Cyclosporin A Inhibits Rotavirus Replication and Restores Interferon-Beta Signaling Pathway In Vitro and In Vivo

Rotavirus (RV) is the most common cause of severe diarrhea among infants and young children. Currently, there is no specific drug available against rotavirus, largely due to the lack of an ideal target molecule which has hampered drug development. Our previous studies have revealed that cyclosporin A (CsA) might be potentially useful as an anti-RV drug. We therefore used both cellular and mouse models to study the immunological safety and effectiveness of CsA as an anti-RV drug. We found that CsA treatment of HT-29 cells before, during, and after viral infection efficiently inhibited Wa strain RV replication and restored IFN-β expression in a HT-29 cell line model. Exploring the underlying mechanisms showed that CsA promoted Interferon Regulatory Factor-5 (IRF-5) expression (a key positive regulator of the type I IFN signaling pathway), but not IRF-1, IRF-3, or IRF-7. Additionally, CsA inhibited SOCS-1 expression (the key negative regulator of IFN-α/β), but not SOCS-2 or SOCS-3. The antiviral effect of CsA was confirmed in an RV-infected neonatal mouse model by evaluation of antigen clearance and assessment of changes in intestinal tissue pathology. Also, no differences in T cell frequency or proliferation between the CsA- and vehicle-treated groups were observed. Thus, both our in vitro and in vivo findings suggest that CsA, through modulating the expression of key regulators in IFN signaling pathway, promote type I IFN-based intracellular innate immunity in RV host cells. These findings suggest that CsA may be a useful candidate to develop a new anti-RV strategy, although further evaluation and characterization of CsA on RV-induced diarrhea are warranted.     

Polygonum cuspidatum and Its Active Components Inhibit Replication of the Influenza Virus through Toll-Like Receptor 9-Induced Interferon Beta Expression

Influenza virus infection is a global public health issue. The effectiveness of antiviral therapies for influenza has been limited by the emergence of drug-resistant viral strains. Therefore, there is an urgent need to identify novel antiviral therapies. Here we tested the effects of 300 traditional Chinese medicines on the replication of various influenza virus strains in a lung cell line, A549, using an influenza-specific luciferase reporter assay. Of the traditional medicines tested, Polygonum cuspidatum (PC) and its active components, resveratrol and emodin, were found to attenuate influenza viral replication in A549 cells. Furthermore, they preferentially inhibited the replication of influenza A virus, including clinical strains isolated in 2009 and 2011 in Taiwan and the laboratory strain A/WSN/33 (H1N1). In addition to inhibiting the expression of hemagglutinin and neuraminidase, PC, emodin, and resveratrol also increased the expression of interferon beta (IFN-β) through Toll-like receptor 9 (TLR9). Moreover, the anti-viral activity of IFN-β or resveratrol was reduced when the A549 cells were treated with neutralizing anti-IFN-β antibodies or a TLR9 inhibitor, suggesting that IFN-β likely acts synergistically with resveratrol to inhibit H1N1 replication. This potential antiviral mechanism, involving direct inhibition of virus replication and simultaneous activation of the host immune response, has not been previously described for a single antiviral molecule. In conclusion, our data support the use of PC, resveratrol or emodin for inhibiting influenza virus replication directly and via TLR-9–induced IFN-β production.     

HSP70 and Its Cochaperone CPIP Promote Potyvirus Infection in Nicotiana benthamiana by Regulating Viral Coat Protein Functions

This study demonstrates that heat shock protein 70 (HSP70) together with its cochaperone CPIP regulates the function of a potyviral coat protein (CP), which in turn can interfere with viral gene expression. HSP70 was copurified as a component of a membrane-associated viral ribonucleoprotein complex from Potato virus A–infected plants. Downregulation of HSP70 caused a CP-mediated defect associated with replication. When PVA CP was expressed in trans, it interfered with viral gene expression and replication-associated translation (RAT). However, CP produced in cis interfered specifically with RAT. CPIP binds to potyviral CP, and overexpression of CPIP was sufficient to restore RAT inhibited by expression of CP in trans. Restoration of RAT was dependent on the ability of CPIP to interact with HSP70 since expression of a J-domain mutant, CPIP^Δ66, had only a minor effect on RAT. CPIP-mediated delivery of CP to HSP70 promoted CP degradation by increasing its ubiquitination when assayed in the absence of virus infection. In conclusion, CPIP and HSP70 are crucial components of a distinct translation activity that is associated with potyvirus replication.     

