Papaya Mosaic Virus Research Articles

The primary structure of papaya mosaic virus coat protein

The amino acid sequence of the coat protein of the potexvirus papaya mosaic virus (PMV) has been determined, using a combination of the Edman degradation procedure (manual and automated) and mass spectrometry. The amino acid sequence is compared with that of the coat protein of potato virus X (PVX) as reported (S. Yu. Morozov, V. M. Zakhariev, B. K. Chernov, V. S. Prasolov, Yu. V. Kozlov, J. G. Atabekov, and K. G. Skyabin, Dokl. Acad. Nauk, SSSR 271, 211–215,1983). PMV has 73 of 211 amino acid residues in common with PVX and has 25 fewer residues in the polypeptide chain. Also 10 of a possible 16 proline residues are in similar positions in both proteins, including a sequence of 3 prolines at the carboxyl end. Furthermore, the 2 cysteine residues in PMV correspond with 2 of the 3 cysteines in PVX.

Synthesis in Vitro of the coat protein of papaya mosaic virus

Papaya mosaic virus (PMV) RNA directs the synthesis in vitro in either the wheat germ or reticulocyte lysate translation systems of numerous products. Three polypeptides are most prominent: pA (155,000 molecular weight), pB (73,000), and pC (22,000). The last, pC, displays the same molecular weight as coat protein and is precipitated by antibodies raised against PMV. The identities of pA and pB are less clear, although peptide mapping suggests that they and many of the minor products of translation are related to each other, but not to pC. The ability of partially encapsidated PMV ribonucleoprotein particles to direct protein synthesis has been assessed. As the extent of encapsidation increased, the relative synthesis of pA and pB decreased markedly. In contrast, the synthesis of pC persisted until a substantial fraction of the PMV RNA was completely encapsidated. In view of the polarity of assembly of PMV in vitro (M. Abouhaidar and J. B. Bancroft, (1978), Virology 90, 54–59) this finding indicates that the coding sequences for PMV coat protein are localized toward the 3′ end of PMV RNA. A kinetic analysis of coat protein synthesis, coupled with Northern blot analysis of PMV RNA during translation suggest that PMV RNA must be cleaved to activate it for in vitro coat protein synthesis.

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.

Viruses detected in Ullucus tuberosus (Basellaceae) from Peru and Bolivia

SUMMARY Ullucus tuberosus (Basellaceae) plants from 12 locations in the Andean highlands of Peru and Bolivia contained complexes of either three or four viruses. Specimens from six sites in Peru contained a potexvirus, a tobamovirus, a potyvirus and a comovirus, but those from another location lacked the potexvirus. All samples from five sites in Bolivia lacked the tobamovirus.The potexvirus (PMV/U) is a strain of papaya mosaic virus differing slightly from the type strain (PMV/T) in inducing milder symptoms in some common hosts and failing to infect a few other species. It symptomlessly infected U. tuberosus, and infected 15 of 29 species from seven of nine other families. PMV/U showed a close serological relationship to PMV/T and to boussingaultia mosaic virus and a distant relationship to commelina virus X, but it is apparently unrelated to any of ten other potexviruses.The tobamovirus (TMV/U) induced symptomless or inconspicuous infection in U. tuberosus, and infected 21 of 30 species from six of eight other families. It showed a very distant serological relationship to some strains of ribgrass mosaic, tobacco mosaic and tomato mosaic viruses, but failed to react with antisera to cucumber green mottle mosaic, frangipani mosaic, odontoglossum ringspot and sunn‐hemp mosaic viruses.The potyvirus, tentatively designated ullucus mosaic virus (UMV), alone in U. tuberosus induced leaf symptoms indistinguishable from the chlorotic mottling and distortion found in naturally infected plants. UMV infected 12 of 20 species from four other families, and was transmitted in the non‐persistent manner by Myzus persicae. It showed a distant serological relationship to only two (bidens mottle and alstroemeria mosaic) of 25 members or possible members of the potyvirus group tested.Some hosts and properties of the comovirus are described in an accompanying paper.None of the four viruses infected potato (Solanum tuberosum) and, with the possible exception of UMV, they differed from viruses reported previously to infect three other vegetatively propagated Andean crops (Oxalis tuberosa, Arracacia xanthorrhiza and Tropaeolum tuberosum).

