Detection, discrimination and discovery of a new Tobacco streak virus strain.

Soybean plants that exhibited symptoms of virus infection were sampled from different counties of Oklahoma. These plants were tested serologically for 15 viruses known to infect soybean plants. Fifty-seven samples that exhibited typical virus-like symptoms did not test positive for any of the 15 viruses used in a dot-immunobinding assay (DIBA). Four samples were pooled and used for next generation sequencing using the 454-Roche protocol. Sequence and phylogenetic analysis of the sequences obtained revealed infection with a distinct strain of Tobacco streak virus (TSV). TSV was one of the 15 viruses initially tested for using DIBA and had tested negative. TSV belongs to the genus Ilarvirus and has been reported as a causal agent of bud blight in soybean crops in Brazil and the United States. Out of 10 reported primer pairs for TSV reverse transcription-polymerase chain reaction (RT-PCR), only two had the potential, based on sequence similarity, to amplify part of the genome of the distinct strain of TSV found in Oklahoma and only one was actually able to amplify the region. In this study, a new primer pair, specific to all known TSV and capable of amplifying the Oklahoma strain (TSV-OK), was designed from a highly conserved region of coat protein (CP) sequences and end-point PCR and quantitative RT-PCR detection methods were developed and their sensitivity assayed. This is the first report of specific primers designed from this highly conserved region in the CP of TSV for detection of TSV. Twenty-three of the 57 DIBA soybean samples that initially tested negative were retested with the new specific end-point PCR method and found positive for TSV infection.

The '30K' superfamily of viral movement proteins.

Relationships among the amino acid sequences of viral movement proteins related to the 30 kDa ('30K') movement protein of tobacco mosaic virus - the 30K superfamily - were explored. Sequences were grouped into 18 families. A comparison of secondary structure predictions for each family revealed a common predicted core structure flanked by variable N- and C-terminal domains. The core consisted of a series of beta-elements flanked by an alpha-helix on each end. Consensus sequences for each of the families were generated and aligned with one another. From this alignment an overall secondary structure prediction was generated and a consensus sequence that can recognize each family in database searches was obtained. The analysis led to criteria that were used to evaluate other virus-encoded proteins for possible membership of the 30K superfamily. A rhabdoviral and a tenuiviral protein were identified as 30K superfamily members, as were plant-encoded phloem proteins. Parsimony analysis grouped tubule-forming movement proteins separate from others. Establishment of the alignment of residues of diverse families facilitates comparison of mutagenesis experiments done on different movement proteins and should serve as a guide for further such experiments.

Limitations to tobacco mosaic virus infection of turnip.

Turnip vein-clearing virus (TVCV) and tobacco mosaic virus (TMV) represent subgroups of tobamoviruses infecting cruciferous and solanaceous plants, respectively. To identify adaptations that may have been necessary in the evolution of the TVCV subgroup from a TMV-like ancestor, the infection of turnip plants by TMV and by chimeras between TMV and TVCV was explored. TMV accumulated at spatially limited sites on inoculated turnip leaves as determined by leaf skeleton hybridization. A plasmid DNA containing a complete TVCV cDNA, when transcribed in vitro, produced RNA that was infectious to tobacco and turnip plants. TVCV-TMV chimeric genomes with junctions within coding regions were not infectious to tobacco, though the movement protein (MP) chimera was infectious to tobacco with a TMV MP transgene. Reciprocal chimeras with junctions between genes were infectious to tobacco. TVCV with a TMV MP gene infected turnips. The other tested chimeras were not detected in non-inoculated leaves, but were found in the inoculated leaves. Thus, the TMV MP is not responsible for the limitation of TMV spread in turnips.

Completion of a cDNA sequence from a tobamovirus pathogenic to crucifers.

Turnip vein-clearing virus (TVCV) is a tobamovirus related to ribgrass mosaic virus. We report the nucleotide (nt) sequences of the 5'-untranslated region (UTR) and the 3'-half of the TVCV genome (the 3' region of the 182-kDa protein-encoding gene, as well as the movement protein and coat protein genes and the 3'-UTR). The determination completes the nt sequence of the cDNA of TVCV.

Resistance of Spiroplasma citri Lines to the Virus SVTS2 Is Associated with Integration of Viral DNA Sequences into Host Chromosomal and Extrachromosomal DNA.

