Endogenous viral sequences and their potential contribution to heritable virus resistance in plants

Tobacco endogenous pararetroviruses (TEPRVs) represent the first virus-derived repetitive sequence family found in plants. The sequence conservation of TEPRVs and the lack of an exogenous form of the virus suggest that TEPRVs serve a beneficial function, perhaps by furnishing virus resistance via homologous sequence interactions. This hypothesis is supported by the observation that TEPRVs are methylated and negligibly transcribed. Moreover, transgenes driven by the TEPRV enhancer are silenced and methylated when introduced into tobacco, but remain active and unmethylated in non-host species devoid of sequences homologous to TEPRVs. In transgenic Arabidopsis, the TEPRV enhancer is active primarily in shoot meristems. This suggests that the virus giving rise to TEPRVs could infect germ cell precursors, a prerequisite for meiotically heritable insertions into host chromosomes. The copy number, organization and methylation of TEPRVs in tetraploid tobacco and one of its diploid ancestors, Nicotiana sylvestris, the presumed original host for the virus, have remained constant since polyploid formation. The remarkable conservation of these features in two independently evolving species further supports a role for TEPRVs in viral immunity.     

Complete genome sequence of a new circular DNA virus from grapevine

A novel circular DNA virus sequence is reported from grapevine. The corresponding genomic organization, coding potential, and conserved origin of replication are similar to those of members of the family Geminiviridae, but the genome of 3,206 nucleotides is 4% larger than the largest reported geminiviral genome and shares only 50% overall sequence identity.     

Clustered repeat sequences in the genome of Epstein Barr virus

The genome of Epstein-Barr virus is composed of unique DNA interspersed with repetitive sequences. This organization suggests that Epstein-Barr virus provides a useful model for studying the function(s) of repetitive sequences in eukaryotic chromosomes. The primary structure of two of the repeat sequences, the 3072 bp large internal repeat, or BamHI-W repeat, and a smaller 125 bp, G, C-rich NotI repeat, are presented here. Their structures and possible functions are discussed.     

Adeno-associated virus Rep proteins target DNA sequences to a unique locus in the human genome.

We have developed a system for site-specific DNA integration in human cells, mediated by the adeno-associated virus (AAV) Rep proteins. In its normal lysogenic cycle, AAV integrates at a site on human chromosome 19 termed AAVS1. We describe a rapid PCR assay for the detection of integration events at AAVS1 in whole populations of cells. Using this assay, we determined that the AAV Rep proteins, delivered in cis or trans, are required for integration at AAVS1. Only the large forms of the Rep protein, Rep78 and Rep68, promoted site-specific integration. The AAV inverted terminal repeats, present in cis, were not essential for integration at AAVS1, but in cells containing Rep, they increased the efficiency of integration. In the presence of the Rep proteins, the integration of a plasmid containing AAV inverted terminal repeats occurred at high frequency, such that clones containing the plasmid could be isolated without selection. In two of the five clones analyzed by fluorescence in situ hybridization, the plasmid DNA was integrated at AAVS1. In most of the clones, at least one copy of the entire plasmid was integrated in a tandem array. Detailed analysis of the integrated plasmid structure in one clone suggested a complex mechanism producing rearrangements of the flanking genomic DNA, similar to those observed with wild-type AAV.     

Polymorphic nuclear gene sequences indicate a novel genome donor in the polyploid genus Thinopyrum

For decades, the wheatgrass genus Thinopyrum has been of interest to plant breeders as a source of genes that confer competitive traits. This genus has been a considerable challenge to plant systematists because of the impacts of polyploidization on the evolution of this group. This study was aimed to augment existing cytogenetic data with a sequence-based investigation of the genomes of these species. Sequences of the internal transcribed spacer 1 (ITS1), introns 9 through 11 of the granule-bound starch synthase (GBSSI) gene and intron III of the beta-amylase gene (Bmy1) were isolated from the genomes of polyploid Thinopyrum species by PCR, cloning and sequencing and the evolutionary distances between these species and putative diploid ancestors were estimated with Kimura's two-parameter method. Phylogenetic analysis of these sequences largely agrees with what has been established through cytogenetic means for the Th. caespitosum (Koch) Liu & Wang and Ps geniculata (Trin.) á. L?ve, and suggests a contribution of the St genome of Ps. spicata (Pursh) á. L?ve to the tetraploids Th. scirpeum (Presl) Dewey and Th. junceiforme (á. L?ve & D. L?ve) á. L?ve. A unique Bmy1 allele, divergent from other Triticeae but shared between Th. caespitosum, Th. intermedium (Host) Barkworth & Dewey, Th. junceum (L.) á. L?ve and Th. ponticum Barkworth & Dewey, implies a connection between these species. Distinct oligonucleotide polymorphisms and distance calculations based on the three loci implicate Crithopsis delileana (Schult.) Roshev. and Taeniatherum caput-medusae (L.) Nevski in the evolution of the hexaploid Th. intermedium and the decaploid Th. ponticum and also suggest a potential connection of these polyploids with Elytrigia repens (L.) Desv. ex Nevski. None of these species have been previously associated with the Thinopyrum genus. Allele-specific PCR was employed to detect the putative Crithopsis allele of ITS1 in a number of accessions. Real-time PCR indicates that two of six genomes of the hexaploid Th. intermedium have the Crithopsis-type ITS1 allele and that all ITS1 loci in the decaploid Th. ponticum are of this type. These results are supportive of the hypothesis that concerted evolution has homogenized the rDNA of Th. ponticum to the allele derived from the Crithopsis or Taeniatherum ancestor. Discovery of these novel alleles, with close homology to Ta. caput-medusae, may represent a fundamental change in the view of the evolution of Th. intermedium and Th. ponticum.     

