David S. Turner

Molecular Properties of Bari 1, a Mild Strain of Cauliflower Mosaic Virus

Summary We studied aspects of the structure and expression of the genome of Bari 1, a mild strain of cauliflower mosaic virus. Differences were observed between gene products of Bari 1 detected in inclusion body preparations and those of the more typically severe strain, Cabb B-JI. The most striking difference was the gel mobility of the Bari 1 gene VI polypeptide (apparent M r 70K) which contrasted with that of Cabb B-JI (M r 62K). This difference was also observed between products of in vitro translation of viral mRNA suggesting that it was not due to post-translational modification. The open reading frame in the nucleotide sequence of the Bari 1 gene VI region was very similar in size to that of other CaMV strains but corresponded to an amino acid sequence with a much lower overall homology and diverged greatly in a 40 base pair sequence in the 3′ region compared to gene VI sequences of other strains. The level of the Bari 1 aphid transmission polypeptide P18, the product of gene II, was much lower than that of Cabb B-JI. Some of the possible subcellular consequences resulting from the molecular properties of Bari 1 were examined by electron microscopy. Differences were observed in the composition and intactness of Bari 1 cytoplasmic inclusion bodies compared with those of a severe strain, and the presence of nuclear inclusions.

Hairpin DNAs of cauliflower mosaic virus generated by reverse transcription in vivo.

Cauliflower mosaic virus (CaMV) is a DNA plant virus which replicates by reverse transcription. During our examination of CaMV replication intermediates by 2‐D gel electrophoresis, we have discovered a population of bizarre linear double‐stranded hairpin DNAs. The largest hairpin is the size of the CaMV genome; hairpin loop ends of smaller molecules map to several sites around the genome but the open ends are all located close to the origin of reverse transcription at the primer binding site. We believe that the hairpin DNAs are generated in vivo by reverse transcription of CaMV RNA followed by self‐primed second strand synthesis. The accumulation of hairpin DNAs in vivo might represent a side reaction of the CaMV reverse transcriptase although an essential role for them in the virus replication cycle cannot be discounted. The structure of the hairpin DNAs provides further evidence for the location of the start site and of the polarity of reverse transcription in CaMV.

Pararetrovirus–crucifer interactions: attack and defence or modus vivendi?

Abstract The compatible infection of plants by viruses usually leads to the development of systemic symptoms. Symptom expression of this kind is generally understood to be a host response that indicates an inability of the host to defend itself from attack. We have been studying compatible interactions between the plant pararetrovirus cauliflower mosaic virus (CaMV) and its crucifer hosts in order to understand the relationship between viral activity, symptom expression and plant defence. A CaMV protein (P6) appears to play a major role in eliciting symptom expression. This host response leads to a regulation of the viral multiplication cycle that is associated with leaf mosaics. The host regulation of CaMV appears to operate at the transcriptional level through an effect on the 35S promoter, or at the post-transcriptional level by a process that is akin to gene silencing, and can lead to host recovery depending upon the genetic background of the host. The plant apex is a focus for antiviral defence mechanisms, presumably because viral infection of the apical meristem would rapidly compromise the ability of the plant to generate new leaves and flowers for reproduction. The balance of interactions between CaMV and crucifers can provide a sustainable source of host plants to ensure viral propagation and viral exposure allows the host to adapt and develop its repertoire of defence mechanisms.

Changes in populations of cauliflower mosaic virus DNA and RNA forms during turnip callus proliferation.

Cauliflower mosaic virus (CaMV) nucleic acids accumulate in the cell in different structural conformations related to their roles in gene expression, replication and virion assembly. We have characterized changes in the population CaMV DNA and RNA replication products which occur following culture of infected turnip leaves under conditions where callus proliferates. After only 5 days in culture, a significant increase in the level of genome-length and subgenomic supercoiled (SC) DNA forms was observed by two-dimensional (2D) gel electrophoresis. Open circular (OC) molecules, corresponding to these SC DNAs, with mobilities consistent with the presence of a single break in each strand, were also detected after 5 days culture. By 10 days culture, the proportion of OC molecules with only one break per double-stranded molecule had increased. After 34 days culture, SC DNA with a range of sizes predominated in the unencapsidated DNA fraction. The change in pattern of OC and SC DNA forms during callus proliferation suggests a possible precursor/product relationship involving generation of deleted molecules from gap-containing virion DNA-like molecules followed by sequential repair of the gaps to produce SC DNA. Moreover, heterogeneity in the mobility of OC DNAs in the neutral dimension of 2D electrophoresis, a feature exhibited by twisted CaMV virion DNA, changed during the time-course suggesting that untwisting occurs during gap repair. Although the relative abundance of SC DNA increased during callus proliferation, CaMV polyadenylated 35S and 19S transcripts declined together with immediate reverse transcription products. We suggest that cellular changes during callus growth lead to a decline in authentic CaMV transcripts in the cytoplasm resulting in cessation of synthesis of viral products and progeny DNA genomes. In consequence, pre-existing virion DNAs return to the nucleus, possibly as a result of a relaxation in a cytoplasmic control mechanism, where they are assembled into various forms of SC DNA. The presence of CaMV SC DNAs in replicating cells might also enhance illegitimate integration into host chromosomes, as hybridization of CaMV DNA to high M(r) DNA was observed.

