CXCL10 production induced by high levels of IKKε in nasal airway epithelial cells in the setting of chronic inflammation.

The airway is the major entry route of pathogens due to continuous gas exchange with the environment. In particular, the nasal epithelial layer is the common site of airborne mucotropic virus infections. The inflammatory response to such infections must be tightly controlled due to its non-specific nature. Unrestrained inflammation breaks down the physiological mucosal defense system and leads to secondary bacterial or fungal infections. Chronic rhinosinusitis (CRS) is a prevalent inflammatory disease that compromises quality of life. In spite of its importance in the initiation of inflammation, the role of interferon signaling in nasal airway epithelial cells is largely unknown. We analyzed the expression of interferon signaling genes using clinical lavage specimens and nasal airway epithelial cells collected from CRS patients and controls. Basal expression of IFNAs, IKBKE, STAT1, and some CXC chemokines was elevated in samples from CRS patients. In subsequent in vitro studies, we found IKKε to be the key molecule and augmented CXCL10 secretion. Based on our findings and review of the literature, we hypothesized that high levels of IKKε might induce intractable inflammation via CXCL10. We tested the hypothesis in an animal model and found not only that IKKε induced severe eosinophilic inflammation with CXCL10 over-production, but also that inhibition of IKKε resolved the inflammation.

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 (αß) 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-ß 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.

Lysine 63-linked TANK-binding kinase 1 ubiquitination by mindbomb E3 ubiquitin protein ligase 2 is mediated by the mitochondrial antiviral signaling protein.

UNLABELLED: Beta interferon (IFN-ß) 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-ß 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-ß 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.

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.

Gene therapeutic approach for inhibiting hepatitis C virus replication using a recombinant protein that controls interferon expression.

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.

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.

Treatment with hydroxyurea and tyrphostin-1 significantly improves the transduction efficiency of recombinant adeno-associated viruses in human cancer cells.

To enhance the transduction efficiency (TE) of a recombinant adeno-associated virus 2 (rAAV2) in human cancer cells, we examined the combined effects of various chemicals known to influence the rAAV2 transduction process at distinct steps. Among the agents tested were trichostatin A, a histone deacetylase inhibitor, MG-132, a proteosome inhibitor, the genotoxic agents hydroxyurea, aphidicolin, etoposide and camptothecin, and tyrphostin-1, an epidermal growth factor receptor inhibitor. During or after chemical treatment, various human cancer cells were infected with rAAV2 expressing beta-galactosidase. Treatment with hydroxy-urea or etoposide plus tyrphostin-1 dramatically increased the TE in most cell lines. The combination of hydroxyurea plus tyrphostin-1 increased TE to 37.7+/-7.9%, 32.8+/-2.0% and 31.8+/-2.1% in SK-Hep1, HeLa, and HCT116 cells, respectively. In addition, following rAAV2 infection and treatment with hydroxyurea plus tyrphostin-1, long-term transgene expression was observed for up to 6 months, with no damage to the transduced cells. These results indicate that rAAV2 transgene expression can be significantly enhanced by a combination of chemical agents with distinct activity and prolonged gene expression can occur following rAAV2 gene transfer into human cancer cells.

Degenerate PCR primer design for the specific identification of rhinovirus C.

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.

Analysis of the production efficiency and titration of various recombinant adeno-associated viruses.

Recombinant adeno-associated virus type 2 (rAAV2) viral vector, a non-pathogenic human parvovirus, has recently emerged as a gene transfer vehicle for cancer gene therapy. To utilize rAAV2 properly and safely while carrying out preclinical and clinical studies, it is crucial to exactly titer the virus. We therefore compared biological infectious rAAV2 titers with physical titers of rAAV2 vectors encoding various transgenes with different sized viral genomes. Biological rAAV2 infectivity was assayed by measuring the number of virus particles able to transduce Hela cells using several detection methods, including X-gal staining and immunocytostaining. Physical titers of rAAV2 were determined using a commercially available rAAV2 particle-specific enzyme-linked immunosorbent assay. We found that total rAAV2 particle production was consistent within the limited size variations of the rAAV2 genome, regardless of the difference in transgenes. In contrast, the infectious titer of rAAV2 differed greatly, even for the same viruses, due to variation in the sensitivity of the relevant assays. Thus, the results suggest that both infectious virus titer and total virus particle should be precisely measured for rAAV2 vector utilized in each study.