Budesonide and formoterol effects on rhinovirus replication and epithelial cell cytokine responses

Background

Combination therapy with budesonide and formoterol reduces exacerbations of asthma, which are closely associated with human rhinovirus (RV) infections in both children and adults. These data suggest that budesonide and formoterol inhibit virus-induced inflammatory responses of airway epithelial cells.

Methods

To test this hypothesis, bronchial epithelial (BE) cells were obtained from airway brushings of 8 subjects with moderate-to-severe allergic asthma and 9 with neither asthma nor respiratory allergies. Cultured BE cells were incubated for 24 hours with budesonide (1.77 μM), formoterol (0.1 μM), both, or neither, and then inoculated with RV-16 (5×10⁶ plaque forming units [PFU]/mL). After 24 hours, viral replication (RV RNA), cytokine secretion (CXCL8, CXCL10, TNFα, IFN-β, IL-28) and mRNA expression (CXCL8, CXCL10, TNF, IFNB1, IL28A&B) were analyzed.

Results

RV infection induced CXCL10 protein secretion and IFNB1 and IL28 mRNA expression. Drug treatments significantly inhibited secretion of CXCL10 in mock-infected, but not RV-infected, BE cells, and inhibited secretion of TNFα under both conditions. Neither budesonide nor formoterol, alone or in combination, significantly affected viral replication, nor did they inhibit RV-induced upregulation of IFNB1 and IL28 mRNA. Overall, RV replication was positively related to CXCL10 secretion and induction of IFNB1 and IL28 mRNA, but the positive relationship between RV RNA and CXCL10 secretion was stronger in normal subjects than in subjects with asthma.

Conclusions

Budesonide and formoterol can inhibit BE cell inflammatory responses in vitro without interfering with viral replication or production of interferons. These effects could potentially contribute to beneficial effects of budesonide/formoterol combination therapy in preventing RV-induced asthma exacerbations.     

The zinc finger antiviral protein acts synergistically with an interferon-induced factor for maximal activity against alphaviruses

Type I interferons (IFNs) signal through specific receptors to mediate expression of genes, which together confer a cellular antiviral state. Overexpression of the zinc finger antiviral protein (ZAP) imparts a cellular antiviral state against Retroviridae, Togaviridae, and Filoviridae virus family members. Since ZAP expression is induced by IFN, we utilized Sindbis virus (SINV) to investigate the role of other IFN-induced factors in ZAP's inhibitory potential. Overexpressed ZAP did not inhibit virion production or SINV-induced cell death in BHK cells deficient in IFN production (and thus IFN signaling), suggesting a role for an IFN-induced factor in ZAP's activity. IFN pretreatment in the presence of ZAP resulted in greater inhibition than IFN alone. Using mouse embryo fibroblast (MEF) cells deficient in Stat1, we showed that signaling through the IFN receptor is necessary for IFN′s enhancement of ZAP activity. Unlike in BHK cells, however, overexpressed ZAP exhibited antiviral activity in the absence of IFN. In wild-type MEFs with an intact Stat1 gene, IFN pretreatment synergized with ZAP to generate a potent antiviral response. Despite failing to inhibit SINV virion production and virus-induced cell death in BHK cells, ZAP inhibited translation of the incoming viral RNA. IFN pretreatment synergized with ZAP to further block protein expression from the incoming viral genome. We further show that silencing of IFN-induced ZAP reduces IFN efficacy. Our findings demonstrate that ZAP can synergize with another IFN-induced factor(s) for maximal antiviral activity and that ZAP's intrinsic antiviral activity on virion production and cell survival can have cell-type-specific outcomes.     

