Influenza Neuraminidase Assembly

Home , M1 protein, Neuraminidase, NS2 (HCV), Viral envelope

I nfluenza neuraminidase assembly Evolution of domain cooperativity

Diogo da Silveira Vieira da Silva

Influenza Neuraminidase assembly

Evolution of domain cooperativity

Diogo da Silveira Vieira da Silva

Cover: Influenza virus: a global virus

©Diogo da Silveira Vieira da Silva, Stockholm University 2016 Copyright information: The earth globe was designed by Freepik

ISBN print 978-91-7649-553-7 ISBN pdf 978-91-7649-554-4

Printed in Sweden by Holmbergs, Malmö 2016 Distributor: Department of Biochemistry and Biophysics, Stockholm University

Ao meu irmão.

Dedicated to my brother.

List of publications

I. da Silva DV*, Nordholm J*, Madjo U, Pfeiffer A, Daniels R (2013) Assembly of subtype 1 influenza neuraminidase is driven by both the transmembrane and head domains. J Biol Chem.;288(1):644-53.

II. Nordholm J*, da Silva DV*, Damjanovic J, Dou D, Daniels R. (2013) Polar residues and their positional context dictate the transmembrane domain interactions of influenza A neuraminidase. J Biol Chem.;288(15):10652-60.

III. da Silva DV, Nordholm J, Dou D, Wang H, Rossman JS, Daniels R. (2015) The influenza virus neuraminidase protein transmembrane and head domains have coevolved. J Virol.;89(2):1094-104.

IV. Östbye H, da Silva DV, Revol R, Nordholm J, Daniels R. (2016) Assembly co-cooperativity between the influenza NA stalk and transmembrane domain defines the insertion deletion boundary. Manuscript.

* Both authors contributed equally to this work.

Additional publications

Dou D, da Silva DV, Nordholm J, Wang H, Daniels R. (2014) Type II transmembrane domain hydrophobicity dictates the cotranslational dependence for inversion. Mol Biol Cell;25(21):3363-74.

Abstract

Influenza A virus (IAV) is one of the most common viruses circulating in the human population and is responsible for seasonal epidemics that affect millions of individuals worldwide. The need to develop new drugs and vaccines against IAVs led scientists to study the main IAV surface antigens hemagglutinin (HA) and neuraminidase (NA). In contrast to HA, which fa- cilitates cell binding and entry of IAVs, NA plays a critical role in the release and spreading of the viral particles. The aim of this thesis was to study how the enzymatic head domain, the stalk and transmembrane domains have evolved to facilitate NA assembly into an enzymatically active homotetramer, and to determine how these regions have evolved together over time. Initially, we observed that the NA transmembrane domain (TMD) assists in the assembly of the head domain by tethering the stalk to the membrane in a tetrameric conformation. Upon examination of the available sequences for NA, we found that the subtype 1 (N1) TMDs have become more polar since 1918 while the subtype 2 (N2) TMDs have consistently retained the expected hydrophobicity of a TMD. Further analysis of the amino-acid sequences revealed a characteristic indicative of an amphipathic assembly for the N1 TMDs that were absent in the TMDs from N2. The function of the amphipathic assembly was exam- ined by creating two viral chimeras, where the original TMD was replaced by another more polar or an engineered hydrophobic TMD. In both cases the viruses carrying the NA TMD chimeras showed reduced growth indicating that the TMD changes created an incompatibility with the head domain of NA. After prolonged passaging of these viruses, natural occurring mutations were observed in the TMD that were able to rescue the defects in viral growth, head domain folding and budding by creating a TMD with the ap- propriate polar or hydrophobic assembly properties. Interestingly, we observed that N1 and N2 have a great difference in the localization and length of amino-acid deletions occurring in the stalk region. In line with this observation, our data suggests that N1 supports large stalk deletions due to its strong TMD association, whereas N2 requires the presence of a strong oligomerizing stalk region to compensate for its weak TMD interaction. These results have demonstrated how important the NA TMD is for viral infectivity and how the three different domains have evolved in a cooperative manner to promote proper NA assembly.

Contents

List of publications ...... vi Additional publications ...... vii Abstract ...... viii Contents ...... ix Introduction ...... 12 Overview of influenza classification and morphology ...... 13 Influenza subtypes ...... 13 Influenza virus morphology ...... 13 Influenza virus life cycle ...... 15 Influenza virus host cell binding and penetration ...... 15 Transcription and replication of the influenza genome ...... 15 Influenza virus assembly ...... 16 Budding of influenza viral particles ...... 18 Functions of the influenza membrane proteins: HA, M2 and NA ...... 20 Hemagglutinin ...... 20 M2 protein ...... 21 Neuraminidase ...... 22 Membrane protein maturation ...... 24 ER targeting sequences ...... 24 Cleavable signal sequences ...... 24 Uncleaved signal sequences ...... 25 Endoplasmic reticulum insertion ...... 26 The transmembrane domain ...... 28 Protein glycosylation ...... 30 Neuraminidase ...... 33 Neuraminidase structure ...... 33 The head domain ...... 33 The stalk domain ...... 36 Neuraminidase maturation and oligomerization ...... 37 The transmembrane domain – Papers I, II and III ...... 39 Paper I – Contribution of TMD to NA assembly ...... 40

Paper II – Amphipathicity dictates neuraminidase TMD interactions ...... 41 Paper III – The influenza NA TMD and head domain coevolved ...... 43 The stalk domain – Manuscript IV ...... 45 Conclusions and future perspectives ...... 47 Sammanfattning på svenska ...... 49 Acknowledgmentes ...... 51 References ...... 54

Abbreviations

IAV – Influenza A virus IBV – Influenza B virus HA – Hemagglutinin NA – Neuraminidase vRNPs – Viral ribonucleoprotein vRNA – Viral RNA NP – Nucleoprotein NS1/2 – Non-structural protein 1/2 NLS – Nuclear Localization Signal TMD – Transmembrane domain ER – Endoplasmic reticulum OST – Oligosaccharyltransferase RBD – Receptor binding domain

Introduction

Influenza viruses are one of the most common viruses circulating among the human population. Every year, seasonal flu infects 10 to 20% of the world’s population, resulting in three to five million cases of illness and up to 500,000 deaths [1]. This causes an additional economic burden, due to medical costs and lost earnings, which in the USA alone is estimated at 16.3 billion US dollars per year [2]. Influenza belongs to the Orthomyxoviridae family of viruses and in humans, the majority of disease is caused by type A and type B influenza viruses [3]. Both are responsible for the seasonal influenza epidemics. Howev- er, type B is less frequent and causes milder symptoms [4]. Whereas humans are the predominant host for influenza type B viruses (IBVs), birds are con- sidered the primary reservoir for influenza type A viruses (IAVs), however these viruses may also circulate in other species such as pigs, horses, and bats [5, 6]. The transfer of influenza viruses from one species to another requires certain adaptations for the virus to overcome specific species barriers [7, 8]. These barriers are quite important because they decrease the fre- quency of avian or swine IAVs entering the human population, which may have little immunity against these ‘new’ viruses. In the rare cases where these ‘new’ viruses cross the species barrier and gain the ability to efficiently transmit from human-to-human, it raises the possibility for global spreading of the virus and the potential for a pandemic. In the past, three major human IAV pandemics have occurred that caused high mortality worldwide: the 1918 H1N1, the 1957 H2N2 and the 1968 H3N2 viruses [9]. More recently, in 2009, a new H1N1 virus lineage composed of a unique combination of genes was found to circulate in humans. An evolutionary analysis of the pathway that crafted this new virus, showed that the genes originated from a triple reassortment H1N2 virus of North American swine-origin and an H1N1 virus of Eurasian swine-origin [10, 11]. Due to the high transmissibility of the new virus, it began circulating across the world in a matter of weeks, and this marked the emergence of first potential pandemic of the 21st century [12].

12 Overview of influenza classification and morphology

Influenza subtypes

IAVs can be categorized into subtypes based on the diverse antigenicity of their two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA) [13]. HA and NA, due to their exposure at the surface of the virus, are the main targets of the protective immune response of the host and can vary as a result of antigenic drift (mutations within the gene over time) or antigenic shift (two or more strains reassort to form a new mixture of the surface antigens) [14]. To date, 18 HA subtypes and 11 NA subtypes have been identified and many different combinations of the two proteins subtypes are possible [15]. In theory, IAVs can make 198 combinations (18x11) based on the HA and NA subtypes alone, but only H1N1 and H3N2 are currently found circulating in humans. In contrast, a large number of the possible IAV subtypes have been found in birds, with the exception of H17N10 and H18N11, which appear to be exclusive to bats [16]. The reasons why only a few IAV subtypes commonly circulate in humans still remain unclear.

