Aging is amongst the strongest risk factors for neurodegenerative disease and elevated Type I interferon (IFN) signaling has been associated with both normal aging and central nervous system (CNS) diseases. Type I IFN is normally produced by nucleated cells in response to the detection of viral pathogen associated molecular patterns (PAMPs) by pathogen recognition receptors (PRRs). More recently it has been appreciated that Type I IFNs are also produced in response to endogenous stimuli, in the absence of viral pathogens. While Type I IFN signaling has been shown to be elevated in human and murine brains during normal aging, the underlying cause was unknown. Here we demonstrate by flow cytometry that aging results in increased size and numbers of mitochondria in the murine brain. Despite identifying increased mitochondrial number and mitochondrial DNA content, we found no change to mitochondrially-encoded transcripts, suggesting either deficits in mitophagy or augmented biogenesis due to insufficient oxidative phosphorylation. Interestingly, mitochondrial numbers correlated with elevated Type I IFN signaling in aging, linking mitochondria to the age-dependent innate immune response in the CNS. Using genetically engineered mice, we excluded roles for two critical innate immune pathways, STING and IRAK4, in the age-dependent increase in Type I IFN signaling in the brain. Notably, we subsequently identified a mitochondrially restricted innate immune protein, mitochondrial antiviral signaling (MAVS) as an essential molecular mediator of the age-dependent Type I IFN response; MAVS deficiency in aged mice restored Type I IFN signaling in the CNS to the levels observed in adult wildtype mice. Further, using intracerebroventricular (icv) administration of antisense oligonucleotides (ASO) as an orthogonal approach, we reduced MAVS transcript and protein expression within the CNS and thereby reduced Type I IFN signaling. Our data demonstrate a specific and selective role of MAVS expression in the CNS in Type I IFN signaling in aging. To investigate the relationship between mitochondrial aging and MAVS activation, we isolated cytoplasmic and mitochondrial RNA from young and aged animals as MAVS is most studied for its response to RNA ligands. Upon transfection into reporter cells, we found that mitochondrial RNA, but not cytoplasmic RNA, from both young and aged mice was sufficient to induce Type I IFN reporter activity in a MAVS-dependent manner. Furthermore, we attempted to mimic the increase of mitochondria observed in the aging CNS by transferring mitochondria from young and aged animals to recipient cells. Mitochondrial transfer also induced MAVS-dependent Type I IFN signaling in wildtype, but not MAVS null, mouse embryonic fibroblasts (MEFs). Collectively, our findings suggest that the accumulation of mitochondria in aging serves as a robust source of MAVS pathway ligands and implicate a novel link between mitochondrial aging and MAVS-mediated innate immune signaling in the CNS.

Mechanisms involved in the establishment of HCMV latency are poorly understood, however, work in our laboratory has demonstrated the ULb' encoded protein, pUL138, as the first viral determinant to function in the establishment of HCMV latency in CD34+ hematopoietic progenitor cells (HPCs). This work characterizes the transcripts that encode pUL138, identifies three novel ULb' proteins (pUL133, pUL135, and pUL136) and represents the first demonstration of an internal ribosome entry site (IRES) mediated expression of pUL138. pUL138 is encoded on three polycistronic transcripts of 3.6-, 2.7- and 1.4-kb in length. pUL133, pUL135 and truncated pUL136, are expressed on the 3.6-, 2.7- and 1.4-kb transcripts, respectively, in addition to pUL138. We demonstrate that pUL138 expression is inducible from the IRES on the 3.6- and 2.7-kb transcripts under conditions of cellular stress, whereas pUL138 expression from the 1.4-kb transcript is inhibited under these same conditions. Differential utilization of the UL138 transcripts and their respective encoded proteins may regulate the outcome of viral infection in a cell type or cell context dependent manner. The interaction of these proteins during HCMV latency is the focus of ongoing research. In addition, this work represents preliminary data regarding the type I interferon (IFN) response during HCMV during productive infection in MRC5 fibroblasts and during the establishment of HCMV latency in CD34+ HPCs.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Raymond R. R. Rowland / Interferons (IFNs) comprise a group of antiviral cytokines; however, their expression at the porcine maternal-fetal interface and in fetal tissues has not previously been investigated. The purpose of this study was to analyze the expression of type I IFNs and their receptors in maternal and fetal tissues from sows infected with PRRSV. The approach was to use real-time RT-PCR to identify the expression of different subtypes of type I IFN genes. The results show that the constitutive gene expression of some subtypes including IFN-[alpha] and IFN-[alpha][omega] were detected in fetal lymphoid nodes (IFN-[alpha][omega]), placenta (several IFN-[alpha] subtypes and IFN-[omega]5) and particularly, thymus (multiple IFN-[alpha], IFN-[delta] and IFN-[omega]5). The results demonstrate that porcine type I IFNs are differentially expressed at the maternal-fetal interface and in fetal tissues in response to PRRSV infection.

