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1.  Anomaly Detection in Host Signaling Pathways for the Early Prognosis of Acute Infection 
PLoS ONE  2016;11(8):e0160919.
Clinical diagnosis of acute infectious diseases during the early stages of infection is critical to administering the appropriate treatment to improve the disease outcome. We present a data driven analysis of the human cellular response to respiratory viruses including influenza, respiratory syncytia virus, and human rhinovirus, and compared this with the response to the bacterial endotoxin, Lipopolysaccharides (LPS). Using an anomaly detection framework we identified pathways that clearly distinguish between asymptomatic and symptomatic patients infected with the four different respiratory viruses and that accurately diagnosed patients exposed to a bacterial infection. Connectivity pathway analysis comparing the viral and bacterial diagnostic signatures identified host cellular pathways that were unique to patients exposed to LPS endotoxin indicating this type of analysis could be used to identify host biomarkers that can differentiate clinical etiologies of acute infection. We applied the Multivariate State Estimation Technique (MSET) on two human influenza (H1N1 and H3N2) gene expression data sets to define host networks perturbed in the asymptomatic phase of infection. Our analysis identified pathways in the respiratory virus diagnostic signature as prognostic biomarkers that triggered prior to clinical presentation of acute symptoms. These early warning pathways correctly predicted that almost half of the subjects would become symptomatic in less than forty hours post-infection and that three of the 18 subjects would become symptomatic after only 8 hours. These results provide a proof-of-concept for utility of anomaly detection algorithms to classify host pathway signatures that can identify presymptomatic signatures of acute diseases and differentiate between etiologies of infection. On a global scale, acute respiratory infections cause a significant proportion of human co-morbidities and account for 4.25 million deaths annually. The development of clinical diagnostic tools to distinguish between acute viral and bacterial respiratory infections is critical to improve patient care and limit the overuse of antibiotics in the medical community. The identification of prognostic respiratory virus biomarkers provides an early warning system that is capable of predicting which subjects will become symptomatic to expand our medical diagnostic capabilities and treatment options for acute infectious diseases. The host response to acute infection may be viewed as a deterministic signaling network responsible for maintaining the health of the host organism. We identify pathway signatures that reflect the very earliest perturbations in the host response to acute infection. These pathways provide a monitor the health state of the host using anomaly detection to quantify and predict health outcomes to pathogens.
doi:10.1371/journal.pone.0160919
PMCID: PMC4988711  PMID: 27532264
2.  The 1918 Influenza Virus PB2 Protein Enhances Virulence through the Disruption of Inflammatory and Wnt-Mediated Signaling in Mice 
Journal of Virology  2016;90(5):2240-2253.
ABSTRACT
The 1918-1919 influenza pandemic remains the single greatest infectious disease outbreak in the past century. Mouse and nonhuman primate infection models have shown that the 1918 virus induces overly aggressive innate and proinflammatory responses. To understand the response to viral infection and the role of individual 1918 genes on the host response to the 1918 virus, we examined reassortant avian viruses nearly identical to the pandemic 1918 virus (1918-like avian virus) carrying either the 1918 hemagglutinin (HA) or PB2 gene. In mice, both genes enhanced 1918-like avian virus replication, but only the mammalian host adaptation of the 1918-like avian virus through reassortment of the 1918 PB2 led to increased lethality. Through the combination of viral genetics and host transcriptional profiling, we provide a multidimensional view of the molecular mechanisms by which the 1918 PB2 gene drives viral pathogenicity. We demonstrate that 1918 PB2 enhances immune and inflammatory responses concomitant with increased cellular infiltration in the lung. We also show for the first time, that 1918 PB2 expression results in the repression of both canonical and noncanonical Wnt signaling pathways, which are crucial for inflammation-mediated lung regeneration and repair. Finally, we utilize regulatory enrichment and network analysis to define the molecular regulators of inflammation, epithelial regeneration, and lung immunopathology that are dysregulated during influenza virus infection. Taken together, our data suggest that while both HA and PB2 are important for viral replication, only 1918 PB2 exacerbates lung damage in mice infected with a reassortant 1918-like avian virus.
