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As a new class of cancer therapeutic agents, oncolytic viruses (OVs) have gained much attention not only due to their ability to selectively replicate in and lyse tumor cells, but also for their potential to stimulate antitumor immune responses. As a result, there is an increasing need for in vitro modeling systems capable of recapitulating the 3D physiological tumor microenvironment. Here, we investigated the potential of our recently developed microphysiological system (MPS), featuring a vessel-like channel to reflect the in vivo tumor microenvironment and serving as culture spaces for 3D multicellular tumor spheroids (MCTSs). The MCTSs consist of cancer A549 cells, stromal MRC5 cells, endothelial HUVECs, as well as the extracellular matrix. 3D MCTSs residing in the MPS were infected with oncolytic VSV expressing GFP (oVSV-GFP). Post-infection, GFP signal intensity increased only in A549 cells of the MPS. On the other hand, HUVECs were susceptible to virus infection under 2D culture and IFN-β secretion was quite delayed in HUVECs. These results thus demonstrate that OV antitumoral characteristics can be readily monitored in the MPS and that its behavior therein somewhat differs compared to its activity in 2D system. In conclusion, we present the first application of the MPS, an in vitro model that was developed to better reflect in vivo conditions. Its various advantages suggest the 3D MCTS-integrated MPS can serve as a first line monitoring system to validate oncolytic virus efficacy., Competing Interests: The authors declare no competing interests.

Immune reactions are controlled by the delicate spatiotemporal orchestration of multiple cells communicating by cytokines. Studies of cytokines that began with the discovery of IFN focused on positive regulatory mechanisms that induce secretion in response to harmful stimuli. However, there is a growing awareness that negative regulatory mechanisms that stop secretion of cytokines at specific times and spaces are also important for a successful immune reaction. Type I IFN is the primary cytokine in innate immunity. Although its induction is a prerequisite for the consequent adaptive immune reaction, its oversecretion can cause destructive tissue damage. IFN regulatory factor 7 (IRF7) is a master transcription factor of type I IFN, and multiple observations indicate the key role of IRF7 and the importance of its negative regulation. In this study, we found that the inducible heat shock protein 70 (HSP70) regulated the early type I IFN response by using mice knockout for HSP70. HSP70 dampened IRF7 activation; the inhibitory effect of HSP70 over IKKε-mediated IRF7 activation originated from simple competitive binding. This suggests the possibility of blocking the feed-forward loop between IRF7 and type I IFN in stress environments with increased expression of HSP70., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Replicable oncolytic viruses (OVs) induce tumor cell lysis and release viral progeny. The released progeny virions and cell debris can spread within surrounding tumor cells or blood vessels. These released molecules may also induce bystander damage in additional tumor cells through spreading within surrounding tumor cells or blood vessels. However, this effect has not been clearly demonstrated due to the difficulty of direct observation. Here, the bystander infection of OVs by vessel delivery and selective infection in 3D multicellular tumoroids (MCTs) in an in vitro microphysiological system (MPS) with integrated medium flow is demonstrated. This study uses replicable vesicular stomatitis virus (VSV)-green fluorescence protein (GFP) to identify the location of infection in 3D MCTs. Using this MPS, the oncoselective infection by VSV-GFP and the spreading by delivery of OVs through flow via block-to-block linkage of the primary infected MPS with uninfected 3D MCTs in an integrated MPS is observed. This MPS enables real-time monitoring and various analysis for the bystander infection of OVs. It is expected that the 3D in vitro MPS can be suitable to investigate the oncoselective spreading and bystander infection of OVs., (© 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Human interferon alpha-2b (IFNα-2b) has therapeutic applications as an antiviral and antiproliferative drug and has been used for a wide range of indications. Efficient production of IFNα-2b in Escherichia coli has been difficult because the protein tends to form inclusion bodies. This obstacle has garnered interest in efficiently expressing IFNα-2b and overcoming its poor solubility. In this study, seven N-terminal fusion partners - hexahistidine (His6), thioredoxin, glutathione S-transferase (GST), maltose-binding protein (MBP), N-utilization substance protein A, protein disulfide bond isomerase (PDI), and b'a' domain of PDI - were tested for soluble overexpression of codon-optimized IFNα-2b in E. coli. Low temperature increased the expression level of all of the tagged proteins except for the GST fusion. All the tags, except for His6 and GST, improved solubility. We purified IFNα-2b from the MBP-tagged fusion using immobilized metal affinity chromatography and anion exchange chromatography, and obtained a final yield of 7.2 mg from an initial 500-ml culture. The endotoxin level was 0.46 EU/µg. Biological activity was demonstrated using a luciferase assay, which showed a dose-dependent response with a calculated EC50 of 10.3 ± 5.9 pM. Our results demonstrate that using an MBP-tagged fusion is an efficient way to produce pure IFNα-2b., (© 2016 S. Karger AG, Basel.)

