Immunochemical studies of tobacco mosaic virus--VI. Attempts to localize viral epitopes with monoclonal antibodies.

The specificity of 18 monoclonal antibodies directed to tobacco mosaic virus (TMV) was studied by measuring their ability to bind to viral mutants, to other tobamoviruses, to dissociated viral subunits and to peptide fragments of the viral coat protein. The apparent binding specificity of the antibodies was dependent on the type of enzyme-linked immunosorbent assay used, probably because the antigens were disrupted or denatured when attached to the plastic surface of microtiter wells. The capacity of different monoclonal antibodies to detect single substitutions in the viral coat protein was used to delineate some of the topographic epitopes of TMV.

Antigenic structure of histone H2B.

Antigenic determinants of histone H2B were localized using a series of 23 overlapping fragments of H2B obtained either by chemical and enzymatic cleavage of the histone or by solid-phase peptide synthesis. The ability of peptides to bind H2B antibodies was measured in an enzyme-linked immunosorbent assay, using antisera directed against calf thymus and chicken erythrocyte H2B as well as four anti H2B monoclonal antibodies obtained from autoimmune mice. Seven antigenic determinants were localized in the H2B molecule in the vicinity of residues 1-11, 6-18, 15-25, 26-35, 50-65, 94-113 and 114-125.

Comparative sensitivity of different ELISA methods for detecting monoclonal antibodies to viruses.

Five different ELISA procedures using alkaline phosphatase conjugates were compared for their sensitivity in detecting binding activity in nine monoclonal antibodies raised against tobacco mosaic virus (TMV). The two procedures in which antigen was used to coat the microtiter plates were found to be unreliable for testing the activity of monoclonal antibodies. The two most sensitive procedures utilized a multilayered sandwich of which one component imperatively had to be an avian antibody devoid of serological cross-reactivity with mammalian globulins.

Serological differentiation of tobamoviruses by means of monoclonal antibodies.

Several serologically related plant viruses have been differentiated for the first time by means of monoclonal antibodies. Two orchid strains of tobacco mosaic virus that were previously considered to be serologically identical with the type strain were shown to be distinguishable with certain monoclonal antibodies. In indirect ELISA, none of the antibodies showed any binding to tobamoviruses that differed from TMV (common strain) by a serological differentiation index larger than 2.

Large-scale purification of the 3'-OH-terminal tRNA-like sequence (n = 159) of turnip-yellow-mosaic-virus RNA.

In order to undertake structural and functional studies on the 3'-terminal part of turnip yellow mosaic virus RNA, a structure which can be specifically aminoacylated by valyl-tRNA synthetase, we have developed large-scale methods for purifying the tRNA-like sequence. Several experimental approaches were tested. One procedure was retained enabling us to purify large quantities of the homogeneous tRNA-like fragment.

The tRNA-like structure of turnip yellow mosaic virus RNA: structural organization of the last 159 nucleotides from the 3' OH terminus.

The secondary structure of the isolated tRNA-like sequence (n=159) present at the 3' OH terminus of turnip yellow mosaic virus RNA has been established from partial nuclease digestion with S1 nuclease and T1, CL(3), and Naja oxiana RNases. The fragment folds into a 6-armed structure with two main domains. The first domain, of loose structure and nearest the 5' OH terminus, is composed of one large arm which extends into the coat protein cistron.

Monoclonal antibodies as probes of the antigenic structure of tobacco mosaic virus.

The antigenic structure of tobacco mosaic virus has been analysed by measuring the ability of nine monoclonal antibodies to distinguish between wild-type virus and 13 mutants showing single and double amino acid substitutions in the coat protein. Although the majority of antibodies detected those substitutions that were located at the outer surface of the virion, some of them also recognized conformation alterations induced by exchanges occurring deep inside the subunit. In the case of five mutants, the antibody reactivity was reduced compared with wild-type virus, while in the case of three others, it was significantly higher.

Investigation of the requirements for O-glycosylation by bovine submaxillary gland UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosamine transferase using synthetic peptide substrates.

