Retained Scfv a Master's Thesis Prese

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

Zhenyu Li

May 2020

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.

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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

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.

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

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

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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.

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 of the viral and endosomal membranes, a key step in viral entry. HA is a homotrimer protein with two domains, HA1 and

HA2. HA1, the globular head domain, recognizes and binds cell surface sialic acid receptors. The stem domain, HA2, mediates membrane fusion (Figure 3A, top panel). The HA C-terminal

Figure 1. EM image of HIV and influenza. A) EM image of HIV. Black arrow points to HIV glycoprotein B) EM image of influenza. Black arrow points to influenza hemagglutinin (HA)

transmembrane domain anchors the protein to the viral membrane. Each HA is translated as an inactivated precursor, HA0, which is then cleaved into the metastable prefusion conformation by an exogenous peptidase20-22(Figure 3A, bottom panel). Following cleavage of HA0 into HA1 and

HA2, the N-terminus of HA2, termed the fusion peptide (Figure 3A, shown in red), translocate to

Figure 2. Influenza Virus Structure and Replication. A) Influenza Virus Life Cycle. 1) virus enters the cell by endocytosis. 2) during late endosome pH drop, HAs mediate viral and endosomal membrane fusion. 3) genomic materials are released into the cytosol and got imported into the nucleus. In the nucleus, transcription of mRNA begins. 4) New HA monomers are translated in the cytosol. HA monomers are transported into the ER where they trimerize. Then, HA trimers are transported onto cell surface. 5) New virus exits host cell by budding off from the cell. B) Cross Section Representation of Influenza Virus Particle. C) Schematic Representation of Influenza virus RNA.

a pocket in the α-helical core, termed the fusion peptide pocket. Once endocytosed into a host cell, the positively charged HA1 heads separate as the endosome acidifies due to increased electrostatic repulsion. Separation of the HA1 heads exposes the HA1-HA2 interface to water. Water molecules interact with and trigger a loop to α-helix conformational change in HA2, translocating the fusion peptide over 100Å to the endosomal membrane and allowing it to insert. The resulting extended

α-helices of the HA2 trimer form a central coiled -oil structure20,22,24. Then, a portion of the C- terminal half of the central α-helix undergoes a helix-to-loop conformational change. The C- terminal loop then inverts 180 degrees and binds to the central groove of the coiled coil structure, shortening HA2 and bringing the viral and endosomal membranes together25. HA assumes the stable post-fusion conformation after this inversion step (Figure 3A, top panel, right). The lipids in the outer leaflets of the two membranes mix in a fusion intermediate termed hemifusion, and, ultimately, a membrane pore opens up23 (Figure 3A, top panel). The virus surface, which is in direct contact with the endosomal membrane, termed the contact patch, houses about a hundred of

HAs (Figure 3B). Each HA within the contact patch has a 50% intrinsic probability to insert into the endosomal membrane24. HAs which successfully insert into the endosomal membrane are considered productive. An HA which fails to insert into the endosomal membrane and becomes inactivated is considered unproductive. If the fusion peptide fails to embed in the endosomal membrane, it will insert into the viral membrane and the HA will foldback into the post-fusion conformation. The current model suggests that membrane fusion requires three to five adjacent productive HAs to occur because of the large kinetic barrier to bring the viral and endosomal membranes together. The combined energy released from adjacent HA2 conformational changes is enough to overcome this kinetic barrier (Figure 3A, bottom panel).

Figure 3. HA Mediates Membrane Fusion. A) Low pH induced HA conformational change. Top panel: HA cleavage and conformational change. HAs are synthesized as precursor HA0. HA0 are activated by protease cleavage into HA1 and HA2. Low pH triggers HA conformational change, which moves its membrane fusion peptide sequence toward target membrane. Green ribbon diagram represents HA1. Gray ribbon diagram represents HA2. Red ribbon represents membrane fusion peptide, which is N-terminus of HA2. Bottom panel: free energy diagram of low pH induced HA conformational change. ΔE represents free energy released from HA2 conformational change. B) Representation of contact patch. After pH drop, HAs are in mixed state of prefusion, participating and non-participating. Membrane fusion occurs due to three adjacent participating HAs. Green denotes pre-fusion HA. Purple denotes participating HA. Black denotes non-participating HA. Triangle represents HAs that carries out membrane fusion.

With emerging drug resistance to current antiviral treatments, HA targeting antiviral drugs are been developed. One way for the virus to evade HA inactivation by inhibitors is resistance mutations. We recently published a manuscript exposing another viral strategy for evading HA inhibition without requiring mutation. Influenza virus produces pleomorphic particles, ranging in size from small, spherical particles about 100-nanometer in diameter, to long, filamentous particles

Figure 4. Influenza Virus HA Knockdown Approach. A) Influenza Virus Life Cycle to Produce Viruses with less HA Incorporation. ER-retained HA-targeting intrabody are synthesized in cytosol. It binds to HA in the ER and sequesters in the ER. Less HAs are transported onto cell surface so that less HAs are incorporated onto new virus. Light blue circles represent ER-retained HA-targeting intrabody. Light blue boxes represent intrabody encoding viral RNA. B) Schematic Representation of PAWT-eGFP Segment. C) Schematic Representation of PAWT-F045 Segment.

tens of micrometers in length25-28. Filamentous virus particles serve as HA pressure reservoir, so that the virus don’t have to mutate when facing extreme HA pressure29. This can provide critical information in potential drug discovery. For example, inhibitors for filamentous particles production can be used in combination with HA inhibitors to better counter influenza virus infection.

