Mechanistic Analysis of Polymer-Attached Inhibitors of Influenza Virus and Their Effect on Minimizing Drug Resistance

Mechanistic analysis of polymer-attached inhibitors of influenza virus and their effect on minimizing drug resistance

Chia Min Lee

B.Sc. Life Sciences (Biomedical Science) National University of Singapore, Singapore, 2006

SUBMITTED TO THE PROGRAM IN COMPUTATIONAL AND SYSTEMS BIOLOGY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY IN COMPUTATIONAL AND SYSTEMS BIOLOGY

at the

MASSACHUSETTS INSTITUTE OF TECHNOLOGY

September 2012

Signature of author Program in Computa onal and Systems Biology July 27, 2012

Certified by Alexander M. Klibanov, Ph.D. Firmenich Chair Professor of Chemistry & Bioengineering Thesis co-advisor

Certified by Jianzhu Chen, Ph.D. Ivan R. Cottrell Professor of Immunology and Professor of Biology Thesis co-advisor

Accepted by Christopher B. Burge, Ph.D. Professor of Biology and Biological Engineering Director, Computational and Systems Biology Graduate Program

Mechanistic analysis of polymer-attached inhibitors of influenza virus and their effect on minimizing drug resistance

Chia Min Lee

Submitted to the Computational and Systems Biology Program August 31st, 2012 in partial fulfillment of the requirements for the degree of

Doctor of Philosophy in Computational and Systems Biology

Abstract

With the emergence of the 2009 A(H1N1) pandemic influenza virus and the rapid spread of drug resistance in recent years, the need to develop new anti- influenza drugs that can reduce the emergence of new resistant viruses is both urgent and important. This thesis explores the use of polymer-attached inhibitors as a new approach in the development of anti-influenza drugs, with particular focus on polymer-attached zanamivir (ZA). We have previously shown that covalently conjugating multiple copies of ZA via a flexible linker to poly-L-glutamine greatly enhances antiviral potency.

In the first study, we have elucidated the mechanism of this phenomenon. Like ZA itself, the polymer-attached inhibitor binds specifically to viral neuraminidase and inhibits both its enzymatic activity and the release of newly synthesized virions from infected cells. In contrast to monomeric ZA, however, the polymer-attached drug also synergistically inhibits virus-endosome fusion, thus contributing to the dramatically increased antiviral potency.

Next, we went on to investigate polymer-attached ZA's effect on the emergence of drug resistance. We found that viruses adapted rapidly to growing in high concentrations of monomeric ZA, whereas viral growth remained inhibited by low concentrations of polymer-attached ZA even after 23 passages in cell culture. Sequencing analysis established the emergence of an amino acid substitution known to confer ZA resistance (E119G in neuraminidase) after 8 passages of monomeric ZA selection. In contrast, virus grown in polymer-attached ZA remained free of substitutions in E119, and other known resistance-associated residues. We instead found novel substitutions in hemagglutinin (R220G, D241G) and neuraminidase (G111D), which emerged during passages 14-17. Importantly, although the drug- selected variants were resistant to monomeric ZA, the viruses remained susceptible to low pM concentrations of polymer-attached ZA itself.

3 Taken together, these data demonstrate that attaching the drug to a polymeric chain (i) confers a new mechanism of antiviral action; (ii) significantly delays the emergence of drug resistance; and (iii) enhances potency against the selected ZA-resistant variants. The studies presented in this thesis provide further impetus for the use of polymer-attached inhibitors as influenza therapy, and as tools for better understanding of influenza biology.

Thesis co-advisor: Alexander M. Klibanov, Ph.D. Title: Firmenich Chair Professor of Chemistry & Bioengineering, M.I.T.

Thesis co-advisor: Jianzhu Chen, Ph.D. Title: Ivan R. Cottrell Professor of Immunology and Professor of Biology, M.I.T.

4 ACKNOWLEDGEMENTS

This thesis work would not have been possible without the support of many to whom I am deeply indebted.

First and foremost, I would like to thank my advisors Professor Alexander M. Klibanov and Professor Jianzhu Chen for giving me the invaluable opportunity to work with them. Both Alexander and Jianzhu are incredible mentors, and have always been generous with advice and support. I have learned so much from Alexander's experience, valuable insights into science and life, and witty humor, and from Jianzhu's knowledge in biology, patience, and optimism. My interactions with them have been instrumental in my personal growth, and in shaping my career as a scientist.

I thank my thesis committee members, Professor Jonathan A. King and Professor Darrell J. Irvine, for their valuable feedback and advice for my thesis work, and for their insight into biology and polymer research, which I deeply appreciate.

I am immensely grateful to our resident chemists Prof. Jayanta Haldar and Alisha K. Weight, without whom this project would not have taken off, and Prof. Ling Wang, for her enthusiasm and tireless help in the drug resistance project.

I am very thankful to all past and present members of the Chen and Klibanov labs. They are the most brilliant, helpful, and kindest colleagues one could ever ask for. Thank you Steve Chen, whom I have had a privilege of sharing a bay with for all this time. Thank you Guangan Hu, Yingzhong Li, and Adam Drake for being ever so generous with suggestions, feedback, and technical advice. Thank you Camille Jusino and Irene Chang for being such enthusiastic food and chat buddies. Thank you Zhuyan Guo and Bettina Iliopoulou for their friendship, motherly care, and advice. Thank you Pete Bak, Feng Shi, Ching Hung Shen, Amanda Souza, Marisha Mikell, Ryan Phennicie, Bryan Hsu, Alyssa Larson, Roger Nassar, Carol Koh, Mobolaji Olurinde, Eileen Higham, Oezcan Talay and Ed Browne, for all their help and for making the lab such a great environment to work in.

My life in graduate school would not have been complete without my dear friends- Alice Lo, Leah Octavio, and Mei Lyn Ong. I am enormously grateful for their unconditional friendship, support, and belief in me, through good times and bad. I will always miss our times together in MIT.

Finally, a special thank you to my husband Jeffrey Tiong and my family. Words cannot express my gratitude for all their love and support throughout the countless Skype calls and long-distance flights.

5 TABLE OF CONTENTS

Title Page ...... 1

Abstract ...... 3

Acknow ledgem ents ...... 5

List of Figures ...... 8

List of Tables ...... 10

Chapter 1: Introduction ...... 11

1.1 Influenza virus ...... 11

1.2 Prophylaxis and treatm ent of influenza virus ...... 15

1.2.1 Licensed prophylactics and treatm ent ...... 15

1.2.2 Antivirals under clinical evaluation ...... 15

1.2.3 Conclusion ...... 18

1.3 M otivation of this thesis and thesis outline...... 20

1.4 Significance...... 21

References...... 24

Chapter 2: Polymer-attached zanamivir synergistically inhibits both early and late steps in influenza virus infection ...... 29

Abstract ...... 29

Introduction ...... 30

Results ...... 31

Discussion ...... 37

M aterials and M ethods ...... 40

Figures and Tables ...... 47

Supplem entary Figures and Tables ...... 59

6 References ...... 60

Chapter 3: Polymer-attached zanamivir delays the emergence of drug resistance ...... 67 Abstract ...... 67

Introduction ...... 69

Results ...... 71

Discussion ...... 77

Materials and Methods ...... 81

Figures and Tables ...... 86

Supplem entary Figures and Tables ...... 93

References ...... 95

Chapter 4: Summary of Findings, Discussion and Future Directions ...... 98

Sum m ary of Findings and Discussion ...... 98

Future Directions ...... 102

References ...... 104

7 LIST OF FIGURES

Chapter 1:

Figure 1 Schematic diagram of influenza A virus ...... 13

Figure 2 Stages of influenza virus replication ...... 14

Figure 3 Influenza antivirals ...... 19 Figure 4 Zanam ivir derivatives ...... 23

Chapter 2:

Figure 1 PGN-ZA binding to influenza virus...... 47

Figure 2 PGN-ZA inhibits the release of newly synthesized viruses from in fected cells ...... 4 8

Figure 3 PGN-ZA inhibits an early step of influenza virus replication...... 49

Figure 4 PGN-ZA synergistically inhibits early and late steps of influenza virus in fection ...... 50

Figure 5 PGN-ZA causes neither viral aggregation nor any direct virucidal activ ity ...... 5 1 Figure 6 PGN-ZA does not cause viral aggregation ...... 52

Figure 7 PGN-ZA does not inhibit binding and endocytosis of influenza virus in fectio n ...... 5 3

Figure 8 PGN-ZA inhibits virus-endosomal fusion ...... 55 Figure 9 PGN-ZA protects influenza virus from acidic (pH 5) inactivation ...... 57

Figure S1 PGN-ZA binds to neuram inidase ...... 59

Figure S2 PGN-ZA does not cause viral aggregation ...... 60

Figure S3 PGN-ZA does not inhibit hemagglutination ...... 61 Figure S4 pH 7 controls for acidic (pH 5) inactivation assay ...... 62

8 Chapter 3:

Figure 1 Viral growth in the presence of increasing concentrations of ZA and P GN -ZA ...... 8 6

Figure 2 Amino acid changes in HA and NA genes of selected passages of virus grow n in ZA or PGN-ZA ...... 87

Figure 3 Localization of amino acid changes identified in this study on the three-dimensional structure of HA and NA ...... 88

Figure 4 Plaque reduction assays of parental wild type virus, and MDCK- passaged viruses in the presence or absence of PGN-ZA or ZA...... 89

Figure S1 NA enzymatic activity of the parental wild type and the MDCK- passaged viruses ...... 93

9 LIST OF TABLES

Chapter 2:

Table 1 Inhibition constants (Ki) of viral neuraminidase by ZA-linker and PGN- ZA against WSN and PR8 influenza strains ...... 58 Chapter 3:

Table 1 List of amino acid substitutions in the final passages of virus cultured in the presence or absence of either PGN-ZA or ZA and their receptor binding specificity ...... 90

Table 2 Plaque reduction assay with ZA and PGN-ZA ...... 91

Table 3 Inhibition of NA enzyme activity by ZA and PGN-ZA ...... 92

Table S1 List of prim er sequences ...... 94

10 Chapter 1: Introduction

Chapter 1 INTRODUCTION

1.1 Influenza A virus Influenza A virus is a negative-sense, single-stranded RNA virus from the

Orthomyxoviridae family. It causes highly contagious infections of the human respiratory tract (1). Millions of people are struck by influenza infection each year, resulting in more than 500,000 deaths worldwide. In the US alone, annual influenza epidemics affect 6-15% of the population, causing severe suffering and economic loss (2, 3). During a pandemic, the effects are far more deadly. An estimated 50 million lives were lost in the 1918 "Spanish flu" pandemic, widely regarded as the most devastating pandemic in recorded world history (4).

Influenza virus is an enveloped virus containing eight genome segments which encodes for ten proteins (Figure 1) (1). The three largest RNA segments encode for the proteins which make up the RNA-dependent RNA polymerase complex: PB1, PB2, and PA.

The next three segments encode for hemagglutinin (HA), nucleoprotein (NP) and neuraminidase (NA). Other proteins encoded by the remaining segments are the M1 matrix protein, M2 ion-channel protein and non-structural proteins NS1 and NS2. There are three proteins on the viral surface: glycoproteins HA and NA, the two principal viral antigens (5), and the M2 ion channel. The main functions of HA are binding to the host receptor sialic acid and mediating viral and endosomal membrane fusion (6), whereas NA is known to facilitate the release of newly synthesized viruses from infected cells by cleavage the sialic

11 Chapter 1: Introduction acid receptor (7, 8). These roles of HA and NA, and that of other viral proteins in the life cycle of influenza virus are depicted in Figure 2.

The widespread 2009 A(H1N1) pandemic, constant antigenic drift of seasonal influenza, and the rapid emergence of antiviral resistance in recent years highlight the ability of this pathogen in adapting to the human population and in evading antiviral drugs

(9). The survival and persistence of influenza virus can be attributed to several of its unique properties. First, the virus transmits easily from person to person by aerosol, and spreads globally through travel and migratory birds. Second, the error-prone viral RNA polymerase lacks proofreading ability, resulting in a high mutation rate of 1.5 x 10-s per nucleotide per infection cycle (10). Given the size of the influenza genome of 15,000 bases, this corresponds to an average of one mutation for every 10 viruses produced by an infected cell. This error-prone replication, coupled with the selective pressure of the immune system readily promotes the antigenic variation in the viral proteins HA and NA amongst seasonal influenza strains (11). The high mutation rate is also the driving force behind the appearance of drug resistant strains, either in naturally occurring variants, or by drug selection pressure in treated patients (12-14). Third, the segmented nature of the influenza genome allows for mixing or reassortment of the eight viral gene segments, which can occur in cells infected with two different influenza viruses (15, 16). The resulting hybrid may contain gene segments from influenza viruses of different species, and can be especially virulent as the population lacks previous exposure to the newly introduced protein subtypes. Termed antigenic shift, this mechanism underlies the emergence of all three influenza pandemics since the 1918 Spanish flu (17).

12 Chapter 1: Introduction

N S2

PB1, PB2, PA (Transcriptase complex) NP (Nucleocapsid)

Figure 1. Schematic diagram of influenza A virus. Influenza A virus is an enveloped, negative-sense, single-stranded RNA virus. Within the lipid bilayer enveloped, the virus has eight gene segments, which encode for the ten proteins. The viral surface is decorated with spike-like projections of the glycoproteins HA and NA, which are inserted in the lipid bilayer.

13 Chapter 1: Introduction

Packaging and budding Release Adsorption

N t t.Receptor containing M2 sialic acid

coalting RNA(+) Endocytosis an fuIon y f

supply

Figure 2. Stages of influenza virus replication. (i) Attachment: Influenza viruses attach to host cells via multivalent interactions of HA with sialic acids on cell surface glycoproteins to initiate infection and replication. (ii) Entry: The virus is then internalized by endocytic compartments. (iii) Fusion and uncoating: The H+ions enter the virus through the M2 ion channel, and the low pH triggers a conformational change in HA. This activates virus-endosome fusion, and releases the genomic contents into the cytosol. (iv) Intracellular processes: The viral genome is transported into the nucleus, where viral RNA synthesis is carried out by the RNA-dependent RNA polymerase complex, and the viral proteins are expressed through the resulting mRNA. Copies of the negative-strand RNA are also made and packaged into ribonucleoprotein complexes for packaging into new viruses. (v) Assembly and release: The viruses assemble, bud, and are released from the cell membrane. NA cleaves sialic acids from the cell surface proteins to release the virions from the host cell (7). Figure adapted with permission from Nature Publishing Group (18).

14 Chapter 1: Introduction

1.2 Treatment and Prophylaxis for Influenza A Virus

1.2.1 Licensed prophylactics and treatment

There are currently two distinct strategies in use to control the spread of influenza: vaccines and conventional small-molecule antiviral drugs. Vaccination with the trivalent inactivated or live attenuated vaccines offers only limited protection (19), and is hampered by logistical issues, such as prediction of future circulating strains, and reliability of supply

(7). In the event of a pandemic, rapid production of sufficient quantities of vaccine can be a challenging task (20). Antivirals present an attractive alternative, and can potentially inhibit viruses of different subtypes or genetic variation. There are four clinically approved antivirals for influenza treatment and prophylaxis (Figure 3). Amantadine and rimantadine are inhibitors of the M2 ion channel, and interferes with viral fusion (21). Zanamivir (ZA) and oseltamivir inhibit NA enzymatic activity, thus blocking the release of newly made virions from infected cells (22, 23). Despite their efficacy, these antivirals suffer from limitations such as a short therapeutic window, high dosage, side effects, and high costs

(18, 24, 25). In addition, all the circulating viruses (both H3N2 and H1N1 strains) are already resistant to the M2 inhibitors (26-28), and resistance to the NA inhibitors has been appearing at an alarming rate in recent years (12, 28).

1.2.2 Investigational Antivirals under Clinical Trials

In response to the need for potent new antivirals to bolster our defense against influenza, there has been intense focus in both academia and industry to identify novel

15 Chapter 1: Introduction candidates for influenza therapy. Candidates currently undergoing clinical trials will be discussed herein.

NA inhibitors

Two additional NA inhibitors, peramivir (29, 30) and laninamivir (31), have been identified for treatment and prophylaxis for influenza (Figure 3). During the recent 2009

H1N1 pandemic, the US Food and Drug Admistration (FDA) even had to issue an

Emergency Use Authorization for intravenous peramivir in cases where current therapy were ineffective or unavailable (32). Nonetheless, since peramivir also inhibits NA, cross- resistance has already been observed with some oseltamivir- and ZA-resistant strains (33).

Laninamivir is structurally related to ZA, with the advantage of a longer half-life. A single dose of inhaled laninamivir has been shown to be comparable to a standard course of oseltamivir in the elderly in reducing the time of recovery (34). However, the activity of laninamivir against existing oseltamivir- or ZA-resistant viruses and its own resistance profile however remains largely unknown.

Polymerase inhibitors

Other influenza antiviral drugs under clinical trials include inhibitors of transcription and genome replication, T-705 (favipiravir) (35, 36) and ribavirin (37)

(Figure 3). T-705 is a pyrazinecarboxamide derivative, and its active form, T-705-4- ribofuranosyl-5'-triphosphate, has been postulated to selectively inhibit the influenza RNA- dependent RNA polymerase (38). T-705 has been shown to inhibit both oseltamivir- or amantadine-resistant 2009 H1N1 influenza at pM concentrations, however, it is

16 Chapter 1: Introduction unexpectedly much less effective against dually resistant viruses (39). In addition, there are few published data on resistance to T-705 (9).

Ribavirin inhibits the host cellular enzyme inosine 5'-monophosphate dehydrogenase, a key enzyme in GTP biosynthesis and viral RNA synthesis (18). Besides limitations in the design of the clinical trials for ribavirin (40), data from animal studies and clinical trials has been inconsistent with the observed in vitro anti-influenza efficacy

(37, 41). In recent years, influenza-related ribavirin studies have mostly focused on investigating ribavirin as an adjunct in combination therapy (42-44).

Host targeting inhibitors

A search on the ClinicalTrials.govwebsite with key words 'influenza' and 'antivirals' reveal that all other candidates under clinical evaluation target host factors (45). DAS181

(FludaseTM) is a fusion sialidase that can be inhaled to remove the viral receptor sialic acid from the airway epithelium, thus preventing the attachment of influenza virus onto host cells (46). DAS181 has shown broad-spectrum anti-influenza activity (47-49), but some unstable resistant mutants have been identified in drug selection experiments (personal communication from the developers of DAS181, cited in (9)).

