NS3 of Bluetongue Virus Interferes with the Induction of Type I Interferon

Upon infection with Bluetongue virus (BTV), an arthropod-borne virus, type I interferon (IFN-I) is produced in vivo and in vitro. IFN-I is essential for the establishment of an antiviral cellular response, and most if not all viruses have elaborated strategies to counteract its action. In this study, we assessed the ability of BTV to interfere with IFN-I synthesis and identified the nonstructural viral protein NS3 as an antagonist of the IFN-I system.     

Constitutively Active MAVS Inhibits HIV-1 Replication via Type I Interferon Secretion and Induction of HIV-1 Restriction Factors

Type I interferon is known to inhibit HIV-1 replication through the induction of interferon stimulated genes (ISG), including a number of HIV-1 restriction factors. To better understand interferon-mediated HIV-1 restriction, we constructed a constitutively active form of the RIG-I adapter protein MAVS. Constitutive MAVS was generated by fusion of full length MAVS to a truncated form of the Epstein Barr virus protein LMP1 (ΔLMP1). Supernatant from ΔLMP1-MAVS-transfected 293T cells contained high levels of type I interferons and inhibited HIV replication in both TZM-bl and primary human CD4+ T cells. Supernatant from ΔLMP1-MAVS-transfected 293T cells also inhibited replication of VSV-G pseudotyped single cycle SIV in TZM-bl cells, suggesting restriction was post-entry and common to both HIV and SIV. Gene array analysis of ΔLMP1-MAVS-transfected 293T cells and trans-activated CD4+ T cells showed significant upregulation of ISG, including previously characterized HIV restriction factors Viperin, Tetherin, MxB, and ISG56. Interferon blockade studies implicated interferon-beta in this response. In addition to direct viral inhibition, ΔLMP1-MAVS markedly enhanced secretion of IFN-β and IL-12p70 by dendritic cells and the activation and maturation of dendritic cells. Based on this immunostimulatory activity, an adenoviral vector (Ad5) expressing ΔLMP1-MAVS was tested as a molecular adjuvant in an HIV vaccine mouse model. Ad5-Gag antigen combined with Ad5-ΔLMP1-MAVS enhanced control of vaccinia-gag replication in a mouse challenge model, with 4/5 animals showing undetectable virus following challenge. Overall, ΔLMP1-MAVS is a promising reagent to inhibit HIV-1 replication in infected tissues and enhance vaccine-mediated immune responses, while avoiding toxicity associated with systemic type I interferon administration.     

IFIT1负反馈上调干扰素β表达促进抗HSV-1病毒保护

Ⅰ型单纯疱疹病毒（HSV-1）是危害人类健康的常见病原体之一,能够通过受损皮肤或黏膜感染宿主细胞并引起多种疾病。HSV-1的侵入激活先天免疫模式识别受体,诱导干扰素β（IFN-β）的产生,通过表达干扰素刺激基因（ISG）发挥抗病毒功能。近年来,干扰素诱导的四肽重复蛋白1（IFIT1）在病毒感染过程中的作用引起了广大研究者的关注。然而,其具体机制尚未完全清楚。本研究利用CRISPR/Cas9技术构建了小鼠成纤维细胞（L929）IFIT1敲除细胞株,免疫印迹方法检测敲除细胞株IFIT1在蛋白质水平的表达。转染HT-DNA和Poly［I:C］刺激L929 WT和IFIT1敲除细胞株,实时定量PCR技术检测发现,HT-DNA刺激敲除细胞时,IFN-β及下游ISGs的表达量显著升高。IFN-β的表达量比 L929-WT组平均高出13.4倍,IFIT1和趋化因子10（CXC chemokine ligand-10,CXCL10）的表达量比L929 WT组分别平均高出 6.7倍和21倍(P<0.001）,而Poly［I:C］刺激无明显变化（P>0.05）,表明IFIT1是通过DNA信号通路来行使其负反馈调节作用。为研究IFIT1基因的抗病毒作用,利用CRISPR/Cas9技术改造的HSV-1-VP26 mCherry 病毒感染该敲除细胞株,通过测定病毒荧光数及病毒拷贝数,发现IFIT1敲除细胞株与L929 WT细胞相比,存活率提高了60%（P<0.001）,病毒增殖能力在48 h后降低28.6倍（P<0.001）。该结果表明,IFIT1基因的缺失有利于抵抗HSV-1的感染。综上所述,IFIT1通过DNA信号通路负反馈上调IFN-β及ISG的表达,IFIT1的缺失对病毒入侵发挥了保护作用。该结果为后续研究开发治疗HSV-1感染相关的治疗药物提供了一个新思路。     

