Abstract
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.
Highlights
The membrane proteins of enveloped viruses, which include Ebola, HIV, influenza, and SARS Coronavirus, play critical roles in viral entry and they are attractive targets for antiviral therapeutics (Kuhn et al 2007; Lagoja and De Clercq 2008; Caffrey 2011; Yermolina et al 2011)
We characterized the interaction of a small molecule inhibitor of influenza entry, MBX2329, with recombinant H5 HA by WL and Saturation Transfer Difference (STD) NMR (Basu et al 2014)
In this work we demonstrated the high potential of using Virus-like particles (VLP)-based systems to study viral membrane proteins by NMR methods
Summary
The membrane proteins of enveloped viruses, which include Ebola, HIV, influenza, and SARS Coronavirus, play critical roles in viral entry and they are attractive targets for antiviral therapeutics (Kuhn et al 2007; Lagoja and De Clercq 2008; Caffrey 2011; Yermolina et al 2011). The use of virus-like particles (VLP) has become a very powerful tool to study the membrane proteins of many dangerous viruses without the necessity for biosafety level 3 or 4 facilities (Yonezawa et al 2005; Zhang et al 2008; Garcia and Lai 2011; Qian et al 2013). In these experiments VLP are prepared by co-transfection of plasmid encoding the desired viral membrane protein with a plasmid encoding a capsid background (e.g. that of HIV or influenza), often with the addition of a reporter construct (e.g. GFP or luciferase). Current applications of viral membrane proteins embedded in VLP include studies of processing, kinetics and activity (Jacobs et al 2005; Jalaguier et al 2011; Ku et al 2013; Antanasijevic et al 2014a; Tedbury et al 2015), drug screening trials (Wang et al 2014; Kouznetsova et al 2014; Basu et al 2014), and use as immunogens (McBurney et al 2007; Haynes 2009; Carra et al 2015)
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