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Transient expression of cellular polypyrimidine-tract binding protein stimulates cap-independent translation directed by both picornaviral and flaviviral internal ribosome entry sites In vivo 总被引:6,自引:0,他引:6
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Gosert R Chang KH Rijnbrand R Yi M Sangar DV Lemon SM 《Molecular and cellular biology》2000,20(5):1583-1595
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The open reading frame 4 (ORF 4) gene product of barley yellow dwarf virus (BYDV) may act as a movement protein (MP) by assisting the transport of viral genomic RNA across the nuclear envelope (NE) of host plant cells. To investigate interactions between BYDV MP and the NE, wild-type and mutant open reading frame (ORF 4)-green fluorescent protein (GFP) fusion cistrons were expressed in insect cells. A fusion protein expressed by the wild-type ORF 4-GFP cistron associated with the NE and caused protrusions from its surface. The fusion protein expressed by the mutant ORF 4-GFP cistron lacked a putative amphiphilic alpha-helix at its N-terminus and although associating with the NE, showed decreased levels of protrusions. A peptide homologue of this putative alpha-helix induced an increase of 7 degrees C in the phase transition temperature of dimyrystoyl phosphatidylserine (DMPS) membranes, accompanied by a decrease in membrane fluidity, but exhibited no significant interaction with either dimyristoyl phosphatidylcholine (DMPC) or dimyristoyl phosphatidylethanolamine (DMPE) membranes. These results strongly support the view that BYDV MP may interact with the NE to help transport viral genomic RNA into the nuclear compartment. This function of BYDV MP appears to involve protrusions on the surface of the NE and may require the presence of an N-terminal amphiphilic alpha-helix, which is speculated to destabilize membranes, thereby assisting the entry of BYDV-GAV into the nuclear compartment. 相似文献
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Multiple widely spaced elements determine the efficiency with which a distal cistron is expressed from the polycistronic pregenomic RNA of figwort mosaic caulimovirus.
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The polycistronic expression mechanism of the plant pararetrovirus figwort mosaic caulimovirus (FMV) depends upon cis-acting elements present in its pregenomic RNA and a trans-acting protein (P6) which is expressed from a monocistronic subgenomic RNA. Using transient expression of FMV-derived polycistronic reporter constructs in Nicotiana edwardsonii cell suspension protoplasts, we further analyzed the cis-acting elements involved in polycistronic expression. A cis-acting element located within the first 74 nucleotides of the 7,954-nucleotide pregenomic RNA appears to be essential for P6 to transactivate expression of an internal cistron. Expression of this internal cistron, in the presence of P6, is greatly enhanced by the combined presence of two cis-acting elements located at the 3' end of the polycistronic RNA. Surprisingly, deletion of the most upstream of these two 3' cis-acting elements exposed a negative-acting element located internally on the polycistronic RNA, at the 3' end of open reading frame I. The action of both this negative-acting internal element and the positive-acting 3' elements is more pronounced when the large 5' untranslated leader region is present. This indicates that the 5' untranslated leader region is central to regulation of the FMV gene expression mechanism. Although a limited set of elements suffices to direct polycistronic expression in this eukaryotic system, a complex interplay between elements is involved in the spatial regulation of the genes present on the pregenomic RNA of FMV. 相似文献
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Kappei Kobayashi Seiji Tsuge Hitoshi Nakayashiki Kazuyuki Mise Iwao Furusawa 《Microbiology and immunology》1998,42(4):329-334
Studies have indicated that cauliflower mosaic virus (CaMV) gene expression is mediated by the translation of polycistronic 35S pregenomic RNA, but the involvement of some minor subgenomic RNA species is also suspected. We examined the involvement of the 35S promoter in the expression of CaMV open reading frames (ORFs) I and IV using both 35S RNA-driven and promoter-less ORF I- and ORF IV-β-glucuronidase (GUS) fusion constructs. In addition to the 35S promoter-dependent expression of both ORF I- and IV-GUS fusions, we detected the 35S promoter-independent expression of both fusion genes via subgenomic mRNAs, which were detected by Northern blotting in the protoplasts transfected with the 35S promoter-driven constructs as well as in those transfected with the promoter-less constructs. These results suggest the involvement of subgenomic RNAs in the expression of CaMV ORFs I and IV, and the operation of a dual strategy in the expression of two viral genes. 相似文献