Influenza virus morphology

Influenza viruses can display different shapes according to the strain. It can present a spherical shape ranging from 100 to 150 nm in diameter or it can be a long filamentous particle with several micrometers in length [17, 18]. Despite the antigenic and shape differences found within influenza viruses and strains, the localization, structure, organization and protein components of each viral particle are quite conserved. All IAVs contain a so called viral or lipid envelope derived from the host cell plasma membrane, which is rich in cholesterol-lipid rafts and non-raft lipids [19]. The primary function of the envelope is to enclose and protect the eight negative sense single-stranded RNA segments that compose the genome. The eight vRNA molecules typically encode for a total of 11 or 12 viral proteins depending on the strain [5, 20, 21]. The viral envelope also houses HA and NA, which facilitate viral entry and release, respectively (Figure 1) [22] .

Figure 1- Schematic influenza A virus particle. An influenza viral particle is delimited by a lipid layer (red circular line) derived from the host cell membrane, which functions as the scaffold for the viral envelope. This lipid envelope contains two glycoproteins that are the major influenza antigens, neuraminidase (NA) and hemagglutinin (HA) and also an ion channel protein (M2). Underlying the viral envelope is a structural protein called matrix protein 1 (M1). Inside the viral particle are eight ribonucleoprotein (RNP) complexes that encode all viral proteins. They are composed of a negative- sense RNA molecule twisted around a string of nucleoproteins (NP) and associated with a complex of three viral polymerases, PA, PB1 and PB2 [14].

From a surface perspective, the viral envelope is enriched with HA, which is responsible for receptor binding and membrane fusion, and the receptor- destroying enzyme NA [23]. A third integral membrane protein with proton- selective ion channel activity called M2, is also present in the viral envelope. The matrix protein (M1) concedes the structure to the virion by peripherally associating with the internal membrane of the envelope. Together with M2, M1 has been shown to be a defining factor in whether the envelope takes on a spherical or filamentous shape [24, 25]. In addition, M1 is also thought to interact with the viral ribonucleoprotein (vRNPs) gene segments [26, 27], which contain the negative single-stranded vRNA, three RNA polymerase proteins (PB1, PB2 and PA) and the nucleocapsid protein (NP) [21].

14 Influenza virus life cycle

The life cycle of an influenza virus can be divided into four main stages: i- host cell binding and penetration; ii- viral genome transcription and replication; iii- viral assembly at the plasma membrane, and iv- viral budding.

Influenza virus host cell binding and penetration

In order to initiate the entry process, influenza viruses need to adhere to the host cell. In this process, HA which is projected away from the viral surface, binds to a-2,3 or a-2,6-linked sialic acid moieties on glycoproteins presented at the plasma membrane of the host cell [28]. The binding of HA to sialic acid initiates endocytosis of the virion, which may vary depending on the morphology of the virus. For spherical influenza viruses, it can occur either in a clathrin or non-clathrin-dependent manner, while for larger filamentous particles, it can occur in a non-clathrin-dependent manner, such as macropinocytosis [29]. Once inside the cell, the virion traffics in the newly formed endosome until its acidification triggers a conformational change in HA, which is proteolytically separated into two domains (HA1 and HA2). During the conformational change, HA1, which contains the receptor binding domain, is shifted down to expose the fusion peptide on HA2 that mediates the fusion of the viral and endosomal membrane [28, 30]. Prior to fusion, the low pH inside the endosome also causes M2 to open and protons are allowed to enter the viral particle. This lowers the pH in the viral core causing M1 to dissociate from the vRNPs, which helps increase the efficiency of vRNP release into the cytoplasm during fusion [31, 32].

Transcription and replication of the influenza genome

After the release of the vRNAs into the host cell cytoplasm, the next step in the influenza viral life cycle is the import of the vRNAs into the nucleus, where they transcribe viral mRNAs to initiate replication. In the cytoplasm, each vRNA molecule remains associated with several NP oligomers and a polymerase complex (PA, PB1 and PB2) [33]. Both of these possess nuclear localization signals (NLS), which are thought to direct the import of the vRNAs into the nucleus via their association with the host nuclear import

15 machinery [33]. Interestingly, influenza viruses are the only RNA viruses without a DNA stage that seems to use the nucleus for replication and why they have evolved to use the nucleus is currently not known [32]. Once inside the nucleus, the viral RNA-dependent RNA polymerase attached to the vRNA begins to transcribe and replicate the (-)vRNA. This gives rise to viral mRNAs necessary for viral protein expression and a complementary (+)cRNA, which is used as a template used to produce more (-)vRNA and (-) small viral RNAs that help regulating the change from transcription to replication. After translation of the viral mRNAs in the cytoplasm, the newly synthesized surface proteins HA, NA and M2 are trafficked through the secretory pathway for transport and insertion into the plasma membrane. After synthesis, NP, PA, PB1 and PB2 are imported back into the nucleus where they function together to amplify vRNA and mRNA transcription, and to assemble vRNPs. M1 and NS2 are also sent back to the nucleus to aid in vRNP nuclear export and trafficking to the plasma membrane for packaging [5, 33, 34].

Influenza virus assembly

In the process of viral assembly two major steps need to be accomplished in an orderly manner. First, all viral components need to be targeted and delivered to the plasma membrane of non-polarized cells or the apical plasma membrane of polarized epithelial cells. Second, all viral components need to interact in an organized fashion to correctly assemble an infectious virion.

Influenza surface proteins HA, NA and M2 are sorted to the plasma membrane due to the presence of an N-terminal targeting sequence that di- rects them to the endoplasmic reticulum (ER), and a transmembrane domain that contains the information for lipid raft association and apical transport. Following ribosomal synthesis at the ER, these proteins use the secretory pathway to move from the ER through the cis, medial and trans-Golgi network (TGN) to reach the plasma membrane [21, 35]. Since M1 is a soluble protein that associates with the envelope, it is speculated that M1 is recruited to the correct place on the plasma membrane by associating with the HA and NA cytoplasmic tails, as well as M2. Alternatively, it has been proposed that

16 M1 may bind to specific lipids that cluster in the region of viral assembly [26, 36]. Besides the envelope proteins, the eight different vRNPs should also traffic to the plasma membrane and pack into the new forming virion, a process that is initiated during export from the nucleus. Currently, it is postulated that export of the vRNPs from the nucleus to the cytoplasm is mediated by a CRM1-dependent nuclear export pathway. CRM1 recognizes nuclear export signals (NES) [32] that are reported to be present in NP and M1. However, in the absence of NS2 the export is ceased, indicating that NP and M1 do not directly interact with CRM1 to facilitate the export process [37]. Despite being an important player in the transport of the vRNPs into the cytoplasm, NS2 does however not directly interact with the vRNPs, but has been shown to interact with nuclear export machinery [38]. Thus, NS2 is hypothesized to work as a connecting bridge between the M1-vRNP complex and the CRM1 export pathway, resulting in the nucleocytoplasmic transport of the eight IAV genome segments [33].

After leaving the nucleus, the vRNPs need to be transported through the cytoplasm to the viral assembly site on the plasma membrane. Although it is not fully understood how vRNPs migrate through the cells, different studies have demonstrated that several mechanisms can be involved. Carrasco et al., showed that NP can traffic to the plasma membrane independently of other viral proteins, potentially through interactions with lipid rafts. Therefore NP, which composes the scaffold of the vRNPs, could function to localize the gene segments at the plasma membrane for integration into new virions [39]. A study from Ali et al., hypothesized that vRNPs are transported to the plasma membrane in a complex with M1. Since M1 interacts with the cytoplasmic tails of NA and HA, the vRNPs could use a piggy-back interaction with M1 to be transported together with NA and HA [40]. Also, Avalos et al., showed that NP and M1 associate with the cellular cytoskeleton and that this interaction could be used by vRNPs for transport on microtubule net- works, that could deliver them to the plasma membrane [33, 41]. Finally, a more recent trend of studies shows that Rab11, a pericentriolar recycling endosome marker colocalizes with vRNPs, which might play a role in membrane trafficking and actin dynamics[42]. In this pathway, the vRNPs that exit the nucleus are diverted into the pericentriolar recycling endosome where they access and use the Rab11 transport pathway to reach the cell periphery and integrate in the budding process [43].

17 While it currently is not clear how the viral proteins and eight vRNPs associate at the plasma membrane to form a new virion, once the virion is assembled, it can be released from the host cell in a process called budding.

Budding of influenza viral particles

Bud formation and release, mark the last stages of viral replication and production of new virions. Budding from the plasma membrane of the host cell is initiated by the accumulation of NA and HA in lipid rafts domains [27]. The concentration of these two proteins at the surface seems to alter membrane curvature, which can be seen as the triggering event for the budding process [26]. Polymerization of the M1 protein, elongates the forming virion and results in a polarized localization of the vRNPs. The M2 protein is then recruited to the proximity of the budding region via interaction with M1. Insertion of the M2 amphipathic helix into the lipid membrane causes an increase in curvature at the neck of the forming virus, resulting in membrane scission and release of the budding virus (Figure 2) [27, 44]. M1 that is associated with the cytoplasmic tails of HA and NA is used by vRNPs as docking sites in order to be packaged [27]. The presence of unique packaging signals in the 5’ and 3’ untranslated regions on each segment allows an ordered and efficient virion incorporation [32]. Each virion incorporates one copy of each of the eight segments, spatially distributed in a “7+1” pattern that is sustained by specific segment interactions [45]. From this moment, the new virus is ready to infect cells leading to proliferation.

