Partial sequence of the N-terminal portion of the protein of cowpea chlorotic mottle virus

The action of trypsin on cowpea chlorotic mottle virus (CCMV) at pH 7.4 caused the release of 25 amino acid residues from the N-terminal of the protein. Separation of peptides released during this digestion on cation exchange resins and determination of their N-termini gave 6 major peptides: the N-terminal peptide CH₃C(0) NH(Lys, Thr₂, Ser, Gly₂, Val₂); AlaAlaAlaArg; AsnThrArg; AlaGlnArg; LeuThrArg; and LysAsnLysArg. Some of these peptides were cleaved by nontryptic enzymes in the digestion. Peptides cleaved by trypsin from chymotrypsintreated virus were Lys, Arg, and AsnThrArg.     

Swelling of isometric and of bacilliform plant virus nucleocapsids is required for virus-specific protein synthesis in vitro

Encapsidated RNA of cowpea chlorotic mottle virus (CCMV), brome mosaic virus (BMV), alfalfa mosaic virus, and southern bean mosaic virus can act as templates for protein synthesis when preswollen virions are added to a wheat germ extract, or when unswollen virions swell in the extract. Encapsidated RNA of turnip yellow mosaic virus, whose particles are unlikely to swell under the conditions used here, did not act as a template. CCMV and BMV, swollen in the extract, gave relatively more RNA 1 and 2 products than when preswollen by dialysis.     

Characterization of monoclonal antibodies specific for the pre-S2 region of the hepatitis B virus envelope protein

Monoclonal antibodies (McAb) specific for the pre-S region of the hepatitis B virus (HBV) envelope protein were prepared using HBV particles of hepatitis B surface antigens (HBsAg) as immunogens. The antibodies reacted in Western blot analyses and in ELISA with pre-S2 sequences of the HBV envelope protein. Pepsin or protease V8 treatment of the antigen abolished reactivity. The fine specificity of one of the McAb (F376) was established by immunoassays using synthetic peptides and a pre-S2-beta-galactosidase fusion protein expressed in E. coli. The shortest peptide recognized by F376 is demarcated by residues pre-S(132) at the N-terminal and pre-S(140)-pre-S(145) at the C-terminal. The corresponding amino acid sequence (for HBV subtype adw2) is: QDPRVRGLY(LPAGG). Additional amino acid residues at the N-terminal, and possibly at the C-terminal ends contribute to the binding of McAb, probably due to conformational influences. The McAb was applied to immunoassays of pre-S2 sequences in purified HBsAg and in human sera containing HBsAg.     

Complete nucleotide sequence of spring beauty latent virus,a bromovirus infectious to Arabidopsis thaliana

Summary.  Spring beauty latent virus (SBLV), a bromovirus, systemically and efficiently infected Arabidopsis thaliana, whereas the well-studied bromoviruses brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV) did not infect and poorly infected A. thaliana, respectively. We constructed biologically active cDNA clones of SBLV genomic RNAs and determined their complete nucleotide sequences. Interestingly, SBLV RNA3 contains both the box B motif in the intercistronic region, as does BMV, and the subgenomic promoter-like sequence in the 5’ noncoding region, as does CCMV. Sequence comparisons of SBLV, BMV, CCMV, and broad bean mottle virus demonstrated that SBLV is closely related to BMV and CCMV. Received May 20, 2002; accepted August 20, 2002     

The serology of the bromoviruses. I. Serological interrelationships of the bromoviruses

The serological relationships between brome mosaic (BMV), cowpea chlorotic mottle (CCMV), and broad bean mottle (BBMV) viruses, and their coat proteins, were studied by gel precipitin, "rocket immunoelectrophoresis" (RIE), and enzyme-linked immunosorbent assay (ELISA) techniques. Gel precipitin and RIE tests indicated that capsid swelling altered the antigenicity of both BMV and CCMV, as although the coat proteins were serologically related at both pH 6.0 and pH 7.0, the viruses appeared to be related only when swollen at pH 7.0. Fixation of the viruses with 2% formaldehyde at pH 6.0 appeared to remove the relationship. BBMV was not related by precipitin tests to either BMV or CCMV but was related to both by direct and indirect ELISA at both pH 6.0 and pH 7.4, though less closely than BMV and CCMV were related to each other. Indirect ELISA showed the three coat proteins to be more closely related than the parent viruses. Formalinized BMV and CCMV appeared less related than the native viruses at pH 6.0. Sandwich ELISA proved too strain specific to show any group relationships. The implications of these results and a serological map proposed for the bromoviruses are discussed.     

