Testing the Capacity to Over-Stabilize Viruses by

Engineering Mutations into the Capsid of Bacteriophage ɸX174

Natalya 1
Usachenko , LuAnn Emma R. 1
Scott ,
James T. Van 1
Altman , 1,2
Leuven ,
2 1,2
Jagdish Patel , Craig R. Miller , and Holly A. Wichman 1,2
1Department of Biological Sciences, 2Institute for Collaboration Modeling and Innovation, University of Idaho, Moscow, Idaho 83843

Can a virus be over-stabilized through the Overarching Context Methods

alteration of its capsid proteins? Understanding basic mechanisms of viral adaptation
& best methods of attenuation for vaccine design A B
Hypothesis
ΦX174
Combination of two or more stabilizing mutations in a viral 5386 bases XXX
capsid protein will have an additive effect on the overall circular

stability of the protein, potentially reducing viral fitness in

single-stranded
11 over-lapping genes
C
two ways:
gene G

1. Making capsid assembly more difficult and

thereby reducing the number of viable offspring. XXX

2. Increasing the overall stability of the capsid 1. Design matching primers with desired mutation
2. PCR 1 - amplify two halves (A)
itself, reducing its ability to attach to and infect a host
3. PCR 2 – connects the two halves
cell.
.
4. Transform new genome with desired mutation (B) into host
cells
5. Pick plaque and sequence
Protein Stability Experimental Design 6. Repeat with primers for another mutation, using B as template
• Combine stabilizing mutations in protein G of φX174, 7. Transform new genome with both mutations (C) into host cells
• Important determining factor in protein evolution 8. Perform fitness assays for growth and stability
the minor capsid/major spike protein (Fig.1).
• Used in predictive modeling (Doore et al.) • Choose 5 phage each with a single amino acid mutation
• Increased capsid stability seen in high temperature phage predicted to stabilize binding of the protein (Fig. 2). Current Results
• Add each amino acid mutation to each of the other four • 9 double mutants created out of 20 proposed
mutants with higher fitness than wild type (Lee et al.) phage by site directed mutagenesis (Fig. 3). • Only 2 have both intended mutations & no others in genome
• Assess the change in fitness – measured by growth rate • Stability assay being developed looks promising (Fig. 4)
→ Correlations between climate change and viral evolution? or plaque size and perform stability assays.
1.2 Wild Type 1.2
G128
Figure 4. Stability
Figure 2. FoldX predictions for A B C 0.7 0.7
assays of wildtype

Survival
Survival
Bacteriophage ΦX174 changes in stability for binding (x
axis) and folding (y axis) when
phage, mutant G117,
mutant G128, and
0.2 0.2
30.5°C

replacing the wild type amino -0.3 2 4 8 16 -0.3 2 4 8 16

36.0°C
double mutant
An efficient virus model acid at A) G11, B) G4, C) G45, D)
G117/128 under a Time, hrs Time, hrs
G128, and E) G117. The
range of different G117 G128/117 43.9°C
predicted ∆∆G (change from wt 1.2 1.5
temperatures. The
• Non-pathogenic change in free energy) for the 19
other amino acids is represented
D E
double mutant appears 0.7 1
49.6°C

Survival

Survival
• Small genome size by dots. Negative values are
more stable than the
wild type phage or 0.5
0.2
• Large populations stabilizing.
either single mutant. 0
2 4 8 16
• Ease of manipulation of genome -0.3
Time, hrs
2 4 8
Time, hrs
16

o Site directed mutagenesis Figure 3. Location of stability

o Engineered fragments inserted using assembly
mutations to be combined. A) A
monomer of G (light blue) and a
Conclusions
platform monomer of F (purple) shown in • The variation in survivability rates and fitness of phage under
• Assays available for fitness, attachment, thermal stability,
relationship to the pentamers of A B
different temperatures could be indictive of similar patterns of viral
G/F (grey). B) Ribbon structure
behavior under fluctuating climatic conditions.
burst time, burst size, length of eclipse, length of of protein G with the locations of
the five stability mutants shown in • Broader implications of this research involve the exploration of this
assembly, rate of assembly and mortality rate dark blue. phenomenon – will continue to add stabilizing mutations to the virus
capsid until a decrease in fitness is observed.

References Acknowledgements
Doore, Sarah M., Fane, Bentley A. (2016) “The microviridae: Diversity, assembly, and experimental evolution”. Virology vol 491, p. 45-55 Phage image by Ben Darby
Lee KH, Miller CR, Nagel AC, Wichman HA, Joyce P, et al. (2011) “First-Step Mutations for Adaptation at Elevated Temperature Increase Capsid Stability in a Virus”. PLoS ONE 6(9): e25640 Funding provided by NSF EPSCoR OIA-1736253, NIH COBRE P20GM104420 and NIH R01 GM076040.
McKenna, R., L. L. Ilag, and M. G. Rossmann. 1994. Analysis of the single-stranded DNA bacteriophage phi X174, refined at a resolution of 3.0 A. J Mol Biol 237:517-543.