Inactivation Mechanism and Efficacy of Grape Seed Extract for Human Norovirus Surrogate.

Proper disinfection of harvested food and water is critical to minimize infectious disease. Grape seed extract (GSE), a commonly used health supplement, is a mixture of plant-derived polyphenols. Polyphenols possess antimicrobial and antifungal properties, but antiviral effects are not well-known. Here we show that GSE outperformed chemical disinfectants (e.g., free chlorine and peracetic acids) in inactivating Tulane virus, a human norovirus surrogate. GSE induced virus aggregation, a process that correlated with a decrease in virus titers. This aggregation and disinfection were not reversible. Molecular docking simulations indicate that polyphenols potentially formed hydrogen bonds and strong hydrophobic interactions with specific residues in viral capsid proteins. Together, these data suggest that polyphenols physically associate with viral capsid proteins to aggregate viruses as a means to inhibit virus entry into the host cell. Plant-based polyphenols like GSE are an attractive alternative to chemical disinfectants to remove infectious viruses from water or food. IMPORTANCE Human noroviruses are major food- and waterborne pathogens, causing approximately 20% of all cases of acute gastroenteritis cases in developing and developed countries. Proper sanitation or disinfection are critical strategies to minimize human norovirus-caused disease until a reliable vaccine is created. Grape seed extract (GSE) is a mixture of plant-derived polyphenols used as a health supplement. Polyphenols are known for antimicrobial, antifungal, and antibiofilm activities, but antiviral effects are not well-known. In studies presented here, plant-derived polyphenols outperformed chemical disinfectants (i.e., free chlorine and peracetic acids) in inactivating Tulane virus, a human norovirus surrogate. Based on data from molecular assays and molecular docking simulations, the current model is that the polyphenols in GSE bind to the Tulane virus capsid, an event that triggers virion aggregation. It is thought that this aggregation prevents Tulane virus from entering host cells.

A novel approach to concentrate human and animal viruses from wastewater using receptors-conjugated magnetic beads.

Viruses are present at low concentrations in wastewater; therefore, an effective method for concentrating virus particles is necessary for accurate wastewater-based epidemiology (WBE). We designed a novel approach to concentrate human and animal viruses from wastewater using porcine gastric mucin-conjugated magnetic beads (PGM-MBs). We systematically evaluated the performances of the PGM-MBs method (sensitivity, specificity, and robustness to environmental inhibitors) with six viral species, including Tulane virus (a surrogate for human norovirus), rotavirus, adenovirus, porcine coronavirus (transmissible gastroenteritis virus or TGEV), and two human coronaviruses (NL63 and SARS-CoV-2) in influent wastewater and raw sewage samples. We determined the multiplication factor (the ratio of genome concentration of the final solution to that of the initial solution) for the PGM-MBs method, which ranged from 1.3 to 64.0 depending on the viral species. Because the recovery efficiency was significantly higher when calculated with virus titers than it was with genome concentration, the PGM-MBs method could be an appropriate tool for assessing the risk to humans who are inadvertently exposed to wastewater contaminated with infectious viruses. Furthermore, PCR inhibitors were not concentrated by PGM-MBs, suggesting that this tool will be successful for use with environmental samples. In addition, the PGM-MBs method is cost-effective (0.5 USD/sample) and has a fast turnaround time (3 h from virus concentration to genome quantification). Thus, this method can be implemented in high throughput facilities. Because of its strong performance, intrinsic characteristics of targeting the infectious virus, robustness to wastewater, and adaptability to high throughput systems, the PGM-MBs method can be successfully applied to WBE and ultimately provides valuable public health information.

Synergistic Combination of Oncolytic Virotherapy and Immunotherapy for Glioma.

