Michael Shanks

Analysis of the nucleotide sequence of bean pod mottle virus middle component RNA.

The complete nucleotide sequence of the middle component RNA (M RNA) of the comovirus bean pod mottle virus (BPMV) has been determined. The sequence consists of 3662 nucleotides and contains a single long open reading frame sufficient to code for a protein of 113,353 Da. The proteolytic processing sites within this protein have been identified by comparison with the known three-dimensional structure of the virion and cleavage at these sites would lead to a range of products consistent with those observed during processing of the M RNA-encoded polyproteins in vitro. We have performed computer-aided searches for reiterated sequences within BPMV M RNA which might explain why ordered RNA is visible in the electron density map of BPMV middle component particles (Chen, Z., Stauffacher, C. V., Li, Y., Schmidt, T., Bomu, W., Kamer, G., Shanks, M., Lomonossoff, G., and Johnson, J. E., 1989, Science 245, 154-159). These searches revealed both the presence of overrepresented pentameric sequences and a consensus sequence which was repeated 15 times within the RNA sequence.

Structural Fingerprinting: Subgrouping of Comoviruses by Structural Studies of Red Clover Mottle Virus to 2.4-Å Resolution and Comparisons with Other Comoviruses

ABSTRACT Red clover mottle virus (RCMV) is a member of the comoviruses, a group of picornavirus-like plant viruses. The X-ray structure of RCMV strain S has been determined and refined to 2.4 Å. The overall structure of RCMV is similar to that of two other comoviruses, Cowpea mosaic virus (CPMV) and Bean pod mottle virus (BPMV). The sequence of the coat proteins of RCMV strain O were modeled into the capsid structure of strain S without causing any distortion, confirming the close resemblance between the two strains. By comparing the RCMV structure with that of other comoviruses, a structural fingerprint at the N terminus of the small subunit was identified which allowed subgrouping of comoviruses into CPMV-like and BPMV-like viruses.

Co-expression of the capsid proteins of cowpea mosaic virus in insect cells leads to the formation of virus-like particles.

The regions of RNA-2 of Cowpea mosaic virus (CPMV) that encode the Large (L) and Small (S) coat proteins were expressed either individually or together in Spodoptera frugiperda (sf21) cells using baculovirus vectors. Co-expression of the two coat proteins from separate promoters in the same construct resulted in the formation of virus-like particles whose morphology closely resembled that of native CPMV virions. No such particles were formed when the individual L and S proteins were expressed. Sucrose gradient centrifugation of the virus-like particles showed that they had the sedimentation characteristics of empty (protein-only) shells. The results confirm that the 60 kDa L-S fusion is not an obligate intermediate in the virion assembly pathway and indicate that expression of the coat proteins in insect cells will provide a fruitful route for the study of CPMV morphogenesis.

Cowpea mosaic virus VPg: sequencing of radiochemically modified protein allows mapping of the gene on B RNA

A partial amino acid sequence of cowpea mosaic virus (CPMV) VPg radiochemically modified by chloramine‐T and Bolton‐Hunter reagent has been determined. VPg covalently bound to viral RNA chains (VPg‐RNA) was iodinated with chloramine‐T and Bolton‐Hunter reagent to label tyrosine and lysine residues, respectively. [125I]VPg‐RNA was digested with nuclease P1 and the resulting [125I]VPg‐pU was purified by SDS‐polyacrylamide gel electrophoresis and subjected to automated Edman degradation. Control experiments with chemically synthesized poliovirus VPg showed the feasibility of radiochemical microsequence analysis of protein that had been radiochemically modified by chloramine‐T and Bolton‐Hunter reagent. Analysis of CPMV [125I]VPg‐pU revealed the presence of tyrosine residues at position 12 and 14, and of lysine residues at position 3 and 20, respectively. In combination with Edman degradation of unlabeled CPMV VPg, which showed serine and arginine residues to be present at position 1 and 2, respectively, the data obtained allow the precise positioning of VPg within the 200 000 dalton (200 K) polyprotein encoded by CPMV B RNA and the prediction of its entire amino acid sequence. VPg is located at the COOH terminus of its 60 K, membrane‐bound,precursor and proximal to the amino terminus of the protease‐polymerase domain of the polyprotein. A processing scheme for the 200 K polyprotein is discussed in which Gln‐Ser amino acid pairs act as the major signal for proteolytic cleavage.