IMPORTANCE As viral pathogenesis is determined in part by the host response, understanding the key host molecular driver(s) of virus-mediated disease, in relation to individual viral genes, is a promising approach to host-oriented drug efforts in preventing disease. Previous studies have demonstrated the importance of host adaptive genes, HA and PB2, in mediating disease although the mechanisms by which they do so are still poorly understood. Here, we combine viral genetics and host transcriptional profiling to show that although both 1918 HA and 1918 PB2 are important mediators of efficient viral replication, only 1918 PB2 impacts the pathogenicity of an avian influenza virus sharing high homology to the 1918 pandemic influenza virus. We demonstrate that 1918 PB2 enhances deleterious inflammatory responses and the inhibition of regeneration and repair functions coordinated by Wnt signaling in the lungs of infected mice, thereby promoting virus-associated disease.
doi:10.1128/JVI.02974-15
PMCID: PMC4810726  PMID: 26656717
3.  Antiviral activity of human oligoadenylate synthetases-like (OASL) is mediated by enhancing retinoic acid-inducible gene I (RIG-I) signaling 
Immunity  2014;40(6):936-948.
SUMMARY
Virus infection is sensed in the cytoplasm by retinoic acid-inducible gene I (RIG-I, also known as DDX58), which requires RNA and polyubiquitin binding to induce type I interferon (IFN), and activate cellular innate immunity. We show that the human IFN-inducible oligoadenylate synthetases-like (OASL) protein had antiviral activity and mediated RIG-I activation by mimicking polyubiquitin. Loss of OASL expression reduced RIG-I signaling and enhanced virus replication in human cells. Conversely, OASL expression suppressed replication of a number of viruses in a RIG-I-dependent manner and enhanced RIG-I-mediated IFN induction. OASL interacted and colocalized with RIG-I, and through its C-terminal ubiquitin-like domain specifically enhanced RIG-I signaling. Bone marrow derived macrophages from mice deficient for Oasl2 showed that among the two mouse orthologs of human OASL; Oasl2 is functionally similar to human OASL. Our findings show a mechanism by which human OASL contributes to host antiviral responses by enhancing RIG-I activation.
doi:10.1016/j.immuni.2014.05.007
PMCID: PMC4101812  PMID: 24931123
4.  SV40 Large T antigen induces ISGs through ATR Kinase 
Polyomaviruses encode a Large T antigen (LT), a multifunctional protein essential for the regulation of both viral and host cell gene expression and productive viral infection. Previously, we have shown that stable expression of LT protein results in upregulation of genes involved in the interferon induction and signaling pathway. In this study, we focus on the cellular signaling mechanism that leads to the induction of interferon (IFN) responses by LT. Our results show that ectopic expression of Simian virus 40 (SV40) LT results in the induction of interferon-stimulated genes (ISGs) in human fibroblasts and confers an antiviral state. We describe a LT-initiated DNA-damage response (DDR) that activates IRF1 causing IFNβ production and consequent ISG expression in human cells. This IFNβ and ISG induction is dependent on ATM and Rad3 related (ATR) kinase, but independent of ataxia-telangiectasia mutated (ATM). ATR kinase inhibition using a selective kinase inhibitor (ETP-46464) caused a decrease in IRF1 stabilization and ISG expression. Furthermore, expression of a mutant LT that does not induce DDR, does not induce IFNβ and ISGs. These results show that in the absence of viral infection, LT-initiated activation of ATR-dependent DDR is sufficient for the induction of an IFNβ-mediated innate immune response in human cells. Thus, we have uncovered a novel and critical role for ATR as a mediator of antiviral responses utilizing LT.
doi:10.4049/jimmunol.1303470
PMCID: PMC4078001  PMID: 24799566
Large T antigen; interferon; ISG; IRF1; ATR
5.  Downregulation of IRF4 induces lytic reactivation of KSHV in primary effusion lymphoma cells✩ 
Virology  2014;0:4-10.
Primary effusion lymphoma (PEL), associated with the latent infection by KSHV, constitutively expresses interferon-regulatory factor 4 (IRF4). We recently showed that IRF4 differentially regulates expression of cellular interferon-stimulated genes (ISGs) and viral genes (Forero et al., 2013). Here, using inducible IRF4 knockdown, we demonstrate that IRF4 silencing results in enhanced transcription of KSHV replication transactivator RTA. As a result viral transcription is increased leading to virus reactivation. Taken together, our results show that IRF4 helps maintain the balance between latency and KSHV reactivation in PEL cells.
