Effect of amino acid replacement on the stability of the tobacco mosaic virus protein structure.

A comparative polarographic study on the alkaline degradation of tobacco mosaic virus (TMV) strain vulgare and its mutant TMV 483, having histidine instead of glutamine at position 9 in the polypeptide chain, was performed. In the course of alkaline degradation and subsequent incubation in the supporting electrolyte at 0 degrees C TMV 483, unlike TMV vulgare, showed a polarographic effect indicating the unfolding of the TMV polypeptide. It was concluded that the replacement of glutamine-9 by histidine causes a decrease in the stability of the three-dimensional structure of the TMV protein subunit. A polarographic study of untreated virions as well as denatured proteins of both TMV strains showed that histidine, when incorporated into the polypeptide chain, is not active polarographically at the conditions used.     

Monoclonal antibodies reactive with myelin basic protein

New monoclonal antibodies (MAbs) to myelin basic protein (BP) reveal epitopes to be in sequences 22–34, 75–82, 83–96, 118–131 and 125–131. Comparison of these results with those previously reported suggest that almost every sequence of about 10 amino acid residues may be sufficiently antigenic to make a single MAb but that certain regions are immunodominant, strong enough to make practically the same MAb repeatedly. One of these new MAbs (clone 3) has especially interesting reactivity, sharply limited to residues 75–82 in bovine and porcine BP: Lys-Ala-Gln-His-Gly-Arg-Pro. Whales presumably have the same sequence, since their BPs are fully reactive with clone 3 MAb, but all other species of BP, with known sequences of BP, have at least two changes in this sequence. Deletion of Lys⁷⁵ (as in a tryptic peptide of porcine BP) reduces reactivity with the MAb about 10-fold, whereas substitution of Ala⁷⁶ by Ser (as in all other species of BP) and either deletion of Gln⁷⁷ (as in human, monkey and rabbit BP) or His⁷⁸ (as in the guinea pig and rat BP) or substitution of Pro⁸² by Thr (as in human, monkey, rat and mouse BP) eliminates reactivity. We speculate that woodchuck and prairie dog BPs in this region closely resemble chicken BP, which has about 2% of the original reactivity. However, squirrel BP is unique, probably having only one of the changes in this region of BP, since it possesses 10–20 times the reactivity of chicken BP but still only 20–50% of the original reactivity with clone 3 MAb, a degree of reactivity not seen with any other species of BP.     

The specific involvement of coat protein in tobacco mosaic virus cross protection

Nicotiana sylvestris infected by strains of tobacco mosaic virus (TMV) causing mosaic can be superinfected in the dark green leaf tissue, but not light green tissue, by necrotizing strains of TMV. The dark green tissue, however, is much less susceptible than healthy tissue, to some extent, even to unrelated viruses. The RNA of necrotizing strains of TMV was relatively more infectious than intact virus on mosaic than on healthy leaves and caused lesions in both light and dark green tissues. The same relationship was found in Nicotiana longiflora and, when the protecting strain in N. sylvestris could be used as a challenge, in Capsicum baccatum. The efficiency of superinfection by RNA was not found with viruses unrelated to TMV. When bentonite at 1 mg/ml, which is known to strip protein from TMV, was included in the inoculum of intact TMV it superinfected in the same manner as RNA. RNA of a necrotizing strain of TMV, encapsidated in brome mosaic virus protein and used as a challenge, superinfected in the same manner as RNA. When encapsidated in common TMV protein, however, it behaved as native virus. Cross protection apparently results from the prevention of uncoating of related challenge virus in light green tissue of N. sylvestris. Locally inoculated N. sylvestris leaves were insusceptible to challenge RNA or intact virus when the protecting virus was increasing. After increase ceased, RNA was more infectious than intact virus.     

Reinvestigation of intracellular localization of the 30K protein in tobacco protoplasts infected with tobacco mosaic virus RNA.

