The nucleotide sequence of cloned wheat dwarf virus DNA

Restriction analysis and cloning of virus-specific double-stranded DNA isolated from plants infected with wheat dwarf virus (WDV) indicated that the virus genome, like that of maize streak virus (MSV), consists of a single DNA circle. The complete nucleotide sequence of cloned WDV DNA (2749 nucleotides) has been determined. Comparison of the potential coding regions in WDV DNA with those in the DNA of two strains of MSV suggests that these viruses encode at least two functional proteins, the coat protein read in the virion (+) DNA sense and a composite protein, formed from two open reading regions, in the complementary (-) DNA sense. Although WDV and MSV are serologically unrelated their coat proteins showed 35% direct amino acid sequence and their DNAs showed 46% nucleotide sequence homology. There was too little homology between the DNAs of WDV and those of two geminiviruses with bipartite genomes, cassava latent virus (CLV) and tomato golden mosaic virus (TGMV), to align the sequences. However comparison of the amino acid sequences of predicted proteins of WDV, MSV, TGMV and CLV revealed clear relationships between these viruses and suggested that the monopartite and the bipartite geminiviruses have a common ancestral origin. Four inverted repeat sequences which have the potential to form hairpin structures of deltaG >/= -14 kcal/mol were detected in WDV DNA. The sequence TAATATTAC present in the loop of one of these hairpins is conserved in similar putative structures in MSV DNA and in both DNA components of CLV and TGMV and may function as a recognition sequence for a protein involved in virus DNA replication.     

Synthesis of viral DNA forms in Nicotiana plumbaginifolia protoplasts inoculated with cassava latent virus (CLV); evidence for the independent replication of one component of the CLV genome.

Totipotent leaf mesophyll protoplasts of Nicotiana plumbaginifolia, Viviani were inoculated with cassava latent virus (CLV) or with full length copies of CLV genomic DNAs 1 and 2 excised from replicative forms of M13 clones. Virus specific DNAs began to appear 48-72h after inoculation with virus or cloned DNAs, coincident with the onset of host cell division. Infected cells accumulated supercoiled forms of DNAs 1 and 2 as well as progeny single-stranded (ss) virion (+) sense DNAs representing each component of the genome. Both supercoiled and ss molecules were synthesised by cells inoculated with cloned DNA 1 alone but DNA 2 failed to replicate independently.     

Characterisation of DNA forms associated with cassava latent virus infection.

In addition to the major encapsidated DNA species found in preparations of cassava latent virus (genomic DNAs 1 and 2) there are minor DNA populations of twice (dimeric) and approximately half genome length. Both minor species resemble the genomic DNAs in that they are composed of predominantly circular single-stranded DNA. All of these size groups have a corresponding covalently-closed circular double-stranded DNA form in infected tissue. Infectivity studies using cloned DNAs 1 and 2 show that dimeric DNA routinely appears, suggesting it to be an intermediate in the DNA replicative cycle that can be encapsidated at low efficiency. In contrast, half unit length DNA has not yet been detected after multiple passaging of virus derived from the cloned DNA inoculum. Half unit length DNAs appear to be derived exclusively from DNA 2 and consist of a population of molecules exhibiting a relatively specific deletion. As they have an inhibitory effect on virus multiplication, their encapsidated forms are analogous to defective interfering particles associated with other eukaryotic DNA containing viruses. Small primer molecules associated with the genomic single-stranded DNAs, as reported for another geminivirus, have not been detected in CLV.     

Expression of a bacterial gene in plants mediated by infectious geminivirus DNA

A viable coat protein deletion mutant of cassava latent virus (CLV) DNA 1 has been isolated, suggesting that this geminivirus might be exploited as a gene replacement vector. An extensive deletion of 727 nucleotides within the coat protein gene renders DNA 1 non-infectious. Chimeric clones have been constructed in which the deleted coat protein open reading frame has been replaced by the coding region of the bacterial chloramphenicol acetyl transferase (CAT) gene. Infectivity is restored to DNA 1 when the CAT gene is inserted in either orientation, producing symptoms typical of CLV infection. The results demonstrate that the coat protein plays no essential role in virus spread throughout the host. Levels of CAT expression of 80 U/mg soluble protein occur in systemically infected Nicotiana benthamiana leaves when the CAT gene is fused in-frame to the amino terminus of the coat protein, providing a sensitive assay for viral DNA replication.     

