For most dsRNA viruses, the genome-enclosing capsid comprises 120 copies of a single capsid protein (CP) organized into 60 icosahedrally equivalent dimers, generally identified as 2 nonsymmetrically interacting CP molecules with extensive lateral contacts. The crystal structure of a partitivirus, Penicillium stoloniferum virus F (PsV-F), reveals a different organization, in which the CP dimer is related by almost-perfect local 2-fold symmetry, forms prominent surface arches, and includes extensive structure swapping between the 2 subunits. An electron cryomicroscopy map of PsV-F shows that the disordered N terminus of each CP molecule interacts with the dsRNA genome and probably participates in its packaging or transcription. Intact PsV-F particles mediate semiconservative transcription, and transcripts are likely to exit through negatively charged channels at the icosahedral 5-fold axes. Other findings suggest that the PsV-F capsid is assembled from dimers of CP dimers, with an arrangement similar to flavivirus E glycoproteins.capsid assembly ͉ mycovirus ͉ Partitiviridae ͉ partitivirus

Stearoyl-acyl carrier protein-desaturase (SACPD)-catalyzed synthesis of oleic acid (18:1) is an essential step in fatty acid biosynthesis. Arabidopsis mutants (ssi2) with reduced SACPD activity accumulate salicylic acid (SA) and exhibit enhanced resistance to multiple pathogens. We show that reduced levels of 18:1 induce similar defense-related phenotypes in soybean. A Bean pod mottle virus (BPMV)-based vector was employed to effectively silence soybean SACPDs. The silenced plants contained reduced 18:1 and increased stearic acid, developed spontaneous cell death lesions, increased SA accumulation, and constitutively expressed pathogenesis-related genes. These plants also expressed elevated levels of resistance-like genes and showed resistance to bacterial and oomycete pathogens. Exogenous application of glycerol induced similar phenotypes, mimicking the effect of silencing SACPDs in healthy soybean plants. Overexpression of a soybean SACPD increased 18:1 levels in ssi2 but not in wild-type Arabidopsis plants, suggesting that the soybean enzyme was under feedback regulation similar to that of the Arabidopsis isozymes. These results suggest that soybean and Arabidopsis respond similarly to 18:1-derived cues by inducing a novel broad-spectrum resistance-conferring pathway, even though they differ significantly in their lipid biosynthetic pathways. We also demonstrate the efficacy of BPMV-induced gene silencing as a tool for functional studies in soybean.

Flexible filamentous viruses make up a large fraction of the known plant viruses, but in comparison with those of other viruses, very little is known about their structures. We have used fiber diffraction, cryo-electron microscopy, and scanning transmission electron microscopy to determine the symmetry of a potyvirus, soybean mosaic virus; to confirm the symmetry of a potexvirus, potato virus X; and to determine the low-resolution structures of both viruses. We conclude that these viruses and, by implication, most or all flexible filamentous plant viruses share a common coat protein fold and helical symmetry, with slightly less than 9 subunits per helical turn.Flexible filamentous plant viruses include at least 19 recognized genera (22), almost all in three families of singlestranded, positive-sense RNA viruses, the Potyviridae, the Flexiviridae, and the Closteroviridae. Members of the family Potyviridae account for almost a third of the total known plant virus species (22) and are responsible for more than half the viral crop damage in the world (37), infecting most economically important crops (32). Members of the family Flexiviridae (2), and particularly of the large genus Potexvirus, are also of considerable significance to agriculture (42). Both families show great potential for biotechnological applications, including protein expression and vaccine production (12, 54). Despite their importance, however, little is known about the structures of any of the flexible filamentous plant viruses, in sharp contrast to the amount of data on the rigid tobamoviruses (48,63) or the icosahedral plant viruses (15); flexibility, instability, and in many cases low levels of expression have made these viruses particularly intractable to structural studies. Structural and evolutionary relationships among the flexible filamentous plant viruses have been suggested (18,47,56,60), but there is very little sequence homology between the coat proteins of viruses in the different families, and there has hitherto been no structural support for such relationships at the level of either viral symmetry or coat protein folding. Indeed, reports of viral symmetry until now appeared to contradict hypotheses of evolutionary relationships.Soybean mosaic virus (SMV) is a potyvirus, that is, a member of the genus Potyvirus, the largest genus in the family Potyviridae (3). SMV is a major pathogen of soybeans, transmitted efficiently through seed and by aphids in a nonpersistent manner; yield losses as high as 35% have been reported (30). Despite dramatic morphological differences, members of the family resemble the icosahedral plant comoviruses and animal picornaviruses in genomic organization and replication strategy (32). Early electron microscopic observations found the potyviruses to be about 7,500 Å long and 120 Å in diameter, with helical pitches of about 34 Å (44, 60). A fiber diffraction study (51) of the tritimovirus wheat streak mosaic virus (WSMV) suggested that WSMV has 6.9 subunits per turn of the viral helix, but there was consider...

