signaling Limits Middle-East Respiratory Syndrome (MERS) Coronavirus Propagation in Cells from an Insectivorous Bat

Insectivorous bats are speculated to be ancestral hosts of Middle-East respiratory syndrome (MERS) coronavirus (CoV). MERS-CoV causes disease in humans with thirty-five percent fatality, and has evolved proteins that counteract human antiviral responses. Since bats experimentally infected with MERS-CoV do not develop signs of disease, we tested the hypothesis that MERS-CoV would replicate less efficiently in bat cells than in human cells because of its inability to subvert antiviral responses in bat cells. We infected human and bat (Eptesicus fuscus) cells with MERS-CoV and observed that the virus grew to higher titers in human cells. MERS-CoV also effectively suppressed the antiviral interferon beta (IFNβ) response in human cells, unlike in bat cells. To determine if IRF3, a critical mediator of the interferon response, also regulated the response in bats, we examined the response of IRF3 to poly(I:C), a synthetic analogue of viral double-stranded RNA. We observed that bat IRF3 responded to poly(I:C) by nuclear translocation and post-translational modifications, hallmarks of IRF3 activation. Suppression of IRF3 by small-interfering RNA (siRNA) demonstrated that IRF3 was critical for poly(I:C) and MERS-CoV induced induction of IFNβ in bat cells. Our study demonstrates that innate antiviral signaling in E. fuscus bat cells is resistant to MERS-CoV-mediated subversion

Luman, the Cellular Counterpart of Herpes Simplex Virus VP16, Is Processed by Regulated Intramembrane Proteolysis

Luman is a human basic leucine zipper transcription factor that, like the herpes simplex virus transcription factor VP16, requires the host cell factor, HCF, for activity. Although both HCF and Luman have been implicated in cell growth, their biological roles have not been clearly defined. Luman conforms to a type II membrane-associated glycoprotein with its carboxyl terminus embedded in cellular membranes and its amino terminus, which contains all its identified functional domains, in the cytoplasm. Here we show that Luman is processed by regulated intramembrane proteolysis (RIP). The site 1 protease (S1P), a Golgi apparatus-resident enzyme responsible for catalyzing the first step in the RIP pathway of the sterol regulatory element binding proteins (SREBPs) and ATF6, may also be involved in the processing of Luman. Thus, processing of Luman was highly stimulated by brefeldin A, a compound that causes the reflux of Golgi apparatus enzymes to the endoplasmic reticulum (ER). In addition, coexpression of Luman with S1P containing a KDEL ER retrieval signal resulted in virtually quantitative cleavage of Luman in the absence of any treatment. Finally, Luman contains a sequence, RQLR, immediately downstream from the transmembrane domain which bears similarity to the consensus S1P cleavage site identified by others. Substitution of arginine residues within this motif abolished S1P cleavage, providing robust evidence that S1P is involved in Luman processing. We observed that following S1P cleavage, the majority of the cleaved Luman was retained in cytoplasmic membranes, indicating that an additional step or enzymes yet to be identified are involved in complete cleavage and release to yield the product which ultimately enters the nuclei of cells

Zhangfei/CREB-ZF – A Potential Regulator of the Unfolded Protein Response

<div><p>Cells respond to perturbations in the microenvironment of the endoplasmic reticulum (ER), and to the overloading of its capacity to process secretory and membrane-associate proteins, by activating the Unfolded Protein Response (UPR). Genes that mediate the UPR are regulated by three basic leucine-zipper (bLZip) motif-containing transcription factors – Xbp1s, ATF4 and ATF6. A failure of the UPR to achieve homeostasis and its continued stimulation leads to apoptosis. Mechanisms must therefore exist to turn off the UPR if it successfully restores normalcy. The bLZip protein Zhangfei/CREBZF/SMILE is known to suppress the ability of several, seemingly structurally unrelated, transcription factors. These targets include Luman/CREB3 and CREBH, ER-resident bLZip proteins known to activate the UPR in some cell types. Here we show that Zhangfei had a suppressive effect on most UPR genes activated by the calcium ionophore thapsigargin. This effect was at least partially due to the interaction of Zhangfei with Xbp1s. The leucine zipper of Zhangfei was required for this interaction, which led to the subsequent proteasomal degradation of Xbp1s. Zhangfei suppressed the ability of Xbp1s to activate transcription from a promoter containing unfolded protein response elements and significantly reduced the ability to Xbp1s to activate the UPR as measured by RNA and protein levels of UPR-related genes. Finally, specific suppression of endogenous Zhangfei in thapsigargin-treated primary rat sensory neurons with siRNA directed to Zhangfei transcripts, led to a significant increase in transcripts and proteins of UPR genes, suggesting a potential role for Zhangfei in modulating the UPR.</p> </div

<i>Zhangfei suppresses</i> the ability of Xbp1s to activate transcription and requires its leucine zipper to do so.