The primary structure of cowpea chlorotic mottle virus coat protein

The amino acid sequence of the coat protein of wild-type cowpea chlorotic mottle virus has been nearly completely determined by direct methods. The sites of amino acid replacements were identified in the coat proteins of an oxidation-sensitive mutant, a temperature-sensitive mutant, and a salt-stable mutant. The replacements are a cysteinyl residue for the arginyl residue at position 25 in the oxidation-sensitive mutant, glutamic acid and alanine, respectively, for lysine and valine at positions 21 and 87 in the temperature-sensitive mutant, and arginine for lysine at position 105 in the salt-stable mutant. The substitutions are consistent with point mutations.     

Functional analysis of brome mosaic virus coat protein RNA-interacting domains

Summary The coat proteins (CP) of cowpea chlorotic mottle (CCMV) and brome mosaic virus (BMV), two members of the genus Bromovirus, share 70% identity at the amino acid (aa) level and contain four highly conserved regions, identified as putative RNA-interacting domains (RIDs). To assess the contribution of the conserved aa sequence within each RID and the structural features contained therein toward virion assembly and RNA packaging, we engineered a set of fourteen independent mutations (deletions and substitutions) encompassing all four RIDs. The effect of each mutation on viral biology, pathogenesis, and RNA packaging was analyzed in whole-plant infection assays. Among the four RIDs, two mutations engineered into the N-proximal domain (RID I) and two of the four mutations engineered into the C-proximal domain (RID IV) proved to be more debilitating (compared to wild-type) while only selected regions in the central domains (RID II or III) showed a detectable effect. Neutral effects were observed when aa residues that are predicted to affect calcium binding were mutated. To further analyze the importance of N and C terminal interactions leading to virus assembly and RNA packaging, four CP hybrids were constructed by precisely exchanging either the N-terminal 77 or the C-terminal 113/112aa between BMV and CCMV. Despite the fact that the CP composition of the hybrid viruses is distinct from either of the parents, the symptom phenotype in Chenopodium quinoa, migration pattern of CP in Western blots and virion mobility in agarose gels was indistinguishable from the respective parent providing the genetic background. Collectively, the data provide insight for assessing the relative importance of each RID during genome packaging and in molecular processes regulating the overall architecture of the assembled virions. Correspondence: A. L. N. Rao, Department of Plant Pathology, University of California, Riverside, CA 92521-0122, U.S.A.     

The methyltransferase domain of the 1a protein of cowpea chlorotic mottle virus controls local and systemic accumulation in cowpea

The type strain of cowpea chlorotic mottle virus (CCMV-T) induces a local and systemic infection in California Blackeye cowpea (Vigna unguiculata (L.) Walp. subs. unguiculata cv. California Blackeye), but accumulates to low levels in inoculated leaves and fails to accumulate systemically in the cowpea plant introduction (PI) 186465. CCMV-R, a mutant strain derived from CCMV-T, accumulates to higher levels than CCMV-T in inoculated leaves and systemically infects PI 186465 plants. The phenotypic determinant of CCMV-R was previously mapped to viral RNA1, but the location of the determinant within RNA1 was not identified. Pseudorecombinants generated from genomic cDNA clones of CCMV-T and CCMV-R indicated that the phenotypic differences on PI 186465 were independent of replication. Through the use of chimeric RNA1 cDNA clones containing portions of CCMV-T and CCMV-R and site-directed mutagenesis, two nucleotides, 299 (amino acid residue 77) and 951 (amino acid residue 294), were identified as being independently critical for the local and systemic accumulation patterns of CCMV-R in PI 186465 plants. A second independently derived CCMV-R-like mutant, identified nucleotide 216 (amino acid residue 49) as being critical for induction of the CCMV-R infection phenotype. Amino acid residues 49, 77, and 294 are within the methytransferase domain of the CCMV 1a protein, suggesting that the methytransferase domain has a role in cell-to-cell and systemic accumulation of the virus that is independent of replication.     

