Inhibition of host gene expression associated with plant virus replication.

Pea seed-borne mosaic virus (PSbMV) RNA replication in pea cotyledonary tissues was restricted largely to a zone of cells close to the infection front. In situ hybridization probes representing nine genes from two pathways of metabolism failed to detect RNA transcripts within this zone, although transcripts were found in similar amounts in tissues on either side of the zone. Thus, in common with some animal viruses, PSbMV transiently suppresses the expression of host genes. Host protein accumulation was also affected. These observations provide insights into virus-plant interactions and symptom expression.

A Model for Seed Transmission of a Plant Virus: Genetic and Structural Analyses of Pea Embryo Invasion by Pea Seed-Borne Mosaic Virus.

Pea seed-borne mosaic virus (PSbMV), a seed-transmitted virus in pea and other legumes, invades pea embryos early in development. This process is controlled by maternal genes and, in a cultivar that shows no seed transmission, is prevented through the action of multiple host genes segregating as quantitative trait loci. These genes control the ability of PSbMV to spread into and/or multiply in the nonvascular testa tissues, thereby preventing the virus from crossing the boundary between the maternal and progeny tissues. Immunocytochemical and in situ hybridization studies suggested that the virus uses the embryonic suspensor as the route for the direct invasion of the embryo. The programmed degeneration of the suspensor during embryo development may provide a transient window for embryo invasion by the virus and could explain the inverse relationship between the age of the mother plant for virus infection and the extent of virus seed transmission.

A systematic review of quantitative and qualitative research on the role and effectiveness of written information available to patients about individual medicines.

OBJECTIVES: To establish the role and value of written information available to patients about individual medicines from the perspective of patients, carers and professionals. To determine how effective this information is in improving patients' knowledge and understanding of treatment and health outcomes. DATA SOURCES: Electronic databases searched to late 2004, experts in information design, and stakeholder workshops (including patients and patient organisations). REVIEW METHODS: Data from selected studies were tabulated and the results were qualitatively synthesised along with findings from the information design and stakeholder workshop strands. RESULTS: Most people do not value the written information they receive. They had concerns about the use of complex language and poor visual presentation and in most cases the research showed that the information did not increase knowledge. The research showed that patients valued written information that was tailored to their individual circumstances and illness, and that contained a balance of harm and benefit information. Most patients wanted to know about any adverse effects that could arise. Patients require information to help decision-making about whether to take a medicine or not and (once taking a medicine) with ongoing decisions about the management of the medicine and interpreting symptoms. Patients did not want written information to be a substitute for spoken information from their prescriber. While not everyone wanted written information, those who did wanted sufficient detail to meet their need. Some health professionals thought that written information for patients should be brief and simple, with concerns about providing side-effect information. They saw increasing compliance as a prime function, in contrast to patients who saw an informed decision not to take a medicine as an acceptable outcome. CONCLUSIONS: The combination of a quantitative and qualitative review, an exploration of best practice in information design, plus the input of patients at stakeholder workshops, allowed this review to look at all perspectives. There is a gap between currently provided leaflets and information which patients would value and find more useful. The challenge is to develop methods of provision flexible enough to allow uptake of varying amounts and types of information, depending on needs at different times in an illness. This review has identified a number of areas where future research could be improved in terms of the robustness of its design and conduct, and the use of patient-focused outcomes. The scope for this research includes determining the content, delivery and layout of statutory leaflets that best meet patients' needs, and providing individualised information, which includes both benefit and harm information. In particular, studies of the effectiveness and role and value of Internet-based medicines information are needed.

Seed transmission of plant viruses: a lesson in biological complexity.

Seed transmission of plant viruses results from the three-way interplay of the genetic components of the virus, the maternal host and its progeny. The challenge is to use genetic and cell biological analyses, integrated with our knowledge of plant reproduction and embryo development, to dissect this complex and rather poorly understood process.

A Spatial Analysis of Physiological Changes Associated with Infection of Cotyledons of Marrow Plants with Cucumber Mosaic Virus.