PURPOSE: We hypothesized that the combination of a local stimulus for activating tumor-specific T cells and an anti-immunosuppressant would improve treatment of gliomas. Virally encoded IL15Rα-IL15 as the T-cell activating stimulus and a prostaglandin synthesis inhibitor as the anti-immunosuppressant were combined with adoptive transfer of tumor-specific T cells. EXPERIMENTAL DESIGN: Two oncolytic poxviruses, vvDD vaccinia virus and myxoma virus, were each engineered to express the fusion protein IL15Rα-IL15 and a fluorescent protein. Viral gene expression (YFP or tdTomato Red) was confirmed in the murine glioma GL261 in vitro and in vivo. GL261 tumors in immunocompetent C57BL/6J mice were treated with vvDD-IL15Rα-YFP vaccinia virus or vMyx-IL15Rα-tdTr combined with other treatments, including vaccination with GARC-1 peptide (a neoantigen for GL261), rapamycin, celecoxib, and adoptive T-cell therapy. RESULTS: vvDD-IL15Rα-YFP and vMyx-IL15Rα-tdTr each infected and killed GL261 cells in vitro. In vivo, NK cells and CD8+ T cells were increased in the tumor due to the expression of IL15Rα-IL15. Each component of a combination treatment contributed to prolonging survival: an oncolytic virus, the IL15Rα-IL15 expressed by the virus, a source of T cells (whether by prevaccination or adoptive transfer), and prostaglandin inhibition all synergized to produce elimination of gliomas in a majority of mice. vvDD-IL15Rα-YFP occasionally caused ventriculitis-meningitis, but vMyx-IL15Rα-tdTr was safe and effective, causing a strong infiltration of tumor-specific T cells and eliminating gliomas in 83% of treated mice. CONCLUSIONS: IL15Rα-IL15-armed oncolytic poxviruses provide potent antitumor effects against brain tumors when combined with adoptive T-cell therapy, rapamycin, and celecoxib.

A cautionary note on the selectivity of oncolytic poxviruses.

BACKGROUND: Oncolytic viruses selectively infect cancer cells while avoiding infection of normal cells. Usually, selectivity is demonstrated by injecting a virus into tumor-bearing mice and observing infection and lysis of tumor cells without infection of other tissues. The general view is that this selectivity is due to tropisms of the virus. However, apparent selectivity could be due to accessibility. For example, intravenously injected virus may not gain access to cells within the central nervous system (CNS) because of the blood-brain barrier. PURPOSE: We tested the CNS safety of two oncolytic poxviruses that have been demonstrated to be safe for treatment of peripheral tumors (vaccinia virus vvDD-IL15-Rα and myxoma virus vMyx-IL15Rα-tdTr). METHODS: Two poxviruses were tested for selectivity in vitro and in vivo. RESULTS: Both viruses infected glioma cells in vitro. In vivo, both viruses infected glioma cells and did not infect neurons when injected into a tumor or into the normal striatum. However, viral gene expression was observed in ependymal cells lining the ventricles, implying that these poxviruses were not as selective as originally predicted. For vvDD-IL15-Rα, some tumor-bearing mice died soon after virus treatment. If the same titer of vvDD-IL15-Rα was injected directly into the lateral cerebral ventricle of nontumor-bearing mice, it was uniformly fatal. Infection of ependymal cells, subventricular cells, and meninges was widespread. On the other hand, vMyx-IL15Rα-tdTr only transiently infected ependymal cells and was safe even when injected directly into the lateral cerebral ventricles. The two poxviruses also differed in their infection of dendritic cells; vvDD-IL15-Rα infected dendritic cells and lysed them but vMyx-IL15Rα-tdTr did not. CONCLUSION: Vaccinia virus vvDD-IL15-Rα is very promising for treating cancer types outside of the brain. However, for cancers located within the brain, myxoma virus vMyx-IL15Rα-tdTr offers a safer alternative.

A comparison of the effect of molluscum contagiosum virus MC159 and MC160 proteins on vaccinia virus virulence in intranasal and intradermal infection routes.

Molluscum contagiosum virus (MCV) causes persistent, benign skin neoplasm in children and adults. MCV is refractive to growth in standard tissue culture and there is no relevant animal model of infection. Here we investigated whether another poxvirus (vaccinia virus; VACV) could be used to examine MCV immunoevasion protein properties in vivo. The MCV MC159L or MC160L genes, which encode NF-κB antagonists, were inserted into an attenuated VACV lacking an NF-κB antagonist (vΔA49), creating vMC159 and vMC160. vMC160 slightly increased vΔA49 virulence in the intranasal and intradermal routes of inoculation. vMC159 infection was less virulent than vΔA49 in both inoculation routes. vMC159-infected ear pinnae did not form lesions, but virus replication still occurred. Thus, the lack of lesions was not due to abortive virus replication. This system provides a new approach to examine MCV immunoevasion proteins within the context of a complete and complex immune system.