Background: Chronic rhinosinusitis (CRS) can be aggravated by viral upper respiratory infections. We aimed to investigate whether any specific human rhinovirus (HRV) serotype is more common in the mucosa of CRS patients, and to find any defining clinical characteristics, according to the various HRV serotypes., Methods: A prospective case-control study was conducted to determine HRV serotypes in 111 CRS patients and 51 non-CRS controls. No participant had a history of upper respiratory infection over a 4-week period. Nasal lavage fluids and turbinate epithelial cells were collected prospectively. When HRV was detected with multiplex polymerase chain reaction (PCR), strains were further characterized by sequencing the VP4/VP2 region of the HRV genome., Results: HRV was detected in 40 CRS subjects (36%) and 11 non-CRS controls (21%). The overall detection rates of HRV in CRS patients were higher than in non-CRS controls (p < 0.05). Of the 8 serotypes detected in CRS patients, 5 belonged to HRV-A and 3 belonged to HRV-B; HRV-C was not detected. In non-CRS controls, only HRV-A was identified, with only 2 serotypes detected (HRV-A13 and HRV-A16). HRV-B and C were not detected., Conclusion: The high prevalence of HRV in CRS patients was confirmed in our study. However, we were unable to determine whether certain HRV serotypes are more predominant in CRS patients than non-CRS controls. HRV-A13 was the most common serotype in both CRS patients and non-CRS controls. We could not find any differences in the clinical characteristics according to the HRV serotypes in CRS patients., (© 2014 ARS-AAOA, LLC.)

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., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Unlabelled: 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., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

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., (Copyright © 2014 by The American Association of Immunologists, Inc.)

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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The assessment of the biological activity of capsaicin, the compound responsible for the spicy flavor of chili pepper, produced controversial results, showing either carcinogenicity or cancer prevention. The innate immune system plays a pivotal role in cancer pathology and prevention; yet, the effect of capsaicin on natural killer (NK) cells, which function in cancer surveillance, is unclear. This study found that capsaicin inhibited NK cell-mediated cytotoxicity and cytokine production (interferon-γ and tumor necrosis factor-α). Capsaicin impaired the cytotoxicity of NK cells, thereby inhibiting lysis of standard target cells and gastric cancer cells by modulating calcium mobilization in NK cells. Capsaicin also induced apoptosis in gastric cancer cells, but that effect required higher concentrations and longer exposure times than those required to trigger NK cell dysfunction. Furthermore, capsaicin inhibited the cytotoxicity of isolated NK cells and of an NK cell line, suggesting a direct effect on NK cells. Antagonists of transient receptor potential vanilloid subfamily member 1 (TRPV1), a cognate capsaicin receptor, or deficiency in TRPV1 expression failed to prevent the defects induced by capsaicin in NK cells expressing functional TRPV1. Thus, the mechanism of action of capsaicin on NK cells is largely independent of TRPV1. Taken together, capsaicin may have chemotherapeutic potential but may impair NK cell function, which plays a central role in tumor surveillance., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Natural killer (NK) cells are multicompetent lymphocytes of the innate immune system that play a central role in host defense and immune regulation. Although increasing evidence suggests that innate immunity plays a key role in the pathogenesis of chronic rhinosinusitis (CRS), the role of NK cells in CRS has been poorly studied. This study aimed to characterize the peripheral blood NK cells from patients with CRS, and to compare the functions of these cells with those from non-CRS controls. The correlation between NK cell functional activity and prognosis was also assessed. Eighteen CRS patients and 19 healthy non-CRS controls were included. The patients with CRS were classified into two subgroups, namely a treatment-responsive group and recalcitrant group. NK cell degranulation was determined by measuring the cell surface expression of CD107a against 721.221 and K562 cells. Intracytoplasmic cytokine production was determined by flow cytometry. Compared to the controls, the NK cells of CRS group had an impaired ability to degranulate and to produce cytokines such as IFN-γ and TNF-α. The recalcitrant subgroup showed the most severe defects in NK cell effector functions. Moreover, the decreased NK cell functions in patients with CRS were associated with poor prognostic factors such as concomitant asthma and peripheral blood eosinophilia. NK cells, which were originally named for their ability to mediate spontaneous cytotoxicity towards diseased cells including infected cells, may play an important role in regulating the inflammatory process in CRS pathogenesis.