The nucleotide sequence of red clover mottle virus bottom component RNA

The complete nucleotide sequence of the bottom component RNA (B RNA) of red clover mottle virus strain S has been determined. The sequence consists of 6033 nucleotides and contains a single long open reading frame sufficient to encode a protein of M(r) 210,258. The proteolytic processing sites within this protein have been deduced by comparison of its sequence with that of the B RNA-encoded protein of cowpea mosaic virus. Comparison of the amino acid sequences of the individual proteins confirms that the two viruses have a similar genome organization.

Epitope tagging of legume root nodule extensin modifies protein structure and crosslinking in cell walls of transformed tobacco leaves

Root nodule extensins (RNEs) are highly glycosylated plant glycoproteins localized in the extracellular matrix of legume tissues and in the lumen of Rhizobium-induced infection threads. In pea and other legumes, a family of genes encode glycoproteins of different overall length but with the same basic composition. The predicted polypeptide sequence reveals repeating and alternating motifs characteristic of extensins and arabinogalactan proteins. In order to monitor the behavior of individual RNE gene products in the plant extracellular matrix, the coding sequence of PsRNE1 from Pisum sativum was expressed in insect cells and in tobacco leaves. RNE products extracted from tobacco tissues were of high molecular weight (in excess of 80 kDa), indicating extensive glycosylation similar to that in pea tissues. Epitope-tagged derivatives of PsRNE1 could be localized in cell walls. However, the introduction of epitope tags at the C-terminus of RNE altered the behavior of RNE in the extracellular matrix, apparently preventing intermolecular crosslinking of RNE molecules and their covalent association with other cell wall components. These observations are discussed in the light of a computational model for the RNE glycoprotein that is consistent with an extended rod-like structure. It is proposed that RNE can undergo three classes of tyrosine-based crosslinking. Intramolecular crosslinking of vicinal Tyr residues is rod stiffening, end-to-end linkage is rod lengthening, and side-to-side intermolecular crosslinking is rod bundling. The control of these interconversions could have important implications for the biomechanics of infection thread growth.

Characterisation of a maize-infecting potyvirus from Spain

In order to characterise and classify an unknown maize-infecting potyvirus isolated from fields in northeast Spain, the entire coat protein gene and the C-terminal twothirds of the large nuclear inclusion protein (NIb) gene were cloned and sequenced. Protein sequencing enabled the cleavage site between the two proteins to be deduced and also revealed that on storage the viral coat protein undergoes a specific degradation in which the N-terminal 39 amino acids are removed. Comparison of the nucleotide sequence of the 3′ non-coding region of the viral RNA and the predicted amino acid sequence of the coat protein with the equivalent regions of other members of the potyvirus group revealed that the Spanish virus is closely related to maize dwarf mosaic virus strain A.

Red clover mottle virus from Ukraine is an isolate of RCMV strain S

The relationship between red clover mottle virus (RCMV) isolated in the Ukraine (designated RCMV-Uk) and well-characterised strains from Sweden has been investigated. Nucleic acid hybridisation indicate that both RNAs from RCMV-Uk are highly homologous to their counterparts from RCMV strain S, a conclusion supported by protein sequence analysis of the two viral capsid proteins. Nucleic acid sequence analysis of a portion of RCMV-Uk RNA2 confirmed the high degree of similarity between RCMV-Uk and RCMV strain S. This information suggests that RCMV-Uk should be considered an isolate of RCMV strain S.