Adenovirus Replication Cycle Disruption from Exposure to Polychromatic Ultraviolet Irradiation.

Polychromatic ultraviolet (UV) light with bandwidth of 20 nm and peak emission centered at 224, 254, or 280 nm (UV224, UV254, and UV280, respectively) were used to inactivate human adenovirus type 2 (HAdV-2). Quantitative polymerase chain reaction (qPCR) and reverse transcriptase qPCR assays were used to elucidate the step in the HAdV-2 replication cycle that was disrupted after UV exposure. UV treatment at any of the wavelengths analyzed did not inhibit association of HAdV-2 to the host cells even after exposure to a fluence (UV dose) that would produce a virus inactivation efficiency, measured by plaque assay, greater than 99.99%. In contrast, UV irradiation at all three peak emissions disrupted early E1A gene transcription and viral DNA replication, but different mechanisms appeared to be dominating such disruptions. UV224 seemed to have little effect on the integrity of the viral genome but produced a structural transformation of the viral capsid that may inhibit the delivery of viral genome into the host cell nucleus. On the other hand, UV254 and UV280 did not affect the integrity of the viral capsid, but the mutations they produced on the viral genome might cause the inhibition of the early gene transcription and DNA replication after the viral genome successfully translocated into the host cell nucleus.

Characterizing the effects of insertion of a 5.2 kb region of a VACV genome, which contains known immune evasion genes, on MVA immunogenicity.

Modified Vaccinia virus Ankara (MVA) is an attenuated Vaccinia virus (VACV) that is a popular vaccine vector candidate against many different pathogens. Its replication-restricted nature makes it a safe vaccine. However, higher doses or multiple boosts of MVA are necessary to elicit an immune response similar to wild-type VACV. Multiple strategies have been used to create modified MVA viruses that remain safe, but have increased immunogenicity. For example, one common strategy is to delete MVA immunomodulatory proteins in hopes of increasing the host immune response. Here, we take the opposite approach and examine, for the first time, how re-introduction of a 5.2 kb region of VACV DNA (that codes for multiple immunomodulatory proteins) into MVA alters viral immunogenicity. Since antigen presenting cells (APCs) are critical connectors between the innate and adaptive immune system, we examined the effect of MVA/5.2 kb infection in these cells in vitro. MVA/5.2 kb infection decreased virus-induced apoptosis and virus-induced NF-κB activation. MVA.5.2 kb infection decreased TNF production. However, MVA/5.2 kb infection did not alter APC maturation or IL-6 and IL-8 production in vitro. We further explored MVA/5.2 kb immunogenicity in vivo. VACV-specific CD8+ T cells were decreased after in vivo infection with MVA/5.2 kb versus MVA, suggesting that the MVA/5.2 kb construct is less immunogenic than MVA. These results demonstrate the limitations of in vitro studies for predicting the effects of genetic manipulation of MVA on immunogenicity. Although MVA/5.2 kb did not enhance MVA's immunogenicity, this study examined an unexplored strategy for optimizing MVA, and the insight gained from these results can help direct how to modify MVA in the future.

Cellular FLIP long isoform (cFLIPL)-IKKα interactions inhibit IRF7 activation, representing a new cellular strategy to inhibit IFNα expression.

Interferon α (IFNα) is important for antiviral and anticancer defenses. However, overproduction is associated with autoimmune disorders. Thus, the cell must precisely up- and down-regulate IFNα to achieve immune system homeostasis. The cellular FLICE-like inhibitory protein (cFLIP) is reported to inhibit IFNα production. However, the mechanism for this antagonism remained unknown. The goal here was to identify this mechanism. Here we examined the signal transduction events that occur during TLR9-induced IRF7 activation. The cFLIP long isoform (cFLIPL) inhibited the expression of IRF7-controlled natural or synthetic genes in several cell lines, including those with abundant IRF7 protein levels (e.g. dendritic cells). cFLIPL inhibited IRF7 phosphorylation; however, cFLIPL-IRF7 interactions were not detectable, implying that cFLIPL acted upstream of IRF7 dimerization. Interestingly, cFLIPL co-immunoprecipitated with IKKα, and these interactions correlated with a loss of IKKα-IRF7 interactions. Thus, cFLIP appears to bind to IKKα to prevent IKKα from phosphorylating and activating IRF7. To the best of our knowledge, this is the first report of a cellular protein that uses this approach to inhibit IRF7 activation. Perhaps this cFLIP property could be engineered to minimize the deleterious effects of IFNα expression that occur during certain autoimmune disorders.

