Subcellular immunolocalization of the coat protein of two potexviruses in infected Chenopodium quinoa.

The production of coat protein is necessary for cell-to-cell transport of potexviruses in plants. To investigate the role of coat protein in the movement process, the intracellular distribution of coat protein in tissues infected with either of two potexviruses was studied using immunocytochemical procedures. We report that coat protein antigens are associated with the plasmodesmata of infected cells.

Purification, properties, and subcellular localization of foxtail mosaic potexvirus 26-kDa protein.

The open reading frame 2 (ORF2) of the potexviral genome encodes a 24- to 26-kDa protein which is part of the "triple gene block," a group of overlapping ORFs also present in the genomes of the carla-, hordei-, and furoviruses. The product of these ORFs is believed to play a role in the cell-to-cell movement of the viruses in host plants. The amino acid sequences of the homologous ORF2 products encoded by these related viruses suggest that they specify NTP binding and possibly helicase activities. We have used an Escherichia coli expression system to produce significant amounts of the 26-kDa protein (p26) encoded by foxtail mosaic potexvirus ORF2. p28 was purified to near homogeneity by conventional purification methods and some of its biochemical properties were determined. We present evidence that p26 is an ATP, CTP, and RNA binding protein with apparent ATPase activity. Western blot analysis of infected plant extracts using a polyclonal antiserum produced against p26 indicates that it is a relatively stable protein maintained at high levels for at least 6 days following its peak level of expression. Moreover, it is found predominantly in the soluble fraction of infected tissues. An immunocytochemical analysis of infected Chenopodium quinoa leaves reveals that p26 is exclusively associated with cytoplasmic inclusions in proximity to but distinct from aggregates of viral particles.

Evidence that fimbriae of the smut fungus Microbotryum violaceum contain RNA.

The cells of the fungus Microbotryum violaceum produce many long, fine surface hairs that are similar in size and morphology to bacterial pili or fimbriae. These fungal fimbriae are assembled from 74 kDa glycoprotein subunits. We now present evidence that these fimbriae also have a RNA component. Isopycnic centrifugation of fimbriae in caesium chloride produced one band at a density intermediate to that of protein and nucleic acid. The absorbance spectrum of the intact fimbriae was consistent with that of a nucleoprotein. After extraneous RNAs were enzymically removed from the purified fimbrial preparation, disruption of the fibrils resulted in the release of not only the 74 kDa glycoprotein subunits, but also a 30 base single-stranded RNA species. To our knowledge, this is the first example of extracellular RNA as a component of a surface appendage.

Partial purification and characterization of foxtail mosaic potexvirus RNA-dependent RNA polymerase.

The RNA-dependent RNA polymerase (RdRp) of foxtail mosaic virus (FMV) was partially purified from infected leaves of Chenopodium quinoa. The membrane fraction of crude plant extracts contained most of the FMV RdRp activity. Additional purification was obtained by solubilization of the RdRp using KCl and dodecyl-sucrose and by centrifugation through a glycerol gradient. The RNA template endogenous to RdRp preparations could be removed using micrococcal nuclease but the resulting fraction was unable to copy added template purified from FMV virions. However, supplementation of fractions containing RdRp activity with FMV RNA resulted in a significant decrease in the level of RNA synthesis. This effect was specific to potexviral RNAs since a similar interference was also observed with clover yellow mosaic virus RNA but not with brome mosaic virus RNA or yeast RNA. RNA transcripts corresponding to various regions of the FMV genome were tested for their ability to inhibit RNA synthesis on endogenous template. The simultaneous presence of both 5' and 3' terminal regions of the viral genome was necessary to interfere with RNA synthesis suggesting that this inhibition resulted from competition for the binding of component(s) of the RdRp complex.

Mutagenesis of a hexanucleotide sequence conserved in potexvirus RNAs.