The genome-length, dicistronic mRNA of the double-stranded RNA fungal virus Helminthosporium victoriae virus 190S (genus Victorivirus, family Totiviridae) contains two long open reading frames (ORFs) that overlap in the tetranucleotide AUGA. Translation of the downstream ORF, which encodes the RNA-dependent RNA polymerase (RdRp), has been proposed to depend on ribosomal reinitiation following termination of the upstream ORF, which encodes the capsid protein. In the current study, we examined the RNA sequence determinants for RdRp translation in this virus and demonstrated that a coupled termination-reinitiation (stop-restart) strategy is indeed used. Signals for termination-reinitiation are found within a 32-nucleotide stretch of RNA immediately upstream of the AUGA motif, including a predicted pseudoknot structure. The close proximity in which this predicted structure is followed by the upstream ORF's stop codon appears to be especially important for promoting translation of the downstream ORF. The normal strong preferences for an AUG start codon and the canonical sequence context to favor translation initiation appear somewhat relaxed for the downstream ORF. Similar sequence motifs and predicted RNA structures in other victoriviruses suggest that they all share a related stop-restart strategy for RdRp translation. Members of the genus Victorivirus thus provide new and unique opportunities for exploring the molecular mechanisms of translational coupling, which remain only partly understood in this and other systems.The vast majority of eukaryotic mRNAs are monocistronic and, hence, contain only one translated open reading frame (ORF). Some, however, contain two or more ORFs and have an associated mechanism, such as ribosomal frameshifting or coupled termination-reinitiation (stop-restart), to promote regulated translation of the downstream ORF(s). Such multicistronic mRNAs are especially common among eukaryotic RNA viruses.In coupled termination-reinitiation, ribosomes translate the upstream ORF of an mRNA, but upon termination, a proportion of the 40S subunits remain bound to the mRNA and go on to reinitiate at the start codon of the downstream ORF (see review in reference 39). This strategy has been shown to promote the translation of downstream ORFs in the mRNAs of several different plus-or minus-stranded RNA mammalian viruses, including those that encode protein VP2 or VP10 of certain caliciviruses (30,33,34), the BM2 protein of influenza B virus (21, 40), and the M2-2 proteins of respiratory syncytial virus (RSV) and other pneumoviruses (1,15,16). One example of a plus-stranded RNA fungal virus that employs such translational coupling has also been described (17). A stop-restart strategy has been previously proposed for downstream ORF translation from the dicistronic mRNA of the double-stranded RNA (dsRNA) fungal virus Helminthosporium victoriae virus 190S (HvV190S) (45), and identifying RNA sequence determinants for that strategy in HvV190S and related viruses is the focus of this report.HvV190S is the pr...

Two distinct partitiviruses, Penicillium stoloniferum viruses S and F, can be isolated from the fungus Penicillium stoloniferum. The bisegmented dsRNA genomes of these viruses are separately packaged in icosahedral capsids containing 120 coat-protein subunits. We used transmission electron cryomicroscopy and three-dimensional image reconstruction to determine the structure of Penicillium stoloniferum virus S at 7.3 A resolution. The capsid, approximately 350 A in outer diameter, contains 12 pentons, each of which is topped by five arched protrusions. Each of these protrusions is, in turn, formed by a quasisymmetric dimer of coat protein, for a total of 60 such dimers per particle. The density map shows numerous tubular features, characteristic of alpha helices and consistent with secondary structure predictions for the coat protein. This three-dimensional structure of a virus from the family Partitiviridae exhibits both similarities to and differences from the so-called "T = 2" capsids of other dsRNA viruses.