The Mechanism Of Paramyxovirus Entering Host Cell

Membrane fusion between the virus envelope and host cells is the first step of the enveloped virus entry into the host cells. This process involves the interaction of viral envelope proteins and their cellular receptors (proteins or sialic aids), which leads to the conformational changes of the envelope proteins. Paramyxovirus might adopt a similar molecular mechanism of membrane fusion with other class I viruses, such as human immunodeficiency virus (HIV), in which the hemagglutinin-neuraminidase (HN) glycoprotein binds receptor/s and triggers the conformational changes of the fusion (F) protein. In this thesis, two paramyxiviruses, Sendai virus (SeV) and Avian paramyxovirus-2 (APMV-2), were selected to study the function and structure of the F and HN proteins to further our understanding of the molecular mechanism of paramyxovirus fusion or entry. In both cases, the heptad repeat (HR) regions of HRl and HR2 from the F protein were expressed in E coli and the purified proteins were tested for their interaction to form 6-helix bundle (trimer-of-hairpins), the post-fusion conformation of the fusion core of the paramyxovirus. In the meanwhile the inhibition test of the HRl and HR2 of the APMV-2 was carried out. Moreover the binding domains between HN and F HR1/HR2 of APMV-2 were tested as well. The CD and Gel-filtration results showed that the HRl-linker-HR2 protein of SeV formed a stable 6-helix bundle structure (denatured Tm 95℃), and the protein crystal grew in the condition of 0.1 M citric acid pH 2.5, the obtained data of crystal diffracting X-rays testified that the protein formed typical trimer-of-hairpins structure. The study showed that only soluble HR2 peptide of APMV-2 had potent and specific virus-cell fusion inhibition activity, and had not cross-inhibiting activity with APMV-1, Newcastle disease virus (NDV). The cleaved HRl and HR2 peptides of APMV-2 formed a stable 6-helix bundle structure judged by CD and Gel-filtration (denatured Tm over 90℃). The secondary structure and hydrophobicity analysis of glycoproteins of APMV-2 were predicted by the bio-software. These results were helpful for identification of interaction domain using interacting experiment. The study demonstrated that the HR2 had a more hydrophilic region, HRl and the globular head domain of HN had a hydrophilic and hydrophobic regions, HRl had a-helix structure, and the HR2 and the globular head domain of HN had 3 -sheet folding tendency. The study showed that the globular head domain of HN could interact with HRl and HR2 of F protein.This study obtained data of postfusional trimer-of-hairpins conformation of functional region of SeV fusion protein, and pointed out the HR2 peptide of APMV-2 was potent and specific virus-cell fusion inhibitor, these testified that the SeV and APMV-2 adopt a similar molecular mechanism of membrane fusion with HIV. The studies sought interaction domain of the two glycoproteins of the APMV-2, and this completed the molecular mechanism in which HN protein activate the F protein inducing membrane fusion.