Aichi virus (AiV) is a small, nonenveloped RNA virus categorized to Picornaviridae. AiV infection causes mild gastroenteritis, but in neonates, AiV usually causes the risk of certain enterovirus-related clinical syndromes, such as fever, nausea, vomiting and diarrhea. The first case of AiV infection in Taiwan was diagnosed from a young patient with diarrhea in Kaohsiung Veterans General Hospital, and the AiV was successfully isolated. Antiviral innate immune system of our body plays the major role to defense virus invasion. Because AiV is an emerging picornavirus, the knowledge about its pathogenesis and the interaction with host innate immunity were totally absent. This study aims to investigate the mechanism of AiV regulating innate immune response. We first demonstrated that AiV is a type I IFN sensitive virus. IFN-£\2 treatment potently inhibited AiV replication. Real-time quantitative PCR data indicated that AiV induced only small amout of type I IFN gene expression, and the similar result was observed using IFN-£] luciferase reporter assay. In addition, the AiV triggered IFN-£] luciferase activity was progressively decreased in the late phase of infection. Immunoblotting assay showed that AiV evidently activated IRF-3 and IRF-7, the transcription factors of type I IFN induction. However, the retinoic acid inducible gene I (RIG-I) protein was cleavaged and its activity was downregulated by AiV. This data suggested that AiV triggered low level of type I IFN response may due to the negative feedback regulation of RIG-I activity. This immune evasion might be important for AiV replication in cells. Our study first reveals the status of innate immune response of AiV infection, and provides the basic virological theory for the development of anti-AiV drugs and vaccines in the future.

Recognition of non-self, pathogen-associated molecular patterns is a central component of host immune response to pathogens like viruses. Intracellular detection of viral nucleic acids leads to the production of type I interferons (IFN-I) and subsequent establishment of an antiviral state in infected and neighboring cells. Viruses have evolved multiple mechanisms to counteract IFN-I responses in infected cells, however, viral nucleic acids released from dying cells can stimulate IFN-I production in surrounding or distal uninfected cells. This thesis examines the mechanisms by which cells recognize extracellular viral nucleic acids and the subsequent downstream antiviral signaling. Class A scavenger receptors (SR-As) internalize extracellular viral double-stranded RNA (dsRNA) to mediate IFN-I responses, but little is known about extracellular viral DNA. We observed that extracellular DNA is recognized and internalized by SR-As in a manner like extracellular dsRNA. Furthermore, we established that SR-A1 is sufficient in mediating extracellular dsRNA-induced cellular responses and other nucleic acid receptors like SR-J1 and DEC-205 are dispensable. Finally, a direct interaction of RNA and DNA species was demonstrated with the coiled-coil collagenous domain of SR-A1, but not the scavenger receptor cysteine rich domain of SR-A6.We elaborated the role of SR-A1 by identifying the cellular processes activated through SR-A1 following uptake of extracellular dsRNA. Cytosolic sensors are essential in mediating an antiviral response to the endocytosed dsRNA, but the mechanism of endoplasmic release and cytoplasmic entry of dsRNA remains an enigma. We demonstrated that the lack of a dsRNA-channel, SIDT2, impaired the ability of the cells to mediate an antiviral response to extracellular dsRNA. Understanding host responses to extracellular viral nucleic acids will enable the development of novel vaccines and antiviral therapeutics against RNA and DNA viruses that efficiently counteract these responses in infected cells. / Thesis / Doctor of Philosophy (PhD) / Viral infections remain a threat to global health as new diseases continue to emerge. To develop effective vaccines and antivirals to combat viruses and alleviate human disease require a deeper understanding of virus-host interactions. Host cells identify virus-associated molecules to detect viruses and eliminate them whereas, viruses employ tactics to prevent the activation of the immune system. However, virus-induced cell lysis releases viral molecules that can stimulate immune responses in neighbouring uninfected cells. This thesis examines the mechanism by which cells respond to extracellular viral nucleic acids. We showed that a protein present at the cell surface called ‘class A scavenger receptor 1’ is sufficient to internalize extracellular viral nucleic acids, leading to immune responses. The response is impaired when a channel protein, SIDT2, is absent in the cells. Further work is necessary to understand how this knowledge can be harnessed to develop vaccines and antiviral therapeutics.

