Regulation of MSV and WDV virion-sense promoters by WDV nonstructural proteins: a role for their retinoblastoma protein-binding motifs

In this work we demonstrate that wheat dwarf virus (WDV) RepA can activate WDV and maize streak virus (MSV) virion (V)-sense expression in plant tissues. Rep alone does not have any effect on the silent WDV promoter and it represses the basal MSV promoter activity. MSV promoter activation by RepA depends on an intact RepA retinoblastoma protein (RB)-binding domain. Promoter repression by Rep also depends on this domain to some extent. Mutation of the RepA RB-binding domain has no effect on WDV promoter activation. The WDV promoter contains two sites that fit the consensus E2F-binding site. One, WDV1, binds human E2F-1 in one-hybrid assays in yeast. It also binds specifically to maize and wheat proteins in vitro and, when fused to a minimal 35S promoter, it confers responsiveness to RepA only when the RepA RB-binding domain and the WDV1 site are intact. In the whole WDV V-sense promoter context, mutations of this sequence have no effect, suggesting that additional sequences are important for RepA-mediated promoter activation.     

Glycosylation of the dengue 2 virus E protein at N67 is critical for virus growth in vitro but not for growth in intrathoracically inoculated Aedes aegypti mosquitoes

To determine the importance of dengue 2 virus (DEN2V) envelope (E) protein glycosylation, virus mutants in one or both of the N-linked glycosylation motifs were prepared. We found that while the E2 mutant virus (N153Q) replicated in mammalian and mosquito cells, the E1 (N67Q) and E1/2 (N67Q and N153Q) mutant viruses were unable to grow in mammalian cells. Infection of C6/36 mosquito cells with either the E1 or E1/2 mutants resulted in the introduction of a compensatory mutation, K64N, restoring glycosylation in the area. All mutants replicated similarly in inoculated Aedes aegypti mosquitoes, with no change in their mutations. These results suggest that N-linked glycosylation of the E protein is not necessary for DEN2V replication in mosquitoes, however N-linked glycosylation at amino acid N67 (or nearby N64) is critical for the survival of the virus in either mammalian or insect cell culture.     

The minor outer capsid protein P2 of rice gall dwarf virus has a primary structure conserved with, yet is chemically dissimilar to, rice dwarf virus P2, a protein associated with virus infectivity

Summary. The nucleotide sequence of the genome segment 2 (S2) of rice gall dwarf virus (RGDV), a phytoreovirus, when compared with the amino acid sequence of a component protein of the virus, showed that S2 potentially encoded a 127K minor outer capsid protein. This 127K protein designated as P2 and the 127K minor outer capsid protein (also termed P2) of rice dwarf virus (RDV) are similar in size, located in the outer capsid, and have well-conserved predicted polypeptide sequences, suggesting similar functions. Infectivity to insect vector cell monolayers of RGDV was maintained and the P2 protein was retained irrespective of carbon tetrachloride (CCl₄) treatment. This is in contrast to the infectivity of RDV which is removed along with P2 protein following CCl₄ treatment. RGDV with P2 was acquired by vector insects and transmitted to host plants, although RDV lacking P2 could not be transmitted to plants as previously published. These results imply that RDV and RGDV require P2 proteins for virus infectivity to vector insects. Received January 6, 1997 Accepted May 14, 1997     

Engineered Tobacco mosaic virus mutants with distinct physical characteristics in planta and enhanced metallization properties

Tobacco mosaic virus mutants were engineered to alter either the stability or surface chemistry of the virion: within the coat protein, glutamic acid was exchanged for glutamine in a buried portion to enhance the inter-subunit binding stability (E50Q), or a hexahistidine tract was fused to the surface-exposed carboxy terminus of the coat protein (6xHis). Both mutant viruses were expected to possess specific metal ion affinities. They accumulated to high titers in plants, induced distinct phenotypes, and their physical properties during purification differed from each other and from wild type (wt) virus. Whereas 6xHis and wt virions contained RNA, the majority of E50Q protein assembled essentially without RNA into rods which frequently exceeded 2 μm in length. Electroless deposition of nickel metallized the outer surface of 6xHis virions, but the central channel of E50Q rods, with significantly more nanowires of increased length in comparison to those formed in wtTMV.     

