Jonathan Donson

The nucleotide sequence of maize streak virus DNA.

The nucleotide sequence of the DNA of maize streak virus (MSV) has been determined. The data were accommodated into one DNA circle of 2687 nucleotides, in contrast to previously characterised geminiviruses which have been shown to possess two circles of DNA. Comparison of the nucleotide sequences of the DNA of MSV with those of cassava latent virus (CLV) and tomato golden mosaic virus (TGMV) showed no detectable homology. Analysis of open reading frames revealed seven potential coding regions for proteins of mol. wt. greater than or equal to 10 000, three in the viral (+) sense and four in the complementary (‐) sense. The position of likely transcription signals on the MSV DNA sequence would suggest a bidirectional strategy of transcription as proposed for CLV and TGMV. Nine inverted repeat sequences which have a potential of forming hairpin structures of delta G greater than or equal to ‐14 kcal/mol have been detected. Three of these hairpin structures are in non‐coding regions and could be involved in the regulation of transcription and/or replication.

The nucleotide sequence of a geminivirus from Digitaria sanguinalis

The encapsidated single-stranded circular DNA of a geminivirus isolated from Digitaria sanguinalis has been sequenced. The data obtained are consistent with there being one DNA circle of 2701 nucleotides. Comparison of the nucleotide sequence with those of maize streak virus (MSV) and wheat dwarf virus showed 64 and 47% DNA homology, respectively. The sequence has four potential coding regions for proteins of greater than 10 kDa, two in the viral (+) sense and two in the complementary (-) sense. Each of these potential coding regions has a highly homologous counterpart among the seven open reading frames previously described for MSV. Virion DNA contained, in addition to the circular single-stranded DNA, a population of small DNA molecules similar to those associated with MSV particles. A comparison with MSV DNA of the region complementary to these small DNA molecules revealed conserved sequences, which may have a role in defining the limits of these primer-like molecules.

A putative primer for second-strand DNA synthesis of maize streak virus is virion-associated.

We have isolated, from maize streak virus (MSV) preparations, a population of ‘nested’ DNA molecules. These molecules have ribonucleotides covalently linked to the DNA species’ discrete 5′ deoxyribonucleotide terminus. The major species has a DNA sequence of 80 nucleotides which is complementary to a region 5′ of two hairpin structures on the MSV genome, almost exclusively in an intergenic region. These molecules have been used to prime the synthesis in vitro of a complementary strand to virion DNA, initiating this reaction at one site on the genome.

Mapping of Digitaria streak virus transcripts reveals different RNA species from the same transcription unit.

All, except 19 [corrected] bp, of the Digitaria streak virus (DSV) genome is transcribed. Two RNA transcripts (1+ and 2+) are encoded by the virion DNA strand and up to five (1‐ to 5‐) by the complementary DNA strand [corrected]. Detailed mapping of these RNAs has revealed evidence for splicing in one species (RNA 4‐), which together with its more abundant unspliced counterpart (RNA 2‐) could synthesize both a 30.5 and 41 kd polypeptide from the same transcription unit. This extensive overlapping of spliced and unspliced RNAs could indicate that the initiation and splicing of transcripts is temporally regulated. At least one transcript (RNA 1‐) may have a non‐translational role. Transcription of the DSV genome shows similarities to some animal DNA viruses, particularly the papovaviruses.

Agrobacterium-mediated infectivity of cloned digitaria streak virus DNA

A monomeric clone of double-stranded DNA synthesized in vitro DNA of the geminivirus Digitaria streak (DSV) was subcloned as a tandem dimeric unit into a binary vector of Agrobacterium tumefaciens, creating a plasmid pDS2. Inoculation of digitaria sanguinalis with A. tumefaciens carrying pDS2 resulted in viral infection. The symptoms, virus particles, and DNA forms obtained were indistinguishable from those of a natural DSV infection of D. sanguinalis. Inoculations have also induced infections in Zea mays and Avena sativa. The sequence of the Agrobacterium-mediated infectious clone of DSV has been determined.

Detection of a non-structural protein of M r 11 000 encoded by the virion DNA of maize streak virus.

