Coalescence of the sites of cowpea mosaic virus RNA replication into a cytopathic structure

Cowpea mosaic virus (CPMV) replication induces an extensive proliferation of endoplasmic reticulum (ER) membranes, leading to the formation of small membranous vesicles where viral RNA replication takes place. Using fluorescent in situ hybridization, we found that early in the infection of cowpea protoplasts, CPMV plus-strand RNA accumulates at numerous distinct subcellular sites distributed randomly throughout the cytoplasm which rapidly coalesce into a large body located in the center of the cell, often near the nucleus. The combined use of immunostaining and a green fluorescent protein ER marker revealed that during the course of an infection, CPMV RNA colocalizes with the 110-kDa viral polymerase and other replication proteins and is always found in close association with proliferated ER membranes, indicating that these sites correspond to the membranous site of viral replication. Experiments with the cytoskeleton inhibitors oryzalin and latrunculin B point to a role of actin and not tubulin in establishing the large central structure. The induction of ER membrane proliferations in CPMV-infected protoplasts did not coincide with increased levels of BiP mRNA, indicating that the unfolded-protein response is not involved in this proces

Tomato spotted wilt virus glycoproteins induce the formation of endoplasmic reticulum- and Golgi-derived pleomorphic membrane structures in plant cells

Tomato spotted wilt virus (TSWV) particles are spherical and enveloped, an uncommon feature among plant infecting viruses. Previous studies have shown that virus particle formation involves the enwrapment of ribonucleoproteins with viral glycoprotein containing Golgi stacks. In this study, the localization and behaviour of the viral glycoproteins Gn and Gc were analysed, upon transient expression in plant protoplasts. When separately expressed, Gc was solely observed in the endoplasmic reticulum (ER), whereas Gn was found both within the ER and Golgi membranes. Upon co-expression, both glycoproteins were found at ER-export sites and ultimately at the Golgi complex, confirming the ability of Gn to rescue Gc from the ER, possibly due to heterodimerization. Interestingly, both Gc and Gn were shown to induce the deformation of ER and Golgi membranes, respectively, also observed upon co-expression of the two glycoproteins. The behaviour of both glycoproteins within the plant cell and the phenomenon of membrane deformation are discussed in light of the natural process of viral infectio

Hardware/Software Co-design Applied to Reed-Solomon Decoding for the DMB Standard

This paper addresses the implementation of Reed- Solomon decoding for battery-powered wireless devices. The scope of this paper is constrained by the Digital Media Broadcasting (DMB). The most critical element of the Reed-Solomon algorithm is implemented on two different reconfigurable hardware architectures: an FPGA and a coarse-grained architecture: the Montium, The remaining parts are executed on an ARM processor. The results of this research show that a co-design of the ARM together with an FPGA or a Montium leads to a substantial decrease in energy consumption. The energy consumption of syndrome calculation of the Reed- Solomon decoding algorithm is estimated for an FPGA and a Montium by means of simulations. The Montium proves to be more efficient

Secoviridae: a proposed family of plant viruses within the order Picornavirales that combines the families Sequiviridae and Comoviridae, the unassigned genera Cheravirus and Sadwavirus, and the proposed genus Torradovirus

The order Picornavirales includes several plant viruses that are currently classified into the families Comoviridae (genera Comovirus, Fabavirus and Nepovirus) and Sequiviridae (genera Sequivirus and Waikavirus) and into the unassigned genera Cheravirus and Sadwavirus. These viruses share properties in common with other picornavirales (particle structure, positive-strand RNA genome with a polyprotein expression strategy, a common replication block including type III helicase, a 3C-like cysteine proteinase and type I RNA-dependent RNA polymerase). However, they also share unique properties that distinguish them from other picornavirales. They infect plants and use specialized proteins or protein domains to move through their host. In phylogenetic analysis based on their replication proteins, these viruses form a separate distinct lineage within the picornavirales branch. To recognize these common properties at the taxonomic level, we propose to create a new family termed “Secoviridae” to include the genera Comovirus, Fabavirus, Nepovirus, Cheravirus, Sadwavirus, Sequivirus and Waikavirus. Two newly discovered plant viruses share common properties with members of the proposed family Secoviridae but have distinct specific genomic organizations. In phylogenetic reconstructions, they form a separate sub-branch within the Secoviridae lineage. We propose to create a new genus termed Torradovirus (type species, Tomato torrado virus) and to assign this genus to the proposed family Secoviridae