Changes in host primary metabolism associated with the compatible interaction between cucumber mosaic virus and cotyledons of the marrow plant (Cucurbita pepo L.) have been localized, first by measuring activities of key enzymes in infected and uninfected regions of the cotyledon, and second by histochemical techniques applied to tissue prints of the infected region. A series of progressive metabolic changes occurs within the expanding infected lesion. Virus replication and the synthesis of viral protein at the periphery creates a strong sink demand associated with increased activities of anaplerotic enzymes, increased photosynthesis, and starch accumulation. Inside the lesion, when the synthesis of virus has declined, photosynthesis is reduced, starch is mobilized, and the emphasis of metabolism is shifted toward glycolysis and mitochondrial respiration. These changes are associated spatially with the onset of chlorosis. A decrease in total protein synthesis in this inner zone could be instrumental in some or all of these changes, leading to symptoms of viral infection.

Aiding Lay Decision Making Using a Cognitive Competencies Approach.

Two prescriptive approaches have evolved to aid human decision making: just in time interventions that provide support as a decision is being made; and just in case interventions that educate people about future events that they may encounter so that they are better prepared to make an informed decision when these events occur. We review research on these two approaches developed in the context of supporting everyday decisions such as choosing an apartment, a financial product or a medical procedure. We argue that the lack of an underlying prescriptive theory has limited the development and evaluation of these interventions. We draw on recent descriptive research on the cognitive competencies that underpin human decision making to suggest new ways of interpreting how and why existing decision aids may be effective and suggest a different way of evaluating their effectiveness. We also briefly outline how our approach has the potential to develop new interventions to support everyday decision making and highlight the benefits of drawing on descriptive research when developing and evaluating interventions.

Evidence for proteolytic processing of tobacco mosaic virus movement protein in Arabidopsis thaliana.

Two ecotypes of Arabidopsis thaliana were transformed with the gene encoding tobacco mosaic virus (TMV) movement protein (P30). P30 accumulated largely in a subcellular fraction containing cell wall components and as a soluble protein. The protein migrated in denaturing gels with an M(r) of 30K, significantly faster than P30 (M(r) approximately 34K) accumulating after expression in transgenic tobacco, Escherichia coli or Spodoptera frugiperda cells, or after virus multiplication in tobacco. The P30 from A. thaliana infected with TMV for 14 days comigrated with that from E. coli, but that from A. thaliana infected for 49 days was of the smaller size. The use of antisera specific for the N- or C-termini of P30 showed that in A. thaliana P30 was proteolytically processed at the N-terminus, a region essential for P30 function. The failure of these plants to complement a TMV P30 mutant indicated that processed P30 was nonfunctional, although the processing was not so rapid that it prevented the development of systemic infections with wild type TMV. The absence of detectable P30 phosphorylation in A. thaliana demonstrated that phosphorylation was not essential for movement protein function and suggested that this species may use proteolytic cleavage of the N-terminus as an alternative strategy to tobacco for deactivating P30.

The application of spot hybridization to the detection of DNA and RNA viruses in plant tissues.

A solid-phase nucleic acid hybridization technique for the detection of DNA and RNA viruses in plant tissues is described. The method involves spotting crude samples onto nitrocellulose and using 12P-labelled DNA hybridization probes. The limit of sensitivity is 5-20 pg virus/spot or approximately 5 micrograms/g leaf tissue. The method is quantitative for DNA viruses in crude homogenates, but not for RNA viruses. The amount of cauliflower mosaic virus in infected leaves and protoplasts was estimated. The amplitude of spot hybridization to screening plant material from glasshouses and field is discussed.

Procedures for the efficient purification of pea seed-borne mosaic virus and its genomic RNA.

An efficient protocol for the purification of pea seed-borne mosaic potyvirus (PSbMV) particles was developed. This led to the purification of 10 PSbMV isolates by a single procedure. Virus aggregation during purification did not occur and consequently, high virus yield was consistently obtained. The virus thus purified was suitable for preparing viral genomic RNA, although conventional methods for RNA extraction resulted in RNA degradation. An alternative method was adopted which yielded reproducibly full length and infectious RNA. This was applied to three isolates of PSbMV and the RNA used to direct complementary DNA synthesis which in turn yielded nearly full length cDNA products.