Cloning of an orange-spotted grouper Epinephelus coioides heat shock protein 90AB (HSP90AB) and characterization of its expression in response to nodavirus

The heat shock proteins (HSPs) family which consists of HSP90, HSP70, and low molecular mass HSPs are involved in chaperone activity. Here, we report the cloning and characterization of HSP90AB gene from orange-spotted grouper, Epinephelus coioides. The full-length of grouper HSP90AB was 727 amino acids and possessed an ATPase domain as well as an evolutionarily conserved molecular chaperone. The HSP90AB-green fluorescent protein fusion protein was evenly distributed in the cytoplasm. Immunohistochemistry (IHC) and real-time polymerase chain reaction (PCR) analyses indicated that the expression of grouper HSP90AB was marginally increased following nodavirus infection. Grouper E. coioides that received HSP90 inhibitor geldanamycin (GA) showed an increase in HSP90AB expression and growth of nodavirus supporting nodavirus replication.     

Cellular growth kinetics distinguish a cyclophilin inhibitor from an HSP90 inhibitor as a selective inhibitor of hepatitis C virus

During antiviral drug discovery, it is critical to distinguish molecules that selectively interrupt viral replication from those that reduce virus replication by adversely affecting host cell viability. In this report we investigate the selectivity of inhibitors of the host chaperone proteins cyclophilin A (CypA) and heat-shock protein 90 (HSP90) which have each been reported to inhibit replication of hepatitis C virus (HCV). By comparing the toxicity of the HSP90 inhibitor, 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG) to two known cytostatic compounds, colchicine and gemcitabine, we provide evidence that 17-AAG exerts its antiviral effects indirectly through slowing cell growth. In contrast, a cyclophilin inhibitor, cyclosporin A (CsA), exhibited selective antiviral activity without slowing cell proliferation. Furthermore, we observed that 17-AAG had little antiviral effect in a non-dividing cell-culture model of HCV replication, while CsA reduced HCV titer by more than two orders of magnitude in the same model. The assays we describe here are useful for discriminating selective antivirals from compounds that indirectly affect virus replication by reducing host cell viability or slowing cell growth.     

Gene- and Protein-Delivered Zinc Finger–Staphylococcal Nuclease Hybrid for Inhibition of DNA Replication of Human Papillomavirus

Previously, we reported that artificial zinc-finger proteins (AZPs) inhibited virus DNA replication in planta and in mammalian cells by blocking binding of a viral replication protein to its replication origin. However, the replication mechanisms of viruses of interest need to be disentangled for the application. To develop more widely applicable methods for antiviral therapy, we explored the feasibility of inhibition of HPV-18 replication as a model system by cleaving its viral genome. To this end, we fused the staphylococcal nuclease cleaving DNA as a monomer to an AZP that binds to the viral genome. The resulting hybrid nuclease (designated AZP–SNase) cleaved its target DNA plasmid efficiently and sequence-specifically in vitro. Then, we confirmed that transfection with a plasmid expressing AZP–SNase inhibited HPV-18 DNA replication in transient replication assays using mammalian cells. Linker-mediated PCR analysis revealed that the AZP–SNase cleaved an HPV-18 ori plasmid around its binding site. Finally, we demonstrated that the protein-delivered AZP–SNase inhibited HPV-18 DNA replication as well and did not show any significant cytotoxicity. Thus, both gene- and protein-delivered hybrid nucleases efficiently inhibited HPV-18 DNA replication, leading to development of a more universal antiviral therapy for human DNA viruses.     