Polyprotein processing in the expression of the cowpea mosaic virus genome

In our study on the proteolytic processing of the 200K polyprotein encoded by CPMV B-RNA we first examined the types of cleavage sites present in this polyprotein. Previously Zabel etal . (1984) had shown that VPg is released from its 60K precursor by cleavage between a glutamine-serine dipeptide sequence, and the question was whether all cleavages in the 200K polyprotein occurred at glutamine-serine sites or not. The determination of partial amino acid sequences of isolated B-RNA- encoded proteins and alignment of these sequences with the open reading frame in B-RNA revealed that three types of cleavage sites are used to process the 200K polyprotein namely glutamine-serine (2x), glutamine- glycine and glutamine-methionine amino acid pairs (Chapter 3). A glutamine-methionine and glutamine-glycine site are also present in the M-RNA-encoded polyprotein, as revealed by partial amino acid sequencing of the capsid proteins (Franssen etal ., 1986). A common feature of the sequences surrounding the different cleavage sites is that they have alanine at position -4 and alanine or proline at position -2, but beyond that there is no obvious homology among the cleavage sites. Since there occur several glutamine-glycine, glutamine-serine and glutaminemethionine dipeptide sequences in the polyproteins which are not cleaved, probably the secondary and tertiary structure of the polypeptide chains are also important factors in determining the cleavage sites involved In the processing.The processing model of the 200K polyprotein, based on analysis of B-RNA-encoded proteins found invivo and invitro translation studies, postulated the formation of a 24K protein as a final cleavage product (Rezelman etal ., 1980; Franssen etal ., 1984a). To demonstrate the actual presence of such protein in infected cells we have used antibodies raised against a synthetic peptide with an amino acid sequence corresponding to part of this hypothetical protein. The antibodies indeed reacted with a 24K protein in CPMV infected protoplasts and also with the 84K, 110K and 170K precursors which contain the sequence of the 24K protein (Chapter 4). In view of the results of processing of the invitro translation products of B-RNA and its homology to the picornavirus encoded proteases it was previously suggested that the 24K protein possesses proteolytic activity. The protease activity of the 24K protein, was examined by expressing a hybrid cDNA construct, containing the 24K coding region linked to the coding region of both capsid proteins, in E . coli using the T7 promoter/polymerase system of Tabor and Richardson (1985) (Chapter 5). This resulted In the production of several virus-specific proteins which were characterized using specific antibodies. Pulse-chase experiments showed that two primary products were produced (the smaller one was probably the result of internal initiation of translation) which underwent faithful cleavage at two glutamine-glycine sites. One of the cleavage products represented the small capsid protein VP23. When a construct was used in which the 24K coding sequence contained a small deletion only two large proteins could be detected. These results unequivocally indicate that the 24K protein catalyzes the cleavages at the glutamine-glycine sites in the CPMV polyproteins (Chapter 5).Recent experiments described by Verver etal . (1987) have shown that the 24K protein (or proteins containing the 24K sequence) is also able to cleave one of the glutamine -serine sites in the 200K polyprotein. Furthermore, Franssen etal . (1984b) had obtained evidence that the B-RNA-encoded 32K protein was involved in the cleavage at the glutamine- methionine site in the M-RNA-encoded polyprotein. Therefore it was proposed that the 24K protein is probably catalyzing the cleavages at all glutamine-serine and glutamine-glycine sites, while on the other hand the 32K protein would be involved in the cleavage of both glutaminemethionine sites in the CPMV polyproteins. Indeed, serine and glycine are similar amino acids with respect to their side groups (small polar) whereas methionine is very different (large non-polar side group), further supporting the idea that two different proteases would be necessary to cleave all cleavage sites. However, recently Vos etal . (1987a) have definitely shown that the 24K protein is able to catalyze all cleavages in the CPMV polyproteins but that for the cleavage of the glutamine-methionine site in the M-polyprotein the 32K protein is essential as a cofactor (see Vos etal ., 1987a).Another question we have addressed in our study on the expression of the CPMV RNAs Is the expression of M-RNA invivo . Invitro , M-RNA is translated into two carboxy-terminal overlapping polyproteins (105K and 95K) the smaller one as a result of initiation at an internal AUG codon (Vos etal ., 1984). These proteins are cleaved by a B-RNA-encoded activity into 58K and 48K proteins and a 60K capsid protein precursor (Franssen etal ., 1982). At the other hand in CPMV-infected cells the two capsid proteins VP23 and VP37 are the only M-RNA-encoded proteins readily detectable. To elucidate the expression mechanism of M-RNA invivo we have searched for the 60K capsid precursor and for the 58K and 48K proteins in CPMV-infected cells. Using antibodies against the capsid proteins and ZnCl 2 the 60K capsid precursor protein was detected in CPMV-inoculated protoplasts incubated In the presence of ZnCl 2 (Chapter 6). Zn ++ions are known to inhibit the proteolytic processing of several viral polyproteins and probably this has caused the 60K protein to accumulate in these cells.Using antibodies against synthetic peptides, corresponding to the common carboxy-terminus of the 48K and 58K proteins, a 48K protein was detected in the membrane fractions of infected cells. A viral 58K protein could not be detected (Chapter 6). The presence of the 48K and 60K proteins in infected cells links the invitro translation results with the invivo situation and shows that also invivo CPMV M-RNA is expressed via proteolytic processing of a polyprotein. As sofar direct evidence for the occurrence of a M-RNA-encoded 58K polypeptide invivo is lacking it remains unknown whether invivo M-RNA, besides being translated into a 95K polypeptide starting at the initiation codon at position 524, is also expressed by translation, starting at the AUG codon at position 161, into a 105K polypeptide. Invitro the 105K protein is usually produced in considerable smaller amounts than the 95K protein and it seems plausible that this also occurs invivo and perhaps the amount of 58K protein is very low and remains below the present level of detection. Another possibility is that the 58K protein is unstable and rapidly degraded in infected cells. It is a striking fact that the M-RNAs of all comoviruses studied sofar produce two polyproteins upon invitro translation (Goldbach and Krijt, 1982), supporting the idea that the presence of two AUG initiation codons in the same reading frame has some functional significance. In our opinion it seems therefore likely that invivo some 58K protein will be produced.Previously it was suggested that the M-RNA-encoded 48K (and 58K) protein has a function in cell to cell transport of the virus (Rezelman etal ., 1982). We have now shown that the 48K protein is present in the membrane fraction of infected cells and is excreted in the incubation medium of CPMV infected protoplasts (Chapter 6). These findings are consistent with a possible function of the 48K protein in virus transport.Infection of cells with CPMV is accompanied with the production of membranous vesicles and electron-dense amorphous material. We have demonstrated that the electron-dense material contains (if not represents) non-structural B-RNA-encoded proteins. This was established by immunocytochemical labeling of sections of CPMV infected protoplasts with specific antibodies and protein Agold (Chapter 7). The data collected correlate well with the observation that during a CPMV infection a considerable amount of non-structural proteins is produced (Goldbach and Van Kammen, 1985). Apparently these proteins are located in the cytophatic structure as a part of the electron-dense material. The membranous vesicles have been implicated with viral RNA replication (De Zoeten etal ., 1974) but the question whether the electron-dense material also has a function In virus multiplication remains to be answered.With the detection of the 60K, 48K and 24K proteins in infected cells probably all major proteins expressed from the CPMV RNAs have now been identified. It is also clear now that all these proteins are produced by proteolytic processing of two polyproteins between three different pairs of amino acids and that the 24K protein As the protease catalyzing these cleavages. Future research will concentrate on the functions of these proteins. The synthesis of infectious transcripts from complete cDNA clones of both M- and B-RNA as recently described by Vos etal . (1987) opens the way to introduce specific mutations in the RNAs and this will, together with the techniques which have been described in this thesis, form the basis for further research on the elucidation of the replication mechanism of CPMV and its pathogenic action in the host plant