Packaging and structural phenotype of brome mosaic virus capsid protein with altered N-terminal β-hexamer structure

The first 45 amino acid region of brome mosaic virus (BMV) capsid protein (CP) contains RNA binding and structural domains that are implicated in the assembly of infectious virions. One such important structural domain encompassing amino acids ²⁸QPVIV³², highly conserved between BMV and cowpea chlorotic mottle virus (CCMV), exhibits a β-hexamer structure. In this study we report that alteration of the β-hexamer structure by mutating ²⁸QPVIV³² to ²⁸AAAAA³² had no effect either on symptom phenotype, local and systemic movement in Chenopodium quinoa and RNA profile of in vivo assembled virions. However, sensitivity to RNase and assembly phenotypes distinguished virions assembled with CP subunits having β-hexamer from those of wild type. A comparison of 3-D models obtained by cryo electron microscopy revealed overall similar structural features for wild type and mutant virions, with small but significant differences near the 3-fold axes of symmetry.     

Assembly and crystallization of a T = 1 icosahedral particle from trypsinized southern bean mosaic virus coat protein

The N-terminal 61 amino acid residues of the coat protein of southern bean mosaic virus were removed by mild trypsinolysis, resulting in a 22,000 MW fragment, P22. In the absence of nucleic acid, the cleaved protein could be assembled into 175 A diameter, T = 1, spherical particles. These particles were formed between pH 4 and 7, at low ionic strength (I 相似文献   

Double infection of tobacco protoplasts with brome mosaic virus and cowpea chlorotic mottle virus

Mixed infections of tobacco protoplasts with Cowpea chlorotic mottle virus (CCMV) and brome mosaic virus (BMV) have been studied. Fluorescent antibody staining showed that capsids of both viruses were made in doubly infected protoplasts but only infectious BMV could be recovered, i.e., BMV dominated in mixed infections and prevented synthesis of infectious CCMV. Healthy protoplasts could be infected after several hours in culture but the susceptibility to infection decreased with time in culture; the decrease was much faster with BMV than with CCMV, probably because the difference in electrical charge of the two viruses affects the method of entry into the protoplasts. If inoculation with the second virus was delayed there was an increasing resistance to superinfection (interference or cross-protection) so that when the delay between inoculations was more than 8 hr BMV was no longer able to infect protoplasts previously infected with CCMV and vice versa. No recombinants between BMV and CCMV were observed. Attempts to rescue a temperature-sensitive strain of CCMV with BMV under restrictive conditions (35 degrees ) were unsuccessful; dominance by BMV prevented synthesis of infectious CCMV RNA.     

A mutant of cowpea chlorotic mottle virus with a perturbed assembly mechanism.

A mutant of cowpea chlorotic mottle virus has been obtained in which the assembly mechanism operates about 1.5 pH units below that of wild-type virus. The structural behavior of the mutant is described as is the inheritance of its coat and density properties.     

Sequence of cowpea chlorotic mottle virus RNAs 2 and 3 and evidence of a recombination event during bromovirus evolution