Liposome-encapsulated polyinosinic-polycytidylic acid (Poly-ICLC), first discovered

40 years ago (50), is being revisited as a possible way to control influenza infection. Poly-

ICLC activates the host immunity to fight influenza virus infections (51), likely by inducing the expression of immune factors like toll-like receptor 3, interferon, and cytokines (52).

Yet, much care has to be exercised with optimizing the administration regime (patient

17 Chapter 1: Introduction selection and dosage) as serious side effects has been reported in humans and animal models (53-56).

1.2.3 Conclusion

The antiviral drugs under clinical development look promising. Nevertheless, it is expected that resistant strains will eventually develop if these antivirals are used as monotherapy clinically, as that observed with the M2 and NA inhibitors (57). The emergence of drug resistance in seasonal and pandemic influenza viruses in recent years remind us not to rest on our laurels; especially given the unpredictability of this pathogen and the paucity of options in controlling its spread. Particular emphasis should be placed on exploring combination therapy, and developing novel antivirals with a different mechanism of action that can significantly reduce drug resistance (58, 59).

18 Chapter 1: Introduction

Amantadine Rimantadine

HO OH O H13C J ., HO H 0 H3C HN,.,

N N Hi" / OH O O-CH2 HN . OH

CH 3 HN' CH 3 ,OH O==< H

HN NH 2 H2N~ CH3 OH 3 CH \2NH 3

Zanamivir Oseltamivir Peramivir

0 OH

NH H OH F N CONH 2 N NH2

O Hi- O11

H2 OH N HO OH HHN

Laninamivir T-705 (Favipiravir) Ribavirin

Figure 3. Antiviral inhibitors. Structures of (i) M2 inhibitors amantadine and rimantadine; (ii) NA inhibitors zanamivir, oseltamivir, peramivir, and laninamivir; and (iii) inhibitors of transcription and genome replication ,T-705 and ribavirin.

19 Chapter 1: Introduction

1.3 Motivation for this thesis and thesis outline

Facing the challenges mentioned above- limitations of current therapeutics, rapid spread of drug resistance, and the looming threat of the next pandemic- we have established a new strategy to develop potent influenza antivirals that can significantly minimize drug resistance. The strategy is based on (i) the observation that attaching monomeric inhibitors to a polymer makes them much more potent (60), and (ii) the principle of combination therapy, where two or more distinct targets are inhibited. Small molecular weight inhibitors covalently conjugated to a biocompatible polymer have been reported to inhibit human influenza strains (61), and prevent influenza binding of red blood cells (62, 63).

My thesis work is based on our lead candidate, ZA covalently attached via a flexible linker to water-soluble, biodegradable poly-L-glutamine (PGN), hereforth termed as PGN-

ZA (Figure 4). We have previously shown that PGN-ZA was 1,000- to 10,000-fold more potent than monomeric ZA in plaque reduction assays, and importantly, PGN-ZA remains effective against both ZA- and oseltamivir-resistant influenza viruses (64). We have also shown that attaching two different inhibitors, ZA and sialic acid, onto the same polymeric chain further enhances antiviral potency (65). My work will be divided into the following two chapters in this thesis:

1) Polymer-attached zanamivir synergistically inhibits both early and late steps of influenza virus infection

In Chapter 2, 1 present results on elucidating the mechanism of action underlying the dramatically increased antiviral potency of this polymeric inhibitor. I will show that, similar to monomeric ZA, the polymeric inhibitor binds specifically to viral NA and inhibits

20 Chapter 1: Introduction its enzymatic activity and the release of newly synthesized virions from infected cells. By using time-of-addition assays, I will also show that, in contrast to the monomeric ZA, the polymer-attached drug also synergistically inhibits an early step of influenza virus infection, accounting for the dramatically increased antiviral potency. By exploring each step of early virus replication, I will show that the inhibition of the early step of influenza infection is not by direct virucidal effect, aggregation of viruses, or inhibition of viral attachment to target cells and the subsequent endocytosis, but rather is due to inhibition of virus-endosome fusion.

2) Polymer-attached zanamivir delays the emergence of influenza drug resistance

In Chapter 3, I present the findings on investigating the ability of the multivalent drug conjugate to minimize drug resistance. By passaging influenza virus in vitro under drug selection pressure, I will show that the PGN-ZA is able to significantly delay the emergence of drug resistance compared to monomeric ZA. This will be further supported genotypic and phenotypic characterization of variant viruses selected under PGN-ZA and monomeric

ZA drug pressure.

1.4 Significance

All current antivirals have only one mechanism of action, and the rapid emergence of drug resistance amongst circulating influenza strains is a major health concern (28, 58,

59), highlighting the urgent need for development of novel influenza antivirals that can minimize drug resistance. The thesis presents new mechanistic information and drug resistance profile for polymer-attached ZA, the lead candidate in our influenza drug development work. The data in the subsequent chapters will show, for the first time, a

21 Chapter 1: Introduction single antiviral drug with dual synergistic mechanisms of inhibition, and the ability to delay the emergence of drug resistance. Results from Chapter 3 also indicate the potential use of polymer-attached drugs as tools to identify critical amino acids involved in certain viral processes, and to better understand the essential balance between HA and NA functions in viral replication. These findings provide new insight on the design of influenza antivirals and further validate polymer-attached inhibitors as a new framework of drug development in overcoming drug resistance.

22 Chapter 1: Introduction

HN-(CH26) NH2 HO- 0 H0

H3COCHN,

x/(x+y) = 0.10 ZA-linker -N H

ZA-linker Polymer-attached ZA

Figure 4. Zanamivir derivatives. Structures of monomeric ZA derivative (ZA-linker) and multivalent polymer-attached ZA (PGN-ZA) are shown as above. PGN-ZA used in the following two chapters of the thesis has 10% degree loading.

23 Chapter 1: Introduction

References

1. Lamb RA & Krug RM (2001) Orthomyxoviridae: The viruses and their replication. Fundamental Virology, ed David M Knipe PMH (Lippincott Williams & Wilkins), pp 725-770. 2. Harper SA, Fukuda K, Uyeki TM, Cox NJ, & Bridges CB (2004) Prevention and control of influenza: recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR Recomm Rep 53(RR-6):1-40. 3. Fiore AE, Shay DK, Haber P, Iskander JK, Uyeki TM, Mootrey G,Bresee JS, & Cox NJ (2007) Prevention and control of influenza. Recommendations of the Advisory Committee on Immunization Practices (ACIP), 2007. MMWR Recomm Rep 56(RR- 6):1-54. 4. Johnson NPAS & Mueller J (2002) Updating the accounts: global mortality of the 1918-1920 "Spanish" influenza pandemic. Bull Hist Med 76(1):105-115. 5. Gamblin SJ & Skehel JJ (2010) Influenza hemagglutinin and neuraminidase membrane glycoproteins. J Biol Chem 285(37):28403-28409. 6. Peter Palese MLS (2007) Orthomyxoviridae: The Viruses and Their Replication (Lippincott Williams & Wilkins) 5 Ed. 7. Palese P & Compans RW (1976) Inhibition of influenza virus replication in tissue culture by 2-deoxy-2,3-dehydro-N-trifluoroacetylneuraminic acid (FANA): mechanism of action.J Gen Virol 33(1):159-163. 8. Palese P, Tobita K, Ueda M, & Compans RW (1974) Characterization of temperature sensitive influenza virus mutants defective in neuraminidase. Virology 61(2):397- 410. 9. Moss RB, Davey RT, Steigbigel RT, & Fang F (2010) Targeting pandemic influenza: a primer on influenza antivirals and drug resistance.jAntimicrob Chemother 65(6):1086-1093. 10. Parvin JD, Moscona A, Pan WT, Leider JM, & Palese P (1986) Measurement of the mutation rates of animal viruses: influenza A virus and poliovirus type 1.j Virol 59(2):377-383. 11. Smith F.L. PP (1989) Variation in influenza virus genes:epidemiology, pathogenic, and evolutionary consequences. The Influenza Viruses, ed Krug RM (Plenum). 12. Moscona A (2009) Global transmission of oseltamivir-resistant influenza. New Englj Med 360(10):953-956. 13. Bloom JD, Gong LI, & Baltimore D (2010) Permissive secondary mutations enable the evolution of influenza oseltamivir resistance. Science 328(5983):1272-1275. 14. Simonsen L, Viboud C, Grenfell BT, Dushoff J, Jennings L, Smit M, Macken C, Hata M, Gog J,Miller MA, & Holmes EC (2007) The genesis and spread of reassortment human influenza A/H3N2 viruses conferring adamantane resistance. Mol Bio Evol 24(8):1811-1820. 15. Webster RG, Laver WG, Air GM, & Schild GC (1982) Molecular mechanisms of variation in influenza viruses. Nature 296(5853):115-121.

24 Chapter 1. Introduction

16. Webby RJ & Webster RG (2001) Emergence of influenza A viruses. Philosophical Transactionsof the Royal Society of London. Series B, Biological Sciences 356(1416):1817-1828. 17. Morens DM, Taubenberger JK, & Fauci AS (2009) The persistent legacy of the 1918 influenza virus. New Englj Med 361(3):225-229. 18. De Clercq E (2006) Antiviral agents active against influenza A viruses. Nature Rev Drug Disc 5(12):1015-1025. 19. Osterholm MT, Kelley NS, Sommer A, & Belongia EA (2012) Efficacy and effectiveness of influenza vaccines: a systematic review and meta-analysis. The Lancet Infect Dis 12(1):36-44. 20. Osterhaus AD (2007) Pre- or post-pandemic influenza vaccine? Vaccine 25(27):4983-4984. 21. Pinto LH & Lamb RA (2007) Controlling influenza virus replication by inhibiting its proton channel. Mol Biosyst 3(1):18-23. 22. Kim CU, Lew W, Williams MA, Liu H, Zhang L, Swaminathan S, Bischofberger N, Chen MS, Mendel DB, Tai CY, Laver WG, & Stevens RC (1997) Influenza neuraminidase inhibitors possessing a novel hydrophobic interaction in the enzyme active site: design, synthesis, and structural analysis of carbocyclic sialic acid analogues with potent anti-influenza activity.J Am Chem Soc 119(4):681-690. 23. von Itzstein M, Wu WY, Kok GB, Pegg MS, Dyason JC, Jin B, Van Phan T, Smythe ML, White HF, Oliver SW, & et al. (1993) Rational design of potent sialidase-based inhibitors of influenza virus replication. Nature 363(6428):418-423. 24. von Itzstein M (2007) The war against influenza: discovery and development of sialidase inhibitors. Nature Rev Drug Disc 6(12):967-974. 25. Lipatov AS, Govorkova EA, Webby RJ, Ozaki H, Peiris M, Guan Y, Poon L, & Webster RG (2004) Influenza: emergence and control.J Virol 78(17):8951-8959. 26. Bright RA, Medina MJ, Xu X, Perez-Oronoz G,Wallis TR, Davis XM, Povinelli L, Cox NJ, & Klimov Al (2005) Incidence of adamantane resistance among influenza A (H3N2) viruses isolated worldwide from 1994 to 2005: a cause for concern. Lancet 366(9492):1175-1181. 27. Fiore AE, Fry A, Shay D, Gubareva L, Bresee JS, & Uyeki TM (2011) Antiviral Agents for the Treatment and Chemoprophylaxis of Influenza: Recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR Recomm Rep, ed CDC, pp 1-24. 28. WHO (2011) Global monitoring of antiviral resistance in currently circulating human influenza viruses, November 2011. Weekly EpidemiologicalRecord, pp 497- 508. 29. Smee DF, Huffman JH, Morrison AC, Barnard DL, & Sidwell RW (2001) Cyclopentane neuraminidase inhibitors with potent in vitro anti-influenza virus activities. Antimicrob Agents Chemother 45(3):743-748. 30. Sidwell RW, Smee DF, Huffman JH, Barnard DL, Bailey KW, Morrey JD, & Babu YS (2001) In vivo influenza virus-inhibitory effects of the cyclopentane neuraminidase inhibitor RJW-270201. Antimicrob Agents Chemother 45(3):749-757. 31. Yamashita M, Tomozawa T, Kakuta M, Tokumitsu A, Nasu H, & Kubo S (2009) CS- 8958, a prodrug of the new neuraminidase inhibitor R-125489, shows long-acting anti-influenza virus activity. Antimicrob Agents Chemother 53(1):186-192.

25 Chapter 1: Introduction

32. Administration USFaD (2009) FDA Authorizes Emergency Use of Intravenous Antiviral Peramivir for 2009 H1N1 Influenza for Certain Patients, Settings. (U.S. Food and Drug Administration), FDA NOTE TO CORRESPONDENTS. 33. Gubareva LV, Webster RG, & Hayden FG (2001) Comparison of the activities of zanamivir, oseltamivir, and RWJ-270201 against clinical isolates of influenza virus and neuraminidase inhibitor-resistant variants. Antimicrob Agents Chemother 45(12):3403-3408. 34. Biota. LANI Phase III Clinical Trials in Asia Prove Successful. http://www.biota.com.au/uploaded/154/1021542_251aniphaseiiiclinicaltri.pdf. Date accessed: 07/04/2012 35. Furuta Y, Takahashi K, Fukuda Y, Kuno M, Kamiyama T, Kozaki K, Nomura N, Egawa H, Minami S, Watanabe Y, Narita H, & Shiraki K (2002) In vitro and in vivo activities of anti-influenza virus compound T-705. Antimicrob Agents Chemother 46(4):977- 981. 36. Takahashi K, Furuta Y, Fukuda Y, Kuno M, Kamiyama T, Kozaki K, Nomura N, Egawa H, Minami S, & Shiraki K (2003) In vitro and in vivo activities of T-705 and oseltamivir against influenza virus. Antivir Chem & Chemother 14(5):235-241. 37. Graci JD & Cameron CE (2006) Mechanisms of action of ribavirin against distinct viruses. Rev Med Virol 16(1):37-48. 38. Furuta Y, Takahashi K, Kuno-Maekawa M, Sangawa H, Uehara S, Kozaki K, Nomura N, Egawa H, & Shiraki K (2005) Mechanism of action of T-705 against influenza virus. Antimicrob Agents Chemother 49(3):981-986. 39. Sleeman K, Mishin VP, Deyde VM, Furuta Y, Klimov Al, & Gubareva LV (2010) In vitro antiviral activity of favipiravir (T-705) against drug-resistant influenza and 2009 A(H1N1) viruses. Antimicrob Agents Chemother 54(6):2517-2524. 40. Chan-Tack KM, Murray JS, & Birnkrant DB (2009) Use of ribavirin to treat influenza. New EngiJ Med 361(17):1713-1714. 41. Riner A, Chan-Tack KM, & Murray JS (2009) Original research: Intravenous ribavirin--review of the FDA's Emergency Investigational New Drug Database (1997-2008) and literature review. PostgradMed 121(3):139-146. 42. Smee DF, Hurst BL, Wong M-H, Bailey KW, & Morrey JD (2009) Effects of Double Combinations of Amantadine, Oseltamivir, and Ribavirin on Influenza A (H5N1) Virus Infections in Cell Culture and in Mice. Antimicrob Agents Chemother 53(5):2120-2128. 43. Sidwell RW, Bailey KW, Wong MH, & Huffman JH (1995) In vitro and in vivo sensitivity of a non-mouse-adapted influenza A (Beijing) virus infection to amantadine and ribavirin. Chemother 41(6):455-461. 44. Smee DF, Bailey KW, Morrison AC, & Sidwell RW (2002) Combination treatment of influenza A virus infections in cell culture and in mice with the cyclopentane neuraminidase inhibitor RWJ-270201 and ribavirin. Chemother 48(2):88-93. 45. NIH US (2012) http://clinicaltrials.gov/ct2/home. (U.S. National Institutes of Health). Date accessed: 07/04/2012 46. Nicholls JM, Aschenbrenner LM, Paulson JC, Campbell ER, Malakhov MP, Wurtman DF, Yu M, & Fang F (2008) Comment on: concerns of using sialidase fusion protein as an experimental drug to combat seasonal and pandemic influenza.JAntimicrob Chemother 62(2):426-428; author reply 428-429.

26 Chapter 1: Introduction

47. Belser JA, Lu X, Szretter KJ, Jin X, Aschenbrenner LM, Lee A, Hawley S, Kim do H, Malakhov MP, Yu M, Fang F, & Katz JM (2007) DAS181, a novel sialidase fusion protein, protects mice from lethal avian influenza H5N1 virus infection.J Infect Dis 196(10):1493-1499. 48. Triana-Baltzer GB, Gubareva LV, Klimov Al, Wurtman DF, Moss RB, Hedlund M, Larson JL, Belshe RB, & Fang F (2009) Inhibition of neuraminidase inhibitor- resistant influenza virus by DAS181, a novel sialidase fusion protein. PloS One 4(11):e7838. 49. Triana-Baltzer GB, Gubareva LV, Nicholls JM, Pearce MB, Mishin VP, Belser JA, Chen LM, Chan RW, Chan MC, Hedlund M, Larson JL, Moss RB, Katz JM, Tumpey TM, & Fang F (2009) Novel pandemic influenza A(H1N1) viruses are potently inhibited by DAS181, a sialidase fusion protein. PloS One 4(11):e7788. 50. Field AK, Tytell AA, Lampson GP, & Hilleman MR (1967) Inducers of interferon and host resistance. II. Multistranded synthetic polynucleotide complexes. Proc Natl Acad Sci USA 58(3):1004-1010. 51. Saravolac EG, Sabuda D, Crist C,Blasetti K, Schnell G,Yang H, Kende M, Levy HB, & Wong JP (2001) Immunoprophylactic strategies against respiratory influenza virus infection. Vaccine 19(17-19):2227-2232. 52. Ichinohe T, Watanabe I, Ito S, Fujii H, Moriyama M, Tamura S, Takahashi H, Sawa H, Chiba J, Kurata T, Sata T, & Hasegawa H (2005) Synthetic double-stranded RNA poly(I:C) combined with mucosal vaccine protects against influenza virus infection.j Virol 79(5):2910-2919. 53. Adamson RH & Fabro S (1969) Embryotoxic effect of poly I. poly C.Nature 223(5207):718. 54. Lefkowitz E, Worthington M, Conliffe MA, & Baron S (1976) Comparative effectiveness of six antiviral agents in Herpes simplex type 1 infection of mice. Proc Soc Exp Biol Med 152(3):337-342. 55. Robinson RA, DeVita VT, Levy HB, Baron S, Hubbard SP, & Levine AS (1976) A phase I-II trial of multiple-dose polyriboinosic-polyribocytidylic acid in patieonts with leukemia or solid tumors. J Natl Cancer Inst 57(3):599-602. 56. Steinberg AD, Baron S, & Talal N (1969) The pathogenesis of autoimmunity in New Zealand mice, I. Induction of antinucleic acid antibodies by polyinosinic- polycytidylic acid. Proc Natl Acad Sci USA 63(4):1102-1107. 57. Boltz DA, Aldridge JR, Jr., Webster RG, & Govorkova EA (2010) Drugs in development for influenza. Drugs 70(11):1349-1362. 58. Das K, Aramini JM, Ma LC, Krug RM, & Arnold E (2010) Structures of influenza A proteins and insights into antiviral drug targets. Nature Struct Mol Biol 17(5):530- 538. 59. Hayden F (2009) Developing new antiviral agents for influenza treatment: what does the future hold? Clin Infect Dis: An official publication of the Infectious Diseases Society ofAmerica 48 Suppl 1:S3-13. 60. Mammen M, Choi S-K, & Whitesides GM (1998) Polyvalent interactions in biological systems: Implications for design and use of multivalent ligands and inhibitors. Angew Chemie Int Ed 37(20):2754-2794.

