番茄黄化曲叶病毒的快速分子检测

番茄黄化曲叶病毒是当前世界范围内危害番茄生产的毁灭性病害。文章针对番茄黄化曲叶病毒全基因组序列的特异区段自主设计了1对特异性PCR引物(上游引物TYLCV-F:5′-ACGCATGCCTCTAATCCAGTGTA-3′,下游引物TYLCV-R:5′-CCAATAAGGCGTAAGCGTGTAGAC-3′),依据PCR扩增特异片段543 bp的有无可以快速、准确、高效、特异地检测出是否感染了TYLCV病毒,这项技术可以方便地应用到工厂化育苗的带毒性检测、蔬菜大规模生产中植株发病情况的快速检测以及抗病毒育种,从而为蔬菜安全可持续生产提供科技支撑。     

番茄黄化曲叶病毒的快速分子检测

番茄黄化曲叶病毒是当前世界范围内危害番茄生产的毁灭性病害。文章针对番茄黄化曲叶病毒全基因组序列的特异区段自主设计了1对特异性PCR引物(上游引物TYLCV-F：5′-ACGCATGCCTCTAATCCAGTGTA-3′, 下游引物TYLCV-R：5′-CCAATAAGGCGTAAGCGTGTAGAC-3′), 依据PCR扩增特异片段543 bp的有无可以快速、准确、高效、特异地检测出是否感染了TYLCV病毒, 这项技术可以方便地应用到工厂化育苗的带毒性检测、蔬菜大规模生产中植株发病情况的快速检测以及抗病毒育种, 从而为蔬菜安全可持续生产提供科技支撑。     

Replication of tomato yellow leaf curl virus (TYLCV) DNA in agroinoculated leaf discs from selected tomato genotypes

The leaf disc agroinoculation system was applied to study tomato yellow leaf curl virus (TYLCV) replication in explants from susceptible and resistant tomato genotypes. This system was also evaluated as a potential selection tool in breeding programmes for TYLCV resistance. Leaf discs were incubated with a head-to-tail dimer of the TYLCV genome cloned into the Ti plasmid ofAgrobacterium tumefaciens. In leaf discs from susceptible cultivars (Lycopersicon esculentum) TYLCV single-stranded genomic DNA and its double-stranded DNA forms appeared within 2–5 days after inoculation. Whiteflies (Bemisia tabaci) efficiently transmitted the TYLCV disease to tomato test plants following acquisition feeding on agroinoculated tomato leaf discs. This indicates that infective viral particles have been produced and have reached the phloem cells of the explant where they can be acquired by the insects. Plants regenerated from agroinfected leaf discs of sensitive tomato cultivars exhibited disease symptoms and contained TYLCV DNA concentrations similar to those present in field-infected tomato plants, indicating that TYLCV can move out from the leaf disc into the regenerating plant. Leaf discs from accessions of the wild tomato species immune to whitefly-mediated inoculation,L. chilense LA1969 andL. hirsutum LA1777, did not support TYLCV DNA replication. Leaf discs from plants tolerant to TYLCV issued from breeding programmes behaved like leaf discs from susceptible cultivars.The Hebrew University of Jerusalem, Faculty of Agriculture, Department of Field and Vegetable Crops     

SARS coronavirus papain-like protease inhibits the type I interferon signaling pathway through interaction with the STING-TRAF3-TBK1 complex

SARS coronavirus (SARS-CoV) develops an antagonistic mechanism by which to evade the antiviral activities of interferon (IFN). Previous studies suggested that SARS-CoV papain-like protease (PLpro) inhibits activation of the IRF3 pathway, which would normally elicit a robust IFN response, but the mechanism(s) used by SARS PLpro to inhibit activation of the IRF3 pathway is not fully known. In this study, we uncovered a novel mechanism that may explain how SARS PLpro efficiently inhibits activation of the IRF3 pathway. We found that expression of the membrane-anchored Plpro domain (PLpro-TM) from SARS-CoV inhibits STING/TBK1/IKK?-mediated activation of type I IFNs and disrupts the phosphorylation and dimerization of IRF3, which are activated by STING and TBK1. Meanwhile, we showed that PLpro-TM physically interacts with TRAF3, TBK1, IKK?, STING, and IRF3, the key components that assemble the STING-TRAF3-TBK1 complex for activation of IFN expression. However, the interaction between the components in STING-TRAF3-TBK1 complex is disrupted by PLpro-TM. Furthermore, SARS PLpro-TM reduces the levels of ubiquitinated forms of RIG-I, STING, TRAF3, TBK1, and IRF3 in the STING-TRAF3- TBK1 complex. These results collectively point to a new mechanism used by SARS-CoV through which Plpro negatively regulates IRF3 activation by interaction with STING-TRAF3-TBK1 complex, yielding a SARS-CoV countermeasure against host innate immunity.     