Inducible Priming Phosphorylation Promotes Ligand-independent Degradation of the IFNAR1 Chain of Type I Interferon Receptor

Phosphorylation-dependent ubiquitination and ensuing down-regulation and lysosomal degradation of the interferon α/β receptor chain 1 (IFNAR1) of the receptor for Type I interferons play important roles in limiting the cellular responses to these cytokines. These events could be stimulated either by the ligands (in a Janus kinase-dependent manner) or by unfolded protein response (UPR) inducers including viral infection (in a manner dependent on the activity of pancreatic endoplasmic reticulum kinase). Both ligand-dependent and -independent pathways converge on phosphorylation of Ser⁵³⁵ within the IFNAR1 degron leading to recruitment of β-Trcp E3 ubiquitin ligase and concomitant ubiquitination and degradation. Casein kinase 1α (CK1α) was shown to directly phosphorylate Ser⁵³⁵ within the ligand-independent pathway. Yet given the constitutive activity of CK1α, it remained unclear how this pathway is stimulated by UPR. Here we report that induction of UPR promotes the phosphorylation of a proximal residue, Ser⁵³², in a pancreatic endoplasmic reticulum kinase-dependent manner. This serine serves as a priming site that promotes subsequent phosphorylation of IFNAR1 within its degron by CK1α. These events play an important role in regulating ubiquitination and degradation of IFNAR1 as well as the extent of Type I interferon signaling.     

Abrogation of the Interferon Response Promotes More Efficient Human Cytomegalovirus Replication

The effect of abrogating the interferon (IFN) response on human cytomegalovirus (HCMV) replication was investigated using primary human cells engineered to block either the production of or the response to type I IFNs. In IFN-deficient cells, HCMV produced larger plaques and spread and replicated more rapidly than in parental cells. These cells demonstrate the vital role of IFNs in controlling HCMV replication and provide useful tools to investigate the IFN response to HCMV.     

Disruption of Type I Interferon Induction by HIV Infection of T Cells

Our main objective of this study was to determine how Human Immunodeficiency Virus (HIV) avoids induction of the antiviral Type I Interferon (IFN) system. To limit viral infection, the innate immune system produces important antiviral cytokines such as the IFN. IFN set up a critical roadblock to virus infection by limiting further replication of a virus. Usually, IFN production is induced by the recognition of viral nucleic acids by innate immune receptors and subsequent downstream signaling. However, the importance of IFN in the defense against viruses has lead most pathogenic viruses to evolve strategies to inhibit host IFN induction or responses allowing for increased pathogenicity and persistence of the virus. While the adaptive immune responses to HIV infection have been extensively studied, less is known about the balance between induction and inhibition of innate immune defenses, including the antiviral IFN response, by HIV infection. Here we show that HIV infection of T cells does not induce significant IFN production even IFN I Interferon production. To explain this paradox, we screened HIV proteins and found that two HIV encoded proteins, Vpu and Nef, strongly antagonize IFN induction, with expression of these proteins leading to loss of expression of the innate immune viral RNA sensing adaptor protein, IPS-1 (IFN-β promoter stimulator-1). We hypothesize that with lower levels of IPS-1 present, infected cells are defective in mounting antiviral responses allowing HIV to replicate without the normal antiviral actions of the host IFN response. Using cell lines as well as primary human derived cells, we show that HIV targeting of IPS-1 is key to limiting IFN induction. These findings describe how HIV infection modulates IFN induction providing insight into the mechanisms by which HIV establishes infection and persistence in a host.     