Peptides containing a triprolyl sequence carboxyl to a threonine residue can be O-glycosylated by a crude Triton x-100 extract of porcine submaxillary glands (Young, J. D., Tsuchiya, D.

Identification of binding sites of turnip yellow mosaic virus protein and RNA by crosslinks induced in situ.

Turnip yellow mosaic virus RNA and protein could be crosslinked in situ by ultraviolet irradiation at pH 4.8 but not at pH 7.3, and by bisulphite treatment at pH 7.

Nucleotide sequence of turnip yellow mosaic virus coat protein mRNA.

The primary structure of the coat protein messenger RNA of turnip yellow mosaic virus is presented. This sequence is the first complete nucleotide sequence of the coat protein messenger of a plant virus to be reported. The coding region, consisting of 567 nucleotides, is flanked by a 5' noncoding region of 19 nucleotides (not including the initiation codon and the cap structure) and by a 3' noncoding region of 109 nucleotides (including the termination signal).

Nucleotide sequence at the 5' extremity of turnip yellow mosaic virus genome RNA.

The sequence of the first 110 nucleotides at the 5' extremity of turnip yellow mosaic virus genome RNA has been determined. The sequence is blocked at its 5' terminus with the group pppm(7)G and contains two AUG triplets. The determined sequence bears a strong resemblance to the 5' noncoding region of rabbit beta-globin mRNA.

Valylation of the two RNA components of turnip-yellow mosaic virus and specificity of the tRNA aminoacylation reaction.

A comparative study of the aminoacylation of the two RNA components of turnip yellow mosaic virus, of yeast tRNAVal, tRNAfMet and of tRNAPhe by purified yeast valyl-tRNA synthetase is reported. Aminoacylations were performed in the presence of pure yeast tRNA nucleotidyltransferase, since 85% of the viral RNA molecules lacked the 3'-adenosine. We find that aminoacylation of the viral RNAs, like tRNA aminoacylation, reflects an equilibrium between the acylation and deacylation reactions.

Nucleotide sequence (n=159) of the amino-acid-accepting 3'-OH extremity of turnip-yellow-mosaic-virus RNA and the last portion of its coat-protein cistron.

The experiments described in this paper and the following one establish the sequence of the 3'-OH terminal 159 nucleotides of turnip yellow mosaic virus RNA. Uniformly 32P-labeled turnip yellow mosaic virus RNA was partially digested with T1 ribonuclease and the fragments were fractionated by polyacrylamide gel electrophoresis. Fragments originating from the 3'-OH end of the RNA molecule were identified by testing for the 3'-terminal oligonucleotide, C-COH, after total U2 ribonuclease hydrolysis.

Nature and specificity of the RNA-protein interaction in the case of the tymoviruses.

Dissociation-reassociation experiments performed with turnip yellow mosaic virus in the presence of various RNAs and polynucleotides were used to investigate the degree of specificity and the contribution of the associated RNA moiety to the stability of TYMV. The results emphasize the importance of strategic cytosine residues spread along the RNA chain. Some insight into the contribution of the protein could be gained from comparison of TYMV and eggplant mosaic virus (EMV), a virus similar to TYMV although its top component contains low molecular mass RNA's able to bind various amino acids.

Physical and functional heterogeneity in TYMV RNA: evidence for the existence of an independent messenger coding for coat protein.

Turnip yellow mosaic virus RNA can be separated into two distinct components of 2 times 10(6) and 300 000 daltons molecular weight after moderate heat treatment in the presence of SDS or EDTA. The two species cannot have arisen by accidental in vitro degradation of a larger RNA, as they both possess capped 5' ends. Analysis of the newly synthesized proteins resulting from translation of each RNA by a wheat germ extract shows that the 300 000 molecular weight RNA can be translated very efficiently into coat protein.

Stucture of the amino-acid accepting 3'-end of high-molecular-weight eggplant mosaic virus RNA.

The sequence of the first 59 nucleotides from the 3'-OH terminus of high-molecular-weight eggplant mosaic virus RNA has been determined by standard radio-chemical techniques. The fragment was identified among the products of partial T1 RNase digestion by making use of the reverse migration, at pH 2.5, of the 3'-OH terminal oligonucleotide.