27 Chapter 1: Introduction

61. Honda T, Yoshida S, Arai M, Masuda T, & Yamashita M (2002) Synthesis and anti- influenza evaluation of polyvalent sialidase inhibitors bearing 4-guanidino- Neu5Ac2en derivatives. Bioorg Med Chem Lett 12(15):1929-1932. 62. Mammen M, Dahmann G,& Whitesides GM (1995) Effective inhibitors of hemagglutination by influenza virus synthesized from polymers having active ester groups. Insight into mechanism of inhibition.J Med Chem 38(21):4179-4190. 63. Lees WJ, Spaltenstein A, Kingery-Wood JE, & Whitesides GM (1994) Polyacrylamides bearing pendant alpha-sialoside groups strongly inhibit agglutination of erythrocytes by influenza A virus: multivalency and steric stabilization of particulate biological systems. j Med Chem 37(20):3419-3433. 64. Weight AK, Haldar J, Alvarez de Cienfuegos L, Gubareva LV, Tumpey TM, Chen J, & Klibanov AM (2011) Attaching zanamivir to a polymer markedly enhances its activity against drug-resistant strains of influenza a virus.J Pharm Sci 100(3):831- 835. 65. Haldar J, Alvarez de Cienfuegos L, Tumpey TM, Gubareva LV, Chen J, & Klibanov AM (2010) Bifunctional polymeric inhibitors of human influenza A viruses. Pharm Res 27(2):259-263.

28 Chapter 2: PGN-ZA inhibits synergistically both early and late steps of influenza virus replication

Chapter 2

POLYMER-ATTACHED ZANAMIVIR INHIBITS SYNERGISTICALLY

BOTH EARLY AND LATE STEPS OF INFLUENZA VIRUS

REPLICATION

Abstract

Covalently conjugating multiple copies of the drug zanamivir (ZA) (the active ingredient in

RelenzaTM) via a flexible linker to poly-L-glutamine greatly enhances the anti-influenza- virus activity. In this study, we have elucidated the mechanism of this phenomenon. Like ZA itself, the polymer-attached drug binds specifically to viral neuraminidase and inhibits both its enzymatic activity and the release of newly synthesized virions from infected cells. In contrast to monomeric ZA, however, the polymer-attached drug also synergistically inhibits an early step of influenza virus infection, thus contributing to the dramatically increased antiviral potency. This inhibition is not caused by a direct viricidal effect, aggregation of viruses, or inhibition of viral attachment to target cells and the subsequent endocytosis, but rather is due to reducing the virus-endosome fusion. These findings rationalize the enhanced anti-influenza potency of polymer-conjugated ZA and reveal that attaching the drug to a polymeric chain confers a new mechanism of antiviral action potentially useful for minimizing drug resistance.

29 Chapter 2: PGN-ZA inhibits synergistically both early and late steps of influenza virus replication

Introduction

Influenza A viruses cause epidemics and pandemics in human populations, inflicting enormous suffering and economic losses (1). Currently, two distinct strategies - vaccines and small-molecule drugs - are utilized to control the spread of influenza (1). Vaccination offers limited protection and is hampered by logistical challenges, such as accurate prediction of future circulating strains and production of sufficient quantities of vaccine for large populations within a short time (2, 3). Two of the four antiviral drugs approved in the

United States for the treatment and prophylaxis of influenza, amantadine and rimantadine, inhibit the viral M2 ion channel protein (4), while and the other two, zanamivir (ZA) and oseltamivir, inhibit the viral neuraminidase (NA) activity (5, 6). These drugs have limited therapeutic windows, side effects, and high costs (7-9), and most circulating viruses are already resistant to the M2 inhibitors (10, 11). Furthermore, resistance to the NA inhibitors is spreading rapidly (12, 13). Thus, the need to develop novel influenza therapeutics that can prevent viral resistance or significantly reduce its incidence is urgent (14, 15).

An alternative approach to conventional antivirals is the use of multivalent polymeric inhibitors (16). In particular, small-molecule inhibitors covalently conjugated to a biocompatible polymer have been reported to inhibit human influenza strains (17) and prevent influenza binding to red blood cells (18, 19). We have previously shown that the antiviral efficacy of ZA is dramatically enhanced when multiple copies thereof are attached via a flexible linker to the benign and biodegradable polymer poly-L-glutamine (PGN) (20): the resultant PGN-ZA is 1,000- to 10,000-fold more potent than monomeric ZA in plaque

30 Chapter 2: PGN-ZA inhibits synergistically both early and late steps of influenza virus replication reduction assays and, importantly, is effective even against ZA- and oseltamivir-resistant influenza viruses.

Herein we have elucidated the mechanisms underlying the dramatically higher antiviral potency of this multivalent drug conjugate. We show that, like ZA itself, PGN-ZA binds to NA and inhibits its activity and the release of newly synthesized virions from the infected cells. In addition, PGN-ZA inhibits virus-endosome membrane fusion during initial virus infection. Thus attaching ZA to PGN results in a new mode of drug action. The synergistic inhibition of both the early and late steps of influenza virus infection accounts for the markedly enhanced antiviral potency of PGN-ZA compared to the monomeric ZA precursor.

Results

PGN-ZA binds to and inhibits viral NA

Influenza virus has two main surface glycoproteins, hemagglutinin (HA) and NA

(21). Both of them bind to the terminal sialic acid of cell-surface glycans (22-24). Since ZA is a sialic acid (SA) derivative and inhibits the enzymatic activity of NA, we sought to (i) determine why its conjugation to PGN via a flexible linker raised its binding and inhibitory activities and (ii) exclude non-specific effects by the PGN chain itself. To characterize binding of PGN-ZA to whole virions, we performed whole-virus ELISA binding assays where PGN-ZA or PGN were immobilized to 96-well plates by UV cross-linking, incubated with influenza A/WSN33 (H1N1) (WSN), and then quantified using HRP-conjugated anti-

H1 antibodies. PGN-ZA exhibited a concentration-dependent binding with saturation to the

31 Chapter 2: PGN-ZA inhibits synergistically both early and late steps of influenza virus replication whole H1N1 viruses in the therapeutic range, as determined by plaque reduction assays

(Figure 1A), whereas PGN itself showed no significant virus binding under the same conditions.

We examined PGN-ZA's specific site of action by measuring its binding to purified

HA and NA proteins by means of ELISA. The polymer-attached drug displayed a dose- dependent binding to NA, but not to HA (Figures 1B, 1C, and Si). In contrast, multivalent polymeric SA conjugates (PGN-SA) exhibited specific binding to HA, as SA is the cognate ligand of HA (Fig. 1C). PGN by itself did not bind to either HA or NA. PGN-ZA was 3- and 10- fold more potent than ZA modified with the linker (ZA-linker) (ZA-linker's antiviral activity is similar to that of ZA itself (20)) in inhibiting NA activity of WSN and influenza

A/PR/8/34 (PR8) viruses, respectively (Table 1). These data indicate that bare PGN has no appreciable interaction with HA, NA, or whole virions and that PGN-ZA specifically binds to

NA and inhibits its enzymatic activity.

PGN-ZA synergistically inhibits both early and late steps of influenza virus infection

Since PGN-ZA inhibits NA, as does the monomeric ZA, we expected PGN-ZA to inhibit the release of newly synthesized virions. MDCK cells were infected at a multiplicity of infection (moi) of 2. Because newly synthesized viruses were released after about 4 h, PGN-

ZA and ZA-linker were added 3 h post-infection (p.i.) to restrict inhibitory activity to the late phase of virus replication (Figure 2A). At 7 h p.i., the culture supernatant was harvested, and the viral titer was measured by the plaque assay. Compared to the PBS control, addition of PGN-ZA and ZA-linker reduced the virus titer by some 90% and 80%, respectively (Figure 2B). To control for the presence of leftover inhibitors in the collected

32 Chapter 2: PGN-ZA inhibits synergistically both early and late steps of influenza virus replication

supernatants (albeit at concentrations below IC50 upon serial dilution), some PBS control samples were spiked with the same concentration of PGN-ZA just prior to the plaque assay.

No significant reduction of virus titer was detected in those cases compared to the PBS control, confirming no interference from low concentrations of inhibitors remaining in the supernatants. These results show that PGN-ZA specifically inhibits the release of newly synthesized viruses from infected cells.

To test whether PGN-ZA inhibits early events of influenza virus infection, we performed time-of-addition experiments in a single-cycle infection (Figure 3A). MDCK cells were infected with WSN virus at a moi of 20, and the inhibitors were added at different time points p.i.: -1 h, 0 h, or 1 h. The cell culture supernatants were harvested at 3 h p.i. before the completion of a single infection cycle. The cells were fixed, and expression of the viral proteins NP and M1 was quantified by flow cytometry. The fraction of infected cells decreased by 30-50% upon the addition of PGN-ZA (Figure 3B). In contrast, for all the conditions tested, ZA-linker did not affect the fraction of cells infected. Thus PGN-ZA, unexpectedly, also specifically inhibits an early step of influenza virus infection.

To explore the relationship between PGN-ZA's inhibitory effects in the early and late steps of virus infection, we performed a time-of-addition plaque assay with the avian strain

A/Turkey/MN/80 (TKY). The inhibitors were added in different time points of the assay:

(i) early (-1 to 1 h p.i.), (ii) late (1 to 72 h p.i.), or (iii) both early and late (-1 to 72 h p.i.).

When added during the late phase of plaque assay, PGN-ZA significantly reduced the number of plaques with an IC5o of 14.8 nM (Figure 4). Remarkably, when the virus was exposed to PGN-ZA throughout the assay in both the early and late stages, the potency of

PGN-ZA rose almost 100-fold to an ICso of 0.16 nM. The IC5o values for the monomeric ZA

33 Chapter 2: PGN-ZA inhibits synergistically both early and late steps of influenza virus replication and ZA-linker remained the same under both conditions, thereby revealing no additional benefit from introducing the monomeric inhibitors in the early phase of the infection. As expected, a reduction in the IC50 values was also associated with a reduction in the sizes of the plaques (data not shown).

Taken together, the foregoing results indicate that (i) the multivalent PGN-ZA potently inhibits at least two distinct steps in influenza infection: an event early during the infection process, as well as the release of newly synthesized virions; (ii) monomeric ZA inhibits only virus release, and (iii) PGN-ZA's dual mechanism of action produces a synergistic inhibition of virus replication.

PGN-ZA inhibits influenza infection through neither direct virucidal effect nor virus aggregation

PGN-ZA can inhibit an early step of influenza virus infection through a direct viricidal effect and/or by aggregating viruses and thus preventing them from infecting target cells. To test these mechanisms, we used transmission electron microscopy (TEM) imaging to look for changes in viral envelope integrity and morphology upon PGN-ZA treatment. Purified WSN virus was filtered through a 0.2-prm filter and treated with either

PGN-ZA or PBS for 1 h prior to staining with uranyl formate, followed by TEM imaging. As seen in high-magnification micrographs, PGN-ZA did not affect the morphology or envelope integrity of viral particles (Figure 3A, lower panel). In addition, low-magnification micrographs (Figure 5A, upper panel) were taken to determine the distribution of viral particles in clusters. From over 5,000 viral particles analyzed, no significant increase was observed in virus aggregation (clustering of two or more viruses together) upon PGN-ZA treatment (Figure 5B), consistent with dynamic light scattering results (Figure S2). To rule

34 Chapter 2: PGN-ZA inhibits synergistically both early and late steps of influenza virus replication out staining artifacts, phosphotungstic acid was also used to visualize the samples, and the data obtained corroborated those of the uranyl formate-stained samples (Figure 6). Thus, somewhat surprisingly, inhibition of the early step of influenza infection by PGN-ZA is not through a direct virucidal effect or aggregation of viral particles.

PGN-ZA does not affect virus attachment and endocytosis

To examine whether PGN-ZA affects virus binding and endocytosis, we performed a flow-cytometry assay using labeled antibodies against viral NP and M1 (Figure 7A). Virus attachment was measured by incubating WSN virus at moi of 20 with MDCK cells at 4*C, at which temperature endocytosis does not occur (Fig. 7A, Group I). To assay for endocytosis, the same cells were incubated at 37*C for 30 min to allow the surface-bound virions to be endocytosed. Bacterial sialidase was later introduced into the system to remove surface- bound virions (Fig. 7A, Groups II and IV). Since internalized viruses are protected from sialidase activity, any cell-associated virus remaining after the sialidase treatment would presumably be that which has been internalized (Fig. 7A, Group IV). As shown in the left panel of Figure 7B, PGN-ZA did not inhibit virus binding to MDCK cells. Expectedly, there was a significant drop in virus-associated cells following sialidase treatment (Fig. 7B, Group

II). PGN-ZA also did not affect virus endocytosis, as evidenced by the similar levels of virus- associated cells with or without sialidase treatment of 37*C-incubated cells (Fig. 7A, Groups

III and IV). Statistical analysis of all four sets of conditions confirmed that the presence of

PGN-ZA does not affect virus attachment and internalization (Figure 7C). Hemagglutination inhibition assays also revealed that PGN-ZA did not affect virus binding to red blood cells

35 Chapter2: PGN-ZA inhibits synergistically both early and late steps of influenza virus replication

(Figure S3). These results indicate that PGN-ZA does not inhibit binding or endocytosis of influenza virus infection.

PGN-ZA inhibits virus-endosome fusion

To investigate PGN-ZA's effect on influenza virus fusion, we imaged individual viral particles in MDCK cells fixed at different time points p.i. by using fluorescence microscopy.

The WSN virus was labeled with amine-reactive Alexa Fluor 647 dye; the virus retained infectivity and binding to red blood cells (data not shown). To synchronize infection, the viruses were first incubated with MDCK cells on ice for 60 min in the absence or presence of PGN-ZA. The mixture was then rapidly warmed to 37*C to initiate infection. After the start of infection, virus fusion begins at about 10 min, with approximately two-thirds of the viruses being fusion-defective (25). The MDCK cells were then fixed at 0, 5, or 15 min p.i. and stained (Figure 8A). No apparent difference in the abundance of labeled viral particles was observed between the samples with or without PGN-ZA at t = 0 min, concordant with the results of the flow cytometry-based binding experiments (Fig. 7B). At t = 15 min, a significant accumulation of viral particles was observed in the perinuclear region with the

PGN-ZA-treated samples, as compared to the PBS control (Figure 8B). Since approximately one-third of the viruses in the PBS control are able to undergo fusion, the observed viral particle accumulation in the PGN-ZA sample at 15 min would reflect a block of fusion by some 85%.

When an influenza virus is exposed to an acidic environment, most HA subtypes undergo a pH-induced conformational change. In the presence of a membrane, fusion occurs; in the absence of a membrane, the HA is irreversibly inactivated abolishing the viral

36 Chapter 2: PGN-ZA inhibits synergistically both early and late steps of influenza virus replication infectivity (26). To investigate the ability of PGN-ZA to inhibit this process, the TKY virus was incubated at pH 5 in the presence or absence of PGN-ZA at 37*C for 15 min. The level of infectious virus remaining after this acidic treatment was determined by serial titrations using the plaque assay. PGN-ZA blocked the pH-5-induced inactivation of virions 2-3 fold compared to the PBS control (Figure 9). In contrast, the viral titer did not change following a pH 7 incubation (Figure S4). These results indicate that PGN-ZA inhibits the early steps of influenza infection by interfering with virus-endosomal membrane fusion.

Discussion

In this report, we have investigated the mechanism underlying the greatly enhanced antiviral potency of a polymer-attached zanamivir. Compared to its small-molecule predecessor, PGN-ZA is three to four orders of magnitude more potent in inhibiting influenza virus infection, as determined by plaque reduction assays (20). We have found that, like ZA, PGN-ZA specifically binds to NA and inhibits its enzymatic activity and the

release of the newly synthesized viruses from infected cells. PGN-ZA is more potent in

inhibiting virus release than ZA itself, likely due to an increased avidity to NA from polymer

binding and hence an increased inhibition of NA's activity. While inhibition of virus release

by PGN-ZA was expected, the observation that PGN-ZA also inhibits an early step of

influenza infection is surprising. Compared to the inhibition of virus release, which reduces virus titer by over 90% (Fig. 2B), inhibition of the early step of influenza infection by PGN-

ZA lowers infection by 30-50% (Figure 3B), indicating that the former process is still the

dominant mechanism of inhibition. More importantly, the two antiviral mechanisms act

37 Chapter 2: PGN-ZA inhibits synergisticallyboth early and late steps of influenza virus replication synergistically (Figure 4), accounting for the greatly enhanced (-1,000 fold) antiviral potency of PGN-ZA over monomeric ZA.