Spiroplasmavirus SVTS2, isolated from Spiroplasma melliferum TS2, produces plaques when inoculated onto lawns of Spiroplasma citri M200H, a derivative of the type strain Maroc R8A2. S. citri strains MR2 and MR3, originally selected as colonies growing within plaques on a lawn of M200H inoculated with SVTS2, were resistant to SVTS2. Genomic DNA fingerprints and electrophoretic protein profiles of M200H, MR2, and MR3 were similar, but three proteins present in M200H were missing or significantly reduced in both resistant lines. None of these three polypeptides reacted with antiserum against S. citri membrane proteins, indicating that they probably are not surface-located virus receptors. Electroporation with SVTS2 DNA produced 1.5 x 10(sup5) transfectants per (mu)g of DNA in M200H but none in MR2 or MR3, suggesting that resistance may result from inhibition of viral replication. The digestion patterns of the extrachromosomal double-stranded (ds) DNA of these lines were similar. Three TaqI fragments of MR2 extrachromosomal DNA that were not present in M200H extrachromosomal DNA hybridized strongly to an SVTS2 probe, and two of these fragments plus an additional one hybridized with the MR3 extrachromosomal DNA, indicating that a fragment of SVTS2 DNA was present in the extrachromosomal ds DNA of MR2 and MR3 but not of M200H. When the restricted genomes of all three lines were probed with SVTS2 DNA, strong hybridization to two EcoRI fragments of chromosomal MR2 and MR3 DNA but not M200H DNA indicated that SVTS2 DNA had integrated into the genomes of MR2 and MR3 but not of M200H. When MR3 extrachromosomal ds DNA containing a 2.1-kb SVTS2 DNA fragment was transfected into M200H, the transformed spiroplasmas were resistant to SVTS2. These results suggest that SVTS2 DNA fragments, possibly integrated into the chromosomal or extrachromosomal DNA of a previously susceptible spiroplasma, may function as viral incompatibility elements, providing resistance to superinfection by SVTS2.

Electron microscopic and molecular characterization of turnip vein-clearing virus.

We recently isolated turnip vein-clearing virus (TVCV), a tobamovirus which causes vein clearing in Brassica rapa (turnip) and a mosaic in Nicotiana tabacum (tobacco). We present an electron microscopic and molecular characterization of TVCV. Viral particles from lower epidermis peel contained rod-shaped viral particles, typical of tobamoviruses. Viral RNA extracted from infected turnip leaves was used as template for cDNA synthesis prior to cloning in a plasmid vector. Inserts of selected cDNA clones were sequenced to obtain the nucleotide sequence of the 126 K replicase component. The nucleotide and predicted amino acid sequences were 56 to 59% identical to those of most other sequenced tobamoviruses. The least related sequence, that of cucumber green mottle mosaic virus, was more related to the TVCV lineage than it was to those of the other sequenced tobamoviruses. UV spectroscopy suggested a tryptophan content characteristic of the ribgrass mosaic virus (RMV) group. Fragmentation of the TVCV coat protein by cyanogen bromide treatment produced a profile of fragments indistinguishable from those generated from the coat protein of RMV. Thus, while symptoms of TVCV infection on Nicotiana tabacum cv. Samsun and Nicotiana clevelandii differ from those reported for RMV, TVCV appears to be closely related to RMV.

HIV-1 proteinase as structural model of intercellular transport proteins of plant viruses.

Intracellular movement of viral infections of plants requires a virus-encoded protein. Alignment of amino acid sequences of central conserved regions of such proteins produced a sequence profile that resembled that of lentiviral proteinases. The known three-dimensional structure of the proteinase encoded by the human immunodeficiency virus-1 (HIV-1) may serve as a model for the three-dimensional structure of the central region of the plant viral proteins. Secondary structures predicted for the plant viral proteins from their amino acid sequences correlate well with those predicted from a proteinase model. In addition, the positions of temperature-sensitive and resistance-breaking mutations in the intercellular transport protein of tobacco mosaic virus are consistent with a structural similarity between the plant viral proteins and the lentiviral proteinases. In a suggested model, the dimeric proteinase-similar domain serves as tether for the attachment of N- and C-terminal domains. The C-terminal domain may be an RNA-binding domain. The similarity was used to assign intercellular transport function to a previously unidentified coding region of the genomes of bacilliform DNA viruses.

A method for the isolation of nuclear DNA from cotton (Gossypium) leaves.

Cotton leaves contain high levels of polyphenolic compounds that irreversibly interact with proteins and nucleic acids during DNA isolation. A procedure to isolate nuclear DNA from cotton (Gossypium hirsutum L.) has been developed. The method is based on the rapid initial isolation of nuclei in a glucose medium designed to stabilize nuclear structure and composition while preventing covalent interactions with polyphenolics. The resulting DNA is high in yield and purity and is suitable for Southern-blot hybridization analysis and restriction fragment length polymorphism analysis.

Similarities between putative transport proteins of plant viruses.

