No abstract

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.Many positive-stranded RNA viruses modify intracellular membranes of their host cells to create a membrane compartment in which RNA replication takes place. Modifications include proliferation and reorganization of different membranes, including the early and late endomembrane system (26,36,44,54), the nuclear envelope (11), the peroxisomal membrane (7), the chloroplasts (49), and the mitochondrial membrane (7). Furthermore, the importance of membranes for viral replication is evident from the observation that the activity of most purified viral RNA-dependent RNA polymerases (RdRps) depends on the presence of membranes and/or phospholipids (27,32,59). It was proposed that the membranes play both a structural role and a functional role in the replication complex. Although the modification of intracellular membranes seems an essential part of the viral replicative cycle, little is known about the mechanisms by which the virus converts intracellular membranes for its own use.Cowpea mosaic virus (CPMV), a bipartite positive-stranded RNA virus, is the type member of the comoviruses and bears strong resemblance to animal picornaviruses both in gene organization and in amino acid sequence of replicative proteins (1, 15). Both RNA1 and RNA2 express large polyproteins, which are proteolytically cleaved into the different cleavage products by the 24-kDa (24K) protease (Fig. 1). The proteins encoded by RNA1 are necessary and sufficient for replication, whereas RNA2 codes for the capsid proteins and the movement protein.Upon infection of cowpea plants with CPMV, a typical cytopathological structure is formed adjacent to the nucleus, consisting of an amorphous matrix of electron-dense material which is traversed by rays of small membranous vesicles (10). The membranous vesicles are closely associated with CPMV RNA replication, as was revealed by autoradiography in conjunction with electron mic...

In cowpea plant cells infected with cowpea mosaic virus, tubular structures containing virus particles are formed in the plasmodesmata between adjacent cells; these structures are supposedly involved in cell-to-cell spread of the virus. Here we show that similar tubular structures are also formed in cowpea protoplasts, from which the cell wall and plasmodesmata are absent. Between 12 and 21 h post-inoculation, tubule formation starts in the periphery of the protoplast at the level of the plasma membrane. Upon assembly, the viruscontaining tubule is enveloped by the plasma membrane and extends into the culture medium. This suggests that the tubule has functional polarity and makes it likely that a tubule 'grows' into a neighbouring cell in vivo. On average, 75 % of infected protoplasts were shown to possess tubular structures extending from their surface. The tubule wall was 3 to 4 nm thick and they were up to 20 gm in length, as shown by fluorescent light microscopy and negative staining electron microscopy. By analogy to infected plant cells, both the viral 58K/48K movement and capsid proteins were located in these tubules, as determined by immunofluorescent staining and immunogold labelling using specific antisera against these proteins. These results demonstrate that the formation of tubules is not necessarily dependent on the presence of plasmodesmata or the cell wall, and that they are composed, at least in part, of virus-encoded components.

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.

Resistance to cowpea mosaic virus (CPMV) in transgenic Nicotiana benthamiana plants is RNA mediated. In resistant CPMV movement protein (MP) gene-transformed lines, transgene steady state mRNA levels were low, whereas nuclear transcription rates were high, implying that a post-transcriptional gene-silencing mechanism is at the base of the resistance. The silencing mechanism can also affect potato virus X (PVX) RNAs when they contain CPMV MP gene sequences.In particular, sequences situated in the 3 ' part of the transcribed region of the MP transgene direct elimination of recombinant PVX genomes. Remarkabty, successive portions of this 3' part, which can be as small as 60 nucleotides; all tag PVX genomes for degradation. These observations suggest that the entire 3' part of the MP transgene mRNA is the initial target of the silencing mechanism. The arrangement of transgenes in the plant genome plays an important role in establishing resistance because the frequency of resistant lines increased from 20 to 60% when transformed with a transgene containing a direct repeat of MP sequences rather than a single MP transgene. Interestingly, we detected strong methylation in all of the plants containing directly repeated MP sequences. In sensitive lines, only the promoter region was found to be heavily methylated, whereas in resistant lines, only the transcribed region was strongly methylated.