A componential investigation of the relation between structural modelling and cognitive accounts of human judgement.

Structural modelling and cognitive process approaches have developed rather different accounts of human judgement and decision making. Two hypotheses to explain these differences were evaluated in the context of a judgement task, and formulated in terms of predictions concerning measurement of attribute importance. First, following suggestions made by Billings and Marcus (1983), it was argued that measures of judgement behaviour based on structural modelling reflect cognitive activity late in the judgement process, whereas measures derived from cognitive process approaches reflect cognitive activity early in the process. A new componential judgement task was developed which not only provided estimates of attribute importance based on structural modelling, but also two sets of cognitive process measures based on cognitive components assumed to occur early and late in the judgement process. A greater degree of convergence between approaches was predicted when the cognitive approach was based on activity in the component occurring later in the judgement process. Second, it was argued that in previous research subjects have had unlimited time to make their judgements, reducing the need for attribute importance to provide the dominant basis for determining processing strategy. The present experiment introduced a time pressure condition and, on the basis of previous research, predicted that this would increase the amount of information processing based on attribute importance, thereby increasing the convergence between estimates of attribute importance derived from the two approaches. The first, but not the second hypothesis was supported and the results were discussed in terms of their implications for understanding previous differences between the two approaches to human judgement.

Effects of time-pressure on decision-making under uncertainty: changes in affective state and information processing strategy.

An experiment is reported that investigated the extent to which affective state, information processing strategy and task structure determine the effects of time-pressure on decision-making. Research participants were presented with risk scenarios involving a choice between safe and risky actions. The scenarios were systematically varied in terms of outcome valence (positive or negative) and effort associated with taking the safe action (high or low). Half the participants were given unlimited time to make their decision, the other half were required to choose within a deadline. The findings showed that time-pressured participants were more anxious and energetic and used a number of different strategies to cope with the deadline. These effects, as well as changes in risk-taking, were shown to vary systematically with task structure, particularly the effort manipulation. The findings are discussed in terms of how they contribute to theories of time-pressure and the methodological implications they have for future research in this area.

Partial characterisation of different classes of viral DNA, and kinetics of DNA synthesis in turnip protoplasts infected with cauliflower mosaic virus.

Turnip protoplasts infected with cauliflower mosaic virus (CaMV) have been used to examine the kinetics of CaMV DNA synthesis, and the different classes of CaMV DNA found in vivo partially characterised. Differential extraction techniques for DNA from infected protoplasts has identified several distinct classes of viral DNA. The same approach applied to virus preparations revealed that while the majority of virion DNA was stably encapsidated, some small DNAs and a heterogeneous population 3.8-ca. 5.0 Kb were not. The structural relationship of sa-DNA (3) with the particle is such that only its 5' RNA moeity is susceptible to nuclease attack. Two-dimensional gel electrophoresis of total CaMV DNA from infected protoplasts revealed all the DNA species found in virion DNA, those species representing the 'free' DNA class and a further class of molecules, rich in DNA of (-) polarity (24), to which the role of reverse transcription intermediates has been ascribed. 'Free' DNA contains 8 Kb supercoiled DNA (Form I DNA), an 8 Kb open circle (Form II), an 8 Kb linear (Form III) and a truncated molecule with an extension of the (-) strand previously observed from infected plants (10). Kinetic experiments show that the accumulation of total CaMV-DNA parallels the accumulation of progeny virions to reach a maximum around 72 h post-inoculation and that there is not a separation of CaMV-DNA synthesis into clearly defined early and late stages.

Limitations on the use of fused green fluorescent protein to investigate structure-function relationships for the cauliflower mosaic virus movement protein.

To investigate the process of tubule formation for the cauliflower mosaic virus movement protein (CaMV MP), the green fluorescent protein (GFP) was fused to the MP to provide a vital marker for MP location after expression in insect cells. In contrast to the long tubular structures seen previously following baculovirus-based expression of the wild-type MP, the fusion protein produced only aggregates of fluorescing material in the cytoplasm. However, by co-expressing wild-type MP and GFP-MP, or by engineering their co-accumulation by introducing a foot-and-mouth disease virus 2A cleavage sequence between GFP and MP, long GFP-fluorescing tubules were formed. The experiments suggest that the presence of GFP at the N or C terminus of the tubule-forming domain of the CaMV MP places steric constraints upon the aggregation of the MP into a tubule but that this can be overcome by providing wild-type protein for inclusion in the aggregate.