Molluscum Contagiosum Virus MC159 Abrogates cIAP1-NEMO Interactions and Inhibits NEMO Polyubiquitination.

Molluscum contagiosum virus (MCV) is a dermatotropic poxvirus that causes benign skin lesions. MCV lesions persist because of virally encoded immune evasion molecules that inhibit antiviral responses. The MCV MC159 protein suppresses NF-κB activation, a powerful antiviral response, via interactions with the NF-κB essential modulator (NEMO) subunit of the IκB kinase (IKK) complex. Binding of MC159 to NEMO does not disrupt the IKK complex, implying that MC159 prevents IKK activation via an as-yet-unidentified strategy. Here, we demonstrated that MC159 inhibited NEMO polyubiquitination, a posttranslational modification required for IKK and downstream NF-κB activation. Because MCV cannot be propagated in cell culture, MC159 was expressed independent of infection or during a surrogate vaccinia virus infection to identify how MC159 prevented polyubiquitination. Cellular inhibitor of apoptosis protein 1 (cIAP1) is a cellular E3 ligase that ubiquitinates NEMO. Mutational analyses revealed that MC159 and cIAP1 each bind to the same NEMO region, suggesting that MC159 may competitively inhibit cIAP1-NEMO interactions. Indeed, MC159 prevented cIAP1-NEMO interactions. MC159 also diminished cIAP1-mediated NEMO polyubiquitination and cIAP1-induced NF-κB activation. These data suggest that MC159 competitively binds to NEMO to prevent cIAP1-induced NEMO polyubiquitination. To our knowledge, this is the first report of a viral protein disrupting NEMO-cIAP1 interactions to strategically suppress IKK activation. All viruses must antagonize antiviral signaling events for survival. We hypothesize that MC159 inhibits NEMO polyubiquitination as a clever strategy to manipulate the host cell environment to the benefit of the virus.IMPORTANCE Molluscum contagiosum virus (MCV) is a human-specific poxvirus that causes persistent skin neoplasms. The persistence of MCV has been attributed to viral downregulation of host cell immune responses such as NF-κB activation. We show here that the MCV MC159 protein interacts with the NEMO subunit of the IKK complex to prevent NEMO interactions with the cIAP1 E3 ubiquitin ligase. This interaction correlates with a dampening of cIAP1 to polyubiquitinate NEMO and to activate NF-κB. This inhibition of cIAP1-NEMO interactions is a new viral strategy to minimize IKK activation and to control NEMO polyubiquitination. This research provides new insights into mechanisms that persistent viruses may use to cause long-term infection of host cells.

Vaccinia virus K1 ankyrin repeat protein inhibits NF-κB activation by preventing RelA acetylation.

The vaccinia virus (VACV) K1 protein inhibits dsRNA-dependent protein kinase (PKR) activation. A consequence of this function is that K1 inhibits PKR-induced NF-κB activation during VACV infection. However, transient expression of K1 also inhibits Toll-like receptor (TLR)-induced NF-κB activation. This suggests that K1 has a second NF-κB inhibitory mechanism that is PKR-independent. This possibility was explored by expressing K1 independently of infection and stimulating NF-κB under conditions that minimized or excluded PKR activation. K1 inhibited both TNF- and phorbol 12-myristate 13-acetate (PMA)-induced NF-κB activation, as detected by transcription of synthetic (e.g. luciferase) and natural (e.g. CXCL8) genes controlled by NF-κB. K1 also inhibited NF-κB activity in PKRkd cells, cells that have greatly decreased amounts of PKR. K1 no longer prevented IκBα degradation or NF-κB nuclear translocation in the absence of PKR, suggesting that K1 acted on a nuclear event. Indeed, K1 was present in the nucleus and cytoplasm of stimulated and unstimulated cells. K1 inhibited acetylation of the RelA (p65) subunit of NF-κB, a nuclear event known to be required for NF-κB activation. Moreover, p65-CBP (CREB-binding protein) interactions were blocked in the presence of K1. However, K1 did not preclude NF-κB binding to oligonucleotides containing κB-binding sites. The current interpretation of these data is that NF-κB-promoter interactions still occur in the presence of K1, but NF-κB cannot properly trigger transcriptional activation because K1 antagonizes acetylation of RelA. Thus, in comparison to all known VACV NF-κB inhibitory proteins, K1 acts at one of the most downstream events of NF-κB activation.