Identification of potyvirus isolates from faba bean (Vicia faba L.), and the relationships between bean yellow mosaic virus and clover yellow vein virus

Summary Clover yellow vein virus (CYVV) isolate H and the related potyvirus isolates E178, E197, and E242 could be distinguished from bean yellow mosaic (BYMV) isolates by their wider host range among non-legume test plant species, the peculiar enlargement of the nucleolus in infected plants, and the larger size of their coat protein as evidenced by slower migration in SDS-PAGE. Serologically, they are qualitatively indistinguishable in electro-blot immunoassay (EBIA) also with antibodies specific to the N-terminal part of BYMV-B25 coat protein, implying therefore that CYVV and BYMV coat proteins contain identical amino acid sequences in the N-terminal region.The faba-bean virus isolates from Sudan, Syria, and The Netherlands could be identified as BYMV isolates especially adapted to faba bean. All of them were weakly pathogenic toPhaseolus bean with the exception of SV205, assuming an intermediate position betweenPhaseolus-bean isolates, with low pathogenicity to faba bean, and faba-bean isolates, usually having low pathogenicity toPhaseolus bean. Strains of BYMV are thus hard to delimit.     

Electrophoretic separation of dsRNA genome segments from maize wallaby ear virus and its relationship to other phytoreoviruses

Partially purified maize wallaby ear virus (MWEV) preparations were shown to contain 10 double-stranded (ds) RNA segments. Electrophoretic mobilities of the dsRNAs of MWEV and of other phytoreoviruses have been compared in two buffers. The total genome molecular weight of maize wallaby ear virus was ～18.92 × 10⁶ and ～19.33 × 10⁶ in PTE and Loening's buffer, respectively. Extracts from the virus-free leafhopper vector, Cicadulina bimaculata, contained two dsRNA segments of molecular weight 1.15 × 10⁶ and 1.08 × 10⁶ in PTE buffer. Extracts from viruliferous insects also contained these same 2 dsRNA segments in addition to the 10 segments of maize wallaby ear virus. The electrophoretic pattern of dsRNA components of maize wallaby ear virus was similar to those of maize rough dwarf virus and Fiji disease virus suggesting a relationship between them. The degrees of relationship among these viruses and rice black-streaked dwarf virus, wound tumor virus, and rice dwarf virus were indicated by the electrophoretic patterns of their genome segments in the two buffers.     

Genome structure of tobacco necrosis virus strain A

An almost complete sequence of the RNA genome of tobacco necrosis virus (TNV) strain A has been determined. The genome organization is very similar to that of carnation mottle virus (CarMV) and turnip crinkle virus (TCV). The 5'-proximal open reading frame (ORF) encodes a 23-kDa protein and read-through of its amber codon into the second ORF is presumably used for the translation of a 82-kDa protein. The third large ORF encodes the 30-kDa coat protein. Two small ORFs are located upstream and one immediately downstream of this coat protein cistron. Extensive sequence similarity was found between the TNV 82-kDa protein and the putative polymerases of TCV, CarMV, cucumber necrosis virus (CNV), maize chlorotic mottle virus (MCMV), red clover necrotic mosaic virus (RCNMV), and barley yellow dwarf virus (BYDV). The TNV coat protein is very similar to southern bean mosaic virus (SBMV) capsid protein. Of the predicted small proteins only a 7.9-kDa protein shows some sequence similarity with a corresponding protein of MCMV, CarMV, and TCV. The others are unique to TNV. Except for the first four nucleotides at the 5' end no homology was found with the RNA of STNV (satellite of TNV).     