Identification of inhibitory mutants of Cauliflower mosaic virus movement protein function after expression in insect cells.

Cauliflower mosaic virus (CaMV) encodes a movement protein (MP) which forms tubules in vivo and mediates the translocation of virus particles through plasmodesmata. The relationship between CaMV MP structure and function, in isolation from the complete virus infection, was studied by using MP expression in insect cells. The study allowed the MP domains necessary for tubule formation to be identified and potential MP-MP interactions to be investigated by using double infections with recombinant baculoviruses. Two MP domains which interfered with the ability of the wild-type MP to form tubules were identified. These mutant domains appeared to act as competitive, rather than dominant negative, inhibitors.

Identification of structural domains within the cauliflower mosaic virus movement protein by scanning deletion mutagenesis and epitope tagging.

Plant viruses encode proteins that mediate their movement from cell to cell through plasmodesmata. Currently, the mechanisms of action of these movement proteins (MPs) can be divided broadly into two types, requiring or not requiring the presence of viral capsid protein. Cauliflower mosaic virus encodes a multifunctional MP (P1) that modifies plasmodesmata through the formation of tubules that contain virus particles. To investigate the structure of P1, 26 small deletions (scanning deletions) were used to characterize regions of P1 essential for full biological activity. These deletions identified an N-terminal region and a region close to but not at the C terminus as domains that could tolerate manipulation, although gross deletions of either domain abolished infection. In sequence comparisons with other caulimovirus MPs, these regions coincided with the areas of least amino acid homology. Epitope tags inserted into either of these regions were stably maintained in systemic infections, and in extracts from infected plants, tagged P1 was detected on immunoblots. We predicted that, from the hypervariability of these regions, they would be located on the surface of the native P1 structure. Immunofluorescence of P1-specific tubules formed on the surface of infected protoplasts confirmed that the N-terminal and C terminus-proximal regions were exposed on the surface of the P1 tubule subunit. These findings establish a structure for P1 that is likely to be applicable to other tubule-forming MPs.

Identification of the cauliflower mosaic virus movement protein RNA-binding domain.

The in vitro RNA-binding activity of the movement protein (P1) of cauliflower mosaic virus was studied after its expression in Escherichia coli, purification, and uv-crosslinking to a radioactive probe. It was found that insoluble P1 aggregates were involved in RNA-binding activity. A series of deletion mutants were used to identify a domain within P1 required for binding activity. The RNA-binding domain is located between amino acids 120 and 197 and includes the region of homology between P1 and the movement protein (P30) of tobacco mosaic virus (TMV). The homologous region corresponds to part of RNA-binding domain "A" in TMV P30, but unlike domain A, the P1 domain is able to bind RNA out of the context of the complete protein. The P1 RNA-binding domain shows some structural similarity with RNA-binding domains of other proteins. The conservation of this domain in the caulimo- and badnaviruses provides support for the view that this activity has biological relevance.

Complex spatial responses to cucumber mosaic virus infection in susceptible Cucurbita pepo cotyledons.

Cucumber mosaic virus infection of its susceptible host Cucurbita pepo results in a program of biochemical changes after virus infection. Applying a spatial analysis to expanding infected lesions, we investigated the relationship between the changes in enzyme activity and gene expression. Patterns of altered expression were seen that could not be detected by RNA gel blot analysis. For all the host genes studied, there was a downregulation (shutoff) of expression within the lesion. In addition, two distinct types of upregulation were observed. The expression of heat shock protein 70 (HSP70) and NADP(+)-dependent malic enzyme (NADP-ME) showed induction in apparently uninfected cells ahead of the infection. This response was more localized than the upregulation exhibited by catalase expression, which occurred throughout the uninfected regions of the tissue. The experiments showed that virus infection induced immediate and subsequent changes in gene expression by the host and that the infection has the potential to give advance signaling of the imminent infection.

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.