We investigated whether Korean red ginseng (KRG) and highly active antiretroviral therapy (HAART) affect the frequency of gross deletion in 5'LTR/gag in 20 hemophiliacs. This study is a prospective study in 20 hemophiliacs who were infected with Korean subclade B of HIV-1 from two cash-paid plasma donors in 1990. Over a 13-year period, we obtained 436 amplicons of 5'LTR/gag genes by nested polymerase chain reaction using 147 peripheral blood mononuclear cells. Of the 436 amplicons, 92 (21.1%) showed gross deletion in 5'LTR/gag. Despite of a 2.3-fold higher monthly dose of KRG intake, the frequency of gross deletion in 5'LTR/gag (16.4%) was significantly decreased during HAART compared with 28.1% prior to HAART (p<0.01). Gross deletion in 5'LTR/gag was 10% more detected on KRG-therapy than prior to KRG-therapy (p<0.05). In addition, we also obtained 28 amplicons containing premature stop codon or isoleucine at initiation codon of 254 amplicons sequenced on KRG intake (7.5%) or HAART (13.6%) compared with 0% before KRG intake. These findings indicate that high frequency of gross deletion in 5'LTR/gag and genetic defects prior to HAART are significantly associated with KRG intake and the detection of gross deletion in 5'LTR/gag is decreased by HAART.

Many viruses, including hepatitis C virus (HCV), are major threats to public health, but few treatment options are available. Systemic administration of a combination of interferon-a and ribavirin is the only approved treatment for HCV. However, half of all patients are not cured by such treatment and a wide spectrum of systemic side-effects limits its effectiveness. I developed a gene therapy approach with three goals: 1) the restoration of local interferon secretion in cells infected with HCV, 2) no secretion of interferon in normal cells not infected with the virus, and 3) suppression of the emergence of resistant viral strains. A recombinant transcription factor was constructed, the intracellular localization of which is controlled by a viral protease to stimulate focal interferon secretion at sites of infection. A recombinant adenovirus associated virus expressing the transcription factor based on the described strategy inhibited HCV replication effectively in a HCV in vitro culture system.

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.

A major concern of antiviral therapy using small interfering RNAs (siRNAs) targeting RNA viral genome is high sequence diversity and mutation rate due to genetic instability. To overcome this problem, it is indispensable to design siRNAs targeting highly conserved regions. We thus designed CAPSID (Convenient Application Program for siRNA Design), a novel bioinformatics program to identify siRNAs targeting highly conserved regions within RNA viral genomes. From a set of input RNAs of diverse sequences, CAPSID rapidly searches conserved patterns and suggests highly potent siRNA candidates in a hierarchical manner. To validate the usefulness of this novel program, we investigated the antiviral potency of universal siRNA for various Human enterovirus B (HEB) serotypes. Assessment of antiviral efficacy using Hela cells, clearly demonstrates that HEB-specific siRNAs exhibit protective effects against all HEBs examined. These findings strongly indicate that CAPSID can be applied to select universal antiviral siRNAs against highly divergent viral genomes.

Upon viral infection, the major defense mounted by the host immune system is activation of the interferon (IFN)-mediated antiviral pathway, which is mediated by IFN regulatory factors (IRFs). In order to complete their life cycle, viruses must modulate host IFN-mediated immune responses. Despite its association with significant human health problems, activities of Epstein-Barr virus (EBV), a human tumor-inducing herpesvirus, to evade host IFN-mediated innate immunity have not been well characterized. To search for EBV genes that block IFN signal transduction, we carried out a screening of EBV open reading frames for their abilities to block IFN-alpha/beta-mediated luciferase expression upon Sendai virus infection. This screening demonstrates that EBV LF2 tegument protein specifically interacts with the central inhibitory association domain of IRF7, and this interaction leads to inhibition of the dimerization of IRF7, which suppresses IFN-alpha production and IFN-mediated immunity. This demonstrates a novel immune evasion mechanism of EBV LF2 in blocking cellular IRF7-mediated innate immunity.

