Replication of cauliflower mosaic virus DNA in leaves and suspension culture protoplasts of cotton

Cauliflower mosaic virus (CaMV) replicated in protoplasts and in inoculated leaves of the non-host, cotton (Gossypium hirsutum, L.). Protoplasts prepared from suspension-cultured cotton cells were infected by incubation with liposome-encapsulated CaMV virions. During a 1-week culture period the amount of CaMV nucleic acid as detected by nucleic acid hybridization in the protoplasts increased significantly regardless of whether or not the protoplasts contained vacuoles. In leaves inoculated with CaMV virions or CaMV DNA, viral DNA sequences were found by leaf skeleton hybridization to be located in small circular areas. DNA extracted from ultracentrifugal pellets of homogenates of inoculated leaves contained circular, gapped CaMV DNA only when inocula contained CaMV virions, CaMV DNA, or partial nested dimer CaMV plasmid DNA. When plants had been heavily watered, the CaMV DNA recovered contained degraded CaMV DNA. The results suggest that the host range limitation for CaMV is not due to an inability to replicate or spread locally in inoculated leaves.     

Cauliflower Mosaic Virus replication complexes: characterization of the associated enzymes and of the polarity of the DNA synthesized in vitro

The synthesis of both strands of CaMV-DNA has been studied in vitro using viral replication complexes obtained by hypotonic extraction of infected plant organelles. Hybridization of the DNA synthesized in vitro to single stranded CaMV DNA probes cloned in bacteriophage M 13 confirmed that the 35 S RNA served as a template for the synthesis of the (–) DNA strand. The response of CaMV DNA synthesis to various inhibitors suggests that a single enzyme directs both steps of the replication cycle. A comparative activity gel analysis of the DNA polymerases present in nuclear extracts from healthy and CaMV-infected turnips revealed an increase of a DNA polymerase species migrating in the 75 Kd range in infected tissue. When the enzyme activity associated with the isolated replicative complexes was similarly analyzed, the 75 Kd polymerase was markedly predominant, confirming that DNA polymerases of the -type (MW in the 110 Kd range) are not involved in the aphidicolin-insensitive CaMV DNA replication. It seems therefore increasingly probable that CaMV codes for its own polymerase.     

Nuclei purified from cauliflower mosaic virus-infected turnip leaves contain subgenomic, covalently closed circular cauliflower mosaic virus DNAs

Nuclei isolated from cauliflower mosaic virus (CaMV) infected turnip leaves contain subgenomic CaMV DNA species in addition to the genome length CaMV DNA. These subgenomic CaMV DNA species are present as covalently closed circles (form I), relaxed circles (form II) and linear (form III) molecules. The subgenomic form I DNA species range in size from about 10% of genome length to nearly genome length. These subgenomic DNA species appear in tissue infected with cloned CaMV DNA, indicating that they arise rapidly and have not accumulated in the virus population from serial propagation of CaMV. No specific region of the CaMV genome appears to be preferentially deleted to form the subgenomic CaMV DNA species. At least three distinct subgenomic species appear to accumulate preferentially in nuclei isolated from infected tissue. Two of these abundant subgenomic CaMV DNA species are form I and the other one is form III. Some of the subgenomic CaMV DNA species appear to be minichromosomes.     

Characterisation of cauliflower mosaic virus DNA forms isolated from infected turnip leaves.

Several different forms of cauliflower mosaic virus (CaMV) DNA were detected in nucleic acid preparations from CaMV-infected turnip leaves. As well as supercoiled and open-circular molecules, various linear DNA structures were identified. The relative amounts of these DNA forms varied in plants infected with different CaMV isolates. Restriction enzyme mapping and one- and two-dimensional gel electrophoresis revealed the presence of linear molecules apparently formed by breaks in the second strand at each of the three discontinuities. Two major linear DNA forms are double-stranded over part of their length and appear to have single-stranded extensions of the -strand of variable length. Since these DNA forms are not produced during extraction and probably exist as unencapsidated or partially encapsidated molecules, they may represent intermediates either in DNA replication or in virion assembly.     

