Hyperplastic Phloem and Its Plastids in Spinach Infected with the Curly Top Virus

The hyperplastic growth induced in the phloem tissue by infectionwith the curly top virus was studied in minor veins of leavesof spinach, Spinacia oleracea L., by the use of the electronmicroscope. Proliferation of cells occurs in the phloem andin the parenchyma bordering the phloem. The arrangement of cellsis less orderly when hyperplasia occurs in older than in youngertissue but in both instances the majority of cells differentiateinto sieve elements. As in normal phloem, sieve element plastidshaving a ring of proteinaceous fibrils are a consistent featurein the hyperplastic phloem. Depending on the kind of cell inwhich hyperplasia is initiated, the plastids may originate fromyoung plastids similar to those in normal sieve elements orfrom more or less completely differentiated chloroplasts. Theprotoplasts of the hyperplastic sieve elements, including theplastids, degenerate during differentiation or after maturation.     

Translocation of Photosynthate in Curly Top Virus-infected Tomatoes

Photosynthate translocation in single leaflets of healthy and curly top virus-infected tomatoes was investigated using ¹⁴C as a marker. The amount of radioactivity found in plant parts not exposed to ¹⁴CO₂ was substantially lower in diseased than in healthy plants. The time lag for the appearance of ¹⁴C in the petiole was considerably longer in the infected plants than in the healthy. The kinetics of disappearance of ¹⁴C from the lamina during the 24-hour period following labeling showed a strong retention of recent assimilates within the diseased leaf, not accompanied by increased immobilization into insoluble forms. Sucrose was the predominant compound participating in photosynthate transport in both healthy and diseased leaves. The amount of ¹⁴CO₂ fixed was approximately 40% lower in curly top virus-infected leaves than in healthy leaves.     

丙型肝炎病毒非结构蛋白NS4B诱导细胞非折叠蛋白反应

用RT-PCR和免疫印迹的方法检测稳定表达NS4B的HeLa细胞中的XBP1;通过RT-PCR的方法在表达NS4B的HeLa和Huh-7细胞中检测ATF6,Grp78和caspase-12的转录,并且通过报告基因的方法分析XBP1和Grp78启动子活性。实验结果表明:在表达NS4B的HeLa细胞中检测到XBP1的两种形式(剪接和未剪接),此外,在细胞中ATF6、Grp78的转录水平和XBP1、Grp78启动子的荧光素酶活性较没有表达NS4B的HeLa和Huh-7细胞中的量有所增加;通过染色质免疫沉淀实验(ChIP)分析,这些增加可能是由于XBP1结合到了这些基因的启动子上引起的。总之,实验结果可提示HCVNS4B通过ATF6或XBP1途径引起内质网压力,导致UPR反应。NS4B可能在HCV的致病性中起着重要的作用,特别是在慢性肝炎,甚至肝细胞癌中。     

丙型肝炎病毒非结构蛋白NS4B诱导细胞非折叠蛋白反应

用RT-PCR和免疫印迹的方法检测稳定表达NS4B的HeLa细胞中的XBP1;通过RT-PCR的方法在表达NS4B的HeLa和Huh-7细胞中检测ATF6,Grp78和caspase-12的转录,并且通过报告基因的方法分析XBP1和Grp78启动子活性.实验结果表明在表达NS4B的HeLa细胞中检测到XBP1的两种形式(剪接和未剪接),此外,在细胞中ATF6、Grp78的转录水平和XBP1、Grp78启动子的荧光素酶活性较没有表达NS4B的HeLa和Huh-7细胞中的量有所增加;通过染色质免疫沉淀实验(ChIP)分析,这些增加可能是由于XBP1结合到了这些基因的启动子上引起的.总之,实验结果可提示HCV NS4B通过ATF6或XBP1途径引起内质网压力,导致UPR反应.NS4B可能在HCV的致病性中起着重要的作用,特别是在慢性肝炎,甚至肝细胞癌中.     