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.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent associated with Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. Hypoxia-inducible factor-1 (HIF-1) is the master regulator of both developmental and pathologic angiogenesis, composed of an oxygen-sensitive alpha-subunit and a constitutively expressed beta-subunit. HIF-1 activity in tumors depends on the availability of the HIF-1 alpha subunit, the levels of which are increased under hypoxic conditions. Recent studies have shown that HIF-1 plays an important role in KSHV reactivation from latency and pathogenesis. Here, we report a novel mechanism by which KSHV activates HIF-1 activity. Specific interaction between KSHV viral IFN regulatory factor 3 (vIRF3) and the HIF-1 alpha subunit led to the HIF-1 alpha stabilization and transcriptional activation, which induced vascular endothelial growth factor expression and ultimately facilitated endothelial tube formation. Remarkably, the central domain of vIRF3, containing double alpha-helix motifs, was sufficient not only for binding to HIF-1 alpha but also for blocking its degradation in normoxic conditions. This indicates that KSHV has developed a unique mechanism to enhance HIF-1 alpha protein stability and transcriptional activity by incorporating a viral homologue of cellular IRF gene into its genome, which may contribute to viral pathogenesis.

Upon viral infection, the major defense mounted by the host immune system is activation of the interferon (IFN)-mediated antiviral pathway that is mediated by IFN regulatory factors (IRFs). In order to complete their life cycle, viruses must modulate the host IFN-mediated immune response. Kaposi's sarcoma-associated herpesvirus (KSHV), a human tumor-inducing herpesvirus, has developed a unique mechanism for antagonizing cellular IFN-mediated antiviral activity by incorporating viral homologs of the cellular IRFs, called vIRFs. Here, we report a novel immune evasion mechanism of KSHV vIRF3 to block cellular IRF7-mediated innate immunity in response to viral infection. KSHV vIRF3 specifically interacts with either the DNA binding domain or the central IRF association domain of IRF7, and this interaction leads to the inhibition of IRF7 DNA binding activity and, therefore, suppression of alpha interferon (IFN-alpha) production and IFN-mediated immunity. Remarkably, the central 40 amino acids of vIRF3, containing the double alpha helix motifs, are sufficient not only for binding to IRF7, but also for inhibiting IRF7 DNA binding activity. Consequently, the expression of the double alpha helix motif-containing peptide effectively suppresses IRF7-mediated IFN-alpha production. This demonstrates a remarkably efficient means of viral avoidance of host antiviral activity.

The promising potential of RNA interference-based antiviral therapies has been well established. However, the antiviral efficacy is largely limited by genomic diversity and genetic instability of various viruses, including human enterovirus B (HEB). In this work, the first evidence supporting the anti-HEB activity of the small interfering RNA (siRNA) targeting the highly conserved cis-acting replication element (CRE) within virus coding region 2C is presented. HeLa cells pre-treated with siRNA complementary to the conserved sequence of the loop region of CRE(2C) were effectively rescued from the cytopathic effects of HEBs. Downregulation of virus replication and attenuation of cytotoxicity were consistently observed in various reference strains and clinical isolates. Cells treated with this siRNA were resistant to the emergence of viable escape mutants and showed sustained antiviral ability. Collectively, the data suggest that the siRNA based on the disordered structure within the highly conserved cis-acting coding region has potential as a universal, persistent anti-HEB agent. The same strategy can be successfully applied to the development of siRNA with consistent antiviral effects in other virus groups possessing similar RNA elements.

RNA interference (RNAi) is a 'knock-down' reaction to reduce expression of a specific gene through highly regulated, enzyme-mediated processes. Small interfering RNAs (siRNAs) are RNA molecules that play an effector role in RNAi and can bind the PAZ domains present in Dicer and RISC. We investigated the interaction between the PAZ domain and the siRNA-like duplexes through dissociation molecular dynamics (DMD) simulations. Specifically, we focused on the response of the PAZ domain to various 3'-overhang structures of the siRNA-like duplexes. We found that the siRNA-like duplex with the 3' UU-overhang made relatively more stable complex with the PAZ domain compared to those with 3' CC-, AA-, and GG-overhangs. The siRNA-like duplex with UU-overhang was easily dissociated from the PAZ domain once the structural stability of the complex is impaired. Interestingly, the 3' UU-overhang spent the least time at the periphery region of the binding pocket during the dissociation process, which can be mainly attributable to UU-overhang's smallest number of hydrogen bonds.

