J.B. Bancroft

The Self-assembly Of Papaya Mosaic Virus

Abstract 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 initiation of papaya mosaic virus assembly

Abstract 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 m7GpppGp and were rich in adenosine. Initiation occurs within about the first 200 nucleotides from the 5′-terminus of the virus RNA.

Self-assembly of protein from a flexuous virus

Abstract Potato virus Y was dissociated in LiCl or CaCl 2 . Isolated coat protein assembled into long flexuous stacked-disc particles in 1–100 m M phosphate solutions at pH 6–9. The conditions required for assembly are discussed.

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.

The assembly of clover yellow mosaic virus and its protein

Clover yellow mosaic virus has been reconstituted near neutrality at low ionic strength. Reconstitution is stopped by low temperatures or by 0.1 M KCl or 10(-3) M MgCl2. Assembly is not confined to homologous RNA. Protein assembles into stacked-ring tubes near pH 8.0 or helical structures of two diameters at pH 5.0 to 5.5.

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 specificity of papaya mosaic virus assembly

Abstract 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

Abstract 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.

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 x 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 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.