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A Convenient and Biosafe Replicon with Accessory Genes of SARS-CoV-2 and Its Potential Application in Antiviral Drug Discovery

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Virologica Sinica 2021年第5期36卷 913-923页

作者： Yun-Yun Jin Hanwen Lin Liu Cao Wei-Chen Wu Yanxi Ji Liubing Du Yiling Jiang Yanchun Xie Kuijie Tong Fan Xing Fuxiang Zheng Mang Shi Ji-An Pan Xiaoxue Peng Deyin Guo The center for infection and immunity Study School of MedicineSun Yat-sen UniversityGuangming Science CityShenzhen 518107China

SARS-CoV-2 causes the pandemic of COVID-19 and no effective drugs for this disease are available thus *** to the high infectivity and pathogenicity of this virus,all studies on the live virus are strictly confined in the biosafety level 3(BSL3)laboratory but this would hinder the basic research and antiviral drug development of SARS-CoV-2 because the BSL3 facility is not commonly available and the work in the containment is costly and *** this study,we constructed a reverse genetics system of SARS-CoV-2 by assembling the viral cDNA in a bacterial artificial chromosome(BAC)vector with deletion of the spike(S)*** of the cDNA into cells results in the production of an RNA replicon that keeps the capability of genome or subgenome replication but is deficient in virion assembly and infection due to the absence of S ***,such a replicon system is not infectious and can be used in ordinary biological *** confirmed the efficient replication of the replicon by demonstrating the expression of the subgenomic RNAs which have similar profiles to the wild-type *** mutational analysis of nsp12 and nsp14,we showed that the RNA polymerase,exonuclease,and cap N7 methyltransferase play essential roles in genome replication and sgRNA *** also created a SARS-CoV-2 replicon carrying a luciferase reporter gene and this system was validated by the inhibition assays with known anti-SARS-CoV-2 ***,such a one-plasmid system is biosafe and convenient to use,which will benefit both fundamental research and development of antiviral drugs.

关键词： SARS-CoV-2 Reverse genetics Replicon Antiviral drug screening

A balancing act between IFITM3 and IRF3

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Cellular & Molecular Immunology 2018年第10期15卷 873-874页

作者： Temet M McMichael Mahesh Chemudupati Jacob S Yount Department of Microbial infection and immunity Center for Microbial Interface BiologyThe Ohio State UniversityColumbusOH 43210USA

Interferon(IFN)-induced transmembrane protein 3(IFITM3)is a potent antiviral factor capable of restricting numerous virus infections.1 Viruses inhibited by IFITM3 generally enter cells via endocytosis,and are trafficked to IFITM3-positive intracellular membrane compartments.1,2 IFITM3-positive compartments are acidic and stain positive for a variety of endosomal,lysosomal and autophagosomal markers.2,3 Although the membrane fusion of many endocytic viruses,such as influenza virus,is triggered by acidic pH,fusion is blocked in the presence of IFITM3,and virions are degraded2,4,5(Figure 1).It has been proposed that IFITM3 alters membrane properties,including rigidity and curvature,to inhibit virus membrane fusion.5 The molecular mechanism underlying these IFITM3-induced membrane alterations is not well characterized,and whether IFITM3 has other cellular functions is also not known.

关键词： IFITM3 inhibited alterations

In search of a pan-coronavirus vaccine:next-generation vaccine design and immune mechanisms

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Cellular & Molecular Immunology 2024年第2期21卷 103-118页

作者： S.Cankat M.U.Demael L.Swadling Division of infection and immunity Institute of Immunity and TransplantationUniversity College LondonPears BuildingLondon NW32PPUK

Members of the coronaviridae family are endemic to human populations and have caused several epidemics and pandemics in recent *** this review,we will discuss the feasibility of and progress toward the ultimate goal of creating a pan-coronavirus vaccine that can protect against infection and disease by all members of the coronavirus *** will detail the unmet clinical need associated with the continued transmission of SARS-CoV-2,MERS-CoV and the four seasonal coronaviruses(HCoV-OC43,NL63,HKU1 and 229E)in humans and the potential for future zoonotic *** will highlight how first-generation SARS-CoV-2 vaccines and natural history studies have greatly increased our understanding of effective antiviral immunity to coronaviruses and have informed next-generation vaccine *** will then consider the ideal properties of a pan-coronavirus vaccine and propose a blueprint for the type of immunity that may offer ***,we will describe a subset of the diverse technologies and novel approaches being pursued with the goal of developing broadly or universally protective vaccines for coronaviruses.

