Keeping It All Together: 2 Projects Testing Stability Predictions in a Virus

Adrienne E. Fairbanks,1 Natalya Usachenko,2

LuAnn Scott, Emma R. Altman, James T. Van Leuven,
Jagdish Patel, Craig R. Miller and Holly A. Wichman
Department of Biological Sciences & the Center for Modeling Complex Interactions, University of Idaho, Moscow Idaho

2Testing the Capacity to Over-Stabilize 1Testing Metrics of Transcript Stability

the Viral Capsid .
What is the relationship between synonymous
Can a virus be over-stabilized through the alteration of mutations engineered to increase or decrease viral
its minor capsid protein? transcript stability and fitness?

Hypothesis Metrics
CAI Codon Adaptation Index
Combination of two or more stabilizing mutations in a viral capsid protein will have
CPB Codon Pair Bias
an additive effect on the overall stability of the protein, potentially reducing viral
sCAI Starvation CAI
fitness in two ways:
tAI tRNA Adaptation Index
1. Making capsid assembly more difficult and thereby reducing the number of
ITE Index of Translation Elongation
viable offspring.
SD Shine-Delgarno
2. Increasing the overall stability of the capsid itself, reducing its ability to infect
mFE 5’ mRNA Folding Energy
a host cell.
Bacteriophage φX174 TPI tRNA Pairing Index
Experimental Design An efficient virus model
• Combine stabilizing mutations in protein G of φX174, the minor capsid/major Proposed Experiment
spike protein (Fig.1). • Non-pathogenic
• Choose 5 phage each with a single amino acid mutation predicted to stabilize
• Design 4 high & 4 low stability mutants for genes F, G, & H, major
• Small genome size
binding of the protein (Fig. 2). capsid, major spike, and pilot proteins, respectively (Fig. 4)
• Large populations
• Add each amino acid mutation to each of the other four phage by site directed • Have gene fragments synthesized & cloned into plasmids
• Ease of manipulation of genome sequence
mutagenesis (Fig. 3). • Use assembly platform to create mutant phage genomes (Fig. 5)
o Site directed mutagenesis
• Assess the change in fitness – measured by growth rate or plaque size. • Transform into host cells, pick plaques and sequence
o Engineered fragments inserted using assembly platform
• Perform fitness assays
A B C
• Assays available for fitness, attachment, thermal stability, burst
Figure 2. FoldX time, burst size, length of eclipse, length of assembly, rate of
predictions for changes
in stability for binding (x assembly and mortality rate
axis) and folding (y
axis) when replacing
the wild type amino acid
Overarching Context
at A) G11, B) G4, C)
G45, D) G128, and E)
Understanding basic mechanisms of viral adaptation
G117. The predicted
D E
∆∆G (change from wt Project 1
change in free energy) Figure 4. The φX174 genome is a single, circular chromosome of 5386 bp and contains 11
for the 19 other amino • This research is part of an effort to develop a genes, some overlapping. Promoters and terminators for the transcripts are below and the cloned
acids is represented by “recipe” for designing viral vaccines. assembly fragments that will be modified in these studies are above.
dots. Negative values • Synonymous mutations could be one
are stabilizing.
ingredient
• But…synonymous mutations affect more than
Figure 3. Location of
just codons
stability mutations to be A B

combined. A) A monomer of
G (light blue) and a Project 2
monomer of F (purple)
shown in relationship to the • Protein stability – important determining factor in
pentamers of G/F (grey). B) protein evolution. Figure 5. Assembly platform for φX174. A) 14 segments of phage genome (black), flanked by
Ribbon structure of protein
G with the locations of the • Used in predictive modeling. (Doore et al.). BsmB1 restriction sites, cloned into a pCRII-TOPO plasmid (grey). One or more fragments is
mutated (gold). B) Phage inserts are excised with BsmB1, which cuts in the phage sequence.
five stability mutants shown • Increased capsid stability previously observed in C) Inserts are ligated together using the unique 4 bp sticky ends created by BsmB1 to
in dark blue. high temperature phage mutants with higher fitness reconstruct the genome with the incorporation of the mutant fragment(s).
than wild type (Lee et al.).
Current Results • Correlations exist between viral evolution and Expected Results
climate change; this study will continue to explore
• Viable double mutants in two phage so far: G4_M/117_M, • Reduced fitness in all 24 phages caused by reduced numbers of
this phenomenon.
G128_V/117_M. protein
• Sequencing and fitness assessment ongoing. o Stabilizing mutations slow down translation
• Plan to add a third stabilizing mutation to all viable doubles. Acknowledgements o Destabilizing mutations cause degradation of mRNA faster than
References Phage image by Ben Darby usual
Doore, Sarah M., Fane, Bentley A. (2016) “The microviridae: Diversity, assembly, and experimental evolution”. Virology vol 491, p. 45-55
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 grant OIA-1736253, NIH COBRE grant • Modelers will evaluate results to better understand the relationship
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 P20GM104420 and NIH R01 grant GM076040. between the metrics and synonymous mutations
Mol Biol 237:517-543.