Young Ran Nam

Lysine 63-Linked TANK-Binding Kinase 1 Ubiquitination by Mindbomb E3 Ubiquitin Protein Ligase 2 Is Mediated by the Mitochondrial Antiviral Signaling Protein

ABSTRACT Beta interferon (IFN-β) is involved in a wide range of cellular functions, and its secretion must be tightly controlled to inhibit viral spreading while minimizing cellular damage. Intracellular viral replication triggers cellular signaling cascades leading to the activation of the transcription factors NF-κB and interferon regulatory factor 3 (IRF3) and IRF7 (IRF3/7), which synergistically bind to the IFN-β gene promoter to induce its expression. The mitochondrial antiviral signaling protein (MAVS) is a governing adaptor protein that mediates signaling communications between virus-sensing proteins and transcription factors. The activity of MAVS in the regulation of IFN-β secretion is affected by many cellular factors. However, the mechanism of MAVS-mediated IRF3/7 activation is not completely understood. Here, we identified a highly conserved DLAIS motif at amino acid positions 438 to 442 of MAVS that is indispensable for IRF3/7 activation. Specifically, the L439S and A440R mutations suppress IRF3/7 activation. Pulldown experiments using wild-type and mutant MAVS showed that mindbomb E3 ubiquitin protein ligase 2 (MIB2) binds to the DLAIS motif. Furthermore, the DLAIS motif was found to be critical for MIB2 binding, the ligation of K63-linked ubiquitin to TANK-binding kinase 1, and phosphorylation-mediated IRF3/7 activation. Our results suggest that MIB2 plays a putative role in MAVS-mediated interferon signaling. IMPORTANCE Mitochondrial antiviral signaling protein (MAVS) mediates signaling from virus-sensing proteins to transcription factors for the induction of beta interferon. However, the mechanism underlying activation of MAVS-mediated interferon regulatory factors 3 and 7 (IRF3/7) is not completely understood. We found a highly conserved DLAIS motif in MAVS that is indispensable for IRF3/7 activation through TANK-binding kinase 1 (TBK1) and identified it as the binding site for mindbomb E3 ubiquitin protein ligase 2 (MIB2). The mutations that targeted the DLAIS motif abolished MIB2 binding, attenuated the K63-linked ubiquitination of TBK1, and decreased the phosphorylation-mediated activation of IRF3/7.

Antiviral potency of a siRNA targeting a conserved region of coxsackievirus A24

Coxsackievirus A24 (CVA24) is responsible for acute hemorrhagic conjunctivitis, a highly contagious eye disease for which no prevention or treatment is currently available. We thus assessed the antiviral potential of a small interfering RNA (siRNA) targeting CVA24. HeLa cells with or without four different siRNAs complementary to 2C or 3D genome region, were challenged with various CVA24s. Among several siRNAs, a siRNA targeting the highly conserved genome region called the cis-acting replication element (CVA24-CRE), was the only siRNA that decreased virus replication and subsequent cytotoxicity by both CVA24 variant and clinical isolates. Furthermore, CVA24-CRE had effective antiviral activity against CVA24 in primary human conjunctival cells. In addition, CVA24-CRE was highly resistant to the emergence of genetically altered escape mutants. Collectively, the present study provides evidence that CVA24-CRE targeting a conserved viral genome region had universal, prolonged anti-CVA24 activity. This siRNA may thus hold a potential to act clinically as a novel anti-CVA24 agent.

Gene Therapeutic Approach for Inhibiting Hepatitis C Virus Replication Using a Recombinant Protein That Controls Interferon Expression

ABSTRACT The hepatitis C virus (HCV) is a continuing threat to public health. The systemic administration of interferon alpha with ribavirin is the only currently approved treatment. However, this treatment is associated with a wide spectrum of systemic side effects that limits its effectiveness; thus, there is an urgent need for new treatment modalities. In this study, we describe a novel anti-HCV strategy employing a recombinant transcription factor that we have engineered in such a way that NS3/4a viral protease controls its intracellular localization, thereby restoring interferon secretion specifically in cells infected with HCV. Proof-of-concept experiments validated the strategy, showing that the recombinant transcription factor was triggered to stimulate interferon promoter by NS3/4A and remained inactive in cells without NS3/4a. Using an adenovirus-associated viral vector delivery system, we found that the recombinant transcription factor inhibited HCV replication effectively in vitro in cultured cells.

