Influenza A virus replication is inhibited in IFN-λ2 and IFN-λ3 transfected or stimulated cells

Interferons lambda (IFN-λ) are the most recently defined members of the class III cytokine family. To investigate whether IFN-λ2 and IFN-λ3 displayed antiviral activity against influenza A virus (IAV), a number of cell lines induced with IFNs - as well as two established cell lines (A549-IFN-λ2 and A549-IFN-λ3) - were infected with IAV. Our results indicate that IFN-λ2 has statistically significant antiviral activity in A549-IFN-λ2 (P=0.0028) although less so than IFN-λ3, which reduced viral titer to 10% (P<0.0001). The reverse was observed for cells treated with IFNs, with IFN-λ2-treated A549 cells inhibiting IAV infection more efficiently than IFN-λ3-treated A549 cells. The antiviral effect on IFN-stimulated cells was most apparent on Vero cells (compared with MDCK and HeLa). Both IFNs significantly inhibited IAV replication and inhibition was observed in a dose-dependent manner, with an optimal IFN concentration of 20 ng/ml. IFN-λ2 was more potent than IFN-λ3 on Vero cells while IFN-λ3 appeared more efficient than IFN-λ2 on MDCK and HeLa cells.     

Antiviral activity of type I interferons and interleukins 29 and 28a (type III interferons) against Apeu virus

Interferons (IFNs) are cytokines with important immunomodulatory activity in vertebrates. Although type I IFNs and interleukins (IL) 29 and 28a (type III IFNs) bind to different cellular receptors and have distinct structures, most of their biological activities are redundant. Apeu virus (APEUV) is a member of the Bunyaviridae family isolated from the Brazilian rain forest. In this paper we evaluated the antiviral activity of type I and type III IFNs against APEUV. All tested IFNs were able to induce an antiviral state against the virus in a dose-dependent way. The activity of type III IFNs did not need the presence of type I IFNs. Mixing both types of IFNs did not improve the biological activity of each type alone. The tested IFNs were also able to protect human peripheral blood mononuclear cells from infection. IFN alpha2, IFN beta, IL-29 and IL-28a induced the expression of 2′,5′-oligoadenylate synthetase (2′5′OAS) and 6–16 genes. Although MxA gene was related to antiviral activity against Bunyaviruses, there was no induction of MxA in our model. We were able to show activity of type I and type III IFNs against a RNA virus, and that this activity is not dependent on MxA gene.     

Antiviral activity of type I interferons and interleukins 29 and 28a (type III interferons) against Apeu virus

Interferons (IFNs) are cytokines with important immunomodulatory activity in vertebrates. Although type I IFNs and interleukins (IL) 29 and 28a (type III IFNs) bind to different cellular receptors and have distinct structures, most of their biological activities are redundant. Apeu virus (APEUV) is a member of the Bunyaviridae family isolated from the Brazilian rain forest. In this paper we evaluated the antiviral activity of type I and type III IFNs against APEUV. All tested IFNs were able to induce an antiviral state against the virus in a dose-dependent way. The activity of type III IFNs did not need the presence of type I IFNs. Mixing both types of IFNs did not improve the biological activity of each type alone. The tested IFNs were also able to protect human peripheral blood mononuclear cells from infection. IFN alpha2, IFN beta, IL-29 and IL-28a induced the expression of 2′,5′-oligoadenylate synthetase (2′5′OAS) and 6–16 genes. Although MxA gene was related to antiviral activity against Bunyaviruses, there was no induction of MxA in our model. We were able to show activity of type I and type III IFNs against a RNA virus, and that this activity is not dependent on MxA gene.     

Influenza A virus replication is inhibited in IFN-λ2 and IFN-λ3 transfected or stimulated cells

Interferons lambda (IFN-λ) are the most recently defined members of the class III cytokine family. To investigate whether IFN-λ2 and IFN-λ3 displayed antiviral activity against influenza A virus (IAV), a number of cell lines induced with IFNs – as well as two established cell lines (A549-IFN-λ2 and A549-IFN-λ3) – were infected with IAV. Our results indicate that IFN-λ2 has statistically significant antiviral activity in A549-IFN-λ2 (P = 0.0028) although less so than IFN-λ3, which reduced viral titer to 10% (P < 0.0001). The reverse was observed for cells treated with IFNs, with IFN-λ2-treated A549 cells inhibiting IAV infection more efficiently than IFN-λ3-treated A549 cells. The antiviral effect on IFN-stimulated cells was most apparent on Vero cells (compared with MDCK and HeLa). Both IFNs significantly inhibited IAV replication and inhibition was observed in a dose-dependent manner, with an optimal IFN concentration of 20 ng/ml. IFN-λ2 was more potent than IFN-λ3 on Vero cells while IFN-λ3 appeared more efficient than IFN-λ2 on MDCK and HeLa cells.     