cFLIPL Interrupts IRF3-CBP-DNA Interactions To Inhibit IRF3-Driven Transcription.

Type I IFN induction is critical for antiviral and anticancer defenses. Proper downregulation of type I IFN is equally important to avoid deleterious imbalances in the immune response. The cellular FLIP long isoform protein (cFLIPL) controls type I IFN production, but opposing publications show it as either an inhibitor or inducer of type I IFN synthesis. Regardless, the mechanistic basis for cFLIPL regulation is unknown. Because cFLIPL is important in immune cell development and proliferation, and is a target for cancer therapies, it is important to identify how cFLIPL regulates type I IFN production. Data in this study show that cFLIPL inhibits IFN regulatory factor 3 (IRF3), a transcription factor central for IFN-ß and IFN-stimulated gene expression. This inhibition occurs during virus infection, cellular exposure to polyinosinic-polycytidylic acid, or TBK1 overexpression. This inhibition is independent of capase-8 activity. cFLIPL binds to IRF3 and disrupts IRF3 interaction with its IFN-ß promoter and its coactivator protein (CREB-binding protein). Mutational analyses reveal that cFLIPL nuclear localization is necessary and sufficient for inhibitory function. This suggests that nuclear cFLIPL prevents IRF3 enhanceosome formation. Unlike other cellular IRF3 inhibitors, cFLIPL did not degrade or dephosphorylate IRF3. Thus, cFLIPL represents a different cellular strategy to inhibit type I IFN production. This new cFLIPL function must be considered to accurately understand how cFLIPL affects immune system development and regulation.

Characterizing Bacteriophage PR772 as a Potential Surrogate for Adenovirus in Water Disinfection: A Comparative Analysis of Inactivation Kinetics and Replication Cycle Inhibition by Free Chlorine.

Elucidating mechanisms by which pathogenic waterborne viruses become inactivated by drinking water disinfectants would facilitate the development of sensors to detect infectious viruses and novel disinfection strategies to provide safe water. Using bacteriophages as surrogates for human pathogenic viruses could assist in elucidating these mechanisms; however, an appropriate viral surrogate for human adenovirus (HAdV), a medium-sized virus with a double-stranded DNA genome, needs to be identified. Here, we characterized the inactivation kinetics of bacteriophage PR772, a member of the Tectiviridae family with many similarities in structure and replication to HAdV. The inactivation of PR772 and HAdV by free chlorine had similar kinetics that could be represented with a model previously developed for HAdV type 2 (HAdV-2). We developed and tested a quantitative assay to analyze several steps in the PR772 replication cycle to determine if both viruses being inactivated at similar rates resulted from similar replication cycle events being inhibited. Like HAdV-2, we observed that PR772 inactivated by free chlorine still attached to host cells, and viral DNA synthesis and early and late gene transcription were inhibited. Consequently, free chlorine exposure inhibited a replication cycle event that was post-binding but took place prior to early gene synthesis for both PR772 and HAdV-2.

Kaposi's Sarcoma-Associated Herpesvirus (KSHV) Induces the Oncogenic miR-17-92 Cluster and Down-Regulates TGF-ß Signaling.

