Limited proteolysis of potato virus X by trypsin and plant proteases

Tremaine, J. H.; Agrawal, Hari Om

doi:10.1016/0042-6822(72)90530-2

Cited by 26 publications

(10 citation statements)

References 13 publications

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“…Other data [2,17] support our suggestion of critical changes in PVX virions on cleavage (or changing) of the CP N‐terminal peptide. In their 1972 electron microscopy study, Tremaine & Agrawal [17] observed unusual twisting of trypsin‐treated PVX particles. In 1992 Chapman et al .…”

Section: Discussionsupporting

confidence: 80%

N‐Terminal segment of potato virus X coat protein subunits is glycosylated and mediates formation of a bound water shell on the virion surface

Baratova

Fedorova

Добров

et al. 2004

European Journal of Biochemistry

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The primary structures of N-terminal 19-mer peptides, released by limited trypsin treatment of coat protein (CP) subunits in intact virions of three potato virus X (PVX) isolates, were analyzed. Two wild-type PVX strains, Russian (Ru) and British (UK3), were used and also the ST mutant of UK3 in which all 12 serine and threonine residues in the CP N-terminal segment were replaced by glycine or alanine. With the help of direct carbohydrate analysis and MS, it was found that the acetylated N-terminal peptides of both wildtype strains are glycosylated by a single monosaccharide residue (galactose or fucose) at NAcSer in the first position of the CP sequence, whereas the acetylated N-terminal segment of the ST mutant CP is unglycosylated. Fourier transform infrared spectra in the 1000-4000 cm )1 region were measured for films of the intact and in situ trypsindegraded PVX preparations at low and high humidity. These spectra revealed the presence of a broad-band in the region of valent vibrations of OH bonds (3100-3700 cm )1 ), which can be represented by superposition of three bands corresponding to tightly bound, weakly bound, and free OH groups. On calculating difference (ÔwetÕ minus ÔdryÕ) spectra, it was found that the intact wild-type PVX virions are characterized by high water-absorbing capacity and the ability to order a large number of water molecules on the virus particle. This effect was much weaker for the ST mutant and completely absent in the trypsin-treated PVX. It is proposed that the surface-located and glycosylated N-terminal CP segments of intact PVX virions induce the formation of a columnar-type shell from bound water molecules around the virions, which probably play a major role in maintaining the virion surface structure.

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Section: Discussionsupporting

confidence: 80%

N‐Terminal segment of potato virus X coat protein subunits is glycosylated and mediates formation of a bound water shell on the virion surface

Baratova

Fedorova

Добров

et al. 2004

European Journal of Biochemistry

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“…The problem of protein degradation during virus purification and storage (Koenig et aL, 1970;Tremaine & Agrawal, 1972;Hiebert & McDonald, 1973;A1 Ani et al, 1979) is especially significant for its effect on estimates of polypeptide number by SDS-gel electrophoresis. Such degradation by plant proteases can often be minimized or prevented by rapid purification of virus from freshly harvested material, by avoiding prolonged incubation of sap extracts and by the use of chelating agents.…”

Section: Analysis Of the Eoat Proteinmentioning

confidence: 99%

Guidelines for the Identification and Characterization of Plant Viruses

Hamilton

Edwardson

Francki

et al. 1981

Journal of General Virology

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“…Plant viruses are not known to require a dynamic capsid to assist in cell entry and are believed to lack this motion. A limited number of techniques have proven useful in the solution phase study of capsid protein dynamics, including the following: ultrasonic absorption (14), Raman spectroscopy (47), nuclear magnetic resonance (48,49,51), site-directed labeling (11), hydrogen-deuterium exchange (29,30,52), time-resolved fluorescence (17), and limited proteolysis (15,37,46). The last technique has proven to be the most generally applicable, and with the advent of matrix-assisted laser desorption ionization (MALDI) mass spectrometry, cleavage sites can be determined with a high degree of certainty.…”

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confidence: 99%

Enhanced Local Symmetry Interactions Globally Stabilize a Mutant Virus Capsid That Maintains Infectivity and Capsid Dynamics

Speir

Bothner

et al. 2006

J Virol

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Structural transitions in viral capsids play a critical role in the virus life cycle, including assembly, disassembly, and release of the packaged nucleic acid. Cowpea chlorotic mottle virus (CCMV) undergoes a well-studied reversible structural expansion in vitro in which the capsid expands by 10%. The swollen form of the particle can be completely disassembled by increasing the salt concentration to 1 M. Remarkably, a single-residue mutant of the CCMV N-terminal arm, K42R, is not susceptible to dissociation in high salt (salt-stable CCMV [SS-CCMV]) and retains 70% of wild-type infectivity. We present the combined structural and biophysical basis for the chemical stability and viability of the SS-CCMV particles. A 2.7-Å resolution crystal structure of the SS-CCMV capsid shows an addition of 660 new intersubunit interactions per particle at the center of the 20 hexameric capsomeres, which are a direct result of the K42R mutation. Protease-based mapping experiments of intact particles demonstrate that both the swollen and closed forms of the wild-type and SS-CCMV particles have highly dynamic N-terminal regions, yet the SS-CCMV particles are more resistant to degradation. Thus, the increase in SS-CCMV particle stability is a result of concentrated tethering of subunits at a local symmetry interface (i.e., quasi-sixfold axes) that does not interfere with the function of other key symmetry interfaces (i.e., fivefold, twofold, quasi-threefold axes). The result is a particle that is still dynamic but insensitive to high salt due to a new series of bonds that are resistant to high ionic strength and preserve the overall particle structure.Assembly, stability, and disassembly of icosahedral virus particles are coordinated by protein-protein and protein-nucleic acid interactions. Cowpea chlorotic mottle virus (CCMV) provides a model system for examining these interactions. The advantages of this system include in vivo and in vitro assembly assays (4, 7, 12, 55), a reversible transition between distinctly different closed and expanded forms (6), a high-resolution crystal structure of the closed particle (42), an electron cryomicroscopy image reconstruction of the swollen particle (42), and a general understanding of the requirements for assembly and stabilization of the capsid (6,53,55,56).CCMV is a small RNA plant virus that belongs to the Bromovirus genus in the Bromoviridae family. The viral genome is composed of four (positive-sense) single-stranded RNA molecules that are encapsidated in three morphologically identical particles (8,21,27). RNA1 and RNA2 encode proteins involved in RNA-dependent RNA replication and are packaged in separate particles. RNA3, encoding the movement and capsid proteins, and RNA 4, a subgenomic RNA of RNA 3 encoding just the capsid protein, are copackaged in a third particle (2). The structure of the CCMV virion has been determined to a 3.2-Å resolution (42) revealing a Tϭ3 truncated icosahedron having a diameter of 286 Å and composed of 180 identical protein subunits arranged as protruding ...

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Limited proteolysis of potato virus X by trypsin and plant proteases

Cited by 26 publications

References 13 publications

N‐Terminal segment of potato virus X coat protein subunits is glycosylated and mediates formation of a bound water shell on the virion surface

N‐Terminal segment of potato virus X coat protein subunits is glycosylated and mediates formation of a bound water shell on the virion surface

Guidelines for the Identification and Characterization of Plant Viruses

Enhanced Local Symmetry Interactions Globally Stabilize a Mutant Virus Capsid That Maintains Infectivity and Capsid Dynamics

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