It has been shown that the 30K protein of tobacco mosaic virus (TMV) is responsible for the cell-to-cell movement function of the virus. It is still obscure how the protein is involved in this function at the molecular level. We formerly found that the 30K protein is localized to the plasmodesmata of TMV-infected plants. We also reported that the 30K protein was detected in a nuclei-rich fraction of TMV-infected protoplasts after biochemical fractionation. To clarify the inconsistency, the 30K protein was immunocytologically localized in TMV-infected protoplasts using a newly prepared antibody against the 30K protein. On some sections, the 30K protein was found near the nucleus but not in or on the nucleus. At later stages of infection a novel electron-transparent structure was detected in the cytoplasm where the 30K proteins were localized. This structure might reflect an intermediate form between its synthesis in the cytoplasm and its targeting to the plasmodesmata in whole plants.     

Monoclonal antibodies specific for the avian sarcoma virus transforming protein pp60src

Monoclonal antibodies to avian sarcoma/leukosis virus-coded transforming, structural, and polymerase proteins have been developed. In this first communication, a monoclonal antibody to pp60src will be characterized. The antibody can bind to pp60src in radioimmunoassays, precipitates this antigen in protein kinase tests, and demonstrates in immunofluorescence investigations that pp60src has different intracellular localizations depending on whether RSV-transformed avian or mammalian cells are studied.     

Functions of the 126- and 183-kDa proteins of tobacco mosaic virus

Tobacco mosaic virus produces two proteins that contain domains similar to the methyltransferase (MT) and helicase (HEL)-like domains of the replicase-associated proteins of other RNA viruses. The more abundant 126-kDa protein contains only the MT and HEL-like domains, whereas the 183-kDa readthrough protein additionally contains the polymerase domain. We examined the functions of these proteins by constructing a bipartite system to express the 126- and 183-kDa proteins from separate RNAs. Mutants expressing the 183-kDa protein recognized promoters for negative- and positive-stranded RNA synthesis, transcribed subgenomic mRNAs, capped RNAs, synthesized proteins, moved cell to cell within the plant, and replicated defective RNAs (dRNAs). The principal function of the 126-kDa protein was to increase the rate of replication approximately tenfold. The 126-kDa protein appeared to function primarily in cis, and production of the 126-kDa protein in trans did not enhance replication of the helper virus. dRNAs producing a functional 126-kDa protein were replicated efficiently by helper viruses that produced only the 183-kDa protein but not by wild-type virus, suggesting that efficient replication required the 183-kDa protein to form a heterodimer with the 126-kDa protein already bound to the target dRNA.     

Accumulation of the 126 kDa protein of tobacco mosaic virus during systemic infection analysed by immunocytochemistry and ELISA

Summary Systemic infection of tobacco with tobacco mosaic virus (TMV) strain WU1, is accompanied by massive accumulation of the virus-coded non-structural 126 kDa protein in X-bodies. The development of X-bodies and the time course of the increase in 126 kDa protein in systemically infected leaves were analyzed by immunocytochemistry and ELISA, respectively, using an antiserum raised against a fusion protein of -galactosidase and part of the 126 kDa protein. The ELISA assay developed enabled routine detection of viral 126 kDa (as well as 183 kDa) protein in samples of less than 5 mg of systemically infected leaves. Plants were inoculated by differential temperature treatment, whereafter the accumulation of 126 kDa protein was related to viral multiplication, the development of X-bodies and the formation of symptoms. Both 126 kDa protein and coat protein became detectable between 40 and 66 h after transfer of the plants and increased in parallel up to 200 h. Vein clearing was visible at 66 h, followed by mosaic in the newly developed leaves at 112 h. By electron microscopical analysis small X-bodies, weakly labelled with antibodies against the 126 kDa protein, were detected as early as 24 h after transfer. At this stage they were not associated with nuclei. Thereafter, however, X-bodies increased in size and 126 kDa labelling density, and were increasingly often observed attached to nuclei. In emerging leaves that developed mosaic symptoms, X-bodies were associated with nuclei already at an early stage. These observations are consistent with the hypothesis that association of X-bodies with nuclei may lead to symptom induction, when the leaf is invaded by the virus early in its development.     