Involvement of Creatine Kinase B in Hepatitis C Virus Genome Replication through Interaction with the Viral NS4A Protein

Persistent infection with hepatitis C virus (HCV) is a major cause of chronic liver diseases. The aim of this study was to identify host cell factor(s) participating in the HCV replication complex (RC) and to clarify the regulatory mechanisms of viral genome replication dependent on the host-derived factor(s) identified. By comparative proteome analysis of RC-rich membrane fractions and subsequent gene silencing mediated by RNA interference, we identified several candidates for RC components involved in HCV replication. We found that one of these candidates, creatine kinase B (CKB), a key ATP-generating enzyme that regulates ATP in subcellular compartments of nonmuscle cells, is important for efficient replication of the HCV genome and propagation of infectious virus. CKB interacts with HCV NS4A protein and forms a complex with NS3-4A, which possesses multiple enzyme activities. CKB upregulates both NS3-4A-mediated unwinding of RNA and DNA in vitro and replicase activity in permeabilized HCV replicating cells. Our results support a model in which recruitment of CKB to the HCV RC compartment, which has high and fluctuating energy demands, through its interaction with NS4A is important for efficient replication of the viral genome. The CKB-NS4A association is a potential target for the development of a new type of antiviral therapeutic strategy.Hepatitis C virus (HCV) infection represents a significant global healthcare burden, and current estimates suggest that a minimum of 3% of the world''s population is chronically infected (4, 19). The virus is responsible for many cases of severe chronic liver diseases, including cirrhosis and hepatocellular carcinoma (4, 16, 19). HCV is a positive-stranded RNA virus belonging to the family Flaviviridae. Its ∼9.6-kb genome is translated into a single polypeptide of about 3,000 amino acids (aa), in which the nonstructural (NS) proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B reside in the C-terminal half region (6, 34, 44). NS4A, a small 7-kDa protein, functions as a cofactor for NS3 to enhance NS3 enzyme activities such as serine protease and helicase activities. The hydrophobic N-terminal region of NS4A, which is predicted to form a transmembrane α-helix, is responsible for membrane anchorage of the NS3-4A complex (8, 44, 50), and the central region of NS4A is important for the interaction with NS3 (10, 44). A recent study demonstrated the involvement of the C terminus of NS4A in the regulation of NS5A hyperphosphorylation and viral replication (28).The development of HCV replicon technology several years ago accelerated research on viral RNA replication (7, 44). Furthermore, a robust cell culture system for propagation of infectious HCV particles was developed using a viral genome of HCV genotype 2a, JFH-1 strain, enabling us to study every process in the viral life cycle (27, 47, 54). RNA derived from genotype 1a, HCV H77, containing cell-culture adaptive mutations, also produces infectious viruses (52). Using these systems, it has been reported that the HCV genome replicates in a distinct, subcellular replication complex (RC) compartment, which includes NS3-5B and the viral RNA (2, 14, 33). The RC forms in a distinct compartment with high concentrations of viral and cellular components located on detergent-resistant membrane (DRM) structures, possibly a lipid-raft structure (2, 41), which may protect the RC from external proteases and nucleases. Almost all processes in viral replication are dependent on the host cell''s machinery and involve intimate interaction between viral and host proteins. However, the functional roles of host factors interacting with the HCV RC in viral genome replication remain ambiguous.To gain a better understanding of cellular factors that are components of the HCV RC and that function as regulators of viral replication, a comparative proteomic analysis of DRM fractions from HCV replicon and parental cells and subsequent RNA interference (RNAi) silencing of selected genes were performed. We identified creatine kinase B (CKB) as a key factor for the HCV genome replication. CKB catalyzes the reversible transfer of the phosphate group of phosphocreatine (pCr) to ADP to yield ATP and creatine and is known to play important roles in local delivery and cellular compartmentalization of ATP (48, 51). The findings obtained here suggest that recruitment of CKB to the HCV RC, through CKB interaction with NS4A, is essential for maintenance or enhancement of viral replicase activity.     