Interference of porcine reproductive and respiratory syndrome virus replication on MARC-145 cells using DNA-based short interfering RNAs

Porcine reproductive and respiratory syndrome (PRRS) is an economically important disease in swine-producing areas of the world. Many vaccine strategies developed to control the disease are not yet completely successful. The objective of this study was to determine if RNA interference (RNAi) could be utilized to inhibit PRRSV replication on MARC-145 cells. Four short interfering RNA (siRNA) sequences (N95, N179, N218 and N294) directed against a well-conserved region of PRRSV genome ORF7 gene were selected. Sense and antisense siRNA encode sequences separated by a hairpin loop sequence were designed as short hairpin RNA (shRNA) expression cassettes driven by mouse U6 promoter. Using a polymerase chain reaction (PCR)-based approach, shRNAs were generated from shRNA expression cassettes. The PCR products were cloned into pEGFP-N1 vector and shRNA expression vectors were constructed. When MARC-145 cells were transfected with shRNA expression vectors and then infected with PRRSV, N179 was found to be the most effective inhibition site in decreasing cytopathic effect (CPE) induced by PRRSV. Western blot, indirect immunofluorescence and fluorescence quantitative PCR (FQ-PCR) confirmed that the expression of ORF7 was reduced both at protein and RNA levels comparing to controls. The results presented here indicated that DNA-based siRNA could effectively inhibit the replication of PRRS virus (approximately 681-fold reduction of viral titers) on MARC-145 cells.     

Toll-like receptor 7-mediated enhancement of contextual fear memory in mice

Toll-like receptor (TLR) 7 recognizes viral single-stranded RNA and triggers production of the type I interferons (IFNs) IFN-α and IFN-β. Imiquimod, a synthetic TLR7 ligand, induces production of type I IFNs and is used clinically as an antiviral and antitumor drug. In the present study, we examined the effect of imiquimod on conditioned and innate fear behaviors in mice. Imiquimod was administered 2, 4, or 15 h before contextual fear conditioning. Imiquimod treatment 4 or 15 h before fear conditioning significantly enhanced context-dependent freezing behavior. This imiquimod-induced enhancement of fear-related behaviors was observed 120 h after fear conditioning. In contrast, imiquimod failed to enhance context-dependent freezing behavior in TLR7 knockout mice. Imiquimod had no significant effect on pain threshold or on innate fear-related behavior, as measured by the elevated plus-maze. The levels of type I IFN mRNA in the brain were significantly increased at 2 h after imiquimod treatment. Imiquimod also increased interleukin (IL)-1β mRNA expression in the brain at 4 h following administration, while mRNA expression of F4/80, a macrophage marker, was unaffected by imiquimod treatment. Our findings suggest that TLR7-mediated signaling enhances contextual fear memory in mice, possibly by inducing the expression of type I IFNs and IL-1β in the brain.     

TANK-binding kinase 1 as a novel therapeutic target for viral diseases

Introduction: TANK-binding kinase 1 (TBK1) is vital for the induction of antiviral innate immune responses. Both RNA and DNA viral infection induces TBK1 activation, triggers phosphorylation of interferon regulatory factor (IRF) 3 and subsequent expression of type I interferons (IFNs; IFN-α/β). Type I IFNs can induce the expression of numerous antiviral genes called interferon-stimulated genes (ISGs) to build a remarkable antiviral state and limit viral replication. Thus, optimal TBK1 activity is crucial for IRF3-induced type I IFNs expression and ISGs-mediated viral elimination.

Areas covered: This review provides an overview of the diverse roles of TBK1 in antiviral innate immune responses, the regulatory mechanisms of TBK1 activity and the implication in antiviral development.

Expert opinion: TBK1 is a key kinase against antiviral infection via inducing type I IFNs expression. Multiple types of post-translational modifications of TBK1 tightly regulate TBK1 activity and subsequent TBK1-dependent antiviral responses. The identified regulators of TBK1 unveil regulatory mechanisms of host antiviral innate immunity and immuno-escape mechanism of virus provide strategies to control viral diseases by modulating TBK1 activity.     

Anti-PRRSV effect and mechanism of sodium tanshinone IIA sulfonate in vitro

This experiment was conducted to study the antiviral activities of sodium tanshinone IIA sulfonate (STS) against porcine reproductive and respiratory syndrome virus (PRRSV) and its mechanism. Anti-PRRSV activities of STS were observed on Marc-145 cells by using visualization of cytopathologic effect assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test, and its antiviral mechanism was determined by time-of-addition assay, adsorption inhibition assay, and virucidal assay. The results showed that STS could reduce the damage of PRRSV to Marc-145 cells, with the inhibition ratio exceeding to 100%, at the maximum non-cytotoxic concentration. The time-of-addition and virucidal assays indicated that the anti-PRRSV activities of STS could be due to inhibiting the virus replication or/and inactivating the virus directly. The inhibition of the virus attachment was not discovered in adsorption inhibition assay. The results proved that STS had strong anti-PRRSV activity and encouraged for further exploration of STS.     