Biologically active in vitro transcripts were synthesized from a cloned cDNA of a defective RNA (D RNA) of clover yellow mosaic virus (CYMV) and were used to determine if a hexanucleotide motif (5'-ACUUAA) conserved in the 3' noncoding region of potexvirus RNAs is essential for accumulation of progeny D RNA in planta. Deletion of rearrangement of the entire hexanucleotide sequence in the D RNA resulted in no detectable accumulation of progeny D RNA when coinoculated with helper CYMV RNA. Single-base substitutions of the four central nucleotides of the hexanucleotide sequence revealed that viable D RNAs can tolerate single residue changes at positions 3 and 5 only. These results suggest that the hexamer motif is involved in the process of D RNA propagation. The hexanucleotide sequence was also identified in the negative strand of potexvirus RNAs in the regions proposed to represent subgenomic RNA (sgRNA) promoters. In addition, the hexamer motif is present in similar regions in carlavirus RNAs. The conservation of this hexanucleotide (in orientation and position) in potexvirus and carlavirus RNAs, which serve as templates for full-length negative-strand synthesis and sgRNA production, strongly suggests that it plays a functional role in the synthesis of viral RNAs. Taken together, our data support our previous proposal (Bancroft et al., 1991. J. Gen. Virol. 72, 2173-2181) that the hexanucleotide sequence acts as a cis element involved in synthesis of full-length negative-sense viral RNA and further implicate the sequence in a similar role in production of positive-sense sgRNA.

Coding capacity determines in vivo accumulation of a defective RNA of clover yellow mosaic virus.

Naturally occurring defective RNAs (D RNAs) derived from the potexvirus clover yellow mosaic virus (CYMV) contain large internal deletions yet maintain a single open reading frame (ORF) representing the in-frame fusion of 5' and 3' terminal ORFs. Capped transcripts of the prototype 1.2-kb D RNA of CYMV were synthesized in vitro and used to inoculate broad bean plants. Progeny D RNA accumulated only if synthetic D RNA transcripts were coinoculated with CYMV RNA. Several experiments showed that helper-dependent accumulation of the D RNA in vivo depended on the maintenance of its encoded fusion ORF. (i) D RNAs with six-residue deletions introduced early in the fusion ORF accumulated, whereas those with four-residue out-of-frame deletions at the same sites were nonviable. (ii) Analysis of D RNAs containing termination codons at different locations showed that only the most 3' stop codon (maintaining over 93% of the fusion ORF) was permissive for D RNA accumulation. (iii) D RNAs with small in-frame deletions and insertions in their 3' coding regions were viable. (iv) Nonviable D RNAs containing disrupted fusion ORFs could not be complemented by the presence in the infection of a D RNA encoding a complete fusion ORF. Taken together, the results indicate that the process of translation, rather than the encoded product, modulates an event(s) which influences the propagation and/or accumulation of this RNA in vivo. This represents a unique requirement among plant virus D RNAs.

The entire nucleotide sequence of foxtail mosaic virus RNA.

The nucleotide sequence of the RNA genome of foxtail mosaic virus (FMV), a member of the potexvirus family, is 6151 nucleotides long, exclusive of a poly(A) tail. The RNA contains five principal open reading frames (ORFs), designated from the 5' terminus as encoding proteins with Mr values of 152.3K (ORF1), 26.4K (ORF2) which overlaps an 11.3K (ORF3) product, 5.8K (ORF4) which overlaps a 28.8K readthrough protein (ORF5A) which leads into the coat protein cistron of 23.7K (ORF5). The sizes and composition of the proteins encoded by the ORFs are generally similar to those found in other potexviruses; the least similar is the coat protein which nonetheless retains apparently critical consensus regions. The 5' terminus of the previously reported 0.9 kb subgenomic (sg) RNA was determined by S1 nuclease mapping and shown to begin with the sequence GAAGA, 43 nucleotides upstream from the first nucleotide of the coat protein initiation codon. The positions of the 5' end of this sgRNA and of that deduced from the nucleotide sequence for a 1.9 kb sgRNA are entirely consistent with the previously published sizes of these sgRNAs.

Defective RNAs of clover yellow mosaic virus encode nonstructural/coat protein fusion products.

A small group of 1.2-kb RNAs present on polyribosomes from clover yellow mosaic virus (CYMV)-infected tissue contains sequences from the genomic RNA (gRNA) of CYMV and is encapsidated by CYMV coat protein. Some features of these RNAs suggest that they are similar to defective interfering (DI) RNAs, and would be the first to be reported for the potexvirus group. The prototype 1.2-kb RNA is 1172 nucleotides in length excluding a probable poly(A) tail and is composed of two noncontiguous regions corresponding to 757 nucleotides of the 5' and 415 nucleotides of the 3' termini of CYMV's gRNA. The sequence of the prototype 1.2-kb RNA reveals that the two terminal gRNA regions present in this RNA encode a single open reading frame (ORF) joining the N-terminus of the 191-kDa nonstructural product and the C-terminus of the coat protein to form a 35-kDa 191-kDa/coat protein fusion product. The coding properties of this prototype RNA have been confirmed by translation in vitro of native and synthetic transcripts of the 1.2-kb RNAs, both of which direct the synthesis of the anticipated 35-kDa product which reacts with anti-CYMV antiserum. Three additional 1.2-kb RNA species, each of which contains a unique junction site, have been characterized. In all cases, a fusion ORF encoding a 191-kDa/coat protein fusion product is encoded on the RNA. The presence of a fusion ORF in all members of the 1.2-kb RNA species analyzed suggests that maintenance of this ORF may be important for the survival of this class of RNA within the plant. This coding strategy represents a novel property of plant virus defective RNAs.