Objective: To understand coxsackievirus B3 (CVB3) virulence at the molecular level., Method: A mutation library was generated from noncardiovirulent CVB3/0. Highly virulent mutation variants were recovered and characterized both phenotypically and genotypically., Results: The variants consistently caused destruction of multiple tissues together with active virus production and induced severe mortality in vivo. The extent of infectious virus generation was directly correlated with that of histopathological aberration. Genotypic analysis of the entire genome indicated that the virulent viruses encode nucleotide substitutions in the 5'-nontranslated region, which have previously been identified in other virulent CVB3s., Conclusion: The present study provides evidence that particular nucleotide substitutions in the 5'-nontranslated region of nonvirulent CVB3 can lead to active virus replication, which is sufficient to induce virulence., ((c) 2008 S. Karger AG, Basel)

Gelsolin consists of six related domains (G1-G6) and the C-terminal half (G4-G6) acts as a calcium sensor during the activation of the whole molecule, a process that involves large domain movements. In this study, we used targeted molecular dynamics simulations to elucidate the conformational transitions of G4-G6 at an atomic level. Domains G4 and G6 are initially ruptured, followed by a rotation of G6 by approximately 90 degrees , which is the dominant conformational change. During this period, local conformational changes occur at the G4 and G5 calcium-binding sites, facilitating large changes in interdomain distances. Alterations in the binding affinities of the calcium ions in these three domains appear to be related to local conformational changes at their binding sites. Analysis of the relative stabilities of the G4-G6-bound calcium ions suggests that they bind first to G6, then to G4, and finally to G5.

Objectives: We investigated the clinical features, pathologic changes, and viral RNA kinetics in the course of acute and subacute experimental coxsackievirus B3 (CVB3) infection in a murine model., Methods: Five-week-old A/J inbred male mice were divided into 5 groups. Four of those groups were inoculated intraperitoneally with 5 x 10(4) (group 1), 1 x 10(5) (group 2), 5 x 10(5) (group 3), or 1 x 10(6) (group 4) PFU of CVB3. Control mice were inoculated with uninfected Vero cell lysate in DMEM. Mice from each group were sacrificed on days 7 or 14 after inoculation., Results: Bloody diarrhea, earlier weight loss, perianal swelling, and death were correlated with higher viral load. One of ten mice in group 3 and 5 of 10 mice in group 4 died spontaneously between days 4 and 12 after inoculation. All of the remaining 34 mice of infected groups demonstrated extensive pancreatic inflammation. Focal myocarditis developed in only 4 (11.8%) of those 34 subjects. Amylase and creatine kinase activities in the serum were increased in the mice of infected groups. CVB3 RNA was detected in the heart and pancreatic tissue in all subjects. The CVB3 RNA copy number in pancreatic tissue was not correlated with the severity of inflammation., Conclusions: In the murine model, viral loading dose determines the clinical features of CVB3-induced infection, and the severity of pancreatitis is not correlated with the viral loading dose or tissue level of viral RNA. ., (Copyright 2006 S. Karger AG, Basel.)

We examined the ability of small interfering RNAs (siRNAs) to disrupt infection by coxsackievirus B3 (CVB3). The incorporation of siRNAs dramatically decreased cell death in permissive HeLa cells in parallel with a reduction in viral replication. Three of four siRNAs had potent anti-CVB3 activity. The present study thus demonstrates that the antiviral effect is due to the downregulation of viral replication. In addition, an effective CVB3-specific siRNA had similar antiviral effects in other related enteroviruses possessing sequence homology in the targeted region. Because the CVB3-specific siRNA is effective against other enteroviruses, siRNAs have potential for a universal anti-enterovirus strategy.