干扰素λ的药理学及临床应用研究进展

干扰素λ（IFN-λ）为一类新发现的Ⅲ型干扰素,隶属于Ⅱ型细胞因子家族,其异二聚体功能性受体复合物IL-28Rα/IL-10Rβ具有组织学分布特异性。IFN-λ主要通过JAK-STAT途径发挥其药理学活性,包括抗病毒、抗增殖及免疫调节作用。在Ⅰ期及Ⅱ期临床试验中IFN-λ产生的不良反应较IFN-α为少,因此在治疗肝炎及特异性抗肿瘤方面可能具有良好的应用前景。本文主要针对近年来IFN-λ的药理学活性及临床应用进行总结。     

Targeting IFN-λ: therapeutic implications

Introduction: Type-III interferons (IFN-λ), the most recently discovered family of IFNs, shares common features with other family members, but also has many distinctive activities. IFN-λ uniquely has a different receptor complex, and a more focused pattern of tissue expression and signaling effects, from other classes of IFNs. Multiple genome-wide association studies (GWAS) and subsequent validation reports suggest a pivotal role for polymorphisms near the IFNL3 gene in hepatitis C clearance and control, as also for several other epithelial cell tropic viruses. Apart from its antiviral activity, IFN-λ possesses anti-tumor, immune-inflammatory and homeostatic functions. The overlapping effects of IFN-λ with type I IFN, with a restricted tissue expression pattern renders IFN-λ an attractive therapeutic target for viral infection, cancer and autoimmune diseases, with limited side effects.

Areas covered: This review will summarize the current and future therapeutic opportunities offered by this most recently discovered family of interferons.

Expert opinion: Our knowledge on IFN-λ is rapidly expanding. Though there are many remaining questions and challenges that require elucidation, the unique characteristics of IFN-λ increases enthusiasm that multiple therapeutic options will emerge.     

Potent anti-HIV (type 1 and type 2) activity of polyoxometalates: structure-activity relationship and mechanism of action

A series of polyoxometalates have been synthesized and evaluated for their inhibitory effects on HIV-1(III(B)) and HIV-1(ROD) replication in MT-4 cells. All compounds showed activity against HIV-1 and HIV-2, but the antiviral potency of the heteropolytungstates varied considerably depending on their chemical structure. The antiviral activity of single, double, and triple Keggin-type of compounds against HIV-1(III(B)) replication was comparable (IC(50): 0.4-0.5 microgram/mL), whereas HIV-2(ROD) appeared to become less sensitive with the increasing number of Keggin structures per compound. The same trend was observed for single and double Dawson structures. Some of these compounds were examined for their inhibitory effect on the replication of HIV-1(RF) and SIV(MAC(251)) in MT-4 cells. Their anti-HIV-1(RF) and anti-SIV(MAC(251)) potencies were comparable to those for the HIV-1(III(B)) or HIV-2(ROD) strain, respectively. The polyoxometalates represent a class of polyanionic compounds, which block the binding of the envelope glycoprotein gp120 of HIV to CD4(+) cells. The compounds interfered with the binding of anti-CD4 mAb to the OKT4A/Leu3a epitope of the CD4 receptor, compound 24 being the most active in this regard, and inhibited the binding of anti-gp120 mAb to infected MT-4 cells. None of the polyoxometalates inhibited the binding of a specific CXCR4 mAb to SUP-T1 cells, suggesting that they do not interact with CXCR4, the main co-receptor for T-tropic HIV strains, and thus act as virus binding, and not as fusion, inhibitors.     

Novel plant-derived recombinant human interferons with broad spectrum antiviral activity

Type I interferons (IFNs) are potent mediators of the innate immune response to viral infection. IFNs released from infected cells bind to a receptor (IFNAR) on neighboring cells, triggering signaling cascades that limit further infection. Subtle variations in amino acids can alter IFNAR binding and signaling outcomes. We used a new gene crossbreeding method to generate hybrid, type I human IFNs with enhanced antiviral activity against four dissimilar, highly pathogenic viruses. Approximately 1400 novel IFN genes were expressed in plants, and the resultant IFN proteins were screened for antiviral activity. Comparing the gene sequences of a final set of 12 potent IFNs to those of parent genes revealed strong selection pressures at numerous amino acids. Using three-dimensional models based on a recently solved experimental structure of IFN bound to IFNAR, we show that many but not all of the amino acids that were highly selected for are predicted to improve receptor binding.     