Compared with HIV surface glycoprotein, HA is densely packed on viral surface (Figure

2A-5 and 1). Both HIV GP and HA go through similar conformational changes to mediate membrane fusion30. However, the reason behind the high density of HA remains unknown. The number of HA trimers scales with particle size so that its density is uniform on particles of different sizes27,31. Published evidence using pseudotyped virus particles suggests that density of HAs on the particles surface can affect the fusion rate32. However, since those experiments are done in a nonnative context, a question of the role of density of HAs on native particles remained open.

Furthermore, our lab’s stochastic simulations make the following prediction that fusion rate decreases with increase in HA inactivation, while fraction of fused particles, termed fusion yield, does not change unless 50% or more HA is inactivated24. I hypothesize that high HA density helps membrane fusion by increasing the probability of formation of the inserted HA cluster. To address my hypothesis, I worked on generating viruses with reduced HA incorporation level. In future single-particle membrane-fusion experiments, I will measure the effect of the reduction in HA incorporation on the membrane-fusion efficiency and kinetics.

Results

I sought to reduce viral HA incorporation by knocking down HA expression on the infected-cell surface. For this, I planned to extend a viral RNA genome segment to additionally express an ER-retained HA-targeting intrabody to interfere with HA trafficking to the cell surface

(Figure 4). Intrabodies are antibodies containing the C-terminal ER-retention signal and have been used successfully to knockdown surface expression of cellular proteins33,34. By having the virus express the intrabody (instead of having it expressed independently in the cell), its expression level should correlate with the extent of infection in a given cell. Consequently, produced virus particles should have a uniform reduction in HA incorporation despite the natural heterogeneity in the extent of infection among cells in culture.

Generation of a replicating influenza virus with an extended genome segment

I first attempted to make a previously reported virus that co-expresses a secreted, HA- specific antibody 9H10 in addition to the full complement of WT PR8 virus proteins35 (Figure 5A).

The reported co-expression of the 9H10 antibody during virus infection was achieved by replacing

WT genome segments for the polymerase subunits PA and PB1 with longer segments that additionally express the 9H10 antibody light and heavy chains, respectively. I term these segments

PAPP-9H10L and PB1PP-9H10H. Hamilton et al. reported that the spherical PR8PPPol virus had

35 unchanged infectivity relative to the WT PR8 virus . Despite obtaining plasmids encoding PAPP-

9H10L and PB1PP-9H10H directly from the authors and using WT PR8 for the remainder of the segments as they have reported, my initial virus rescue attempts were not successful. Three potential explanations could account for that result. First, I found mutations within the open reading frame (ORF) of both the PAPP-9H10L and PB1PP-9H10H relative to both the authors’ maps

and the published PAWT and PB1WT coding sequences (NCBI accession number: ACV49552.1 and

ADX99492.1). Those mutations might affect the polymerase function. Second, since PR8 virus makes small spherical particles36, they might not be large enough to accommodate the extended viral genome segments. Finally, to make the 9H10-expressing genome segments, the authors had to recreate after the inserted 9H10 ORF the segment-specific packaging signals that are normally contained within the gene ORF. Those packaging signals might not have been faithfully reproduced.

Figure 5. Influenza Virus Reverse Genetic System. A) Eight-Plasmid Reverse Genetic System. Plasmids encoding eight viral RNA genomes are transfected into cells. Plasmids are transcribed into viral RNA. Because plasmids contain dual promoters, viral protein are also translated. PB1, PB2 and PA form polymerase complex, which transcribes NP coated viral RNAs into mRNAs. Newly synthesized virus leaves the cell by budding off. B) eGFP Expression by Viral Polymerase. ‘-’eGFP RNA is synthesized. eGFP viral RNA is transcribed into mRNA by polymerase complex (PB1, PB2 and PA) after coated by NP. eGFP mRNA is translated resulting in green fluorescent cells.

I first tested the activity of the PAPP and PB1PP polymerase subunits by monitoring the expression of eGFP under the control of the viral polymerase complex (Figure 5B). In this experiment, plasmids encoding the polymerase proteins PB1, PB2, and PA, and the nucleoprotein,

NP, are co-transfected with a plasmid directing the synthesis of a negative-sense eGFP RNA under the polymerase I promoter (‘-‘eGFP RNA). ‘-‘eGFP RNA is designed as a virus segment and contains the eGFP ORF sequence in place of an HA ORF and retains the authentic HA segment end sequences. The segment-end sequences ensure that the ‘-‘eGFP RNA is recognized by the viral polymerases as an authentic template for the synthesis of both the eGFP mRNA and the antigenome (‘+’eGFP RNA). The ‘+‘eGFP RNA in turn serves as the template for the ‘-' eGFP

RNA synthesis, which then leads to amplification of the eGFP mRNA ensuring robust eGFP protein expression. I compared eGFP expression in an experiment that used all WT PR8 PB1, PB2,

PA, and NP proteins with one in which PB1 and PA were replaced with PAPP and PB1PP. The cells expressing either of the two sets of proteins showed indistinguishable eGFP expression (Figure 6, panel 1 and 2). The mutations I identified in PAPP and PB1PP are thus not the reason for the unsuccessful 9H10-PR8 virus production.