Coxsackievirus B3 (CVB3) has been identified as a major causative agent of acute and chronic myocarditis, but the involvement of other CVB serotypes in myocarditis has not been investigated. To dissect the pathological properties of different CVB serotypes toward primary cardiomyocytes, we tested their effects on primary cardiomyocyte cultures from neonatal rats. Morphological abnormalities were examined by both light and fluorescence microscopy after Hoechst 33342 staining, and loss of cell viability was estimated by MTT assay. All six CVB serotypes showed a similar degree of severe toxicity toward primary cardiomyocytes. CVB clinical isolates had cytopathic effects (CPEs) similar to those of their respective CVB reference strains. Within 1-2 days of infection with multiplicities of infection MOI 50, the cells began to experience morphological changes including cell shrinkage, rounding-up, and slight nuclear condensation. The irreversible loss of cell viability was readily observed within 3-5 days following virus infection. These results suggest that all six CVB serotypes induce direct, irreversible toxicity towards cardiomyocytes, which eventually leads to the death of infected cells. These findings indicate that the variations in CVB serotype are not the limiting factor determining the susceptibility of cardiomyocytes to CVB infection.

Objective: To investigate histopathological changes and distribution of coxsackievirus B5 (CVB5) RNA in mouse heart, liver, and pancreas during the acute phase of infection., Methods: C3H/HeJ male mice, aged 3-4 weeks, were inoculated intraperitoneally with 5 x 10(5) plaque-forming units of CVB5 and sacrificed at 1, 2, 3, 4, 7 and 10 days postinfection (p.i.). Inflammation of the heart, liver, and pancreatic tissue sections was evaluated by hematoxylin and eosin staining, and virus was detected using antibody to viral coat protein VP1. A quantitative real-time RT-PCR method, using primers and probe targeted to the highly conserved sequences in the 5'-untranslated region of the virus, was used to evaluate the kinetics of CVB5 RNA during the development of myocarditis or pancreatitis., Results: Marginal inflammatory changes were observed in the heart tissues although viral RNA was constantly present between 1 and 10 days p.i., peaking at 4 days p.i. The pancreatic tissues displayed massive lymphocyte infiltration and loss of acinar cells at day 4 p.i. and viral RNA was detected between 1 and 10 days p.i., peaking at 2-3 days p.i. In the liver, viral RNA was detected between 1 and 7 days. No mortality was observed., Conclusions: CVB5 induced acute pancreatitis without subsequent development of myocarditis. Clearance of CVB5 RNA from the pancreas and heart was slower than clearance from the liver. Our real-time RT-PCR method, which is more sensitive than conventional plaque assay, may provide valuable insight into viral RNA kinetics during CVB5 infection., (Copyright (c) 2005 S. Karger AG, Basel.)

Objectives: We attempted to characterize nonpolio enteroviruses recovered from Korean patients with aseptic meningitis., Methods: We performed RT-PCR on the 5'-nontranslated region using clinical specimens. Infectious clinical isolates were amplified by infecting Vero cells with RT-PCR-positive clinical specimens. We then investigated the direct effect in primary neuronal cells or cardiomyocytes following virus infection., Results: Total 12 clinical isolates were subtypically analyzed by both RT-PCR/sequencing comparison of the VP-1 region and neutralization assay. 43-2, 43-2S, 57 and 58 were found to be coxsackievirus B1 (CVB1), 312 to be CVB5, 14-2S and 327 to be echovirus 6, 165 to be echovirus 9, 337 to be echovirus 11, and 270 to be echovirus 30. All the clinical isolates tested showed profound cytotoxicity to various degrees in the primary neuronal cells within 24 h postinfection at 10 MOI. By contrast, a significant cytopathic effect was observed in the primary cardiomyocytes at 3-5 days postinfection at 50 MOI., Conclusions: The present study suggests that the clinical isolates recovered from Korean patients belonged to different CVB or echovirus serotypes and that these viruses showed diversities in their virulence in primary neuronal cells and cardiomyocytes., (Copyright (c) 2005 S. Karger AG, Basel.)

