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Alkaline Disassembly of Potato Virus X
Alexander S. Maris BSCI 296
Dr. Gerald Stubbs April 24, 2013
Potato virus X (PVX)
• Flexible, filamentous • Significance – crop
damage, biotechnology • RNA genome • Single coat protein (CP)
type • Diameter – 130 Å, length –
5100 Å • Helical pitch – 34.5 Å
Helical symmetry – 8.9 subunits per turn
Bar = 500 Å
Low Resolution Structure of PVX
• 21 Å resolution 3D model of PVX • Data from fiber diffraction, STEM, cryo-EM, and
helical reconstruction
Kendall, A., Bian, W., Maris, A., Azzo, C., Groom, J., Williams, D., Shi, J., Kim, R., Stewart, P., Wall, J., and Stubbs, G. 2013. A Common Structure for the Potexviruses. Virology 436(1):173-8.
Bar = 50 Å
Goal • Understand PVX disassembly mechanism
Techniques
- Molecular biology - PVX disassembly assay
?
+ RNA
Carboxylate-mediated Disassembly in TMV
H+
Ca2+
• TMV disassembles in vitro under plant intracellular conditions – High pH → strips H+s off carboxyl groups – Low [Ca2+] → prevents chelation of opposing negative
carboxylates
• Negative charges are exposed at CP-CP interface → electrostatic repulsion → CP dissociation from virion
• Identify specific acidic residues at CP-CP interface • Site directed mutagenesis to convert acidic amino acids to neutral
amides • Negative charge abolished • Virus and CP subunits exist in equilibrium • Mutant CPs shift equilibrium to assembly • Digestion of RNA tails → free ribonucleotides to quantify
disassembly spectroscopically
Carboxylate-mediated Disassembly
+ RNA
Stubbs and Kendall, 2012
Carboxylate-mediated Disassembly of TMV
Lu et al., 1996
PVX (UK3) CP sequence
• No high resolution structure available for PVX
• 20 acidic residues in red
Methods: CP Expression and Purification Carboxylate mutant plasmid
Overexpression in E. coli
Cell pellet lysis, extraction of cell lysate
Cleavage of 6x His tag
CP concentration
Ni2+-NTA column passage
Thrombin removal
HHHHHHGLVPR | GSAPASTTQPIGSTTSTTTATA…
PVX Alkaline Disassembly Assay
PVX + KCl → pH 7.4 PVX + KCl → pH 12 PVX + NΔ2 CP → pH 12 PVX + mutant CP → pH 12
pH 8.0-8.5
Micrococcal nuclease digest of RNA tails
(NH4)2SO4 precipitation
Centrifugation Supernatant removal
Spectroscopy
1 hr 1 hr
1 hr & 4 hr
[PVXi]= 2.84 mg/ml [CPi]= 0 mg/ml
2.9
9.3
3.3
7.1
0
1
2
3
4
5
6
7
8
9
10
PVX+KCl pH 7.4 PVX+KCl pH 12
% D
isas
sem
bly
Baseline Alkaline Disassembly Assay
1h
4h
0.54
6.6 6.0
7.3
0.86
8.1 7.6
9.0
0
1
2
3
4
5
6
7
8
9
10
11
PVX+KCl pH 7.4 PVX+KCl pH 12 PVX+NΔ2 pH 12 PVX+D37N pH 12
% D
isas
sem
bly
D37N Alkaline Disassembly Assay
1h
4h
[PVXi]= 2.50 mg/ml [CPi]= 1.50 mg/ml
3.3
9.3 9.7
8.8
3.8
8.1 8.4 7.6
0
1
2
3
4
5
6
7
8
9
10
11
PVX+KCl pH 7.4 PVX+KCl pH 12 PVX+NΔ2 pH 12 PVX+D39N pH 12
% D
isas
sem
bly
D39N Alkaline Disassembly Assay
1h
4h
[PVXi]= 2.50 mg/ml [CPi]= 1.00 mg/ml
2.0
7.5
9.4 9.0
1.8
7.6
9.3
7.8
0
1
2
3
4
5
6
7
8
9
10
11
PVX+KCl pH 7.4 PVX+KCl pH 12 PVX+NΔ2 pH 12 PVX+D100N pH 12
% D
isas
sem
bly
D100N Alkaline Disassembly Assay
1h
4h
[PVXi]= 1.85 mg/ml [CPi]= 1.50 mg/ml
10.1 10.2 9.8 10.3
0123456789
1011
PVX+NΔ2 pH 12 PVX+D58N pH 12
% D
isas
sem
bly
D58N Alkaline Disassembly Assay
1h
4h 11.0 10.0 10.4 10.4
0123456789
101112
PVX+NΔ2 pH 12 PVX+E97Q pH 12
% D
isas
sem
bly
E97Q Alkaline Disassembly Assay
1h
4h
10.1 9.2 9.7 10.1
0
1
2
3
4
5
6
7
8
9
10
11
PVX+NΔ2 pH 12 PVX+E156Q pH 12
% D
isas
sem
bly
E156Q Alkaline Disassembly Assay
1h
4h 9.6 9.5 9.4 9.9
0
1
2
3
4
5
6
7
8
9
10
11
PVX+NΔ2 pH 12 PVX+E186Q pH 12
% D
isas
sem
bly
E186Q Alkaline Disassembly Assay
1h
4h
7.2
8.1 7.5
8.5
0
1
2
3
4
5
6
7
8
9
PVX+NΔ2 pH 12 PVX+WT CP pH 12
% D
isas
sem
bly
WT PVX-CP Alkaline Disassembly Assay
1h
4h
[PVXi]= 2.60 mg/ml [CPi]= 1.00 mg/ml
7.1
6.4 6.4
7.4
0
1
2
3
4
5
6
7
8
PVX+KCl pH 12 PVX+KCl pH 12 with Ca2+
% D
isas
sem
bly
Alkaline Disassembly Assay ± Ca2+
1h
4h
[PVXi]= 3.10 mg/ml [CPi]= 0 mg/ml [Ca2+f]= 14 mM
Results
• Significant PVX disassembly at alkaline pH
• No significant differences in seven assayed carboxylate mutants
• Possible decrease in % disassembly with D39N
• No difference between NΔ2 WT-mimic and WT PVX-CP
• No difference with ± Ca2+ • Ca3(PO4)2 and Ca(OH)2 ppt
likely
3.3
9.3 9.7 8.8
3.8
8.1 8.4 7.6
0
1
2
3
4
5
6
7
8
9
10
11
PVX+KClpH 7.4
PVX+KClpH 12
PVX+NΔ2 pH 12
PVX+D39NpH 12
% D
isas
sem
bly
D39N Alkaline Disassembly Assay
1h
4h
Discussion
• No difference between PVX+KCl and PVX+CP at pH 12
• 6x His tag cleavage complete ?
• Strong electrostatic repulsion
• Incomplete hypothesis ? • TGBp1 and N-terminal phosphorylation
Future Directions • 7/20 carboxylate mutants
assayed • Plasmids → expression →
assay remaining mutants
• Baseline assays at varying pH
• Determine threshold
• ± Ca2+ in soluble buffer
• Double, triple mutants • D39N
• In vivo assays
• Higher resolution PVX structure
Acknowledgements
• Amy Kendall
• Sarah Baumgarten and Joe Groom
• Dr. Stubbs
• This work was supported by NSF grant MCB-0235653.
