Subclone,Expression,Purification And Its Structure Analysis Of Heptad Repeat Regions From The F Protein Of Newcastle Disease Virus

Membrane fusion within the Paramyxoviridae family of viruses is mediated by a surface glycoprotein termed the "F", or fusion protein. Infection by Newcastle Disease Virus (NDV) involves the fusion of viral and cellular membranes with subsequent transfer of viral genetic material into the cell. Study of the molecular mechanism of membrane fusion is targeted for the development of new anti-viruses drugs. Two heptad repeat (HR) regions in the N-terminus and C-terminus of most paramyxoviruses F protein, includes of other enveloped viruses, such as HIV and respiratory syncytial virus (RSV), have been identified as important to fusion, named HRland HR2. The two heptad repeat regions can form coiled coil heterotrimer in other envelope viruses. The formation of coiled coil heterotrimer could play a key role in virus mediated fusion. But it is not clear if NDV has the same fusion mechanism as other envelope viruses. In this thesis, we have clone, expressed fusion form of HRlLinkerHR2 (HR.1,2) protein. And the fusion and free form of HR1,2 were purified. Chemical crosslinking assay and gel filtration confirmed that HR1,2 can form homeotrimer. The Circular Dichroism (CD) spectroscopy showed that HR1,2 has a -helix characteristic, exhibiting double minima at 208nm and 222nm, and has high thermal stability. All those indicated that the a -helix homeotrimer formed by HR1,2 of NDV F protein is the most stable form during fusion process and NDV mediate fusion with the same mechanism as other envelope viruses.Different to the other enveloped viruses, there are 250 amino acids between HR1 and HR2. Ghosh et al, have recently noted that NDV F protein sequences contain another heptad repeat region located between HR1 and HR2. Single point mutations assy showed that a single amino acid change in the HR3 sequences of NDV F protein can alter the stringent requirement for HN protein expression in syncytium formation. To farther study the structure and function of HR3, we also have cloned, expressed the fusion form of HRl,3LinkerHR2 (HR1,3.2) protein. And, the fusion form of HR1,3,2 were purified.