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

Summary The 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 M_r polypeptide of CLV-1 (3) and the 40 285 M_r polypeptide of TGMV-A (4) show extensive homologies with the 17 768 M_r and 31 388 M_r 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.     

Immunocytochemical investigation of the causal agent of cassava mosaic disease in southern Africa

Cassava mosaic disease (CMD) exists throughout Africa, and cassava latent virus (CLV) has been implicated as the etiological agent in Kenya and West Africa. However, in Southern Africa, the causal agent of CMD was not until recently associated with CLV, and the possibility of a second flexuous virus particle has not been ignored. Attempts to isolate and visualize CLV antigen have been successful with Nicotiana benthamiana, an indicator host plant of CLV, but all efforts to isolate and visualize particles in infected cassava plants have failed. Immunocytochemical studies were undertaken in an attempt to localize virus antigen in infected cassava tissue.Cytochemical staining (light microscope) of infected cassava leaf material revealed the presence of inclusion bodies in epidermal and palaside mesophyll cells, and in epidermal collenchyma and outer parenchyma cells from the petiole and stem. However, transmission electron-microscopical (TEM) investigations revealed electron dense bodies in the cytoplasm, and no characteristic CLV nuclear inclusion bodies were evident. Transmission experiments to N. benthamiana and N. tabacum were attempted and leaves, exhibiting symptoms, examined microscopically. The nuclei appeared swollen (in comparison to uninfected leaves), a characteristic of CLV- infected N. benthamiana. However at the TEM level, no characteristic fibrillar-ring inclusion bodies or particles, could be visualized.Further immunocytochemical investigations were initiated, employing antisera raised against CLV isolated from N. benthamiana, and antisera for cassava common mosaic virus (CCMV), cassava brown streak virus (CBSV) and cassava X virus (CsXV). Goat anti-rabbit IgG-gold was used as a direct stain. No labelling occurred with CCMV and CBSV antisera. Intense gold labelling was located in the cytoplasm of phloem, mesophyll and epidermal cells of infected cassava and to a lesser extent in N. tabacum and N. benthamiana using affinity chromatography purified CLV antiserum. Little labelling was observed in nuclei of infected cells. Inconclusive results were obtained with CsXV antiserum.Immunogold labelling located CLV viral antigens in infected cassava leaf tissue. This observation, together with positive ELISA, transmission and DNA hybridization experiments, proves conclusively that CLV viral antigen is present in infected cassava in Southern Africa. However, most viral antigen in infected cassava, unlike N. benthamiana (fibrillar and granular nuclear inclusions) appears to be in the cytoplasm. This may tentatively suggest that the CLV protein is synthesized in the cytoplasm of its natural host, cassava, even though the virus may assemble in the nucleus at the appropriate time. However, as yet no virus inclusions have been observed in nuclei of infected cassava. Due to previous isolation of a flexuous rod and ambiguous staining results, the possibility of two viruses in cassava cannot be ruled out.     

Characterization of avian myeloblastosis-associated virus DNA intermediates.

