Abstract
The gamma(1)-peptide is a 21-residue lipid-binding domain from the non-enveloped Flock House virus (FHV). Unlike enveloped viruses, the entry of non-enveloped viruses into cells is believed to occur without membrane fusion. In this study, we performed NMR experiments to establish the solution structure of a membrane-binding peptide from a small non-enveloped icosahedral virus. The three-dimensional structure of the FHV gamma(1)-domain was determined at pH 6.5 and 4.0 in a hydrophobic environment. The secondary and tertiary structures were evaluated in the context of the capacity of the peptide for permeabilizing membrane vesicles of different lipid composition, as measured by fluorescence assays. At both pH values, the peptide has a kinked structure, similar to the fusion domain from the enveloped viruses. The secondary structure was similar in three different hydrophobic environments as follows: water/trifluoroethanol, SDS, and membrane vesicles of different compositions. The ability of the peptide to induce vesicle leakage was highly dependent on the membrane composition. Although the gamma-peptide shares some structural properties to fusion domains of enveloped viruses, it did not induce membrane fusion. Our results suggest that small protein components such as the gamma-peptide in nodaviruses (such as FHV) and VP4 in picornaviruses have a crucial role in conducting nucleic acids through cellular membranes and that their structures resemble the fusion domains of membrane proteins from enveloped viruses.
Highlights
Structure of a Membrane-binding Domain from a Non-enveloped Animal Virus: Insights into the Mechanism of Membrane Permeability and Cellular Entry
We show the solution nuclear magnetic resonance (NMR) structure of a membrane-binding peptide contained in the protein shell of a small, nonenveloped icosahedral virus
The threedimensional structure of Flock House virus (FHV) γ1-domain was determined at pH 6.5 and 4.0 in a hydrophobic environment
Summary
Peptides - The synthetic γ1-peptide composed of 21 residues (ASMWERV-KSIIKSS-LAAASNI) was purchased from Genemed Synthesis, Inc (San Francisco, CA, USA). The samples of γ1-peptide (200 μM) were prepared in 10 mM of Na phosphate buffer, pH 7.0 in different concentrations of trifluoroethanol, in 60 mM of SDS micelles or containing lipid vesicles. Resonances of the sample at pH 6.5 were assigned from TOCSY (spin lock time of 70 ms) using the MLEV-17 pulse sequence [21], COSY-GP and NOESY (mixing time of 150 ms) spectra Resonances of the sample at pH 4.0 were assigned from TOCSY (MLEV-17 pulse sequence, spin lock time of 70 ms) and NOESY (mixing time 160 ms). SDS- TOCSY (spin lock time of 70 ms) using the MLEV-17 pulse sequence [21] and NOESY (mixing time of 120 ms) spectra [22, 23]. Release was initiated by the addition of peptide and monitored by the fluorescence intensity increase after the addition of the peptide as described for the ANTS/DPX assay
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