Oas1b-dependent Immune Transcriptional Profiles of West Nile Virus Infection in the Collaborative Cross

The oligoadenylate-synthetase (Oas) gene locus provides innate immune resistance to virus infection. In mouse models, variation in the Oas1b gene influences host susceptibility to flavivirus infection. However, the impact of Oas variation on overall innate immune programming and global gene expression among tissues and in different genetic backgrounds has not been defined. We examined how Oas1b acts in spleen and brain tissue to limit West Nile virus (WNV) susceptibility and disease across a range of genetic backgrounds. The laboratory founder strains of the mouse Collaborative Cross (CC) (A/J, C57BL/6J, 129S1/SvImJ, NOD/ShiLtJ, and NZO/HlLtJ) all encode a truncated, defective Oas1b, whereas the three wild-derived inbred founder strains (CAST/EiJ, PWK/PhJ, and WSB/EiJ) encode a full-length OAS1B protein. We assessed disease profiles and transcriptional signatures of F1 hybrids derived from these founder strains. F1 hybrids included wild-type Oas1b (F/F), homozygous null Oas1b (N/N), and heterozygous offspring of both parental combinations (F/N and N/F). These mice were challenged with WNV, and brain and spleen samples were harvested for global gene expression analysis. We found that the Oas1b haplotype played a role in WNV susceptibility and disease metrics, but the presence of a functional Oas1b allele in heterozygous offspring did not absolutely predict protection against disease. Our results indicate that Oas1b status as wild-type or truncated, and overall Oas1b gene dosage, link with novel innate immune gene signatures that impact specific biological pathways for the control of flavivirus infection and immunity through both Oas1b-dependent and independent processes

Selective Packaging in Murine Coronavirus Promotes Virulence by Limiting Type I Interferon Responses

Selective packaging is a mechanism used by multiple virus families to specifically incorporate genomic RNA (gRNA) into virions and exclude other types of RNA. Lineage A betacoronaviruses incorporate a 95-bp stem-loop structure, the packaging signal (PS), into the nsp15 locus of ORF1b that is both necessary and sufficient for the packaging of RNAs. However, unlike other viral PSs, where mutations generally resulted in viral replication defects, mutation of the coronavirus (CoV) PS results in large increases in subgenomic RNA packaging with minimal effects on gRNA packaging in vitro and on viral titers. Here, we show that selective packaging is also required for viral evasion of the innate immune response and optimal pathogenicity. We engineered two distinct PS mutants in two different strains of murine hepatitis virus (MHV) that packaged increased levels of subgenomic RNAs, negative-sense genomic RNA, and even cellular RNAs. All PS mutant viruses replicated normally in vitro but caused dramatically reduced lethality and weight loss in vivo. PS mutant virus infection of bone marrow-derived macrophages resulted in increased interferon (IFN) production, indicating that the innate immune system limited the replication and/or pathogenesis of PS mutant viruses in vivo. PS mutant viruses remained attenuated in MAVS−/− and Toll-like receptor 7-knockout (TLR7−/−) mice, two well-known RNA sensors for CoVs, but virulence was restored in interferon alpha/beta receptor-knockout (IFNAR−/−) mice or in MAVS−/− mice treated with IFNAR-blocking antibodies. Together, these data indicate that coronaviruses promote virulence by utilizing selective packaging to avoid innate immune detection.Coronaviruses (CoVs) produce many types of RNA molecules during their replication cycle, including both positive- and negative-sense genomic and subgenomic RNAs. Despite this, coronaviruses selectively package only positive-sense genomic RNA into their virions. Why CoVs selectively package their genomic RNA is not clear, as disruption of the packaging signal in MHV, which leads to loss of selective packaging, does not affect genomic RNA packaging or virus replication in cultured cells. This contrasts with other viruses, where disruption of selective packaging generally leads to altered replication. Here, we demonstrate that in the absence of selective packaging, the virulence of MHV was significantly reduced. Importantly, virulence was restored in the absence of interferon signaling, indicating that selective packaging is a mechanism used by CoVs to escape innate immune detection