Host GRXC6 restricts Tomato yellow leaf curl virus infection by inhibiting the nuclear export of the V2 protein

Geminiviruses cause serious symptoms and devastating losses in crop plants. With a circular, single-stranded DNA genome, geminiviruses multiply their genomic DNA in the nucleus, requiring the nuclear shuttling of viral proteins and viral genomic DNAs. Many host factors, acting as proviral or antiviral factors, play key roles in geminivirus infections. Here, we report the roles of a tomato glutaredoxin (GRX), SlGRXC6, in the infection of Tomato yellow leaf curl virus (TYLCV), a single-component geminivirus. The V2 protein of TYLCV specifically and preferentially interacts with SlGRXC6 among the 55-member tomato GRX family that are broadly involved in oxidative stress responses, plant development, and pathogen responses. We show that overexpressed SlGRXC6 increases the nuclear accumulation of V2 by inhibiting its nuclear export and, in turn, inhibits trafficking of the V1 protein and viral genomic DNA. Conversely, the silenced expression of SlGRXC6 leads to an enhanced susceptibility to TYLCV. SlGRXC6 is also involved in symptom development as we observed a positive correlation where overexpression of SlGRXC6 promotes while knockdown of SlGRXC6 expression inhibits plant growth. We further showed that SlGRXC6 works with SlNTRC80, a tomato NADPH-dependent thioredoxin reductase, to regulate plant growth. V2 didn’t interact with SlNTRC80 but competed with SlNTR80 for binding to SlGRXC6, suggesting that the V2-disrupted SlGRXC6-SlNTRC80 interaction is partially responsible for the virus-caused symptoms. These results suggest that SlGRXC6 functions as a host restriction factor that inhibits the nuclear trafficking of viral components and point out a new way to control TYLCV infection by targeting the V2-SlGRXC6 interaction.     

Nuclear import of the capsid protein of tomato yellow leaf curl virus (TYLCV) in plant and insect cells

The tomato yellow leaf curl virus (TYLCV) found in Israel is a whitefly-transmitted monopartite geminivirus. Although geminiviruses have been found in the nuclei of phloem-associated cells, the mechanism of viral invasion is poorly understood. The possible role of the TYLCV capsid protein (CP), the only known component of the viral coat, in virus transport into the host cell nucleus was investigated by monitoring its specific nuclear accumulation in plant and insect cells. CP was fused to the β-glucuronidase (GUS) reporter enzyme to assay nuclear import in petunia protoplasts, and micro-injection of purified fluorescently labeled CP was used to examine its nuclear uptake in Drosophila embryos. Both assays demonstrated that TYLCV CP is transported into plant-and insect-cell nuclei by an active process of nuclear import via a nuclear localization signal (NLS)-specific pathway. Using the GUS assay and deletion analysis, the TYLCV CP NLS sequence was identified in the amino-terminus of the protein.     

A型流感病毒PB1-F2蛋白与MOAP-1的相互作用

【目的】探讨A型流感病毒PB1-F2蛋白和人类凋亡调节因子1(MOAP-1)之间的相互作用。【方法】构建pACT2-MOAP-1重组质粒,与pGBKT7-PB1-F2质粒共转化酵母AH109,检测转化菌在四缺培养基的生长情况及β半乳糖苷酶报告基因的活性;利用GST pull-down和免疫共沉淀(Co-IP)技术进一步验证PB1-F2与宿主细胞蛋白MOAP-1的相互作用;通过过表达PB1-F2和MOAP-1,检测PB1-F2对MOAP-1蛋白表达水平的影响。【结果】酵母双杂交结果表明,PB1-F2和MOAP-1可以在酵母细胞内特异性结合。GST pull-down和Co-IP实验也进一步证实了这两种蛋白的相互作用,而且PB1-F2可上调外源MOAP-1的蛋白水平。【结论】流感病毒PB1-F2与MOAP-1存在相互作用,PB1-F2可能通过与MOAP-1的相互作用参与调控细胞生长及凋亡过程。     