To investigate the presence of infectious agents in human atherosclerotic arterial tissues. Atherosclerotic plaques were removed from 128 patients undergoing carotid endarterectomy or other bypass procedures for occlusive disease, and from twenty normal arterial wall samples, obtained from transplant donors with no history of diabetes, hypertension, smoking, or hyperlipidemia. Using the polymerase chain reaction (PCR) or reverse transcription-PCR, these samples were analyzed for the presence of Chlamydia pneumoniae, cytomegalovirus, enterovirus, adenovirus, herpes simplex viruses types 1 and 2, and Epstein-Barr virus. The amplicons were then sequenced, and phylogenetic analyses were performed. Enteroviral RNA was found in 22 of 128 atherosclerotic vascular lesions (17.2%), and C. pneumoniae and cytomegalovirus were each found in 2 samples (1.6%). In contrast, adenovirus, herpes simplex viruses, and Epstein-Barr virus were not identified in any of the atherosclerotic samples. Enterovirus was detected in 6/24 (25.0%) aortas, 7/33 (21.2%) carotid arteries, 6/40 (15.0%) femoral arteries, and 3/31 (9.7%) radial arteries of patients with chronic renal failure. There were no infectious agents detected in any of the control specimens. Using phylogenetic analysis, the enterovirus isolates were clustered into 3 groups, arranged as echovirus 9 and coxsackieviruses B1 and B3. Enteroviral RNA was detected in 17.2% of atherosclerotic plaques, but was not observed in any of the control specimens. This suggests a connection between enteroviral infection and atherosclerosis. These findings differ from those of other studies, which found more frequent incidence of C. pneumoniae and cytomegalovirus infection in atherosclerotic plaques.

Coxsackievirus B (CVB) is often associated with aseptic meningitis and encephalitis, but the six serotypes of CVB vary in their relative disease severity. To elucidate the detailed mechanisms of CVB-induced cytopathological effects, the morphological and biochemical characteristics caused by the CVB serotypes in mouse primary cortical neuronal cells were investigated. By 24 h post-infection, all CVB serotypes except CVB2 induced severe cytotoxic alterations, including a loss of neurites. Both fluorescence and transmission electron microscopy revealed CVB-induced morphological changes indicative of apoptosis, including heavily condensed nuclei, subsequent chromatin condensation into the periphery of the nuclei and oligonucleosomal DNA fragmentation. It was also found that infection with all six CVB serotypes led to productive virus replication, which was completed prior to an apoptotic signal. The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone significantly inhibited nuclear changes associated with virus-induced apoptosis, but had less effect on virus-associated cytopathic effects and no effect on virus production. In contrast, the transcription inhibitor actinomycin D profoundly inhibited all three virus-induced events. Taken together, these findings demonstrate that all six CVB serotypes can efficiently replicate in mouse cortical neuronal cells and that productive replication of these CVBs, except for CVB2, induces multiple cytopathological effects, including apoptotic alterations.

Objective: To investigate the relationship between enteroviral infection and myocardial tissue apoptosis during the development of viral myocarditis in a murine model., Methods: C3H/HeJ mice were inoculated with two strains of coxsackievirus B3, specifically CVB3 (cardiovirulent Nancy strain) and CVB3/0 (noncardiovirulent strain). Mice were sacrificed at 4, 7 and 10 days postinfection (p.i.). Hearts were removed, and plaque assays and RT-PCR were performed to detect the presence of viruses. Myocardial tissue sections were additionally evaluated by hematoxylin and eosin staining for inflammation, VP1 and Bax immunohistochemical staining for detection of virus and Bax expression, and TUNEL and Apostain for localization of apoptosis., Results: CVB3 replicated to significantly higher titers than CVB3/0 at all time points. Histopathological analyses revealed significant inflammatory changes at all time points in CVB3-infected mice, in contrast to minimal changes in CVB3/0-infected mice. TUNEL and Apostain assays of myocardial tissues from mice infected with CVB3 disclosed maximum apoptotic lesions at 4 days p.i. and to a lesser extent at 7 and 10 days p.i. Moreover, CVB3-infected myocardial tissues displayed significantly enhanced Bax expression at 4 days p.i., and lesions overlapped with VP1-stained areas., Conclusions: These data indicate that (1) the cardiovirulent strain CVB3 induces more severe inflammation and apoptosis than the noncardiovirulent CVB3/0 strain, (2) viral replication is localized in inflammatory and apoptotic lesions in myocardial tissues, (3) apoptotic changes are observed in the early stages of myocarditis and (4) Bax may be associated with the apoptosis process in CVB3-induced myocarditis., (Copyright 2003 S. Karger AG, Basel)