Role of Phosphatidylinositol 4-Phosphate (PI4P) and Its Binding Protein GOLPH3 in Hepatitis C Virus Secretion

Hepatitis C virus (HCV) RNA replicates within the ribonucleoprotein complex, assembled on the endoplasmic reticulum (ER)-derived membranous structures closely juxtaposed to the lipid droplets that facilitate the post-replicative events of virion assembly and maturation. It is widely believed that the assembled virions piggy-back onto the very low density lipoprotein particles for secretion. Lipid phosphoinositides are important modulators of intracellular trafficking. Golgi-localized phosphatidylinositol 4-phosphate (PI4P) recruits proteins involved in Golgi trafficking to the Golgi membrane and promotes anterograde transport of secretory proteins. Here, we sought to investigate the role of Golgi-localized PI4P in the HCV secretion process. Depletion of the Golgi-specific PI4P pool by Golgi-targeted PI4P phosphatase hSac1 K2A led to significant reduction in HCV secretion without any effect on replication. We then examined the functional role of a newly identified PI4P binding protein GOLPH3 in the viral secretion process. GOLPH3 is shown to maintain a tensile force on the Golgi, required for vesicle budding via its interaction with an unconventional myosin, MYO18A. Silencing GOLPH3 led to a dramatic reduction in HCV virion secretion, as did the silencing of MYO18A. The reduction in virion secretion was accompanied by a concomitant accumulation of intracellular virions, suggesting a stall in virion egress. HCV-infected cells displayed a fragmented and dispersed Golgi pattern, implicating involvement in virion morphogenesis. These studies establish the role of PI4P and its interacting protein GOLPH3 in HCV secretion and strengthen the significance of the Golgi secretory pathway in this process.     

Involvement of Calcium-dependent Protein Kinases in ABA regulation of Stomatal Movement

Patch-clamp techniques were employed to investigate if calcium-dependent protein kinases (CDPKs) be involved in the signal transduction pathways of stomatal movement regulation by the phytohormone abscisic acid (ABA) in Vicia faba. Stomatal opening was completely inhibited by external application of 1 μmol/L ABA, and such ABA inhibition was significantly reversed by the addition of CDPK inhibitor trifluoper- azine (TFP). The inward whole-cell K+-currents were inhibited by 60% in the presence of 1 μmoL/L intracellular ABA, and this inhibition was completely abolished by the addition of CDPK competitive substrate histone Ⅲ-S. The results suggest that CDPKs may be involved in the signal transduction cascades of ABA-regulated stomatal movements.     

Quantification and Localization of Watermelon Chlorotic Stunt Virus and Tomato Yellow Leaf Curl Virus (Geminiviridae) in Populations of Bemisia tabaci (Hemiptera,Aleyrodidae) with Differential Virus Transmission Characteristics

Bemisia tabaci (Gennadius) is one of the economically most damaging insects to crops in tropical and subtropical regions. Severe damage is caused by feeding and more seriously by transmitting viruses. Those of the genus begomovirus (Geminiviridae) cause the most significant crop diseases and are transmitted by B. tabaci in a persistent circulative mode, a process which is largely unknown. To analyze the translocation and to identify critical determinants for transmission, two populations of B. tabaci MEAM1 were compared for transmitting Watermelon chlorotic stunt virus (WmCSV) and Tomato yellow leaf curl virus (TYLCV). Insect populations were chosen because of their high and respectively low virus transmission efficiency to compare uptake and translocation of virus through insects. Both populations harbored Rickettsia, Hamiltonella and Wolbachia in comparable ratios indicating that endosymbionts might not contribute to the different transmission rates. Quantification by qPCR revealed that WmCSV uptake and virus concentrations in midguts and primary salivary glands were generally higher than TYLCV due to higher virus contents of the source plants. Both viruses accumulated higher in insects from the efficiently compared to the poorly transmitting population. In the latter, virus translocation into the hemolymph was delayed and virus passage was impeded with limited numbers of viruses translocated. FISH analysis confirmed these results with similar virus distribution found in excised organs of both populations. No virus accumulation was found in the midgut lumen of the poor transmitter because of a restrained virus translocation. Results suggest that the poorly transmitting population comprised insects that lacked transmission competence. Those were selected to develop a population that lacks virus transmission. Investigations with insects lacking transmission showed that virus concentrations in midguts were reduced and only negligible virus amounts were found at the primary salivary glands indicating for a missing or modified receptor responsible for virus attachment or translocation.     