To test the hypothesis that filamentous virus particles might permit packaging of the extended genome segments, I mixed and matched genome segments to make reassortant viruses that produce filamentous virus particles. Unlike the PR8 strain of influenza that makes 100nm-in- diameter isometric particles, Udorn strain produces pleomorphic particles, with lengths ranging from about 100 nanometers to tens of micrometers. Viral M1 protein and its interaction with the

M2 and NP proteins are key determinants of particle morphology25-28. I first attempted to generate

Figure 6. eGFP Expression by Viral Polymerases Result. Panel 1, eGFP expression by PAWT and PB1WT polymerases. Panel 2, eGFP expression by PAPP and PB1PP Ud polymerases. Panel 3, eGFP expression by PAWT and PB1WT with NP .

a reassorted PR8 virus with Udorn M and NP genome segments (filamentous PR8pol virus). I was able to generate the virus, but its spread was attenuated as evidenced by its smaller plaque size compared to that of the WT PR8 virus in a standard infectivity assay (Figure 7). Two reasons might account for that result. First, the PR8 polymerase complex might not efficiently recognize Udorn

NP-coated RNA (nucleocapsid) as its template. Second, the PR8 HA and/or NA might not efficiently assemble into a virus particle with Udorn M1. To test if Udorn nucleocapsid is compatible with PR8 polymerase complex, I co-transfected plasmids expressing WT PR8

Figure 7. Images of Plaque Result from Infection from Different Viruses. Plaque assays are performed with MDCK 2 cells and fixed and stained 2 days after infection. Top panel, Images of plaques by WT PR8 virus. Middle panel, Images of plaques by filamentous PR8Pol virus. Bottom panel, Images of plaques by filamentous PR8Pol HAUd Virus.

polymerase subunits, ‘-’eGFP RNA, and either the Udorn or the PR8 NP. The source of NP did not make a difference for eGFP expression (Figure 6, panel 1 and 3), showing that Udorn NP is compatible with the PR8 polymerase activity and is likely not the reason for the small-plaque phenotype of the filamentous PR8pol virus. To test if the strain origin of HA and/or NA needs to match M1, I generated a virus that additionally replaces the PR8 HA and NA segments with those

Figure 8. Filamentous PA/eGFP PR8Pol Viruses. A) Schematic representation of PAWT-eGFP genome. B) Relative Infectivity of viruses. Relative infectivity is shown as plaque forming unit (PFU) per hemagglutinin unit (HAU). PFU is determined using MDCK 2 and MDCK HA cells at 34°C.

of Udorn in the filamentous PR8Pol HAUd virus. Indeed, this virus showed normal plaque phenotype (Figure 7, top and bottom panel). Furthermore, X31 HA (another H3 HA related to

Udorn) was also compatible with WT infectivity in filamentous PR8Pol HAX31 virus. I measured the specific infectivity of both viruses as the ratio of plaque forming units to hemagglutination units (PFU/HAU). Both filamentous PR8Pol viruses have slightly lower (two to five-fold) infectivity than WT PR8 virus (Figure 8B) but within the normal range for WT Udorn or X31 viruses37. Thus, I created two gene combinations that allowed for filamentous virus particles production (data not shown) and retained PR8 polymerase segments, thus enabling me to test packaging of the extended PAPP-9H10 and PB1PP-9H10 segments.

My attempt to make filamentous virus incorporating PAPP-9H10L and PB1PP-9H10H was unsuccessful, leaving open a possibility that one or both of the extended genome segments did not recapitulate correct packaging signals. In order to enable testing of one extended segment at a time, and to facilitate the detection of the inserted-ORF expression, I decided to generate analogous

Figure 9. Cell Images after filamentous PA/eGFP PR8Pol HAUd PR8Pol Virus Infection. Virus was infecting at a multiplicity of infection of 2*10-4 PFU per cell. Top panel, Images of cells 36 hours post infection. Bottom panel, Images of cells 50 hours post infection.

constructs to PAPP-9H10L and PB1PP-9H10H that replace each antibody ORF with that of eGFP, and that use WT PR8 PA or PB1 sequences (Figure 8A). I tried to produce filamentous PR8Pol viruses incorporating either PAWT-eGFP or PB1WT-eGFP alone, or both eGFP segments together.