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第24329号 / 生博第488号 / 新制||生||65(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 野田 岳志, 教授 杉田 昌彦, 教授 垣塚 彰 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Differential Display technique was used for gene profiling in trnasformed human umbilical vein endothelial cell line (ECV 304) and primary human umbilical vein endothelial cells (HUVECs) to study the cellular response to viral infection. After screening the mRNA from uninfected and infected HUVECs and ECV 304 cells with 16 different random primers we identified 8 gene targets. These genes included the human inhibitor of apoptosis-1 (h-IAP1), 2'-5' oligoadenylate synthetase (2'-5' OAS), 2'-5' oligoadenylate synthetase-like (2'-5' OAS-like), Galectin-9 (Gal-9), MxA, Mx1, Regulator of g-protein signaling (RGS2) and endothelial and smooth muscle cell-derived neuropilin-like protein (ESDN). We found that HUVECs were a better model to study gene expression dureing dengue 2 virus infection but not the transformed cell line, ECV 304. Of the 41 primer combinations utilized in ECV 304 cells detected only one upregulated gene, h-IAP1 and 8 out of the 16 primer combinations tried for HUVECs. We hypothesize the activation of two novel signaling pathways (Tumor necrosis factor- alpha (TNF-alpha), Interleukin1-beta (IL1-beta) in endothelial cells during D2V infection. ALso, our data detected genes that are activated in the Type-I IFN (IFN alpha/beta) signaling pathway during dengue 2 virus infection in HUVEC.

The adenosine deaminase acting on RNA (ADAR) enzymes catalyse the hydrolytic deamination of adenosine (A) to inosine (I) in double stranded (ds) RNA. Mutations in ADAR1 underlie the autoimmune disorder Aicardi Goutiѐres syndrome (AGS). Patients with AGS display heightened levels of type I interferon (IFN) and IFN stimulated genes (ISGs). The first aim of my thesis was to determine whether the mutations found in the human ADAR1 gene affected RNA editing. I found that the ADAR1 mutants identified in the AGS patients have reduced editing activity. Interestingly, the mutations have a greater effect on the IFN-inducible cytoplasmic isoform, ADAR1p150 than on the constitutive ADAR1p110 isoform. These results imply that A-to-I editing plays a role in regulating the type I IFN response. The Adar1 null mouse dies by E12.5 with a type I IFN signature similar to that observed in the AGS patients. The second aim of my thesis was to characterize the immune signalling pathway aberrantly activated in the absence of Adar1. A colleague in our research group rescued the Adar1 null mouse to birth by blocking the cellular response to cytoplasmic dsRNA by generating a double mutant with the mitochondrial antiviral signalling adaptor, Mavs. In the Adar1-/-; Mavs-/- mutant I found that the aberrant immune response is rescued at E11.5. This indicates that MAVS is the downstream adaptor in the aberrant immune response that underlies the embryonic lethality in the Adar1-/- mouse. The third aim of my thesis was to determine if the lack of inosine modification within cellular RNA was triggering the aberrant immune response in the Adar1-/- mouse. To study this, Adar1-/-; p53 -/- mouse embryonic fibroblasts (MEFs) were generated. By reintroducing various ADAR isoforms into the Adar1-/-; p53 -/- MEFs I found that to rescue the aberrant immune response requires both catalytic activity and the location of an ADAR protein within the cytoplasm. Moreover, I demonstrated that transfecting inosine-containing dsRNA oligonucleotides into Adar1-/-; p53 -/- MEFs suppresses the aberrant immune response. Overall my results suggest that A-to-I editing by ADAR1 is an essential RNA modification that is required by the cell to distinguish between ‘self’ and ‘non-self’ RNA. Editing of cellular RNAs prevents an autoimmune response whereas editing of viral RNA may act to suppress a heightened antiviral immune response and prevent long-term damage to the cell.