Degenerate PCR primer design for the specific identification of rhinovirus C

Abstract Human rhinovirus (HRV)-A and -B is a common cause of upper respiratory tract infections. Recently, a third species, HRV-C, was categorized based on molecular typing studies. The results showed that the HRV-C genome had diverged from that of HRV-A and -B. Despite its late identification, increasing evidence suggests that HRV-C causes more severe pathogenic infections than HRV-A or -B; however, a large amount of epidemiological data is required to confirm this association in different clinical settings. Consequently, a simple and rapid method for identifying HRV-C is required to expedite such epidemiological studies. Here, two degenerate primer sets (HEV and HRVC) were designed based on bioinformatic analyses. The HEV set targeting the fifth IRES domain sequence within the 5′-UTR, which is highly conserved among enteroviruses, was designed to detect all enteroviruses, whereas the HRVC set, which targeted the VP2 coding region, was designed to detect HRV-C alone. Both primer sets were tested against a panel of standard enteroviruses and clinical lavage samples. HEV detected all enteroviruses tested whereas HRVC was specific for HRV-C. Although the primer design strategy was confirmed with a limited number of samples, extensive tests are required to be applied in clinical settings.

Serine Cluster Phosphorylation Liberates the C-terminal Helix of IFN Regulatory Factor 7 To Bind Histone Acetyltransferase p300

IFN regulatory factor 7 (IRF7) is a major regulator of type I (αβ) IFN secretion. A growing body of evidence shows that IRF7 is involved in a wide variety of pathologic conditions in addition to infections; however, the detailed mechanism of IRF7 transactivation remains elusive. Our current knowledge of IRF7 transactivation is based on studies of IRF3, another major regulator of IFN-β secretion. IRF3 and IRF7 are closely related homologs with high sequence similarity in their C-terminal regions, and both proteins are activated by phosphorylation of a specific serine cluster (SC). Nevertheless, the functional domains of the two proteins are arranged in an inverted manner. We generated a model structure of the IRF7 C-terminal region using homology modeling and used it to guide subsequent functional domain studies. The model structure led to the identification of a tripod-helix structure containing the SC. Based on the model and experimental data, we hypothesized that phosphorylation-mediated IRF7 transactivation is controlled by a tripod-helix structure. Inducible IκB kinase binds a tripod-helix structure. Serial phosphorylation of the SC by the kinase liberates C-terminal helix from an inhibitory hydrophobic pocket. A histone acetyltransferase P300 binds the liberated helix. The difference in the P300 binding sites explains why the domain arrangement of IRF7 is inverted relative to that of IRF3.

Cysteine-rich secretory protein 3 inhibits hepatitis C virus at the initial phase of infection.

Hepatitis C virus (HCV) affects 2-3% of the global population. Approximately one-quarter of acute infections cause chronic hepatitis that leads to liver cirrhosis or hepatocellular carcinoma. The major obstacle of current research is the extremely narrow host tropism of HCV. A single HCV strain can replicate in the Huh7 human hepatoma cell line. Huh7 cells can be adapted under selective pressure in vitro to identify host factors that influence viral replication. Here, we extended this strategy to the in vivo condition and generated a series of cell lines by multiple rounds of adaptation in immunocompromised mice. Adaptation increased the cellular resistance to HCV infection. Microarray analyses revealed that the expression levels of several genes were associated with HCV resistance. Notably, up-regulation of the mRNA encoding cysteine-rich secretory protein 3 (CRISP3), a glycoprotein with unknown function that is secreted from multiple exocrine glands, was correlated with HCV resistance. The presence of CRISP3 in the culture medium limited HCV replication at the early phase of infection.