The structure of tubes of bovine rotavirus nucleocapsid protein (VP6) assembled in vitro.

The structure of tubes of reassembled nucleocapsid protein (VP6) from bovine rotavirus (BRV) was determined using optical diffraction of electron micrographs. The tubes consist of a five-start helix of hexagons, with 38 hexagons per helix in a true repeat of three turns. The morphological subunits comprising the hexagons are probably elongated trimers. The structure of naturally occurring tubes (D. Chasey and J. Labram, 1983, J. Gen. Virol. 64, 863-872) was also examined and shown to be similar but not identical to that of tubes assembled in vitro. Considerations of the assembly process are discussed.

Single- and double-stranded viral RNAs in plants infected with the potexviruses papaya mosaic virus and foxtail mosaic virus.

Three classes of viral RNA were recovered from polyribosomes purified from papaya leaves infected with papaya mosaic virus (PapMV) and from barley leaves infected with foxtail mosaic virus (FoMV): full-length viral RNAs [6.8 and 6.2 kilobases (kb), respectively]; less abundant intermediate subgenomic RNAs (2.2 and 1.9 kb), and abundant, small subgenomic RNAs (1 and 0.9 kb). Small amounts of the PapMV-specified 1.0-kb subgenomic RNA were encapsidated, whereas no encapsidated subgenomic RNAs could be found in preparations of FoMV. Immunoprecipitation of the products of in vitro translation of the small subgenomic RNA of both viruses showed that it codes for the corresponding viral coat protein. FoMV genomic RNA isolated from polyribosomes also directed the efficient synthesis of a 37- to 38-kilodalton protein which was immunoprecipitated by an antiserum raised against the coat protein. We presume this product to be a readthrough protein initiated to the 5' side of and in the same reading frame as the coat protein-coding sequences in FoMV RNA. The predominant double-stranded viral-specified RNAs in tissues infected with PapMV, FoMV, and clover yellow mosaic virus were genome sized (6.8, 6.2, and 7.0 kb pairs, respectively). If double-stranded RNAs corresponding to coat protein subgenomic RNAs exist, they must be present in much lower relative abundances.

Molecular cloning of clover yellow mosaic virus RNA: identification of coat protein coding sequences in vivo and in vitro.

We have prepared double-stranded cDNA from clover yellow mosaic virus (CYMV) RNA and have created a library of cloned CYMV cDNAs in plasmid vectors. Cloned fragments ranging from 0.2 to 2.0 kbp have been mapped relative to the CYMV genome and to each other by digestion with restriction enzymes and by colony hybridization. Together, these cloned DNAs constitute almost 90% of the CYMV genome. Two overlapping plasmids whose inserts originate from the 3' portion of CYMV RNA arrest the synthesis of CYMV coat protein in vitro. A 0.44-kbp PstI fragment from one of these anneals to three CYMV-specific RNAs comprising the genomic RNA and two subgenomic RNAs of 2.1 and 1.0 kb isolated from polyribosomes from CYMV-infected broad bean leaves. RNA was enriched in the 1.0-kb subgenomic species by fractionating RNA extracted from purified preparations of CYMV or from polyribosomes isolated from infected leaves. Such RNA fractions directed the synthesis of CYMV coat protein in vitro indicating that the 1.0-kb subgenomic RNA is likely to be the coat protein messenger in vivo.

The longer RNA species in narcissus mosaic virus encodes all viral functions.

