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.

Antiviral protection against enterovirus 71 mediated by autophagy induction following FLICE-inhibitory protein inactivation.

Even with the recent awareness of enterovirus 71 (EV71) as a major public health issue, there are no preventive or therapeutic agents that are effective against EV71 infection. Although FLICE-like inhibitory protein (FLIP) has been identified as a factor that modulates virus pathogenesis, there are no reports regarding its effects on EV71 infection. The aim of the present study was to identify whether FLIP influences EV71 pathogenesis and to understand the underlying mechanisms. Virus replication was markedly reduced in MRC5 cells preincubated with anti-FLIP peptides, and infected cells were rescued from the cytopathic effects of the virus. The anti-FLIP peptides induced autophagy by disrupting intrinsic FLIP functions. The antiviral activity of these peptides was reduced when autophagy was inhibited by treatment with siRNA targeted to beclin-1. Thus, the present study provides evidence that anti-FLIP peptides induce autophagy by inactivating cFLIP, and that this is associated with antiviral effects against EV71.

Antiviral effects of small interfering RNA simultaneously inducing RNA interference and type 1 interferon in coxsackievirus myocarditis.

Antiviral therapeutics are currently unavailable for treatment of coxsackievirus B3, which can cause life-threatening myocarditis. A modified small interfering RNA (siRNA) containing 5'-triphosphate, 3p-siRNA, was shown to induce RNA interference and interferon activation. We aimed to develop a potent antiviral treatment using CVB3-specific 3p-siRNA and to understand its underlying mechanisms. Virus-specific 3p-siRNA was superior to both conventional virus-specific siRNA with an empty hydroxyl group at the 5' end (OH-siRNA) and nonspecific 3p-siRNA in decreasing viral replication and subsequent cytotoxicity. A single administration of 3p-siRNA dramatically attenuated virus-associated pathological symptoms in mice with no signs of toxicity, and their body weights eventually reached the normal range. Myocardial inflammation and fibrosis were rare, and virus production was greatly reduced. A nonspecific 3p-siRNA showed relatively less protective effect under identical conditions, and a virus-specific OH-siRNA showed no protective effects. We confirmed that virus-specific 3p-siRNA simultaneously activated target-specific gene silencing and type I interferon signaling. We provide a clear proof of concept that coxsackievirus B3-specific 3p-siRNA has 2 distinct modes of action, which significantly enhance antiviral activities with minimal organ damage. This is the first direct demonstration of improved antiviral effects with an immunostimulatory virus-specific siRNA in coxsackievirus myocarditis, and this method could be applied to many virus-related diseases.

Characterization of infections of human leukocytes by non-polio enteroviruses.

To elucidate the detailed susceptibilities of leukocytes to clinically important non-polio enteroviruses (EVs), primary monocytes and various human leukocyte cell lines were infected with coxsackievirus A24 (CVA24), coxsackievirus B3 (CVB3), and enterovirus 70 (EV70). The permissiveness was then assessed by determining virus replication and resultant cytopathic effects. Different EVs varied markedly in their ability to infect leukocyte cell lines. CVB3 replicated effectively in leukocytes of B-cell, T-cell, and monocyte origin, CVA24 in leukocytes of B-cell and monocyte origin, and EV70 in leukocytes of monocyte origin. Primary monocytes, as well as monocyte-derived U-937 cells, were permissive to all three EVs. We observed a positive correlation between cytotoxicity and active virus replication, except in CVB3-infected monocytes. U-937 cells efficiently generated CVB3 progeny virus without severe cellular damage, including cell death. Moreover, infectivity on leukocytes was not absolutely associated with the availability of viral receptors. These findings suggest that the susceptibility of human leukocytes to non-polio EVs may be responsible for virus transport during the viremic phase, particularly to secondary target organs, and that active replication of CVB3 in all human leukocyte lineages leads to greater dissemination, in agreement with the ability of CVB to cause systemic diseases.

An ex vivo model of coxsackievirus infection using multilayered human conjunctival epithelial cells.

BACKGROUND: The purpose of this study was to establish an ex vivo model of coxsackievirus infection since there seems to be no suitable disease model currently. METHODS: Human conjunctival epithelial cells (HCECs) were cultured for 2 weeks in a serum-free air-liquid interface system to produce a multilayered structure. The cells were infected with coxsackievirus A24 (CVA24). Histological changes were investigated by staining the cells with H&E and DAPI, and apoptosis was evaluated using the TUNEL technique. Virus replication was measured in HeLa cells infected with viral progeny from multilayered HCECs, after 1 and 3 days, using the TCID(50) method. RESULTS: Cultured HCECs formed multiple layers. The cells showed characteristics of conjunctival epithelial cells and goblet cells, being immunohistochemically positive for CK19 and MUC5AC, respectively. CVA24 replicated readily in cultured multilayered HCECs. A mild cytopathic effect was noted 1 day after viral inoculation. Cell damage was extensive at 3 days. TUNEL imaging confirmed that the cytopathology was attributable to apoptosis. The TCID(50) data from HeLa cells indicated that the virus was actively replicating at both 1 and 3 days after inoculation. CONCLUSIONS: This novel infection model may be useful in investigating the pathogenesis of acute hemorrhagic conjunctivitis and the effectiveness of antiviral treatments.