Our observations afford further mechanistic insights. A PGN-ZA-induced viral aggregation may lead to a direct virucidal effect or interfere with infection. However, we detected no obvious deformation of virus integrity or significant aggregation of viruses caused by PGN-ZA. Nor did we see any significant effect of PGN-ZA on attachment of viruses to cell surface and their subsequent endocytosis into target cells. What we did observe was the prolonged accumulation of viruses in the perinuclear region. Normally, following exposure to the low pH in the endosomes, viral HA undergoes a conformation change leading to fusion of the viral envelope with the endosomal membrane and subsequent release of viral genome into the cytosol (27). The unexpected observation that PGN-ZA inhibits virus-endosome fusion is further supported by our finding that PGN-ZA protects influenza virus from low pH-induced inactivation. Therefore, although PGN-ZA does not interact with HA directly (Figure 1), its interaction with NA somehow prevents HA from a pH-induced conformation change and, in turn, virus-endosome fusion. To our knowledge, this is the first report showing that attaching monomeric inhibitors to a polymeric backbone confers new mechanisms of action.

How does PGN-ZA inhibit virus-endosome fusion? Influenza viruses have 300-500

HA and 30-50 NA per virion (27). HA is required for viral attachment to cell surface glycan receptors, as well as virus-endosome fusion (28). Our finding that virus attachment to target cells and endocytosis are not affected by PGN-ZA is consistent with no interaction between PGN-ZA and HA, as measured by ELISA. Although ZA has been reported to inhibit fusion of HA-expressing cells with erythrocytes, the experiment was performed with 2-10

38 Chapter2: PGN-ZA inhibits synergistically both early and late steps of influenza virus replication mM of ZA, which is some 10s- to 106-fold higher than the therapeutic range used in our study (29). Therefore, the effect of PGN-ZA on HA is probably indirect. It is possible that the multivalent binding of PGN-ZA to NA blocks the conformational change of HA through steric hindrances without affecting HA binding to surface glycan receptors. It is more likely, however, that NA is directly involved in virus-endosome fusion. A role of NA in early virus infection is less clear and defined than its essential function in virus release. Although it has been suggested that viral NA plays a role in early infection by removing SA in mucin to allow virions to reach the lung epithelial cells (30, 31) and/or enhancing late endosome/lysosome trafficking (32), other studies suggest that NA is not required for virus entry, replication, or assembly (33). Because inhibition of the early step of influenza infection contributes less than inhibition of virus release to overall reduction of infection, the virus-endosome fusion may be less dependent on NA activity. As a result, while not inhibited by less potent monomeric ZA, this fusion event is inhibited by the more potent polymeric inhibitor PGN-ZA.

All existing influenza antivirals have only one mode of action, and a rapid emergence of drug-resistant variants is a major public health challenge in the control of influenza (13-

15). The data presented here show that PGN-ZA can synergistically inhibit both viral fusion and release at sub-nM concentrations of ZA. This dual mechanism of inhibition is novel among known influenza antivirals and consistent with our previous observation that PGN-

ZA remains effective against ZA- or oseltamivir-resistant influenza virus isolates (20).

Multivalent antivirals thus offer an alternative to conventional combination therapy by not only protecting against influenza virus infection but also minimizing the emergence of drug resistance.

39 Chapter 2: PGN-ZA inhibits synergistically both early and late steps of influenza virus replication

Materials and Methods

Inhibitors.

Poly-L-glutamic acid (molecular weight of 50,000-100,000 Da) and all other chemicals, biochemicals, and solvents were from Sigma-Aldrich Chemical Co. (St. Louis,

MO). 4-Guanidino-Neu5Ac2en (4-guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid) was purchased from Bioduro (Beijing, China). The ZA-linker derivative was synthesized as described previously (34). PGN-ZA and the bare PGN were prepared from poly-L-glutamic acid and characterized as described previously (20). Concentrations of inhibitors used in the mechanistic studies were 100 x ICso, unless indicated otherwise.

Viruses and cells.

Influenza virus A/WSN/33 (WSN), subtype H1N1, was kindly provided by Dr. Peter

Palese (Mount Sinai School of Medicine, New York City). Influenza A/Turkey/MN/80 (TKY), subtype H4N2, was obtained from the Centers for Disease Control and Prevention (Atlanta,

GA). Influenza A/PR/8/34 virus was purchased from Charles River Laboratories

(Wilmington, MA). The WSN virus was cultured in Madin-Darby canine kidney (MDCK) cells from the ATCC. The cells were routinely passaged in Eagle's minimal essential medium

(MEM) containing 10% fetal bovine serum. The A/Turkey/MN/80 virus was propagated in

11-day-old embryonated chicken eggs. The grown viruses were clarified by low-speed centrifugation and concentrated before sucrose gradient purification using a Beckman

SW41 rotor at 24,000 rpm. Viruses were resuspended in phosphate-buffered saline (PBS) and stored at -80'C.

40 Chapter 2: PGN-ZA inhibits synergistically both early and late steps of influenza virus replication

Virus binding assay and HA/NA specificity assays.

ELISA to measure the direct binding activity of influenza A/WSN/33 virus was performed using a modified literature procedure (35). Briefly, microtiter plates (Corning

Polystyrene Universal-BIND Microplate, Corning, NY) were incubated with 50 pL of varying dilutions of the multivalent inhibitor in PBS at 4*C overnight and irradiated with 254-nm

UV light for 5 min. The solution was then aspirated, and the plates were washed thrice with

2% BSA (Sigma) and 0.05% (v/v) Tween 20 in PBS (PBST), followed by a further 3-h blocking step with 0.3 mL of PBST at RT. The plates were then washed thrice each with

PBST and 2% BSA in PBS (PBS-BSA), followed by incubation with a solution containing influenza virus in PBS-BSA at 4*C overnight. Polyclonal antibodies to the virus diluted in

PBS-BSA were subsequently added to the plates and incubated for 5 h at 4*C. The plates were then washed with PBS-BSA thrice and incubated with horseradish peroxidase (HRP)- conjugated secondary antibodies in PBS-BSA for 2 h at 4*C. The plates were washed as above with PBS-BSA before addition of substrate. Colorimetric development of 50 ptL of 1-

Step Ultra TMB (Thermo Scientific) at RT was stopped with 50 p.L of 0.2 M H2SO 4 after incubation for 30 min, and the absorbance was determined at 450 nm.

For the HA/NA binding specificity assays, the microtiter plates were covalently conjugated with 50 pL of 10 ig/mL polymeric inhibitor and blocked as above. For the HA specificity assay, His-tagged A/WSN/33 (H1N1) HA protein (eEnzyme, Gaithersburg, MD), primary (mouse anti-His tag IgG, Abcam, Cambridge, MA), and secondary (HRP-conjugated goat anti-mouse IgG, Biolegend, San Diego, CA) antibodies were mixed in the ratio 4:2:1 and incubated on ice for 20 min. Likewise, His-tagged A/Cal/04/2009 (HiN1) NA (Sino

Biological, Beijing, China), primary and secondary antibodies were mixed in the ratio 4:2:1,

41 Chapter 2: PGN-ZA inhibits synergistically both early and late steps of influenza virus replication and incubated on ice for 20 min in the NA specificity assay. The mixtures of pre-complexed

HA or NA were then diluted to varying concentrations with PBS-BSA, and 50 ptL was added to each well and incubated for 2 h at RT. The wells were washed four times with PBST, and

HRP activity was measured as in the whole virus binding assay above. The experiment was repeated with varying dilutions of the multivalent inhibitor conjugated to the plate, with concentrations of the pre-complexed proteins constant at 5 ptg/mL.

NA inhibition assay was performed and analyzed as described previously (20). Briefly, 20 ptL of virus and 15 ptL of inhibitor dilutions were pre-incubated for 1 h at RT. The fluorogenic NA substrate 4-methylumbelliferyl-a-D-N-acetylneuraminic acid (at a concentration of 3-4 pM) was added, and the enzyme kinetics was measured by monitoring the generation of 4-methylumbelliferone for 1 h. Values of Ki were determined with nonlinear regression.

Virus release assay.

MDCK cells were incubated with the WSN virus on ice for 60 min to allow binding, and the cells were washed thrice with PBS to remove unbound virus. The cells were then moved to 37*C to begin the infection process. After 3 h p.i., the infection media was replaced with that containing either PGN-ZA, ZA-linker, or PBS. After 4 h, the supernatant was harvested, and the viral titer quantified by virus plaque assay.

42 Chapter 2: PGN-ZA inhibits synergistically both early and late steps of influenza virus replication

Flow cytometry.

To quantify single-cycle infection by flow cytometry, MDCK cells were infected with

WSN virus at moi of 20 for 1 h on ice, followed by washing thrice with ice-cold PBS to remove unbound virus. Infection medium was then added and the temperature raised to

37*C to allow infection to begin. The inhibitors were added at -1, 0, or 1 h p.i. To remove them, the cells were washed 4 times with pre-warmed PBS. Mock-infected and WSN- infected/untreated (PBS) samples acted as controls. At 3 h p.i., the MDCK cells were trypsinized, washed with PBS twice, and fixed with 2% paraformaldehyde in PBS. The fixed cells were washed with PBS containing 2% FBS (PBS-FBS) twice and resuspended in 0.1% saponin in PBS-FBS (permeabilization buffer). After 10 min at RT, the samples were centrifuged and resuspended in 80 tL of the permeabilization buffer containing 1 pg/mL anti-NP (AbD Serotec, Raleigh, NC) and anti-Mi (Abcam) monoclonal antibodies. Following a 1-h incubation in the dark at RT, unbound antibodies were removed by two washes with

1 mL of the permeabilization buffer. The cells were then incubated with 50 p1 of phycoerythrin-linked anti-mouse IgG antibody (Biolegend) for 30 min at RT. Unbound antibodies were again removed by two washes of 1 mL of the permeabilization buffer.

Finally, the cell pellets were resuspended in PBS-FBS and analyzed on the Accuri C6 flow cytometer. The analytical gatings between infected and uninfected cells were determined from the PE fluorescence intensity histograms of the mock-infected negative controls. The extent of influenza infection was quantified as the fraction of cells with fluorescence intensity above the analytical gating. All samples were normalized to the mean of 3 infected, untreated (PBS) controls.

43 Chapter 2: PGN-ZA inhibits synergistically both early and late steps of influenza virus replication

For the flow cytometry-based binding and internalization studies, MDCK cells were

trypsinized, resuspended in DMEM, and exposed to WSN virus at moi of 20 on ice for 1 h.

For internalization studies, the cells were then moved to 37*C for 30 min to allow

endocytosis of the bound virions. To differentiate between internalized and surface-bound

virions, bacterial sialidase was introduced to remove surface-bound virions. The cells were

washed twice with DMEM to remove unbound viruses and inhibitor and treated with

Arthrobacter ureafaciens (20 mU/100 pL) and Vibrio cholera (25 mU/100 ptL)

neuraminidase for 1 h at 37*C. For subsequent flow cytometry analysis, the cells were

fixed, processed, and analyzed as described above.

Virus plaque assays were performed using a modified literature procedure (20, 36, 37).

Briefly, for the early-stage inhibition samples (-1 to 1 h), equal volumes of viruses and

inhibitors of various concentrations were pre-incubated for 1 h prior to cell inoculation.

Thereafter the inoculum was aspirated, and the cells were washed 4 times with pre-

warmed PBS to remove any residual inhibitor or viruses. The cells were then overlaid with

agar solution with no inhibitor. In the case of the late-stage inhibition samples (1 to 72 h),

there was no pre-treatment, and the initial 1-h infection was also done in the absence of

inhibitors. After infection, the cells were overlaid with agar solution containing the

appropriate concentrations of inhibitor. For the combination samples (-1 to 72 h), the

inhibitor was present throughout the assay, from pre-treatment through the agar overlay.

44 Chapter 2: PGN-ZA inhibits synergistically both early and late steps of influenza virus replication

Transmission electron microscopy (TEM).

The WSN virus was sonicated, and remaining viral aggregates were removed using a

0.2-ptm-pore filter. The virus was then incubated at RT for 1 h with either PBS or PGN-ZA at a concentration exceeding 10 x ICso. The surface of a carbon/formvar film supported on a

Cu grid was treated with a drop of the influenza virus solution for 1 min. The surface was washed by successively dipping the grid in 3 drops of water and stained with either 0.75% uranyl acetate or 1% phosphotungstic acid. A drop of the stain was placed on the surface of the grid for 45 s and then removed by absorption onto a piece of filter paper. The samples were allowed to dry overnight and analyzed using a Tecnai G2 Spirit Biotwin TEM instrument.

Fluorescence microscopy.

The labeling process was modified from a published protocol (25). The WSN virus was labeled with Alexa Fluor 647 carboxylic acid succinimidyl ester dye (Invitrogen, Grand

Island, NY) in a carbonate buffer (pH 9.3) at RT for 1 h with gentle shaking. Unbound dye was removed by a buffer exchange with 50 mM Hepes buffer (pH 7.4, 145 mM NaCl) using

NapS gel filtration columns (GE Healthcare, Waukesha, WI). Viral aggregates were removed by filtration immediately prior to experiments using a 0.2-pm filter. MDCK cells were exposed to the dye-labeled WSN at moi of 20 on ice for 1 h to allow binding. Unbound virus and inhibitors were removed by 3 washes of cold PBS, and the samples were immediately moved to a 37'C water bath to begin infection. Samples were washed, fixed at

0, 5, and 15 min p.i. with 2% paraformaldehyde, and cured overnight with DAPI Prolong

Gold (Invitrogen). Images were taken on Applied Precision DeltaVision Ultimate Focus

45 Chapter 2: PGN-ZA inhibits synergistically both early and late steps of influenza virus replication

Microscope with a 60x objective. The images taken were deconvolved to visualize individual virus peaks. For quantitative analysis, the number of viruses per cell was quantified using ImageJ and normalized to the PBS controls.

Inactivation of virus by acidic treatment.

The low-pH inactivation of influenza virus was performed as previously described

(38), and the virus was titrated with the plaque assay.

Statistical Analysis was performed using two-tailed t-tests (39).

46 Chapter 2: PGN-ZA inhibits synergisticallyboth early and late steps of influenza virus replication

Figures and Tables

)0 A * PGN-ZA O PGN INC

01 2 10 50 300 1x10 3 6x10 3 3x10 4 2x10 58x10 54x106 [Inhibitor] (pg/mL) B 100 * PGN-ZA OPGN aso 0 0

[NA] (pg/mL) C f100 * PGN-ZA 10 PGN-SA [PGN

0 0.1 0.5 1 2.5 5 7.5 [HA] (pg/mL)

Figure 1. PGN-ZA binding to influenza virus. PGN-ZA and PGN were first covalently attached to 96-well plates via UV cross-linking. The relative binding of whole influenza A/WSN/33 virions (A), and the two major influenza surface proteins, NA (B) and HA (C), to the inhibitors were determined using ELISA assays. Bare PGN was included as a control of non-specific binding and PGN-SA as a positive control for HA binding. Error bars represent the standard errors of means (SEM) from two independent experiments.

47 Chapter 2: PGN-ZA inhibits synergistically both early and late steps of influenza virus replication

A + Inhibitor V ii I I Oh 3h 4h 7h

Infection Analysis

B ( 1.6

0.8-

PBS PGN-ZA PGN-ZA ZA-linker PBS control + Inhibitor

Figure 2. PGN-ZA inhibits the release of newly synthesized viruses from infected cells. (A) Experimental design to detect the release of newly synthesized viruses from infected cells. (B) MDCK cells were inoculated with WSN virus in a synchronized infection, and unbound virus was then removed. The inhibitors were added 3 h p.i. The supernatant was collected at 7 h p.i., serially diluted, and titrated using the plaque assay. As a control for any remaining inhibitor in the plaque assay, some PBS controls were spiked with the same concentration of PGN-ZA and titrated in parallel with the original PBS controls. Error bars in B represent standard errors of means (SEM) from 3-4 independent experiments. *p<0.05; **p<0.01.

48 Chapter 2: PGN-ZA inhibits synergistically both early and late steps of influenza virus replication

A +Inhibitor Th IT -1 h 0 h 1 h 2 h 3 h

Infection Analysis

0 142 E E0** *PGN-ZA 0.8 - ,,, o ZA-linker E 0 a 0

PBS control -1 to 1 h 0 to 1 h 1 to 3 h -1 to 3 h Infected Uninfected

Figure 3. PGN-ZA inhibits an early step of influenza virus replication. (A) Scheme of time-of-addition experiment to assay inhibition in the early phase of virus infection in a single replication cycle assay. (B) MDCK cells were infected with WSN, and the inhibitors were added in at -1, 0, or 1 h p.i.. The cells were trypsinized, fixed at 3 h p.i., stained for intracellular viral NP and M1, and analyzed by flow cytometry. The gating for virus-infected cells was drawn based on the expression level of a mock-infected control, and the fraction of the cells infected was normalized to the untreated, infected sample. Error bars in B represent standard errors of means (SEM) from 3-5 independent experiments. ***p <

0.001.

49 Chapter2: PGN-ZA inhibits synergistically both early and late steps of influenza virus replication

106-

105 E 10. C E PGN-ZA 103, N o ZA-linker 102. * Zanamivir C 10- 0 .1-

0.1 -1 to 1 h 1 to 72 h -1 to 72 h Time of inhibitor treatment

Figure 4. PGN-ZA synergistically inhibits early and late steps of influenza virus infection. MDCK cells were infected with A/Turkey/MN/80, and the inhibitors were added either -1 to 1 h p.i. or 1 to 72 h p.i. The bars represent IC50 values, i.e., the concentration of inhibitor reducing the plaque number in untreated controls by half. For the samples in the -

1 to 1 h condition, the ICsos are higher than the values indicated, as we did not observe any significant plaque number reduction at this concentration. Error bars in represent standard errors of means (SEM) from 3 independent experiments. ***p < 0.001.

50 Chapter 2: PGN-ZA inhibits synergisticallyboth early and late steps of influenza virus replication

A PBS control PGN-ZA

B 0.8

. *PBS control 0 PGN-ZA 0.4-

0 2 0. U. 1 2 3 4 25 Distribution of viral particles

Figure 5. PGN-ZA causes neither viral aggregation nor any direct virucidal activity.

