Comparison of barley stripe mosaic virus strains

BSMV (barley stripe mosaic virus) particles were obtained in a pure state from infected host plant tissues of Hordeum vulgare. The three genomic parities (alpha, beta and gamma) were amplified by PCR using specific primers for each particle; each was cloned. Partial sequence of the alpha, beta and gamma segments was determined for the Egyptian isolate of barley stripe mosaic virus (BSMV AE1). Alignment of nucleotide sequences with that of other known strains of the virus, BSMV type strains (CV17, ND18 and China), and the generation of phylogenetic trees was performed. A low level of homology was detected comparing 467 bp of the a and 643 bp of the segments to that of the other strains, and thus BSMV alpha and beta segments were in separate clusters. However, 1154 bp of the gamma segments of BSMV AE1 showed a high level of homology especially to strain BSMV ND18, as they both formed a distinct cluster. Northern blotting of pure BSMV AE1 virus and H. vulgare-infected tissue were compared using an alpha ND18 specific probe. Western blotting using antibodies specific for the coat protein (CP) and the triple gene block 1 (TGB1) protein, which are both encoded by the beta ND18 segment, still indicated a high level of similarity between proteins produced by BSMV ND18 and AE1. We suggest that the BSMV AE1 isolate is a distinct strain of BSMV which reflects the genetic evolutionary divergence among BSMV strains and members of the Hordeivirus group.     

A high throughput barley stripe mosaic virus vector for virus induced gene silencing in monocots and dicots

Barley stripe mosaic virus (BSMV) is a single-stranded RNA virus with three genome components designated alpha, beta, and gamma. BSMV vectors have previously been shown to be efficient virus induced gene silencing (VIGS) vehicles in barley and wheat and have provided important information about host genes functioning during pathogenesis as well as various aspects of genes functioning in development. To permit more effective use of BSMV VIGS for functional genomics experiments, we have developed an Agrobacterium delivery system for BSMV and have coupled this with a ligation independent cloning (LIC) strategy to mediate efficient cloning of host genes. Infiltrated Nicotiana benthamiana leaves provided excellent sources of virus for secondary BSMV infections and VIGS in cereals. The Agro/LIC BSMV VIGS vectors were able to function in high efficiency down regulation of phytoene desaturase (PDS), magnesium chelatase subunit H (ChlH), and plastid transketolase (TK) gene silencing in N. benthamiana and in the monocots, wheat, barley, and the model grass, Brachypodium distachyon. Suppression of an Arabidopsis orthologue cloned from wheat (TaPMR5) also interfered with wheat powdery mildew (Blumeria graminis f. sp. tritici) infections in a manner similar to that of the A. thaliana PMR5 loss-of-function allele. These results imply that the PMR5 gene has maintained similar functions across monocot and dicot families. Our BSMV VIGS system provides substantial advantages in expense, cloning efficiency, ease of manipulation and ability to apply VIGS for high throughput genomics studies.     

萤光素酶表达质粒pEE14.1-luc的构建及表达

目的:为研究10 kb以上DNA疫苗质粒的体内电穿孔递送,构建萤光素酶报告基因表达质粒p EE14.1-luc,并验证其表达。方法:从质粒p GL3-CMV中通过酶切、补平和纯化的方法获得萤光素酶基因luc,克隆入p EE14.1载体,构建重组表达质粒p EE14.1-luc,瞬时转染293T细胞,采用Western印迹、流式细胞术和免疫荧光等方法对萤光素酶基因在体外的表达情况进行验证,并运用活体成像仪检测萤光素酶基因在小鼠活体内的表达。结果:经菌液PCR鉴定和测序验证,p EE14.1-luc与预期设计完全一致;流式细胞术检测luc阳性表达率为22.41%,免疫荧光检测可见绿色荧光表达,Western印迹检测在相对分子质量为62×103处显现目的蛋白条带;同时,利用活体成像技术也检测到萤光素酶基因在小鼠活体内的表达。结论:p EE14.1-luc表达质粒构建成功,为研究DNA疫苗体内表达机制和体内电穿孔递送条件优化奠定了基础。     

Rapid obtention of stable,bioluminescent tumor cell lines using a tCD2-luciferase chimeric construct

Background  

Bioluminescent tumor cell lines are experimental tools of major importance for cancer investigation, especially imaging of tumors in xenografted animals. Stable expression of exogenous luciferase in tumor cells combined to systemic injection of luciferin provides an excellent signal/background ratio for external optical imaging. Therefore, there is a need to rationalize and speed up the production of luciferase-positive tumor cell lines representative of multiple tumor phenotypes. For this aim we have designed a fusion gene linking the luciferase 2 protein to the c-terminus of a truncated form of the rat CD2 protein (tCD2-luc2). To allow simultaneous assessment of the wild-type luciferase 2 in a context of tCD2 co-expression, we have made a bicistronic construct for concomitant but separate expression of these two proteins (luc2-IRES-tCD2). Both the mono- and bi-cistronic constructs were transduced in lymphoid and epithelial cells using lentiviral vectors.     

