Keith H. Gough

Expression of potyvirus coat protein in Escherichia coli and yeast and its assembly into virus-like particles.

When the full-length coat protein (CP) of the potyvirus, Johnsongrass mosaic virus (JGMV), was expressed in Escherichia coli or yeast, it assembled to form potyvirus-like particles. The particles were heterogeneous in length with a stacked-ring appearance and resembled JGMV particles in their flexuous morphology and width. This cell-free assembly system should permit analysis of the mechanisms of particle assembly and genome encapsidation. Two mutant forms of CP produced by site-directed mutagenesis failed to assemble into virus-like particles.

Coat protein of potyviruses. 2. Amino acid sequence of the coat protein of potato virus Y.

The amino acid sequence of the coat protein of potato virus Y (PVY), the type member of the potyvirus group, has been determined by protein sequencing techniques. The protein contains 267 amino acid residues with a calculated mol wt of 29,945. A comparison of the PVY coat protein sequence with those of tobacco etch virus (TEV) and pepper mottle virus (PeMV) predicted from nucleotide sequence data (R. F. Allison, J. G. Sorenson, M. E. Kelly, F. B. Armstrong, and W. G. Dougherty, Proc. Natl. Acad. Sci. USA82, 3969-3972, 1985; W. G. Dougherty, R. F. Allison, T. D. Parks, R. E. Johnston, M. J. Feild, and F. B. Armstrong, Virology 146,282-291, 1985) shows that sequence homology between the coat proteins from PVY and PeMV is 92% and that between PVY and TEV is 62%. These data suggest that PVY and PeMV are much more closely related than previously believed from serological studies.

The Use of Protein A, from Staphylococcus aureus, in Immune Electron Microscopy for Detecting Plant Virus Particles

Summary An immune electron microscopic technique for detecting plant viruses is described which involves pre-coating electron microscope grids with protein A (a wall protein of Staphylococcus aureus) before coating them with specific anti-serum. The method trapped 339 and 51 times more sugarcane mosaic virus and tobacco mosaic virus particles, respectively, than untreated grids. It appears particularly suitable for virus particles occurring in plant extracts in small numbers.

Systematic Mapping of Potential Binding Sites for Shc and Grb2 SH2 Domains on Insulin Receptor Substrate-1 and the Receptors for Insulin, Epidermal Growth Factor, Platelet-derived Growth Factor, and Fibroblast Growth Factor

Multipin peptide synthesis has been employed to produce biotinylated 11-mer phosphopeptides that account for every tyrosine residue in insulin receptor substrate-1 (IRS-1) and the cytoplasmic domains of the insulin-, epidermal growth factor-, platelet-derived growth factor- and basic fibroblast growth factor receptors. These phosphopeptides have been screened for their capacity to bind to the SH2 domains of Shc and Grb in a solution phase enzyme-linked immunosorbent assay. The data revealed new potential Grb2 binding sites at Tyr-1114 (epidermal growth factor receptor (EGFR) C-tail); Tyr-743 (platelet-derived growth factor receptor (PDGFR) insert region), Tyr-1110 from the E-helix of the catalytic domain of insulin receptor (IR), and Tyr-47, Tyr-939, and Tyr-727 in IRS-1. None of the phosphopeptides from the juxtamembrane or C-tail regions of IR bound Grb2 significantly, and only one phosphopeptide from the basic fibroblast growth factor receptor (Tyr-556) bound Grb2 but with medium strength. Tyr-1068 and −1086 from the C-tail of EGFR, Tyr-684 from the kinase insert region of PDGFR, and Tyr-895 from IRS-1 were confirmed as major binding sites for the Grb2 SH2 domain. With regard to Shc binding, the data revealed new potential binding sites at Tyr-703 and Tyr-789 from the catalytic domain of EGFR and at Tyr-557 in the juxtamembrane region of PDGFR. It also identified new potential Shc binding sites at Tyr-764, in the C-tail of basic fibroblast growth factor receptor, and Tyr-960, in the juxtamembrane of IR, a residue previously known to be required for Shc phosphorylation in response to insulin. The study confirmed the previous identification of Tyr-992 and Tyr-1173 in the C-tail of EGFR and several phosphopeptides from the PDGFR as medium strength binding sites for the SH2 domain of Shc. None of the 34 phosphopeptides from IRS-1 bound Shc strongly, although Tyr-690 showed medium strength binding. The specificity characteristics of the SH2 domains of Grb2 and Shc are discussed. This systematic peptide mapping strategy provides a way of rapidly scanning candidate proteins for potential SH2 binding sites as a first step to establishing their involvement in kinase-mediated signaling pathways.

Nucleotide Sequence of the Capsid and Nuclear Inclusion Protein Genes from the Johnson Grass Strain of Sugarcane Mosaic Virus RNA

Summary The nucleotide sequence of the 3′ terminal 1782 nucleotides of sugarcane mosaic virus (SCMV) genome has been determined. There is an open reading frame, from the 5′ end, of 1307 nucleotides upstream from a 475 nucleotide 3′ non-coding region that is polyadenylated. The open reading frame encodes a polypeptide of 435 amino acids. The segment of the genome encoding the viral capsid protein (mol. wt. 34200) is adjacent to the 3′ non-coding region. The predicted capsid protein is similar in sequence to the capsid protein sequence predicted for tobacco etch virus (TEV). Part of another protein encoded in the same reading frame, similar to the predicted nuclear inclusion protein from TEV, has been identified upstream from the coat protein gene. The results indicate that the genome of SCMV encodes one or more large proteins that are processed to the mature proteins.