副黏病毒F蛋白融合活性位点中病毒特异性氨基酸基因突变分析

目的 了解副黏病毒融合蛋白(F)融合活性位点中的病毒特异性氨基酸在细胞融合中的作用.方法 以新城疫病毒(NDV)和人副流感病毒(hPIV)为例,在已确定的F蛋白融合活性位点中对病毒特异性氨基酸进行定点突变,然后将突变体F基因与同源或异源的血凝素.神经氨酸酶(HN)基因共转染BHK21细胞后,在真核细胞中表达.Giemsa染色和指示基因法检测细胞融合功能,荧光强度分析(FACS)检测F蛋白的表达效率.结果 在NDV F的突变体中,N150D-L152D的融合功能达到野毒株的46.31%;而N257D-N258D-Q259E、G271D-N272D和Q279E-Q281E的融合活性却几乎消失,分别只有野毒株的1.25%、3.14%和2.23%;N296D-N297D的融合功能是野毒株的97.68%.在hPIV F的突变体中,D143A-E145A的融合功能达到野毒株的32.63%;E223Q-K224A几乎不能形成合胞体,其融合活性只有野毒株的1.91%;K263A-R265A、D268A-D270A和R475A-R476A的融合功能分别是野毒株的14.63%、19.52%和28.95%.FACS结果表明,NDV F的N257D-N258D-Q259E、G271D-N272D和Q279E-Q281E突变体及hPIVF的E223Q-K224A突变体F蛋白在细胞表面几乎没有表达;其余所有突变体F蛋白的表达效率与野毒株相比,基本不变.结论 对于NDV F来说,N257、N258、Q259、G271、N272、Q279、Q281对NDV F的特异性细胞融合功能起重要作用;N150和L152也起一定的作用,但是N296和N297却没有作用.对于hPIV F来说,E223和K224对hPIV F的特异性细胞融合功能起非常重要的作用;D143、E145、K263、R265、D268、D270、R475、R476的作用也很重要.     

副黏病毒F蛋白融合活性位点中病毒特异性氨基酸基因突变分析

目的 了解副黏病毒融合蛋白(F)融合活性位点中的病毒特异性氨基酸在细胞融合中的作用.方法 以新城疫病毒(NDV)和人副流感病毒(hPIV)为例,在已确定的F蛋白融合活性位点中对病毒特异性氨基酸进行定点突变,然后将突变体F基因与同源或异源的血凝素.神经氨酸酶(HN)基因共转染BHK21细胞后,在真核细胞中表达.Giemsa染色和指示基因法检测细胞融合功能,荧光强度分析(FACS)检测F蛋白的表达效率.结果 在NDV F的突变体中,N150D-L152D的融合功能达到野毒株的46.31%;而N257D-N258D-Q259E、G271D-N272D和Q279E-Q281E的融合活性却几乎消失,分别只有野毒株的1.25%、3.14%和2.23%;N296D-N297D的融合功能是野毒株的97.68%.在hPIV F的突变体中,D143A-E145A的融合功能达到野毒株的32.63%;E223Q-K224A几乎不能形成合胞体,其融合活性只有野毒株的1.91%;K263A-R265A、D268A-D270A和R475A-R476A的融合功能分别是野毒株的14.63%、19.52%和28.95%.FACS结果表明,NDV F的N257D-N258D-Q259E、G271D-N272D和Q279E-Q281E突变体及hPIVF的E223Q-K224A突变体F蛋白在细胞表面几乎没有表达;其余所有突变体F蛋白的表达效率与野毒株相比,基本不变.结论 对于NDV F来说,N257、N258、Q259、G271、N272、Q279、Q281对NDV F的特异性细胞融合功能起重要作用;N150和L152也起一定的作用,但是N296和N297却没有作用.对于hPIV F来说,E223和K224对hPIV F的特异性细胞融合功能起非常重要的作用;D143、E145、K263、R265、D268、D270、R475、R476的作用也很重要.     

副黏病毒F蛋白融合活性位点中病毒特异性氨基酸基因突变分析

目的 了解副黏病毒融合蛋白(F)融合活性位点中的病毒特异性氨基酸在细胞融合中的作用.方法 以新城疫病毒(NDV)和人副流感病毒(hPIV)为例,在已确定的F蛋白融合活性位点中对病毒特异性氨基酸进行定点突变,然后将突变体F基因与同源或异源的血凝素.神经氨酸酶(HN)基因共转染BHK21细胞后,在真核细胞中表达.Giemsa染色和指示基因法检测细胞融合功能,荧光强度分析(FACS)检测F蛋白的表达效率.结果 在NDV F的突变体中,N150D-L152D的融合功能达到野毒株的46.31%;而N257D-N258D-Q259E、G271D-N272D和Q279E-Q281E的融合活性却几乎消失,分别只有野毒株的1.25%、3.14%和2.23%;N296D-N297D的融合功能是野毒株的97.68%.在hPIV F的突变体中,D143A-E145A的融合功能达到野毒株的32.63%;E223Q-K224A几乎不能形成合胞体,其融合活性只有野毒株的1.91%;K263A-R265A、D268A-D270A和R475A-R476A的融合功能分别是野毒株的14.63%、19.52%和28.95%.FACS结果表明,NDV F的N257D-N258D-Q259E、G271D-N272D和Q279E-Q281E突变体及hPIVF的E223Q-K224A突变体F蛋白在细胞表面几乎没有表达;其余所有突变体F蛋白的表达效率与野毒株相比,基本不变.结论 对于NDV F来说,N257、N258、Q259、G271、N272、Q279、Q281对NDV F的特异性细胞融合功能起重要作用;N150和L152也起一定的作用,但是N296和N297却没有作用.对于hPIV F来说,E223和K224对hPIV F的特异性细胞融合功能起非常重要的作用;D143、E145、K263、R265、D268、D270、R475、R476的作用也很重要.     