Potyviral VPg enhances viral RNA Translation and inhibits reporter mRNA translation in planta

Viral protein genome-linked (VPg) plays a central role in several stages of potyvirus infection. This study sought to answer questions about the role of Potato virus A (PVA; genus Potyvirus) VPg in viral and host RNA expression. When expressed in Nicotiana benthamiana leaves in trans, a dual role of VPg in translation is observed. It repressed the expression of monocistronic luciferase (luc) mRNA and simultaneously induced a significant upregulation in the expression of both replicating and nonreplicating PVA RNAs. This enhanced viral gene expression was due at least to the 5' untranslated region (UTR) of PVA RNA, eukaryotic initiation factors 4E and iso 4E [eIF4E/eIF(iso)4E], and the presence of a sufficient amount of VPg. Coexpression of VPg with viral RNA increased the viral RNA amount, which was not the case with the monocistronic mRNA. Both mutations at certain lysine residues in PVA VPg and eIF4E/eIF(iso)4E depletion reduced its ability to upregulate the viral RNA expression. These modifications were also involved in VPg-mediated downregulation of monocistronic luc expression. These results suggest that VPg can titrate eIF4Es from capped monocistronic RNAs. Because VPg-mediated enhancement of viral gene expression required eIF4Es, it is possible that VPg directs eIF4Es to promote viral RNA expression. From this study it is evident that VPg can serve as a specific regulator of PVA expression by boosting the viral RNA amounts as well as the accumulation of viral translation products. Such a mechanism could function to protect viral RNA from being degraded and to secure efficient production of coat protein (CP) for virion formation.     

The barley stripe mosaic virus 58-kilodalton beta(b) protein is a multifunctional RNA binding protein.

The barley stripe mosaic virus (BSMV) beta(b) gene product is the major viral nonstructural protein synthesized during early stages of the infection cycle and is required for systemic movement of the virus. To examine the biochemical properties of beta(b), a histidine tag was engineered at the amino terminus and the protein was purified from BSMV-infected barley tissue by metal affinity chromatography. The beta(b) protein bound ATPs in vitro, with a preference for ATP over dATP, and also exhibited ATPase activity. In addition, beta(b) bound RNA without detectable sequence specificity. However, binding was selective, as the beta(b) protein had a strong affinity for both single-stranded (ss) and double-stranded (ds) RNAs but not for tRNA or DNA substrates. Mutational analyses of beta(b) purified from Escherichia coli indicated that the protein has multiple RNA binding sites. These sites appear to contribute differently, because mutants that were altered in their binding affinities for ss and ds RNA substrates were recovered.     

Sensitivity of virally-driven luciferase reporter plasmids to members of the steroid/thyroid/retinoid family of nuclear receptors

During a series of transfection experiments, the pRSV-luc plasmid used as an internal control was found to be sensitive to co-transfection with expression vectors for several members of the steroid/thyroid/retinoid superfamily of nuclear receptors. Therefore, a survey of the effect of these expression vectors on the activity of four reporter plasmids was conducted. In CV-1 cells, the activity of pRSV-luc, which contains the P. pyralis luciferase gene, was repressed by co-transfection of PPAR and ARP-1 and was activated by COUP-TFI. Expression of pSV40-luc, containing the same luciferase gene, was repressed by PPAR and HNF-4 and activated by both COUP-TFI and ARP-1. All four of these expression vectors reduced the expression of the pRL-TK plasmid, which contains the luciferase gene from Renilla reniformis. RXR expression vectors had no effect on luciferase activity in CV-1 cells but induced luciferase activity in H4IIEC3 hepatoma cells. This activation was blocked by the addition of ligand, 9-cis retinoic acid. pSV2-CAT, which contains the chloramphenicol acetyltransferase gene, was insensitive to all receptor expression vectors tested. Both the P. pyralis and R. reniformis luciferase genes appear to contain sequences that render them responsive to steroid/thyroid/retinoid nuclear receptors.     

食品级乳酸乳球菌pNZ8149-luc质粒的构建及表达

目的:构建能够稳定表达萤火虫荧光素酶报告基因(luc)的乳酸乳球菌(Lactococcus lactis, L.lactis)食品级表达系统,以便后续研究对目的基因进行示踪。方法:从pGL4.10质粒中PCR扩增萤火虫荧光素酶报告基因,测序,克隆至载体pNZ8149,构建pNZ8149-luc表达质粒;电击转化宿主乳酸乳球菌NZ3900,采用乳糖筛选法获得重组的乳酸乳球菌,Nisin诱导,采用微孔板发光检测仪检测荧光素酶的存在,Western Blot检测目标蛋白luc的表达。结果:PCR扩增的荧光素酶报告基因成功克隆至pNZ8149质粒,并电击转化宿主乳酸乳球菌NZ3900,得到乳酸乳球菌表达系统NZ3900/pNZ8149-luc。Nisin诱导后,检测到荧光素酶随诱导时间的延长活性逐渐增强,时间超过24 h之后荧光素酶活性逐渐下降。Western Blot检测到目标蛋白luc在胞内表达。结论:成功构建了p NZ8149-luc表达载体,并能够在乳酸乳球菌体内稳定表达。     