Novel plant-derived recombinant human interferons with broad spectrum antiviral activity

Type I interferons (IFNs) are potent mediators of the innate immune response to viral infection. IFNs released from infected cells bind to a receptor (IFNAR) on neighboring cells, triggering signaling cascades that limit further infection. Subtle variations in amino acids can alter IFNAR binding and signaling outcomes. We used a new gene crossbreeding method to generate hybrid, type I human IFNs with enhanced antiviral activity against four dissimilar, highly pathogenic viruses. Approximately 1400 novel IFN genes were expressed in plants, and the resultant IFN proteins were screened for antiviral activity. Comparing the gene sequences of a final set of 12 potent IFNs to those of parent genes revealed strong selection pressures at numerous amino acids. Using three-dimensional models based on a recently solved experimental structure of IFN bound to IFNAR, we show that many but not all of the amino acids that were highly selected for are predicted to improve receptor binding.     

Ubiquitin specific protease 5 negatively regulates the IFNs-mediated antiviral activity via targeting SMURF1

Type I interferons are broadly used for antiviral therapy in clinical. However, the IFNs-mediated antiviral efficacy is commonly restricted by negative regulators. Here, we show that the ubiquitin-specific protease 5 (USP5) inhibits the IFNs-induced p-STAT1 activation (phosphorylation at tyrosine site of STAT1) and its downstream antiviral genes expression. We clarify that USP5 physically interacts with SMURF1 (Smad ubiquitination regulating factor 1) and IFNs signaling regulates the interaction and turnover of both proteins. USP5 enhances the stability and turnover of SMURF1 via decreasing its polyubiquitin expression level, which caused STAT1 to decrease. Importantly, USP5 is also involved in the SMURF1-mediated antiviral response, and its small-molecule inhibitor PYR41 remarkably enhances the IFNs antiviral efficacy. These findings reveal a previously unrecognized function of the USP5 and USP5-SMURF1 axis in regulating the IFNs-mediated antiviral activity.     

CK2 inhibitors increase the sensitivity of HSV-1 to interferon-β

Herpes simplex virus type 1 (HSV-1) requires the activities of cellular kinases for efficient replication. The host kinase, CK2, has been shown or is predicted to modify several HSV-1 proteins and has been proposed to affect one or more steps in the viral life cycle. Furthermore, potential cellular and viral substrates of CK2 are involved in antiviral pathways and viral counter-defenses, respectively, suggesting that CK2 regulates these processes. Consequently, we tested whether pharmacological inhibitors of CK2 impaired HSV-1 replication, either alone or in combination with the cellular antiviral factor, interferon-β (IFN-β). Our results indicate that the use of CK2 inhibitors results in a minor reduction in HSV-1 replication but enhanced the inhibitory effect of IFN-β on replication. This effect was dependent on the HSV-1 E3 ubiquitin ligase, infected cell protein 0 (ICP0), which impairs several host antiviral responses, including that produced by IFN-β. Inhibitors of CK2 did not, however, impede the ability of ICP0 to induce the degradation of two cellular targets: the promyelocytic leukemia protein (PML) and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Notably, this effect was only apparent for HSV-1, as the CK2 inhibitors did not enhance the antiviral effect of IFN-β on either vesicular stomatitis virus or adenovirus type 5. Thus, our data suggest that the activity of CK2 is required for an early function during viral infection that assists the growth of HSV-1 in IFN-β-treated cells.     

Anti-PRRSV effect and mechanism of tetrahydroaltersolanol C in vitro

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important arterivirus that causes substantial economic losses to the swine industry. Current control strategies against PRRSV are still inadequate and there is an urgent need for new antiviral therapies. Tetrahydroaltersolanol C (TD-C) is a new anthraquinone derivative isolated from the marine-derived fungi. In the present study, we first demonstrated its anti-PRRSV activity in vitro through assessing the inhibition of TD-C on cytopathic effect, viral ORF7 gene and N protein expressions, progeny virions production by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, relative-quantitative RT-PCR, Western blotting, and indirect immunofluorescence assay. Our experimental results showed that TD-C could significantly inhibit PRRSV replication in a dose-dependent manner. The 50% effective concentration, 50% cytotoxic concentration and the selectivity index were 12.11, 395.31 μM, and 32.64, respectively. Furthermore, the possible anti-PRRSV mechanism was explored by virucidal assay, virus adsorption inhibition assay, and the time-of-addition assay. The results showed that TD-C might inhibit the internalization and replication of PRRSV, but did not directly inactivate the virus or block its adsorption to cell surface. In conclusion, our findings indicated that TD-C possessed a significant anti-PRRSV activity, and provided a strong basis for further exploration of this compound as an antiviral agent against PRRSV.