Coxsackievirus B (CVB) causes a wide spectrum of human diseases which are closely associated with direct destruction of infected cells. We investigated the morphological and biochemical characteristics of CPEs in permissive Vero cells caused by different CVB serotypes. Regardless of serotype, the infected cells experienced similar degrees of CPEs within 24 h postinfection (p.i.). Using both Hoechst 33342 staining and transmission electron microscopy, we consistently observed morphological properties of apoptosis, heavily condensed nuclei and subsequent chromatin condensation into the periphery of the nuclei within 12 h p.i. Moreover, we noticed typical oligonucleosomal DNA fragmentation, while productive CVB multiplication was accomplished within 6 h p.i. prior to an apoptotic signal. Caspase inhibitor significantly prohibited nuclear changes due to apoptosis with no influence on virus production and cell death, demonstrating that all the CVBs induced more than one type of pathological effect, including apoptotic alteration in permissive Vero cells., (Copyright 2003 S. Karger AG, Basel)

Advanced gastric cancer cannot be treated with surgery or conventional cancer therapy, which has prompted a search for new therapeutic modalities. Previously, we and other groups showed that E1B 55 kDa-deleted recombinant adenoviruses, such as YKL-1, effectively replicate and induce cytotoxicity in p53-deficient cancer cells while sparing normal cells. Here, we investigated selective YKL-1 replication and resultant cytolysis in human gastric cancer cells. The cytopathic effects were obvious in all five gastric cancer cell lines we examined. Evaluation of p53 expression indicated that only the AGS cell line retained functionally normal p53. Nevertheless, AGS was 10-fold more sensitive to YKL-1 than the other cell lines. Transmission electron microscopy showed typical morphological alterations along with efficient replication of YKL-1 in AGS cells. Therefore, YKL-1 induces preferential cytotoxic effects in human gastric cancer cells in a p53-independent manner, making YKL-1 a promising therapeutic agent for human gastric cancers.

Enterovirus infections of the central nervous system (CNS) are common and important causes of morbidity in immunocompromised children and adults. In this study we identify and characterize coxsackievirus B4-induced neuronal death. To investigate the CNS pathophysiology resulting from this viral infection, cultured rat neurons were infected with coxsackievirus B4 (CVB4) and nuclear morphology, phosphatidylserine (PS) externalization, and the effects of Actinomycin D or cycloheximide (CHX) were examined. CVB4 induced neuronal cell death within 24 h while PS externalization was apparent in cell bodies 16 h after CVB4 infection. Actinomycin D or CHX significantly reduced CVB4 induced-neuronal cell death in a dose-dependent manner. Pretreatment with CHX or actinomycin D also inhibited nuclear condensation, which occurred after CVB4 infection. However, the changes were relatively unresponsive to zVAD-fmk. These results suggest that CVB4 induces CHX- and actinomycin D-sensitive, but zVAD-fmk-insensitive neuronal apoptosis.

Selectively replicating recombinant adenovirus has emerged as a novel strategy for the treatment of incurable human cancers. One of the major characteristics of hepatocellular carcinoma is the transcriptional reactivation of alpha-fetoprotein (AFP). In this study, we evaluated the liver cancer-specific oncolytic potential of E1B 55kDa-deleted recombinant adenovirus (YKL-1001), which retained other E1 genes driven by the AFP promoter. Transient transfection study using luciferase indicated the selective activation of the AFP promoter only in human liver cancer cells secreting AFP (HepG2, Hep3B). YKL-1001 induced both cytopathic effects exclusively in AFP-positive liver cancer cells and the growth inhibition of pre-established Hep3B xenografts. Finally, hematoxylin-eosin staining and the immunohistochemistry to the adenoviral hexon showed a large distributed necrotic area and this implied a wide spread of YKL-1001. Therefore, the present study demonstrated that YKL-1001 holds significant promise as an oncolytic agent for hepatocellular carcinoma.

A major problem with reverse transcription-polymerase chain reaction (RT-PCR) is that the products amplified from RNA and DNA are not distinguishable. The use of a primer set targeted for an intron-containing sequence or RNA extract without contaminating DNA has been established to overcome this problem. However, an intron sequence does not always exist in the target region and complete removal of DNA during RNA extraction is not practical. For these reasons, we developed differential amplifying RT-PCR (DART-PCR) based on differences in reaction temperatures between RT and PCR, as well as in architectures between RNA and DNA. DART-PCR was designed to differentiate products amplified from RNA and DNA regardless of the presence of introns. DART-PCR can be readily applied for diagnosis of active infection of human cytomegalovirus by detection of glycoprotein B mRNA, which gene lacks introns. Other advantages of DART-PCR were as follows: there is no need for separation of RNA from DNA, simultaneous/differential detection in a single tube and possibility of determination of relative amounts of RNA and DNA.