Isolation of a fraction from cauliflower mosaic virus-infected protoplasts which is active in the synthesis of (+) and (-) strand viral DNA and reverse transcription of primed RNA templates

Sub-cellular fractions, isolated from cauliflower mosaic virus (CaMV)-infected turnip protoplasts, are capable of synthesising CaMV DNA in vitro on an endogenous template and of reverse transcribing oligo dT-primed cowpea mosaic virus RNA. The activity was not detected in mock-inoculated protoplasts. In vitro-labelled DNA hybridized to single-stranded M13 clones complementary to the putative origins of (-) and (+) strand CaMV DNA synthesis and to restriction endonuclease fragments encompassing more than 90% of the CaMV genome. The synthesis of (-) and (+) strand DNA appeared asymmetric. The template(s) for in vitro CaMV DNA synthesis are in a partially nuclease-resistant form. Fractions capable of in vitro CaMV DNA synthesis contained CaMV RNA both heterogeneous and as discrete species; they also contained a range of different sizes of CaMV DNA. Several lines of evidence indicate that this range of in vitro-labelled CaMV DNA, extending from 0.6kb to 8.0kb in length, represents elongating (-) strand DNA. These are discussed in relation to their role as possible replicative intermediates.     

Interaction of the DNA untwisting enzyme with the SV40 nucleoprotein complex.

The SV40 nucleoprotein complex which was isolated from infected CV-1 cells did not possess an active DNA untwisting enzyme. The superhelix density of the DNA in the chromatin complex was unchanged after treatment with purified rat liver DNA untwisting enzyme. However, in the presence of ethidium bromide (1 microgram/ml) the superhelix density was changed. Moreover, the nicked intermediate in the DNA untwisting reaction could be detected using the chromatin DNA as a substrate. These results show that the DNA in the SV40 chromatin which is accessible to the DNA untwisting enzyme is under no topological strain.     

A small DNA molecule containing covalently-linked ribonucleotides originates from the large intergenic region of the cauliflower mosaic virus genome.

We have detected a small DNA molecule (sa-DNA), 725 nucleotides long, in cauliflower mosaic virus (CaMV)-infected, but not non-infected, turnip leaves. Alkali and RNase A treatments shortened sa-DNA by 100 nucleotides and we conclude that it contains covalently-linked ribonucleotides. This DNA co-purified with cellular polyadenylated RNA. It is complementary to the beta-strand of CaMV DNA and of opposite polarity to RNAs transcribed from the alpha-strand. Hybridisation studies suggest that sa-DNA originates from the large intergenic region (IR1) of the CaMV genome. A small double-stranded DNA with three single-stranded components, which co-purifies with cellular DNA, appears to be related to sa-DNA but lacks detectable ribonucleotides.     

Evidence from cauliflower mosaic virus virion DNA for additional discontinuities in the plus strand.

Two-dimensional electrophoresis of cauliflower mosaic virus (CaMV) virion DNA and analysis of Southern blots using (+) strand-specific probes to the 5' termini of the beta (5.4 Kb) and alpha (2.6 Kb) strands, revealed the presence of molecules in addition to those predicted from the known structure of CaMV DNA. The presence of 8 Kb molecules of (+) sense after denaturation suggested that a small proportion of circular molecules have only a single discontinuity in the (+) strand. Other molecules, probably 5' coterminal with the beta strand but smaller than 5.4 Kb, indicated that a minority of the circular full length CaMV DNA contain additional gaps in the (+) strand. Consequently, molecules equivalent to the remainder of the beta strand could be identified using a single strand probe for a region towards the 3'-end of the beta strand. Computer analysis of the nucleotide sequence of CaMV DNA in the region of the proposed additional discontinuities revealed regions displaying some homology with the major (+) strand priming sites at the 5' ends of the beta and alpha strands. It is our contention that the additional (+) strand molecules of beta specificity are a consequence of minor (+) strand priming sites.     

The sequence of carnation etched ring virus DNA: comparison with cauliflower mosaic virus and retroviruses

Carnation etched ring virus (CERV) DNA comprises 7932 bp. CERV primer binding sites and overall genome organization are similar to those of the related cauliflower mosaic virus (CaMV). The six open reading frames of CERV showed amino acid homology (50-80%) with CaMV ORFs I-VI; no homologues of CaMV ORFs VII or VIII were found. CERV ORFs 1-5 interface each other with the sequence ATGA. The comparison of CERV ORF5 with CaMV ORFV highlighted regions which show homologies to retrovirus gag/pol protease, RNase H and DNA polymerase domains; the possibility that the DNA polymerase domain comprises two subdomains, operating off different templates, is discussed. Both CERV and CaMV ORFs I have sequence homology to tobacco mosaic virus P30 and plastocyanin.     