Amino acid sequences of alpha-helical segments from S-carboxymethylkerateine-A. Complete sequence of a type-II segment.

1. The helical fragments obtained by partial chymotryptic digestion of S-carboxymethylkeratine-A, the low-sulphur fraction from wool, were fractionated into type-I and type-II helical segments in aqueous urea under conditions limiting carbamoylation. 2. The amino acid sequence of a 109-residue type-II segment was completed by using the sequenator. 3. When the data were incorporated into a helical model of 3.6 residues per turn the hydrophobic residues generated a band aligned at a slight angle to the helical axis. This result is in accord with the postulated coiled-coil structure of the crystalline regions of alpha-keratin.

Coat protein of potyviruses. 3. Comparison of amino acid sequences of the coat proteins of four Australian strains of sugarcane mosaic virus.

SummaryThe amino acid sequence of the coat protein of the Johnsongrass (JG) strain of sugarcane mosaic virus (SCMV) has been determined by protein sequencing techniques. The protein contains 303 amino acid residues corresponding to a molecular weight of 33,510 and when compared to the coat proteins of other potyviruses that have been characterized (263–267 residues) is found to have additional residues at its N-terminus. The N-terminus is acetylated as shown by fast atom bombardment mass spectrometry. Partial amino acid sequences of the coat proteins of the other three Australian SCMV strains, sugarcane (SC), Queensland blue couch grass (BC) and sabi grass (Sabi) have also been obtained. The sequence data and the comparative tryptic peptide HPLC profiles showed that the JG coat protein was substantially different from those of the other three SCMV strains, the sequence homology being around 66 per cent. This is in marked contrast to the high sequence homology between SC, BC and Sabi strains (95–100 per cent) but similar to that (51–62 per cent) found between coat proteins of distinct members of the potyvirus group. On the basis of these structural findings and other information on major differences in serological, biological and biochemical properties we believe that the present JG strain should not be considered a strain of SCMV but should be regarded as an independent member of the potyvirus group. The name “Johnsongrass mosaic virus” is proposed for this new member.

Bean yellow mosaic, clover yellow vein, and pea mosaic are distinct potyviruses: evidence from coat protein gene sequences and molecular hybridization involving the 3′ non-coding regions

SummaryThe sequences of the 3′ 1019 nucleotides of the genome of an atypical strain of bean yellow mosaic virus (BYMV-S) and of the 3′ 1018 nucleotides of the clover yellow vein virus (CYVV-B) genome have been determined. These sequences contain the complete coding region of the viral coat protein followed by a 3′ non-coding region of 173 and 178 nucleotides for BYMV-S and CYVV-B, respectively. When the deduced amino acid sequences of the coat protein coding regions were compared, a sequence identity of 77% was found between the two viruses, and optimal alignment of the 3′ untranslated regions of BYMV-S and CYVV-B gave a 65% identity. However, the degree of homology of the amino acid sequences of coat proteins of BYMV-S with the published sequences for three other strains of BYMV ranged from 88% to 94%, while the sequence homology of the 3′ untranslated regions between the four strains of BYMV ranged between 86% and 95%. Amplified DNA probes corresponding to the 3′ non-coding regions of BYMV-S and CYVV-B showed strong hybridization only with the strains of their respective viruses and not with strains of other potyviruses, including pea mosaic virus (PMV). The relatively low sequence identities between the BYMV-S and CYVV-B coat proteins and their 3′ non-coding regions, together with the hybridization results, indicate that BYMV, CYVV, and PMV are distinct potyviruses.

Differentiation of potyviruses and their strains by high performance liquid chromatographic peptide profiling of coat proteins.

Summary Comparison by HPLC of tryptic digests of coat proteins from six biologically and serologically distinct potyviruses, namely bean yellow mosaic virus, Johnson grass mosaic virus, passion-fruit woodiness virus (PWV), potato virus Y (PVY), sugarcane mosaic virus (SCMV) and water-melon mosaic virus II, demonstrated that each potyvirus can be distinguished from the others. HPLC of tryptic peptides from coat proteins of four strains of PVY, two strains of PWV and three strains of SCMV showed that peptide patterns of strains from the same potyvirus were very similar. These findings were supported by amino-terminal amino acid sequence analysis of the peptides. The use of enzymes from different sources and variation in the temperature (35 °C to 40 °C) and time (16 to 20 h) of digestion caused small variations in the profiles but did not change the main features of the peptide patterns of each potyvirus. The results suggest that HPLC profiles of tryptic digests of the coat proteins of potyviruses could be useful criteria for the identification and classification of potyviruses.

Coat protein of potyviruses. I. Comparison of the four Australian strains of sugarcane mosaic virus

As an aid to a more rational classification of the potyvirus group, we have examined the molecular weight, amino acid composition, and tryptic peptide map of the coat protein of four Australian sugarcane mosaic virus strains: Johnson grass (JG), sugarcane (SC), Queensland blue couch grass (BC), and sabi grass (Sabi). The proteins migrated as a single band in SDS-polyacrylamide gel electrophoresis with molecular weights of 33,700 (SC), 34,200 (JG), 39,100 (BC), and 40,300 (Sabi). The BC and Sabi strains have identical amino acid compositions and tryptic peptide maps. The SC strain has a similar amino acid composition and tryptic peptide map but these two properties of the JG strain show very little similarity to the three other strains. On the basis of the coat protein properties investigated, we propose that the four sugarcane mosaic virus strains be subdivided into two groups: the sugarcane group containing SC, BC, and the Sabi strains and the Johnson grass group containing the JG strain.