关键词： Vaccine Coronavirus Cross-reactive immunity Cross-protection Universal

Identification of a CD4 T-cell epitope in the hemagglutinin stalk domain of pandemic H 1N 1 influenza virus and its antigen-driven TCR usage signature in BALB/c mice

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Cellular & Molecular Immunology 2017年第6期14卷 511-520页

作者： I-Na Lu Sophie Farinelle Aurelie Sausy Claude P Muller Department of infection and immunity Luxembourg Institute of Health Esch-sur-Alzette L-4354 Luxembourg

The stalk region of the influenza virus hemagglutinin is relatively well conserved compared with the globular head domain, which makes it a potential target for use as a universal vaccine against influenza. However, the role of CD4 T cells in the hemagglutinin stalk-specific immune response is not clear. Here we identified a mouse CD4 T-cell epitope that encompasses residues HA2113-131 from the hemagglutinin stalk domain after a sub-lethal infection of influenza. In response to stimulation with the identified epitope, splenocytes derived from the infected mice showed significant polyfunctionality as shown by IL-2, TNF-a and IFN-7 production as well as degranulation. Moreover, mice immunized with the peptide corresponding to this CD4 T-cell epitope exhibited interindividual sharing of the CD4 T-cell receptor I~ sequences, and they had a higher survival rate following a challenge with a lethal dose of pandemic H IN 1 influenza virus. Thus, our data demonstrated a crucial role of hemagglutinin stalk-specific CD4 T cells in the host immune resoonse a^ainst influenza virus infection.

关键词： H1N1 hemagglutinin influenza T-cell receptor

Analyses of SELEX-derived ZAP-binding RNA aptamers suggest that the binding specificity is determined by both structure and sequence of the RNA

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Protein & Cell 2010年第8期1卷 752-759页

作者： Zhi Huang Xinlu Wang Guangxia Gao Key Laboratory of infection and immunity Institute of BiophysicsChinese Academy of SciencesBeijing 100101China

The zinc-finger antiviral protein(ZAP)is a host factor that specifically inhibits the replication of certain viruses,including murine leukemia virus,Sindbis virus and Ebola virus,by targeting the viral mRNAs for *** directly binds to the target viral mRNA and recruits the cellular RNA degradation machinery to degrade the *** significant sequence similarity or obvious common motifs have been found in the so far identified target viral *** minimum length of the target sequence is about 500 nt *** workable ZAP-binding RNAs should facilitate further studies on the ZAP-RNA interaction and characterization of such RNAs may provide some insights into the underlying *** this study,we used the SELEX method to isolate ZAP-binding RNA *** 21 rounds of selection,ZAP-binding aptamers were *** analysis revealed that they are G-rich RNAs with predicted stem-loop structures containing conserved“GGGUGG”and“GAGGG”motifs in the loop *** of the aptamer sequence into a luciferase reporter failed to render the reporter sensitive to ***,overexpression of the aptamers modestly but significantly reduced ZAP’s antiviral *** of the conserved motifs of the aptamers significantly impaired their ZAP-binding ability and ZAP-antagonizing activity,suggesting that the RNA sequence is important for specific interaction between ZAP and the target *** aptamers identified in this report should provide useful tools to further investigate the details of the interaction between ZAP and the target RNAs.

关键词： zinc-finger antiviral protein(ZAP) SELEX RNA aptamers

Generation and Application of a Luciferase Reporter Virus Based on Yellow Fever Virus 17D

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Virologica Sinica 2021年第6期36卷 1456-1464页

作者： Dan Li Jing-Long Ye Zhong-Yu Liu The Centre for infection and immunity Studies School of MedicineSun Yat-Sen UniversityGuangzhou 510006China

Yellow fever virus(YFV) is a re-emerging virus that can cause life-threatening yellow fever disease in *** the availability of an effective vaccine,little is known about the replication mechanism of YFV,and there are still no available specific anti-YFV ***,by introducing the Renilla luciferase gene(Rluc) into an infectious clone of YFV vaccine strain 17 D,we generated a recombinant virus 17 D-Rluc.2 A via reverse genetics *** 17 D-Rluc.2 A had similar plaque morphology and comparable in vitro growth characteristics with its parental ***,the reporter luciferase was efficiently expressed in 17 D-Rluc.2 A-infected mammalian and mosquito cells,and there was a good linear correlation between intracellular luciferase expression and extracellular infectious virion ***,by a combination of the 17 D-Rluc.2 A reporter virus and selective 2’-hydroxyl acylation analyzed by primer extension(SHAPE)technology,the conserved 5’-SLA element was shown to be essential for YFV replication,highlighting the capability of17 D-R1 uc.2 A in the investigation of YFV *** last,we demonstrated that two compounds with distinct anti-viral mechanisms can effectively inhibit the viral propagation in 17 D-Rluc.2 A-infected cells,demonstrating its potential application in the evaluation of anti-viral *** together,the 17 D-Rluc.2 A serves as a useful tool for the study of YFV replication and anti-YFV medicine development.

