Statistical analysis of functional region(s) of the TSH receptor.

In this study, we apply recently developed methods to evaluate the thyrotropin receptor (TSH-R). These methods are called deviation (DEV) model, deviation decrease (DD) and DEV/DD analyses, and are based on deviation of amino acid sequences. A 3-dimensional structure model of TSH-R was graphically constructed, and found to possess a large central cavity (donut-like structure). The N-terminus was found to be in the center of the whole extracellular structure and to form a part of the bottom of the cavity. High DEV values indicate deviated amino acid compositions in the protein and were seen in 7 regions, 6 of which were found to be in regions with hydrophilic and acrophilic character. On the basis of the analysis of intra-molecular cis-acting relationships, 7 pairs of regions were presumed to be closely related. Further, when 3 exoplasmic loop lesions were analyzed similarly, 3 other regions were shown to have a close relationship with the cell surface. DEV/DD values were applied to predict the interface of TSH-R with trans-acting molecules such as TSH-R antibody or TSH. The regions in association with trans-acting molecules were seen in 14 regions, 11 of which included the high DEV regions. Both of the TSH-R specific regions in the N- and C-terminal side, especially the latter, were found to be the major components.

Immunoglobulin G that interferes with thyroid-stimulating antibody measurements can be eliminated specifically by incubation with synthetic peptides corresponding to partial sequences of the human thyrotropin receptor.

Two IgG preparations out of more than 100 tested, distinct from the typical Graves' disease IgG, were shown specifically to enhance the cAMP production of FRTL-5 cells by the addition of a synthetic peptide, P-218, corresponding to the partial amino acid sequence from No. 354 to 367 of the h thyroid-stimulating hormone (TSH) receptor. IgG obtained from a patient with Graves' disease revealed a serial alteration of the enhancement; negative in July, 1989, potent in January, 1991, and weak in September 1991. During this time there was no remarkable change in the patient's serum protein components or TSH receptor antibody activities. A peptide with a completely reverse sequence of P-218 showed little effect, and P-218 in combination with bTSH or forskolin did not affect cAMP production by these ligands, and did not alter the inhibitory activity of thyroid-stimulation-blocking antibody. High concentrations of P-218 resulted in reduction of such enhancing effects of cAMP by thyroid-stimulating antibody. P-218 affinity chromatography showed almost complete absorption and recovery of thyroid-stimulating antibody and P-218 reactivity. In the 15 synthesized peptides with proximal sequences of P-218 (from 338 to 378), regions thought to be involved with the enhancement were defined as follows: 354-367 (P-218) is a critical unit; 354-357 and 364-367 are considered to be the essential sites; several amino acid extensions on both N- and C-terminal sides of P-218 show additional enhancement. In conclusion, evidence was shown to indicate the presence of IgG that interferes with thyroid-stimulating antibody measurements.

Binding of viral glycoprotein with trypsin and its relation to virulency. I. Initial step of binding.

The interaction between surface proteins of some enveloped viruses and trypsin was studied by computer analysis. Prior to the cleavage of the viral protein by trypsin, hydrophobic interaction between them at the vicinity of their active sites may occur. An exposed hydrophobic portion was found there which theoretically could stimulate the interaction. This interaction would be rather non-specific: according to the analysis, trypsin could bind equally well with weakly virulent virus and virulent viruses. Following this interaction, a specific reaction between their active sites would occur. The specificity was found to be related to the virulency of the virus.

Functional structure of protein: exposed vs hidden function.

The dev analysis was used to construct the functional structure of protein (3). Based on this principle, the intramolecular interaction of dev peak was investigated. Two types of peak were observed: one is the hidden peak which interacts strongly with another region of the molecule and the other is the exposed peak whose interaction was weak. Analysis was made with trypsin and its inhibitor and on the intermolecular interaction between their exposed peaks. One interaction between their exposed peaks was found to coincide with one which had been already known to be authentic between their active sites.

Protein folding: construction of functional structure.

The problem of protein folding was studied with trypsin inhibitor by deviation analysis (1). The results showed that: i) Qualitatively, the main features of the structure, determined by this method, coincided with the structure determined by X-ray crystallography (3). This structure is, however, not topological but functional, and may elucidate the functional relations between various parts of the protein.

Binding of viral glycoprotein with trypsin and its relation to virulency. II. Comparison between bovine and Streptomyces griseus trypsins.

The two step nature of the binding reaction between trypsin and viral glycoproteins was further investigated using two types of trypsin, bovine trypsin and Streptomyces griseus trypsin. The experimental results were explained by the van der Waals energy operating in the second step, suggesting that the conformational aspects, in addition to the electrostatic nature, of the interacting peptides are decisive in this specific process.

Prediction of the virulencies of some enveloped viruses from the structure of the cleavage recognition site of viral glycoproteins essential for infectivity. I. Calculation of interaction energy.