A Leafhopper-Transmissible DNA Virus with Novel Evolutionary Lineage in the Family Geminiviridae Implicated in Grapevine Redleaf Disease by Next-Generation Sequencing

A graft-transmissible disease displaying red veins, red blotches and total reddening of leaves in red-berried wine grape (Vitis vinifera L.) cultivars was observed in commercial vineyards. Next-generation sequencing technology was used to identify etiological agent(s) associated with this emerging disease, designated as grapevine redleaf disease (GRD). High quality RNA extracted from leaves of grape cultivars Merlot and Cabernet Franc with and without GRD symptoms was used to prepare cDNA libraries. Assembly of highly informative sequence reads generated from Illumina sequencing of cDNA libraries, followed by bioinformatic analyses of sequence contigs resulted in specific identification of taxonomically disparate viruses and viroids in samples with and without GRD symptoms. A single-stranded DNA virus, tentatively named Grapevine redleaf-associated virus (GRLaV), and Grapevine fanleaf virus were detected only in grapevines showing GRD symptoms. In contrast, Grapevine rupestris stem pitting-associated virus, Hop stunt viroid, Grapevine yellow speckle viroid 1, Citrus exocortis viroid and Citrus exocortis Yucatan viroid were present in both symptomatic and non-symptomatic grapevines. GRLaV was transmitted by the Virginia creeper leafhopper (Erythroneura ziczac Walsh) from grapevine-to-grapevine under greenhouse conditions. Molecular and phylogenetic analyses indicated that GRLaV, almost identical to recently reported Grapevine Cabernet Franc-associated virus from New York and Grapevine red blotch-associated virus from California, represents an evolutionarily distinct lineage in the family Geminiviridae with genome characteristics distinct from other leafhopper-transmitted geminiviruses. GRD significantly reduced fruit yield and affected berry quality parameters demonstrating negative impacts of the disease. Higher quantities of carbohydrates were present in symptomatic leaves suggesting their possible role in the expression of redleaf symptoms.     

Host-Specific Involvement of the HC Protein in the Long-Distance Movement of Potyviruses

Plum pox virus (PPV) is a member of the Potyvirus genus that, in nature, infects trees of the Prunus genus. Although PPV infects systemically several species of the Nicotiana genus, such as N. clevelandii and N. benthamiana, and replicates in the inoculated leaves of N. tabacum, it is unable to infect systemically the last host. The long-distance movement defect of PPV was corrected in transgenic tobacco plants expressing the 5"-terminal region of the genome of tobacco etch virus (TEV), a potyvirus that infects systemically tobacco. The fact that PPV was unable to move to upper noninoculated leaves in tobacco plants transformed with the same TEV transgene, but with a mutation in the HC protein (HC-Pro)-coding sequences, identifies the multifunctional HC-Pro as the complementing factor, and strongly suggests that a defect in an HC-Pro activity is responsible for the long-distance movement defect of PPV in tobacco. Whereas PPV HC-Pro strongly intensifies the symptoms caused by potato virus X (PVX) in the PPV systemic hosts N. clevelandii and N. benthamiana, it has no apparent effect on PVX pathogenicity in tobacco, supporting the hypothesis that long-distance movement and pathogenicity enhancement are related activities of the potyviral HC proteins. The movement defect of PPV in tobacco could also be complemented by cucumber mosaic virus in a mixed infection, demonstrating that at least some components of the long-distance machinery of the potyviruses are not strictly virus specific. A general conclusion of this work is that the HC-Pro might be a relevant factor for controlling the host range of the potyviruses.     

Demonstration of the Specific Involvement of Coat Protein in Tobacco Mosaic Virus (TMV) Cross Protection using a TMV Coat Protein Mutant

The DT-1G mutant of tobacco mosaic virus (TMV) which has no coat protein was used to study the specific involvement of coat protein in TMV cross protection in N. sylvestris. Leaves of N. sylvestris previously inoculated with the mutantor the common strain of TMV were challenged with either turnip mosaic virus (TuMV) or a strain of TMV (TMV-N). Both TuMV and TMV-N produce necrotic lesions on N. sylvestris. About one-half as many lesions were produced by TuMV and TMV-N on leaves, inoculated with the DT-1G mutant compared with lesions produced by the same inoculum on control leaves. When leaves of N. sylvestris previously inoculated with the common strain of TMV were challenged with either TuMV or TMV-N, TuMV produced about one-half as many lesions as on control leaves whereas TMV-N produced about one-tenth as many lesions as on control leaves. A high level of non-specific resistance was induced by the mutant without coat protein, but it did not specifically protect against TMV.     