KSHV is a DNA tumor virus that causes Kaposi's sarcoma. Upon KSHV infection, only a limited number of latent genes are expressed. We know that KSHV infection regulates host gene expression, and hypothesized that latent genes also modulate the expression of host miRNAs. Aberrant miRNA expression contributes to the development of many types of cancer. Array-based miRNA profiling revealed that all six miRNAs of the oncogenic miR-17-92 cluster are up-regulated in KSHV infected endothelial cells. Among candidate KSHV latent genes, we found that vFLIP and vCyclin were shown to activate the miR-17-92 promoter, using luciferase assay and western blot analysis. The miR-17-92 cluster was previously shown to target TGF-ß signaling. We demonstrate that vFLIP and vCyclin induce the expression of the miR-17-92 cluster to strongly inhibit the TGF-ß signaling pathway by down-regulating SMAD2. Moreover, TGF-ß activity and SMAD2 expression were fully restored when antagomirs (inhibitors) of miR-17-92 cluster were transfected into cells expressing either vFLIP or vCyclin. In addition, we utilized viral genetics to produce vFLIP or vCyclin knock-out viruses, and studied the effects in infected TIVE cells. Infection with wildtype KSHV abolished expression of SMAD2 protein in these endothelial cells. While single-knockout mutants still showed a marked reduction in SMAD2 expression, TIVE cells infected by a double-knockout mutant virus were fully restored for SMAD2 expression, compared to non-infected TIVE cells. Expression of either vFLIP or vCycIin was sufficient to downregulate SMAD2. In summary, our data demonstrate that vFLIP and vCyclin induce the oncogenic miR-17-92 cluster in endothelial cells and thereby interfere with the TGF-ß signaling pathway. Manipulation of the TGF-ß pathway via host miRNAs represents a novel mechanism that may be important for KSHV tumorigenesis and angiogenesis, a hallmark of KS.

Analysis of the viral replication cycle of adenovirus serotype 2 after inactivation by free chlorine.

Free chlorine is effective at inactivating a wide range of waterborne viral pathogens including human adenovirus (HAdV), but the mechanisms by which free chlorine inactivates HAdV and other human viruses remain to be elucidated. Such advances in fundamental knowledge are key for development of new disinfection technologies and novel sensors to detect infectious viruses in drinking water. We developed and tested a quantitative assay to analyze several steps in the HAdV replication cycle upon increasing free chlorine exposure. We used quantitative polymerase chain reaction (qPCR) to detect HAdV genomic DNA as a means to quantify attachment and genome replication of untreated and treated virions. Also, we used quantitative reverse-transcription PCR (RT-qPCR) to quantify the transcription of E1A (first early protein) and hexon mRNA. We compared these replication cycle events to virus inactivation kinetics to determine what stage of the virus replication cycle was inhibited as a function of free chlorine exposure. We observed that adenovirus inactivated at levels up to 99.99% by free chlorine still attached to host cells; however, viral DNA synthesis and early E1A and late hexon gene transcription were inhibited. We conclude that free chlorine exposure interferes with a replication cycle event occurring postbinding but prior to early viral protein synthesis.

Myxoma virus expressing a fusion protein of interleukin-15 (IL15) and IL15 receptor alpha has enhanced antitumor activity.

Myxoma virus, a rabbit poxvirus, can efficiently infect various types of mouse and human cancer cells. It is a strict rabbit-specific pathogen, and is thought to be safe as a therapeutic agent in all non-rabbit hosts tested including mice and humans. Interleukin-15 (IL15) is an immuno-modulatory cytokine with significant potential for stimulating anti-tumor T lymphocytes and NK cells. Co-expression of IL15 with the α subunit of IL15 receptor (IL15Rα) greatly enhances IL15 stability and bioavailability. Therefore, we engineered a new recombinant myxoma virus (vMyx-IL15Rα-tdTr), which expresses an IL15Rα-IL15 fusion protein plus tdTomato red fluorescent reporter protein. Permissive rabbit kidney epithelial (RK-13) cells infected with vMyx-IL15Rα-tdTr expressed and secreted the IL15Rα-IL15 fusion protein. Functional activity was confirmed by demonstrating that the secreted fusion protein stimulated proliferation of cytokine-dependent CTLL-2 cells. Multi-step growth curves showed that murine melanoma (B16-F10 and B16.SIY) cell lines were permissive to vMyx-IL15Rα-tdTr infection. In vivo experiments in RAG1-/- mice showed that subcutaneous B16-F10 tumors treated with vMyx-IL15Rα-tdTr exhibited attenuated tumor growth and a significant survival benefit for the treated group compared to the PBS control and the control viruses (vMyx-IL15-tdTr and vMyx-tdTr). Immunohistological analysis of the subcutaneous tumors showed dramatically increased infiltration of NK cells in vMyx-IL15Rα-tdTr treated tumors compared to the controls. In vivo experiments with immunocompetent C57BL/6 mice revealed a strong infiltrate of both NK cells and CD8+ T cells in response to vMyx-IL15Rα-tdTr, and prolonged survival. We conclude that delivery of IL15Rα-IL15 in a myxoma virus vector stimulates both innate and adaptive components of the immune system.