The major species of unintegrated linear viral DNA identified in chicken embryonic fibroblasts infected with either the avian myeloblastosis-associated viruses (MAV-1, MAV-2) or the standard avian myeloblastosis virus complex (AMV-S) has a mass of 5.3 X 10(6) daltons. An additional minor DNA component observed only in AMV-S-infected cells has a mass of 4.9 X 10(6) daltons. The unintegrated linear viral DNAs and integrated proviruses of MAV-1 and MAV-2 have been analyzed by digestion with the restriction endonucleases EcoRI and HindIII. MAV-2 lacks a HindIII site present in MAV-1. These fragments have been compared to those generated by EcoRI and HindIII digestion of linear viral DNAs of AMV-S. Restriction enzyme digestion of AMV-S viral DNA produced unique fragments not found with either MAV-1 or MAV-2 viral DNAs. The major viral component present in AMV-S stocks has the HindIII restriction pattern of MAV-1. Restriction enzyme analysis of the 5.3 X 10(6)-dalton unintegrated MAV viral DNAs and their integrated proviruses suggests that the DNAs have a direct terminal redundancy of approximately 0.3 megadaltons and integrate colinearly with respect to the unintegrated linear DNA.     

Cloning of human papilloma virus genomic DNAs and analysis of homologous polynucleotide sequences.

The complete DNA genomes of four distinct human papilloma viruses (human papilloma virus subtype 1a [HPV-1a], HPV-1b, HPV-2a, and HPV-4) were molecularly cloned in Escherichia coli, using the certified plasmid vector pBR322. The restriction endonuclease patterns of the cloned HPV-1a and HPV-1b DNAs were similar to those already published for uncloned DNAs. Physical maps were constructed for HPV-2a DNA and HPV-4 DNA, since these viral DNAs had not been previously mapped. By using the cloned DNAs, the genomes of HPV-1a, HPV-2a, and HPV-4 were analyzed for nucleotide sequence homology. Under standard hybridization conditions (Tm = --28 degrees C), no homology was detectable among the genomes of these papilloma viruses, in agreement with previous reports. However, under less stringent conditions (i.e., Tm = --50 degrees C), stable DNA hybrids could be detected between these viral DNAs, indicating homologous segments in the genomes with approximately 30% base mismatch. By using specific DNA fragments immobilized on nitrocellulose filters, these regions of homology were mapped. Hybridization experiments between radiolabeled bovine papilloma virus type 1 (BPV-1) DNA and the unlabeled HPV-1a, HPV-2a, or HPV-4 DNA restriction fragments under low-stringency conditions indicated that the regions of homology among the HPV DNAs are also conserved in the BPV-1 genome with approximately the same degree of base mismatch.     

Nucleic acid renaturation and restriction endonuclease cleavage analyses show that the DNAs of a transforming and a nontransforming strain of Epstein-Barr virus share approximately 90% of their nucleotide sequences.

Viral DNA molecules were purified from a nontransforming and a transforming strain of Epstein-Barr virus. Each viral DNA was labeled in vitro and renatured in the presence of an excess of either one or the other unlabeled viral DNA. Both viral DNAs were also digested with the Eco R1 restriction endonuclease and subsequently labeled by using avian myeloblastosis virus DNA polymerase to repair either the EcoR1 nuclease-generated single-stranded ends of the DNAs or their single-stranded ends produced by a second digestion with exonuclease III after the first EcoR1 nuclease digestion. The results of these experiments support three general conclusions: (i) the DNAs of these two strains of Epstein-Barr virus share approximately 90% of their nucleotide sequences; (ii) both viral DNA populations are reasonably homogenous; and (iii) both DNAs contain repetitions or inverted repetitions of some of their nucleotide sequences.     

The high affinity binding site on polyoma virus DNA for the viral large-T protein.

In order to map the high affinity binding site for the viral large-T protein on polyoma virus DNA, we have developed an assay which does not require purified protein. It is based on the specific elution of the large-T ATPase activity from calf thymus DNA cellulose by recombinant DNA molecules including known sequences of the viral DNA. Using this assay, a high affinity binding site has been mapped on the early region side of the ori region. Binding requires the integrity of a sequence /AGAGGC/TTCC/AGAGGC/ (nucleotides 49 to 64 in the DNA sequence of the A2 strain). Similar repeats of a PuGPuGGC sequence within less than 20 bases are not found within the viral coding regions, but are strikingly common in the control regions of papovaviruses and other eukaryotic DNAs.