871. rAAV2-Mediated Gene Expression in Primary Cortical Neural Cells Following Inhibition of DNA Synthesis and/or EGFR Tyrosine Kinase

Recombinant adeno-associated virus vectors, derived from a dependent human parvovirus, can transduce non-dividing normal cells to yield long-term transgene expression associated with a minimal immunological response. Among these vectors, a recombinant adeno-associated virus serotype 2 vector (rAAV2) is capable of transducing neural cells, making it an important potential vector for delivery of therapeutic genes into neural cells for treatment of CNS disease. However, the transduction efficiency (TE) of rAAV2 in neural cells is not optimal for most therapeutic needs. Recently, we showed that co-treatment with genotoxic agent hydroxyurea (HU) and EGFR tyrosine kinase inhibitor Tyr-1 dramatically increased the TE of rAAV2 in various human cancer cells of different origins. Here, we investigated whether these same chemical co-treatments could enhance rAAV2 transduction efficiency in primary cortical neural cells. Unexpectedly, pretreatment of cells with HU and/or Tyr-1, did not improve TE. SK-Hep1 human hepatocellular carcinomar cells expressing dramatic increase in rAAV2 transduction following chemical combination, experienced both significant release from G1/G0 phase and phosphorylation of p38, whereas no in neural cells. Thus, our results suggest that chemical pretreatment with HU/Tyr-1 is unable to increase rAAV2 TE in neural cells, indicating that other strategies should be investigated.

122. Enhanced Expression of Glutamate Decarboxylase 65 Significantly Improves Rat Parkinsonian Symptom

Glutamate decarboxylase (GAD) is the rate-limiting enzyme in GABA production, which has been intensively investigated as a mean to increase inhibitory synaptic activity. By expressing GAD65 encoded in a recombinant adeno-associated virus 2 (rAAV2) with a modified CMV promoter (JDK) in the subthalamic nucleus (STN), we investigated the degree of the amelioration of parkinsonian symptoms in a rat Parkinsons disease (PD) model. Behavioral improvement was evaluated by apomorphine-induced rotational and forepaw-adjusting tests. Additionally, the alteration of neuronal activities was investigated by monitoring electrophysiological changes.

757. Enhancement of Gene Expression by a Recombinant Adeno-Associated Virus by Various Chemical Treatments

A recombinant adeno-associated virus (rAAV) has drawn attention as a novel gene delivery system with the advantage of long-term and efficient gene expression, particularly in neuronal and skeletal muscle cells. However, transduction efficiency (TE) by rAAV is still low, which may explain its limited application, even in neuronal cells in vitro. To overcome this drawback of rAAV system, we attempted to enhance the transduction efficacy using many different kinds of chemicals, mostly modulating DNA synthesis, cell cycle or rAAV receptor expression. To investigate the effects of various chemical treatments on TE, we infected several human cancer cells lines (cervical Hela, colon HCT 116, breast MCF-7, hepatocellular carcinoma SK-Hep1, and glioma U 251 cancer cells) with rAAV-expressing b-galactosidase. We then examined the degree of x-gal staining 48 h after post-infection. Total 7 chemicals were employed to enhance TE by rAAV of Trichostatin A as an inducer of rAAV receptor expression, Aphidicolin and Hydroxyurea as a DNA damaging agent, Etopocide and Camptothecin as a DNA synthesis blocker, MG-132 as a proteosome inhibitor, and Tyrphostin-1 as a EGFR inhibitor. Hydorxyurea increased TE by 25- or 5-fold in Hela or HCT 116 cells, respectively. MG-132 increased TE by 8.5-fold in SK-Hep1 cells. Co-treatment of MG-132 and camptothecin enhanced TE by 12-fold in Hela cells. The combined treatment of hydroxyurea and tyrphostin-1 also enhanced the transduction efficiency by 12-fold in Hela cells. Finally, MTT assay consistently implied that the cytotoxicity was not observed in any concentrations of each chemical used in this study.