Some evidences have been found that virulency in paramyxoviruses depends on the sensitivity of the cleavage recognition site of the F glycoprotein to serine type proteases. In this report, the interaction energies between the active site of trypsin and the cleavage recognition sites in paramyxoviruses are calculated. Results show that van der Waals energy and electrostatic energy contribute to the sensitivity. The virulencies of some myxo- and retro-viruses are then predicted on the basis of the two calculated interaction energy values.

Prediction of the virulencies of some enveloped viruses from the structure of the cleavage recognition site of viral glycoprotein essential for infectivity. II. Deviation analysis.

Analysis of the amino acid sequence in protein (deviation analysis) suggests that the binding between trypsin and the enveloped virus is the first step of their interaction, which occurs in a specified configuration. It is possible that the distance between their active sites is important for the viral sensitivity to trypsin, which is related to the virulency of the enveloped virus.

Antibodies to Asp-Asp-Glu-Asp can inhibit transport of nuclear proteins into the nucleus.

The signal sequence of simian virus 40 (SV40) large T-antigen for translocation into the nucleus is composed of positively charged amino acids Lys-Lys-Lys-Arg-Lys. Rabbit antibodies to a synthetic peptide containing the negatively charged amino acid sequence Asp-Asp-Asp-Glu-Asp were obtained. Indirect immunofluorescence of the antigens recognized by the antibody was punctate at the nuclear rim or the nuclear surface, depending on the plane of focus. The antibody blocked transport of nuclear proteins into the nucleus. The antigens recognized by the antibody were predominantly localized to the nuclear pores.

Regional variation and function of nucleotide and amino acid sequence.

Two methods of qualitative analysis of sequence distribution in DNA and protein are presented. The first method is based on the finding that the frequency of occurrence of each nucleotide in a defined sequence with functional significance more or less deviates from uniform distribution. The deviation found in this defined sequence seems to parallel the function of this sequence. In the second method, two model compounds (trypsin and its inhibitor) have been used to see the topological fit between their local structures. Acrophilicity parameter for amino acid was used to construct the topological structure. Both methods may find practical application in algorithms to design functional DNA and protein molecules.

Molecular basis of plant viral virulence; the complete nucleotide sequence of an attenuated strain of tobacco mosaic virus.

The total genome sequence of L11A, an attenuated strain of tobacco mosaic virus (TMV), has been determined. This strain is able to multiply in tomato plants without inducing any remarkable symptoms, but to protect them from later infection with virulent TMV strains. When compared with the recently published total genome sequence of TMV L (the virulent ancestral strain of L11A) ten base substitutions were found in the L11A genome. Seven of these occurred in the third letters of in-phase codons and did not influence amino acids. Only three, which were in the common reading frame for both the 130K and 180K proteins, resulted in amino acid changes. Together with the result of the partial sequence of RNA of L11, an intermediate strain in sequential isolation from L to L11A, it is observed that one base at the nucleotide position 1117 is changed from L to L11 and two bases at the positions 2349 and 2754 are changed from L11 to L A11.

Unique nature of an attenuated strain of tobacco mosaic virus: autoregulation.

An attenuated strain L11A of tobacco mosaic virus (TMV) multiplied like wild type strain L at an early stage of infection in tomato leaves. Four days after inoculation, however, multiplication of L11A was drastically reduced (autoregulation) compared with the constant multiplication of L. In mixed infections, L11A strongly inhibited the multiplication of homologous strain L. Experiments with cucumber mosaic virus (CMV) or tobacco plants revealed that the inhibitory mechanism of L11A is not host-specific but virus-specific, and the autoregulatory mechanism is effective only for TMV. RNA synthesis in L11A infected leaves 4 days after inoculation was studied by polyacrylamide gel electrophoresis. Synthesis of TMV-RNA and its replicative intermediate were strongly inhibited, whereas the replicative form of TMV-RNA and ribosomal RNA were synthesized as in the case of L infection. Synthesis of non-coat-protein was studied by the incorporation of radioactive histidine into subcellular fractions derived from leaves infected with L or L11A for 4 days. Different patterns of the two strains in protein synthesis were noted. At least three proteins were predominantly synthesized in L11A infection. One of them was observed in the mitochondria fraction. From its position in polyacrylamide gel, it could be viral coded 165K protein which is considered to be involved in viral RNA replication. These results suggest that the unique nature of attenuated virus L11A, i.e. autoregulation, resulted from the inhibitory mechanism of viral RNA synthesis due to overproduction of 165K protein and is quite distinct from interferon, intrinsic interference or interference by defective virus.

Single amino acid substitution in 30K protein of TMV defective in virus transport function.