B virus (Cercopithecine herpesvirus 1), which causes an often fatal zoonotic infection in humans, shares extensive homology with human herpes simplex virus type 1 (HSV-1). The ƒ×134.5 gene of HSV-1 plays a major role in counteracting dsRNA-dependent protein kinase (PKR) activity. HSV-1 Us11 protein, if expressed early as a result of mutation, binds to PKR and prevents PKR activation. The results of experiments in this dissertation revealed that although B virus lacks a ƒ×134.5 gene homolog, it is able to inhibit PKR activation, and subsequently, eIF2ƒÑ phosphorylation. The initial hypothesis was that B virus Us11 protein substitutes for the function of ƒ×134.5 gene homolog by blocking cellular PKR activation. Using western blot analysis, Us11 protein (20 kDa) of B virus was observed early following infection (3 h post infection). Expression of B virus Us11 protein was not blocked by phosphonoacetic acid (PAA), an inhibitor of DNA replication, confirming Us11 is not a ¡§true late¡¨ gene of B virus as it is in HSV-1. Analysis of these results suggested that B virus Us11 protein compensates for the lack of the ƒ×134.5 gene homolog and prevents PKR activation. Next, the results demonstrated that B virus Us11 recombinant protein prevented PKR activation by dsRNA in vitro. A B virus Us11 protein stable expression cell line (U373-BVUs11) was established to investigate whether Us11 protein inhibited PKR activation in vivo. Experiments revealed that B virus Us11 protein stably expressed in U373 cells prevented PKR activation and subsequent eIF2ƒÑ phosphorylation induced by the infection of these cells with ƒ´ƒ×134.5 of HSV-1. As the consequence of preventing PKR activation and subsequent eIF2ƒÑ phosphorylation, B virus Us11 protein complemented ƒ´ƒ×134.5 HSV-1 in U373 cells as evidenced by restoration of virus protein synthesis and replication in U373 cells. Furthermore, pull-down assays showed that B virus Us11 protein binds to PKR. In addition, the results demonstrated that B virus Us11 protein stably expressed in U373 cells counteracted the inhibiting effect of IFN-ƒÑ on HSV-1 replication by preventing PKR activation. These data suggested that B virus and HSV-1, two closely related viruses, use different mechanisms to counteract PKR activity.

Antigen-specific CD8 T cells provide an important protective role in response to infection by viruses, intracellular bacteria, and parasites. Pathogen-specific CD8 T cells render this protection by undergoing robust expansion in numbers while gaining the ability to produce cytokines and cytolytic machinery. Following expansion and effector differentiation, pathogen-specific CD8 T cells will contract in number while further differentiating into a highly functional population of memory CD8 T cells. These antigen-experienced cells persist in secondary lymphoid organs and the periphery in order to rapidly respond to repeated infection. Creating optimal CD8 T cell responses to infection can be critical for raising sufficient armament to provide protection against invading intracellular pathogens. Although CD8 T cells have protective value, many vaccine strategies tend to focus on creating productive B cell antibody responses to promote immunological protection. Even though antibody responses can be highly protective, coupling optimal CD8 T cell responses with B cell responses could provide higher orders of protection than either one on their own. Therefore, a deeper understanding of the pathways that ultimately guide the magnitude of CD8 T cell responses is required to achieve this potential therapeutic benefit. My studies evaluate the role of receptor signaling events in guiding the expansion of activated CD8 T cells during primary and secondary responses. Specifically, the first portion of my studies dissect the mechanism by which direct IL-12 and Type I IFN stimulation can substantially bolster primary CD8 T cell responses in vivo. Within this context, I demonstrate that direct IL-12 and Type I IFN signaling increases CD8 T cell accumulation during primary expansion by prolonging division without altering survival. IL-12/Type I IFN signaling promoted prolonged division of activated CD8 T cells by maintaining high-affinity IL-2 receptor subunit (CD25) expression and IL-2 signaling. The other portion of my work was dedicated to understanding the expression and role of the inhibitory FcgR (FcgRIIB) during primary and secondary CD8 T cell responses. FcgRIIB expression could be detected as early as the peak of the CD8 T cell response and marked activated CD8 T cells that were highly sensitive to antigen stimulation. Although FcgRIIB did not appear to play a substantial role in regulating the magnitude of primary CD8 T cell responses, it played an important role in inhibiting the expansion and cytotoxicity of memory CD8 T cells during homologous challenge. Collectively, these data highlight potential avenues that could be exploited by future therapies that aim to achieve appropriately sized CD8 T cell responses.