两个入侵烟粉虱隐种MEAM1与MED对番茄黄曲叶病毒水平传播能力的比较

【目的】烟粉虱 Bemisia tabaci 是番茄黄曲叶病毒（Tomato yellow leaf curl virus, TYLCV）在自然界的唯一传播媒介,除了可以直接取食获取TYLCV,烟粉虱还可以通过交配获取此植物病毒。虽然前人研究证明了烟粉虱Middle East-Asia Minor 1 （MEAM1）和Mediterranean （MED）隐种都可以通过交配在两性个体之间水平传播TYLCV,但有关MEAM1与MED隐种水平传播TYLCV的能力是否存在差异,不同研究的结果却并不一致。另外,目前尚无关于烟粉虱的水平传播行为是否有助于TYLCV在田间扩散的相关研究。【方法】从浙江、广东、云南和河南4个省份的田间采集MEAM1和MED隐种烟粉虱种群,在室内应用分子标记对各种群所属隐种鉴定后分别建立7个供试种群,然后观察每个种群内带毒成虫与不带毒成虫通过交配在不同性别个体之间水平传播TYLCV的能力,并选用采自广东的MEAM1隐种烟粉虱模拟群体交配实验探究通过交配获毒的烟粉虱个体是否具有传播TYLCV致健康番茄植株发病的能力。【结果】4个省内的MEAM1和MED隐种烟粉虱都可通过交配对TYLCV进行水平传播,但传播频率一般在10%以下。不同省份的MEAM1隐种种群之间在水平传播TYLCV的能力上无显著差异,不同省份的MED隐种种群之间也不存在显著差异。另外,同一省份的MEAM1隐种与MED隐种之间在水平传播TYLCV的能力上也不存在显著差异,而且在两个隐种中,带毒雄虫将病毒水平传播给不带毒雌虫与带毒雌虫将病毒水平传播给不带毒雄虫的频率没有显著差异。研究还表明,采自广东的MEAM1隐种烟粉虱个体通过交配水平获毒后不能致健康番茄植株发病。【结论】综合以上结果,我们推测TYLCV在中国境内两个入侵烟粉虱隐种MEAM1和MED各自种群内个体之间的水平传播概率较低,对该病毒在田间的扩散可能没有作用或作用不大。     

Characterization of the cysteine protease domain of Semliki Forest virus replicase protein nsP2 by in vitro mutagenesis

The function of Semliki Forest Virus nsP2 protease was investigated by site-directed mutagenesis. Mutations were introduced in its protease domain, Pro39, and the mutated proteins were expressed in Escherichia coli, purified and their activity in vitro was compared to that of the wild type Pro39. Mutations M781T, A662T and G577R, found in temperature-sensitive virus strains, rendered the enzyme temperature-sensitive in vitro as well. Five conserved residues were required for the proteolytic activity of Pro39. Changes affecting Cys⁴⁷⁸, His⁵⁴⁸, and Trp⁵⁴⁹ resulted in complete inactivation of the enzyme, whereas the replacements N600D and N605D significantly impaired its activity. The importance of Trp⁵⁴⁹ for the proteolytic cleavage specificity is discussed and a new structural motif involved in substrate recognition by cysteine proteases is proposed.     

Human DDX56 protein interacts with influenza A virus NS1 protein and stimulates the virus replication

Influenza A viruses (IAV) are enveloped viruses carrying a single-stranded negative-sense RNA genome. Detection of host proteins having a relationship with IAV and revealing of the role of these proteins in the viral replication are of great importance in keeping IAV infections under control. Consequently, the importance of human DDX56, which is determined to be associated with a viral NS1 with a yeast two-hybrid assay, was investigated for IAV replication. The viral replication in knocked down cells for the DDX56 gene was evaluated. The NS1 was co-precipitated with the DDX56 protein in lysates of cells transiently expressing DDX56 and NS1 or infected with the viruses, showing that NS1 and DDX56 interact in mammalian cells. Viral NS1 showed a tendency to co-localize with DDX56 in the cells, transiently expressing both of these proteins, which supports the IP and two-hybrid assays results. The data obtained with in silico predictions supported the in vitro protein interaction results. The viral replication was significantly reduced in the DDX56-knockdown cells comparing with that in the control cells. In conclusion, human DDX56 protein interacts with the IAV NS1 protein in both yeast and mammalian cells and has a positive regulatory effect on IAV replication. However, the mechanism of DDX56 on IAV replication requires further elucidation.     

Novel protein kinase interacts with the Cucumber mosaic virus 1a methyltransferase domain

The Cucumber mosaic virus (CMV)-encoded 1a protein has been implicated to play a role in replication of the viral genome along with 2a and one or more host factors. To identify the host cell factors interacting with CMV 1a, we used the yeast two-hybrid system using tobacco cDNA library. One of the cDNA clones encoded a protein homologous to the Arabidopsis putative protein kinase and was designated as Tcoi2 (Tobacco CMV 1a interacting protein 2). Tcoi2 specifically interacted with methyltransferase (MT) domain of CMV 1a protein in yeast cell. In vitro analyses using recombinant proteins showed that Tcoi2 also specifically interacted with CMV 1a MT domain. Tcoi2 did not have autophosphorylation activity but phosphorylated CMV 1a MT domain. Analysis of the subcellular localization of the Tcoi2 fused to GFP demonstrated that it is targeted to the endoplasmic reticulum. These results suggest Tcoi2 as a novel host factor that is capable of interacting and phosphorylating MT domain of CMV 1a protein.     