A polypeptide of approximately 11 000 daltons (11 kDa protein) encoded by an open reading frame (10.9 ORF) from the virion sense of maize streak virus (MSV) DNA has been detected among the products of in vitro translation reactions programmed with RNA from infected maize plants and also in total protein extracts from infected leaves. The 11 kDa protein has not been detected in virions and is therefore proposed to have a nonstructural role.Viral DNA with an additional in-frame translation stop codon in the 10.9 ORF was not infectious when transmitted to maize plants via Agrobacterium tumefaciens “agroinfection”, suggesting that the 10.9 ORF may be essential for virus function. Computer comparison data show that equivalent ORFs in wheat dwarf virus (WDV) and digitaria streak virus (DSV) have some sequences in common with the 10.9 ORF of MSV. Further-more, the absence of similar sequences in geminiviruses which infect dicotyledonous plants suggests that the 11 kDa protein and its putative homologs in WDV and DSV have a function necessary only for those geminiviruses which infect the Gramineae.The significance of the 11 kDa protein in relation to expression of the virion sense DNA of MSV is discussed.

Computer analysis identifies sequence homologies between potential gene products of Maize Streak Virus and those of Cassava Latent Virus and Tomato Golden Mosaic Virus.

SummaryThe amino acid sequences of the putative polypeptides of maize streak virus (MSV) have been systematically compared with those of cassava latent virus (CLV) and tomato golden mosaic virus (TGMV) using the programme DIAGON (8).Conserved sequences have been detected between peptides encoded by the complementary (-) sense of MSV and those of CLV and TGMV, viz; the 40 200 Mr polypeptide of CLV-1 (3) and the 40 285 Mr polypeptide of TGMV-A (4) show extensive homologies with the 17 768 Mr and 31 388 Mr polypeptides of MSV (6).Distant and variable homologies have been detected between the putative coat protein of MSV when compared with those of CLV and TGMV. No other relationships between the potential gene products of MSV and those of CLV and TGMV have been detected.The extensive homologies detected between the complementary sense encoded peptides suggest that they are derived from functional genes, and that the directly conserved sequences may contain amino acids essential to the function of these proteins. The less extensive homologies among the putative coat proteins are considered in relation to their possible structures and functions.

Physical Mapping and Molecular Cloning of Caulimovirus DNA

Summary Native DNA of both mirabilis mosaic virus (MMV) and the previously undescribed thistle mottle virus (ThMV) formed multiple bands when analysed by gel electrophoresis, thereby resembling DNA from other caulimoviruses. Denaturation showed that ThMV DNA had three discontinuities (one in one strand and two in the other) and that MMV DNA had four discontinuities which mapped in the same relative positions as those in DNA of figwort mosaic virus (FMV). DNA from carnation etched ring virus (CERV), FMV, MMV and ThMV was cloned in bacterial plasmids. Cloned full-length DNA of CERV, FMV or MMV was infective for plants. Comparisons by restriction mapping and Southern transfer hybridization among DNA from cauliflower mosaic virus, CERV, FMV, MMV and ThMV suggest that these viruses are distinct members of the caulimovirus group.

Physical mapping of the DNAs of Carnation Etched Ring and Figwort Mosaic Viruses

Summary The native DNAs of both carnation etched ring virus (CERV) and figwort mosaic virus (FMV) formed multiple bands on gel electrophoresis thus resembling cauliflower mosaic virus (CaMV) DNA; there were some minor differences in banding patterns. Denaturation and nuclease S1 digestion showed that CERV DNA had three discontinuities (two in one strand and one in the other) and FMV DNA had four discontinuities (three in one strand and one in the other). These discontinuities and the sites of restriction endonuclease digestion were mapped on CERV and FMV DNAs and the maps compared with those of CaMV DNA. The maps of the three virus DNAs were very different.

Structure and Replication of Geminivirus Genomes

SUMMARY The geminiviruses are a group of plant viruses containing single-stranded (ss) DNA in particles comprising two quasi-icosahedral units. Some are transmitted by whiteflies, others by leafhoppers. Comparisons were made of the genome organization and expression of cassava latent virus (CLV) and maize streak virus (MSV) and beet curly top virus (BCTV), each with distinct host range and insect vector species characteristics. From these studies, several indications as to the replication mechanism(s) are suggested.