Figure 2 - Budding of an influenza virus particle. The insertion of NA and HA transmembrane domains into the host cell membrane and the resulting accumulation of these two proteins start to form a bud in the plasma membrane. The M1 protein that migrates to the membrane together with NA and HA is responsible for the recruitment of vRNPs to the forming bud. The association of NA and HA with lipid-raft domains promotes a high accumulation of cholesterol in the budding region. The difference in cholesterol amount to the surrounding plasma membrane creates a tension that initiates constriction of the bud neck. Accumulation of the M2 protein in this region promotes membrane fission via the amphipathic helix on M2 leading to the budding of the viral particle [44].

19 Functions of the influenza membrane proteins: HA, M2 and NA

Hemagglutinin

HA is the major glycoprotein and one of the most important IAV antigens because it is an active participant in viral entry with two important roles: -i- binding to sialic acid at the surface of the host cell enabling endocytosis of the virus and -ii- promoting fusion of the viral membrane with the endosome membrane. Structurally, HA projects outwards as a spike at the viral surface and is presented as a homotrimer with non-covalently linked subunits, a conserved trait among all HA subtypes [46]. HA can be further subdivided into two main regions: a long membrane-proximal stem region and a globular head that comprises the receptor binding domain and the main antigenic sites [47]. By proteolytic cleavage at a specific cleavage site in HA, each HA0 monomer is converted to two polypeptides, HA1 and HA2. For low pathogenic strains, the cleavage occurs mainly extracellularly at a monobasic Ar- ginine or Lysine residue and can be performed by several trypsin-like proteases [48, 49]. However, recently TMPRSS2 was also found to cleave HA already during trafficking through the secretory pathway [50]. For highly pathogenic strains, multiple basic residues are present at the cleavage site and the processing of HA occurs mainly intracellularly during ER-Golgi trafficking by the action of serine proteases such as furin and PC6 [51]. The HA receptor binding domain (RBD) that associates with sialic acid is located within HA1. The HA2 peptide is commonly known as the “fusion peptide” and its C-terminus is anchored to the viral envelope. It mediates fusion between the viral membrane and the membrane of the endosome that contains the viral particle. This enables penetration and uncoating of the virus into the cytoplasm [52].

20 The HA-sialic acid interaction is species specific as it is linked to the richness in the particular sialic acids on the human and swine respiratory epithelial or the avian respiratory and intestinal epithelium [53]. For this reason, human viruses have a higher preference for a2,6-linked sialic acids, avian viruses for a2,3-linked sialic acids and swine viruses are able to bind sialic acid with both types of linkages [54]. This observation led to the proposal that pigs could work as a host for human and avian IAV coinfections that can produce novel antigenic reassortant viruses and potential influenza pandemics [55] Besides the RBD, it is also important to highlight the presence and the role of N-linked glycans that exist on the surface of HA. Roberts et al. and Gallagher et al., showed that when the N-linked glycans on Asn12, Asn28 and Asn478 (H7 numbering) were absent, the folding and transport of the HA trimer was impaired resulting in premature degradation [56, 57]. The glycans can also partially fill the RBD, changing the specificity and sialic acid binding affinity [58, 59]. Similar to the HIV g120 envelope protein, the presence of glycans in specific places of HA could also provide a mechanism of antibody evasion by difficult antibody binding to the epitope [60].

M2 protein

In contrast to the HA gene segment, the M segment of the influenza genome encodes two distinct proteins named M1 and M2. M2 is synthesized from a spliced version of the M segment mRNA and it behaves like a membrane-anchored protein in the viral envelope. M2 is a homotetramer with an

Nin-Cout orientation and functions as an ion channel [61]. The ion channel activity enables M2 to equilibrate the pH across the viral membrane during cell entry and across the trans-Golgi membrane of infected cells during viral replication [62]. In the closed state, the four transmembrane helices of M2 pack tightly resulting in a narrow gated channel with a tryptophan residue blocking proton passage [63, 64]. At low pH, a histidine residue activates the channel by destabilizing the well-packed transmembrane helices and repositioning the tryptophan gate. The repositioning unlocks the channel and allows water to conduct protons, while the lower C-terminal part of the channel helps maintain a tetrameric conformation [65, 66]. For many years the ion channel activity of M2 was the major drug target in influenza with high efficiency of inhibition [67], and these drugs (aman-

21 tadine and rimantidine) were used as a first-choice during early influenza outbreaks [68]. However, a drug resistant mutation spread rapidly among seasonal influenza strains and even exists in the majority of 2009 H1N1 pandemic strains [68-70]. The high prevalence of these drug resistant mutations has minimized the reliability and efficiency of drugs that target M2. For this reason, HA and NA are now used as the major drug targets to fight influenza infections [23, 71, 72].

Neuraminidase

The IAV neuraminidase glycoprotein is trafficked to the surface of infected host cells where it becomes a structural component of the virion envelope and an important antigen of the virus [73]. While HA mediates attachment to the host cell via sialic acid-containing glycoconjugate receptors, NA has an opposing function as it removes terminal sialic acids from oligossacharide residues [28]. More specifically, it acts on the a-ketosidic linkage between the terminal N-acetylneuraminic acid and a neighboring saccharide, usually galactose [74]. In the context of infection, NA sialidase activity is required in three important steps. First, NA acts in the process of viral penetration through the ciliated epithelium of the human airways by removing sialic acid decoy receptors from mucins, cilia and the cellular glycocalyx [75]. Second, NA aids in viral release by removing sialic acid residues from the surface of the infected cell, which enables the virions to detach and spread to neighbouring cells [76, 77]. Third, it prevents viral aggregation by removing sialic acid residues from the carbohydrate moieties of HA and NA from the viral surface [78]. In a broad perspective, NA is responsible for viral mobility, allowing it to reach the site of infection and leave the same site after viral replication, which explains why NA inhibitors can limit the normal progression of the infection [79].

Since HA and NA have opposing functions, it is likely that balanced activities of these two proteins is critical for efficient IAV replication. For in- stance, the HA affinity for sialic acid needs to be strong enough to ensure virus binding, but weak enough to enable NA to release new virions from the infected cell. To examine the relationship between HA and NA, Mitnaul et al., created several IAV mutants with defective NA and viral growth was

22 impaired in chicken eggs [80]. By repeated passaging these viruses, different mutants were rescued and the resulting viruses acquired substitutions in the HA receptor binding pocket that decreased the affinity for sialic acid conju- gates. In addition, Yen et al., used reverse genetics to assess 2009 pandemic H1N1 transmissibility and linked this property to high NA activity. Howev- er, when this NA was paired with a non-pandemic HA, respiratory droplet transmission was lost, proposing once more that balanced HA-NA activity is required for infection viability [81]. In another work by Gen et al., it was shown that a specific mutation that increases HA affinity was able to decrease viral replication in mice lungs when compared to the parental strain in cell culture. The authors concluded the lost fitness was likely due to a stronger decoy receptor binding that was not counteracted by NA, demon- strating another stage where the functional relationship of HA and NA is important for IAV infection [82].

From what is known about NA function, it is commonly accepted that NA is completely dispensable during viral entry as HA is the main player in this process. However, recent findings suggest that NA is also capable of receptor-binding activity just like HA. Lin et al., reported that several NAs from H3N2 viruses isolated from cell culture were able to bind to receptors on red blood cells mimicking HA function. A substitution of an aspartic acid to a glycine, alanine or asparagine at position 151 was the key factor behind this new feature. This mutation seemed to alter NA specificity, being used by the virus to attach to sialic acid receptors while retaining some sialic acid cleavage capacity [83]. Hooper et al., also found that NA could serve as the receptor-binding protein on a H1N1 strain. In this background, substitution of a glycine residue at position 147 into an arginine rendered NA the binding capability of HA. Changing and/or deleting conserved residues in the HA RBD in the presence of the mutated NA did not significantly impair viral replication of this strain in mice or cell culture, but it was completely dependent on NA for viral entry. Surprisingly, the G147R substitution is also found in several sequences of the 2009 pandemic H1N1, seasonal H1N1 and chicken H5N1 sequences, pointing to a possible natural evolution of NA over time [84, 85]. However, it is relevant to point out that HA was still required for the fusion of the viral and endosomal membranes.

23 Membrane protein maturation

HA, NA and M2 are all integral membrane proteins. In eukaryotic cells this class of proteins is quite diverse and represents up to 30 % of the proteins encoded by the genome. Functionally, integral membrane proteins are associated with signalling, trafficking, and mediating transport of molecules across membranes. Based on particular characteristics of the membrane protein they can be found in the plasma membrane or in the intracellular organelles [86]. The majority of the membrane proteins use the secretory pathway for both integration into the membrane and trafficking to their point of function in the cell. The first step to enter the secretory pathway is to trans- locate the newly synthesized membrane protein or secretory protein across the endoplasmic reticulum (ER) membrane. During this process the transmembrane domains are integrated into the ER membrane and the proteins fold within the ER lumen prior to any further trafficking [87].