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Dry I Krake L Mullineaux P Rezaian A 《Molecular plant-microbe interactions : MPMI》2000,13(5):529-537
The regulation of expression of the two virion-sense (V1 and V2) and four complementary-sense (C1, C2, C3, and C4) open reading frames (ORFs) of Tomato leaf curl virus (TLCV) was studied in both stably and transiently transformed Nicotiana tabacum tissues with fusions with the beta-glucuronidase (GUS) reporter gene. GUS-expressing transgenic lines were obtained with each of the four complementary-sense gene-GUS fusion constructs and with truncated versions of the virion-sense gene-GUS fusion constructs (V1GUSdeltaC and V2GUSdeltaC) lacking complementary-sense sequences encoding the C1, C2, and C3 ORFs. However, little or no GUS expression was observed in kanamycin-resistant plants transformed with full-length, virion-sense gene constructs (V1GUS and V2GUS) constituting the complete viral genome. In contrast, V1GUS and V2GUS were found to direct high-level GUS expression in transient assays with tobacco protoplasts, suggesting that integration of viral constructs containing functional, complementary-sense genes may lead to repression or deletion of the introduced constructs in transgenic tissues. V2GUS expression in the transient protoplast assay was found to be severely curtailed by specific mutation of the C2 ORF, supporting a role for the C2 protein in transactivation of TLCV virion-sense gene expression. TLCV ORF-GUS constructs displayed distinctive tissue expression patterns in transgenic tobacco plants that could be divided into constitutive (C1, C4, and V2GUSdeltaC), predominantly vascular (C2, C3), or reduced expression in cells associated with the vascular bundles (V1GUSdeltaC). The significance of these results is discussed in terms of current models of gene function and regulation in geminiviruses. 相似文献
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Replication of poliovirus RNA and subgenomic RNA transcripts in transfected cells. 总被引:10,自引:7,他引:3
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P S Collis B J O''''Donnell D J Barton J A Rogers J B Flanegan 《Journal of virology》1992,66(11):6480-6488
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Proteolytic processing of the coronavirus infectious bronchitis virus 1a polyprotein: identification of a 10-kilodalton polypeptide and determination of its cleavage sites. 总被引:2,自引:1,他引:1
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Proteolytic processing of the polyprotein encoded by mRNA 1 is an essential step in coronavirus RNA replication and gene expression. We have previously reported that an open reading frame (ORF) 1a-specific proteinase of the picornavirus 3C proteinase group is involved in processing of the coronavirus infectious bronchitis virus (IBV) 1a/1b polyprotein, leading to the formation of a mature viral protein of 100 kDa. We report here the identification of a novel 10-kDa polypeptide and the involvement of the 3C-like proteinase in processing of the ORF 1a polyprotein to produce the 10-kDa protein species. By using a region-specific antiserum, V47, raised against a bacterial-viral fusion protein containing IBV sequence encoded between nucleotides 11488 and 12600, the 10-kDa polypeptide was detected in lysates from both IBV-infected and plasmid DNA-transfected Vero cells. Coexpression, deletion, and mutagenesis studies showed that this novel polypeptide was encoded by ORF 1a from nucleotide 11545 to 11878 and was cleaved from the 1a polyprotein by the 3C-like proteinase domain. Evidence presented suggested that a previously predicted Q-S (Q3783 S3784) dipeptide bond encoded by ORF 1a between nucleotides 11875 and 11880 was responsible for the release of the C terminus of the 10-kDa polypeptide and that a novel Q-N (Q3672 N3673) dipeptide bond encoded between nucleotides 11542 and 11547 was responsible for the release of the N terminus of the 10-kDa polypeptide. 相似文献
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甜菜黑色焦枯病毒外壳蛋白与病毒致病性的关系 总被引:1,自引:0,他引:1
利用RT-PCR方法,构建获得了由T7RNA聚合酶启动子驱动的甜菜黑色焦枯病毒(BBSV)全长cDNA克隆pUBF52.摩擦接种苋色藜(Chenopodiumamaranticolor)后,体外转录产物可导致与野生病毒相同的枯斑症状,蛋白质印迹和RNA印迹检测也都证明了转录产物的侵染活性.构建了BBSVp24基因的原核表达载体pECP1,转化大肠杆菌BL21后的诱导表达产物能够与BBSV的抗血清呈现特异性反应,表明该基因编码产生BBSV的外壳蛋白(CP).以pUBF52为模板,分别构建了BBSVCP基因的移码突变体和不同程度的缺失突变体.侵染性检测表明,CP基因的移码突变对BBSV在苋色藜上所导致的枯斑症状及病毒RNA在寄主体内的积累基本没有影响,但CP基因的大部或完全缺失会使体内病毒RNA的积累水平大大降低,其中CP基因完全缺失的突变体转录物接种苋色藜后仅能够产生很轻的枯斑症状.将绿色荧光蛋白(GFP)基因和葡糖苷酸酶(GUS)基因分别与BBSVCP基因的5′端融合,构建了表达载体pBGFP和pBGUS.摩擦接种苋色藜叶片后可观察到GFP或GUS基因的表达,为探索利用BBSV作为外源蛋白的表达载体奠定了基础. 相似文献
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Szu-Cheng Kuo Ying-Ju Chen Yu-Ming Wang Pei-Yi Tsui Ming-Der Kuo Tzong-Yuan Wu Szecheng J Lo 《Journal of biomedical science》2012,19(1):44