Inhibition of interferon gene activation by death-effector domain-containing proteins from the molluscum contagiosum virus.

Apoptosis, NF-κB activation, and IRF3 activation are a triad of intrinsic immune responses that play crucial roles in the pathogenesis of infectious diseases, cancer, and autoimmunity. FLIPs are a family of viral and cellular proteins initially found to inhibit apoptosis and more recently to either up- or down-regulate NF-κB. As such, a broad role for FLIPs in disease regulation is postulated, but exactly how a FLIP performs such multifunctional roles remains to be established. Here we examine FLIPs (MC159 and MC160) encoded by the molluscum contagiosum virus, a dermatotropic poxvirus causing skin infections common in children and immunocompromised individuals, to better understand their roles in viral pathogenesis. While studying their molecular mechanisms responsible for NF-κB inhibition, we discovered that each protein inhibited IRF3-controlled luciferase activity, identifying a unique function for FLIPs. MC159 and MC160 each inhibited TBK1 phosphorylation, confirming this unique function. Surprisingly, MC159 coimmunoprecipitated with TBK1 and IKKε but MC160 did not, suggesting that these homologs use distinct molecular mechanisms to inhibit IRF3 activation. Equally surprising was the finding that the FLIP regions necessary for TBK1 inhibition were distinct from those MC159 or MC160 regions previously defined to inhibit NF-κB or apoptosis. These data reveal previously unappreciated complexities of FLIPs, and that subtle differences within the conserved regions of FLIPs possess distinct molecular and structural fingerprints that define crucial differences in biological activities. A future comparison of mechanistic differences between viral FLIP proteins can provide new means of precisely manipulating distinct aspects of intrinsic immune responses.

The MC159 protein from the molluscum contagiosum poxvirus inhibits NF-κB activation by interacting with the IκB kinase complex.

Molluscum contagiosum virus (MCV) causes persistent neoplasms in healthy and immunocompromised people. Its ability to persist likely is due to its arsenal of viral immunoevasion proteins. For example, the MCV MC159 protein inhibits TNF-R1-induced NF-κB activation and apoptosis. The MC159 protein is a viral FLIP and, as such, possesses two tandem death effector domains (DEDs). We show in this article that, in human embryonic kidney 293 T cells, the expression of wild-type MC159 or a mutant MC159 protein containing the first DED (MC159 A) inhibited TNF-induced NF-κB, or NF-κB activated by PMA or MyD88 overexpression, whereas a mutant protein lacking the first DED (MC159 B) did not. We hypothesized that the MC159 protein targeted the IκB kinase (IKK) complex to inhibit these diverse signaling events. Indeed, the MC159 protein, but not MC159 B, coimmunoprecipitated with IKKγ. MC159 coimmunoprecipitated with IKKγ when using mouse embryonic fibroblasts that lack either IKKα or IKKß, suggesting that the MC159 protein interacted directly with IKKγ. MC159-IKKγ coimmunoprecipitations were detected during infection of cells with either MCV isolated from human lesions or with a recombinant MC159-expressing vaccinia virus. MC159 also interacts with TRAF2, a signaling molecule involved in NF-κB activation. However, mutational analysis of MC159 failed to reveal a correlation between MC159-TRAF2 interactions and MC159's inhibitory function. We propose that MC159-IKK interactions, but not MC159-TRAF2 interactions, are responsible for inhibiting NF-κB activation.

Viral double-stranded RNAs from vaccinia virus early or intermediate gene transcripts possess PKR activating function, resulting in NF-kappaB activation, when the K1 protein is absent or mutated.