A virus-derived short hairpin RNA confers resistance against sugarcane mosaic virus in transgenic sugarcane

RNA interference (RNAi) is commonly used to produce virus tolerant transgenic plants. The objective of the current study was to generate transgenic sugarcane plants expressing a short hairpin RNAs (shRNA) targeting the coat protein (CP) gene of sugarcane mosaic virus (SCMV). Based on multiple sequence alignment, including genomic sequences of four SCMV strains, a conserved region of ~ 456 bp coat protein (CP) gene was selected as target gene and amplified through polymerase chain reaction (PCR). Subsequently, siRNAs2 and siRNA4 were engineered as stable short hairpin (shRNA) transgenes of 110 bp with stem and loop sequences derived from microRNA (sof-MIR168a; an active regulatory miRNA in sugarcane). These transgenes were cloned in independent RNAi constructs under the control of the polyubiquitin promoter. The RNAi constructs were delivered into two sugarcane cultivars ‘SPF-234 and NSG-311 in independent experiments using particle bombardment. Molecular identification through PCR and Southern blot revealed anti-SCMV positive transgenic lines. Upon mechanical inoculation of transgenic and non-transgenic sugarcane lines with SCMV, the degree of resistance was found variable among the two sugarcane cultivars. For sugarcane cultivar NSG-311, the mRNA expression of the CP–SCMV was reduced to 10% in shRNA2-transgenic lines and 80% in shRNA4-transgenic lines. In sugarcane cultivar SPF-234, the mRNA expression of the CP–SCMV was reduced to 20% in shRNA2-transgenic lines and 90% in shRNA4 transgenic lines, revealing that transgenic plants expressing shRNA4 were almost immune to SCMV infection.     

Some genetic aspects of the resistance of Spodoptera frugiperda to a nuclear polyhedrosis virus

A probit analysis of the dosage-response of two isolates of Spodoptera frugiperda to a single nuclear polyhedrosis virus revealed a greater than 5-fold difference in the LD₅₀. The response of F₁ progeny of reciprocal crosses between the two parental isolates demonstrated that resistance to the virus is not sex-linked. The LD₅₀ values of F₁ and F₂ backcrosses suggest that the major variation in resistance is due to a single gene or genes that lack dominance.     

Identification of resistance gene analogs linked to a powdery mildew resistance locus in grapevine

Oligonucleotide primers, designed to conserved regions of nucleotide binding site (NBS) motifs within previously cloned pathogen resistance genes, were used to amplify resistance gene analogs (RGAs) from grapevine. Twenty eight unique grapevine RGA sequences were identified and subdivided into 22 groups on the basis of nucleic acid sequence-identity of approximately 70% or greater. Representatives from each group were used in a bulked segregant analysis strategy to screen for restriction fragment length polymorphisms linked to the powdery mildew resistance locus, Run1, introgressed into Vitis vinifera L. from the wild grape species Muscadinia rotundifolia. Three RGA markers were found to be tightly linked to the Run1 locus. Of these markers, two (GLP1–12 and MHD145) cosegregated with the resistance phenotype in 167 progeny tested, whereas the third marker (MHD98) was mapped to a position 2.4 cM from the Run1 locus. The results demonstrate the usefulness of RGA sequences, when used in combination with bulked segregant analysis, to rapidly generate markers tightly linked to resistance loci in crop species. Received: 2 May 2001 / Accepted: 3 August 2001     

The GLI gene encodes a nuclear protein which binds specific sequences in the human genome.