The nucleic acids of many plant viruses encode proteins with one or more of the following properties: an Mr of approximately 30,000, localization in the cell wall of the infected plant and a demonstrated role in cell-to-cell transport of infection. A progressive alignment strategy, aligning first those sequences known to be similar, and then aligning the resulting groups of sequences, was used to examine further the relatedness of the amino acid sequences of putative transport proteins of caulimoviruses, of proteins similar to the putative transport protein of alfalfa mosaic virus (A1MV) and of those similar to the tobacco mosaic virus (TMV) 30K protein. The strategy first identified regions in which multiple dipeptides of one group were similar to those of another group. The regions of similarity were brought into alignment by the conservative introduction of gaps. The positions of the introduction of gaps were adjusted to optimize similarity. Statistical significances of the resulting alignments, determined both by comparison with shuffled amino acid sequences and with the sequence alignment off-set by 1 to 15 residues in each direction, suggest that the amino acid sequences of the three groups of viruses are distantly related. Nevertheless, significant relationships between members of the caulimoviral group of sequences and members of each of the A1MV-like and TMV-like groups were found. These relationships and the analysis of the number of insertions/deletions between present sequences and a hypothetical common ancestor suggest that the sequences of the caulimoviral proteins are less diverged from the ancestor than either the A1MV-like or TMV-like proteins. The alignment identified common regions of predicted secondary structure and regions of similar hydropathy, regions possibly crucial for proper functioning of the proteins.

A readable and space-efficient DNA sequence representation: application to caulimoviral DNAs.

A new representation of nucleotide sequence information has been devised to allow the clear presentation of large amounts of sequence information in a limited amount of space. Nucleotide sequences of DNA are represented by squares in three vertically aligned positions for each nucleotide. The squares represent, from top to bottom: a purine, a guanine or cytosine and a pyrimidine. The representation provides a simultaneous representation of both strands of double-stranded DNA and can be easily scanned visually for a variety of sequence features. Biologically important features of the DNA sequence near the origin of SV40 replication are easily recognized. The representation was also used to identify possibly base-paired stems in caulimoviral transcripts that may be of significance for viral replication.

Infectious and non-infectious mutants of cauliflower mosaic virus DNA.

Mutants of cauliflower mosaic virus (CaMV), generated in vitro by modification of recombinant DNA plasmids containing the viral genome, either retained the ability to induce disease symptoms on turnip plants, produced less severe symptoms or failed to induce symptoms. Wild-type symptoms were produced by a variant CaMV DNA of the Cabbage S isolate that had 4 bp in open reading frame (ORF) III replaced with a 16 bp sequence. Less severe symptoms, due to a delay in symptom appearance relative to inoculation with wild-type DNA, were induced by a mutant with a frameshift mutation in ORF II (pSA103). CaMV DNA, recovered from plants infected with pSA103, contained a second mutation which restored the original translation reading frame. Nucleic acid hybridization to 'squishes' of leaf tissue from plants that had been inoculated with mutant DNAs that included DNAs modified in each of the six major ORFs of CaMV DNA revealed that only those plants that appeared diseased had detectable CaMV nucleic acid in uninoculated leaves. Replicated CaMV DNA was also not detected in non-encapsidated and virion DNA fractions from inoculated leaves of non-diseased plants.

Helper component for aphid transmission encoded by region II of cauliflower mosaic virus DNA.

A deletion mutant of cauliflower mosaic virus (CaMV) isolate NY8153 deficient in aphid transmissibility was constructed by BAL-31 exonuclease treatment of XhoI linearized pCMS31 (a plasmid containing the entire CaMV genome cloned in the SalI site of pBR322), followed by ligation. The resulting mutant, pSA103, lacked about 100 by from the putative protein-coding region II of CaMV DNA. In turnips there was no difference in the number and appearance of starch lesions or hybridization lesions, or in the nature of symptoms in systemically infected leaves induced by pSA103 DNA, pCMS31 DNA, and NY8153 DNA. Systemic symptoms appeared later in plants infected with pSA103 (27 +/- 2 days) than in those infected with the parental pCMS31 DNA (19 +/- 2 days). Aphids fed on virus SA103-infected mustard plants were unable to transmit the virus to healthy plants while 30-70% transmission was observed from plants infected by the parent virus. Virions extracted from turnips infected with the mutant had DNA still containing the deletion. In addition, the single-stranded discontinuity in region II of NY8153 DNA was missing in this DNA. The results suggest that region II codes for a "helper component" required for aphid transmission of CaMV.

Polypeptides associated with inclusion bodies from leaves of turnip infected with cauliflower mosaic virus.