Modulation of Hepatitis C Virus Genome Encapsidation by Nonstructural Protein 4B

Hepatitis C Virus (HCV) NS4B protein has many roles in HCV genome replication. Recently, our laboratory (Q. Han, J. Aligo, D. Manna, K. Belton, S. V. Chintapalli, Y. Hong, R. L. Patterson, D. B. van Rossum, and K. V. Konan, J. Virol. 85:6464–6479, 2011) and others (D. M. Jones, A. H. Patel, P. Targett-Adams, and J. McLauchlan, J. Virol. 83:2163–2177, 2009; D. Paul, I. Romero-Brey, J. Gouttenoire, S. Stoitsova, J. Krijnse-Locker, D. Moradpour, and R. Bartenschlager, J. Virol. 85:6963–6976, 2011) have also reported NS4B''s function in postreplication steps. Indeed, replacement of the NS4B C-terminal domain (CTD) in the HCV JFH1 (genotype 2a [G2a]) genome with sequences from Con1 (G1b) or H77 (G1a) had a negligible impact on JFH1 genome replication but attenuated virus production. Since NS4B interacts weakly with the HCV genome, we postulated that NS4B regulates the function of host or virus proteins directly involved in HCV production. In this study, we demonstrate that the integrity of the JFH1 NS4B CTD is crucial for efficient JFH1 genome encapsidation. Further, two adaptive mutations (NS4B N216S and NS5A C465S) were identified, and introduction of these mutations into the chimera rescued virus production to various levels, suggesting a genetic interaction between the NS4B and NS5A proteins. Interestingly, cells infected with chimeric viruses displayed a markedly decreased NS5A hyperphosphorylation state (NS5A p58) relative to JFH1, and the adaptive mutations differentially rescued NS5A p58 formation. However, immunofluorescence staining indicated that the decrease in NS5A p58 did not alter NS5A colocalization with the core around lipid droplets (LDs), the site of JFH1 assembly, suggesting that NS5A fails to facilitate the transfer of HCV RNA to the capsid protein on LDs. Alternatively, NS4B''s function in HCV genome encapsidation may entail more than its regulation of the NS5A phosphorylation state.     

甜菜坏死黄脉病毒外壳蛋白基因在甜菜转基因植株中的表达

甜菜坏死黄脉病毒(Beet Necrotic Yellow Vein Virus,BNYVV)是一种由甜菜多粘菌(Polymyxo be tae)传播的多分体植物病毒．基因组由4～5条单链正意RNA构成^[1]。60年代末,由Tamada首次报道^[2],这种病毒可对甜菜造成严重危害,侵染甜菜后产生丛根症状(Rhizomania),并导致甜菜产量和含糖 量的大幅度下降。除欧洲、北美及日本的严重发生以外,我国自70年代以来在东北、内蒙古及西北许多省区也有大量甜菜丛根病的发生报道^[3]。由于尚无有效药剂及措施用于甜菜丛根病或病毒传播介体的防治．在我国也无法采用大面积轮作作为防治手段,所以目前在世界各地及我国上述地区甜菜丛根病的发病面积逐年扩展,对甜菜生产和制糖业造成直接威胁。针对这一情况．本文报道了含有甜菜坏死黄脉病毒外壳蛋白基因的甜菜植株的转化再生工作,以期在甜菜亲本育种中获得新的抗性材料．为抗病毒品种的培育打下基础。     

Poly(C)-Binding Protein 1, a Novel Npro-Interacting Protein Involved in Classical Swine Fever Virus Growth

N^pro is a multifunctional autoprotease unique to pestiviruses. The interacting partners of the N^pro protein of classical swine fever virus (CSFV), a swine pestivirus, have been insufficiently defined. Using a yeast two-hybrid screen, we identified poly(C)-binding protein 1 (PCBP1) as a novel interacting partner of the CSFV N^pro protein and confirmed this by coimmunoprecipitation, glutathione S-transferase (GST) pulldown, and confocal assays. Knockdown of PCBP1 by small interfering RNA suppressed CSFV growth, while overexpression of PCBP1 promoted CSFV growth. Furthermore, we showed that type I interferon was downregulated by PCBP1, as well as N^pro. Our results suggest that cellular PCBP1 positively modulates CSFV growth.