ER targeting sequences

Cleavable signal sequences

Membrane or secreted proteins have sequences that target them to the ER. These are commonly called signal sequences and are usually N-terminal extensions that direct nascent or complete proteins from the cytosol to the translocation sites in the ER membrane [88]. Upon translocation across the ER membrane, signal sequences are proteolytically cleaved by the signal peptidase located on the luminal side of the ER membrane [89]. While the signal sequence is not present in the mature form of the protein, the timing of the cleavage can vary between proteins [90].

24 Signal sequences were originally predicted to have a distinctive sequence motif [91], but sequencing of many different secretory proteins confirmed that these sequences were in fact quite diverse. Despite this variation, signal sequences were later found to share three distinct regions that compose the overall structure: i- a positively charged N-terminal region, ii- a central hydrophobic core (h-region) and iii- a more polar C-terminal region where the proteolytic cleavage occurs based on the presence of small uncharged residues at positions -1 and -3 in the signal sequence [89]. Although these are common regions to all signal sequences, they generally vary in length and amino-acid composition [92, 93]. The N-region varies greatly with the overall length, representing one half of the total variation. In the h-region, residues -6 to -13 are the most essential and constitute the minimal length required for protein targeting [94].

A minimal functional signal sequence can be defined as: a c-region with five residues, a seven residues long h-region and a one residue n-region. Also, a specific role can be attributed to each region in the targeting process. The c-region defines the cleavage site for the signal peptidase, the h-region is possibly the binding site for the signal recognition particle that is involved in trafficking nascent secretory proteins to the ER and the n-region contributes to translation initiation as well as the topology required for cleavage, which is dictated by the presence of positive charged residues [95-97].

Uncleaved signal sequences

Targeting sequences that conduct many single-spanning membrane proteins into the ER are not cleaved. In these cases, if the targeting sequence has a certain length and hydrophobicity it can anchor the protein in the ER membrane by forming a transmembrane domain. It is then called a signal- anchor sequence [98]. Signal-anchor sequences have the interesting feature of being inserted into the ER membrane in different orientations normally based on the charge of the n-region and hydrophilic regions [99, 100]. After insertion of the signal anchor sequence into the ER, if the N-terminal is located in the cytoplasm (Nout-Cin) it is called a type II membrane protein, and if it is not the N-terminus will remain positioned toward the ER lumen (Nin-

Cout) it is defined as a type I. It is more the hydrophobic character rather than the amino-acid sequence of the N-terminus that is capable of membrane insertion and protein anchor-

25 ing [101]. However, the targeting segment hydrophobicity by itself might not be enough to dictate if it can anchor the protein. The presence of hydrophilic regions flanking the N- and C-terminal sides of the hydrophobic segment can also influence the translocation and insertion of the membrane protein [102]. Signal-anchor sequences commonly have charged amino-acids residues at their NH2-terminal end. Positively charged residues are proposed to prevent the translocation of the NH2-terminus across the ER membrane resulting in the inversion of the signal anchorsequence while negatively charged residues do not seem to interfere with translocation of the NH2- terminus across the ER membrane [103-105]. From these premises it is possible to conclude that a typical signal-anchor sequence requires: i- NH2- terminal region positively charged, ii- central segment composed of hydrophobic amino-acid residues and iii- the absence of a signal peptidase cleavage site in the C-terminal region.

In the case of IAVs, both types of ER-targeting sequences and topology can be found in HA and NA. HA possesses a cleavable signal sequence and a C-terminal transmembrane domain (TMD) that tethers it to the membrane, while NA has a signal-anchor sequence in its N-terminus that functions as a transmembrane domain to keep the protein anchored in the ER membrane. For HA, the cleavable signal sequence results in the N-terminus being positioned in the ER lumen and the C-terminus facing towards the cytoplasm resulting in an Nin-Cout conformation on the ER membrane [46]. On the other side, NA which possesses positively charged residues on the N-terminal juxtamembrane region, inverts its TMD resulting in a Nout-Cin conformation [106-108].

Endoplasmic reticulum insertion

Entering the ER is not only a way for the membrane proteins to reach the plasma membrane, but it is also the beginning of the protein biogenesis and maturation. For many proteins, maturation and folding occurs co- translationally. In this process, the N-terminal region of the polypeptide chain starts to fold even before the C-terminus has finished being translated by the ribosome [109]. By folding in this manner, it decreases the number of intermediates a nascent peptide can sample and creates a more controlled maturation environment that increases the folding efficiency [110].

26 The co-translational folding of many secretory proteins is coupled to the translocation across the ER membrane [111]. The process starts when the N- terminus of the polypeptide chain is recognized by the signal recognition particle (SRP), which halts synthesis and guides the ribosome-nascent chain complex to the SRP receptor in the ER membrane. Upon docking to the receptor, the ribosome-nascent chain complex is transferred to the Sec61 translocon, which acts as a channel for the translocation of the nascent chain across the membrane and into the ER lumen once protein synthesis resumes. During this process, the N-terminal signal sequence is cleaved by the signal peptidase, and transmembrane domains (TMDs) are inserted into the ER membrane through a lateral gate in the side of the translocon (Figure 3) [112, 113].

Figure 3 – Protein translocation across the ER membrane. During the translation process of secretory protein mRNAs, a polypeptide nascent chain emerges from the ribosome and is bound by the signal recognition particle (SRP). By interacting with its receptor at the ER membrane, SRP transfers the translating complex to the Sec61 complex, a protein-conducting channel, and translocation occurs. In the case that the translating polypeptide contains a signal peptide, this N-terminal sequence is cleaved in the ER lumen by the signal peptidase. For secretory proteins, the translocated protein is released to the ER lumen while for membrane proteins, the presence of a TMD di- rects the protein laterally out of the conducting channel, inserting the protein in the ER membrane [114].

27 Whether or not a polypeptide segment is inserted into the bilayer as a TMD is highly dependent on its physical properties as the environment in the lipid bilayer is substantially different from an aqueous environment. Li- pid bilayers are anisotropic, varying from a hydrophilic portion to a hydrophobic core rich in hydrocarbons and changing back again to a hydrophilic portion [115]. Thus a particular polypeptide segment must possess adequate hydrophobicity in order to be integrated into the lipid environment as a TMD. For bitopic proteins that have only one TMD, this domain generally possesses high hydrophobicity due to the fact there is only one TMD to promote membrane insertion. However, in multi-spanning membrane proteins, TMDs with low hydrophobicity can be compensated by interacting with other neighbouring TMDs [116]. Our studies have shown that in addition to the role in membrane integration, the TMD from NA also contributes to folding and assembly of the enzymatic head domain.

The transmembrane domain

A transmembrane domain can be simply described as an a-helix that spans the hydrophobic region of a lipid bilayer [117, 118]. In terms of amino-acid composition, a TMD has a central hydrophobic segment that is very rich in aliphatic residues and phenylalanine. It has short border regions containing mostly tryptophan and tyrosine residues and polar “caps”. These “caps” often include asparagine and glycine residues that function in N- and C-terminal helix capping [119].

Due to their role in protein anchoring to the membrane, TMDs suffer different constraints. The primary one is the partitioning out of the translocon into the ER membrane. To help with this step, TMDs are rich in hydrophobic residues that promote partitioning out of the sec61 translocon and into the membrane [120]. Besides the translocon, the physical properties of the bilayer where the protein is inserted imposes other constraints on the TMD and these are different depending on which organelle the membrane is derived from. For example, the length of the TMD sequence, which typically vary between 20 and 30 amino-acids, is indicative of the compartment where it resides [119]. This is easily observed when comparing membrane proteins from the Golgi and the plasma membrane and indicative of a step-change in bilayer thickness that occurs in the secretory pathway [121, 122]. This change in thickness is most probably associated with membrane asymmetry

28 and the fact that the plasma membrane is very rich in sterols and sphingo- lypids, which increases the size of the bilayer and longer TMDs are required to span the whole membrane [123]. This way, the TMDs can be used as a signature of specific organelles and also for intracellular transport mechanisms [124].

Transmembrane domains are also very important for protein-protein interactions of integral membrane proteins. They can establish a homo- or hetero- interaction with another TMD that can be partial or fully responsible for the respective proteins interactions [125]. TMDs associate non- covalently by a variety of conserved motifs, for example: i) GxxxG motifs, which are the most common and help facilitate hydrophobic packing [126], ii) polar residues that establish hydrogen bonds [127], iii) glycine zippers [128], iv) leucine zippers or v) charged residues [129].

Contacts between the TMDs start when the proteins are inserted into the membrane followed by formation of the secondary structure. This leads to tertiary contacts between the TMD a-helices. These contacts are mainly driven by Van der Waals forces but hydrogen-bonds that form between one or more polar residues of pairing TMDs can also help stabilize inter-helical associations [130, 131]. Different studies tried to verify the importance of polar residues to TMD associations. It was concluded that two polar side- chain atoms in an amino-acid gives it a greater tendency for TMD interaction acting simultaneously as a hydrogen bond donor and acceptor creating a more stable oligomer. This does not occur so easily when only one polar side-chain is present [132]. However, just by having polar residues within the TMD may not be enough to drive association, it also depends on the exact position of these residues in the TMD [133].