Monoclonal antibodies as structural probes of southern bean mosaic virus

The reactivity of six monoclonal antibodies with native southern bean mosaic virus particles, swollen virus, swollen virus contracted by divalent cations or by pH adjustment to 5.0, and virus coat protein at pH 7.5 and 5.0 were assessed by antigen inhibition enzyme-linked immunosorbent assays. Clones B4, B7, and B11 did not react with native virus but were inhibited by low levels of all nonnative virus antigens. B6 had a high reactivity only with the native virus. B5 and B10 reacted equally with native virus and swollen virus contracted by Ca2+ or by pH adjustment, but reacted only weakly with swollen virus or swollen virus contracted by Mg2+. B5 reacted with viral protein at pH 5.0 and weakly with coat protein at pH 7.5, whereas B10 did not react with protein at pH 7.5 and had only limited reactivity with viral protein at pH 5.0. The reactivity of B5 and B10 with swollen virus increased as the pH was gradually decreased between 7.3 and 6.5. Similarly, their reactivity with swollen-contracted virus decreased as the pH was increased within the same range. Reactivity versus pH plots of these two transition forms showed hysteresis with B10 but did not with B5.     

Monoclonal antibodies reactive with idiotypic and variable-region specific determinants on human immunoglobulins

Monoclonal antibody JG-B1, specific for the human VKIIIb sub-subgroup of L chains, and JG-B4 specific for an idiotypic determinant on Glo, a monoclonal human IgM-VKIIIb anti-IgG, were produced and characterized. The VKIIIb determinant was detected on L chains alone and intact immunoglobulins with VKIIIb L chains. However, the idiotypic determinant was expressed only on IgM-Glo and required association of H and L chains. Binding of the immunogen Glo, to its antigen-IgG partially inhibited anti-idiotype and anti-VKIIIb binding. Cross-inhibition experiments demonstrated that intact pentameric IgM-Glo expressed one-half the number of idiotypic sites as VKIIIb determinants. However, Glo half-molecules expressed equal numbers of idiotypic and VKIIIb determinants. This is the first described monoclonal antibody produced by hybridoma technology which recognizes an antigenic determinant specific for a single variable region in intact immunoglobulin.     

The tryptic peptides and terminal sequences of the protein from the cowpea strain of tobacco mosaic virus

The use of ion exchange chromatography, gel filtration, and paper electrophoresis for the separation of the peptides obtained by tryptic digestion of the cowpea strain of tobacco mosaic virus protein is described. The amino acid compositions of the 13 tryptic peptides obtained were determined and accounted for 159 residues compared with the 158 found for type tobacco mosaic virus protein. The only tryptic peptide that cowpea tobacco mosaic virus protein and type tobacco mosaic virus protein have in common is asparaginyl-arginine despite the fact that the former is very similar to type tobacco mosaic virus protein in a number of its properties. The amino terminal sequence was found to be acetyl alanyl-tyrosine and the carboxyl terminal sequence was confirmed as alanine preceded by threonine.     

Identification of a novel tobacco DnaJ-like protein that interacts with the movement protein of tobacco mosaic virus

The movement protein (MP) of tobacco mosaic virus (TMV) mediates the transport of viral RNA from infected cells to neighboring uninfected cells via plasmodesmata by interacting with putative host factors. To find such host factors, we screened tobacco proteins using the yeast two-hybrid system. NtMPIP1, a novel subset of DnaJ-like proteins, was identified from a tobacco cDNA library, and its specific interaction with TMV MP was confirmed with an in vitro filter-binding assay. In a deletion analysis, using a series of truncated TMV MPs and NtMPIP1s, at least two regions of TMV MP, amino acid residues 65–86 and 120–185, conferred the ability to interact with the C-terminal domain of NtMPIP1, which is thought to be involved in substrate binding. Virus-induced gene silencing of NtMPIP1 significantly inhibited the spread of TMV. Therefore, it is reasonable to consider that endogenous NtMPIP1 is a host factor involved in virus cell-to-cell spread by interacting with TMV MP. Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession number AB092334.