Involvement of the tobacco mosaic virus (TMV) coded 30K protein in a virus transport function within the infected plant has been suggested. Previously a temperature sensitive mutant, TMV Ls 1, that is defective in cell-to-cell movement at a restrictive temperature, was reported. To demonstrate a relationship between the 30K protein and the transport function, the nucleotide sequences of the 30K and coat protein cistrons of the mutant, TMV Ls 1, and the wild type, TMV L (tomato strain) were compared. A single base substitution which causes replacement of a proline codon in the L strain by a serine codon was found in the Ls 1 mutant. Results support the notion that the 30K protein is responsible for the virus transport function.

Inhibitory effect of hydroquinone and related agents on multiplicity reactivation and genetic recombination of bacteriophage T4.

The multiplicity reactivation of T4D phage irradiated with UV was inhibited by treatment of infected cells with 0.5 mM hydroquinone (HQ). HQ also inhibited genetic recombination of T4B rII mutants. The recombination frequency was reduced by treatment of infected cells with HQ in a buffer when compared with an untreated control. In addition to HQ, the effects of quinone, an oxide form of HQ, mercaptoethanol and alpha-tocopherol, antioxidants, were also investigated. Quinone and mercaptoethanol inhibited recombinations but alpha-tocopherol did not.

Sensitivity to ultraviolet light of tobacco mosaic virus modified by cetyltrimethylammonium bromide.

Cetyltrimethylammonium bromide (CTAB) modified tobacco mosaic virus (TMV) virions so that the intrinsic fluorescence changed, viral infectivity decreased, sensitivity to RNase or UV irradiation increased, and coat protein subunits were released by the addition of Triton X-100. The change in fluorescence emission at 320 nm shifted to 340 nm was observed at 100 micrograms of CTAB per ml. This represents a change in the tryptophan environment inside the virion. At a lower concentration of CTAB, intersubunit contact was weakened, resulting in the release of coat protein subunits and an increase in RNase sensitivity. The release of coat protein took place gradually and two relatively stable intermediates were observed. Increase in UV sensitivity was observed at a lower concentration of CTAB and formation of pyrimidine hydrate was involved in this inactivation. The nature of the minor structural change leading to UV inactivation is discussed.

Modification of tobacco mosaic virus by polyornithine and lecithin.

Combined action of polyornithine and lecithin modified tobacco mosaic virus (TMV) virions making them sensitive to ribonuclease (RNase), pronase or Triton X-100. Sedimentational analysis and examination of the fluorescence spectrum revealed that the reaction product obtained after RNase treatments of modified TMV was a three-component complex made of coat protein, polyornithine and lecithin. The minimum requirement for the modification was completely fulfilled by cetyltrimethylammonium bromide, suggesting that a positively charged nitrogen group and an alkyl group of moderate size, C10--18, are necessary components. These components react with the surface region of TMV which is considered to have an important role in connecting coat protein subunits in TMV virions.

Infectivity suppressing and virus-binding activities of a membrane material isolated from tobacco leaves.

TMV binding substance (R) was isolated from a tobacco leaf membrane fraction and was purified by extraction with organic solvents and by column chromatography. Experimental results suggest that the binding of R with TMV results in inactivation of TMV. When tobacco leaves were inoculated with the R-TMV complex, it was found that the formation of polysome containing infecting viral RNA was inhibited. Model experiments showed that the mode of R-TMV adsorption to the membrane is different from that of TMV adsorption and that stripping of coat protein from TMV by SDS was inhibited by R. A possible explanation for the mechanism of this inhibition by R is that the R-TMV complex follows a pathway which does not lead to establishment of infection. Although less efficient, R was still active when it was applied after virus inoculation. Due to its affinity to coat protein, R might also interfere with a later process of viral multiplication.

Disassembly of tobacco mosaic virus by membrane lipid isolated from tobacco leaves and polyornithine.

In vitro disassembly of tobacco mosaic virus (TMV) virions occurred in the presence of both polyornithine and a lipid fraction isolated from tobacco leaf membrane. The latter could be replaced by lecithine. Disassembly of 10 microgram of TMV virions was attained in the presence of a 500-mg leaf equivalent of membrane lipid and 20 microgram of polyornithine in 1 ml of 0.01 M Tris-HCl buffer, pH 7.4 at 30 C. Similarity and dissimilarity between the in vitro disassembly and the in vivo uncoating mechanisms are discussed.

Binding of tobacco mosaic virus to membrane material isolated from tobacco leaves.

Binding of tobacco mosaic virus (TMV) to disrupted tobacco leaf membrane was studied. Membrane isolated from tobacco leaves was treated successively with (NH4)2SO4, Li-diiodosalicylate and then pronase. TMV-binding substance was thus isolated in a soluble form. From enzymatic digestion experiments, it was suggested that the binding substance was composed of lipid and carbohydrate.