Positively charged residues also have a role in TMD-TMD interaction. These amino-acids that can be found within the TMD are known to have structural and functional roles in integral membrane proteins, like substrate recognition [134]. Mutations leading to the introduction of positively charged residues in a TMD have been shown to be linked to some human genetic diseases [135]. Since TMDs are involved in the assembly of membrane proteins, most probably, these residues affect the structure of the protein by impairing TMD-TMD interactions. One relevant aspect is that the low dielectric core of the membrane changes the physical and chemical properties of polar or charged amino-acids [127]. In certain instances these

29 changes can result in altered protonation states that can stabilize dimerization through hydrogen bonding and then favour dissociation by inducing repulsion [136].

Membrane properties such as fluidity also influence TMD-TMD interactions. Anbazhagan et al., showed that phenylethanol can decrease the order of the acyl chains that compose the membrane bilayer, resulting in an increase in fluidity and a reduction in some TMD interactions [137]. Lipid solvation is also capable of influencing TMDs interactions. Cunningham et al., showed that dimerization increases with decreasing lipid accessibility. This was determined by substituting two valine residues to other hydrophobic residues and the mutations substantially changed an association via a GxxxG motif. As a conclusion they argue that an increased ability or inabil- ity of the lipid environment to solvate the a-helical structure may decrease or increase helix-helix interactions. More simply put, there is a constant competition between lipid-TMD interactions and TMD-TMD interactions and this contributes to the fluidity in membrane protein complexes and enables complexes to assemble and disassemble.

Protein glycosylation

Part of the process of membrane protein maturation within the ER involves glycosylation. The addition of N-linked glycans to secretory proteins in the ER lumen is catalyzed by a membrane enzyme complex called the oligosaccharyltransferase (OST) [138]. When the ribosome is docked to the ER membrane and the nascent chain is translocated through the translocon or Sec61 protein conducting channel, any Asn-X-Ser/Thr sequence (where X cannot be a Pro) is recognized and glycosylated at the Asn residue by OST once the sequence is 12-15 amino-acids away from the membrane [139]. The co-translational addition of N-linked glycans to a protein is highly relevant to the maturation process as they can promote favourable folding energetics and reduce the risk of aggregation. Hydrogen bonds between the glycans and the protein are thought to reduce the entropy of the unfolded conformations. They can also increase the free energy of the folding inter- mediate resulting in a higher propensity to achieve the native state [138, 140]. During the folding process in the ER lumen, N-linked glycans also

30 interact with lectin chaperones such as calnexin and calreticulin, which associate with the oxidoreductase ERp57. Thus, N-linked glycans can be positioned to recruit the lectin chaperone machinery to specific regions of the protein to assist in the folding process and the formation of disulphide bonds. The recruitment is dictated by the trimming of the N-linked glycan to a monoglucosylated state by glucosidase I and II, which acts as a regulator for when and where the chaperones are recruited to protect specific domains from deleterious interactions that can lead to misfolding (Figure 4) [141, 142].

Figure 4 - N-linked glycan structure. N-linked glycans are added to polypeptide chains when the glycosylation sequence, Asn-XXX-Ser/Thr is recognized by OST in the ER. The primary structure of the glycan is composed by three glucose residues (Glc), nine manose residues (Man) and two N- acetylglucosamine residues (GlcNAc). Glycan residues are linked to each other by a and b-linkages [138].

After a period of time in the ER lumen, glucosidase II removes the end glucose residue on the N-linked glycan to release calnexin and calreticulin, which enables the protein to fold. If the protein is misfolded, the N-linked glycan is reglucosylated by Glucosyltransferase to enable another round of chaperone-mediated folding [141]. Due to the lectin chaperone association with ERp57, N-linked glycans can also regulate the formation of disulfide bonds in the oxidizing environment of the ER lumen. Daniels et al., using HA as a model, showed that by positioning a glycan near a specific cysteine

31 residue, an improper and premature disulfide bond could be prevented during synthesis until the protein was released from the lectin chaperones. Thus, one can hypothesize that the location of the N-linked glycans in HA is not merely random, but rather an evolutionary process that allowed to be regu- lated the maturation of complex folding domains [142]. After the glycosylation and maturation in the ER lumen, glycoproteins are loaded into transport vesicles and directed to the Golgi complex. The Golgi apparatus is composed of different stacks of membrane-bound sacs called cisternae that are involved in shuttling proteins to different parts of the cell [143]. The vesicles that arrive to the Golgi through the cis cisternae unload the recently synthesized and folded glycoproteins. From there they are trafficked through the medial to the trans cisternae, where the proteins will con- tact a set of enzymes that further modify the glycosylation moieties produced in the ER [144]. In most cases, the enzymes in the cis cisternae remove the mannose core of the N-linked glycan to leave only three mannose residues. As the protein moves towards the trans compartment, the N-linked glycans receive a subsequent addition of: two N-acetylglucosamines, followed by two galactoses, two N-acetylneuraminic acids and a single fucose residue, which marks the end of the N-linked glycan chain [145, 146]. Once the N- linked glycans are properly processed, membrane proteins like HA, NA or M2 bud off from the trans-golgi membrane in transport vesicles that are directed to the plasma membrane. Upon reaching the plasma membrane, the vesicle fuses with the lipid bilayer releasing any soluble proteins into the extracellular space and positioning any membrane proteins on the cell surface [147].

At this point, a general introduction was done with the intent of covering different aspects of membrane protein biogenesis. Now the focus will be directed to neuraminidase which was the basis for all the studies in this thesis.

32 Neuraminidase

Neuraminidase structure

NA morphology was first proposed by Laver et al., almost 40 years ago. Based on its appearance in electron micrographs they described NA in a very simple way: “the neuraminidase subunits were elongated objects, many showing a centrally attached fiber which possessed a diffuse tail or knob” [148]. Later, the NA heads were isolated from viral particles by digestion with pronase resulting in the identification of a 200 kDa protein composed by four identical polypeptides [149]. The subsequent crystallization and structure determination of these heads confirmed that NA is presented at the surface of the viral particle as a homotetramer [150, 151]. Nowadays, we know that NA is composed of three main domains: i) a highly conserved cytoplasmic tail followed by a hydrophobic transmembrane domain, which both targets NA to the ER and anchors it the viral envelope; ii) a filamentous stalk domain and iii) a globular head domain containing the active site for sialic acid cleavage and the main antigenic determinants [152].

The head domain

The enzymatic head domain has always been the major interest in NA. The fact that it harbours the enzymatic active site makes it a preferred target for drugs, has resulted in a large amount of biochemical and structural data being available. The active site is located in a deep central cavity in each of the four monomers that constitute the active form of NA [23]. It presents a rigid catalytic center and is fundamental for NA function [73]. The eight functional charged and polar amino-acids that constitute the active site are highly conserved among the eleven NA subtypes and these are supported by eleven residues that form a structural framework (Figure 5) [153]. Substitu-

33 tion of any of these residues, commonly results in the loss of enzymatic activity, which is not surprising given their conservation [154].

Figure 5 - Neuraminidase active site. The structure of the head domain of a NA (N2 subtype) monomer is represented in complex with Neu5Ac2en (2-deoxy-2,3-didehydro-N-acetylneuraminic acid). In red are highlighted the conserved and functional amino-acid residues of the active site, Arg118, Asp151, Arg152, Arg224, Glu276, Arg292, Arg371 and Tyr406 [155].

The fact that the active site is so important for viral spreading and is highly conserved, makes it a desirable target for neuraminidase inhibitors that can be used in antiviral therapies. The design of inhibitors tries to mimic the natural substrates of NA (sialic acids) and compete with higher affinity for the binding of the active site, which prevents the cleavage of the natural ones. This inhibition greatly affects NA activity, blocking the release of new virions from the cell surface and causing them to aggregate, which limits the progression of the infection to neighbouring cells. At the moment, oseltamivir, zanamivir and peramivir are the three NA inhibitors approved by the US Food and Drug Administration. However, many circulating IAV strains are already resistant to oseltamivir [156, 157].

Figure 6 - Representation of the crystal structure of the tetrameric NA head domain. Four identical monomers compose the tetrameric head of the 1918 N1 NA. Each monomer is composed of six four-stranded, antiparal- lel-sheets with the active site located on top of the molecule. Nine calcium ions bound to NA are shown in magenta and in the N-linked glycans in gold are represented [153].

From the above structure of the NA enzymatic head domain (Figure 6), calcium ions (Ca2+) that are coordinated with NA through specific metal binding sites are visible. Dimmock et al., showed that in the absence of calcium, the activity of an H1N1 strain could drop up to 99 % of its original activity while an H3N2 lost less than 50 % [158]. Brett et al., also showed that these ions are important for maintaining the tetrameric structure as extensive dialysis with EDTA, lowered NA activity and immunogenicity in an irreversible manner [159]. These results indicate that Ca2+ has an active role in supporting conformational integrity of the tetrameric molecule.

