Journal of Biomolecular NMR

, Volume 64, Issue 3, pp 255–265 | Cite as

Application of virus-like particles (VLP) to NMR characterization of viral membrane protein interactions

  • Aleksandar Antanasijevic
  • Carolyn Kingsley
  • Arnab Basu
  • Terry L. Bowlin
  • Lijun Rong
  • Michael Caffrey


The membrane proteins of viruses play critical roles in the virus life cycle and are attractive targets for therapeutic intervention. Virus-like particles (VLP) present the possibility to study the biochemical and biophysical properties of viral membrane proteins in their native environment. Specifically, the VLP constructs contain the entire protein sequence and are comprised of native membrane components including lipids, cholesterol, carbohydrates and cellular proteins. In this study we prepare VLP containing full-length hemagglutinin (HA) or neuraminidase (NA) from influenza and characterize their interactions with small molecule inhibitors. Using HA-VLP, we first show that VLP samples prepared using the standard sucrose gradient purification scheme contain significant amounts of serum proteins, which exhibit high potential for non-specific interactions, thereby complicating NMR studies of ligand-target interactions. We then show that the serum contaminants may be largely removed with the addition of a gel filtration chromatography step. Next, using HA-VLP we demonstrate that WaterLOGSY NMR is significantly more sensitive than Saturation Transfer Difference (STD) NMR for the study of ligand interactions with membrane bound targets. In addition, we compare the ligand orientation to HA embedded in VLP with that of recombinant HA by STD NMR. In a subsequent step, using NA-VLP we characterize the kinetic and binding properties of substrate analogs and inhibitors of NA, including study of the H274Y-NA mutant, which leads to wide spread resistance to current influenza antivirals. In summary, our work suggests that VLP have high potential to become standard tools in biochemical and biophysical studies of viral membrane proteins, particularly when VLP are highly purified and combined with control VLP containing native membrane proteins.


Antiviral agent Drug resistance Influenza STD Virus entry WaterLOGSY 



This research was supported by DHHS/NIH Grants 1R21AI101676 and 5R44AI072861 and the Chicago Biomedical Consortium with support from the Searle Funds at the Chicago Community Trust.


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Copyright information

© Springer Science+Business Media Dordrecht 2016

Authors and Affiliations

  • Aleksandar Antanasijevic
    • 1
  • Carolyn Kingsley
    • 1
  • Arnab Basu
    • 2
  • Terry L. Bowlin
    • 2
  • Lijun Rong
    • 3
  • Michael Caffrey
    • 1
  1. 1.Department of Biochemistry and Molecular GeneticsUniversity of Illinois at ChicagoChicagoUSA
  2. 2.Microbiotix Inc.WorcesterUSA
  3. 3.Department of Microbiology and ImmunologyUniversity of Illinois at ChicagoChicagoUSA

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