A fractal model of chromosomes and chromosomal DNA replication

With the aim of clarifying topological problems involved in the process of chromosomal DNA replication, a fractal model of chromosomes was built based on the assumption that a part of a chromosome, e.g. a radial loop, is similar in shape to a whole chromosome and each radial loop represents structures in the lower-order organization (an assumption of self-similarity). Several other assumptions used include (i) one continuous DNA fiber makes a whole chromosome (a unineme hypothesis), (ii) in situ DNA exists in the form of a double duplex or a tetraplex which is made of two duplex DNAs, although a duplex DNA may appear transiently in S-phase (multi-strandedness hypothesis) and (iii) torsional stress on a DNA fiber causes the fiber to supercoil and thus stabilizes chromosome structure (torque-based stabilization). This model allowed to calculate of a fractal dimension of a representative metaphase chromosome (e.g. d = 2.34), to predict the mode of replication of double duplex and to furnish a topological basis for the decondensation unit hypothesis. It must also be admitted that all the arguments in this report except for the possible existence of split telomeres hold true without assuming a tetraplex organization of chromosomes. Implications of this model was discussed and the importance of the fractal dimension as a measure of chromatin condensation stressed.     

Atomic force microscopic study on topological structures of pBR322 DNA

Plasmid pBR322 DNA (0.5mg/mL) isolated from Escherichia coli HB101 was suspended in Tris-HCl-EDTA (1 mol/L - 0.1 mol/L, pH8.5); then a drop of the above solution was deposited on freshly cleaved mica substrate. After adsorption for about 1 min, the sample was stained with phosphotungstic acid. The residua] solution was removed with a piece of filter paper. Afterwards the sample was imaged with a home-made atomic force microscope (AFM) in air. The AFM images of pBR322 DNA with a molecular resolution have been obtained. These images show that pBR322 DNA exists in several different topological structures: (i) relaxed circular DNA with a different diameter; (ii) supercondensed DNA with different particle sizes; (iii) dimeric catenane connected by one relaxed circular molecule and another dose-compacted molecule which might be either supercoiled or intramolecular knotted form; (iv) oligomeric catenane with multiple irregular molecules in which DNA is interlocked into a complex oligomer; (v) possibly-existing     

A restriction map of cauliflower mosaic virus DNA (strain PV 147). Mapping of the cleavage sites of HhaI, SacI, AvaI, PvuII, PstI, XbaI, EcoRI, Bg/II, HincII, HpaII and HindII + III.

The virion-extracted DNA (Mr5 x 10(6)) of cauliflower mosaic virus (CaMV) has three single-stranded interruptions. The mapping of this DNA using eleven restriction endonucleases (HhaI, SacI, AvaI, PvuII, PstI, XbaI, EcoRI, Bg/II, HincII, HpaII and HindII + III) is reported here. The existence of the three single-stranded breaks complicates the identification and the molecular weight determination of fragments produced by HpaII, HindIII and HindII + III. Indeed the electrophoretic mobility of some fragments in which a single-stranded discontinuity is located is modified, and the fluorescence of ethidium bromide complexed with these fragments is reduced as compared to that observed for the other fragments existing in a molar ratio. These drawbacks were overcome by performing experiments of nick-translation of CaMV DNA with Escherichia coli DNA polymerase I. FRom the data it follows that the CaMV DNA molecule bears bears 1 site for HhaI and SacI, 2 for AvaI and PvuII, 3 for PstI, 4 for XbaI, 5 for EcoRI, 6 for Bg/II and HincII, 11 for HpaII and 15 for HindII + III. The corresponding fragments have all been ordered and precisely located providing a suitable map for further investigations connected with the study of the fine structure and the function of the CaMV genome.     

Maize streak virus coat protein is karyophyllic and facilitates nuclear transport of viral DNA.

Transport of maize streak virus (MSV) DNA into the nucleus of host cells is essential for virus replication and the presence of virus particles in the nuclei of infected cells implies that coat protein (CP) must enter the nucleus. To see if CP is imported into the nucleus in the absence of other viral gene products, the MSV CP gene was expressed in insect cells with a baculovirus vector system, and also in tobacco protoplasts with a cauliflower mosaic virus (CaMV) 35S promoter-driven transient gene expression vector. Immunofluorescent staining showed that the CP accumulated in the nuclei of both insect and tobacco cells. Mutagenesis of a potential nuclear localization signal in the CP resulted in cytoplasmic accumulation of the mutant protein. We have shown previously that the CP binds to single-stranded (ss) and double-stranded (ds) viral DNA. To investigate if CP might also be involved in viral DNA nuclear transport, Escherichia coli-expressed CP, together with TOTO-1-labeled viral ss or ds DNA, was microinjected into maize and tobacco epidermal cells. Both ss and ds DNA moved into the nucleus when co-injected with the CP but not with E. coli proteins alone. These results suggest that, in addition to entering the nucleus where it is required for encapsidation of the viral ss DNA, the MSV CP facilitates the rapid transport of viral (ss or ds) DNA into the nucleus.