35 The stalk domain

From the first studies on NA made by Laver et al., it was possible to identify a region that connected the four head subunits to the TMD. This visible thin fiber less than a 1.5 nm in diameter was the stalk region [148]. Besides serving as a linking element between the two distant domains of NA, Blok et al., identified disulfide bond linkage between monomers within the stalk domain and also potential glycosylation sites in this region. The fact that the crystal structures used to study the NA head domain lacked the stalk region, made the properties and function of this domain unknown. Extensive sequence analysis of the stalk region, showed variability and flexibility between NA subtypes. This analysis led to the proposal that the stalk domain is in an extended conformation or in a very loosely coiled form [160]. One curious observation made at that time was that the length of the stalk region was also quite variable. Based on sequence comparison of many IAV NA genes, it was observed that the stalk region could vary between 25 and 57 amino-acids long [161].

With the purpose of understanding the importance of the length of the stalk region, Luo et al., used reverse genetics to assess how deletions and insertions in the NA stalk domain of the WSN strain impacts NA function and viral infectivity. From their data it was possible to observe that a great flexibility in the length of the stalk is allowed. Deletions up to 28 amino- acids and insertions up to 41 residues did not abolish viral infectivity, but in some instances it resulted in slower viral growth and lower titers. They also found that these deletions are limited to residues between the TMD and the cysteine at position 76. This cysteine residue appeared to be vital to viral growth since its substituition would impair viral infectivity. These findings were one of the first to suggest that the stalk domain of NA could be a region of viral adaptation since it can modulate the biological characteristics of the influenza virus [162].

Castrucci et al., also did a similar work by creating different NA stalk mutants with varying lengths in WSN background. Their results were important to shed more light of how this region can have a great impact on the host range of IAVs. When a full stalk-deleted virus was used to infect eggs, viral growth was abolished, but in contrast, viruses with longer NA stalks were viable and able to grow better. In mice, the insertions of 28 residues did

36 not have a great impact in viral titer but once more the lack of a stalk domain highly attenuated viral growth. Growth differences registered between tissue culture, eggs and mice might indicate that the stalk domain has a relevant functional role in natural settings [163]. It can be hypothesized that if the stalk domain does not have a minimum length, the NA head might not reach glycoconjugates like mucins during viral movement in the respiratory tract. The lack of NA function would trap the viral particle and prevent its attachment to the cell surface receptors for viral entry. More recently, the stalk domain has been associated with pathogenicity and transmissibility of different strains. Zhou et al., identified stalk-motifs that increase virulence and pathogenesis of the avian H5N1 [164]; Blumenkrantz et al., found that short NA stalks of highly pathogenic avian (HPA) H5N1 reduce transmission of the 2009 H1N1 pandemic in ferrets [165]; Stech et al,. showed that the presence of a stalk deletion is an important determinant to increase virulence of HPA H5N1 in chicken [166] and finally Bi et al., concluded that by changing the length of the stalk region in a H7N9 strain, virulence in mice was enhanced [167].

Neuraminidase maturation and oligomerization

Neuraminidase is initially synthesized co-translationally as a monomeric protein that matures and oligomerizes in the secretory pathway. We can track down the starting point of NA maturation during the cotranslation oxi- dation, when NA polypeptide crosses the translocon [111]. By using several monoclonal antibodies that were specific for different conformations of a N8 head domain, Saito et al., were able to follow NA oligomerization over time (Figure 7). The inclusion of the transport inhibitor BFA, the authors confirmed that NA oligomerzation occurs within the ER lumen, and the conformational specific antibodies revealed that NA folding starts shortly after translation begins. Specifically, dimerization occurs with a t1/2 of 2.5 minutes through the formation of intermolecular disulfide bond, and this is followed by two additional conformational changes one at 8 minutes and another at 13 minutes when the tetramer-specific epitope can be recognized. 30 minutes later, NA is detected at the plasma membrane reaching its maximal level at 1 hour when budding starts [168, 169].

Figure 7 - Neuraminidase maturation. In 1995, Saito et al., proposed a model for the complete maturation and tetramerization of NA. With the use of monoclonal antibodies that recognized different NA conformations, they were able to track NA folding from monomer to tetramer and determine how long this process lasts [169].

In a more detailed study, Hoge et al. showed that NA co-translationally binds to the ER resident chaperone GRP78-BiP. This transient association allows NA folding to be assisted from the early steps of synthesis until oligomerization begins and it is transported out of ER [168]. Wang et al., observed that glycan addition and chaperone binding were all required for proper NA maturation. More important, they verified that dimerization of NA occurs co-translationally before the complete synthesis of the second monomer, which implies that NA dimerization is an N-terminal mediated process [111]. Despite all the information collected about NA over the years, it is still not clear why NA is presented at the viral surface as a tetramer and why it loses enzymatic activity when tetramerization is disrupted [170]. It is possible that intracellular monomeric NA probably lacks a functionally active conformation that can only be achieved when in the tetrameric oligomer, allowing NA to be functional [169].

38 The transmembrane domain – Papers I, II and III

No structural data is available for the NA TMD (Figure 8). Thus, we and others have performed a series of biochemical studies to assess the importance of the NA TMD and to identify certain phases of viral replication where it might be involved. The results obtained showed the influence of NA TMD at a protein, cellular and viral levels. Deletions in TMD impaired NA tetramerization and function [171]; insertion of the NA TMD into a basolat- eral membrane protein sorted it to the apical membrane [172]; amino-acids between position 19 and 35 are significantly related with lipid raft association [173]; mutations within the TMD and close to the stalk domain reduced viral growth up to 250 times by reducing NA activity [174]; replacing the NA TMD from an IAV with a homologous TMD from an IBV impaired IAV growth in MDCK cells and only 14 % of NA was present in the virion but replacing the whole NA did not affect these two parameters [175]

Figure 8 - Schematic representation of a NA tetramer. Three distinct domains compose a NA tetramer. A head domain (Protein Data Bank code 3BEQ; WSN33 numbering), a length-variable stalk domain and a TMD within the signal anchor sequence [Paper III]

39 All of these studies produced relevant information regarding the NA TMD and indicated that it may contribute to the structure and assembly of NA, its function and to virus viability. However, most of these studies used deletions, which are highly unpredictable and biologically non-relevant substitutions.

Paper I – Contribution of TMD to NA assembly

We systematically addressed if the TMD contributes to the assembly of the NA head domain, and if so how could the TMD assist in the folding of the distal head domain. Using a molecular and biochemical approach, this study had the objective of determining the role of the TMD in the folding and oligomerization of NA.

Initially, the N-terminal signal anchor sequence of NA was replaced by a cleavable signal sequence. Instead of being inserted into the membrane, NA remains a soluble protein that is secreted from the cell. Although it efficiently accumulated in the cell growth media, blue-native electrophoresis analysis revealed that the soluble form of NA aggregated, explaining the residual sialidase activity. If NA is only active as a tetramer, how could the TMD contribute to its oligomerization? Our original hypothesis was that the TMD is also capable to oligomerize into a tetramer. By adding a second cysteine to the stalk region in an NA construct where the head domain was absent (to avoid positive influence on tetramerization) it was possible to observe that the TMD and the stalk domain can tetramerize independently of the head domain. So, could it be possible to recover soluble NA activity by N-terminal oligomerization? Insertion of a dimeric, trimeric and tetrameric GCN4 coiled-coil domains between the signal sequence cleavage site and the stalk domain [176], progressively increased NA activity and it registered maximum re- covery when the tetramerization domain was inserted.

Thus, optimal NA assembly involves TMD and head domain cooperativity by matching the oligomeric state of both domains. This data also proves that a large soluble domain can promote assembly with a poorly compatible TMD, but an optimal assembly requires coordinated oligomerization between the TMD and the soluble domain.

40 Paper II – Amphipathicity dictates neuraminidase TMD interactions

The data obtained in paper I, rose our interest on the conservation of TMD oligomerization among the two human (N1 and N2) and the nine avian (N1-N9) subtypes. Using the GALLEX system that enables in vivo measurement of TMD associations [177], we analysed the interaction between the consensus sequences of the TMDs (residues 7-35) from the nine avian IAV subtypes and N1 and N2 from the humans (Figure 9).

Figure 9 - Measurement of TMDs interaction strength. The Gallex system placed in the inner membrane of Escherichia coli is used to quantify the association between TMDs. In this system, the degree of interaction is meas- ured by the TMD-mediated dimerization of LexA, which represses LacZ transcription, resulting in a decrease in b-galactosidase activity. NA TMD consensus sequences of all human and avian subtypes were analyzed to determine their interaction [Paper II].

It was clear that for avian subtypes N1, N3-N9 there is a high degree of TMD association except for the N2 subtype where the interaction between TMDs is very weak. In the case of the human TMDs, N2 had a very low association strength (similar to the avian N2 TMD), when compared to the reasonably strong interacting TMD of the N1 subtype. By plotting the relative interaction strength of each TMD with respect to their predicted hydrophobicity it was possible to observe that the association between TMDs increases in a linear manner with decreasing hydrophobicity.

