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.

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 infection.

Identification of distinct steps during tubule formation by the movement 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.

Subcellular location of the helper component-proteinase of Cowpea aphid-borne mosaic virus.

The helper component-proteinase (HC-Pro) of Cowpea aphid-borne mosaic virus (CABMV) was expressed in Escherichia coli and used to obtain HC-Pro antiserum that was used as an analytical tool for HC-Pro studies. The antiserum was used in immunofluorescence assays to study the subcellular location of HC-Pro expressed with other viral proteins in cowpea protoplasts in a natural CABMV infection, or in protoplasts transfected with a transient expression construct expressing HC-Pro separately from other viral proteins under the control of the 35S promoter. In both cases the protein showed a diffuse cytoplasmic location. Similar localisation patterns were shown in live protoplasts when the transient expression system was used to express HC-Pro as a fusion with the green fluorescent protein as a reporter. In an alternative expression system, the HC-Pro coding region was subcloned in-frame between the movement protein and large coat protein genes of RNA2 of Cowpea mosaic virus (CPMV). Upon transfection of protoplasts with this construct, HC-Pro was expressed as part of the RNA2 encoded polyprotein from which it was fully processed. In this case, the protein localised in broad cytoplasmic patches reminiscent of the typical CPMV induced cytopathic structures in which CPMV replication occurs, suggesting an interaction of HC-Pro with CPMV proteins or host factors in these structures. Finally, recombinant CPMV expressing HC-Pro showed a strongly enhanced virulence on cowpea and Nicotiana benthamiana consistent with the role of HC-Pro as a pathogenicity determinant, a phenomenon now known to be linked to its role as a suppressor of host defense responses based on post-transcriptional gene silencing.

Transgenic plants expressing HC-Pro show enhanced virus sensitivity while silencing of the transgene results in resistance.

Nicotiana benthamiana plants were engineered to express sequences of the helper component-proteinase (HC-Pro) of Cowpea aphid-borne mosaic potyvirus (CABMV). The sensitivity of the transgenic plants to infection with parental and heterologous viruses was studied. The lines expressing HC-Pro showed enhanced symptoms after infection with the parental CABMV isolate and also after infection with a heterologous potyvirus, Potato virus Y (PVY) and a comovirus, Cowpea mosaic virus (CPMV). On the other hand, transgenic lines expressing nontranslatable HC-Pro or translatable HC-Pro with a deletion of the central domain showed wild type symptoms after infection with the parental CABMV isolate and heterologous viruses. These results showed that CABMV HC-Pro is a pathogenicity determinant that conditions enhanced sensitivity to virus infection in plants, and that the central domain of the protein is essential for this. The severe symptoms in CABMV-infected HC-Pro expressing lines were remarkably followed by brief recovery and subsequent re-establishment of infection, possibly indicating counteracting effects of HC-Pro expression and a host defense response. One of the HC-Pro expressing lines (h48) was found to contain low levels of transgenic HC-Pro RNA and to be resistant to CABMV and to recombinant CPMV expressing HC-Pro. This indicated that h48 was (partially) posttranscriptionally silenced for the HC-Pro transgene inspite of the established role of HC-Pro as a suppressor of posttranscriptional gene silencing. Line h48 was not resistant to PVY, but instead showed enhanced symptoms compared to nontransgenic plants. This may be due to relief of silencing of the HC-Pro transgene by HC-Pro expressed by PVY.

The cytoskeleton and the secretory pathway are not involved in targeting the cowpea mosaic virus movement protein to the cell periphery.

The movement protein (MP) of cowpea mosaic virus (CPMV) forms tubules on infected protoplasts and through plasmodesmata in infected plants. In protoplasts the MP fused to GFP (MP-GFP) was shown to localize in peripheral punctate structures and in long tubular structures extending from the protoplast surface. Using cytoskeletal assembly inhibitors (latrunculin B and oryzalin) and an inhibitor of the secretory pathway (brefeldin A), targeting of the MP to the peripheral punctate structures was demonstrated not to be dependent on an intact cytoskeleton or functional secretion pathway. Furthermore it was shown that a disrupted cytoskeleton had no effect on tubule formation but that the addition of brefeldin A severely inhibited tubule formation. The results presented in this paper suggest a role for a plasma membrane host factor in tubule formation of plant viral MPs.

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 process.

Cowpea mosaic virus 32- and 60-kilodalton replication proteins target and change the morphology of endoplasmic reticulum membranes.

Cowpea mosaic virus (CPMV) replicates in close association with small membranous vesicles that are formed by rearrangements of intracellular membranes. To determine which of the viral proteins are responsible for the rearrangements of membranes and the attachment of the replication complex, we have expressed individual CPMV proteins encoded by RNA1 in cowpea protoplasts by transient expression and in Nicotiana benthamiana plants by using the tobacco rattle virus (TRV) expression vector. The 32-kDa protein (32K) and 60K, when expressed individually, accumulate in only low amounts but are found associated with membranes mainly derived from the endoplasmic reticulum (ER). 24K and 110K are freely soluble and accumulate to high levels. With the TRV vector, expression of 32K and 60K results in rearrangement of ER membranes. Besides, expression of 32K and 60K results in necrosis of the inoculated N. benthamiana leaves, suggesting that 32K and 60K are cytotoxic proteins. On the other hand, during CPMV infection 32K and 60K accumulate to high levels without causing necrosis.

Characterization of plant proteins that interact with cowpea mosaic virus '60K' protein in the yeast two-hybrid system.

Cowpea mosaic virus (CPMV) replication occurs in close association with small membranous vesicles in the host cell. The CPMV RNA1-encoded 60 kDa nucleotide-binding protein ('60K') plays a role in the formation of these vesicles. In this study, five cellular proteins were identified that interacted with different domains of 60K using a yeast two-hybrid search of an Arabidopsis cDNA library. Two of these host proteins (termed VAP27-1 and VAP27-2), with high homology to the VAP33 family of SNARE-like proteins from animals, interacted specifically with the C-terminal domain of 60K and upon transient expression colocalized with 60K in CPMV-infected cowpea protoplasts. eEF1-beta, picked up using the central domain of 60K, was also found to colocalize with 60K. The possible role of these host proteins in the viral replicative cycle is discussed.

Cowpea mosaic virus: effects on host cell processes.

SUMMARY Taxonomy: Cowpea mosaic virus (CPMV) is the type member of the Comoviridae and bears a strong resemblance to animal picornaviruses, both in gene organization and in the amino acid sequence of replication proteins. Little systematic work has been done to compare isolates of the virus from different parts of the world. Physical properties: Purified preparations of virus contain three centrifugal components; empty protein shells without RNA (T) and two nucleoprotein components (M and B), containing 24% and 34% RNA, respectively. The icosahedral particles have with a diameter of 28 nm, consist of 60 copies of two coat proteins, and are heat stable. Hosts: CPMV causes one of the most commonly reported virus diseases of cowpea (Vigna unguiculata), in which it produces chlorotic spots with diffuse borders in inoculated primary leaves. Trifoliate leaves develop a bright yellow or light green mosaic of increasing severity in younger leaves. The host range is rather limited, and few hosts are known outside the Leguminosae. The virus is transmitted by various beetles with biting mouthparts. Reported in Africa, the Philippines and Iran. Is apparently absent from North and South America. Useful website: http://mmtsb.scripps.edu/viper/1cpmv.html (structure); http://image.fs.uidaho.edu/vide/descr254.htm (general information).