Immunity to Rice black streaked dwarf virus, a plant reovirus, can be achieved in rice plants by RNA silencing against the gene for the viroplasm component protein

The nonstructural protein P9-1 of Rice black streaked dwarf virus has been confirmed to accumulate in viroplasms, the putative sites of viral replication, in infected plants and insects. We transformed rice plants by introducing an RNA interference construct against the P9-1-encoding gene. The resultant transgenic plants accumulated short interfering RNAs specific to the construct. All progenies produced by self-fertilization of these transgenic plants with induced RNA interference against the gene for P9-1 were resistant to infection by the virus. Our results demonstrated that interfering with the expression of a viroplasm component protein of plant reoviruses, which plays an important role in viral proliferation, might be a practical and effective way to control plant reovirus infection in crop plants.     

副黏病毒F蛋白融合活性位点中病毒特异性氨基酸基因突变分析

目的 了解副黏病毒融合蛋白(F)融合活性位点中的病毒特异性氨基酸在细胞融合中的作用.方法 以新城疫病毒(NDV)和人副流感病毒(hPIV)为例,在已确定的F蛋白融合活性位点中对病毒特异性氨基酸进行定点突变,然后将突变体F基因与同源或异源的血凝素.神经氨酸酶(HN)基因共转染BHK21细胞后,在真核细胞中表达.Giemsa染色和指示基因法检测细胞融合功能,荧光强度分析(FACS)检测F蛋白的表达效率.结果 在NDV F的突变体中,N150D-L152D的融合功能达到野毒株的46.31%;而N257D-N258D-Q259E、G271D-N272D和Q279E-Q281E的融合活性却几乎消失,分别只有野毒株的1.25%、3.14%和2.23%;N296D-N297D的融合功能是野毒株的97.68%.在hPIV F的突变体中,D143A-E145A的融合功能达到野毒株的32.63%;E223Q-K224A几乎不能形成合胞体,其融合活性只有野毒株的1.91%;K263A-R265A、D268A-D270A和R475A-R476A的融合功能分别是野毒株的14.63%、19.52%和28.95%.FACS结果表明,NDV F的N257D-N258D-Q259E、G271D-N272D和Q279E-Q281E突变体及hPIVF的E223Q-K224A突变体F蛋白在细胞表面几乎没有表达;其余所有突变体F蛋白的表达效率与野毒株相比,基本不变.结论 对于NDV F来说,N257、N258、Q259、G271、N272、Q279、Q281对NDV F的特异性细胞融合功能起重要作用;N150和L152也起一定的作用,但是N296和N297却没有作用.对于hPIV F来说,E223和K224对hPIV F的特异性细胞融合功能起非常重要的作用;D143、E145、K263、R265、D268、D270、R475、R476的作用也很重要.     