Narcissus mosaic virus (NMV) contains two nucleoprotein particles (M. N. Short and J. W. Davies, 1983, Biosci. Rep. 3, 837-846). We have fractionated the viral particles and have assayed the long (550 nm) and short (100 nm) rods for infectivity singly or in combination in several ways. The longer particle is biologically competent, as are its progeny. The RNA from the long particle encodes several products in vitro, including one which is electrophoretically and immunologically identical to coat protein. The RNA from the short particle contains about 1250 residues and, in agreement with the previous report, encodes coat protein. The sequence of this RNA is nested within that of the genomic RNA. Since the template properties of NMV can be reconciled readily with those of other potexviruses which we have examined, we believe that NMV differs from these in the encapsidation of a subgenomic mRNA for coat protein, rather than in its translational strategy.

The assembly of barrel cactus virus protein.

The coat protein of barrel cactus virus, a member of the potexvirus family, self-assembles into long tubular particles in the absence of the RNA. The particles have the same structure as the virus. The conditions under which they form suggest a new control mechanism not involving carboxyl-carboxylate interactions.

The semipermeability of simple spherical virus capsids.

Hydrogen-ion titration curves are reported for tomato bushy stunt virus, two strains of cowpea chlorotic mottle virus, and turnip crinkle virus, with particular attention to the hysteresis loops associated with the swelling and contraction of virions. There appears to be an archetypal shape of hysteresis loops, which is shared by viruses in several groups, suggestive of many intermediate states in the swelling of any one particle. In contrast, eggplant mosaic virus behaves as if its protein capsid is impermeable to small ions in mild conditions; its cation-binding sites were revealed by treatment with high concentrations of salt or urea, or at raised temperatures. Putting these observations together with the fact that a spherical virus capsid is a closed, holey, charged surface leads to a theory of titration hysteresis: its key feature is that the protein capsids of simple viruses are inherently semipermeable, with many of the ion-handling properties usually attributed only to complex lipid membranes.

Subassembly aggregates of papaya mosaic virus protein.

An examination of the number of subunits in small aggregates of papaya mosaic virus (PMV) coat protein is presented based on a model system which gives results consistent with the experimental observation that the 14 S subassembly species is a double disc, composed of two rows of nine subunits each. The estimated hydration of the disc, about 0.85 g 1H20/9 protein, is unusually large and indicates a cavitated structure for the disc. Comparison with other rod-shaped viruses suggests that the flexuous nature of PMV is a consequence of sparse axial inter-subunit contacts at high radius.

The structure of an unusual ordered aggregate of papaya mosaic virus protein.

The structure of unusual rod-shaped particles made from papaya mosaic virus protein in methylpentanediol is described. It is shown how the structural subunits group to form the morphological subunits observed on the particles.

The architecture of the potexviruses.

The gross structural parameters describing particles of viruses of the Potex group are presented with particular reference to five hitherto unanalyzed definitive or putative members of the group. The new evidence in conjunction with published results for other members of the group leads to the hypothesis that all Potexviruses have essentially the same architecture. In particular, each particle has almost the same number of subunits per turn and that number is probably 9. The different viruses mainly differ in the fractional departure from n = 9 and in the size of the true repeats, values of 8 3/4 , 8 4/5 , 8 5/6 , 8 5/7 , 8 7/8 , 8 9/10 , and 8 9/11 subunits per turn of the helices of the various viruses described here and/or previously being obtained.

The generality of cation-binding sites in rod-shaped viruses.

Hydrogen-ion titration curves have been measured for two filamentous plant viruses (clover yellow mosaic virus and potato virus X) and two filamentous bacterial viruses (fd and Pf1) with and without Ca2+ or Mg2+ ions present, and for the protein of the PM6 mutant of tobacco mosaic virus. The bacterial viruses do not possess the 'strong' cation-binding sites found in all plant viruses, but they have 'weak' sites that can be assigned to juxtaposed carboxylate groups on their external surfaces. The strong sites in plant viruses still cannot be assigned to any particular amino-acid side chains, but they must be located in the region of high electronegativity near the axis.

Melting of viral RNA by coat protein: assembly strategies for elongated plant viruses.

The coat proteins of two rod-shaped plant viruses, papaya mosaic virus (PMV) and tobacco mosaic virus (TMV), have been tested for RNA melting activity. Under reconstitution conditions, PMV protein melts RNA in a noncooperative fashion. This activity is aspecific and is inhibited by low concentrations of NaCl as is virus reconstitution. TMV protein does not melt RNA either in the absence of NaCl or under reconstitution conditions at moderate ionic strength levels. The results suggest that elongated plant viruses have evolved at least two different assembly strategies in order to satisfy the requirement that the RNA within these viruses be in a melted configuration.