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The Zika journey continues – have you been bitten by the mosquito?

Our blog

29 June, 2018

drug-discovery-2Missed part 1? Click here

Over the last 5 years Zika virus infection has become a prevalent issue globally of which epidemiology spread has caused concern for residents of and visitors to endemic areas. The apparent connection between infection and fetal abnormalities and transmission via bodily fluids has set the virus apart from other members of the flavivirus family. The seriousness of infectious outcomes and its uniqueness have demanded significant resource to support discovery of suitable treatments and to explore possible preventative measures. In the hunt for treatments, structural biology has become a critical approach to better understand the dynamics and interactions of the viral and host proteins during infection. In the recent paper by Shang et al., (2018) the crystal structure of the ZIKV capsid (C) protein was determined to elucidate the full scope of functions possessed by this component. Enabled by nanolitre dropsetting with the mosquito LCP liquid handler (yes, we understand that’s just a tad ironic), the ZIKV C protein samples were crystallized via the lipidic cubic phase (LCP) method – see table 1 below for more information about different crystallization techniques. The resulting crystal structure of ZIKV C protein has a similar overall fold to the capsid protein structures of the Dengue and West Nile, with some obvious distinctions. Previous confocal microscopy studies have demonstrated that the ZIKV C protein is capable of association with lipid droplets though to be important for membrane association. The unique long pre-α1 loop enables tight dimeric assembly that underpins the functional divergence from other flaviviruses. ZIKV C is also fascinating for its apparent functions beyond viral assembly, including nucleotide and RNA binding.

Environment and context are key in understanding protein structure and function relationships. The study of protein complexes and their function as part of whole-virus interactions with host cells benefit from study in states closely resembling native environments. This work is not achievable through protein crystallization alone. Are there other techniques or methods that could lend a hand in this endeavour? Could new advances in Cryo-EM technologies be the way forward? Have other Nobel prize winning methods unveiled insights into the eradication of Zika infection? Find out next week in our next instalment of the Zika virus blog series!

Technique Description Advantages Limitations Solution
Microbatch under oil (sitting drop)
  • Protein, buffer and precipitant combined into a single supersaturated protein solution under inert oil
  • Oil prevents sample evaporation
  • Limited amounts of sample needed
  • Airborne contamination free
  • Easy crystal harvesting
  • Can be adapted for HTS systems
  • Detergent solubilization affects sample purity
  • Chemical and biochemichal conditions need to be optimised for specific crystallization experiments
mosquito cyrstal or mosquito LCP
Vapour diffusion (sitting drop or hanging drop)
  • Physical separation (coverslip/micro-bridge) of protein and precipitant
  • Protein supersaturation achieved through concentration gradients
  • Limited amount of sample required
  • Easy access to crystals
  • Mutiple experiments with a single reservoir
  • Can be adapted for HTS systems
  • Chemical and biochemichal conditions need to be optimised for specific crystallization experiments
Lipidic cubic phase
  • 3D hydrophobic confinement of protein in a  self-assembled lipid bilayer
  • Protein supersaturation trigered by precipitant addition
  • Protein stabilization in its native lipid environment and conformation
  • High sample purity achieved
  • Robust methodology
  • Can be adaptaped for HTS  systems
  • High viscosity degree
  • Sample preparation
  • Not adaptable for traditional methods
mosquito LCP