副黏病毒F蛋白融合活性位点中病毒特异性氨基酸基因突变分析

目的 了解副黏病毒融合蛋白(F)融合活性位点中的病毒特异性氨基酸在细胞融合中的作用.方法 以新城疫病毒(NDV)和人副流感病毒(hPIV)为例,在已确定的F蛋白融合活性位点中对病毒特异性氨基酸进行定点突变,然后将突变体F基因与同源或异源的血凝素.神经氨酸酶(HN)基因共转染BHK21细胞后,在真核细胞中表达.Giemsa染色和指示基因法检测细胞融合功能,荧光强度分析(FACS)检测F蛋白的表达效率.结果 在NDV F的突变体中,N150D-L152D的融合功能达到野毒株的46.31%;而N257D-N258D-Q259E、G271D-N272D和Q279E-Q281E的融合活性却几乎消失,分别只有野毒株的1.25%、3.14%和2.23%;N296D-N297D的融合功能是野毒株的97.68%.在hPIV F的突变体中,D143A-E145A的融合功能达到野毒株的32.63%;E223Q-K224A几乎不能形成合胞体,其融合活性只有野毒株的1.91%;K263A-R265A、D268A-D270A和R475A-R476A的融合功能分别是野毒株的14.63%、19.52%和28.95%.FACS结果表明,NDV F的N257D-N258D-Q259E、G271D-N272D和Q279E-Q281E突变体及hPIVF的E223Q-K224A突变体F蛋白在细胞表面几乎没有表达;其余所有突变体F蛋白的表达效率与野毒株相比,基本不变.结论 对于NDV F来说,N257、N258、Q259、G271、N272、Q279、Q281对NDV F的特异性细胞融合功能起重要作用;N150和L152也起一定的作用,但是N296和N297却没有作用.对于hPIV F来说,E223和K224对hPIV F的特异性细胞融合功能起非常重要的作用;D143、E145、K263、R265、D268、D270、R475、R476的作用也很重要.     

副黏病毒F蛋白融合活性位点中病毒特异性氨基酸基因突变分析

目的 了解副黏病毒融合蛋白(F)融合活性位点中的病毒特异性氨基酸在细胞融合中的作用.方法 以新城疫病毒(NDV)和人副流感病毒(hPIV)为例,在已确定的F蛋白融合活性位点中对病毒特异性氨基酸进行定点突变,然后将突变体F基因与同源或异源的血凝素.神经氨酸酶(HN)基因共转染BHK21细胞后,在真核细胞中表达.Giemsa染色和指示基因法检测细胞融合功能,荧光强度分析(FACS)检测F蛋白的表达效率.结果 在NDV F的突变体中,N150D-L152D的融合功能达到野毒株的46.31%;而N257D-N258D-Q259E、G271D-N272D和Q279E-Q281E的融合活性却几乎消失,分别只有野毒株的1.25%、3.14%和2.23%;N296D-N297D的融合功能是野毒株的97.68%.在hPIV F的突变体中,D143A-E145A的融合功能达到野毒株的32.63%;E223Q-K224A几乎不能形成合胞体,其融合活性只有野毒株的1.91%;K263A-R265A、D268A-D270A和R475A-R476A的融合功能分别是野毒株的14.63%、19.52%和28.95%.FACS结果表明,NDV F的N257D-N258D-Q259E、G271D-N272D和Q279E-Q281E突变体及hPIVF的E223Q-K224A突变体F蛋白在细胞表面几乎没有表达;其余所有突变体F蛋白的表达效率与野毒株相比,基本不变.结论 对于NDV F来说,N257、N258、Q259、G271、N272、Q279、Q281对NDV F的特异性细胞融合功能起重要作用;N150和L152也起一定的作用,但是N296和N297却没有作用.对于hPIV F来说,E223和K224对hPIV F的特异性细胞融合功能起非常重要的作用;D143、E145、K263、R265、D268、D270、R475、R476的作用也很重要.     

副黏病毒F蛋白融合活性位点中病毒特异性氨基酸基因突变分析

目的 了解副黏病毒融合蛋白(F)融合活性位点中的病毒特异性氨基酸在细胞融合中的作用.方法 以新城疫病毒(NDV)和人副流感病毒(hPIV)为例,在已确定的F蛋白融合活性位点中对病毒特异性氨基酸进行定点突变,然后将突变体F基因与同源或异源的血凝素.神经氨酸酶(HN)基因共转染BHK21细胞后,在真核细胞中表达.Giemsa染色和指示基因法检测细胞融合功能,荧光强度分析(FACS)检测F蛋白的表达效率.结果 在NDV F的突变体中,N150D-L152D的融合功能达到野毒株的46.31%;而N257D-N258D-Q259E、G271D-N272D和Q279E-Q281E的融合活性却几乎消失,分别只有野毒株的1.25%、3.14%和2.23%;N296D-N297D的融合功能是野毒株的97.68%.在hPIV F的突变体中,D143A-E145A的融合功能达到野毒株的32.63%;E223Q-K224A几乎不能形成合胞体,其融合活性只有野毒株的1.91%;K263A-R265A、D268A-D270A和R475A-R476A的融合功能分别是野毒株的14.63%、19.52%和28.95%.FACS结果表明,NDV F的N257D-N258D-Q259E、G271D-N272D和Q279E-Q281E突变体及hPIVF的E223Q-K224A突变体F蛋白在细胞表面几乎没有表达;其余所有突变体F蛋白的表达效率与野毒株相比,基本不变.结论 对于NDV F来说,N257、N258、Q259、G271、N272、Q279、Q281对NDV F的特异性细胞融合功能起重要作用;N150和L152也起一定的作用,但是N296和N297却没有作用.对于hPIV F来说,E223和K224对hPIV F的特异性细胞融合功能起非常重要的作用;D143、E145、K263、R265、D268、D270、R475、R476的作用也很重要.     