Only viruses carrying PAWT-eGFP segment alone could be produced, and they were produced in the context of either HA (filamentous PA/eGFP PR8Pol HAUd virus and filamentous PA/eGFP

X31 PR8Pol HA virus). In other words, neither virus including PB1WT-eGFP was produced, suggesting that the packaging signal used for PB1 genome might not be functional. Filamentous

PA/eGFP PR8Pol viruses with either X31 or Udorn HAs have indistinguishable specific infectivity from their corresponding filamentous PR8Pol viruses (Figure 8B). To test if filamentous PA/eGFP

PR8Pol HAUd virus can spread, I infected MDCK cells at a low multiplicity of infection (2*10-4

PFU/cell). Green cells were apparent in culture at 36 hours post infection, then increased in intensity as more cells turned green by 50hours post infection (Figure 9). This shows that filamentous PA/eGFP PR8Pol HAUd virus can replicate. It retained eGFP expression and unchanged specific infectivity with subsequent passages in culture (data not shown). Furthermore, filamentous PA/eGFP PR8Pol HAX31 virus also showed retained eGFP expression in infection assay and further passages (data no shown). Taken together, I have produced replicating viruses expressing eGFP and retaining WT infectivity. These viruses thus form the genetic contexts in which to express an ER-retained scFv by swapping out the eGFP coding sequence.

Generation of a functional HA-targeting scFv

I obtained expression vectors for a panel of antibodies broadly reactive against H3 HAs

(F045-092, BH151, HC19, CR8020 and C05) or against both H3 and H1 HAs (MEDI8852 and

FI6v3)38-46. I initially focused the scFv generation on the first set of antibodies because successful

downstream HA knockdown in a virus using an H3-HA binder could be complemented with H1-

HA expression during virus amplification. To facilitate the expression screen, my initial constructs all excluded the ER-retention tag, so that scFvs would be secreted into the cell culture supernatant to be purified and analyzed from there. Each scFv consisted of an antibody light (L) and heavy (H) chains connected with one of six linkers (short, medium, long-1, long-2, (G4S)3 and Yol) either in

Figure 10. Knockdown of HA Transfection on Cell Surface. A) HA transient expression by transfection. Left panel, Cross section of HA transfected cell. Right panelHA expression Western Blot. HA are probed by A2 antibody. Trypsin is added to cleave HA precursor one hour before cell lysing. Purified viruses are loaded as loading control. Total viral protein amount is determined by Bradford assay. Loading amount represents total viral protein loaded. B) co-transfection of HA and ER-retained F045-092 scFv. Left panel, Cross section of HA and F045 scFv transfected cell Light blue circles represent ER retained F045-092 ScFv. Right Panel, HA knockdown western blot. 293T cells are co-transfected with HA and ER-retained F045-092 ScFv expression plasmid. F045 represents transfected ER-retained F045-092 expression plasmid amount. HA expression plasmid amount is fixed at 2.5ug. eGFP expression plasmid is used so that total plasmid transfected is 5ug. GAPDH is used as loading control.

L to H, or H to L orientation (see Methods). Of the 5 antibodies that I tested initially for the total of 10 constructs, only one expressed sufficiently to be purified and tested for HA-binding activity.

The successful scFv derived from the F045-092 antibody and was assembled as H-yol-L (Figure

12A, top panel). In this experiment, I transfected 50ml 293F suspension cells and harvested supernatant five days later. I purified F045 scFv by affinity chromatography. Coomassie gel revealed high purity of the eluted protein (Figure 12B and C), and size exclusion chromatography revealed the expected ~35 kDa peak. I obtained about 8.5mg of purified F045 scFv from another

200ml cell culture.

I tested the F045 scFv for binding to HA on virus particles and measured its effects on HA functions in receptor attachment or membrane fusion. To test F045 scFv effects on attachments, I performed an HA-inhibition assay in the presence of an increasing F045 scFv concentration form

1.25nM to 40nM. 40nM F045 caused 2-fold reduction in measured HAU (Figure 12E). To test

F045 scFv effects on membrane fusion, I performed single-particle fusion experiment on supported lipid bilayer. Virus particles were fluorescently labeled with DiD and visualized using total internal reflection fluorescence (TIRF) microscope. Membrane fusion results in diffusion of partially quenched DiD into the supported lipid bilayer, which causes dequenching of the fluorophore.

320nM F045 scFv reduced binding of virus particles to supported membrane bilayers and inhibited membrane fusion of the successfully bound particles (Figure 12F). Both assays show that the purified F045 scFv functions to prevent HA1-receptor interaction. This experiment thus set the stage for adding the ER retention tag to the F045 scFv expression construct in order to test its ability to knockdown surface levels of co-expressed HA.

Surface HA knockdown in transfected cells by an ER-retained scFv

To avoid weeks-long process of making and passaging virus, I developed a transfection assay, which allowed me to semi-quantitatively determine the levels of both the surface exposed and intracellular HA. Without the addition of an exogenous protease, HA is produced as an inactive

Figure 11. Filamentous PA/F045 PR8Pol Viruses showed Reduced HA Incorporation. A) HA Knockdown Western Blot of Virus. PAWT and PAWT-F045 represents PA segment that the virus is carrying. Same amount of virus is loaded onto the western blot. HA are probed by A2 antibody. M1 protein is probed by GA2B antibody. B) HA Incorporation Level from Western Blot. Densitometry is performed on western blot bands. HA incorporation level is determined by HA1 density divided by M1 density. Unperturbed virus is set to have 100% HA incorporation level. Percent HA incorporation level on reassorted virus expressing F045 is determined. Results are gathered from at least three independent experiments.