The cytoplasmic domain of influenza M2 protein interacts with caveolin-1

The cytoplasmic domain of influenza M2 protein (M2c) consists of 54 amino acid (aa) residues from aa44 to aa97. In this paper, M2c and its deletion mutant M2c_Δ47-55 were expressed using prokaryotic expression system. First, glutaraldehyde crosslinking assay showed that M2c had multimerization potential mediated by aa47-55. Then, M2c, instead of M2c_Δ47-55, directed eGFP from the whole cell localization to a predominately perinuclear region in CHO cells, which indicated that aa47-55 of M2c mediated the localization. Moreover, M2c colocalized with caveolin-1 (Cav) when CHO cells were cotransfected with Cav. A caveolin-1 binding motif ΦxxxxΦxxΦ (Φ represents aromatic amino acid residues) in aa47-55 of M2c was found by sequence alignment and analysis. Further overlay ELISA result showed that M2c, but not M2c_Δ47-55, bound to prokaryotically expressed cholesterol-free Cav_2-101, which illustrated the interaction could be cholesterol-independent. That was the first report of cellular protein bound to M2c.     

Influenza virus matrix protein M1 interacts with SLD5 to block host cell cycle

Influenza virus matrix 1 protein (M1) is highly conserved and plays essential roles at many stages of virus life cycle. Here, we used a yeast two‐hybrid system to identify the host protein SLD5, a component of the GINS complex, which is essential for the initiation of DNA replication in eukaryotic cells, as a new M1 interacting protein. M1 from several different influenza virus strains all interacted with SLD5. Overexpression of SLD5 suppressed influenza virus replication. Transient, stable, or inducible expression of M1 induced host cell cycle blockade at G0/G1 phase. Moreover, SLD5 partially rescued M1 expression‐ or influenza virus infection‐induced G0/G1 phase accumulation in cell lines and primary mouse embryonic fibroblasts. Importantly, SLD5 transgenic mice exhibited higher resistance and improved lung epithelial regeneration after virus infection compared with wild‐type mice. Therefore, influenza virus M1 blocks host cell cycle process by interacting with SLD5. Our finding reveals the multifunctional nature of M1 and provides new insight for understanding influenza virus–host interaction.     

FMDV-2A sequence and protein arrangement contribute to functionality of CYP2B1-reporter fusion protein

Two optimized forms of green fluorescence proteins (GFP), enhanced GFP (EGFP) and humanized Renilla GFP (hrGFP), were used to track expression of cytochrome P450 2B1 (CYP2B1), an endoplasmic reticulum membrane-bound protein. In transiently expressing HEK293 cells we show that CYP2B1-GFP fusion proteins are stable and functional, whereas the vice-versa-arranged GFP-CYP2B1 fusions are not. The CYP2B1-hrGFP fusion protein is characterized by reduction in mean fluorescence intensity (MFI) to less than 20% of that of the hrGFP protein alone, accompanied by a 50% loss of CYP2B1 activity. Exchanging the linker for an alpha-helical peptide structure between CYP2B1 and hrGFP does not improve fusion protein activity. Insertion of a short linker (five amino acids) increases reporter protein fluorescence intensity twofold without improving CYP2B1 activity. Introduction of the foot and mouth disease virus 2A sequence providing cotranslational cleavage led to an unstable hrGFP-2A protein, whereas the corresponding EGFP-2A protein was stable and yielded an MFI superior to those of all other fusion constructs tested. CYP2B1 activity of the EGFP-2A-CYP2B1 protein was in the range of that of the unmodified CYP2B1. These data indicate that the protein arrangement EGFP-2A-CYP2B1 is superior to others, since it is most active and visible, which is essential for an effective tracking of the CYP2B1 enzyme.     

Photodamaged D1 protein is degraded in Arabidopsis mutants lacking the Deg2 protease

In plants exposed to high irradiances of visible light, the D1 protein in the reaction center of photosystem II is oxidatively damaged and rapidly degraded. Earlier work in our laboratory showed that the serine protease Deg2 performs the primary cleavage of photodamaged D1 protein in vitro. Here, we demonstrate that the rate of D1 protein degradation under light stress conditions in Arabidopsis mutants lacking the Deg2 protease is similar to those in wild-type plants. Therefore, we propose that several redundant D1 protein degradation pathways might exist in vivo.