Investigation of Potato Virus X Disassembly

1
Investigation of Potato Virus X Disassembly

• Sarah Baumgarten
• Research Advisor Dr. Gerald Stubbs

2
Potexviruses

• Potato virus X (PVX) is a member of the
  potexvirus genus
• Flexible, filamentous plant viruses
• Single strand of messenger- sense RNA surrounded
  by over 1000 coat protein subunits
• Approximately 5000Å long and about 130 Å in
  diameter

Negative stain electron micrograph of PVX. Scale
bar 100nm
3
Why Study PVX?

• PVX can have important consequences for
  agriculture and biotechnology
• Knowing more details about the structure and
  function of PVX will allow for advances in both
  fields
• Virus resistant transgenic plants
• Protein expression and harvesting in plant hosts

4
PVX Disassembly

• Two methods by which PVX is known to disassemble
• 1. Phosphorylation of the N-terminus of the
  coat protein at the region of the virion
  containing the 5 end of RNA
• 2. Binding of TGBp1 proteins to the end of PVX
  containing the 5 end of RNA
• - results in linear destabilization of the
  viral helix
• Could there be other factors playing a role in
  PVX disassembly?

5
Carboxylate Disassembly

• Carboxylate interactions between coat protein
  subunits are known to play a role in disassembly
  of TMV and other viruses

Ribbon drawing of two TMV coat protein subunits
with the detailed skeletal structures of Glu50
and Asp77 shown
6
Carboxylate Mutants
Aspartate
Asparagine
Glutamate
Glutamine
7
TMV Disassembly Studies

• Carboxylate mutant coat proteins engineered
• Mutant coat proteins added in excess to solution
  of wild-type virus
• Solution adjusted to a basic pH to promote
  disassembly
• Aliquots removed at particular time intervals and
  treated with nuclease to digest free RNA
• Percent degradation calculated as a ratio of the
  concentration of free nucleotides to the total
  concentration of nucleotides present initially in
  the sample

8
Project Goals

• Engineer carboxylate mutant PVX coat protein
• 2. Develop an assay that can consistently and
  reliably measure the degree of PVX viral
  disassembly

9
Carboxylate Mutants

• A high-resolution three-dimensional structure of
  the PVX coat protein subunit is not available

10
Carboxylate Mutants

• A high-resolution three-dimensional structure of
  the PVX coat protein subunit is not available

PVX (UK-3) coat protein sequence. All nineteen
carboxylate group residues to be mutated are
highlighted in red.
11
Carboxylate Mutants

• Strategenes QuikChange? Site-directed
  Mutagenesis Kit was used to make nineteen
  carboxylate mutant coat proteins
• D37N, D39N, E57Q, D58N, E63Q, D68N, D74N, D82N,
  D89N, E97Q, D100N, E119Q, E156Q, D163N, E178Q,
  E186Q, E188Q, D209N, D214N
• The mutant plasmids were amplified in XL1-Blue
  cells, isolated using a Qiagen miniprep
  procedure, and sequenced at the Vanderbilt DNA
  Sequencing Core Facility

12
Carboxylate Mutants

• Strategenes QuikChange? Site-directed
  Mutagenesis Kit was used to make nineteen
  carboxylate mutant coat proteins
• D37N, D39N, E57Q, D58N, E63Q, D68N, D74N, D82N,
  D89N, E97Q, D100N, E119Q, E156Q, D163N, E178Q,
  E186Q, E188Q, D209N, D214N
• The mutant plasmids were amplified in XL1-Blue
  cells, isolated using a Qiagen miniprep
  procedure, and sequenced at the Vanderbilt DNA
  Sequencing Core Facility

13
Conclusions and Future Plans

• Carboxylate mutant PVX coat proteins
• Work to make the five carboxylate mutations that
  were unsuccessful
• Express the mutant coat protein in BL21(DE3)pLysS
  cells, harvest, and purify for use in disassembly
  assays

14
Project Goals

• Engineer carboxylate mutant PVX coat protein
  plasmids
• 2. Develop an assay that can consistently and
  reliably measure the degree of PVX viral
  disassembly

15
Disassembly Assay

• PVX dialyzed against distilled, deionized water
  overnight at 4C
• Three samples of virus were placed on ice
• one was adjusted to pH 9.8, another to pH 10.6,
  and the pH of the last was not adjusted
• Aliquots taken after zero, 1, 2, and 4 hours
  incubation