The GLI gene is amplified in a subset of human tumors and encodes a protein product with five zinc finger DNA-binding motifs. In this study, we show that the GLI gene product has a predominantly nuclear localization and binds DNA in a sequence-specific fashion. Three GLI binding sites were identified by using a novel procedure in which total human DNA was bound to a GLI recombinant fusion protein, and the polymerase chain reaction was used to amplify and recover the bound sequences. The GLI protein protected a 23- to 24-base region within all three binding sites, and the protected region in each case included the 9-base-pair sequence 5'-GACCACCCA-3'. One of the binding sites was contained within a 63-base-pair repeat of the variable number of tandem repeat type, whereas the other two sites were represented once in the genome. The approach used here to identify GLI binding sites should be applicable to the characterization of other zinc finger proteins.     

Heterologous human immunodeficiency virus type 1 lentiviral vectors packaging a simian immunodeficiency virus-derived genome display a specific postentry transduction defect in dendritic cells

Heterologous lentiviral vectors (LVs) represent a way to address safety concerns in the field of gene therapy by decreasing the possibility of genetic recombination between vector and packaging constructs and the generation of replication-competent viruses. Using described LVs based on human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus MAC251 (SIV(MAC251)), we asked whether heterologous virion particles in which trans-acting factors belonged to HIV-1 and cis elements belonged to SIV(MAC251) (HIV-siv) would behave as parental homologous vectors in all cell types. To our surprise, we found that although the heterologous HIV-siv vector was as infectious as its homologous counterpart in most human cells, it was defective in the transduction of dendritic cells (DCs) and, to a lesser extent, macrophages. In DCs, the main postentry defect was observed in the formation of two-long-terminal-repeat circles, despite the fact that full-length proviral DNA was being synthesized and was associated with the nucleus. Taken together, our data suggest that heterologous HIV-siv vectors display a cell-dependent infectivity defect, most probably at a post-nuclear entry migration step. As homologous HIV and SIV vectors do transduce DCs, we believe that these results underscore the importance of a conserved interaction between cis elements and trans-acting viral factors that is lost or suboptimal in heterologous vectors and essential only in the transduction of certain cell types. For gene therapy purposes, these findings indicate that the cellular tropism of LVs can be modulated not only through the use of distinct envelope proteins or tissue-specific promoters but also through the specific combinatorial use of packaging and transfer vector constructs.     

Nucleotide sequences in human chromosomal DNA from nonhepatic tissues homologous to the hepatitis B virus genome

DNA extracted from human nonhepatic tissues (placenta and kidney) have been digested with restriction endonucleases and examined by the Southern procedure with cloned 32P-labelled DNA of hepatitis B virus (HBV). In placental DNAs of women with the history of a hepatitis B infection and in one out of four cases of patients with no known HBV exposure or manifestation, HBV-related chromosomal nucleotide sequences were detected. The integration of HBV-related sequences was observed also in human kidney DNA. Moreover, in the placenta of women who had hepatitis B infection prior to delivery, unusual unintegrated forms of HBV have been found. We conclude that HBV sequences can be found not only in hepatic tissue but also in placental and kidney DNA, both of HBV-exposed and in one case even of a nonexposed patient.     

West Nile virus T-cell ligand sequences shared with other flaviviruses: a multitude of variant sequences as potential altered peptide ligands

Phylogenetic relatedness and cocirculation of several major human pathogen flaviviruses are recognized as a possible cause of deleterious immune responses to mixed infection or immunization and call for a greater understanding of the inter-Flavivirus protein homologies. This study focused on the identification of human leukocyte antigen (HLA)-restricted West Nile virus (WNV) T-cell ligands and characterization of their distribution in reported sequence data of WNV and other flaviviruses. H-2-deficient mice transgenic for either A2, A24, B7, DR2, DR3, or DR4 HLA alleles were immunized with overlapping peptides of the WNV proteome, and peptide-specific T-cell activation was measured by gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) assays. Approximately 30% (137) of the WNV proteome peptides were identified as HLA-restricted T-cell ligands. The majority of these ligands were conserved in ～≥88% of analyzed WNV sequences. Notably, only 51 were WNV specific, and the remaining 86, chiefly of E, NS3, and NS5, shared an identity of nine or more consecutive amino acids with sequences of 64 other flaviviruses, including several major human pathogens. Many of the shared ligands had an incidence of >50% in the analyzed sequences of one or more of six major flaviviruses. The multitude of WNV sequences shared with other flaviviruses as interspecies variants highlights the possible hazard of defective T-cell activation by altered peptide ligands in the event of dual exposure to WNV and other flaviviruses, by either infection or immunization. The data suggest the possible preferred use of sequences that are pathogen specific with minimum interspecies sequence homology for the design of Flavivirus vaccines.     

