Abstract
Membrane fusion is a common process for all enveloped viruses to penetrate host cells. Influenza viral surface glycoprotein, hemagglutinin (HA) mediates viral membrane fusion with the endosomal membrane. Viral surface is densely covered by hundreds of HAs, and about a hundred of them reside at the viral and endosomal contact interface (contact patch). Within the contact patch, three to five adjacent HAs need to insert into the endosomal membrane and fold back cooperatively to induce membrane fusion. However, about half of the HAs fail to insert into the target membrane and instead become inactivated. I hypothesize that the high density of HAs on the virus particle increases the probability that the cluster of adjacent HAs can form and induce membrane fusion. To address this hypothesis, I made an influenza virus which reduces its own HA incorporation into particles by co-expressing in an infected cell an HA-targeting, ER-retained antibody (intrabody) encoded by an extended viral genome segment. This approach ensures uniform HA reduction on the viral surface, because intrabody expression correlates with the extent of viral replication in a given cell. First, I mixed and matched RNA genome segments from the PR8 and Udorn influenza strains to generate reassorted viruses that produce filamentous particles to help incorporate into particles a genome segment extended by the eGFP sequence. These viruses showed similar infectivity compared to viruses carrying genome segments of the normal length and induced eGFP expression in the infected cells. I then co-transfected HA and an ER-retained single-chain antibody (scFv), and my results showed that HA level on the cell surface decreased proportionally to the amount of scFv transfected. Finally, reassorted viruses, expressing ERretained scFv in place of eGFP showed about 20% HA incorporation reduction. Thus, I have created a system to generate virus particles with less HA incorporation. Further reduction in HA incorporation could be achieved by incorporating two copies of the same scFv into the virus genome, or a different scFv with higher expression level and HA-binding affinity. The effect on membrane fusion kinetics of the reduction in HA incorporation into virus particles can then be quantified using single-particle membrane fusion experiments.