PPEY motif within the rabies virus (RV) matrix protein is essential for efficient virion release and RV pathogenicity

Late (L) domains containing the highly conserved sequence PPXY were first described for retroviruses, and later research confirmed their conservation and importance for efficient budding of several negative-stranded RNA viruses. Rabies virus (RV), a member of the Rhabdoviridae family, contains the sequence PPEY (amino acids 35 to 38) within the N terminus of the matrix (M) protein, but the functions of this potential L-domain in the viral life cycle, viral pathogenicity, and immunogenicity have not been established. Here we constructed a series of recombinant RVs containing mutations within the PPEY motif and analyzed their effects on viral replication and RV pathogenicity. Our results indicate that the first proline at position 35 is the most important for viral replication, whereas P36 and Y38 have a lesser but still noticeable impact. The reduction in viral replication was most likely due to inhibition of virion release, because initially no major impact on RV RNA synthesis was observed. In addition, results from electron microscopy demonstrated that the M4A mutant virus (PPEY→SAEA) displayed a more cell-associated phenotype than that of wild-type RV. Furthermore, all mutations within the PPEY motif resulted in reduced spread of the recombinant RVs as indicated by a reduction in focus size. Importantly, recombinant PPEY L-domain mutants were highly attenuated in mice yet still elicited potent antibody responses against RV G protein that were as high as those observed after infection with wild-type virus. Our data indicate that the RV PPEY motif has L-domain activity essential for efficient virus production and pathogenicity but is not essential for immunogenicity and thus can be targeted to increase the safety of rabies vaccine vectors.     

Involvement of p32 and microtubules in alteration of mitochondrial functions by rubella virus

The interaction of the rubella virus (RV) capsid (C) protein and the mitochondrial p32 protein is believed to participate in virus replication. In this study, the physiological significance of the association of RV with mitochondria was investigated by silencing p32 through RNA interference. It was demonstrated that downregulation of p32 interferes with microtubule-directed redistribution of mitochondria in RV-infected cells. However, the association of the viral C protein with mitochondria was not affected. When cell lines either pretreated with respiratory chain inhibitors or cultivated under (mild) hypoxic conditions were infected with RV, viral replication was reduced in a time-dependent fashion. Additionally, RV infection induces increased activity of mitochondrial electron transport chain complex III, which was associated with an increase in the mitochondrial membrane potential. These effects are outstanding among the examples of mitochondrial alterations caused by viruses. In contrast to the preferential localization of p32 to the mitochondrial matrix in most cell lines, RV-permissive cell lines were characterized by an almost exclusive membrane association of p32. Conceivably, this contributes to p32 function(s) during RV replication. The data presented suggest that p32 fulfills an essential function for RV replication in directing trafficking of mitochondria near sites of viral replication to meet the energy demands of the virus.     

The UL13 and US3 Protein Kinases of Herpes Simplex Virus 1 Cooperate to Promote the Assembly and Release of Mature,Infectious Virions

Herpes simplex virus type 1 (HSV-1) encodes two bona fide serine/threonine protein kinases, the US3 and UL13 gene products. HSV-1 ΔUS3 mutants replicate with wild-type efficiency in cultured cells, and HSV-1 ΔUL13 mutants exhibit <10-fold reduction in infectious viral titers. Given these modest phenotypes, it remains unclear how the US3 and UL13 protein kinases contribute to HSV-1 replication. In the current study, we designed a panel of HSV-1 mutants, in which portions of UL13 and US3 genes were replaced by expression cassettes encoding mCherry protein or green fluorescent protein (GFP), respectively, and analyzed DNA replication, protein expression, and spread of these mutants in several cell types. Loss of US3 function alone had largely negligible effect on viral DNA accumulation, gene expression, virion release, and spread. Loss of UL13 function alone also had no appreciable effects on viral DNA levels. However, loss of UL13 function did result in a measurable decrease in the steady-state levels of two viral glycoproteins (gC and gD), release of total and infectious virions, and viral spread. Disruption of both genes did not affect the accumulation of viral DNA, but resulted in further reduction in gC and gD steady-state levels, and attenuation of viral spread and infectious virion release. These data show that the UL13 kinase plays an important role in the late phase of HSV-1 infection, likely by affecting virion assembly and/or release. Moreover, the data suggest that the combined activities of the US3 and UL13 protein kinases are critical to the efficient assembly and release of infectious virions from HSV-1-infected cells.     