Comparison of viral nucleic acid intermediates at early and late stages of cauliflower mosaic virus infection suggests a feedback regulatory mechanism

An important phase of the multiplication cycle of the pararetrovirus cauliflower mosaic virus (CaMV) is transcription of the viral minichromosome in the nucleus. Leaves of infected turnip plants at the vein clearing stage were found to contain a relatively low level of minichromosome DNA, and abundant viral transcripts and characteristic reverse transcription products. In contrast, at the much later stage of severe leaf chlorosis, an elevated level of minichromosome DNA but less RNA, especially the 35S RNA reverse transcription template, was observed. Changes in the composition of virus nucleic acid intermediates were also seen in roots and stems early, compared with late, in infection. A possible feedback mechanism controlling the level of viral minichromosome DNA and its importance in regulation of the CaMV multiplication cycle are discussed in the light of these observations.

Transient expression of a GUS reporter gene from cauliflower mosaic virus replacement vectors in the presence and absence of helper virus

Vectors based upon the genome of cauliflower mosaic virus (CaMV) have only a limited capacity for replicating foreign DNA in plants. A helper virus system has been developed to complement CaMV constructs capable of carrying a large foreign gene (glucuronidase; GUS). GUS replaced part or all of the non-essential CaMV gene II and the essential genes III, IV and V. This construct was co-inoculated mechanically with wild-type CaMV helper virus onto Brassica rapa leaves to promote GUS vector complementation. After 1 week, blue foci of GUS activity were observed in the centres of the local lesions. Leaves inoculated with the GUS construct in the absence of helper virus showed randomly distributed foci of GUS activity that were generally smaller than the lesion-associated GUS foci. Inoculation with a simple non-replicating CaMV 35S promoter-GUS construct also produced small GUS foci. Co-inoculation of helper virus with CaMV gene replacement vectors in which replication was prevented by moving the primer-binding site or by deletion of an essential splice acceptor produced only small, randomly distributed GUS activity foci, demonstrating that the lesion-associated foci were produced by gene expression from replicating constructs. These experiments show that CaMV genes III-V can be complemented by wild-type virus and replacement gene vectors can be used for transient gene expression studies with CaMV constructs that distinguish gene expression associated with a replicating vector from that associated with a non-replicating vector.

Caulimovirus Isolation and DNA Extraction

Members of the cauhmovn-us group (1) each have a circular double-stranded DNA genome of approx 8 kbp that is encapsidated m a spherical, naked nucleocapsid of approx 50 nm diameter (Fig. 1). Caulimovnuses charactertsttcally produce subcellular mclusron bodies m infected tissues that contam most of the virions found in cells, embedded m an apparently random manner. The host ranges of mdtvldual caultmovnuses tend to be restricted to one or a few plant families, and group members are transmitted between plants by aphrd vectors Based on possession of all, or most, of these charactertstrcs, 12 defimte, and 3 possible, members of the group have been identified (2) The best-characterized and type member of the cauhmovu-uses IS cauliflower mosaic virus (CaMV), from which the group name derives. The complete nucleotrde sequence of at least eight different CaMV isolates (3-ZO), and that of four other caultmovn-uses (21-14) has been determmed. The orgamzatton of vu-al genes (Fig. 1) IS mostly conserved m sequenced caulimovn-uses, but one member, cassava vein mosaic virus (CVMV), IS somewhat different from the others (14). Repltcatton of cauhmovnuses mvolves alternation of genomes as DNA and RNA forms, progeny vlrron DNA being generated by reverse transcrtptton of a terminally redundant, genome-length RNA uttltzmg a vn-usencoded polymerase This feature IS shared by another group of plant DNA vtruses, the badnavnuses, and by animal hepatms B vn-uses. Such vu-uses have been termed pararetrovnuses to dtstmgutsh them from animal retrovuuses, which package an RNA form of the genome derived by transcrtptton of an integrated provn-us. Sequence homologtes m putative coding regions of dtfferent cauhmovu-uses are relatively low One short sequence 1s conserved among