Inclusion bodies, the major intracellular site of accumulation of cauliflower mosaic virus (CaMV), have been partially purified by centrifugation onto a saturated sucrose cushion, followed by detergent treatment to lyse chloroplasts, then centrifugation on a saturated sucrose cushion again. The resulting preparation was enriched for inclusion bodies as judged by light and electron microscopic observation and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the polypeptides in the preparation. Two major polypeptides of 61,000 and 43,000 apparent molecular weights were found in inclusion body preparations from leaves infected separately with each of three different isolates of CaMV. Polypeptides of apparent molecular weights 56,000, 50,000, 39,000, 37,000, and 34,000 were also detected in preparations from infected leaves, but not in comparable preparations from healthy leaves. The mobilities of all but the 61,000 and 34,000 molecular weight polypeptides were slightly different from the CM4-184 isolate, as compared with Cabbage B Davis or NY8153. The 61,000 molecular weight polypeptide did not correspond in mobility to any of the virion polypeptides. The relative intensities of polypeptide bands common to inclusion bodies and isolated virions were different in these two preparations, suggesting that considerable degradation took place during virion isolation.

Density comparisons of heavy chains of membrane and secreted immunoglobulins of mouse.

In order to explore structural differences between membrane and secreted immunoglobulins the buoyant densities of mouse immunoglobulin (Ig) heavy (H) chains were compared by isopycnic centrifugation in CsCl containing guanidine hydrochloride. The buoyant densities, under denaturing conditions, of mouse myeloma protein MOPC 21 IgG, MOPC 315 IgA and MOPC 104E IgM H chains were consistent with their carbohydrate contents. Mouse membrane IgM and MOPC 104E-secreted IgM H chains were of equal density. The buoyant densities of MOPC 104E-secreted IgM and spleen-cell-secreted IgM H chains were indistinguishable. The IgD-like membrane H chain was denser than membrane IgM H chain, and its carbohydrate content was calculated to be 15.5%. The resolution of the technique was sufficient to conclude that the apparent 1500 mol.wt. difference, as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, between membrane and secreted IgM H chains was due to peptide rather than to carbohydrate. The results also imply that intact membrane IgM and IgD bind detergent and are thus integral membrane proteins.

In Vitro Synthesis of a Precursor to the Methionine-rich Polypeptide of the Zein Fraction of Corn.

The messenger ribonucleic acid fraction isolated from a protein bodyenriched fraction of developing corn (Zea mays L.) endosperm stimulated the incorporation of radioactive amino acids into at least five polypeptides when added to a wheat germ extract capable of protein synthesis. Of these, the two major polypeptides formed with messenger from freshly frozen corn were identified as precursors to zein A and B, the two major polypeptides of the prolamine fraction of corn meal (21,600 and 19,600 molecular weight). The identification was based on the relative incorporations of radioactive leucine, lysine, and methionine, and the susceptibility of the zein A precursor, but not the zein B precursor to cleavage with cyanogen bromide. Using extracts from stored frozen corn, a third polypeptide of 14,500 molecular weight was identified as a major in vitro product. It was preferentially labeled with methionine and slightly larger than a similar peptide in the zein fraction of corn meal. Two other polypeptides of still lower molecular weight could be detected above the background of probably incomplete polypeptides.

Cell surface immunoglobulin. XVI. Polypeptide chain structure of mouse IgM and IgD-like molecule.

125I-membrane IgM, 125I-membrane IgD-like molecules, and their constituent chains from iodinated murine splenocytes were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The apparent m.w. of the heavy chains decreased as the acrylamide concentration was raised. The membrane mu-chain had a slower mobility than did mu-chain from secreted IgM. Unreduced IgM and IgD-like molecules had mobilities consistent with an H2L2 structure. Intact IgD-like molecules were replaced after overnight dialysis by molecules with the properties of HL. Unreduced surface IgM had a slower mobility than that of monomeric IgM obtained by partial reduction of secreted IgM.

Cell surface immunoglobulin. XI. The appearance of an IgD-like molecule on murine lymphoid cells during ontogeny.

An Ig molecule containing L chains and H chains similar to human delta-chains has been detected on the surface of radioiodinated murine lymphoid cells. Newborn mice have only IgM on their splenocytes. Between 10 and 15 days, the IgD-like molecule appears and increases in amount until 3 mo of age, when it is the predominant cell surface Ig in terms of radioactivity. IgD is found only in peripheral lymphoid tissues and is present in larger amounts on peripheral lymph node cells (approximately 85% of surface Ig) than on splenocytes (approximately 50%). IgD is also present in comparable amounts on cells from both nu/nu and germfree mice, indicating that its expression may be independent of both thymic influence and antigenic stimulation. These studies suggest that there is a switch from cell surface IgM to IgD that occurs during differentiation of virgin B lymphocytes in the spleen.