The amino acid sequences of eleven tryptic peptides of papaya mosaic virus protein by electron ionization mass spectrometry.

Eleven of the fourteen tryptic peptides of papaya mosaic virus protein have been sequenced by electron ionization mass spectrometry using chemical and enzymic hydrolyses and mixture analysis as required. Mid-chain cleavages of N-C bonds produced secondary ion series which allowed up to 16 residues to be sequenced without further hydrolysis. Mixture analysis on hydrolysis products enabled a 24 residue tryptic peptide to be sequenced from the data recorded in a single mass spectrum.

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 polar alkaline disassembly of papaya mosaic virus

The disassembly of papaya mosaic virus (PMV) at alkaline pH (pH 10) was monitored by following the decrease of turbidity at 320 nm and by electron microscopy. Most of the virion particle population undergoes a sequential loss of protein subunits starting exclusively from the 3′OH end of the RNA, but a small fraction resists degradation. The progressive decrease in size distribution suggests a sequential process and the presence of “brush-like” structures at one end of the virus particle favors a polar model for disassembly. Furthermore, after treatment with nucleases to digest the exposed RNA tails, the 5′ cap structure (m 7GpppGp) was found to be conserved in the remaining nucleoprotein intermediates. This indicates that the disassembly of PMV occurs from the 3′ terminus to the 5′ end, a polarity opposite to that of viral assembly. These RNA fragments (containing the 5′ end) retain their ability to reassemble with the viral protein. This result is consistent with earlier evidence that the initiation site for virus assembly is located at the 5′ end of PMV-RNA. Electron microscope examination suggests the presence of meta-stable size classes during the disassembly process. This observation might indicate a variability of the RNA-protein interactions along the RNA molecules.

Circular dichroism studies of papaya mosaic virus coat protein and its polymers

The near-u.v. circular dichroism of papaya mosaic virus coat protein is dominated by intrasubunit interactions involving tryptophan and, to a much lesser extent, phenylalanine. The conformations of the 14 S protein discs at pH 4.0, pH 6.0 and pH 8.0 are judged to be similar by circular dichroism. At pH 8.0, however, the conformation of the protein is influenced by the extent of aggregation, which is reflected in the intensity, but not the shape, of the circular dichroism spectrum. The increase in intensity level that accompanies aggregation at pH 8.0 mainly appears to reflect modifications of tryptophanyl interactions. On the other hand, no spectral changes result when discs polymerize to form helical tubes at pH 4.0, indicating that a major conformational change is not requisite for helix formation. The above results are interpreted in view of the various protein and virus assembly reactions in which papaya mosaic virus coat protein participates. Denaturation of the coat protein results in quenching of optical activity in the near-u.v. region, and also in marked changes in the u.v. spectrum of the protein. These results indicate that the majority of the tryptophan and tyrosine residues are buried within a hydrophobic environment in the undenatured protein.

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.

The polarity of assembly of papaya mosaic virus and tobacco mosaic virus RNAs with PMV-protein under conditions of nonspecificity

The problem of the rapid multiinitiation of papaya mosaic virus or tobacco mosaic virus RNA by PMV-protein near pH 7.0 at low ionic strength has been overcome. If NaCl is added to 0.1 M, both RNAs are first encapsidated at their respective 5′ ends. This shows that the initial site of helix formation depends on the protein rather than the RNA.

Diffraction studies of papaya mosaic virus

X-ray and optical diffraction studies of the flexuous papaya mosaic virus are described. The virus is constructed so that there are 35 coat protein subunits in 4 turns of the helix. The virus contains about 1410 protein subunits and 6800 nucleotides and has a molecular weight of about 33 × 10 6. The structure of tubes assembled in vitro from coat protein both in the presence and absence of nucleic acid resembles that of the native virus.

The inhibition of papaya mosaic virus assembly related to the effect of cations on its RNA

Levels of MgCl 2 and spermidine which have no effect on the equilibrium of papaya mosaic virus (PMV) coat protein, as measured by analytical centrifugation, or on its configuration, as judged by circular dichroism, inhibit reconstitution. This inhibition can be correlated with enhanced secondary structure of PMV-RNA as indicated by melting profiles. It is inferred that the inhibition of PMV assembly by NaCl or KCl results from the same effect, which is not found with glycine.