Non-specific interactions are sufficient to explain the position of heterochromatic chromocenters and nucleoli in interphase nuclei

The organization of the eukaryote nucleus into functional compartments arises by self-organization both through specific protein–protein and protein–DNA interactions and non-specific interactions that lead to entropic effects, such as e.g. depletion attraction. While many specific interactions have so far been demonstrated, the contributions of non-specific interactions are still unclear. We used coarse-grained molecular dynamics simulations of previously published models for Arabidopsis thaliana chromatin organization to show that non-specific interactions can explain the in vivo localization of nucleoli and chromocenters. Also, we quantitatively demonstrate that chromatin looping contributes to the formation of chromosome territories. Our results are consistent with the previously published Rosette model for Arabidopsis chromatin organization and suggest that chromocenter-associated loops play a role in suppressing chromocenter clustering

Redox-active ferrocene-modified Cowpea mosaic virus nanoparticles

A naturally occurring nanoparticle, the plant virus Cowpea mosaic virus, can be decorated with ferrocene derivatives, of various linker lengths with amine and carboxylategroups, on the external surface using a range of conjugation strategies. The multiple, organometallic, redox-active ferrocene moieties on the outer surface of the virus are electrochemically independent with reduction potentials that span a potential window of 0.16 V that are dependent on the site of modification and the nature of the ferrocene derivative. The number of ferrocenes coupled to each virus ranges from about 100 to 240 depending upon the conjugation site and the linker length and these redox active units can provide multielectron reservoirs