In silico identification of coffee genome expressed sequences potentially associated with resistance to diseases

Sequences potentially associated with coffee resistance to diseases were identified by in silico analyses using the database of the Brazilian Coffee Genome Project (BCGP). Keywords corresponding to plant resistance mechanisms to pathogens identified in the literature were used as baits for data mining. Expressed sequence tags (ESTs) related to each of these keywords were identified with tools available in the BCGP bioinformatics platform. A total of 11,300 ESTs were mined. These ESTs were clustered and formed 979 EST-contigs with similarities to chitinases, kinases, cytochrome P450 and nucleotide binding site-leucine rich repeat (NBS-LRR) proteins, as well as with proteins related to disease resistance, pathogenesis, hypersensitivity response (HR) and plant defense responses to diseases. The 140 EST-contigs identified through the keyword NBS-LRR were classified according to function. This classification allowed association of the predicted products of EST-contigs with biological processes, including host defense and apoptosis, and with molecular functions such as nucleotide binding and signal transducer activity. Fisher's exact test was used to examine the significance of differences in contig expression between libraries representing the responses to biotic stress challenges and other libraries from the BCGP. This analysis revealed seven contigs highly similar to catalase, chitinase, protein with a BURP domain and unknown proteins. The involvement of these coffee proteins in plant responses to disease is discussed.     

Homologous terminal sequences of the genome double-stranded RNAs of bluetongue virus.

The 3'-terminal sequences of the 10 double-stranded RNA genome segments of bluetongue virus (serotypes 10 and 11) were determined. The double-stranded RNAs were 3' labeled with [5'-32P]pCp and resolved into 10 segments by electrophoresis. After denaturation, the two complementary strands of segments 4 through 10 were resolved into fast- and slow-migrating species by polyacrylamide gel electrophoresis, and their 3' end sequences were determined. Complete RNase T1 digestion of the individual 3'-labeled double-stranded RNA segments yielded two labeled oligonucleotides, one of which migrated faster than the other on 20% polyacrylamide-7 M urea gels. Sequence analyses of the two oligonucleotides of segments 4 through 10 confirmed the corresponding RNA sequence data. For RNA segments 1 through 3 the oligonucleotide analyses gave comparable results. The 3'-terminal sequences of the fast-migrating RNA species were HOCAAUUU. . . ; those of the slow-migrating RNA species were HOCAUUCACA. . . . Similar results were obtained for double-stranded RNA from bluetongue virus serotypes 10 and 11. Beyond the common termini, the sequences for each segment varied considerably.     

Role of pyrimidine salvage pathway in the maintenance of organellar and nuclear genome integrity

Nucleotide biosynthesis proceeds through a de novo pathway and a salvage route. In the salvage route, free bases and/or nucleosides are recycled to generate the corresponding nucleotides. Thymidine kinase (TK) is the first enzyme in the salvage pathway to recycle thymidine nucleosides as it phosphorylates thymidine to yield thymidine monophosphate. The Arabidopsis genome contains two TK genes ?TK1a and TK1b? that show similar expression patterns during development. In this work, we studied the respective roles of the two genes during early development and in response to genotoxic agents targeting the organellar or the nuclear genome. We found that the pyrimidine salvage pathway is crucial for chloroplast development and genome replication, as well as for the maintenance of its integrity, and is thus likely to play a crucial role during the transition from heterotrophy to autotrophy after germination. Interestingly, defects in TK activity could be partially compensated by supplementation of the medium with sugar, and this effect resulted from both the availability of a carbon source and the activation of the nucleotide de novo synthesis pathway, providing evidence for a compensation mechanism between two routes of nucleotide biosynthesis that depend on nutrient availability. Finally, we found differential roles of the TK1a and TK1b genes during the plant response to genotoxic stress, suggesting that different pools of nucleotides exist within the cells and are required to respond to different types of DNA damage. Altogether, our results highlight the importance of the pyrimidine salvage pathway, both during plant development and in response to genotoxic stress.     

Complete genome sequence of a novel vitivirus isolated from grapevine

A novel virus-like sequence from grapevine was identified by Illumina sequencing. The complete genome is 7,551 nucleotides in length, with polyadenylation at the 3' end. Translation of the sequence revealed five open reading frames (ORFs). The genomic organization was most similar to those of vitiviruses. The polymerase (ORF1) and coat protein (ORF4) genes shared 31 to 49% nucleotide and 40 to 70% amino acid sequence identities, respectively, with other grapevine vitiviruses. The virus was tentatively named grapevine virus F (GVF).