Toscana Virus NSs Protein Inhibits the Induction of Type I Interferon by Interacting with RIG-I

Toscana virus (TOSV) is a phlebovirus, of the Bunyaviridae family, that is responsible for central nervous system (CNS) injury in humans. Previous data have shown that the TOSV NSs protein is a gamma interferon (IFN-β) antagonist when transiently overexpressed in mammalian cells, inhibiting IRF-3 induction (G. Gori Savellini, F. Weber, C. Terrosi, M. Habjan, B. Martorelli, and M. G. Cusi, J. Gen. Virol. 92:71–79, 2011). In this study, we investigated whether an upstream sensor, which has a role in the signaling cascade leading to the production of type I IFN, was involved. We found a significant decrease in RIG-I protein levels in cells overexpressing TOSV NSs, suggesting that the nonstructural protein interacts with RIG-I and targets it for proteasomal degradation. In fact, the MG-132 proteasome inhibitor was able to restore IFN-β promoter activation in cells expressing NSs, demonstrating the existence of an evasion mechanism based on inhibition of the RIG-I sensor. Furthermore, a C-terminal truncated NSs protein (ΔNSs), although able to interact with RIG-I, did not affect the RIG-I-mediated IFN-β promoter activation, suggesting that the NSs domains responsible for RIG-I-mediated signaling and interaction with RIG-I are mapped on different regions. These results contribute to identify a novel mechanism for bunyaviruses by which TOSV NSs counteracts the early IFN response.     

A Versatile Vector for In Vivo Monitoring of Type I Interferon Induction and Signaling

Development of reporter systems for in vivo examination of IFN-β induction or signaling of type I interferon (IFN-I) pathways is of great interest in order to characterize biological responses to different inducers such as viral infections. Several reporter mice have been developed to monitor the induction of both pathways in response to different agonists. However, alternative strategies that do not require transgenic mice breeding have to date not been reported. In addition, detection of these pathways in vivo in animal species other than mice has not yet been addressed. Herein we describe a simple method based on the use of an adeno-associated viral vector (AAV8-3xIRF-ISRE-Luc) containing an IFN-β induction and signaling-sensitive promoter sequence controlling the expression of the reporter gene luciferase. This vector is valid for monitoring IFN-I responses in vivo elicited by diverse stimuli in different organs. Intravenous administration of the vector in C57BL/6 mice and Syrian hamsters was able to detect activation of the IFN pathway in the liver upon systemic treatment with different pro-inflammatory agents and infection with Newcastle disease virus (NDV). In addition, intranasal instillation of AAV8-3xIRF-ISRE-Luc showed a rapid and transient IFN-I response in the respiratory tract of mice infected with the influenza A/PR8/34 virus lacking the NS1 protein. In comparison, this response was delayed and exacerbated in mice infected with influenza A/PR/8 wild type virus. In conclusion, the AAV8-3xIRF-ISRE-Luc vector offers the possibility of detecting IFN-I activation in response to different stimuli and in different animal models with no need for reporter transgenic animals.     

BUD13 Promotes a Type I Interferon Response by Countering Intron Retention in Irf7

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Gossypol Interferes with Both Type I and Type II Topoisomerase Activities Without Generating Strand Breaks

A considerable number of agents with chemotherapeutic potentials reported over the past years were shown to interfere with the reactions of DNA topoisomerases, the essential enzymes that regulate conformational changes in DNA topology. Gossypol, a naturally occurring bioactive phytochemical is a chemopreventive agent against various types of cancer cell growth with a reported activity on mammalian topoisomerase II. The compounds targeting topoisomerases vary in their mode of action; class I compounds act by stabilizing covalent topoisomerase-DNA complexes resulting in DNA strand breaks while class II compounds interfere with the catalytic function of topoisomerases without generating strand breaks. In this study, we report Gossypol as the interfering agent with type I topoisomerases as well. We also carried out an extensive set of assays to analyze the type of interference manifested by Gossypol on DNA topoisomerases. Our results strongly suggest that Gossypol is a potential class II inhibitor as it blocked DNA topoisomerase reactions with no consequently formed strand breaks.