(A) WSN viruses were stained with uranyl formate and visualized by TEM in the presence or absence of PGN-ZA. Representative TEM images were taken at 4,800x (upper panel) and

49,000x (lower panel) magnifications, respectively. (B) The number of viral particles (1, 2,

3, etc.) in each viral cluster in images of 4,800x magnification were enumerated and normalized to the total number of virions in each image. N is the number of viruses counted for each group (1: N=3303; 2: N=909; 3: N=393; 4: N=208; 5: N= 516). Scale: black, 500 nm; white, 100 nm.

51 Chapter 2: PGN-ZA inhibits synergistically both early and late steps of influenza virus replication

*PBS control o PGN-ZA 1-

.LJ, I Ii . 1 2 3 4 25 Distribution of virus particles per cluster

Figure 6. PGN-ZA does not cause viral aggregation. WSN viruses were stained with phosphotungstic acid and visualized by TEM in the presence or absence of PGN-ZA.

Representative TEM images were taken at 4,800x magnifications. The number of viral particles (1, 2, 3, etc.) in each viral cluster were enumerated and normalized to the total number of virions in each image. N is the number of viruses counted for each group (1:

N=1430; 2: N=401; 3: N=116; 4: N=104; 5: N=152).

52 Chapter 2: PGN-ZA inhibits synergistically both early and late steps of influenza virus replication

A J

+Sialidase i PI, 37*C, 1 h IlIl

B (1) (11) (111) (IV) 4*C 40C 37*C 37*C Binding Binding Internalization Internalization + Sialidase + Sialidase

PBS control

PGN-ZA

Viral NP

c -C 1. U PBS control O PGN-ZA E080O. 4 0

Ub-

4*C 40C 370C 370C + Sialidase + Sialidase

53 Chapter 2: PGN-ZA inhibits synergistically both early and late steps of influenza virus replication

Figure 7. PGN-ZA does not inhibit binding and endocytosis of influenza virus infection. (A) An experimental scheme to study the effect of PGN-ZA on viral binding to cells and the subsequent endocytosis. (B) MDCK cells were inoculated with virus in the absence (top row) or presence of PGN-ZA (middle row) at 4*C for 1 h to allow for virus binding to cells. To study the effect of PGN-ZA on binding, this sample was fixed directly after the 4*C incubation and stained for viral proteins NP and M1 (Group I). For assaying endocytosis, the cells were then incubated at 37*C for 30 min to allow for the bound virus to be internalized. Some samples were treated with sialidase to remove surface-bound virions (Group II and IV). All samples were fixed and stained for viral NP and M1. Flow- cytometry gating was determined based on the uninfected control (shown as gray overlay in Group I PBS control panel), and the percentage of cells exceeding the gate for each sample was normalized to the untreated control to determine virus binding and endocytosis. (C) The results represent the mean ± SEM of the fraction of cells infected from

2 to 4 independent experiments normalized to the mean of untreated Group I samples.

54 Chapter2: PGN-ZA inhibits synergistically both early and late steps of influenza virus replication

A PBS control PGN-ZA

0 min

5 min

15 mi

B 2.0 ** U PBS control 0 PGN-ZA 81.5- C0

I * 0 0

0. 0 min 5 min 15 min

55 Chapter2: PGN-ZA inhibits synergisticallyboth early and late steps of influenza virus replication

Figure 8. PGN-ZA inhibits virus-endosomal fusion. (A) Influenza virus was labeled with

Alexa 647 succinimidyl ester (red) and mixed with PGN-ZA. The mixture was then added to

MDCK cells at 4C for 1 h, washed twice with PBS, and incubated with medium containing either PBS or PGN-ZA, at 37*C for 15 min. At time points of 0, 5, and 15 min post-infection, the cells were washed, fixed, DAPI-stained (green) to show cell nuclei, and imaged by fluorescent microscopy. Representative images are shown. (B) The mean ± SEM of the number of virions per cell was quantified from the microscopy images and normalized to the untreated control. *p < 0.05; **p < 0.01.

56 Chapter 2: PGN-ZA inhibits synergisticallyboth early and late steps of influenza virus replication

2- cc >1

0 25xIC 5 100xlIC5 PBS control PGN-ZA

Figure 9. PGN-ZA protects influenza virus from acidic (pH 5) inactivation. Influenza virus was incubated at pH 5 in the presence or absence of PGN-ZA for 15 min at 37*C. The level of infectious viruses remaining after the low pH treatment was quantified by serial dilution and the plaque assay. The SEM of the normalized viral titer from three independent experiments are shown. **p < 0.01; ***p < 0.001.

57 Chapter 2: PGN-ZA inhibits synergistically both early and late steps of influenza virus replication

Strain Ki (nM, based on ZA)

ZA-linker PGN-ZA

A/WSN/33 0.92 ± 0.19 0.27 ± 0.06

A/PR/8/34 6.8 ± 1.3 0.72 ± 0.04

Table 1. Inhibition constants (Ki) of viral neuraminidase by ZA-linker and PGN-ZA against WSN and PR8 influenza strains. The Ki values, expressed in concentrations of ZA whether free or conjugated to PGN, were obtained from experiments run in triplicate.

58 Chapter 2: PGN-ZA inhibits synergistically both early and late steps of influenza virus replication

Supplementary Figures and Tables

A 100

w PGN-ZA D PGN

04 50

S0 20 80 300 101 5x10 3 2x10 4 8x10 4 3x10 5 106 5x106 2x10 7

_0 100 .O B

" PGN-ZA PGN-SA c PGN 50

0 1 Ja 'k, A M M '-' 6 1, I 20 80 300 103 5x10 3 2x10 4 8x10 4 3x10 5 106 5x10 6 2x10 7 [Inhibitor] (pg/mL) Figure S1. PGN-ZA binds to neuraminidase. Varying dilutions of PGN-ZA, PGN-SA, and

PGN were first covalently attached to 96-well plates via UV cross-linking. The relative binding of NA (A) and HA (B) to the inhibitors was determined using ELISA assays. As with

Figure 1, bare PGN was used as a control for non-specific binding, and PGN-SA was included as a positive control for HA binding. Error bars represent the SEM from two independent experiments.

59 Chapter2: PGN-ZA inhibits synergistically both early and late steps of influenza virus replication

WSN virus 12

0 10 100 103 104

0.01 mg/mL 'I, PGN-ZA C a, 45

0 10 100 103 104

22 WSN virus

0.01 mg/mL 11 PGN-ZA

0 10 100 103 104

Hydronamic radius (nm)

Figure S2. PGN-ZA does not cause viral aggregation. The hydrodynamic radius of WSN viruses was determined by dynamic light scattering in the presence (lower graph) or absence (upper graph) of PGN-ZA. PGN-ZA alone was included as a negative control

(middle graph). Results shown are representative of two independent experiments.

60 Chapter 2: PGN-ZA inhibits synergistically both early and late steps of influenza virus replication

[Inhibitor] (mg/mL) 1 10-3 0

PGN

PGN-ZA + PGN-SA

PGN

. PGN-ZA

Figure S3. PGN-ZA does not inhibit hemagglutination. Hemagglutination inhibition assay was used as an additional method to evaluate the effect of the multivalent inhibitors on virus binding to target cells. Four hemagglutinating units (HAU) of the WSN virus was first pre-incubated with serial dilutions of inhibitor in a U-bottom 96-well plate for 1 h at

RT, and 50 ptL of 1% red blood cells was added to each well. The plate was then placed on ice for 1 h before observing red blood cell agglutination. PGN was included as a control for non-specific binding, and PGN-SA was included as a positive control for hemagglutination inhibition. Results shown are representative of two independent experiments.

61 Chapter 2: PGN-ZA inhibits synergistically both early and late steps of influenza virus replication

1.6

CU o0.8 -

0 25xIC 50 100xIC50 PBS control PGN-ZA

Figure S4. pH 7 controls for acidic (pH 5) inactivation assay. The samples were included as a control for the pH 5 virus inactivation assay in Figure SC. Influenza virus was incubated at pH 7 in the presence or absence of PGN-ZA for 15 min at 370 C. The level of infectious viruses remaining was quantified by serial dilution and the plaque assay. The mean ± SEM of the normalized viral titer from three independent experiments are shown.

62 Chapter 2: PGN-ZA inhibits synergisticallyboth early and late steps of influenza virus replication

References

1. Fiore AE, et al. (2010) Prevention and Control of Influenza with Vaccines: Recommendations of the Advisory Committee on Immunization Practices (ACIP), 2010 in MMWR Recomm Rep, pp 1-62. 2. Webby RJ & Webster RG (2001) Emergence of influenza A viruses. Philosophical transactions of the Royal Society of London. Series B, Biological sciences 356(1416):1817-1828. 3. Osterholm MT, Kelley NS, Sommer A, & Belongia EA (2012) Efficacy and effectiveness of influenza vaccines: a systematic review and meta-analysis. The Lancet Infect Dis 12(1):36-44. 4. Pinto LH & Lamb RA (2007) Controlling influenza virus replication by inhibiting its proton channel. Mol Biosyst 3(1):18-23. 5. Kim CU, et al. (1997) Influenza neuraminidase inhibitors possessing a novel hydrophobic interaction in the enzyme active site: design, synthesis, and structural analysis of carbocyclic sialic acid analogues with potent anti-influenza activity.JAm Chem Soc 119(4):681-690. 6. von Itzstein M, et al. (1993) Rational design of potent sialidase-based inhibitors of influenza virus replication. Nature 363(6428):418-423. 7. De Clercq E (2006) Antiviral agents active against influenza A viruses. Nat Rev Drug Discov 5 (12):1015-1025. 8. von Itzstein M (2007) The war against influenza: discovery and development of sialidase inhibitors. Nat Rev Drug Discov 6(12):967-974. 9. Lipatov AS, et al. (2004) Influenza: emergence and control. J Virol 78(17):8951- 8959. 10. Bright RA, et al. (2005) Incidence of adamantane resistance among influenza A (H3N2) viruses isolated worldwide from 1994 to 2005: a cause for concern. Lancet 366(9492):1175-1181. 11. Fiore AE, et al. (2011) Antiviral Agents for the Treatment and Chemoprophylaxis of Influenza: Recommendations of the Advisory Committee on Immunization Practices (ACIP) in MMWR Recomm Rep, ed CDC, pp 1-24. 12. Moscona A (2009) Global transmission of oseltamivir-resistant influenza. New Englj Med 360(10):953-956. 13. WHO (2011) Global monitoring of antiviral resistance in currently circulating human influenza viruses, November 2011. in Weekly Epidemiological Record, pp 497-508. 14. Das K, Aramini JM, Ma LC, Krug RM, & Arnold E (2010) Structures of influenza A proteins and insights into antiviral drug targets. Nat Struct Mol Biol 17(5):530-538. 15. Hayden F (2009) Developing new antiviral agents for influenza treatment: what does the future hold? Clinical infectious diseases: an official publication of the Infectious DiseasesSociety of America 48 Suppl 1:S3-13. 16. Mammen M, Choi S-K, & Whitesides GM (1998) Polyvalent interactions in biological systems: implications for design and use of multivalent ligands and inhibitors. Angew Chem Int Ed 37(20):2754-2794.

63 Chapter 2: PGN-ZA inhibits synergistically both early and late steps of influenza virus replication

17. Honda T, Yoshida S, Arai M, Masuda T, & Yamashita M (2002) Synthesis and anti- influenza evaluation of polyvalent sialidase inhibitors bearing 4-guanidino- Neu5Ac2en derivatives. Bioorg Med Chem Lett 12(15):1929-1932. 18. Mammen M, Dahmann G, & Whitesides GM (1995) Effective inhibitors of hemagglutination by influenza virus synthesized from polymers having active ester groups. Insight into mechanism of inhibition.J Med Chem 38(21):4179-4190. 19. Lees WJ, Spaltenstein A, Kingery-Wood JE, & Whitesides GM (1994) Polyacrylamides bearing pendant alpha-sialoside groups strongly inhibit agglutination of erythrocytes by influenza A virus: multivalency and steric stabilization of particulate biological systems.J Med Chem 37(20):3419-3433. 20. Weight AK, et al. (2011) Attaching zanamivir to a polymer markedly enhances its activity against drug-resistant strains of influenza a virus. J Pharm Sci 100(3):831- 835. 21. Gamblin SJ & Skehel JJ (2010) Influenza hemagglutinin and neuraminidase membrane glycoproteins. J Biol Chem 285(37):28403-28409. 22. Klenk E, Faillard H, & Lempfrid H (1955) Enzymatic effect of the influenza virus. Hoppe-Seyler'sZeitschrift Physiol Chem 301(4-6):235-246. 23. Gottschalk A (1957) Neuraminidase: the specific enzyme of influenza virus and Vibrio cholerae. Biochim Biophys Acta 23(3):645-646. 24. Gottschalk A (1959) On the mechanism underlying initiation of influenza virus infection. Ergebnisse Mikrobiol Immun Exp Ther 32:1-22. 25. Lakadamyali M, Rust MJ, Babcock HP, & Zhuang X (2003) Visualizing infection of individual influenza viruses. Proc Natl Acad Sci USA 100(16):9280-9285. 26. Scholtissek C (1985) Stability of infectious influenza A viruses at low pH and at elevated temperature. Vaccine 3(3):215-218. 27. Lamb RA & Krug RM (2001) Orthomyxoviridae: the viruses and their replication (Lippincott Williams & Wilkins). 28. Palese P (2007) Orthomyxoviridae: the viruses and their replication (Lippincott Williams & Wilkins) 5 Ed. 29. Greengard 0, Poltoratskaia N, Leikina E, Zimmerberg J, & Moscona A (2000) The anti-influenza virus agent 4-GU-DANA (zanamivir) inhibits cell fusion mediated by human parainfluenza virus and influenza virus HA.j Virol 74(23):11108-11114. 30. Matrosovich MN, Matrosovich TY, Gray T, Roberts NA, & Klenk HD (2004) Neuraminidase is important for the initiation of influenza virus infection in human airway epithelium.J Virol 78(22):12665-12667. 31. Colman PM & Ward CW (1985) Structure and diversity of influenza virus neuraminidase. Current Topics Microbiol Immunol 114:177-255. 32. Suzuki T, et al. (2005) Sialidase activity of influenza A virus in an endocytic pathway enhances viral replication.J Virol 79(18):11705-11715. 33. Liu C, Eichelberger MC, Compans RW, & Air GM (1995) Influenza type A virus neuraminidase does not play a role in viral entry, replication, assembly, or budding. j Virol 69(2):1099-1106. 34. Haldar J, et al. (2010) Bifunctional polymeric inhibitors of human influenza A viruses. Pharm Res 27(2):259-263. 35. Chandrasekaran A, et al. (2008) Glycan topology determines human adaptation of avian H5N1 virus hemagglutinin. Nat Biotechnol 26(1):107-113.

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36. Hayden FG, Cote KM, & Douglas RG (1980) Plaque inhibition assay for drug susceptibility testing of influenza viruses. Antimicrob Agents Chemother 17(5):865- 870. 37. Gubareva LV, et al. (2002) A release-competent influenza A virus mutant lacking the coding capacity for the neuraminidase active site.J Gen Virol 83(11):2683-2692. 38. Plotch SJ, et al. (1999) Inhibition of influenza A virus replication by compounds interfering with the fusogenic function of the viral hemagglutinin. J Virol 73(1):140- 151. 39. Geoff C, Fiona A, & David LV (2007) Error bars in experimental biology. J Cell Biol 177(1): 7-11

65 Chapter 2: PGN-ZA inhibits synergistically both early and late steps of influenza virus replication

66 Chapter3: PGN-ZA delays the emergence of drug resistance

Chapter 3

POLYMER-ATTACHED ZANAMIVIR DELAYS THE EMERGENCE

OF DRUG RESISTANCE

Abstract

Rapid development of resistance to the existing antivirals poses a major challenge in the control of influenza virus infection. Covalently conjugating multiple copies of the drug zanamivir (ZA) via a flexible linker to poly-L-glutamine greatly enhances its antiviral potency, and confers a new mechanism of antiviral action potentially useful for minimizing drug resistance. In this study, we have investigated the effects of this polymer-attached drug on the emergence of drug resistance, by passaging influenza virus at increasing concentrations of monomeric ZA or the polymeric drug. We found that viruses adapted to growing in high concentrations of monomeric ZA (>100 ptM) by passage 8, whereas viral growth remained suppressed in the presence of low concentrations of the polymer (< 0.1 pM) even up to passage 23. Sequence analysis revealed the emergence of an amino acid substitution E119G in neuraminidase at passage 8 of the viruses grown in monomeric ZA; this mutation is known to confer high-level resistance to ZA. In contrast, the virus grown in the polymeric drug remained free of substitutions in residue E119, and other known resistance-associated residues. Interestingly, we instead found novel amino acid substitutions in hemagglutinin (R220G, D241G) and neuraminidase (G111D) emerging during passages 14-17. Next, we explored the effects of these substitutions on virus sensitivity to ZA and polymer-attached ZA. Surprisingly, we found that although the polymeric drug-selected variant was resistant to monomeric ZA, the virus remained

67 Chapter 3: PGN-ZA delays the emergence of drug resistance susceptible to low pM concentrations of the polymeric drug itself. Taken together, these data demonstrate that polymer-attached ZA is able to (i) delay the emergence of drug resistance by at least six passages; and (ii) retain moderate potency against the selected

ZA-resistant variants. These findings not only shed light on amino acid residues that are critical for certain viral processes, but also provides further impetus to explore the use of polymer-attached inhibitors to minimize influenza drug resistance.

68 Chapter 3: PGN-ZA delays the emergence of drug resistance

Introduction

Emergence of antiviral resistance is a major challenge that plagues the clinical management of influenza (1, 2). Not only does antiviral resistance emerge during drug treatment, it can also develop in untreated cases, and spread to replace the susceptible strains in the absence of drug pressure (3).

Four antivirals have been approved for influenza prophylaxis and treatment in the

United States. Amantadine and rimantadine are inhibitors of the influenza M2 ion channel

(4), and oseltamivir and zanamivir (ZA) are inhibitors of the influenza neuraminidase (NA) enzyme (5, 6). Emergence of resistance to the M2 inhibitors is well documented. A single mutation in M2, most often S31N, is sufficient to confer high-level resistance, which develops rapidly during treatment in patients (7-11), and within 1-2 passages with drug in cell culture (12, 13). The mutation does not compromise virus fitness and transmissibility, and we have since observed the global spread of M2 inhibitor resistance in H3N2 viruses since 2003, and the naturally occurring S31N mutation in the 2009 H1N1 pandemic virus

(3, 14, 15). All currently circulating influenza strains are resistant to amantadine and rimantadine (1), and the use of these inhibitors are no longer recommended (16).