Identification of rice black-streaked dwarf fijivirus in maize with rough dwarf disease in China

Summary.  Three virus isolates from maize with rough dwarf in different provinces in China were analyzed at the molecular level. When compared to an isolate from diseased rice plants in Hubei Province, all four isolates had identical genomic RNA electrophoretic profiles, which were composed of ten double-stranded (ds) RNAs. Full-length cDNAs of segment 10 (S10) from each of the four isolates were cloned by RT-PCR and the complete sequences were determined. Analysis of the sequences revealed that each consisted of 1801 nucleotides and contained a single open reading frame (ORF) which potentially encoded a protein with 558 amino acids. Further, the sequences showed more than 97.0% and 98.0% identity atnucleotide and amino acid levels, respectively. In addition, theiridentities to rice black-streaked dwarf virus S10 were significantlyhigher than those to maize rough dwarf virus S10. Based on theseresults, it is suggested that the virus which causes this maize diseasein China is rice black-streaked dwarf virus. Received April 4, 2000 Accepted July 4, 2000     

Simultaneous Analysis of the Bidirectional African Cassava Mosaic Virus Promoter Activity Using Two Different Luciferase Genes

The expression of geminivirus genes is controlled by bidirectional promoters which are located in the large intergenic region of the circular DNA genomes and specifically regulated by virus encoded proteins. In order to study the simultaneous regulation of both orientations of the DNA A and DNA B promoters of African cassava mosaic virus (ACMV), they were cloned between two different luciferase genes with the firefly luciferase gene in complementary-sense and the Renilla luciferase gene in virion-sense orientation. The regulation of the ACMV promoters by proteins encoded by the complete DNA A, as well as by the individually expressed transactivator (TrAP) or replication-associated (Rep) proteins was assessed in tobacco and cassava protoplasts using dual luciferase assays. In addition, the regulation of the DNA A promoter integrated into tobacco genome was also assessed. The results show that TrAP activates virion-sense expression strongly both in cassava and tobacco protoplasts, but not in transgenic tobacco plants. In contrast to this, DNA A encoded proteins activate virion-sense expression both in protoplasts and in transgenic plants. At the same time they reduce the expression of the complementary-sense Rep gene on DNA A but activate the expression of the complementary-sense movement protein (MPB) gene on DNA B. The degree of MBP activation is higher in cassava than in tobacco protoplasts, indicating that the plant host also influences the promoter strength. Transient transformation experiments using linearized DNA indicate that the different regulation of the ACMV DNA A promoter in protoplasts and transgenic plants could be due to different DNA curvature in free plasmids and in genes integrated in plant genomic DNA.     

The coat protein of beet curly top virus is essential for infectivity

We have applied the procedure of Agrobacterium-mediated inoculation to develop a simple, efficient, and reproducible assay for the infectivity of the leafhopper-transmitted geminivirus, beet curly top virus (BCTV). This assay system was used to show that a coat protein mutant of BCTV is not infectious, but could be complemented by coagroinoculation with a second mutant bearing a lethal mutation in the complementary-sense open reading frame, C1. Furthermore, the coat protein mutant retained the ability to replicate and to produce both ssDNA and dsDNA when electroporated into Nicotiana tabacum protoplasts. We conclude that the coat protein of BCTV is essential for spread of the virus. The results are discussed in the light of results with coat protein mutants of other geminiviruses.