The Mechanism Of Canine Distemper Virus And Canine Coronavirus Entering Host Cell

Membrane fusion between the virus envelope and host cells is the key step of the enveloped virus to enter into the host cells. This process involves the interaction between envelope proteins of virus and surface proteins of cell. The research results of Retrovirus, Filovirus, Orthomyxovirus and Paramyxovirus showed that 6-helix bundle formed by heptad repeat regions of fusion proteins was the key to start membrane fusion. The structure of 6-helix bundle of Paramyxovirus was clear, but the other correlative function regions were not clear. The research results of SARS-Cov and MHV showed that it is possible that Coronavirus and other envelope virus have the similar membrane fusion mechanism. Fusion protein of Paramyxovirus consists of fusion peptide (FP) and heptad repeat 3 (HR3), both of which contribute to membrane fusion. Canine distemper virus is a member of genus Morbillivirus in the family Paramyxoviridae. Canine coronavirus is a member of genus Coronavirus in the family Coronaviridae. Here, we get the gene of HRl, HR2, HR3, FP of canine distemper virus (CDV) and the gene of HRl, HR2 of canine coronavirus (CCV) by bridge PCR. All of them were cloned into pGEX-6p-l vector separately and expressed in the GST fusion expression system. Then the expression proteins were digested and purified. The CD and Gel-filtration results showed that HR1/HR2 protein of CDV and CCV formed a stable 6-helix bundle structure with abundance of α-helixes. It is possible that Coronavirus and Paramyxovirus have the similar membrane fusion mechanism. Comparing to SARS-Cov and MHV, the HRs regions of CCV have 14 inserted amino acids which could not affect formed 6-helix bundle. Fusion inhibition tests showed HR2 peptide of CDV could specific inhibit virus-cell fusion, but HRl peptide of CDV could not inhibit virus-cell fusion. Cross-inhibition experiments showed that HRl and HR2 have no cross-inhibition activity with Measles virus(MeV) of same genus. The secondary structure and hydrophobicity analysis of glycoprotein of CDV were predicted by the ExPASy bio-software. The results demonstrated that FP and HR3 had more hydrophilic tendency, HRl and HR2 had both hydrophilic region and hydrophobic region. The CD results showed that FP had abundance of α-helixes and HR3 had ruleless curls. The protein binding experiment showed HR3 could interact with HRl and HR2 of F protein of CDV, FP could not interact with HRl and HR2 of F protein of CDV. Fusion inhibition experiments showed HR3 peptide of CDV could inhibit virus-cell fusion and FP peptide have no inhibition activity.