Although the NA inhibitors appear to have a higher barrier to resistance than the M2

inhibitors (17), oseltamivir-resistant viruses have been recovered in vitro, and from

patients during treatment (18-23). The common mutations identified (R292K, N294S,

H274Y) prevent the rearrangement of amino acids in the NA active site required for the

binding of oseltamivir. Not much attention was given to these oseltamivir-resistant

mutants, as these mutants were believed to have impaired infectivity and transmissibility

69 Chapter 3: PGN-ZA delays the emergence of drug resistance

(24). However, in 2008-2009, the startlingly widespread oseltamivir resistance (H274Y mutation) amongst seasonal H1N1 viruses clearly signals our lack of understanding and defenses against this unpredictable pathogen (24, 25). The resistant seasonal H1N1 were replaced in circulation by the 2009 H1N1 pandemic viruses, which thankfully remain sensitive to the NA inhibitors. Worryingly, a -1% incidence rate of oseltamivir resistance amongst the 2009 H1N1 clinical isolates has been reported so far, and some evidence for human-to-human transmission of these mutants put forth (1, 26).

In theory, since ZA more closely resembles the natural substrate of NA, sialic acid, resistance may not develop as readily as in the case of oseltamivir (17). Although ZA- resistant mutants have yet to be recovered from immunocompetent patients, ZA-resistant mutants have been isolated in cell culture, with amino acid changes found in hemagglutinin

(HA) and/or NA (27). The mutations in HA may compromise receptor binding fitness, and the mutations in NA typically involve previously conserved residues 119 or 292 in the NA enzymatic active site (19, 27, 28). In vitro, these amino acid substitutions typically break through after 5-8 cell culture passages (29-34). ZA resistance has also been observed sporadically amongst circulating viruses in Asia, conferred by naturally occurring mutations Q136K and S247N in NA (35, 36).

Thus, the need to develop antivirals that can reduce the risk of development of resistant influenza viruses is both urgent and important (2). There have been few studies on antivirals that can delay or minimize drug resistance. Combination chemotherapy with

125 IM amantadine and 0.1-1 pM of oseltamivir was shown to suppress viral growth up to passage 5 (37), and triple combination therapy with amantadine, oseltamivir and ribavirin,

70 Chapter3: PGN-ZA delays the emergence of drug resistance used at amounts 3 times higher than the clinically relevant concentration, delayed the emergence of drug resistance up to passage 5 (38).

A promising approach for the next generation of antivirals is the use of multivalent polymeric inhibitors (39). We have previously shown that the antiviral efficacy of ZA is dramatically enhanced when multiple copies of ZA are attached via a flexible linker to the benign and biodegradable polymer poly-L-glutamine (PGN) (20). The resultant PGN-ZA is three to four orders of magnitude more potent than monomeric ZA in plaque reduction assays and, importantly, is effective against NA inhibitor-resistant influenza viruses.

Further investigation also revealed the mechanism underlying this dramatically improved antiviral potency: the multivalent PGN-ZA can inhibit synergistically influenza viral fusion and release.

Herein we have tested in vitro the ability of PGN-ZA in minimizing the emergence of drug resistance. We show that PGN-ZA can delay the emergence of drug-resistant variants, up to at least six passages more than monomeric ZA. Sequencing analysis revealed novel mutations in the HA1 (R220G, D241G) and NA (G111D) in the PGN-ZA selected viruses after 23 passages. Although these mutations confer high-level resistance to monomeric ZA, the variant is still inhibited by pM concentrations of PGN-ZA in plaque reduction assays.

71 Chapter3: PGN-ZA delays the emergence of drug resistance

Results

PGN-ZA suppresses viral growth under drug selection pressure

To investigate the effects of PGN-ZA on the emergence of drug resistance, we passaged A/Turkey/MN/80/833 (H4N2) (TKY) in MDCK cells in the presence of increasing concentrations of either ZA itself, or PGN-ZA, and assayed the ability of the residual viruses to grow (Figure 1A). The viruses were first diluted to MOIs of 0.001-0.1, and pre-incubated with either inhibitors or a PBS control for 1 h at RT. MDCK cells were inoculated with these virus mixtures for 45 min at 37*C, and the cells were then washed with pre-warmed PBS to remove any unbound or weakly bound viruses. The virus was grown for three days in medium containing the appropriate concentrations of PGN-ZA, ZA, or a PBS control. Viral growth for the three different conditions was determined on day three of each passage by a hemagglutination assay (Figure 1B). Virus from the lowest MOI showing hemagglutinating activity will be used for the next passage. The starting concentration for PGN-ZA was determined based on its IC5o value by plaque reduction assay (Chapter 2, Figure 2E), and that of ZA was determined based on previous reports (29, 30, 40). The inhibitor concentration was increased in the subsequent passage if the virus appeared to have adapted to growing in the presence of the inhibitor. We found that influenza virus adapted to growing in high concentrations (>100 pM) of monomeric ZA by passage 8 (Figure 1B), consistent with previous studies (29, 30), whereas the growth of viruses under PGN-ZA selection pressure remained suppressed by low concentrations of PGN-ZA (<0.1 pM)

(Figure 1B) even after 23 passages. These results clearly demonstrate that PGN-ZA

72 Chapter 3: PGN-ZA delays the emergence of drug resistance suppresses viral growth under drug selection pressure, and most likely delay the emergence of drug-resistant influenza viruses.

Sequence analysis of viral supernatants

Influenza virus has two main surface glycoproteins, HA and NA (41). Both HA and

NA bind to the same receptor, sialic acid on the cell-surface glycans (42-45). HA binds to terminal sialic acid on cell-surface glycans to initiate infection, and NA cleaves sialic acid from the cell-surface glycans to release newly generated viruses from cells (45-47). A functional balance exists between these two opposing activities to allow for efficient virus replication (28). Selection pressure by NA inhibitors disrupts this balance, and adaptation can be achieved by compensatory mutations in HA, NA or both proteins (28). Also, viruses grown in eggs or in MDCK cells can show different sensitivities to ZA (48), and host tropism selection has been linked to mutations in HA (49), which affects its binding preference to specific conformations of sialic acid (50, 51).

Thus, to determine if PGN-ZA delays the emergence of drug resistance, we first sequenced the entire HA and NA genes from the day 3 supernatants of parental wild type

(WT), MDCK-passaged, and drug-selected viruses from selected key passages (Table 1 and

Figure 2). Passaging the virus for 23 rounds in the absence of inhibitors (hereforth termed

DF23) resulted in the appearance of a mutation at residue 43 of the HA2 subunit (Ala to

Val) (Table 1). Analysis of NA gene confirmed the emergence of drug resistance after 8 passages in ZA; a mutation was found in residue 119 (Gln to Gly), and this variant

(hereforth termed Z12) took over the entire population by passage 12 (Figure 2). The

73 Chapter 3: PGN-ZA delays the emergence of drug resistance residue 119 substitution was previously discovered in other ZA selection studies, and is well documented to be associated with ZA resistance (29, 30, 34, 40, 52, 53). Additional mutations were also found in HA1 (residue 220: Arg to Lys; residue 149: Gly to Gln) and

HA2 (residue 66: Ile to Val; residue 114: Gln to Lys), although these mutations did not reach full saturation in the population by passage 12 (Figure 2).

Next, for the viruses grown in PGN-ZA, we examined the HA and NA sequence of viral supernatant from passages 8, and 12 to 23. Sequencing analysis revealed that the viruses remained free of changes in residue E119, and other known resistance-associated residues like R292K for all 23 passages. Interestingly, we found novel amino acid substitutions in HA1 (R220G and D241G), and in NA (G111D) appearing subsequently during passage 14-17 (Figure 2). All three substitutions reached 100% saturation by the final passage (hereforth termed P23). To confirm and test for stability of PGN-ZA-selected genotype, we also isolated 20 virus clones from the P23 viral supernatant and cultured each clone in the absence of inhibitors. The sequences of the clones are consistent with that of the primary P23 supernatant.

All the amino acid substitutions for each variant are listed in Table 1, and the variants did not show any obvious defects in viral growth (data not shown) or changes in receptor binding specificity (Table 1). The localizations of the amino acid residues with substitutions selected by ZA and PGN-ZA are shown in Figure 3, and their significance will be further discussed in the next section. Briefly, most of the amino acid substitutions found in HA were located near the primary or secondary receptor binding site. In NA, residue 119

74 Chapter 3: PGN-ZA delays the emergence of drug resistance is part of the NA enzymatic active site, and residue 111 is at the interface of two NA subunits.

Effect of amino acid changes to drug sensitivity in plaque reduction assays

To understand the biological significance of the novel amino acid substitutions identified, we assessed the sensitivity of the viral variants Z12 and P23 to both ZA and

PGN-ZA using plaque reduction assays (Fig. 4 and Table 2). The experiments yielded several interesting observations. Similar to the parental wild type virus (WT), the MDCK- passaged DF23 variant remained sensitive to both inhibitors (Fig. 4 and Table 2, row 2). As expected, virus grown in 12 passages of ZA (Z12), with the E119G substitution, was resistant to ZA (Fig. 4A and Table 2, last row). Even at 150 pM of ZA, neither inhibition of plaque size nor plaque quantity was observed, which is at least 3000 times less sensitive than WT. Importantly, multivalent drug conjugate PGN-ZA was still somewhat effective against Z12; reduction in plaque size and number was observed with ICso of around 5-15

PM.

Next, we examined the inhibition of plaque number and size of virus grown after 23 passages in PGN-ZA (P23) (Figure 4B and Table 2, row 3). Interestingly, P23 was resistant against monomeric ZA; like Z12, it was also at least 3000-fold less sensitive compared to

WT. Although P23 was also more resistant against the multivalent drug conjugate compared to WT and DF23, PGN-ZA was still able to inhibit P23 replication with an IC50 of

0.6 ptM and 5 pM for plaque size and plaque number inhibition respectively. In summary, these plaque reduction data indicate that the ZA-selected variant Z12 was highly resistant

75 Chapter 3: PGN-ZA delays the emergence ofdrug resistance to ZA itself, and less so to PGN-ZA. Similarly, the PGN-ZA-selected variant P23 was highly resistant to monomeric ZA, and less so to PGN-ZA itself.

Inhibition of the NAs of variant viruses

Next, we went on to investigate if the viral variants' reduction in sensitivity to both inhibitors in the plaque reduction assay was predominantly caused by the amino acid substitutions in NA. To determine if their plaque reduction assay phenotype was correlated with the binding affinity of the inhibitors to viral NA, we performed kinetic NA inhibition assays to measure the variants' inhibition constants Ki. The results presented in Table 3 reveal several important observations. First, as expected, the NA of drug-selected variants

P23 and Z12 bind monomeric ZA more weakly compared to the parental WT and MDCK- passaged DF23 (Table 3, column 2). The G111D mutation lowers ZA binding about 10-fold, whereas the E119G variant NA has a 70-fold decrease in ZA binding. Taken together with the plaque reduction results, the binding affinities observed for ZA correlates with its increased IC50 in the plaque reduction assay. Second, in all the strains tested, PGN-ZA is a markedly more potent NA enzyme inhibitor than its monomeric counterpart ZA. Third, surprisingly, the viral NAs of P23 and Z12 bind almost just as strongly to multivalent PGN-

ZA as WT and DF23; with almost two orders of magnitude improvement over that of ZA

(Table 2, last two rows). The polymeric presentation of PGN-ZA completely compensates for the weakened binding in the NA of drug-selected variants, consistent with the observations in our previous study (54). However, this does not reflect the reduction in sensitivity to PGN-ZA observed from the plaque reduction assays (Fig. 4, Table 2).

76 Chapter 3: PGN-ZA delays the emergence of drug resistance

Discussion

In this report, we have investigated the effect of PGN-ZA on the emergence of resistance by culturing the TKY virus at increasing concentrations of either ZA or PGN-ZA.

We have found that under ZA selection, the virus appears to adapt to growing in high ZA concentrations (ICso>100 pM) by passage 8 in vitro (Figure 1A and 1B). By Passage 12, the

ZA-selected variant (Z12) was growing well in the presence of 500 pM ZA. Compared to its

small-molecule predecessor, the viral growth in PGN-ZA was still suppressed by low

concentrations of the inhibitor after 23 passages (Figure 1B).

Sequence analysis of viral supernatant from selected passages of the study afforded

several findings on the timeline and mechanism of drug resistance progression. First, for

the virus grown in the presence of ZA, genotyping established the emergence of the E119G

substitution in NA as early as passage 8; this variant reached 100% saturation by passage

12 (Figure 2). This is consistent with previous ZA selection studies where the same

mutation was also identified, and this mutation is well documented to confer high-level

resistance to ZA (29, 30, 34, 40, 52, 53). Glu 119 is conserved among all influenza viruses

and located in the enzyme active site of NA (Figure 3C) (47). The mutation from Glu to Gly

causes the loss of a stabilizing ionic interaction between the guanidino moiety on ZA and

the carboxylate of residue 119 (29, 55, 56). Changes in Glu 119 can lead to thermal and pH

instability, a 250- to 1000-fold decrease in ZA binding affinity, and a drop of up to five

orders of magnitude in the plaque reduction assay IC5o (27, 31, 40, 53, 57). Similarly, we

have also observed a consistent loss in ZA binding affinity and a large increase in the IC5o

against ZA. Although Z12 was much less sensitive to PGN-ZA compared to the WT and DF23

77 Chapter 3: PGN-ZA delays the emergence ofdrug resistance controls, the virus plaque formation was still susceptible to 5-15 pM concentrations of

PGN-ZA (Table 2). Several other amino acid substitutions were also observed in HA1 and

HA2, but these did not reach 100% saturation at passage 12. Although less is known regarding the biological significance of these residues, they (R220K and G137E) form part of the receptor binding site on HA1, and residue 66 of HA2 is close to the secondary receptor binding site (Figure 3A), and may affect receptor binding efficiency.

From sequence analysis, we found that PGN-ZA did not induce changes in residue

119 or any other known resistance-associated residues like R292K throughout the experiment. Instead, we found that PGN-ZA selected for a set of different, and previously undescribed amino acid substitutions (Figure 2). Amino acid substitutions emerged in HA1

(R220G, D241G) and NA (G111D) during passages 14-17, and all three substitutions reached saturation by passage 21. Next, we asked the question- what were the effects of these novel substitutions on the P23's sensitivity to ZA and PGN-ZA? Surprisingly, P23 showed reduced ZA binding affinity, and its plaque formation remained uninhibited even at

150 p.M concentration ZA (Fig. 4A, Table 2-3). In the case of multivalent PGN-ZA, although its binding to P23's NA was only slightly affected (Table 3), P23 was much less sensitive in the plaque reduction assay (Table 2). Nonetheless, despite the reduction in sensitivity, P23 was still susceptible to 0.6-6 p.M of PGN-ZA in the plaque reduction assay (Table 2).

How does each of the amino acid changes in HA and NA contribute to P23's drug resistance phenotype? The location of Gly 111 is distal from the NA enzymatic active site and faces the other subunits of the NA homotetramer complex (Figure 3C-D), and the

G111D substitution caused a 10-fold and 3-fold decrease in binding affinity to ZA and PGN-

78 Chapter 3: PGN-ZA delays the emergence of drug resistance

ZA, respectively. The amino acid change from the small glycine to the charged aspartic acid may affect the formation or stability of the tetramer, or the presentation of the enzyme active sites to ZA. However, although only a modest change was observed in binding affinity to PGN-ZA, P23 required 2500-fold more PGN-ZA for 50% inhibition in the plaque reduction assay compared to the MDCK passaged control DF23 (Table 2). This lack of correlation alludes to the important role of the mutations in HA1 in conferring resistance to the polymer. The R220G mutation, which first emerged in Passage 15, is situated close to the receptor binding domain (Figure 3A). The D241G mutation first emerged in Passage 17; it is situated at the interface of two HA subunits in the trimeric complex, and may participate in stabilizing salt bridge formation with the receptor binding domain of the adjacent HA (58, 59) (Figure 3A). Interestingly, an R220S mutation had been identified in

H3N2 virus by an inhibitor of influenza virus fusion, and shown to increase the virus fusion pH while maintaining similar receptor binding properties (60). We have previously shown that PGN-ZA has at least two mechanisms of antiviral action; it interferes with viral- endosomal fusion, and inhibits NA enzymatic activity to block viral release. Addition of

PGN-ZA causes an accumulation of viruses in late endosomes, and also protects the viruses from inactivation by pH 5 incubation. In light of PGN-ZA's mechanism of action, in addition to selecting variants with weaker binding to ZA, an additional mechanism of viral escape from PGN-ZA would perhaps be to modulate viral fusion and/or destabilize the HA trimeric complex. By increasing the pH of fusion, the viral genome can be released into the cytosol early on in the endocytic pathway, thus enabling viral escape from PGN-ZA inhibition.

Taken together, results from the sequence analysis and phenotyping assays clearly indicate that PGN-ZA is able to delay the emergence of drug resistance by at least six

79 Chapter 3: PGN-ZA delays the emergence of drug resistance passages, with a significantly better resistance profile than its monomeric predecessor ZA.

Since both the HA1 and NA mutations in the polymer-selected virus emerged and reached saturation together (Figure 2), the virus may simultaneously need two distinct mechanisms of viral escape against PGN-ZA. At least two mutations have to occur for the virus to escape from PGN-ZA's dual mode of antiviral action. The probability of this event occurring is much lower than the single mutations required to gain resistance against all the other antivirals, which can rationalize the delay in the emergence of drug resistance (61). Also, it is noted that the ZA-resistant variants Z12 and P23 are still susceptible to low ptM concentrations of PGN-ZA. Since PGN-ZA was a very potent inhibitor to begin with, their plaque formation was still inhibited by low [tM concentrations of PGN-ZA despite the three orders of magnitude increase in IC50.