<p>A and B. Vero cells were transfected with a plasmid containing the coding sequence for CAT linked to a promoter with three copies of the unfolded protein response element as well as a plasmid expressing Xbp1s and varying amounts of plasmids expressing either Zhangfei (ZF) or a mutant, ZF(L/A) in which all leucine residues in the LZip domain had been replaced with alanines. All samples also contained, as a control, a plasmid expressing β-galactosidase. The CAT activity in each sample was normalized to this internal control and expressed as a percentage of the activity in samples containing no vector expressing either ZF or ZF (L/A). The total amount of DNA in each transfection was made up to 5μg with “empty” expression vector (pcDNA3). Bars indicate standard deviation from the mean. B. ZF(L/A) does not activate a promoter containing UPRE but enhances the activity of Xbp1s. C. ZF interacts with Xbp1 with its leucine zipper. Cells were transfected with a vector with the coding sequence for CAT linked to three copies of a sequence, UAS, that binds the DNA-binding domain of the yeast protein GAL4. Cells also received plasmids expressing either ZF or ZF(L/A) linked to the Gal4 DNA-binding domain and either an “empty” expression vector or vectors expressing Xbp1s. Bars represent the ratio of the relative CAT activity (normalized to the internal control, β-galactosidase) of samples with Xbp1s to samples with no activator (“empty” vector). D. An immunoblot showing that vectors with cloned ZF or ZF (L/A) express the proteins in a dose-dependent manner. The results represent the averages of three experiments assayed in duplicate. Bars in all figures represent standard deviation from the mean and p values are indicated on the figures.</p

Effect of Zhangfei on HERP and GRP78 transcripts and proteins in cells treated with thapsigargin.

<p>ONS76 and Vero cells were treated for 4 hr with thapsigargin either after infection, or mock infection, with an adenovirus vector expressing ZF (lanes 1,2, 5 and 6) or before infection (lanes 3,4, 7 and 8). Cells were harvested 24 hr after infection. Proteins in samples were separated by SDS-PAGE and probed with antibodies against HERP, GRP78 or Zhangfei (ZF) and GAPDH (A) and RNA was extracted from parallel duplicate cultures and assayed for HERP and GRP78 transcripts by qRT-PCR (B). C. ONS-76 and Vero cells were transfected with plasmids expressing FLAG epitope linked to the coding sequences of Zhangfei. Cells were fixed and incubated with a mixture of mouse monoclonal antibody against FLAG and rabbit antibody against GRP78 followed by Alexa 488-linked anti-mouse and Alexa 546-linked anti-rabbit antibodies. Cells were also stained with Hoechst to stain nuclei. Arrows identify Zhangfei (ZF) expressing cells.</p

Endogenous <i>Zhangfei suppresses</i> the activation of UPR genes in rat peripheral neurons.

<p>Primary rat dorsal root neurons were transfected with plasmids expressing either control siRNA or siRNA against Zhangfei (siRNA-ZF). The next day cells were treated with DMSO or thapsigargin and 4hr later RNA was harvested for qRT-PCR analysis using primers designed to amplify Xbp1s, CHOP, GRP78, Xbp1us (A) or Zhangfei (B). A. Fold increases in RNA between DMSO and thapsigargin treated samples comparing siRNA-ZF (black bars) and siRNA control (white bars) expressing cells. B. Effect of si-ZF on transcripts levels of endogenous Zhangfei. Results are expressed as 1/Ct. Columns represent averages of triplicate samples with bars as standard deviation from the mean. C. Suppression of endogenous Zhangfei increases Xbp1s, Grp78 and HERP proteins. Lysates of neurons transfected with either siRNA-ZF or siRNA-control were analyzed by immunoblotting using antibodies against UPR-related proteins. Bands on immunoblots of the left were scanned and band densities relative to the internal standard GAPDH are on the graph on the right. D. Transfection efficiency test of siRNA. Primary rat dorsal root neurons were transfected at a final concentration of 10 nM with the TYE 563 DS Transfection Control duplex, and were imaged 24 hours post transfection (Red: marked siRNA; blue: nuclear).</p

Immunofluorescent images showing the absence of Xbp1 in cells expressing Zhangfei but not <i>Zhangfei with</i> a mutated leucine zipper.

<p>Vero cells were transfected with plasmids expressing either Xbp1 alone or in combination with either Zhangfei or ZF(L/A). Cells were then fixed and incubated with a mixture of rabbit anti-Zhangfei and mouse anti-Xbp1 antibodies. Antibodies were visualized by staining with Alexa546 (red) anti mouse and Alexa488 (green) anti-rabbit antibodies.</p

<i>Zhangfei directs</i> Xbp1 for proteasomal degradation and requires its leucine zipper to do so.

<p>A. Vero cells were transfected as indicated with plasmids expressing Xbp1s and an empty vector or plasmid expressing Zhangfei. Cells either received 5μM MG132 or an equivalent volume of carrier, DMSO. Cells were harvested 24 hr later and proteins detected by immunoblotting. B. Vero cells were transfected with a plasmid expressing Xbp1s alone or increasing amounts of a plasmid expressing Zhangfei or a mutant in which all leucine residues in the zipper had been changed to alanine - pcZF(L/A). Cells were harvested 24 hr later and proteins detected by immunoblotting. The density of each band on the immunoblot was estimated by densitometry and normalized to the density of the GAPDH band in the sample. Average values and standard deviation from three experiments are shown in (C). </p