The genomic sequence of cowpea chlorotic mottle virus (CCMV) was completed by sequencing biologically active cDNA clones of CCMV RNA2 (2774 bases) and RNA3 (2173 bases). While only the central core of the encoded 94-kDa CCMV 2a protein contains features conserved among known and putative RNA replication proteins from many viruses, both flanking regions of CCMV 2a show substantial similarity to the corresponding protein of the related brome mosaic virus (BMV). The 3a proteins of CCMV and BMV, implicated as contributors to the distinct host specificities of the two viruses, show lower levels of conservation but are still discernibly related throughout. Major differences occur in the organization of noncoding sequences in CCMV and BMV RNA3. With respect to an otherwise similar region preceding the BMV 3a gene, the CCMV RNA3 5' noncoding sequence contains a clearly bounded 111-base insertion that must reflect a sequence rearrangement in evolution of at least one of the two viruses. The presence of a subgenomic promoter-like sequence near the end of the novel CCMV sequence makes the organization of genes in CCMV RNA3 reminiscent of the 3' end of tobacco mosaic virus RNA, suggesting that CCMV or its 3a gene might have been derived from an ancestor with fewer genomic RNAs. Sequence similarities between the CCMV and BMV RNA3 intercistronic regions include the subgenomic mRNA promoter and an oligo(A), but not an intercistronic segment required for BMV RNA3 amplification, implying that replication signals on the two RNA3s may be organized quite differently.     

A single amino acid substitution at N-terminal region of coat protein of turnip mosaic virus alters antigenicity and aphid transmissibility

Summary The antigenic activity of the N-terminal region of coat protein of turnip mosaic virus (TuMV) aphid transmissible strain 1 and non-transmissible strain 31 was examined by using a panel of monoclonal antibodies (MAbs) raised against the two virus strains as well as antisera raised against several synthetic peptides from the N-terminal region of the protein. The reactivity of these antibodies was tested in ELISA and in a biosensor system (BIAcore Pharmacia) using virus particles, dissociated coat protein and synthetic peptides as antigens. Substitution of a single amino acid at position 8 in the coat protein of TuMV strain 1 abolished any cross-reactivity between MAbs to strain 1 and the substituted peptide (strain 31) in ELISA although some cross-reactivity was apparent in BIAcore inhibition experiments. In reciprocal tests with MAbs to strain 31 no cross-reactivity with the heterologous peptide was detected in either type of assay. The amino acid residue present at position 8 appears to play a critical role in the binding capacity of MAbs specific for the N-terminal region of TuMV. Antiserum to a synthetic peptide corresponding to residues 1–14 of the protein of TuMV strain 1 was found to react strongly with dissociated coat protein and intact virus particles and was able to inhibit the aphid transmission of the virus. Antiserum to the corresponding peptide of strain 31 did not have this capacity.     

Binding of cowpea chlorotic mottle virus to cowpea protoplasts and relation of binding to virus entry and infection

Cowpea chlorotic mottle virus (CCMV) and cowpea protoplasts were used to study initial interactions between virus and protoplast. Protoplasts and virus were incubated under varying conditions of temperature, pH, ionic strength, and the presence of added compounds. Both the amount of 35S-labeled virus bound to protoplasts and the percentage of infected cells were determined. At 0 and 25 degrees the amount of virus associated with protoplasts increased with the amount of virus added. With inoculum of 25 x 10(6) virus particles per protoplast, 4 x 10(3) and 14 x 10(3) particles per protoplast were bound at 0 and 25 degrees, respectively. In the presence of polyethylene glycol, 85 x 10(3) associated particles per protoplast were bound at both temperatures and ca. 50% of the protoplasts became infected. No infection occurred in the absence of PEG. Variation of pH or ionic strength in the absence of PEG caused little to no change in binding and no infection. In the presence of PEG, increase of pH resulted in lower binding, but infectivity was not affected. Increasing ionic strength, however, increased both binding and infectivity. The presence of unlabeled CCMV, tobacco mosaic virus coat protein, bovine serum albumin, and polycations during inoculation in the absence of PEG decreased the amount of bound CCMV. In contrast, CCMV coat protein, which has a positively charged N-terminal arm, increased binding. In the presence of PEG the effects were similar, although larger amounts of virus were bound. The percentage of infection was reduced by all additives to 5-25%. Addition of ammonium chloride, which inhibits endocytotic virus uptake in animal cells, during inoculation as well as in culture media, did not reduce infectivity. These data do not support a specific receptor-mediated endocytotic uptake of virus but favor a nonspecific mechanism of entry, possibly through membrane lesions. Observations in the electron microscope support the latter mechanism.     

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.     