Heat shock protein 70 is necessary for Rice stripe virus infection in plants

Heat shock proteins 70 (HSP70s) are a highly conserved family of genes in eukaryotes, and are involved in a remarkable variety of cellular processes. In many plant positive‐stranded RNA viruses, HSP70 participates in the construction of a viral replication complex and plays various roles during viral infection. Here, we found increased expression of HSP70 following infection by Rice stripe virus (RSV), a negative‐stranded RNA virus, in both rice (the natural host) and Nicotiana benthamiana (an experimental host). Heat treatment of N. benthamiana (Nb) plants enhanced viral infection, whereas RSV infection was retarded and viral RNAs accumulated at a low level when HSP70 was silenced. In both bimolecular fluorescence complement and in vitro pull‐down assays, the N‐terminus of RSV RNA‐dependent RNA polymerase (RdRp) interacted and co‐localized with the HSP70s of both plants (OsHSP70 and NbHSP70). The localization of the N‐terminus of RdRp when expressed alone was not obviously different from when it was co‐expressed with OsHSP or NbHSP, and vice versa. RSV infection also had no effect on the localization of host HSP70. These results demonstrate that host HSP70 is necessary for RSV infection and probably plays a role in viral replication by interacting with viral RdRp, which provides the first evidence of an interacting host protein related to RSV replication, which has been little studied to date.     

Rotavirus-induced IFN-β promotes anti-viral signaling and apoptosis that modulate viral replication in intestinal epithelial cells

Rotavirus (RV), a leading cause of diarrhea, primarily infects intestinal epithelial cells (IEC). Rotavirus-infected IEC produce IFN-β and express hundreds of IFN-dependent genes. We thus hypothesized that type 1 IFN plays a key role in helping IEC limit RV replication and/or protect against cell death. To test this hypothesis, we examined IEC (HT29 cells) infected with RV (MOI 1) ± neutralizing antibodies to IFN-α/β via microscopy and SDS-PAGE immunoblotting. We hypothesized that neutralization of IFN would be clearly detrimental to RV-infected IEC. Rather, we observed that blockade of IFN function rescued IEC from the apoptotic cell death that otherwise would have occurred 24-48?h following exposure to RV. This resistance to cell death correlated with reduced levels of viral replication at early time points (< 8 h) following infection and eventuated in reduced production of virions. The reduction in RV replication that resulted from IFN neutralization correlated with, and could be recapitulated by, blockade of IFN-induced protein kinase R (PKR) activation, suggesting involvement of this kinase. Interestingly, pharmacologic blockade of caspase activity ablated RV-induced apoptosis and dramatically increased viral protein synthesis, suggesting that IFN-induced apoptosis helps to control RV infection. These results suggest non-mutually exclusive possibilities that IFN signaling is usurped by RV to promote early replication and induction of cell death may be a means by which IFN signaling possibly clears RV from the intestine.     

Resveratrol Inhibits Enterovirus 71 Replication and Pro-Inflammatory Cytokine Secretion in Rhabdosarcoma Cells through Blocking IKKs/NF-κB Signaling Pathway

Polydatin and resveratrol, as major active components in Polygonum cuspidatum, have anti-inflammatory, antioxidant and antitumor functions. However, the effect and mechanism of polydatin and resveratrol on enterovirus 71 (EV71) have not been reported. In this study, resveratrol revealed strong antiviral activity on EV71, while polydatin had weak effect. Neither polydatin nor resveratrol exhibited influence on viral attachment. Resveratrol could effectively inhibit the synthesis of EV71/VP1 and the phosphorylation of IKKα, IKKβ, IKKγ, IKBα, NF-κB p50 and NF-κB p65, respectively. Meanwhile, the remarkably increased secretion of IL-6 and TNF-α in EV71-infected rhabdosarcoma (RD) cells could be blocked by resveratrol. These results demonstrated that resveratrol inhibited EV71 replication and cytokine secretion in EV71-infected RD cells through blocking IKKs/NF-κB signaling pathway. Thus, resveratrol may have potent antiviral effect on EV71 infection.