Structure Biology Research Of Heptad Repeat Regions From Mumps Virus Fusion Protein

Like other enveloped viruses, Mumps virus (MuV) fusion protein (F) may possess two heptad repeat domains (HR). Previous studies on the F protein of other enveloped viruses have proved that the HR plays important role in their infection to host intermediated by the F protein. This finding has illustrated a common mechanism involved in the membrane fusion. However, it is unknown whether the MuV has a membrane fusion mechanism similar to that of the other enveloped viruses in the course of host infection.In the present study, recombinant vectors containing HR sequences named HR1, HR2 and 2-Helix were constructed and used to express HR1, HR2 and 2-Helix in the form of soluble fusion protein with GST in Eschechia coli. Purified proteins were acquired by affinity chromatography and gel filtration chromatography. Pull-down experiment testified an interaction between HRl and HR2 and the mixture of HR1 and HR2 peptides was confirmed as a polymer in gel filtration analysis. Chemical cross-linking trials further demonstrated that an equimolar mixture of HRl and HR2 could form heterotrimer, in which the 2-Helix showed evident concentrations of monomer and polymer as the EGS concentration increased whereas the HR1/HR2 was obviously in the form of heterotrimer despite unclear gradeing concentrations of monomer and polymer due to the low protein concentration. Based on gel-filtration analysis, HRl and HR2 could be eluted at the same volume of 2-Helix, suggesting a possible interaction between HRl and HR2. The elution peaks of the HR1/HR2 and the 2-Helix were located between the corresponding volumes of 52 and 14.4 kDa, indicating that the HR1/HR2 peptides were in the possible form of a 6-helix bundle. Based on mass spectral analysis, molecular weights of HRl, HR2, and 2-Helix were measured as 7.1, 4.3, and 11.8 kDa, respectively (close to their theoretical values). This result was well in accordance with that of gel-filtration analysis. The CD spectra of HRl were minimal at 208 and 222 nm, a characteristic for a-helices, whereas HR2 was not folded very much. However, the interaction product of equimolar HRl and HR2 exhibited a CD spectrum curve with double minima at 208 and 222 nm, and contained a-helices more than HRl or HR2 alone. Thermostabilities of 2-Helix and HR1/HR2 peptides were separately measured on a basis of their CD signal records. Apparently unfolded transition occurred when the temperature increased to 76C. The a-helices werecharacteristic with the refolded polypeptides based upon CD spectroscopy. This confirmed again reversible changes of the helices. Crystal screen kits (Crystal Screen I and II, Hampton Research, Riverside, CA, USA) were used to optimize the growing conditions of 2-Helix protein. High quality crystals were eventually obtained at the optimized regime of 15% PEG 8000 and 1.0 MLi2SO4 buffer, and used for diffusion analysis. With the software AMORE, the central structure of the MuV F protein was determined as SV5 F protein Nl/Cl structure (PDB code: 1SVF). The structure of the 6-helix bundle similar to those of SV5, RSV and NDV suggests that MuV may share a common fusion infection mechanism with the other enveloped viruses.