Inoculation of protoplasts with viruses by electroporation

Optimal conditions for electroporation have been determined using inoculation of brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV) and its RNA into protoplasts of Nicotiana tabacum and N. plumbaginifolia. The most satisfactory medium was 0.5-0.7 M mannitol; calcium ions were toxic and other electrolytes were not helpful during electroporation. Brief pulses (ca. 10 microsec) were less destructive to the protoplasts than longer ones (ca. 10 msec) and gave high percentage infections with CCMV RNA. RNA entered the protoplasts only if present during the voltage pulse. Optimal voltage depended on the sample size, interelectrode distance, and pulse duration. A 50-nF capacitor discharging a 5- to 10-microsec pulse through a 1-ml sample in 0.7 M mannitol with a 4-mm interelectrode distance gave maximum infection with minimal protoplast damage at 2.5 kV/cm. A single pulse was sufficient; multiple pulses slightly increased infection. Electroporation of viral RNA was at least as effective as inoculation in the presence of polyethylene glycol. Positively charged BMV also infected readily but negatively charged CCMV only poorly.     

Nucleotide sequence of the coat protein cistron and the 3' noncoding region of cucumber green mottle mosaic virus (watermelon strain) RNA

Double-stranded cDNA copies of cucumber green mottle mosaic virus (watermelon strain, CGMMV-W) RNA polyadenylated in vitro were cloned into the pBR322 at the PstI site. The sequence of 1071 nucleotides from the Tend of the genomic RNA was determined using two recombinant plasmids and the genomic RNA. The coat protein cistron was located in residues 176-661 from the 3' end. The coat protein was composed of 160 amino acid residues with the molecular weight of 17,261. The 3' noncoding region of the CGMMVW genome was 175 nucleotides long and highly homologous to that of the common strain of TMV. The assembly origin of reconstitution is positioned within the coat protein cistron as predicted previously. In the 5' flanking region of the coat protein cistron a long open frame, probably of 30K protein, was found. The predicted 30K and the coat protein cistron would overlap each other as is the case of the cowpea strain of TMV.     

Foreign complementary sequences facilitate genetic RNA recombination in brome mosaic virus

We have demonstrated that local antisense sequences can mediate genetic recombination within the 3' noncoding region among brome mosaic virus (BMV) RNAs (P. Nagy and J. J. Bujarski, 1993, Proc. Natl. Acad. Sci. USA 90, 6390-6394). Here we show that foreign complementary inserts can direct crossovers between BMV RNA3 components within an internal region. A 170-nt polynucleotide derived from the cowpea chlorotic mottle virus (CCMV) RNA3 was inserted just upstream of the initiation codon of the BMV coat protein open reading frame in either sense or antisense orientations. The resulting respective mutants, BCC+ and BCC-, maintained unchanged CCMV inserts when inoculated separately on leaves of a local lesion host for BMV. In contrast, when a mixture containing both mutated RNAs3 was inoculated, a significant fraction of lesions accumulated the BMV RNA3 lacking the CCMV insert. The presence of a 3' marker mutation confirmed that the BMV RNA3 progeny arose due to crossovers between BCC+ and BCC- within the complementary sequences. The highest frequency of recombinant appearance was observed when the RNA mixtures were annealed prior to inoculation on the host plants. Our results confirm a concept predicting the general nature of the heteroduplex-mediated recombination functioning in RNA viruses. Examples of possible applications of this approach in recombinant RNA technology are discussed.     

Rapid and efficient purification of Cowpea chlorotic mottle virus by sucrose cushion ultracentrifugation

A simple, economical and efficient method for isolation of Cowpea chlorotic mottle virus (CCMV) particles was developed. Abundant viral particles could be obtained with three steps of 10 min duration each of conventional differential centrifugations and one sucrose cushion ultracentrifugation step of 2 h. Characterization of purified virus assessed by transmission electron microscopy, polyacrylamide gel electrophoresis (PAGE) and Western blot revealed the typical isometric particles of CCMV without any visible contamination or degradation. RNA extracted from purified CCMV particles showed all four RNA components by agarose gel electrophoresis. Both purified virus particles and RNA were biologically active and initiated successful infection upon inoculation to cowpea.