HA0 trimer. Upon subsequent addition of the exogenous trypsin, only the surface exposed HA0 can be cleaved into HA1 and HA2. Intracellular HA0 remains uncleaved. I transfected HEK293 cells with an HA expression plasmid (Figure 10A, left panel). 23 hours post transfection, I treated the cells with 10ug/ml TPCK-trypsin or left a duplicate sample untreated. I lysed the cells and ran lysates on an SDS-PAGE gel for immunoblotting with an HA1-specific antibody. Without the exogenous trypsin, all HA was detected as HA0 on the immunoblots (Figure 10A, right panel).

Trypsin addition, on the other hand, resulted in most HA being detected as cleaved HA1, leaving very little unprocessed HA0. This experiment showed that under the unperturbed conditions, majority of expressed HA reached the cell surface, setting the stage for detecting ER-retention of

HA upon co-transfection of the ER-retained F045 scFv. I inserted an ER retention tag (SEKDEL) at the C-terminus of F045 and co-transfected this construct with the HA-expression plasmid

(Figure 10B, left panel). I treated all, except for one, samples with trypsin. Cells expressing the

ER-retained F045 scFv in addition to HA had lower HA1 level and higher HA0 level compared to cells expressing HA alone (Figure 10B, right panel). HA1 level decreased with the increase in the amount of the F045 scFv plasmid transfected. Furthermore, the detected HA0 level remained unchanged for the different amounts of ER-retained F045 scFv transfected, suggesting that some

HA0 might have become degraded after its retention via the ER-associated protein degradation pathway. This experiment demonstrated the proof of principle that the surface HA levels can be knocked down by co-expression of an ER-retained F045 scFv.

Figure 12. Expression of Soluble F045-092 ScFv and Testing its Function. A) Diagram of F045-092 and MEDI-8852 ScFv design. HC represents heavy chain. LC represents light chain. B) FPLC Result of F045-092 and MEDI-8852 ScFv Purification with Affinity column. 50ml transfection culture was loaded onto HisTrapHP 5ml column.

C) Coomassie gel of F045-092 ScFv purified fractions. Fractions 14-18 were run on 10% SDS-PAGE gel. Correct size of F045-092 ScFv is indicated by arrow. D) F045-092 ScFv Size determined by Size Exclusion Chromatography (SEC). Molecular weight of SEC standard vs retention volume plot. I ran size exclusion protein markers with known molecular weight on SEC so that a plot of molecular weight against retention volume can be plotted. By fitting retention volume of F045, I calculated that its native size is 35kDa, which is similar to its molecular weight (30kDa). E) HA Inhibition Assay. F045 scFv was added to inhibit HA-receptor interaction. F) Single Particle Fusion Assay with F045 scFv. Left panel: pre and post fusion images without F045 scFv. Right panel: pre and post fusion images with F045 scFv.

Production of viruses with reduced HA incorporation

In the final set of experiments, I attempted to make viruses with reduced HA incorporation by swapping the eGFP ORF in the filamentous PA/GFP PR8Pol viruses with the that of the ER- retained F045 scFv (Figure 4C). I produced the filamentous PA/F405 PR8Pol virus in the context of either Udorn or X31 HA. Because influenza virus produces mixed-size virus particles, and HA and M1 both scale with particle size27,31, the ratio in HA and M1 levels represents normalized HA

Figure 13. Sortase Labeling Reaction with MEDI-8852 FAb. A) Diagram of MEDI-8852 sortase labeling design. MEDI 8852 heavy chain has sortase tag (LPETGG) and 6xhis tag on its C-terminus. Sortase replaced terminal GG-6xhis-tag with JF549 labeled GGGK peptide. B) Coomassie gel and fluorescent gel of labeling reaction. Top panel, Denaturing gel of MEDI-8852 FAb labeling reaction. Reaction mixture of labeled MEDI and purified labeled MEDI were loaded. Serial dilution of unlabeled MEDI was used to determine the amount of labeled MEDI. Bottom panel, fluorescent image of the same gel shown above. Excessive labeled peptide is shown in reaction mixture. Labeled MEDI is shown in purified labeled MEDI.

incorporation. I ran the partially purified filamentous PA/F405 PR8Pol viruses on an SDS-PAGE gel, and detected HA and M1 by quantitative immunoblot using fluorescently labeled anti-mouse antibody in secondary detection. I loaded 3-4 different input particle amounts in order to ensure that the fluorescent detection occurred in the linear regime. HA and M1 band intensities were plotted against the amount of viral protein loaded and each was fitted with a line. I divided the slope the HA line with that of the M1 line to obtain normalized HA incorporation level.

Filamentous PA/F405 PR8Pol viruses have about 20% less HA incorporation compared to filamentous PR8Pol viruses (Figure 11B). Taken together, I have made viruses with reduced HA incorporation by co-expressing the ER-retained F045 scFv.

Discussion

The goal of my project is to study the effect of high HA density on membrane fusion efficiency and kinetics. The current fusion model suggests that 3-5 adjacent productive HAs are needed to support membrane fusion24. However, within the contact patch, where about a hundred

HAs reside, only about 50% of HAs are productive24. I reasoned that high HA density on virus particles can increase the probability of fusion by increasing the probability of 3-5 productive HA neighbors forming. To study test this hypothesis, I have generated filamentous PA/F405 PR8pol viruses that co-express an ER-retained F045 scFv that reduces viral surface HA incorporation. My results show that I have achieved about 20% reduction in HA incorporation. I will use this virus in single-particle fusion experiments to test my hypothesis. If my hypothesis turns out correct, it will show another strategy for influenza virus to evade the effects of HA inhibitors without requiring adaptive mutations.