PKR is a potent antiviral molecule that can terminate infection by inhibiting protein synthesis and stimulating NF-κB activation and apoptosis. Originally, it was thought that only intermediate and late gene transcription produced double-stranded (ds) RNA to activate PKR during vaccinia virus (VACV) infection. The VACV E3 or K3 proteins squelch this effect by binding to either dsRNA or PKR. However, in the absence of the K1 protein, VACV infection activates PKR at very early times post-infection and despite the presence of E3 and K3. These data suggest that VACV infection induces PKR activation by a currently unknown mechanism. To determine this mechanism, cells were infected with K1L-containing or -deficient VACVs. By using conditions that limited the progression of the poxvirus replication cycle, we observed that early gene transcripts activated PKR in RK13 cells, identifying a new PKR-activating mechanism of poxvirus infection. Using a similar approach for HeLa cells, intermediate gene transcription was sufficient to activate PKR. RNA isolated from infected RK13 or HeLa cells maintained PKR-activating properties only when dsRNA was present. Moreover, viral dsRNA was directly detected in infected cells either by RT-PCR or immunofluorescent microscopy. Interestingly, dsRNA levels were higher in infected cells in which the K1 protein was nonfunctional. Only K1 proteins with PKR inhibitory function prevented downstream NF-κB activation. These results reveal a new PKR activation pathway during VACV infection, in which the K1 protein reduces dsRNA levels early in VACV infection to directly inhibit PKR and several of its downstream antiviral effects, thereby enhancing virus survival.

Mechanistic aspects of adenovirus serotype 2 inactivation with free chlorine.

Free chlorine is an effective disinfectant for controlling adenoviruses in drinking water, but little is known about the underlying inactivation mechanisms. The objective of this study was to elucidate the molecular components of adenovirus type 2 (Ad2) targeted by free chlorine during the inactivation process. The effects of free chlorine treatment on several Ad2 molecular components and associated life cycle events were compared to its effect on the ability of adenovirus to complete its life cycle, i.e., viability. Free chlorine treatment of Ad2 virions did not impair their ability to interact with monoclonal antibodies specific for hexon and fiber proteins of the Ad2 capsid, as measured by enzyme-linked immunosorbent assays, nor did it impair their interaction with recombinant, purified Coxsackie-adenovirus receptor (CAR) proteins in vitro. Free chlorine-treated Ad2 virions also retained their ability to bind to CAR receptors on A549 cell monolayers, despite being unable to form plaques, suggesting that free chlorine inactivates Ad2 by inhibiting a postbinding event of the Ad2 life cycle. DNA isolated from Ad2 virions that had been inactivated by free chlorine was able to be amplified by PCR, indicating that genome damage was not the cause of inactivation. However, inactivated Ad2 virions were unable to express E1A viral proteins during infection of A549 host cells, as measured by using immunoblotting. Collectively, these results indicate that free chlorine inactivates adenovirus by damaging proteins that govern life cycle processes occurring after host cell attachment, such as endocytosis, endosomal lysis, or nuclear delivery.

Kinetics of adenovirus type 2 inactivation with free chlorine.

The objective of this study was to elucidate the effects of pH, temperature, and other relevant water quality parameters on the kinetics of adenovirus serotype 2 inactivation with free chlorine. Over a pH range of 6.5-10, a temperature range of 1-30 degrees C, and in a variety of water types, free chlorine was highly effective against adenovirus type 2. Its disinfection efficacy decreased with increasing pH and decreasing temperature, yet was unaffected by hardness and buffering species. Under the most challenging conditions investigated in this study (pH 10, 1 degrees C), a four-log reduction of adenovirus viability would be achieved at a CT value of 2.6mgCl(2)min/L. The inactivation kinetics was characterized by three phases of inactivation under most conditions. The first phase resulted from a reaction involving primarily the hypochlorous acid species and was characterized by rapid inactivation of viruses to a limit that increased with decreasing pH and increasing temperature. After reaching this limit, adenovirus exhibited two subsequent phases of inactivation at lesser rates that were not affected by temperature or pH. As with the first phase of kinetics, a limit of inactivation was approached in the second phase that decreased with increasing pH, and after which the kinetics was characterized by a third and final phase. An inactivation model consistent with these observations was found to provide adequate representation for the free chlorine inactivation of adenovirus serotype 2 as well as that reported in the literature for other adenovirus serotypes.