In summary, these results presented here demonstrate, to our knowledge, for the first time, an influenza inhibitor can significantly delay the emergence of drug resistance. In addition, we also identified novel mutations associated with ZA resistance, which is of much interest, as few mutations are known to confer resistance to ZA. Surprisingly, these findings also highlight the potential of these dual mechanism inhibitors as novel probes to identify amino acid residues involved in the fusion or viral release processes, and to better understand the functional balance between HA and NA. Our finding that the multivalent presentation of an existing small molecule drug ZA can minimize drug resistance opens up further possibilities in influenza drug design, and provides further impetus for the use of polymer-attached inhibitors in the control of influenza drug resistance.

80 Chapter 3: PGN-ZA delays the emergence of drug resistance

Materials and Methods

Inhibitors.

Poly-L-glutamic acid (molecular weight = 50,000-100,000 Da) and all other chemicals, biochemicals, and solvents were purchased from Sigma-Aldrich Chemical Co (St.

Louis, Missouri, USA). 4-guanidino-Neu5Ac2en (4-guanidino-2,4-dideoxy-2,3-dehydro-N- acetylneuraminic acid) was purchased from Bioduro (Beijing, China). PGN-ZA and the bare poly-L-glutamine was prepared and characterized as described previously (54).

Viruses and cells.

The wild-type A/Turkey/MN/80 (H4N2) (TKY) virus stock was obtained from CDC.

The virus stock was propagated in 11-day-old embryonated chicken eggs. After 3 days of virus growth, the allantoic fluid was collected, clarified with low-speed centrifugation, and concentrated before sucrose gradient purification in a Beckman SW41 rotor at 24,000 rpm.

Viruses were resuspended in phosphate-buffered saline (PBS) and stored at -80'C. The

MDCK cells were routinely passaged in Eagle's minimal essential medium (MEM) containing 10% fetal bovine serum, and only cells that were below Passage 11 were used in this study. In the drug selection study, viruses were grown in high-glucose medium supplemented with BSA, glutamine, and penicillin streptomycin.

Drug selection study.

The procedure used in the drug selection study was modified from previously published reports (30, 31). TKY virus was first diluted to 3 serial logio dilutions,

81 Chapter 3: PGN-ZA delays the emergence of drug resistance appropriate for infecting MDCK cells in a 12-well plate at a multiplicity of infection of

0.001-0.1. Each virus dilution were pre-incubated with either ZA, PGN-ZA, or PBS for 1 h at

RT, after which the virus was allowed to infect the cells for 45 minutes at 370 C. The inoculum was then aspirated, and the cells washed twice with pre-warmed PBS to remove any weakly bound or unbound virus. Medium containing the appropriate amount of ZA,

PGN-ZA or a PBS (drug-free) control was added to the matching wells. Viral growth was assayed 3 days post-infection for each passage by hemagglutination assay. Virus from the lowest MOI showing hemagglutinating activity from the three conditions were each used to make three serial logio dilutions for the subsequent passage. The remaining viruses were stored in -80*C for genotyping and biological characterization. The PBS (drug-free) samples were used as controls for adaptation to cell culture, and for spontaneous mutations. The inhibitor concentration will be gradually increased in the subsequent passages when the virus appeared to have adapted to growing in the presence of the inhibitor. ZA selection of variant viruses were halted at the twelfth passage as the virus adapted to growing in high concentrations of ZA. The virus grown in the presence of PGN-ZA and the drug-free control virus were cultured up to Passage 23.

Hemagglutination assay.

Day 3 supernatants were titered using the hemagglutination assay. Briefly, the viral supernatants were serially diluted, and 50 pL of each titration was mixed with 50 pL of 1% chicken erythrocytes in PBS using a 96-well U bottom plate. The 96-well plate was then incubated on ice for 1 h before observing for signs of hemagglutination.

82 Chapter 3: PGN-ZA delays the emergence ofdrug resistance

Virus cloning.

Viruses from selected passages were cloned by plaque formation in MDCK cells in the presence of the appropriate amount of either PGN-ZA or ZA. Briefly, the viral supernatant was serially diluted and pre-incubated with inhibitor of the appropriate concentration for 1 h at RT. MDCK cells were then inoculated with these mixtures for 45 min at 37*C, and any unbound virus and inhibitor were removed with two PBS washes.

Agar containing the same concentration of inhibitor was then overlaid on the cells. After a

2-3 day incubation at 37*C, individual plaques were picked and extracted by incubating the agar plaque in PBS with 0.3% BSA overnight at 4*C. These clones were each propagated in

MDCK cells in the absence of inhibitor. On Day 3, the viral supernatants were harvested and clarified by low speed centrifugation and 0.45 pm pore size filtration. Aliquots of each clone were then stored in -80'C. The viral titer was then determined using hemagglutination assay and/or plaque assay.

Sequencing of NA and HA genes

Viral RNA was first extracted from allantoic fluid or cell-free supernatant using the

PureLinkT M Viral RNA/DNA Mini Kit (Life Technologies, Grand Island, NY, USA). The RNA was then reverse-transcribed using the SuperScript@ First-Strand Synthesis System for

RT-PCR (Life Technologies) with primers INF-RT and INF-RT2. All primer sequences and the corresponding nucleotide positions are detailed in Table S1. The NA gene was amplified using the primer pairs NAF and NAR. Primers HA-1F and HA-1R were used to amplify the

HA1 region of the HA gene, and primers HA-2F and HA-2R were used to amplify the HA2

83 Chapter3: PGN-ZA delays the emergence of drug resistance region. The PCR product was ran on a 1% agarose gel, and the band with the corresponding size was cut. After the DNA was purified from the agar using the QlAquick Gel Extraction

Kit (Qiagen, Valencia, CA, USA), sequencing was performed on the Applied Biosystems

Model 3730 capillary DNA sequencer with Big Dye Terminator Cycle Sequencing Kit (Life

Technologies).

Plaque reduction assay

For measuring the IC50 of selected clones against PGN-ZA and ZA, the plaque reduction assay was performed using a modified literature procedure (62, 63). Briefly, equal volumes (60 tL) of virus and serial dilutions of inhibitor were mixed and incubated for 1 h at RT. Confluent MDCK monolayers in 12-well plates were then inoculated with 100 ptL of these mixtures at RT, with gentle shaking. After 1 h, the virus-inhibitor solution was aspirated, and the cells were washed three times with PBS. The cells were overlaid with agar containing the corresponding amount of inhibitor, and incubated at 37*C. After 3 days, the plaques were enumerated, and fixed with 1% formaldehyde in PBS at RT for 1 h. After aspirating the formaldehyde, the agar overlay was carefully removed, and the cell monolayer stained with 1% crystal violet in 20% aqueous methanol. The ICso value was determined by the inhibitor concentration at which the plaque quantity or size were halved in comparison to the PBS control.

84 Chapter 3: PGN-ZA delays the emergence of drug resistance

NA inhibition assay

Viral NA activity and their inhibition constants (Ki) for PGN-ZA and ZA were determined using the NA inhibition assay, which was performed and analyzed as described previously (54). Briefly, 20 ptL of virus and 15 ptL of inhibitor dilutions were pre-incubated for 1 h at RT. The fluorogenic NA substrate 4-methylumbelliferyl-a-D-N-acetylneuraminic acid (at a concentration of 16 pM) was added, and the enzyme kinetics was measured by monitoring the generation of 4-methylumbelliferone for 1 h. ZA dilutions used ranged from

0.5 to 200 [M and 2 to 1000 pM, whereas PGN-ZA dilutions ranged from 0.05 to 20 pM and

0.05 to 1000 pM for the WT and drug-selected variants respectively. Values of Ki were determined with nonlinear regression.

85 Chapter 3: PGN-ZA delays the emergence of drug resistance

Figures and Tables

A 1000. - Zanamivir 100- - PGN-ZA 10- O 1

-i= 0.1 0.01-

0.001

0.0001 ...... 1 3 5 7 9 11 13 15 17 19 21 23 Virus passage number B 1400 - Zanamivir -1200 - PGN-ZA - Drug-free .E 1000 800 600- 400 200

1 3 5 7 9 11 13 15 17 19 21 23 Virus passage number Figure 1. Viral growth in the presence of increasing concentrations of ZA and PGN-

ZA. (A) Schema of drug selection experiment showing the concentrations of Zanamivir and

PGN-ZA used in each. The concentration of inhibitor was increased in the subsequent passage by at least two-fold whenever viral appeared to adapt to growing in the presence of the inhibitor. (B) Viral titer on Day 3 at each passage as determined by hemagglutination assay with chicken erythrocytes. Each sample was cultured at least in triplicate.

86 Chapter 3: PGN-ZA delays the emergence of drug resistance

Polymer-Attached Viral Zanamivir Zanamivi (PGN-ZA) passage HA1 HA2 NA HA1 NA number G137E R220K 166V E114K E119G R220G D241G G111D E433D

8 _ _ _ 9 10 11 N.D. 12 0.2

-zz~

Figure 2. Amino acid changes in HA and NA genes of selected passages of virus grown in ZA or PGN-ZA. Amino acid changes are most likely associated with adaptation to drug selection pressured. Changes that were also found in drug-free selected passages are not shown here. The HA amino acid numbering shown here is based on H3 subtype numbering.

N.D.: Sequencing data not available for Passage 9-11 of ZA-selected viruses. Legend: Color scheme shows level of saturation (0: wild-type...1: 100% mutant).

87 Chapter 3: PGN-ZA delays the emergence of drug resistance

A D241G B G137E

R2:

Sialic acid 166V Sialic acid Sialic acid-

130-loop E114K- G137E

C Sialic acid D Sialic acid El19G

G111D -+

Figure 3. Localization of amino acid changes identified in this study on the three- dimensional structure of HA and NA. (A-B) The amino acid changes are shown on the structure of the monomer of A/X-31 HA (Protein Data Bank accession 1HGG). (A) Close-up view of the sialic acid binding sites of HA1. (B) Mutations in HA2 are also shown in this view. (C-D) The amino acid changes are shown on the structure of the monomer (C) and tetramer (D) of A/Tokyo/3/1967NA (Protein Data Bank accession 2BAT). Mutations selected by ZA selection are marked in blue, and mutations selected by PGN-ZA are marked in red.

88 Chapter 3: PGN-ZA delays the emergence of drug resistance

A Zanamivir B Polymeric Zanamivir WT DF23 P23 Z12 WT DF23 P23 Z12 nM (ZA) nM (ZA)

0 0

2 0.02

20 0.2

200 2

2000 20

2x10 4 200

2x10 5 2000

Figure 4. Plaque reduction assays of parental wild type virus, and MDCK-passaged virus in the presence or absence of PGN-ZA, or ZA. Plaque reduction assays were performed with a wide range of both inhibitors, and the cells were fixed and stained with

1% crystal violet on Day 3 post-infection. Concentrations of both inhibitors are expressed in concentrations of ZA (1 s.f.), whether free or conjugated to the polymer. WT: parental wild type virus; DF23: virus passaged in MDCK cells for 23 passages in the absence of either inhibitor (drug-free); P23: virus under PGN-ZA selection pressure for 23 passages; and Z12: virus under ZA selection pressure for 12 passages.

89 Chapter 3: PGN-ZA delays the emergence of drug resistance

Amino acid substitution(s) Ratio of HAU/plaque

forming units

HA1 HA2 NA

DF P23 A43V 0.0013

PGN-ZA P23 R220G, D241G A43V G111D 0.001

ZA P12 G137E, R220K 166V E119G 0.0024

Table 1. List of amino acid substitutions in the final passages of virus cultured in the presence or absence of either PGN-ZA or ZA and their receptor binding specificity.

The ratio shown here indicates the hemagglutinating units of binding to chicken erythrocytes normalized by their plaque forming units. Changes in this ratio would indicate a change in the receptor binding specificity of the MDCK passaged viruses. DF23: virus passaged in MDCK cells for 23 passages in the absence of either inhibitors (drug-free); P23: virus under PGN-ZA selection pressure for 23 passages; and Z12: virus under ZA selection pressure for 12 passages.

90 Chapter3: PGN-ZA delays the emergence of drug resistance

Influenza Am ino acid Plaque quantity Plaque size virus substitution(s) ICso (nM, based on ZA) ICso (nM, based on ZA) strain HA1 HA2 NA ZA PGN-ZA ZA PGN-ZA

WT 56 ±8 0.16 ±0.02 1.3 0.2 1.2 ± 0.8

DF23 A43V 38 ±31 1.9 ±1.8 12 1 1.6 ±0.9

5 P23 R220G, A43V G111D >>1. x 10 (5.7 ± 5.9) x (8.9 ±0.6) x (5.9 1) x 102

D241G 103 104 Z12 R220K 166V E119G (1.5 ±0.2) x >>1.5 x 105 >>1.5 x 105 (5.65 1) x 103 104

Table 2. Plaque reduction assay with ZA and PGN-ZA. The IC50 values were measured in terms of plaque quantity and plaque size reduction. The values are expressed in concentrations of ZA, whether free or conjugated to PGN. WT: parental wild type virus;

DF23: virus passaged in MDCK cells for 23 passages in the absence of either inhibitors

(drug-free); P23: virus under PGN-ZA selection pressure for 23 passages; and Z12: virus under ZA selection pressure for 12 passages.

91 Chapter 3: PGN-ZA delays the emergence of drug resistance

Amino acid Influenza virus Ki (nM, based on zanamivir) substitution(s) strain HA1 HA2 NA ZA PGN-ZA

WT 5.3 ±0.4 1.1 ±0.1

DF23 A43V 8.8 ± 5.6 2.1 ± 0.9

P23 R220G, A43V G111D 85 ±36 5.6 ±0.4 D241G Z12 R220K 166V E119G 340 ±54 5.3 ±0.1

Table 3. Inhibition of NA enzyme activity by ZA and PGN-ZA. The Ki values, expressed in concentrations of ZA, whether free or conjugated to PGN, reflect mean ± STD from two independent experiments. WT: parental wild type virus; DF23: virus passaged in MDCK cells for 23 passages in the absence of either inhibitors (drug-free); P23: virus under PGN-

ZA selection pressure for 23 passages; and Z12: virus under ZA selection pressure for 12 passages.

92 Chapter 3: PGN-ZA delays the emergence of drug resistance

Supplementary Figures and Tables

U Turkey WT --20 e 2A Drug-free Passage 23 (Clone 44) XPGN-ZA Passage 23 (Clone 87) 15 *Zanamivir Passage 12 (Clone 22) 0 10 E

LU 5

0.E+00 1.E+05 2.E+05 3.E+05 4.E+05 5.E+05 6.E+05 Viral titer (pfu/mL)

Figure S1. NA enzymatic activity of the parental wild type and the MDCK passaged viruses. The enzyme velocity was measured using a kinetic inhibition assay, and the

enzyme activity was plotted against viral titer measured using the plaque assay.

93 Chapter 3: PGN-ZA delays the emergence of drug resistance

Primer Sequence Nucleotide position

INF-RT 5'-AGGAAAAGCAGG-3' 1-12

INF-RT2 5'-AGCAAAAGCAGG-3' 1-12

HA-1F 5'-GCAAAAGCAGGGGAAACAATG-3' 2-22

HA-1R 5'-GTGCCTAAATCCATACCAAC-3' 1107-1126

HA-2F 5'-GTGAGAGGCCAAAGCGGCAG-3' 725-744

HA-2R 5'-GAAACAAGGGTGTTTTTTC-3' 1716-1734

NA-F 5'-AGCAAAAGCAGGAGTGAAAATG-3' 1-22

NA-R 5'-AGTAGAAACAAGGAGTTTTTTTC-3' 1444-1466

Table S1. List of primer sequences. This is a list of primers used in the genotyping analysis of both the heterogenous viral supernatant and isolated clones. The INF-RT and

INF-RT2 primers were used for reverse transcribing the viral RNA, and the next three pairs of primers were used for amplification and sequencing of the entire HA1 and HA2 subunits of HA, and NA respectively.

94 Chapter 3: PGN-ZA delays the emergence of drug resistance

References

1. WHO (2011) Global monitoring of antiviral resistance in currently circulating human influenza viruses, November 2011. in Weekly EpidemiologicalRecord, pp 497-508. 2. Hayden F (2009) Developing new antiviral agents for influenza treatment: what does the future hold? Clin Infect Dis: An official publication of the Infectious Diseases Society ofAmerica 48 Suppl 1:S3-13. 3. Hayden FG & de Jong MD (2011) Emerging influenza antiviral resistance threats.] Infect Dis 203(1):6-10. 4. Pinto LH & Lamb RA (2007) Controlling influenza virus replication by inhibiting its proton channel. Mol Biosyst 3(1):18-23. 5. Kim CU, et al. (1997) Influenza neuraminidase inhibitors possessing a novel hydrophobic interaction in the enzyme active site: design, synthesis, and structural analysis of carbocyclic sialic acid analogues with potent anti-influenza activity. jAm Chem Soc 119(4):681-690. 6. von Itzstein M, et al. (1993) Rational design of potent sialidase-based inhibitors of influenza virus replication. Nature 363(6428):418-423. 7. Hayden FG, et al. (1991) Recovery of drug-resistant influenza A virus during therapeutic use of rimantadine. Antimicrob Agents Chemother 35(9):1741-1747. 8. Hayden FG, et al. (1989) Emergence and apparent transmission of rimantadine- resistant influenza A virus in families. New EnglJ Med 321(25):1696-1702. 9. Hall CB, et al. (1987) Children with influenza A infection: treatment with rimantadine. Pediatrics80(2):275-282. 10. Shiraishi K, et al. (2003) High frequency of resistant viruses harboring different mutations in amantadine-treated children with influenza.J Infect Dis 188(1):57-61. 11. Englund JA, et al. (1998) Common emergence of amantadine- and rimantadine- resistant influenza A viruses in symptomatic immunocompromised adults. Clin Inf Dis: An official publication of the Infectious DiseasesSociety ofAmerica 26(6):1418- 1424. 12. Tsunoda A, Maassab HF, Cochran KW, & Eveland WC (1965) Antiviral activity of alpha-methyl-1-adamantanemethylamine hydrochloride. Antimicrob Agents Chemother (Bethesda) 5:553-560. 13. Cochran KW, Maassab HF, Tsunoda A, & Berlin BS (1965) Studies on the antiviral activity of amantadine hydrochloride. Annals of the New York Academy ofSciences 130(1):432-439. 14. Bright RA, et al. (2005) Incidence of adamantane resistance among influenza A (H3N2) viruses isolated worldwide from 1994 to 2005: a cause for concern. Lancet 366(9492):1175-1181. 15. Fiore AE, et al. (2011) Antiviral Agents for the Treatment and Chemoprophylaxis of Influenza: Recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR Recomm Rep, ed CDC, pp 1-24.

