Real-time monitoring of oncolytic VSV properties in a novel in vitro microphysiological system containing 3D multicellular tumor spheroids.

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-ß 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.

Evaluation of Bystander Infection of Oncolytic Virus using a Medium Flow Integrated 3D In Vitro Microphysiological System.

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.

Negative Regulation of IKKε-Mediated IRF7 Phosphorylation by HSP70.

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.

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.

Soluble Prokaryotic Expression and Purification of Human Interferon Alpha-2b Using a Maltose-Binding Protein Tag.

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.

Human rhinovirus serotypes in the nasal washes and mucosa of patients with chronic rhinosinusitis.

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.

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.

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.

Attenuation of natural killer cell functions by capsaicin through a direct and TRPV1-independent mechanism.

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.

Natural killer cells from patients with chronic rhinosinusitis have impaired effector functions.

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.

Recombinant adeno-associated virus expressing a modified transcription factor triggered by the hepatitis C viral protease NS3/4a.

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.

Frequent Genetic Defects in the HIV-1 5' LTR/gag Gene in Hemophiliacs Treated with Korean Red Ginseng: Decreased Detection of Genetic Defects by Highly Active Antiretroviral Therapy.

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.

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.

A novel program to design siRNAs simultaneously effective to highly variable virus genomes.

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.

Epstein-Barr virus LF2: an antagonist to type I interferon.

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.

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.

Kaposi's sarcoma-associated herpesvirus viral IFN regulatory factor 3 stabilizes hypoxia-inducible factor-1 alpha to induce vascular endothelial growth factor expression.

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.