Interaction of tobacco mosaic virus and tobacco mosaic virus protein with bovine serum albumin.

Bovine serum albumin (BSA) causes tobacco mosaic virus (TMV) to crystallize at pH values where both have negative charges. The amount of albumin required to precipitate the virus varies inversely with ionic strength of added electrolyte. At pH values above 5, the precipitating power is greatest when BSA has the maximum total, positive plus negative, charge. Unlike early stages of the crystallization of TMV in ammonium sulfate-phosphate solutions, which can be reversed by lowering the temperature, the precipitation of TMV by BSA is not readily reversed by changes in temperature. The logarithm of the apparent solubility of TMV in BSA solutions, at constant ionic strength of added electrolyte, decreases linearly with increasing BSA concentration. This result and the correlation of precipitating power with total BSA charge suggest that BSA acts in the manner of a salting-out agent. The effect of BSA on the reversible entropy-driven polymerization of TMV protein (TMVP) depends on BSA concentration, pH, and ionic strength. In general, BSA promotes TMVP polymerization, and this effect increases with increasing BSA concentrations. The effect is larger at pH 6.5 than at pH 6. Even though increasing ionic strength promotes polymerization of TMVP in absence of BSA, the effect of increasing ionic strength from 0.08 to 0.18 at pH 6.5 decreases the polymerization-promoting effect of BSA. Likewise, the presence of BSA decreases the polymerization-promoting effect of ionic strength. The polymerization-promoting effect of BSA can be interpreted in terms of a process akin to salting-out. The mutual suppression of the polymerization-promoting effects of BSA and of electrolytes by each other can be partially explained in terms of salting-in of BSA.     

Fluorescent study of tobacco mosaic virus protein

The synthesis of two new variants of gramicidin is described. It is shown that the changes in the aromatic side groups do not influence the single channel conductivity. Experiments in which solutions having different molarities on the two sides of the bilayer lipid membrane are described and their results compared with a rate theory analysis. It is concluded that the gramicidin pore contains approximately 10 equal potential maxima.     

Secondary structure of tobacco mosaic virus protein

A set of rules is proposed for the prediction of α-helices in proteins. These rules lead to the correct assignment to either helical or non-helical regions of over 80% of the amino acid residues in the proteins from which they are derived. Applied to tobacco mosaic virus protein these rules lead to the prediction of five α-helical regions which may be consistent with other data.     

Location of the cistron of the tobacco mosaic virus coat protein.

Treatment of tobacco mosaic virus (TMV) RNA with T1 RNase under mild conditions cuts the RNA molecule into a large number of fragments, only a few of which may be specifically recognized by disks of TMV protein. It has been shown elsewhere that these specifically recognized RNA fragments are a part of the coat protein cistron, the portion coding for amino acids 95 to 129 of the coat protein. It is reported that different size classes of partially uncoated virus particles were prepared by limited reconstitution between TMV RNA and protein or by partial stripping of intact virus with DMSO. Both procedures produce nucleoprotein rods in which the 5'-terminal portion of the RNA is encapsidated and the 3'-terminal region is free. The free and the encapsidated portions of the RNA were each tested for the ability to give rise to the aforesaid specifically recognized fragments of the coat protein cistron upon partial T1 RNase digestion. It was found that only the 3'-terminal third of the virus particle need to be uncoated in order to expose the portion of the RNA molecule from which these fragments are derived. We conclude, therefore, that the coat protein cistron is situated upon the 3'-terminal third of the RNA chain, i.e. within 2000 nucleotides of the 3'-end.     

Hydrogen ion uptake upon tobacco mosaic virus protein polymerization.

To determine the stage at which H⁺ ions are bound during the entropy-driven polymerization of tobacco mosaic virus protein, acid-base titrations were carried out at a concentration of 5 mg/ml in 0.1 m-KCl from pH 8 to pH 5.2 and back to pH 8 at 4, 10, 15 and 20 °C. The titration was always completely reversible when the addition of acid or base was so slow that the experiment required seven hours in each direction. When the titration was started at pH 7 and performed down and up twice as rapidly, a hysteresis loop, indistinguishable from one previously published, was obtained at 20 °C.Ultracentrifugation experiments were carried out at selected pH values at the four temperatures. H⁺ ion uptake, as determined from the reversible titration curves, is correlated with the disappearance of the 4 S component and is independent of whether the polymerized species is in a 20 S or higher state of aggregation. At pH 7, approximately 1 mole of H⁺ ion is bound per mole of monomer. At pH values between 6.56 and 6.05, 1.5 moles of H⁺ ion are bound per mole of monomer upon polymerization. At pH 6.05, 0.5 mole of H⁺ ion is bound before any polymerization takes place.Tobacco mosaic virus protein at 20 °C in an unbuffered 0.1 m-KCl solution at pH 7.18 at a concentration of 41 mg/ml, largely in the 20 S state, was depolymerized entirely to the 4 S state by dilution with 0.1 m-KCl adjusted to the same pH. Under these conditions, there was no pH change, indicating that no H ⁺ ions are released.These seemingly contradictory findings can be explained by assuming that the 4 S component polymerizes to form either double discs without binding H⁺ ions, or, alternatively, two-turn helices accompanied by the binding of H⁺ ions. Both double discs and two-turn helices sediment at approximately 20 S. Whether polymerization in the neighborhood of pH 7 leads to helices or discs depends upon the availability of H⁺ ions.     

Dimerization of recombinant tobacco mosaic virus movement protein

The p30 movement protein (MP) is essential for cell-to-cell spread of tobacco mosaic virus in planta. We used anion-exchange chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to obtain highly purified 30-kDa MP, which migrated as a single band in native PAGE. Analytical ultracentrifugation suggested that the protein was monodisperse and dimeric in the nonionic detergent n-octyl-beta-D-glucopyranoside. Circular dichroism (CD) spectroscopy showed that the detergent-solubilized protein contained significant alpha-helical secondary structure. Proteolysis of the C-tail generated a trypsin-resistant core that was a mixture of primarily monomers and some dimers. We propose that MP dimers are stabilized by electrostatic interactions in the C terminus as well as hydrophobic interactions between putative transmembrane alpha-helical coiled coils.     

Assembly of tobacco mosaic virus.

The assembly of tobacco mosaic virus requires the presence of a particular protein aggregate, the disk. During the nucleation, a specific region of the RNA interacts with a single disk, to bring about a necessarily cooperative transition from the paired two-layer structure to a short segment of nucleo-protein helix. There is a high selectivity for this region of the TMV RNA, because of the many nucleotides bound at once, and other nucleotide sequences appear only to bind by a different mechanism. Elongation of the nucleated rods can continue with either further disks or the less aggregated 'A-protein' as the protein source, but the continued cooperativity inherent with disks would have some advantages. The rates of the two processes have been separately determined and growth is faster when disks are still present. New experiments show that the breakdown of disks to yield A-protein is relatively slow and it is concluded that virus growth from disks could not proceed through a prior breakdown in solution, but must involve the direct interaction of the disk with the growing nucleoprotein rod. The detailed mechanism of disk addition is not understood but it may involve a directed breakdown, since there is also evidence for the existence of a non-equilibrium form of A-protein which has aggregation kinetics distinct from those of equilibrium A-protein. Some implications for the general assembly pathways of viruses both of the specificity and of the assembly/disassembly cycle during the viral infection are considered.