Selective Packaging in Murine Coronavirus Promotes Virulence by Limiting Type I Interferon Responses

This work is licensed under a Creative Commons Attribution 4.0 International License.Selective packaging is a mechanism used by multiple virus families to specifically incorporate genomic RNA (gRNA) into virions and exclude other types of RNA. Lineage A betacoronaviruses incorporate a 95-bp stem-loop structure, the packaging signal (PS), into the nsp15 locus of ORF1b that is both necessary and sufficient for the packaging of RNAs. However, unlike other viral PSs, where mutations generally resulted in viral replication defects, mutation of the coronavirus (CoV) PS results in large increases in subgenomic RNA packaging with minimal effects on gRNA packaging in vitro and on viral titers. Here, we show that selective packaging is also required for viral evasion of the innate immune response and optimal pathogenicity. We engineered two distinct PS mutants in two different strains of murine hepatitis virus (MHV) that packaged increased levels of subgenomic RNAs, negative-sense genomic RNA, and even cellular RNAs. All PS mutant viruses replicated normally in vitro but caused dramatically reduced lethality and weight loss in vivo. PS mutant virus infection of bone marrow-derived macrophages resulted in increased interferon (IFN) production, indicating that the innate immune system limited the replication and/or pathogenesis of PS mutant viruses in vivo. PS mutant viruses remained attenuated in MAVS−/− and Toll-like receptor 7-knockout (TLR7−/−) mice, two well-known RNA sensors for CoVs, but virulence was restored in interferon alpha/beta receptor-knockout (IFNAR−/−) mice or in MAVS−/− mice treated with IFNAR-blocking antibodies. Together, these data indicate that coronaviruses promote virulence by utilizing selective packaging to avoid innate immune detection

Transcriptional profiles of WNV neurovirulence in a genetically diverse Collaborative Cross population

West Nile Virus (WNV) is a mosquito-transmitted virus from the Flaviviridae family that causes fever in 1 in 5 infected people. WNV can also become neuro-invasive and cross the blood-brain barrier leading to severe neurological symptoms in a subset of WNV infected individuals [1]. WNV neuro-invasion is believed to be influenced by a number of factors including host genetics. In order to explore these effects and recapitulate the complex immune genetic differences among individuals, we studied gene expression following WNV infection in the Collaborative Cross (CC) model. The CC is a mouse genetics resource composed of >70 independently bred, octo-parental recombinant inbred mouse lines [2]. To identify the individual host gene expression signatures influencing protection or susceptibility to WNV disease and WNV neuroinvasion, we used the nanostring nsolver platform to quantify gene expression in brain tissue isolated from WNV-infected CC mice at days 4, 7 and 12 post-infection [3]. This nanostring technology provided a high throughput, non-amplification based mRNA quantitation method to detect immune genes involved in neuro-invasion. Data was deposited into the Gene Expression Omnibus (GEO) under accession GSE85999

Comparison of WNV disease models by clinical presentation and neuropathology.