结构域Ⅰ缺失的丙型肝炎病毒5＇NCR调控荧光素酶基因的表达

目的：分析丙型肝炎病毒（HCV）5＇端非编码区（NCR）的结构域Ⅰ序列在其翻译启动活性中的作用.方法：以质粒pCMVNCRluc为模板,PCR扩增分别得到缺失5＇端20nt和43nt的HCV 5＇NCR片段,并分别替换pCMVNCRluc中的完整HCV 5NCR,构建结构域Ⅰ缺失的HCV 5＇NCR调控萤火虫荧光素酶（luc）基因表达的真核表达质粒（pCNl-d1、pCNl-d2）.以脂质体方法转染人肝癌细胞株HepG2,用双荧光素酶报告基因检测系统检测荧光素酶相对于内参考的海肾荧光素酶表达活性,同时采用RT-PCR方法检测转染后细胞中萤火虫荧光素酶基因的相对表达水平.结果：酶切和测序结果表明,各重组质粒构建成功.各重组质粒转染细胞后luc mRNA的相对表达水平与pCMVNCRluc相比差异无显著性（P＞0.05）;pCNl-d1、pCNl-d2表达的荧光素酶活性与pCMVNCRluc差异无显著性（P＞0.05）.结论：HCV 5NCR的5＇端20nt和43nt序列缺失不影响它的翻译启动活性.     

The complete nucleotide sequence of RNA beta from the type strain of barley stripe mosaic virus.

The complete nucleotide sequence of RNA beta from the type strain of barley stripe mosaic virus (BSMV) has been determined. The sequence is 3289 nucleotides in length and contains four open reading frames (ORFs) which code for proteins of Mr 22,147 (ORF1), Mr 58,098 (ORF2), Mr 17,378 (ORF3), and Mr 14,119 (ORF4). The predicted N-terminal amino acid sequence of the polypeptide encoded by the ORF nearest the 5'-end of the RNA (ORF1) is identical (after the initiator methionine) to the published N-terminal amino acid sequence of BSMV coat protein for 29 of the first 30 amino acids. ORF2 occupies the central portion of the coding region of RNA beta and ORF3 is located at the 3'-end. The ORF4 sequence overlaps the 3'-region of ORF2 and the 5'-region of ORF3 and differs in codon usage from the other three RNA beta ORFs. The coding region of RNA beta is followed by a poly(A) tract and a 238 nucleotide tRNA-like structure which are common to all three BSMV genomic RNAs.     

Enhancement of hepatitis C virus RNA replication by cell culture-adaptive mutations

Studies of the Hepatitis C virus (HCV) replication cycle have been made possible with the development of subgenomic selectable RNAs that replicate autonomously in cultured cells. In these replicons the region encoding the HCV structural proteins was replaced by the neomycin phosphotransferase gene, allowing the selection of transfected cells that support high-level replication of these RNAs. Subsequent analyses revealed that, within selected cells, HCV RNAs had acquired adaptive mutations that increased the efficiency of colony formation by an unknown mechanism. Using a panel of replicons that differed in their degrees of cell culture adaptation, in this study we show that adaptive mutations enhance RNA replication. Transient-transfection assays that did not require selection of transfected cells demonstrated a clear correlation between the level of adaptation and RNA replication. The highest replication level was found with an adapted replicon carrying two amino acid substitutions located in NS3 and one in NS5A that acted synergistically. In contrast, the nonadapted RNA replicated only transiently and at a low level. The correlation between the efficiency of colony formation and RNA replication was corroborated with replicons in which the selectable marker gene was replaced by the gene encoding firefly luciferase. Upon transfection of naive Huh-7 cells, the levels of luciferase activity directly reflected the replication efficiencies of the various replicon RNAs. These results show that cell culture-adaptive mutations enhance HCV RNA replication.     

Wheat dwarf virus vectors replicate and express foreign genes in cells of monocotyledonous plants.

Wheat dwarf virus (WDV) is a geminivirus that infects monocotyledonous plants. To exploit the potential of WDV as a replicative gene vector, we developed a transient replication and expression system based on the transfection of protoplasts derived from Triticum monococcum suspension culture cells. Cloned genomic copies of various WDV isolates as well as mutants constructed in vitro were introduced into the protoplasts and assayed for their ability to replicate. As a result, regions of the WDV genome necessary or dispensable for the viral DNA replication could be defined. In addition, the gene encoding the viral capsid protein was replaced by three different bacterial marker genes, neomycin phosphotransferase, chloramphenicol acetyltransferase, and beta-galactosidase. The beta-galactosidase gene doubled the size of the WDV genome. The replication of the recombinant WDV genomes and the expression of these genes were monitored in suspension culture cells of T. monococcum. The potential of replicative expression vectors based on the WDV genome is discussed.