I have successfully made fully replicating infectious viruses incorporating PAWT-eGFP genome segment. Previous attempts in the literatures to make viruses co-expressing a foreign protein resulted in significant reduction in viral infectivity47-50. The filamentous PA/eGFP PR8Pol viruses that I made showed the same infectivity as the filamentous PR8Pol viruses with normal length genome segments. Although the exact reason that these viruses retained their infectivity remains untested for now, the production of the filamentous particles might play a role in the ability of viruses to accommodate the extended genome segment. Filamentous PA/eGFP PR8Pol viruses have also provided a way for our lab to quantify infection by calculating fraction of cells expressing eGFP. They can also help us to evaluate extend of virus particle production by using fluorescent intensity of eGFP. These viruses have started projects to study cellular pathways for filamentous particle entry and determine potential inhibitors for these pathways.

I have successfully generated filamentous PA/F045 viruses that showed 20% less HA incorporation by quantitative western blot. Virus particles with uniform HA incorporation level is needed, because difference in HA incorporation level per particle can cause additional effect on fusion kinetics. However, quantitative western blot only shows average HA incorporation level in a population. Thus, I need to quantify HA incorporation per particle. I can use the single particle

HA quantification method described by Li et al with modifications29. They coated DiD-labeled virus particles with single-fluorophore labeled MEDI8852 Fab and quantified number of

MEDI8852 FAb colocalized with virus particles. However, electron cryotomography showed that

FI6 FAb, which binds to HA2 as MEDI8852 Fab, sterically blocked other neighboring HA binding site31. This suggests that number of MEDI8852 bound per HA trimer follows a distribution, which makes it hard to determine number of HA per virus particle. In order to make sure each HA monomer is bound with inhibitors, smaller scFv can be used to avoid steric hindrance toward

neighboring HA binding site. I have developed a sortase labeling reaction protocol to singly label

MEDI8852 FAb with JF549 fluorophore (Figure 13). I have also expressed and purified F045-092 scFv with sortase tag and labeled GGGK peptide with JF549 fluorophore, necessary for sortase reaction. I would have already performed sortase reaction with F045-092 scFv and single particle quantification experiment if I didn’t have to go home due to COVID-19 outbreak.

My results show that I only have 20% reduction in HA incorporation. Previous research has shown that up to 50% HA inactivation by inhibitors doesn’t change fusion yield but only increases hemifusion time24. In order to have effect on fusion yield, I need to generate viruses with further reduction in HA incorporation. There are two strategies to achieve this. First, I can extend

PAWT with two copies of scFv. Second, I can swap out F045 scFv with other scFvs with higher expression and HA affinity. As a proof of principle for the first strategy, I have extended PAWT segment with eGFP and mCherry ORFs (PAWT-eGFP-mCherry). I can incorporate the genome segment into virus. The virus showed reduce in infectivity and didn’t passage well. I am confident that I can solve these problems and make a virus expressing two stochiometric quantity of scFvs.

In order to achieve the second strategy, I have expressed and purified soluble MEDI8852 scFv.

Compared with F045 scFv, MEDI8852 demonstrated more robust expression (Figure 12B). From

200ml suspension culture, I was able to purify about 30mg of MEDI8852 scFv, while I only got

8.5mg for F045-092 scFv. MEDI8852 FAb also has higher affinity (4 to 40-fold) for HA than

F045-092 FAb45,51. Since scFv has same paratope as FAb, and both FAb and scFv bind HA in one- to-one ratio, MEDI8852 scFv should have higher affinity than F045-092 scFv. Thus, viruses co- expressing ER-retained MEDI8852 scFv should have further reduction in HA incorporation. Using both strategies, I should be able to make virus with more than 50% reduction in HA incorporation.

This allows me to test effect of HA reduction with fusion yield.

Materials and Methods

Plasmids

Protein Expression Plasmids scFv nucleotide sequences (Table 1) were cloned into pVRC8400 vector, with a Tissue plasminogen activator signal peptide sequence (MKRGLCCVLLLCGAVFVSPS) in the N- terminal, and a Sortase tag (LPETGG) followed by a His6 tag in the C-terminal. All nucleotide sequences ware codon optimized (Integrated DNA Technology, Iowa) and cloned in pVRC8400 plasmid generated by NheI and NotI restriction digestion (New England Biolabs, Massachusetts).

Gene block of F045-092 scFv was synthesized (IDT, New Jersey) and inserted into the pVRC8400 vector by HiFi DNA assembly (New England Biolabs, Massachusetts). For HA knockdown, the

Sortase sequence was removed and ER-retention signal (SEKDEL) was inserted after His6 tag to generate the ER-retained F045 scFv plasmid.

Table 1: scFv design.

Antibody Identity Chain Orientation Linker

HC19 H to L* (G4S)3: GGGGSGGGGSGGGGS REF?