All the TMD consensus sequences revealed a pattern of two adjacent hydrophilic faces with the exception of the N2 subtype, which is only predicted to have one hydrophilic face. This suggested these two polar faces might

41 confer amphipathicity to the TMD, which drives its association. To test this hypothesis, the polar faces on the human pandemic N1 (hpN1), human N2 (hN2) and avian N3 (aN3) TMDs were converted into hydrophobic faces by alanine residue substitution. Removal of the polar residues in hpN1 and aN3 TMDs drastically reduced TMD association, reaching the low level of hN2 TMD association that was not affected by the substitution of its single polar face. For the N1 subtype removal of the polar residues in the a- and e-face demonstrated to be synergistic, whereas for the N3 subtype the interaction appeared to be more additive.

The conclusion that NA TMD interactions are dictated by the specific position of polar residues in respect to the a-helix of the TMD and the membrane allowed us to stipulate a general model for NA TMD oligomerization (Figure 10).

Figure 10 – Neuraminidase TMD assembly. In the proposed model, the TMDs from the N1 and N3-N9 subtypes have a similar assembly mechanism. Initially, the TMDs dimerize followed by a rotation and association with another TMD dimer, forming a tetramer. The tetramer formation is dependent on the location of the polar a- and e-faces. N2 does not have a polar a-face and their TMDs interact very weakly and may be unable to tetramerize [Paper II].

42 Paper III – The influenza NA TMD and head domain coevolved

When the hydrophobicity of all N1 TMDs from human IAVs was determined in paper II, it was noticeable that they have become increasingly more polar since 1918 with a stronger TMD association. We hypothesized that this behaviour could be the result of an evolutionary process, where immune pressure on the NA head introduced changes that was compensated by the TMD, since they cooperate for proper NA folding. To test our hypothesis, we substituted the less polar NA TMD from the 1933 H1N1 lab strain WSN with a more polar TMD from the H1N1 2009 pandemic. This resulted in a NA chimera where the head domain and the TMD are not compatible. We assessed the implications of this change on the replication of the virus and any resulting rescue mutations.

Determination of viral titters at optimal growth temperature (37 0C), showed that the new chimera had decreased viral growth when compared to WT-WSN. However, by doing several infection cycles with the chimera, viral titters were restored to WT-WSN levels. This rescue was achieved by the appearance of different mutations within the NA TMD. The initial fitness loss indicates that using a more polar NA TMD in conjunction with the WSN head domain creates a domain incompatibility, leading to folding defects that affect viral budding and reduces viral growth. To counteract, the head domain selects for compensatory mutations in the TMD that are capable of repairing the harmony between both domains.

The adaptive mutations in the NA TMD, were mainly polar changes that decreased the TMD hydrophobicity further. Despite being less identical to the NA TMD of WSN, these substitutions possibly changed the packing of the TMD in the membrane by altering the hydrogen bond network as they lowered of the relative TMD association to WSN levels. In all cases, the amphipathicity of the TMD was preserved indicating that this is fundamental for the proper function of at least N1 subtype.

Knowing that all TMDs from N1 subtypes are amphipathic, we converted the polar a-face to be more hydrophobic. Disruption of this conserved property resulted in little to no viral growth at 37 0C and slow growth at 33 0C, which is in line with previous observations that NA-defective viruses can be rescued at 33 0C, but not at higher temperatures as folding defects lower NA

43 thermal stability [178]. Sequential passaging of the rescued virus at 33 0C showed TMD rescue mutations that increased the TMD hydrophobicity and allowed viral growth at 37 0C. This time, the selection of hydrophobic and not polar substitutions did not alter the relatively weak TMD association, but it might have rearranged the TMDs in a way that was not detected. Thus, we concluded once more that TMD-head compatibility is a requirement for a proper NA assembly and this relationship is under selective pressure that can be alleviated by specific adaptations that are dependent of TMD properties (Figure 11).

Figure 11 - Co-evolution of NA TMDs and head main. In our proposed model, changes in NA head domain can affect the folding compatibility with the respective TMD in the N1 subtype. These changes in the head domain favors polar substitutions in the TMD that can alter the amphipathic TMD assembly and restore the compatibility between the two NA domains [Paper III].

44 The stalk domain – Manuscript IV

The observation that the TMD oligomerization is the driving force for proper assembly of N1, the absence of a strong TMD association for N2 subtypes raised the question of whether this particular subtype had a unique assembly process that diverged from all other NA subtypes [Paper II]. Since all NAs have the same tetrameric structure and function, we hypothesized that for the N2 subtype, the stalk domain can compensate for weak TMD association.

Sequence analysis of N1 and N2 stalks showed that N1 stalks are more prone to have amino-acid deletions than N2. These deletions are longer and cover almost all of the middle part of the stalk, while in N2 they are shorter and more located to the C-terminal side of the stalk. This pattern might point to the need of N2 to keep the stalk central region intact, which seems to be dispensable for N1 subtype.

The introduction of N1 and N2 subtype TMDs in the GALLEX system, with the addition of stalk portions of increasing length, showed a variation in the strength of TMD interaction. The presence of longer portions of the stalk domain did not change N1 TMD association, while for N2, an addition of only 7 stalk amino-acids greatly increased TMD interaction, reaching N1 levels. Elimination of the polar a and e faces in N1 TMD resulted in an impaired TMD association that could be rescued with the addition of the stalk region, confirming the N2 results and need of a stalk when TMD association is weak. Also, both N1 and N2 stalks carry natural cysteines that could contribute to an enhancement of association, but only for N2 they seemed to be relevant, probably by stabilizing the stalk. By reverse genetics we are able to create viral mutants with 20 amino-acids deletion in the stalk region of N1, but unfortunately not yet in N2. Comparison with the viruses carrying the respective wild-type N1, showed that viral mutants were still viable and reached similar growth titters. This confirmed previous observations that for the N1 subtype in tissue culture the presence of a long stalk domain is not mandatory.

Analysis of N1 and N2 stalk sequences revealed a remarkable difference in the number and positioning of cysteine residues in this domain. For the N1 subtype, the majority of the stalks carries only one cysteine residue but in

45 many other sequences this residue is absent. In contrast, the N2 stalk has more cysteine residues. It mainly varies between 2 and 3 residues but there are sequences where 4 residues are present. Once more, in the GALLEX system, and for the N1 subtype, the substitution of the cysteine residue into a serine had no effect on TMD association, while for N2 a small decrease was registered. This shows that the enhancement of the TMD association is more dependent on the presence of a certain stalk length than any cysteine residues. By reverse genetics, the substitution of the cysteine residues in N1 stalk domain did not impair viral growth, and only a minor decrease in the initial growth was detected. Unfortunately, our lab does not have yet a reverse genetic system that can be used to test a N2 protein did not allow us to confirm that the stalk is required by this subtype for viral growth.

One more difference between N1 and N2 stalks is the variation in the number and location of N-linked glycans. Sequence analysis showed that the N1 subtype has several glycosylation sites evenly distributed over the stalk, while N2 has fewer and localized once more in the C-terminal of the stalk. Since there is no structural information on the NA stalk, we can only assume the presence of oligomerizing domains in the stalk. The presence of these complex sugars could in fact disturb these regions preventing oligomerization. This theory could explain the high number of glycans in N1 but not in N2

From our recent and initial observations, it is possible to conclude that the stalk domain in the N2 subtype seems to be quite important for NA assembly since it compensates for the weaker N2 TMD interaction. This way, the formation of the NA tetramer is secured and the protein can be functional. In the N1 subtype, the fact that the TMDs strongly associate minimizes any stalk effect, making this domain dispensable in terms of contributing for NA tetramerization.

46 Conclusions and future perspectives

The presented doctoral work had the primary objective of investigating if the assembly of the neuraminidase protein from the influenza A virus, is a concerted process between the three domains: transmembrane, stalk and the enzymatic head. We proposed a new model for neuraminidase assembly with the understanding of the biophysical force that drives it and concluded that this is an evolving process. Through our studies, we verified that the NA TMD is necessary to tether NA to the membrane, to NA tetramerization and activation as a sialidase but not required for NA synthesis and trafficking. Moreover, we showed that the polar properties of the NA TMD creates a conserved amphipathic pattern in the TMD a-helices that drives TMD association in N1 subtype but not in the N2 subtype. These results show that the positioning of polar residues within the helix and the lipid layer can mediate TMD interactions in a predictable manner. Further, we demonstrated that the cooperativity between the NA TMD and the head domain is vital and has been evolving to maintain viral budding and replication. Disturbing this relationship promotes the introduction of rescue mutations in the NA TMD that relates to the mechanism of TMD assembly. The results from our last study indicate that when TMDs interaction is weak, like in N2, the tetramerization of the NA head domain is held by the presence of the stalk region. Besides the great interest of these results to the influenza field, they can also be extrapolated to most membrane proteins. From what we have showed, it is clear that single-spanning TMDs have many functions besides placing the protein in the membrane. They are related to lipid binding, trafficking, processing and can have a major role in the folding and function of distal domains in bitopic membrane proteins. The assembly of a TMD when coordinated with a distal ectodomain can contribute for the optimal assembly of type II transmembrane protein. But if the TMD is less compatible with the soluble domain, the latter can force the assembly process. Our studies also demonstrate that in natural TMDs, polar residues contribute to TMDs as-