Reverse transcription products generated by defective plus-strand synthesis during cauliflower mosaic virus replication

Abstract Cauliflower mosaic virus (CaMV) replicates by reverse transcription of an RNA template producing DNA (−)-strands on which DNA (+)-strands are initiated at two specific priming sites. We have analysed unencapsidated DNA forms of the CaMV genome isolated from infected leaf tissue to understand detailed aspects of the replicative process. This complex population of molecules contains linear single-stranded (ss), double-stranded (ds) and hairpin (hp) DNAs, and open-circular (oc) forms generated by reverse transcription. Purification by hydroxylapatite chromatography of ss-DNA (−)-strand molecules, revealed heterogeneous DNAs of up to genome length on which priming at both of the recognised (+)-strand priming sites had apparently failed to occur. Analysis by 2-dimensional gel electrophoresis suggested that the ss-DNA molecules had also lost the tRNA primer of reverse transcription necessary for the second strand-switch involving the (+)-strand. Loss of this bridging sequence might be responsible for many of the putative defective linear DNA forms observed in the unencapsidated fraction. Amongst these were DNA molecules in which (+)-strand priming had not occurred at either one or other of the recognised sites and were resolved as linear hairpin DNAs. Defective (+)-strand priming could also produced potentially infectious oc- forms with only one (+)-strand discontinuity. These latter DNAs were similar in structure to virion DNA of a CaMV isolate (CM4-184) with only one authentic (+)-strand priming site and were found preferentially in the unencapsidated dated DNA fraction. The relationship between the oc-forms and CaMV suppercoiled DNA suggest the possibility that defective (+)-strand synthesis might play a regulatory role in the CaMV multiplication cycle.

Contribution of Plant and Virus Genes to Cauliflower Mosaic Virus Pathogenicity

Viruses are useful entities for the molecular biologist since they can be exploited as simple genetic systems to study molecular processes of plant cells, and because they are pathogens and can be used to understand and potentially control plant virus diseases. The development of a plant virus disease typically involves the interaction of virus and host gene products such that the virus exploits host processes to ensure its multiplication and dissemination within the plant. Progeny virus must also become available for transmission to other plants. Methods of studying these processes have centred on the analysis of viral gene products, on changes in host gene expression, and more recently by genetic manipulation techniques. Genetic variability in both virus and host can be used to pinpoint possible sites of interaction and potential targets for ameliorating the outcome of the disease.

Multiple pathogenic factors influence aphid transmission of cauliflower mosaic virus from infected plants

Aphid transmission of cauliflower mosaic virus (CaMV) is mediated by a polypeptide (P18) encoded by the viral gene II. We have investigated the factors which influence acquisition by aphids of CaMV variants from infected plants. Aphid non-transmissible (AT-) CaMV isolates with a full-length gene II sequence share two amino acid changes, gly to arg at position 94 and ile to val at 105, relative to wild type transmissible (AT+) isolates. We have mutated the gly to arg at position 94 in the AT+ isolate Cabb B-JI which then exhibited the AT- phenotype as predicted. However, replacement of a DNA fragment in Cabb B-JI with one containing the gly to arg change from the AT- isolate Campbell to produce hybrid pBJIC1 resulted in a change in symptom phenotype as well as in aphid transmissibility. pBJIC1 also showed characteristics of partial transmissibility related to the stage of infection when it was tested. The level of P18 was measured in plants and showed that recombinants based upon the Campbell (AT-) genome accumulated P18 later than those based upon the Cabb B-JI genome (AT+). However, the Campbell P18 or recombinant proteins like it, were still not able to mediate transmission even when the P18 level in plants was relatively high and by employing large numbers of aphids. We conclude that aphid transmissibility of CaMV is influenced by multiple factors including P18 levels, inherent functionality of the protein, pathogenic characters of the infecting strain, and the number of aphids used to test transmissibility.