Reduction in Viral Surface Hemagglutinin Incorporation by Co-expressing HA-targeting ER- retained scFv A Master’s Thesis Presented to The Faculty of the Graduate School of Arts and Sciences Brandeis University Department of Biochemistry Dr. Tijana Ivanovic, Advisor In Partial Fulfillment of the Requirements for the Degree Master of Science by Zhenyu Li May 2020 Copyright by Zhenyu Li 2020 Acknowledgement I would like to thank Dr. Ivanovic for giving me this wonderful opportunity to work on this project. I would like to thank Dr. Tian Li and Erin Deans for their help for the past two years for my experiments and writing. I want to thank Ellen Nguyen for her virus infectivity data. Lastly, I want to thank the Ivanovic lab for all the support and love. iii ABSTRACT Reduction in Viral Surface Hemagglutinin Incorporation by Co-expressing HA-targeting ER- retained scFv A thesis presented to the Faculty of the Graduate School of Arts and Sciences of Brandeis University Waltham, Massachusetts By Zhenyu Li 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 iv 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 ER- retained 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. v Table of Contents Introduction ..................................................................................................................................... 1 Results ............................................................................................................................................. 8 Generation of a replicating influenza virus with an extended genome segment ....................................... 8 Generation of a functional HA-targeting scFv ........................................................................................ 15 Surface HA knockdown in transfected cells by an ER-retained scFv..................................................... 18 Production of viruses with reduced HA incorporation ........................................................................... 21 Discussion ..................................................................................................................................... 22 Materials and Methods .................................................................................................................. 25 Work Cited .................................................................................................................................... 32 vi List of Figures Figure 1. EM image of HIV and influenza. .................................................................................................. 2 Figure 2. Influenza Virus Structure and Replication. ................................................................................... 3 Figure 3. HA Mediates Membrane Fusion. ................................................................................................... 5 Figure 4. Influenza Virus HA Knockdown Approach. ................................................................................. 6 Figure 5. Influenza Virus Reverse Genetic System. ..................................................................................... 9 Figure 6. eGFP Expression by Viral Polymerases Result. .......................................................................... 11 Figure 7. Images of Plaque Result from Infection from Different Viruses. ................................................ 12 Figure 8. Filamentous PA/eGFP PR8Pol Viruses. ...................................................................................... 13 Figure 9. Cell Images after filamentous PA/eGFP PR8Pol HAUd PR8Pol Virus Infection. ....................... 14 Figure 10. Knockdown of HA Transfection on Cell Surface. ............................................................................. 16 Figure 11. Filamentous PA/F045 PR8Pol Viruses showed Reduced HA Incorporation. ........................... 18 Figure 12. Expression of Soluble F045-092 ScFv and Testing its Function. .............................................. 20 Figure 13. Sortase Labeling Reaction with MEDI-8852 FAb. ................................................................... 21 vii Introduction Influenza virus is an enveloped virus, which incorporates eight, negative-sense RNA genome segments (Figure 2B). During the 1918 H1N1 influenza pandemic, it is estimated that one third of the world’s population was infected, resulting in about 50 million deaths1. The 1918 influenza pandemic was the most devastating influenza pandemic in history2. Today, even with an increased standard of care, a pandemic comparable to the 1918 influenza pandemic is still possible2. Annual vaccination has widely been used as a prevention method for influenza outbreaks. A recent study by the CDC during the 2018-2019 influenza season shows that if the circulating strains are included in the vaccine, vaccination will reduce illness 40% to 60% among all populations3. However, predictions of circulating strains are not always accurate. Thus, vaccination efficacy varies each year. Apart from preventative methods like vaccination, antiviral drugs are used as treatments for influenza virus infection. Current FDA approved anti-viral drugs target neuraminidase (NA). However, drug resistance to current anti-viral treatments has emerged4,5. Thus, more detailed understanding of influenza virus can help us control and prevent potential pandemics such as in 1918. The influenza virus infection begins when the influenza surface glycoprotein hemagglutinin (HA) binds to sialic acid receptors on host cells (Figure 2A-1). Once bound to host cell sialic acid receptors, influenza virus enters the host cell via receptor-mediated endocytosis6. Upon endosomal acidification, M2 proton channels acidify the viral core, causing matrix protein underneath viral membrane (M1 protein) to release the viral ribonucleoproteins (vRNP)7. Each vRNP consists of one viral RNA genome segment coated by nucleoproteins (NP) and one copy of the viral polymerase complex. The viral polymerase complex is composed of the polymerase basic protein 1 (PB1), the polymerase basic protein 2 (PB2) and the polymerase acidic protein (PA)8. 1 Within a vRNP, NP interacts with RNA and the polymerase subunits PB1 and PB29. Acidification of the endosome also triggers HA to undergo large-scale conformational changes, mediating membrane fusion between the endosomal and viral membranes 10,11(Figure 2A-2). Following membrane fusion, the vRNPs are released into the cytoplasm and are actively imported into the nucleus12 (Figure 2A-3). Inside the nucleus, viral RNA is transcribed into mRNA by the viral polymerase complex13. After transcription, mRNAs are transported into the cytoplasm where cellular ribosomes translate them into viral proteins13,14 (Figure 2A-4). Newly formed viral RNA segments are formed into vRNPs. Surface glycoproteins HA and NA are transported to the cell surface. HA and NA bend the local membrane, which marks the start of viral budding15-17 (Figure 2A-5). The matrix-1 (M1) protein is recruited to the budding viral particle. Eight fully formed vRNPs interact with the recruited M1 proteins at the cell surface18. If more than eight vRNPs are incorporated at this step, progeny virions will not be infectious20. NA then cleaves the interaction between HA and a host cell sialic acid receptor, releasing the viral particle19. The influenza surface glycoprotein HA mediates fusion