doi:10.1016/j.virol.2014.04.020
PMCID: PMC4058074  PMID: 24928034
Primary effusion lymphoma; Kaposi's sarcoma-associated herpesvirus; Interferon regulatory factor 4
6.  Role of IRF4 in IFN-stimulated gene induction and maintenance of KSHV latency in primary effusion lymphoma cells 
Interferon regulatory factor (IRF) 4 is a hematopoietic cell-specific transcription factor that regulates the maturation and differentiation of immune cells. Using an inducible expression system, we found that IRF4 directly induced a specific subset of interferon-stimulated genes (ISG) in a type I interferon (IFN)-independent manner in both epithelial and B cell lines. Moreover, Kaposi sarcoma-associated herpesvirus (KSHV)-encoded viral FLICE inhibitory protein (vFLIP) enhances IRF4-mediated gene induction. Co-expression of IRF4 with vFLIP significantly increased ISG60 (IFIT3) and Cig5 (RSAD2) transcription that was dependent on the ability of vFLIP to activate NF-κB. A vFLIP mutant (A57L) – defective in NF-κB activation, failed to enhance IRF4-mediated ISG induction. Thus, we provide a physiologically relevant mechanism where viral protein mediated NF-κB activation modulates specific ISG induction by IRF4. In contrast, IRF4 also acted as a negative regulator of KSHV replication and transcription activator (RTA) expression after induction of KSHV lytic reactivation in KSHV positive primary effusion lymphoma (PEL) cells. Taken together, these results suggest a dual role for IRF4 in regulating ISG induction and KSHV lytic reactivation in PEL cells.
doi:10.4049/jimmunol.1202514
PMCID: PMC3740746  PMID: 23804715
7.  Defective NF-κB signaling in metastatic head and neck cancer cells leads to enhanced apoptosis by dsRNA 
Cancer research  2011;72(1):45-55.
Ligands to several Toll-like receptors (TLR) which mediate innate immune responses and chronic inflammation have been used as adjuvants to immunotherapy to enhance their anti-tumor activity. In particular, double-stranded RNAs that are cognate ligands of TLR3 have been used to trigger pro-apoptotic activity in cancer cells. However, a mechanistic understanding of TLR3-mediated apoptosis and its potential involvement in controlling tumor metastasis has been lacking. In this study we used paired cell lines and fresh tumor specimens, derived from autologous primary and metastatic head and neck squamous cell carcinoma, to investigate the role of TLR3 signaling in metastatic progression. Compared to primary tumor cells, metastatic tumor cells were highly sensitive to TLR3-mediated apoptosis after dsRNA treatment. Enhanced apoptosis in metastatic cells was dependent on dsRNA and TLR3 and also the TLR3 effector signaling protein TRIF. Downstream responses requiring NF-κB were critical for apoptosis in metastatic cells, the defects in which could be resuscitated by alternative pathways of NF-κB activation. By elucidating how TLR3 ligands trigger apoptosis in metastatic cells our findings suggest insights into how to improve their clinical use.
doi:10.1158/0008-5472.CAN-11-1484
PMCID: PMC3251732  PMID: 22058147
8.  Inhibition of Indoleamine-2,3-dioxygenase (IDO) in Glioblastoma Cells by Oncolytic Herpes Simplex Virus 
Advances in Virology  2012;2012:815465.
Successful oncolytic virus treatment of malignant glioblastoma multiforme depends on widespread tumor-specific lytic virus replication and escape from mitigating innate immune responses to infection. Here we characterize a new HSV vector, JD0G, that is deleted for ICP0 and the joint sequences separating the unique long and short elements of the viral genome. We observed that JD0G replication was enhanced in certain glioblastoma cell lines compared to HEL cells, suggesting that a vector backbone deleted for ICP0 may be useful for treatment of glioblastoma. The innate immune response to virus infection can potentially impede oncolytic vector replication in human tumors. Indoleamine-2,3-dioxygenase (IDO) is expressed in response to interferon γ (IFNγ) and has been linked to both antiviral functions and to the immune escape of tumor cells. We observed that IFNγ treatment of human glioblastoma cells induced the expression of IDO and that this expression was quelled by infection with both wild-type and JD0G viruses. The role of IDO in inhibiting virus replication and the connection of this protein to the escape of tumor cells from immune surveillance suggest that IDO downregulation by HSV infection may enhance the oncolytic activity of vectors such as JD0G.
doi:10.1155/2012/815465
PMCID: PMC3424635  PMID: 22924042

Results 1-8 (8)