Cowpea mosaic virus infection induces a massive proliferation of endoplasmic reticulum but not Golgi membranes and is dependent on de novo membrane synthesis

Replication of cowpea mosaic virus (CPMV) is associated with small membranous vesicles that are induced upon infection. The effect of CPMV replication on the morphology and distribution of the endomembrane system in living plant cells was studied by expressing green fluorescent protein (GFP) targeted to the endoplasmic reticulum (ER) and the Golgi membranes. CPMV infection was found to induce an extensive proliferation of the ER, whereas the distribution and morphology of the Golgi stacks remained unaffected. Immunolocalization experiments using fluorescence confocal microscopy showed that the proliferated ER membranes were closely associated with the electron-dense structures that contain the replicative proteins encoded by RNA1. Replication of CPMV was strongly inhibited by cerulenin, an inhibitor of de novo lipid synthesis, at concentrations where the replication of the two unrelated viruses alfalfa mosaic virus and tobacco mosaic virus was largely unaffected. These results suggest that proliferating ER membranes produce the membranous vesicles formed during CPMV infection and that this process requires continuous lipid biosynthesis

Identification of distinct steps during tubule formation by the movement of protein of Cowpea mosaic virus

The movement protein (MP) of Cowpea mosaic virus (CPMV) forms tubules through plasmodesmata in infected plants thus enabling virus particles to move from cell to cell. Localization studies of mutant MPs fused to GFP in protoplasts and plants identified several functional domains within the MP that are involved in distinct steps during tubule formation. Coinoculation experiments and the observation that one of the C-terminal deletion mutants accumulated uniformly in the plasma membrane suggest that dimeric or multimeric MP is first targeted to the plasma membrane. At the plasma membrane the MP quickly accumulates in peripheral punctuate spots, from which tubule formation is initiated. One of the mutant MPs formed tubules containing virus particles on protoplasts, but could not support cell-to-cell movement in plants. The observations that this mutant MP accumulated to a higher level in the cell than wt MP and did not accumulate in the cell wall opposite infected cells suggest that breakdown or disassembly of tubules in neighbouring, uninfected cells is required for cell-to-cell movement

Combining transplant professional's psychosocial donor evaluation and donor self-report measures to optimise the prediction of HRQoL after kidney donation:an observational prospective multicentre study

OBJECTIVES: Living donor kidney transplantation is currently the preferred treatment for patients with end-stage renal disease. The psychosocial evaluation of kidney donor candidates relies mostly on the clinical viewpoint of transplant professionals because evidence-based guidelines for psychosocial donor eligibility are currently lacking. However, the accuracy of these clinical risk judgements and the potential added value of a systematic self-reported screening procedure are as yet unknown. The current study examined the effectiveness of the psychosocial evaluation by transplant professionals and the potential value of donor self-report measures in optimising the donor evaluation. Based on the stress-vulnerability model, the predictive value of predonation, intradonation and postdonation factors to impaired longer term health-related quality of life (HRQoL) of kidney donors was studied. DESIGN: An observational prospective multicentre study. SETTING: Seven Dutch transplantation centres. PARTICIPANTS: 588 potential donors participated, of whom 361 donated. Complete prospective data of 230 donors were available. Also, 1048 risk estimation questionnaires were completed by healthcare professionals. METHODS: Transplant professionals (nephrologists, coordinating nurses, social workers and psychologists) filled in risk estimation questionnaires on kidney donor candidates. Furthermore, 230 kidney donors completed questionnaires (eg, on HRQoL) before and 6 and 12 months after donation. PRIMARY AND SECONDARY OUTCOME MEASURES: HRQoL, demographic and preoperative, intraoperative and postoperative health characteristics, perceived support, donor cognitions, recipient functioning and professionals risk estimation questionnaires. RESULTS: On top of other predictors, such as the transplant professionals’ risk assessments, donor self-report measures significantly predicted impaired longer term HRQoL after donation, particularly by poorer predonation physical (17%–28% explained variance) and psychological functioning (23%). CONCLUSIONS: The current study endorses the effectiveness of the psychosocial donor evaluation by professionals and the additional value of donor self-report measures in optimising the psychosocial evaluation. Consequently, systematic screening of donors based on the most prominent risk factors provide ground for tailored interventions for donors at risk