关键词： Yellow fever virus(YFV) 17D Reporter virus Cis-acting RNA elements Anti-viral medicines

DEXH-Box protein DHX30 is required for optimal function of the zinc-finger antiviral protein

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Protein & Cell 2010年第10期1卷 956-964页

作者： Peiying Ye Shufeng Liu Yiping Zhu Guifang Chen Guangxia Gao center for infection and immunity Institute of BiophysicsChinese Academy of SciencesBeijing 100101China Graduate School of Chinese Academy of Sciences Beijing 100039China

The zinc-finger antiviral protein(ZAP)is a host factor that specifically inhibits the replication of certain viruses by eliminating viral mRNAs in the *** previous studies,we demonstrated that ZAP directly binds to the viral mRNAs and recruits the RNA exosome to degrade the target *** this article,we provide evidence that a DEXH box RNA helicase,DHX30,is required for optimal antiviral activity of ***-down and co-immunoprecipitation assays demonstrated that DHX30 and ZAP interacted with each other via their N terminal *** of DHX30 with shRNAs reduced ZAP’s antiviral *** data implicate that DHX30 is a cellular factor involved in the antiviral function of ZAP.

关键词： zinc-finger antiviral protein RNA helicase DHX30

The hepatitis B virus-associated tumor microenvironment in hepatocellular carcinoma

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National Science Review 2014年第3期1卷 396-412页

作者： Pengyuan Yang Geoffrey J.Markowitz Xiao-Fan Wang Department of Pharmacology and Cancer Biology Duke University Medical Center CAS Key Laboratory of infection and immunity Institute of Biophysics Chinese Academy of Sciences

In contrast to a majority of cancer types, the initiation of hepatocellular carcinoma(HCC) is intimately associated with a chronically diseased liver tissue, with one of the most prevalent etiological factors being hepatitis B virus(HBV). Transformation of the liver in HBV-associated HCC oten follows from or accompanies long-term symptoms of chronic hepatitis, inlammation and cirrhosis, and viral load is a strong predictor for both incidence and progression of HCC. Besides aiding in transformation, HBV plays a crucial role in modulating the accumulation and activation of both cellular components of the microenvironment,such as immune cells and ibroblasts, and non-cellular components of the microenvironment, such as cytokines and growth factors, markedly inluencing disease progression and prognosis. his review will explore some of these components and mechanisms to demonstrate both underlying themes and the inherent complexity of these interacting systems in the initiation, progression, and metastasis of HBV-positive HCC.

关键词： hepatitis B hepatocellular carcinoma tumor microenvironment leukocytes cytokines growth factors

Innate and adaptive T cells in influenza disease

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Frontiers of Medicine 2018年第1期12卷 34-47页

作者： Simone Nfissing Sneha Sant Marios Koutsakos Kanta Subbarao Thi H.O. Nguyen Katherine Kedzierska Department of Microbiology and Immunology University of Melbourne at the Peter Doherty Institute for Infection and Immunity Parkville 3010 Victoria Australia World Health Organisation (WHO) Collaborating Centre for Reference and Research on Influenza at the Peter Doherty Institute for Infection and Immunity Melbourne 3000 Victoria Australia

Influenza is a major global health problem, causing infections of the respiratory tract, often leading to acute pneumonia, life-threatening complications and even deaths. Over the last seven decades, vaccination strategies have been utilized to protect people from complications of influenza, especially groups at high risk of severe disease. While current vaccination regimens elicit strain-specific antibody responses, they fail to generate cross-protection against seasonal, pandemic and avian viruses. Moreover, vaccines designed to generate influenza- specific T-cell responses are yet to be optimized. During natural infection, viral replication is initially controlled by innate immunity before adaptive immune responses （T cells and antibody-producing B cells） achieve viral clearance and host recovery. Adaptive T and B cells maintain immunological memory and provide protection against subsequent infections with related influenza viruses. Recent studies also shed light on the role of innate T- cells （MAIT cells, y~ T cells, and NKT cells） in controlling influenza and linking innate and adaptive immune mechanisms, thus making them attractive targets for vaccination strategies. We summarize the current knowledge on influenza-specific innate MAIT and γδ T cells as well as adaptive CD8＋ and CD4＋ T cells, and discuss how these responses can be harnessed by novel vaccine strategies to elicit cross-protective immunity against different influenza strains and subtypes.

关键词： influenza innate T cells CD4+ and CD8+ T cells vaccination