95 Chapter 3: PGN-ZA delays the emergence of drug resistance

16. Fiore AE, et al. (2008) Prevention and control of influenza: recommendations of the Advisory Committee on Immunization Practices (ACIP), 2008. MMWR Recomm Rep 57(RR-7):1-60. 17. Moscona A (2008) Medical management of influenza infection. Ann Rev of Med 59:397-413. 18. Gubareva LV, Kaiser L, Matrosovich MN, Soo-Hoo Y, & Hayden FG (2001) Selection of influenza virus mutants in experimentally infected volunteers treated with oseltamivir.J Infect Dis 183(4):523-531. 19. Mishin VP, Hayden FG, & Gubareva LV (2005) Susceptibilities of antiviral-resistant influenza viruses to novel neuraminidase inhibitors. Antimicrob Agents Chemother 49(11):4515-4520. 20. Roberts NA (2001) Treatment of influenza with neuraminidase inhibitors: virological implications. Philos Trans R Soc Lond B Biol Sci 356(1416):1895-1897. 21. Kiso M, et al. (2004) Resistant influenza A viruses in children treated with oseltamivir: descriptive study. Lancet 364(9436):759-765. 22. Whitley RJ, et al. (2001) Oral oseltamivir treatment of influenza in children. Pediact Infect DiseaseJ 20(2):127-133. 23. McKimm-Breschkin JL (2005) Management of influenza virus infections with neuraminidase inhibitors: detection, incidence, and implications of drug resistance. Treatments in Respir Med 4(2):107-116. 24. Moscona A (2009) Global transmission of oseltamivir-resistant influenza. New EnglJ Med 360(10):953-956. 25. Anonymous (2009) Update: influenza activity--United States, September 28, 2008- April 4, 2009, and composition of the 2009-10 influenza vaccine. MMWR. Morbidity and mortality weekly report 58(14):369-374. 26. (NISN) NISN (2010) An Overview of Antiviral Drug Resistance Data. Optionsfor the Control of Influenza VII. 27. McKimm-Breschkin JL (2000) Resistance of influenza viruses to neuraminidase inhibitors--a review. Antivir Res 47(1):1-17. 28. Wagner R, Matrosovich M, & Klenk HD (2002) Functional balance between haemagglutinin and neuraminidase in influenza virus infections. Rev Med Virol 12(3):159-166. 29. Blick TJ, et al. (1995) Generation and characterization of an influenza virus neuraminidase variant with decreased sensitivity to the neuraminidase-specific inhibitor 4-guanidino-Neu5Ac2en. Virology 214(2):475-484. 30. Gubareva LV, et al. (1996) Characterization of mutants of influenza A virus selected with the neuraminidase inhibitor 4-guanidino-Neu5Ac2en.j Virol 70(3):1818-1827. 31. McKimm-Breschkin JL, et al. (1996) Generation and characterization of variants of NWS/G70C influenza virus after in vitro passage in 4-amino-Neu5Ac2en and 4- guanidino-Neu5Ac2en. Antimicrob Agents Chemother 40(1):40-46. 32. Tai CY, et al. (1998) Characterization of human influenza virus variants selected in vitro in the presence of the neuraminidase inhibitor GS 4071. Antimicrob Agents Chemother 42(12):3234-3241. 33. Cheam AL, Barr IG,Hampson AW, Mosse J, & Hurt AC (2004) In vitro generation and characterisation of an influenza B variant with reduced sensitivity to neuraminidase inhibitors. Antivir Res 63(3):177-181.

96 Chapter 3: PGN-ZA delays the emergence of drug resistance

34. Barnett JM, et al. (1999) In vitro selection and characterisation of influenza B/Beijing/1/87 isolates with altered susceptibility to zanamivir. Virology 265(2):286-295. 35. Hurt AC, et al. (2011) Increased detection in Australia and Singapore of a novel influenza A(H1N1)2009 variant with reduced oseltamivir and zanamivir sensitivity due to a S247N neuraminidase mutation. Euro Surveillance: Communicable Disease Bulletin 16(23). 36. Hurt AC, Holien JK, Parker M, Kelso A, & Barr IG(2009) Zanamivir-resistant influenza viruses with a novel neuraminidase mutation.J Virol 83(20):10366-10373. 37. Ilyushina NA, Bovin NV, Webster RG, & Govorkova EA (2006) Combination chemotherapy, a potential strategy for reducing the emergence of drug-resistant influenza A variants. Antivir Res 70(3):121-131. 38. Hoopes JD, et al. (2011) Triple combination antiviral drug (TCAD) composed of amantadine, oseltamivir, and ribavirin impedes the selection of drug-resistant influenza A virus. PloS One 6(12):e29778. 39. Mammen M, Choi S-K, & Whitesides GM (1998) Polyvalent Interactions in Biological Systems: Implications for Design and Use of Multivalent Ligands and Inhibitors. Angew Chemie Int Ed 37(20):2754-2794. 40. Gubareva LV, Robinson MJ, Bethell RC, & Webster RG (1997) Catalytic and framework mutations in the neuraminidase active site of influenza viruses that are resistant to 4-guanidino-Neu5Ac2en.J Virol 71(5):3385-3390. 41. Lamb RA & Krug RM (2001) Orthomyxoviridae: The viruses and their replication. Fundamental Virology, ed David M Knipe PMH (Lippincott Williams & Wilkins), pp 725-770. 42. Klenk E, Faillard H, & Lempfrid H (1955) [Enzymatic effect of the influenza virus]. Hoppe-Seyler's Zeitschriftfur physiologische Chemie 301(4-6):235-246. 43. Gottschalk A (1957) Neuraminidase: the specific enzyme of influenza virus and Vibrio cholerae. Biochimica et biophysica acta 23(3):645-646. 44. Gottschalk A (1959) On the mechanism underlying initiation of influenza virus infection. Ergebnisse der Mikrobiologie, Immunitatsforschung und Experimentellen Therapie 32:1-22. 45. Colman P (1989) Neuraminidase: enzyme and antigen. The Influenza Viruses, ed Krug R (Plenum Press, New York), pp 175-210. 46. Palese P, Tobita K, Ueda M, & Compans RW (1974) Characterization of temperature sensitive influenza virus mutants defective in neuraminidase. Virology 61(2):397- 410. 47. Peter Palese MLS (2007) Orthomyxoviridae: the viruses and their replication (Lippincott Williams & Wilkins) 5 Ed. 48. Woods JM, et al. (1993) 4-Guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid is a highly effective inhibitor both of the sialidase (neuraminidase) and of growth of a wide range of influenza A and B viruses in vitro. Antimicrob Agents Chemother 37(7):1473-1479. 49. Schild GC, Oxford JS, de Jong JC, & Webster RG (1983) Evidence for host-cell selection of influenza virus antigenic variants. Nature 303(5919):706-709. 50. Martin J, et al. (1998) Studies of the binding properties of influenza hemagglutinin receptor-site mutants. Virology 241(1):101-111.

97 Chapter 3: PGN-ZA delays the emergence of drug resistance

51. Suzuki Y, et al. (2000) Sialic acid species as a determinant of the host range of influenza A viruses.J Virol 74(24):11825-11831. 52. Blick TJ, et al. (1998) The interaction of neuraminidase and hemagglutinin mutations in influenza virus in resistance to 4-guanidino-Neu5Ac2en. Virology 246(1):95-103. 53. Staschke KA, et al. (1995) Molecular basis for the resistance of influenza viruses to 4-guanidino-Neu5Ac2en. Virology 214(2):642-646. 54. Weight AK, et al. (2011) Attaching zanamivir to a polymer markedly enhances its activity against drug-resistant strains of influenza a virus.J Pharm Sci 100(3):831- 835. 55. Colman PM (2009) New antivirals and drug resistance. Annu Rev Biochem 78:95- 118. 56. Varghese JN, Epa VC, & Colman PM (1995) Three-dimensional structure of the complex of 4-guanidino-Neu5Ac2en and influenza virus neuraminidase. ProtSci:A publication of the Protein Society 4(6):1081-1087. 57. Goto H, Bethell RC, & Kawaoka Y (1997) Mutations affecting the sensitivity of the influenza virus neuraminidase to 4-guanidino-2,4-dideoxy-2,3-dehydro-N- acetylneuraminic acid. Virology 238(2):265-272. 58. DuBois RM, etal. (2011) Acid stability of the hemagglutinin protein regulates H5N1 influenza virus pathogenicity. PLoS Pathogens 7(12):e1002398. 59. Rachakonda PS, et al. (2007) The relevance of salt bridges for the stability of the influenza virus hemagglutinin. FASEB journal: Official publication of the Federation ofAmerican Societiesfor ExperimentalBiology 21(4):995-1002. 60. Vanderlinden E, et al. (2010) Novel inhibitors of influenza virus fusion: structure- activity relationship and interaction with the viral hemagglutinin.J Virol 84(9):4277-4288. 61. Nguyen JT, et al. (2010) Triple combination of amantadine, ribavirin, and oseltamivir is highly active and synergistic against drug resistant influenza virus strains in vitro. PloS One 5(2):e9332. 62. Haldar J, et al. (2010) Bifunctional polymeric inhibitors of human influenza A viruses. Pharm Res 27(2):259-263. 63. Hayden FG, Cote KM, & Douglas RG (1980) Plaque inhibition assay for drug susceptibility testing of influenza viruses. Antimicrob Agents Chemother 17(5):865- 870.

98 Chapter 4: Summary offindings, discussion and future directions

Chapter 4

SUMMARY OF FINDINGS, DISCUSSION AND FUTURE

DIRECTIONS

Summary of Findings and Discussion

With the increasing challenges in the control of influenza virus and our current lack

of therapeutic options, urgent measures should be taken to develop antivirals with the

following characteristics: (i) greater potency that rapidly inhibits viral replication, (ii)

lower risk of developing resistant virus strains, and (iii) new mechanisms of antiviral

action for use in combination therapies (1, 2). In this thesis, the characteristics of a new

antiviral design paradigm were investigated, with focus on our lead candidate, where

zanamivir (ZA) is covalently attached to poly-L-glutamine through a flexible linker (PGN-

ZA). We have previously shown that PGN-ZA is three to four orders of magnitude more

potent over its monomeric predecessor ZA, and is effective against neuraminidase (NA)

inhibitor-resistant viruses (3).

In the first investigation, we elucidated the mechanism of action underlying this

dramatic antiviral potency of PGN-ZA. We show that, similar to the monomeric ZA, PGN-ZA

binds to neuraminidase (NA) and inhibits its enzymatic activity, thus blocking the release

of newly generated virions from infected cells. In addition, we found that PGN-ZA inhibits

an early step in virus replication.

99 Chapter 4: Summary offindings, discussion andfuture directions

How does PGN-ZA affect the early phase of virus replication? The possibility of viral aggregation, virucidal activity, and each step in initial virus infection were investigated systematically using a variety of approaches. Surprisingly, we found that addition of PGN-

ZA reduced viral-endosomal fusion. This was further supported by the observation that

PGN-ZA protected virus from inactivation in acidic pH (pH 5). Therefore, attaching ZA to a polymer results in a new mode of antiviral action. The synergistic inhibition of early and late steps of influenza virus replication forms the basis of the dramatically enhanced antiviral potency of the multivalent drug conjugate over the monomer

In the second study, we embarked on an investigation of the ability of PGN-ZA in minimizing drug resistance. Influenza virus was passaged sequentially in increasing concentrations of PGN-ZA, or monomeric ZA. Amongst the current antivirals, monomeric

ZA is considered to have a strong barrier to resistance. We found that virus adapted quickly to growing in high pM concentrations of ZA by the 8th passage in cell culture. In contrast, even at low nM concentrations, PGN-ZA can suppress viral growth well into 23 passages of cell culture. Next, to investigate the timeline of drug resistance progression, the genotype of viruses from various passages was analyzed by Sanger sequencing. In the ZA-selected virus, amino acid substitution in NA (E119G) emerged in passage 8, a timeline that is consistent with that seen in a previous report (4). This substitution lowers binding affinity to ZA by

70-fold, and in the plaque reduction assay, IC5 0 is >>150 ptM, an increase of at least 3000- fold over the parental wild type.

In contrast, PGN-ZA did not induce any changes at residue Glu 119, or any other residues that were previously associated with NA inhibitor resistance. Instead, we unexpectedly found novel amino acid substitutions R220G and D241G in HA1 and G111D in

100 Chapter 4: Summary offindings, discussion andfuture directions

NA, which emerged by passages 14-15. Interestingly, although G111D substitution only lowered ZA binding affinity by 10-fold, the combination of HA and NA substitutions

unexpectedly conferred high-level resistance to ZA in plaque reduction assays. More

importantly, both variant viruses generated remain susceptible to low pM of PGN-ZA in

plaque reduction assays. Taken together, these results demonstrate that PGN-ZA can delay

the emergence of drug resistance by at least six passages. To our knowledge, this is the first

time an influenza antiviral has been shown to significantly delay the emergence of drug

resistance.

How do the changes in HA and NA contribute to drug resistance? Residue Arg 220

in HA1 had been identified in a drug selection study using influenza virus fusion inhibitors,

and the R220S substitution was found to increase the viral fusion pH in H3N2 viruses. Gly

111 is situated near the interface of two NA subunits, and the change to Asp may affect the

NA homotetramer formation. We postulated that for viral escape from PGN-ZA's two

mechanisms of antiviral action, the virus has to accumulate at least two substitutions that

affect both virus fusion and release, which can rationalize PGN-ZA's higher barrier to

resistance. In addition, the role of these previously undescribed amino acid residues in

virus receptor binding, fusion, and release, and their eventual contribution to drug

resistance are of interest, as few mutations are known to be associated with ZA resistance.

These studies presented here further validate the polymer-attached inhibitors as a

new paradigm of drug development to minimize viral drug resistance, and provide further

impetus for exploring their use in influenza therapy. Furthermore, the investigations also

highlight their potential use as tools to accelerate the understanding of influenza HA and

101 Chapter 4: Summary offindings, discussion and future directions

NA, in particular their roles and functional balance for efficient virus replication and the molecular mechanism of drug resistance.

Future Directions

The work presented in this thesis opens up several avenues for further investigation.

Possible future directions can be divided into the following three categories:

1. The contribution of the amino acid substitutions in HA and NA in conferring ZA and

PGN-ZA resistance

2. Combination therapy with multifunctional polymeric inhibitors

A natural extension of the work in Chapter 3 would be to elucidate the contributions of the novel substitutions identified in HA and NA to the drug resistance phenotype. The

G111D mutation only caused a moderate drop in NA binding affinity to PGN-ZA, but the

P23 virus showed a high resistance level in the plaque reduction assay. This points to the importance of the substitutions identified in HA in conferring resistance. What are the roles of those amino acids in HA and NA, and can we tease apart their individual contributions to the resistance phenotype? We could try to isolate variants possessing only one of the following substitutions: R220G in HA1, and G111D in NA from the Passage 15 viral supernatant. With the single mutation variants in hand, we could analyze their phenotype with different approaches. First, plaque reduction assays can be done to measure their sensitivity to either ZA, or PGN-ZA. Next, we could also measure for any changes in fusion pH using a hemolysis assay.

102 Chapter 4: Summary offindings, discussion andfuture directions

Structural analyses on HA using molecular dynamics could also shed light on the effects of the R220G substitution on the properties of the receptor binding site, viral fusion, and trimer formation. Similarly, the effect of the G111D substitution on NA's enzymatic active site, and tetramer formation could be modeled. Such a study may help in increasing our understanding of the complex interplay between HA and NA for efficient virus replication, and their eventual effect on the molecular mechanism of drug resistance.

If we are unable to isolate variants with single mutations of G111D in NA or R220G in HA1, this would support the hypothesis that both mutations are essential for the virus to begin its escape from polymer-attached drug selection.

Using the polymer-attached drug as a probe, similar drug resistance studies could also be run on influenza viruses of other subtypes such as H1N1 or H5N1. It would be interesting to see if the multivalent inhibitor can also delay emergence in such cases, and we can gain some understanding of the critical amino acid residues for viral fusion and release.

The next step in our development of influenza polymer-attached inhibitors would be to covalently conjugate ZA, and other small molecule inhibitor(s) onto the same polymer.

Since we have shown previously that conjugating two different inhibitors onto the same polymer is superior to a physical mix of two separate mono-functional polymers (5), this opens up further possibilities in the polymeric drug design paradigm. Ideally, this new inhibitor should target a different step in the virus replication cycle than PGN-ZA. In theory, a potent inhibitor of viral entry would complement the existing two mechanisms of antiviral action of PGN-ZA. With three modes of antiviral action in one molecule, it is expected to be even more effective in minimizing influenza drug resistance.

103 Chapter 4: Summary offindings, discussion and future directions

A complicated but potentially more rewarding endeavor would be to develop a broadly active viral inhibitor. Like influenza virus, other common respiratory viruses like respiratory syncytial virus, parainfluenza, rhinovirus, and human metapneumovirus also have multiple copies of viral proteins displayed on their surface. With multivalent interaction, weak inhibitors attached to polymeric chains can have greatly increased antiviral potency. By covalently conjugating ZA and inhibitors of other viruses onto the same polymeric chain, we can potentially develop a broad-spectrum inhibitor for respiratory infections.

References

1. Hayden FG & de Jong MD (2011) Emerging influenza antiviral resistance threats.J Infect Dis 203(1):6-10. 2. Hayden F (2009) Developing new antiviral agents for influenza treatment: what does the future hold? Clin Infect Dis: An official publication of the Infectious Diseases Society ofAmerica 48 Suppl 1:S3-13. 3. Weight AK et al. (2011) Attaching zanamivir to a polymer markedly enhances its activity against drug-resistant strains of influenza a virus.J Pharm Sci 100(3):831- 835. 4. Gubareva LV, et al. (1996) Characterization of mutants of influenza A virus selected with the neuraminidase inhibitor 4-guanidino-Neu5Ac2en.J Virol 70(3):1818-1827. 5. Haldar J, et al. (2010) Bifunctional polymeric inhibitors of human influenza A viruses. Pharm Res 27(2):259-263.

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