16
Disassembly Assay

• The aliquots were titrated to neutral and
  digested with micrococcal nuclease at room
  temperature for 1 hour
• The free coat protein and intact virus were
  precipitated with 5 TCA and centrifuged at 9000
  g for 10 minutes
• The supernatant was titrated to neutral and the
  concentration of free nucleotides was determined
  using UV spectroscopy

17
Disassembly Assay
Percent degradation of PVX in solutions at
different pH conditions at 0, 1, 2, and 4 hours
is shown. The initial disassembly measured at
zero hours was determined from one aliquot of
virus solution before pH adjustment. The control
is a solution of PVX with an unaltered pH of 7.6.
18
Disassembly Assay

• PVX dialyzed against distilled, deionized water
  overnight at 4C
• Is PVX stable in water?
• Virus concentration measured and adjusted to a
  concentration of 2 mg/ml
• Three samples of virus were placed on ice
• one was adjusted to pH 9.8, another to pH 10.6,
  and the pH of the last was not adjusted

19
Disassembly Assay

• Aliquots taken after zero, 1, 2, and 4 hours
  incubation
• The aliquots were titrated to neutral, and
  digested with micrococcal nuclease at room
  temperature for 1 hour
• Are intact virions susceptible to the nuclease?
• The free coat protein and intact virus were
  precipitated with 5 trichloroacetic acid (TCA)
  and centrifuged at 9000 g for 10 minutes
• Is TCA causing PVX to disassemble?
• The supernatant was titrated to neutral and the
  concentration of free nucleotides was determined

20
Virion Stability

• Negative stain transmission electron microscopy

10 mM Tris-HCl pH 8, 5 mM EDTA, and 1 PIC
Distilled, deionized water 0.1M KCl
21
Virus Precipitation

• PVX was dialyzed against 0.1M KCl
• TCA was added to a final w/v of 3, 10, and 20
• Ammonium sulfate was added to a final
  concentration of 1, 1.5, 2, 2.5, 3, 3.5, 4, and
  4.5 M
• Aliquots incubated at 4 ?C for 1 hour
• Centrifuged at 9000 g for 15 minutes and the
  supernatant was collected
• The supernatant was titrated to neutral and the
  concentration of remaining virus was determined

22
Virus Precipitation Results

• TCA interferes with UV absorption

• Final concentration of 2.5 M or higher is
  sufficient to precipitate PVX completely from
  solution

23
Ribonuclease Susceptibility

• PVX dialyzed against 0.1 M KCl
• Treated with micrococcal nuclease, bovine
  pancreatic ribonuclease A, or RNase ONE?
• Samples were removed after 20, 40 and 60 minutes
• The free coat protein and intact virus were
  precipitated with 4.5 M ammonium sulfate and
  centrifuged at 9000 g for 10 minutes
• The supernatant was titrated to neutral and the
  concentration of free nucleotides was determined

24
Ribonuclease Susceptibility Results
The percent of PVX disassembly after treatment
with micrococcal nuclease, bovine pancreatic
ribonuclease A, and RNase ONE? for 20, 40, and 60
minutes
25
Modified Assay

• PVX dialyzed against 0.1 M KCl, rather than
  distilled, deionized water
• Ammonium sulfate was used to precipitate free
  coat protein and intact virions from solution,
  rather than TCA
• Three nucleases were used micrococcal nuclease,
  bovine pancreatic ribonuclease A, and RNase ONE?
• Other steps in the procedure were unchanged

26
Modified Assay Results
Percent of PVX degraded at pH 7.6, 9.6, 10.3
determined from the concentration of free
nucleotides after treatment with micrococcal
nuclease, bovine pancreatic ribonuclease A, and
RNase one.
27
Conclusions and Future Plans

• Viral Disassembly Assay
• Repeat experiments multiple times and with higher
  concentrations of PVX to ensure the significance
  and accuracy of the results
• Determine which pH is appropriate to use in the
  assay
• Experiment with additives, such as phosphate,
  that could lower the necessary pH closer to the
  pH within a plant cell

28
Acknowledgements
I would like to thank -Dr. Stubbs for his
support and the opportunity to conduct research
in his lab -Amy Kendall for knowing everything
and always being there to help when I needed
it -Tim Bowles for answering a wide variety of
questions as they came up in my project -Wen
Bian, Michele McDonald, Hayden Box, Luke
Richards, and Adrianne Eyman for their undying
support -Dr. Krezel and Dr. Eichman for serving
on my honors committee