<p>(A) Representative RIX lines for 3 WNV disease model categories: 1) No disease 2) Chronic disease and 3) Disease. Representative “No Disease” and “Disease” lines were selected from 40+ lines of each category, identified through the screen. Each RIX line’s CC nomenclature and <i>Oas1b</i> allele status is shown (N = null, F = functional allele). Clinical scores and (B) weight loss and survival curves are shown for the seven CC RIX lines over the time course of WNV infection. “No disease” RIX show little to no weight loss and maintain a “0” clinical score across the time course. “Disease” RIX lines show increased weight loss by d8, as well as increase in clinical scores corresponding to neurological weakness and paralysis (see description in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005996#sec009" target="_blank">methods</a>). (C) Brain WNV loads at d12 for the seven lines. p<0.001 for CC(032x013)F1 compared to all disease lines. (D-E) Neuropathology of WNV infection in RIX lines as indicated. (D) The approximate subregions of the brain that were scored for neuropathology are colorized. These regions were chosen due to the quality and consistency of the sections across the high throughput histologic study. (E) Hematoxylin and eosin stained sections of formalin-fixed paraffin-embedded tissues of WNV-infected lines as indicated with subregion noted. <b><i>Thalamus</i></b>: Arrows indicate neurons. In the Chronic and Diseased tissues, the neurons are surrounded by glia (satellitosis). In the Disease tissue there is moderate hemorrhage (arrow), diffuse gliosis and mild malacia. <b><i>Cortex Meninges</i></b>: In the Chronic and Diseased boxed region there is gliosis in the superficial cortex with mononuclear cell infiltrate in the meninges, which is more pronounced and widespread in the Diseased tissue. There are prominent mononuclear cells with rod-shaped nuclei in the Diseased cortex (arrows) are consistent with activated microglia. <b><i>Cortex</i></b>: Arrowheads indicate blood vessels. In the Chronic and Diseased tissues there is mild to marked accumulation of mononuclear inflammatory cells in the vessel wall and perivascular space. In the Disease tissue there is moderate diffuse gliosis and perivascular cuffing. <b><i>Cortex 40X</i></b>: Arrowheads indicate neuron cell bodies. In the Chronic tissue, there are glial nodules and early degeneration of neurons with mild malacia. In the Disease tissue the neurons are shrunken with dark eosinophilic cytoplasm, and pyknotic nuclei consistent with acute neuronal degeneration to apoptosis (left arrow). Note karyorrhectic debris (small basophilic bodies). All panels 20X except where indicated. Data represent three mice per group for all lines in the screen, and six mice per group for d28 and d60 validation time points.</p

CC(032x013)F1 WNV-infected mice maintain a chronic WNV infection, with virus persisting in the CNS at d60.

<p>Cohorts of mice were infected with 100pfu WNV. (A) Compared to the B6 model of infection, CC(032x013)F1 mice do not return to and exceed starting weight by d25 p.i., but maintain weight loss out to d60. (B) PCR for WNV in brain at indicated time points. Results are from two separate experiments: the discovery screen, which included d7, d12, d21, and d28 time points, and validation mice, which included d28 and d60 endpoints. Results were comparable for overlapping endpoints. Viral RNA was detected in the brains of 66% of d28 and 20% of d60 mice, for both CC(032x013)F1 and B6 mice. (C) Spleen WNV-specific and (D) Brain WNV-specific CD8+ T cells in B6 and CC(032x013)F1 mice at corresponding time points. The discovery screen included three mice per time point (d7, 12, 21, and 28) and the validation experiments included six mice per time point.</p

Activation of Mitogen-Activated Protein Kinases by Lysophosphatidylcholine-Induced Mitochondrial Reactive Oxygen Species Generation in Endothelial Cells

Lysophosphatidylcholine (lysoPC) evokes diverse biological responses in vascular cells including Ca(2+) mobilization, production of reactive oxygen species, and activation of the mitogen-activated protein kinases, but the mechanisms linking these events remain unclear. Here, we provide evidence that the response of mitochondria to the lysoPC-dependent increase in cytosolic Ca(2+) leads to activation of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase through a redox signaling mechanism in human umbilical vein endothelial cells. ERK activation was attenuated by inhibitors of the electron transport chain proton pumps (rotenone and antimycin A) and an uncoupler (carbonyl cyanide p-trifluoromethoxyphenylhydrazone), suggesting that mitochondrial inner membrane potential plays a key role in the signaling pathway. ERK activation was also selectively attenuated by chain-breaking antioxidants and by vitamin E targeted to mitochondria, suggesting that transduction of the mitochondrial hydrogen peroxide signal is mediated by a lipid peroxidation product. Inhibition of ERK activation with MEK inhibitors (PD98059 or U0126) diminished induction of the antioxidant enzyme heme oxygenase-1. Taken together, these data suggest a role for mitochondrially generated reactive oxygen species and Ca(2+) in the redox cell signaling pathways, leading to ERK activation and adaptation of the pathological stress mediated by oxidized lipids such as lysoPC