The complete nucleotide sequence of Alternanthera mosaic virus infecting Portulaca grandiflora represents a new strain distinct from phlox isolates.

A southeastern European isolate of Alternanthera mosaic virus (AltMV-MU) of the genus Potexvirus (family Flexiviridae) was purified from the ornamental plant Portulaca grandiflora. The complete nucleotide sequence (6606 nucleotides) of AltMV-MU genomic RNA was defined. The AltMV-MU genome is different from those of all isolates described earlier and is most closely related to genomes of partly sequenced portulaca isolates AltMV-Po (America) and AltMV-It (Italy). Phylogenetic analysis supports the view that AltMV-MU belongs to a new "portulaca" genotype distinguishable from the "phlox" genotype.

Characterization of Alternanthera mosaic virus and its Coat Protein.

A new isolate of Alternantheramosaic virus (AltMV-MU) was purified from Portulaca grandiflora plants. It has been shown that the AltMV-MU coat protein (CP) can be efficiently reassembled in vitro under different conditions into helical RNA-free virus-like particles (VLPs) antigenically related to native virus. The AltMV-MU and VLPs were examined by atomic force and transmission electron microscopies. The encapsidated AltMV-MU RNA is nontranslatable in vitro. However, it can be translationally activated by CP phosphorylation or by binding to the TGB1protein from the virus-coded movement triple gene block.

N-Terminal segment of potato virus X coat protein subunits is glycosylated and mediates formation of a bound water shell on the virion surface.

The primary structures of N-terminal 19-mer peptides, released by limited trypsin treatment of coat protein (CP) subunits in intact virions of three potato virus X (PVX) isolates, were analyzed. Two wild-type PVX strains, Russian (Ru) and British (UK3), were used and also the ST mutant of UK3 in which all 12 serine and threonine residues in the CP N-terminal segment were replaced by glycine or alanine. With the help of direct carbohydrate analysis and MS, it was found that the acetylated N-terminal peptides of both wild-type strains are glycosylated by a single monosaccharide residue (galactose or fucose) at NAcSer in the first position of the CP sequence, whereas the acetylated N-terminal segment of the ST mutant CP is unglycosylated. Fourier transform infrared spectra in the 1000-4000 cm(-1) region were measured for films of the intact and in situ trypsin-degraded PVX preparations at low and high humidity. These spectra revealed the presence of a broad-band in the region of valent vibrations of OH bonds (3100-3700 cm(-1)), which can be represented by superposition of three bands corresponding to tightly bound, weakly bound, and free OH groups. On calculating difference ('wet' minus 'dry') spectra, it was found that the intact wild-type PVX virions are characterized by high water-absorbing capacity and the ability to order a large number of water molecules on the virus particle. This effect was much weaker for the ST mutant and completely absent in the trypsin-treated PVX. It is proposed that the surface-located and glycosylated N-terminal CP segments of intact PVX virions induce the formation of a columnar-type shell from bound water molecules around the virions, which probably play a major role in maintaining the virion surface structure.

Linear remodeling of helical virus by movement protein binding.

Previously we have shown that encapsidated potato virus X (PVX) RNA was nontranslatable in vitro, but could be converted into a translatable form by binding of the PVX-coded movement protein (termed TGBp1) to one end of a polar helical PVX virion. We reported that binding of TGBp1 to coat protein (CP) subunits located at one extremity of the helical particles induced a linear destabilization of the CP helix, which was transmitted along the whole particle. Two model structures were used: (i) native PVX and (ii) artificial polar helical PVX-like particles lacking intact RNA (PVX(RNA-DEG)). Binding of TGBp1 to the end of either of these particles led to their destabilization, but no disassembly of the CP helix occurred. Influence of additional factors was required to trigger rapid disassembly of TGBp1-PVX and TGBp1-PVX(RNA-DEG) complexes. Thus: (i) no disassembly was observed unless TGBp1-PVX complex was translated. A novel phenomenon of TGBp1-dependent, ribosome-triggered disassembly of PVX was described: initiation of translation and few translocation steps were needed to trigger rapid (and presumably cooperative) disassembly of TGBp1-PVX into protein subunits and RNA. Importantly, the whole of the RNA molecule (including its 3'-terminal region) was released. The TGBp1-induced linear destabilization of CP helix was reversible, suggesting that PVX in TGBp1-PVX complex was metastable; (ii) entire disassembly of the TGBp1-PVX(RNA-DEG) complex (but not of the TGBp1-free PVX(RNA-DEG) particles) into 2.8S subunits was triggered under influence of a centrifugal field. To our knowledge, transmission of the linear destabilization along the polar helical protein array induced by a foreign protein binding to the end of the helix represents a novel phenomenon. It is tempting to suggest that binding of TGBp1 to the end of the PVX CP helix induced conformational changes in terminal CP subunits that can be linearly transferred along the whole helical particle, i.e. that intersubunit conformational changes may be transferred along the CP helix.

AFM study of potato virus X disassembly induced by movement protein.

Recently we have reported that a selective binding of potato virus X (PVX)-coded movement protein (termed TGBp1 MP) to one end of a polar coat protein (CP) helix converted viral RNA into a translatable form and induced a linear destabilization of the whole helical particle. Here, the native PVX virions, RNase-treated (PVX(RNA-DEG)) helical particles lacking intact RNA and their complexes with TGBp1 (TGBp1-PVX and TGBp1-PVX(RNA-DEG)), were examined by atomic force microscopy (AFM). When complexes of the TGBp1 MP with PVX were examined by means of AFM in liquid, no structural reorganization of PVX particles was observed. By contrast, the products of TGBp1-dependent PVX degradation termed "beads-on-string" were formed under conditions of AFM in air. The AFM images of PVX(RNA-DEG) were indistinguishable from images of native PVX particles; however, the TGBp1-dependent disassembly of the CP-helix was triggered when the TGBp1-PVX(RNA-DEG) complexes were examined by AFM, regardless of the conditions used (in air or in liquid). Our data supported the idea that binding of TGBp1 to one end of the PVX CP-helix induced linear destabilization of the whole helical particle, which may lead to its disassembly under conditions of AFM.