HC19 H to L Long-1: GSTSGSGKPGSGEGSTKG

HC19 L to H* Medium: GSSRSSSSGGSSRSS

CR8020 H to L (G4S)3

CR8020 H to L Short: GGSSRSS

CR8020 H to L Long-2: SSGGGGSGGGGGGSSRSS

CR8020 H to L Yol: ASTKGPSVKLEEGEFSEARV

F045-92 H to L Yol

BH151 H to L Yol

C05 H to L Yol

MEDI8852 H to L Yol

FI6v3 H to L Yol

*H to L means heavy chain variable region is upstream of light chain variable region *L to H means light chain variable region is upstream of heavy chain variable region

Plasmids encoding viral RNA genome pDZ PA LC 9H10 and pDZ PB1 HC 9H10 plasmids, encoding PR8 PAPP and PR8 PB1PP RNA genome segments respectively47, were generous gifts from Peter Palese. pHW 193 PA plasmid is modified to encode extended PAWT-eGFP segment (pHW 193 PA-eGFP plasmid). To generate plasmid, I’ve amplified the mutated packaging signal and 2A peptides separately from pDZ PA

LC 9H10 plasmid. Both amplified mutated packaging signal and 2A peptide were inserted into linearized pHW 193 PA plasmid. eGFP ORF was ligated after the 2A peptide by HiFi DNA assembly (New England Biolabs, Massachusetts). pHW 193 PA F045-092 KDEL plasmid was modified from pHW 193 PA eGFP plasmid. eGFP ORF was swapped with F045-092 KDEL ORF.

Cell maintenance

MDCK, MDCK 2, MDCK HA and HEK 293T cells were maintained in Dulbecco's Modified

Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) HEK 293 F cells were cultured in suspension in Freestyle293 expression medium (Gibco, Massachusetts).

Virions

Virus RNA Genome Composition

Spherical PR8PPPol virus, expressing 9H10 IgG, contains PR8 PAPP and PR8 PB1PP RNA genome segments with other genomes from A/Puerto Rico/8/34 (PR8) strain. Influenza viral genomes, isolated from A/Aichi/2/1968 (X31), A/Udorn/307/1972 (Udorn) and PR8 strains, were mixed and matched to make reassorted viruses. Filamentous PR8pol virus contains NP and M genomes segments from Udorn strain, and other genome segments from PR8 strain. Filamentous PR8Pol

HAUd virus has PR8 PB2, PB1, PA and NS genome segments and Udorn HA, NA NP and M genome segments. Filamentous PR8Pol HAX31 virus has the same genome composition except for

X31 HA segment in place of Udorn HA segment. Filamentous PA/eGFP PR8Pol viruses contain same genome segments as Filamentous PR8Pol viruses and with PAWT-eGFP segment.

Filamentous PA/F045 PR8Pol viruses contain same genome segments as Filamentous PR8Pol viruses and with PAWT-F045 segment.

Virus Primary Rescue and Passage

Viruses were rescued as described before with modifications52,53 (Figure 5A). 2.4*105 HEK 293T cells and 4*105 MDCK HA cells (per well) were cocultured in 6-well plate one day before the transfection. On the day of transfection, 16ul of TransIt LT was added to 8ug of DNA in 200ul

OptiMeM and incubated at room temperature for 15 minutes, according to manufacturer’s instructions. Cocultured cells were cultured in OptiMEM with 0.1% FBS for transfection. The media was changed to OptiMeM with 1ug/ml TPCK-trypsin 24 hours post transfection. 3-4 days post transfection, virus containing supernatant was harvested by centrifuging at 1,000g for 10 minutes. HA assays and plaque assays (see below for details) were performed for harvested virus.

All viruses were passaged and purified as previously described7. In brief, viruses were passaged at

multiplicity of infection (MOI) 0.001 (plaque forming unit per cell) at 34°C in MDCK or MDCK

HA cells. All viruses were propagated in the presence of 1ug/mL TPCK-trypsin. Infections would continue for 2-3 days before harvesting. Viruses were harvested, and HA assay and plaque assay were performed as described above.

HA assay and plaque assay

In HA assay, HA binds sialic acid receptors on chicken red blood cells. This interaction can form lattice network, which prevent red blood cells from sinking to the bottom of point well and forming a blood dot. 0.5% chicken red blood cells were mixed with 2-fold serial diluted virus in pointy 96- well plate. The plate was stored on ice for 1-2 hours to allow red blood cell to settle.

Hemagglutination unit is determined as highest virus dilution before blood dot appears. For HA inhibition assay (HAI), F045 scFv was added during virus dilution. F045 concentrations were two- fold higher than final concentration. Viruses were preincubated with F045 scFv for 30 mins at room temperature, before 0.5% red blood cells were added. For plaque assays, serial dilution of the virus was used to infect monolayer of cells for 1 hour at room temperature, before overlayed with 0.6% oxoid agar in OptiMem with 2ug/ml TPCK-. 2-3 days post infection, cells were fixed by 3.7% formaldehyde in PBS and stained by crystal violet. Plaques were imaged under 40x magnification with bright field microscope.