An antiviral small-interfering RNA simultaneously effective against the most prevalent enteroviruses causing acute hemorrhagic conjunctivitis.

PURPOSE: Acute hemorrhagic conjunctivitis (AHC), a highly contagious eye disease, is caused primarily by either enterovirus 70 (EV70) or coxsackievirus A24 (CVA24) infection. Yet methods to prevent or cure AHC are not available. Recent evidence has shown that small-interfering RNAs (siRNAs), mediators of posttranscriptional gene knockdown, can act as effective antiviral agents. Thus, the authors attempted to develop a novel siRNA-based anti-AHC agent effective against both EV70 and CVA24. METHODS: Concurrent screening of the entire viral genome sequences of EV70 and CVA24 using the CAPSID program identified five different siRNA candidates complementary to genome regions of both viruses. The antiviral potentials of these siRNAs were assessed by treating MRC5 and primary human conjunctival cells with the siRNAs and following this with viral challenge. RESULTS: Among the five siRNAs, AHCe-3D-3 siRNA showed excellent cytoprotective effects and dramatic decreases in virus replication and virus protein synthesis. This siRNA, targeting the virus polymerase 3D gene, also induced similar antiviral effects in primary human conjunctival cells. CONCLUSIONS: These findings strongly suggest that the AHCe-3D-3 siRNA, homologous to two different AHC-associated enteroviruses, can provide equivalent antiviral activities against both AHC-causing enteroviruses. Such an siRNA may be developed as a clinically valuable AHC control agent.

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.

High frequency of gross deletions in 5' LTR/gag and nef genes in patients infected with CRF02_AG of HIV type 1 who survived for over 20 years: an association with Korean red ginseng.

Abstract We have shown that Korean red ginseng (KRG) intake is associated with gross deletions in the 5' LTR/gag (gDeltaLTR/gag) and nef genes (gDeltanef) of patients infected with subtype B of HIV-1. Here, we investigated these effects in three long-term survivors (LTSs) of subtype CRF02_AG of HIV-1. The three LTSs were diagnosed with HIV in 1987, 1988, and 1989, and have been treated with KRG for 7-15 years. Thirty-two samples of peripheral blood mononuclear cells were obtained from the subjects and used to amplify the 5' LTR/gag and nef genes via nested PCR. We obtained 88 amplicons in 5' LTR/gag and 128 amplicons in nef. The frequency of gDeltaLTR/gag was significantly higher (37.5%) in three LTSs than in control patients (8.6%, p < 0.01). Eight amplicons (9.5%) contained premature stop codon(s) in the gDeltaLTR/gag in three LTSs. Fourteen of the 128 nef amplicons (10.9%) contained the gDeltanef, which was present in only two (7.7%) of the 26 amplicons from control subjects. Interestingly, gDeltanef was detected 7 years after the reinitiation of KRG intake in an LTS and, coincidently, CD4 T cell counts and CD4/CD8 ratios rapidly increased. These data indicate that long-term intake of KRG has the therapeutic potential to induce gross deletions in HIV-1.

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.

Primary neurons become less susceptible to coxsackievirus B5 following maturation: the correlation with the decreased level of CAR expression on cell surface.

Coxsackievirus B (CVB) is one of the major pathogens of aseptic meningitis and meningioencephalitis, particularly in newborn infants. To analyze the influence of neural maturation on susceptibility to CVB infection, we prepared immature and mature neurons from 16-day-old BALB/c embryonic cortex. In contrast to immature neurons, mature neurons were less susceptible to CVB5 infection, as indicated by the decrease of cytopathic features. In mature neurons, progeny virus production was significantly hindered, and virus capsid protein VP1 synthesis and virus genome amplification were concomitantly reduced. In addition, the expression of coxsackievirus and adenovirus receptor (CAR), the major receptor of CVB5, was down-regulated in mature neurons. The antibody treatment specific to CAR significantly attenuated CVB5 susceptibility of immature neurons. These findings demonstrate that mature neurons become less susceptible to CVB by the decrease of CAR level. Thus, the data strongly support the idea that the level of virus receptor in neurons is one of the crucial determinants in the age-dependency of CVB virulence in central nervous system.

Universal and mutation-resistant anti-enteroviral activity: potency of small interfering RNA complementary to the conserved cis-acting replication element within the enterovirus coding region.

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.

Contributions of 3'-overhang to the dissociation of small interfering RNAs from the PAZ domain: molecular dynamics simulation study.

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.

Mutation variants generated from nonvirulent coxsackievirus B3 acquire virulence phenotypes by active virus replication.

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.

Targeted molecular dynamics simulation studies of calcium binding and conformational change in the C-terminal half of gelsolin.

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.

Quantitative analysis of viral RNA in the murine heart and pancreas with different concentration of coxsackievirus B3.

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. .

A small interfering RNA targeting coxsackievirus B3 protects permissive HeLa cells from viral challenge.

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.

Characterization of nonpolio enteroviruses recovered from patients with aseptic meningitis in Korea.

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.

Distribution of viral RNA in mouse tissues during acute phase of coxsackievirus B5 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.

All CVB serotypes and clinical isolates induce irreversible cytopathic effects in primary cardiomyocytes.

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.