Cation binding by papaya mosaic virus and its protein

Hydrogen-ion titration curves of papaya mosaic virus and its protein, with or without added CaCl 2 or MgCl 2 are presented and analysed. The virus particle contains three cation-binding sites per subunit, half protonated at pH values of 5.6, 7.0, and 8.6, respectively. Long protein rods, which are stable below pH 7, probably contain two cation-binding sites per subunit, while more dissociated protein probably contains only one. We calculate that the electric charge on the virus will be almost zero, and almost independent of pH, in biological fluids containing significant amounts of divalent cations.

Sequential encapsidation of heterologous RNAs with papaya mosaic virus protein

Papaya mosaic virus protein encapsidates heterologous RNAs under conditions normally restricted to the encapsidation of homologous RNA if the heterologous RNAs are first initiated with protein under conditions of nonspecificity. This means that initiation, but not maturation, controls the specificity of encapsidation.

The initiation of papaya mosaic virus assembly

Initiation complexes were obtained by reacting papaya mosaic virus protein with its RNA at 1 or 25°. The encapsidated fragments of RNA extracted from the initiation complexes contained m 7GpppGp and were rich in adenosine. Initiation occurs within about the first 200 nucleotides from the 5′-terminus of the virus RNA.

The specificity of papaya mosaic virus assembly

Papaya mosaic virus protein forms sinuous tubular particles with homologous RNA or that from a related virus at pH 8.0, whereas it makes thin extended particles with RNAs from unrelated viruses under the same conditions. The coat protein forms particles with discontinuities with both homologous and heterologous RNAs from pH 6.0 to 7.5. It will also encapsidate DNA under similar conditions. The protein encapsidates poly(A) and poly(C), but not poly(U) or poly(I) at pH 8.0, nor will it form tubular particles at pH 8.0 with homologous RNA in which cytosine has been transformed to uracil.

The kinetics of papaya mosaic virus assembly

Papaya mosaic virus is assembled in two energetically and kinetically distinct steps. There is a rapid initiation phase in which particles about 50 nm long are formed at 1 or 25°, followed by a slow elongation phase which does not proceed at 1° but does at 25°. The kinetics of the elongation phase have been formally described as a second order reaction in which the rate-limiting step is the productive collision of coat protein in some form with the growing end of the helix. The second order rate constant for the elongation reaction was estimated to be approximately 1.8 × 1061 mol −1 min −1. Reconstitution is completed by about 20 min in stoichiometric mixtures (protein to RNA = 20:1) and by about 5 min in protein excess (80:1). The rate of elongation may be influenced by localized regions on the RNA.

The self-assembly of papaya mosaic virus

The conditions for the in vitro reconstitution of papaya mosaic virus (PMV) from its isolated constituents are described and are related to the formation of coat protein subassembly products. PMV assembles best in 0.01 M, pH 8.0, Tris buffer at 25° at a protein to RNA ratio of 20:1 (w/w). At lower pH levels, faulty, segmented particles are formed which are sensitive to ribonuclease. The assembly process at pH 8.0 is composed of two energetically distinct phases corresponding to helix initiation and elongation. Both reactions may be stopped by low levels of NaCl, whereas only elongation requires elevated temperatures. If the growth of elongating particles is stopped by lowering the temperature, long and thin “extended particles” are detected; if NaCl is used, the arrested particles terminate with a “brush” at one end only. Under virus assembly conditions, the coat protein exists in an equilibrium among several polymeric species, most notably 14 S and 25 S polymers. The latter is not required for reconstitution. The equilibrium mixture is very sensitive to changes in pH, ionic strength, temperature, and protein concentration.

The Structure of the 5'-Terminus of Papaya Mosaic Virus RNA

SUMMARY The RNA of the sinuous papaya mosaic virus particles was labelled in vivo with 32P or in vitro after oxidation by periodate and reduction by KB3H4. After digestion with RNases T1, T2 and A, the oligonucleotides were fractionated by electrophoresis or by chromatography on a DEAE-cellulose column. After such fractionation, an oligonucleotide resistant to the RNases and phosphatase with a structure of m7G5′ppp5′Gp was obtained from the 5′-terminus of the RNA.

The assembly of papaya mosaic virus protein.

Abstract Protein isolated from papaya mosaic virus, which is a flexuous virus similar in morphology to potato virus X, will self-assemble into long helical particles in the absence of RNA. The significance of the conditions required for assembly are briefly considered.