GFP expression by viral polymerases

Plasmids encoding viral polymerase and eGFP was co-trasnfected into HEK 293T cells. Same transfection condition was used as virus rescue with modifications. In brief, 4 plasmids encoding

PR8 PA, PB1, PB2 and NP (pHW 191 PB2, pHW 192 PB1, pHW 193 PA and pHW 195 NP) with

pHH21 eGFPORF-HAends plasmid, directing the synthesis of ‘-’eGFP RNA. For experiment

testing PAPP and PB1PP polymerases, pDZ PA LC 9H10 and pDZ PB1 HC 9H10 plasmids were transfected, in place of pHW 192 PB1 and pHW 193 PA plasmids. For experiment testing Udorn

NP, pCDNA Udorn NP plasmid was transfected, instead of pHW 195 PA plasmid. 24 hours post transfection, cells were imaged using EVOS microscope using both brightfield and green fluorescent settings.

Knockdown of transfected HA

One day prior to transfection, 6.5-7.5*105 HEK 293T cells were seeded in each well of a six well plate. On the day of transfection, 5ug of DNA was mixed with 7.5ug of PEI in 140ul of OptiMeM.

2.5ug of pCIneo X31 HA plasmid was used for HA expression, pVRC8400 F045-092 KDEL plasmid was transfected to express ER-retained F045-092 ScFv. pCDNA4 eGFP was added to ensure total DNA per transfection is 5ug. HEK 293T cells were transfected in OptiMeM supplemented with 0.1% FBS for 23 hours, then media was replaced by OptiMeM with 10ug/mL

TPCK-trypsin to cleave HA on cell surface at 37°C for one hour for trypsinized samples. Cells are dislodged by pipetting and harvested by centrifuge at 200g for 2 minutes. Cells were lysed by lysis buffer (10mM HEPES pH 7.5, 150mM NaCl, 0.5% Triton X-100, 1mM PMSF, 1X cOmplete Mini

EDTA-free Protease Inhibitor Cocktail) on ice for 30 minutes and clarified by centrifuge at 500g for 15 minutes. Cell lysate was boiled with sample buffer at 95C for 5 minutes, before separated by 10% SDS-PAGE gel. Purified X31 Virus was loaded as markers for HA1 and HA0. Total protein mount in viruses are determined by Bradford assay. Protein was transferred onto a 0.45um

PVDF membrane. HA was blotted using 1:10,000 diluted A2 antibody (1.75mg/ml stock) in 5% milk in TBST (10mM Tris, 150mM NaCl, 0.1% Tween 20 pH 7.5) at 37°C for 4 hours. GAPDH

was blotted using 1:10,000 dilution of 1E6D9 (Proteintech, Illinois, 1mg/ml) antibody at 4°C overnight. 1;10,000 dilution of anti-mouse-HRP (Promega, Wisconsin, 1mg/ml stock) in 5% non- fat milk in TBST was incubated at room temperature for 1 hour with PVDF membrane. ELC plus

Western Blotting Detection System (GE Healthcare, Illinois) was used to blot.

Virus HA incorporation level western blot

Various HA units of virus were separated by 10% SDS-PAGE gel and transferred onto a 0.45um

PVDF membrane. HA was blotted as previously described. M1 protein was blotted with 1:10,000 diluted GA2B antibody (Abcam, UK, 1mg/ml stock) at 4°C overnight. 1:5,000 dilution of fluorescent-tagged anti-mouse antibody (Invitrogen, California, 2mg/ml stock) was used as secondary antibody. The membrane was imaged using Geldoc imager. Densitometry was performed on HA bands and M1 bands. Band intensity versus virus loading amount was plotted.

HA expression level is calculated by the ratio between slope of HA and slope of M1. At least three independent experiments were performed.

scFv expression and purification

HEK 293F cells were transfected with scFv expression plasmids (MEDI8852 and F045-092) using

PEI as transfection reagent. HEK 293F cells were centrifuged at 200g for 5 minutes to pellet and resuspended in fresh, prewarmed freestyle 293 media at density of 2*106 cells/ml before transfection. 200ug of plasmids were used to transfect 2 *108 cells with 300ug of PEI.

Freestyle293 expression medium was added to reach a final cell density of 1*106 cells/ml at 4 hours post transfection. Cell supernatant was harvested at 5 days post transfection at 2000g for 10 minutes. Recombinantly expressed protein from clarified supernatant was purified by HisTrap HP

(GE Healthcare, Illinois) column. A gradient of 30-350mM imidazole in His elution buffer (10mM phosphate, 500mM NaCl pH 7.5) was used for elution. Fractions was separated on 16% SDS-

PAGE and stained by Coomassie Blue. Fractions with scFv was pooled, concentrated with 10 kDa concentrator (Milliporesigma, Massachusetts), and buffer exchanged into PBS. F045-092 scFv was further purified with Superdex 75 10/300GL (GE healthcare, Illinois). Gel filtration standard

(BIO-RAD, California) was loaded onto Superdex 75 10/300GL according to manufacturer’s instructions. 3 column volume of PBS was used to elute at flow rate of 0.75ml/min. Purified protein stored in 10% glycerol in PBS at -80°C.

Sortase labeling reaction

Sortase labeling reaction is performed as described29.

Single particle membrane fusion assay

Single particle membrane fusion assay is performed as described29.

Work Cited

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