47 sembly in a process dependent on their position and localization in the membrane. This increases the degree of specificity to how proper lateral TMD interactions are achieved. The analysis of the TMD amino-acid properties with respect to their helix localization and favorability toward membrane integration should increase our ability to identify polar-mediated interactions in the large number of uncharacterized TMDs from bitopic proteins. Finally, the fact that some results were surprisingly regarding the plasticity of a bitopic TMD and protein folding, suggests that we should use native biological systems. In the future, it would be interesting to see how the involvement of lipids, the composition of the membrane and other associated proteins can influence the function of the NA TMD. The stalk domain has a high mutation rate, greatly varies in length and has many potential glycosylation sites. Thus, it would be exciting to find if these are mechanisms of epitope masking for antibody binding. Lastly, studying the stability, structure and kinetics of the head domain for several NAs from different years and strains, could indicate that the immune system has been pressuring the head domain to change. This would test whether the decreasing hydrophobicity and increasing interaction of NA TMDs was the solution found by the virus to maintain NA properly folded and functional. The observations made in our work might be a starting point to find new strategies and new targets to successfully fight influenza. By realizing that things that once were thought to be almost irrelevant, like the NA transmembrane domain, are essential to IAVs, the future of antiviral therapeutics might be assured.

48 Sammanfattning på svenska

Influensa är en av de mest smittsamma sjukdomarna som drabbar männi- skor och de flesta kan räkna med att bli infekterade många gånger under sin livstid. Influensaviruset attackerar främst luftvägarna, men kan även leda till t.ex. lunginflammation. De enskilda viruspartiklarna (virionerna) kan komma i olika former, men den vanligaste formen som används för att beskriva viruset är den sfäriska. På en virions yta så finns det två olika typer av membranproteiner, som kan liknas med två olika sorters spikar som sticker ut från viruset. Den ena ”spiken” kallas neuraminidas, eller bara kort för NA, och den andra för hemagglutinin (HA). När man har andats in ett influensavirus så kan viruset ta sig till de övre luftvägarna och vidare ner i luftstrupen för att där använda sig av HA för att ta sig in i en cell. Viruset använder sig sedan av cellen för att skapa många nya virioner, som tar sig ut ur cellen för att infektera fler celler. NA är det protein som virionerna använder sig av för att klyva sig loss från modercellen. Målet för avhandlingen var att studera NA och beskriva hur proteinet måste vara ihopsatt för att vara aktivt. NA har en uppbyggnad liknande en trädklunga, där fyra stycken identiska träd (med tillhörande rötter, stammar och trädkronor) går ihop och bildar en enda aktiv enhet, en s.k. tetramer. ”Rötterna” hos NA är den transmembrana domänen (TMD), den del av proteinet som sitter fast i influenaviruskroppen. ”Stammen”, eller stjälkdelen av NA, binder samman TMD med den största delen, huvuddomänen som mots- varar ”trädkronan”. Det är just huvuddomänen som är ansvarig för att klyva loss viruspartiklar från en modercell. Vi har i våra studier sett att det kan vara väldigt viktigt att TMD- domänerna går ihop i grupper om fyra för att hela NA ska kunna gå ihop i en tetramer och aktivt kunna klyva loss viruspartiklarna. När vi studerade TMD från olika influensavirus så märkte vi att vissa egenskaper hos TMD krävs för att de skulle kunna gå ihop, men också att dessa egenskaper inte fanns hos alla influensavirus. Virusen har evolverat över lång tid och har anpassat sig efter värdorganismerna (inklusive människan) och har hittat olika lös- ningar på problemet med att behöva bilda en tetramer. När vi gjorde änd-

49 ringar i en TMD som vanligtvis gick ihop till en tetramer, och därmed för- hindrade detta, så noterade vi att huvuddomänens funktion påverkades vilket ledde till att influensaviruset hade svårt att spridas. Vidare så har våra på- gående studier på stjälkdelen visat att även denna del kan ha stor betydelse för tetrameriseringen av NA, speciellt i de fall där TM-domänen saknar egenskaper för att gå ihop. Avhandlingen tillför inte bara ny och viktig information till influensa- forskningen, utan även potentiellt för framställandet av nya influensavacci- ner/-mediciner.

50 Acknowledgmentes

During the long journey that is becoming a Doctor, only hard work (spe- cially on weekends) is not enough. During those years, you will need your colleagues, friends and family that will support you to achieve success. The- se are the persons that made my PhD possible and which I am very thankful:

My supervisor Rob. You had no reason to risk accepting me without even knowing me, but you still did it and I will never forget that. If I am what/where I am now it is in great part because of you. Thank you for your scientific guidance and for so many times being a friend.

Prof. Stefan Nordlund for being always available for everyone at DBB. I would also like to thank prof. Pia Ädelroth for her time during all my PhD “check points” and Dan Daley for co-supervising me and always being available for me.

My collaborator Jeremy Rossman at Kent University, that helped us greatly with his valuable input and viral knowledge.

My great colleagues that make working everyday a great and happy expe- rience: Johan, it seems like yesterday that we started to work together in the old lab and we didn’t know how to do anything. We have been having a lot of fun these years! Thanks for all the times that you helped me with comput- er stuff (they were many!!!) and for always having a friendly word to moti- vate me! Soon it’s your turn. Dan, I still remember that when you arrived I was supposed to teach and guide you, but after a couple of days I realized that my job was done! How good can you be?! It was very good to have you in our group to control us. Hao, you are one of the funniest and friendliest persons that I ever met. I am amazed with how much you evolved since you arrived and how hard working you can be! Thank you for all the times that you did things for me without even questioning. I am sure that you will be

51 great in your PhD. Henrik, it was a pleasure to share the lab with you and learn a lot from Swedish politics and culture! Chenge, our honorary mem- ber, it was very fun to have you always around and without you it wouldn’t be the same.

Pedro and Bea, more than colleagues you are great friends that helped me a lot when I arrived to Stockholm and DBB. Thank you so much for all the good moments and for being a great example of how a real scientist should be.

My great lab neighbours Claudio, Kiavash, Patrick and Rageia. You guys created a great environment in that part of the corridor and any time that we needed a laugh it was enough to go to your lab. But seriously guys, practice foosball a bit more, we are getting tired of beating you.

The previous and present members of DBB groups: Isolde, Erika, Bill, Patricia, Anna, Jörg, Candan, Johannes, Rickard, Weihua Ye, Nina, Thomas thank you for creating a great working environment at DBB.

My “Portuguese family at Stockholm”: Ana Borges, Ana Catarina, An- dré, Cátia, Duarte, Gisa, Ivo, Nuno, Oscar and Preto with you Portugal was always closer and all the dinners, parties and good moments that we had were the energy that made me going on so many times!

All the amazing friends that I have made during these years with each I shared great moments: Pilar, Marco, PG, Edurne, Gabriel, Rafa, Dani, Pontus, Nacho, Damiano, Irina. Thank you for your friendship and making life happier. All my friends at Portugal, with a special thanks to Pro and Vi that always made the distance between us to be the shortest as possible! Thank you for your friendship, support during these years and by receiving me in Portu- gal as if I have never left.

My parents-in-law: Nelinha e Pinho, quando pensavam que a Catarina estava cada vez mais perto de regressar a casa eu adiei esse plano por mais uns anos. No entanto, sempre me incentivaram e se interessaram para que eu fosse bem sucedido. Muito obrigado.

52 My parents: Pai e Mãe, sei que este foi um dos maiores sacrifícios que já fizeram na vida ao ver-me sair de casa e do país sem data para regressar. No entanto, o vosso apoio e compreensão foram soberbos e como sempre me ensinaram e demonstraram, temos que fazer de tudo para termos sucesso na vida. Hoje é o dia e sem vocês nunca teria conseguido. Obrigado, vocês são os melhores pais que podia algum dia desejar.

My brother and my idol, Pedro. Desde pequenos que foste sempre o meu melhor amigo e desde aí vi em ti um exemplo a seguir. Todas as vezes que me ajudaste a estudar e que muitas vezes acabava em discussão e choradeira resultaram onde estou hoje. Mostraste-me que tinha sempre que ser melhor. Obrigado por todo o apoio e carinho que sempre me deste!

A minha mulher Catarina que me fez mudar de país, de vida e iniciar esta aventura fantástica!! Não houve um dia que não me apoiasses e me des- ses força para continuar. Obrigado por tornares as minhas tristezas em riso- tas intermináveis e mostrares-me que o empenho e trabalho resultam sempre em grande sucesso. Como vamos continuar a nossa aventura não sei, mas sei que contigo será sempre maravilhosa.

A minha filha Clara. Ainda és muito nova para ler isto, mas sei que um dia o farás e espero que o pai te encha de orgulho. Obrigado pelas tuas brin- cadeiras, pelos teus sorrisos e miminhos quando chegava a casa do trabalho e que fizeram com a minha vida tivesse sempre felicidade para continuar.

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