Characterization of a new carmovirus isolated from an Adonis plant.

An isometric virus was isolated from a cultivated Adonis plant (A. ramosa). The purified virus particle is 28 nm in diameter and is composed of a single coat protein and a single RNA genome of 3,991 nucleotides. Sequence analysis showed that the virus is closely related to carnation mottle virus. The virus was used to mechanically infect healthy A. ramosa plants, resulting in mosaic and leaf curl symptoms; however, attempts to inoculate carnation plants did not result in infection. We propose the virus as a new carmovirus and have named it adonis mosaic virus (AdMV).

Preliminary X-ray data analysis of crystalline hibiscus chlorotic ringspot virus.

Hibiscus chlorotic ringspot virus (HCRSV) is a positive-sense monopartite single-stranded RNA virus that belongs to the Carmovirus genus of the Tombusviridae family, which includes carnation mottle virus (CarMV). The HCRSV virion has a 30 nm diameter icosahedral capsid with T = 3 quasi-symmetry containing 180 copies of a 38 kDa coat protein (CP) and encapsidates a full-length 3.9 kb genomic RNA. Authentic virus was harvested from infected host kenaf leaves and was purified by saturated ammonium sulfate precipitation, sucrose density-gradient centrifugation and anion-exchange chromatography. Virus crystals were grown in multiple conditions; one of the crystals diffracted to 3.2 A resolution and allowed the collection of a partial data set. The crystal belonged to space group R32, with unit-cell parameters a = b = 336.4, c = 798.5 A. Packing considerations and rotation-function analysis determined that there were three particles per unit cell, all of which have the same orientation and fixed positions, and resulted in tenfold noncrystallography symmetry for real-space averaging. The crystals used for the structure determination of southern bean mosaic virus (SBMV) have nearly identical characteristics. Together, these findings will greatly aid the high-resolution structure determination of HCRSV.

The structure of melon necrotic spot virus determined at 2.8 A resolution.

The structure of melon necrotic spot virus (MNSV) was determined at 2.8 A resolution. Although MNSV is classified into the genus Carmovirus of the family Tombusviridae, the three-dimensional structure of MNSV showed a higher degree of similarity to tomato bushy stunt virus (TBSV), which belongs to the genus Tombusvirus, than to carnation mottle virus (CMtV), turnip crinkle virus (TCV) or cowpea mottle virus (CPMtV) from the genus Carmovirus. Thus, the classification of the family Tombusviridae at the genus level conflicts with the patterns of similarity among coat-protein structures. MNSV is one of the viruses belonging to the genera Tombusvirus or Carmovirus that are naturally transmitted in the soil by zoospores of fungal vectors. The X-ray structure of MNSV provides us with a representative structure of viruses transmitted by fungi.

A new strain of Melon necrotic spot virus that is unable to systemically infect Cucumis melo.

We report a new strain of Melon necrotic spot virus (MNSV) that is unable to systemically infect Cucumis melo. A spherical virus (W-isolate), about 30 nm in diameter like a carmovirus, was isolated from watermelons with necrotic symptoms. The W-isolate had little serological similarity to MNSV, and it did not cause any symptoms in six melon cultivars susceptible to MNSV; however, the host range of the W-isolate was limited exclusively to cucurbitaceous plants, and transmission by O. bornovanus was confirmed. Its genomic structure was identical to that of MNSV, and its p89 protein and coat protein (CP) showed 81.6 to 83.2% and 74.1 to 75.1% identity to those of MNSV, respectively. Analysis of protoplast showed that the W-isolate replicated in melons at the single-cell level. Furthermore, chimeric clones carrying the CP of MNSV induced necrotic spots in melons. These results suggested that the absence of symptoms in melons was due to a lack of ability of the W-isolate to move from cell to cell. In view of these findings, we propose that the new isolate should be classified as a novel MNSV watermelon strain.

Isolation of an asymmetric RNA uncoating intermediate for a single-stranded RNA plant virus.

We have determined the three-dimensional structures of both native and expanded forms of turnip crinkle virus (TCV), using cryo-electron microscopy, which allows direct visualization of the encapsidated single-stranded RNA and coat protein (CP) N-terminal regions not seen in the high-resolution X-ray structure of the virion. The expanded form, which is a putative disassembly intermediate during infection, arises from a separation of the capsid-forming domains of the CP subunits. Capsid expansion leads to the formation of pores that could allow exit of the viral RNA. A subset of the CP N-terminal regions becomes proteolytically accessible in the expanded form, although the RNA remains inaccessible to nuclease. Sedimentation velocity assays suggest that the expanded state is metastable and that expansion is not fully reversible. Proteolytically cleaved CP subunits dissociate from the capsid, presumably leading to increased electrostatic repulsion within the viral RNA. Consistent with this idea, electron microscopy images show that proteolysis introduces asymmetry into the TCV capsid and allows initial extrusion of the genome from a defined site. The apparent formation of polysomes in wheat germ extracts suggests that subsequent uncoating is linked to translation. The implication is that the viral RNA and its capsid play multiple roles during primary infections, consistent with ribosome-mediated genome uncoating to avoid host antiviral activity.