NA claw machine

Influenza Neuraminidase Novel Mechanisms of Influenza NA that Enable Adaptation andPromote Diversification

 Hao Wang

Hao W

ang    Influ

enza N

euram

inidase

Doctoral Thesis in Biochemistry at Stockholm University, Sweden 2020

Department of Biochemistry and Biophysics

ISBN 978-91-7911-214-1

Influenza NeuraminidaseNovel mechanisms of influenza NA that enable adaptation andpromote diversificationHao Wang

Academic dissertation for the Degree of Doctor of Philosophy in Biochemistry at StockholmUniversity to be publicly defended on Tuesday 29 September 2020 at 14.00 in via Zoom. A linkwill be published on https://www.dbb.su.se.

AbstractInfluenza A viruses (IAVs) are one of the most common human respiratory pathogens and are largely responsible for the seasonal influenza epidemics that cause mild to severe disease. The two IAV glycoproteins, hemagglutinin (HA or H) and neuraminidase (NA or N), serve as the major surface antigens and also are the main determinants of infectivity, pathogenicity and transmissibility. Due to the high abundance in the IAV envelope and its defined functions of mediating cell binding and viral entry, current influenza vaccines have primarily been developed based on HA. The less abundant NA is a receptor-destroying enzyme that facilitates virion release from the infected cell and the escape from decoy receptors during the entry process. Despite these important roles for infection, NA has been largely neglected in vaccines because of its low abundance and labile properties.

The work in this thesis involves several studies that have primarily focused on establishing a general overview of NAmaturation and providing a biochemical assessment of the enzymatic properties in the NAs from circulating H1N1 IAVs.The results from these studies show that the membrane integration of a class of NAs is dependent on the synthesis of its longC-terminus, NA tetramerization is coordinated by its N-terminal transmembrane domain (TMD) and the distal enzymatichead domain, NA stability changes are related to intrinsic and extrinsic determinants, and that the N-linked glycosylationsites on the NA head domain contribute to viral incorporation. In addition, we demonstrated that NA oligomeric structurepossesses sufficient plasticity to allow the formation of heterotetramers, which increases the tolerance for suboptimalsubstitutions and contributes to the diversification of its enzymatic properties.

Together, these results provide new insights into the NA maturation process and the biochemical mechanisms that areresponsible for the NA property differences that are observed in circulating H1N1 IAVs.

Keywords: Influenza, IAV, neuraminidase, transmembrane domain, the central Ca2+ binding site, heterotetramericformation, viral incorporation, evolution, adaptation, diversification.

Stockholm 2020http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-182612

ISBN 978-91-7911-214-1ISBN 978-91-7911-215-8

Department of Biochemistry and Biophysics

Stockholm University, 106 91 Stockholm

INFLUENZA NEURAMINIDASE 

Hao Wang

Influenza Neuraminidase 

Novel Mechanisms of Influenza NA that Enable Adaptation andPromote Diversification 

Hao Wang

©Hao Wang, Stockholm University 2020 ISBN print 978-91-7911-214-1ISBN PDF  978-91-7911-215-8 Printed in Sweden by Universitetsservice US-AB, Stockholm 2020

Dedication to my family:my parents, mother-in-law, wife, daughter andson.

List of publications

I. Type II Transmembrane Domain Hydrophobicity Dictates the Co-translational Dependence for Inversion. Dou D, da Silva DV, Nordholm J, Wang H, Daniels R. Mol Biol Cell. 2014 Nov 1;25(21):3363-74. doi: 10.1091/mbc.E14-04-0874

II. The Influenza Virus Neuraminidase Protein Transmembrane and

Head Domains Have Coevolved. da Silva DV, Nordholm J, Dou D, Wang H, Rossman JS, Daniels R. J Virol. 2015 Jan 15;89(2):1094-104. doi: 10.1128/JVI.02005-14

III. Structural restrictions for influenza neuraminidase activity promote

adaptation and diversification. Wang H, Dou D, Östbye H, Revol R, Daniels R. Nature Microbiol-ogy. 2019 Aug 26; doi.org/10.1038/s41564-019-0537-z

IV. Conserved N-linked glycans on the influenza NA head domain con-

tribute to viral incorporation but are not essential for H1N1 replica-tion. Manuscript Östbye H*, Wang H*, Martinez MR., Gao J., Daniels R.

* Both authors contributed equally to this work

Publications not included in this thesis

I. Analysis of IAV Replication and Co-infection Dynamics by a Versa-tile RNA Viral Genome Labeling Method. Dou D, Hernández-Neuta I, Wang H, Östbye H, Qian X, Thiele S, Resa-Infante P, Kouassi NM, Sender V, Hentrich K, Mellroth P, Hen-riques-Normark B, Gabriel G, Nilsson M, Daniels R. Cell Rep. 2017 Jul 5;20(1):251-263. doi: 10.1016/j.celrep.2017.06.021.

II. Translational Regulation of Viral Secretory Proteins by the 5' Cod-ing Regions and a Viral RNA-binding Protein. Nordholm J, Petitou J, Östbye H, da Silva DV, Dou D, Wang H, Dan-iels R. J Cell Biol. 2017 Aug 7;216(8):2283-2293. doi: 10.1083/jcb.201702102

III. Multiple nuclear-replicating viruses require the stress- induced pro-tein ZC3H11A for efficient growth. Shady Younis, Wael Kamel, Tina Falkeborn, H Wang, Di Yue, Rob-ert Daniels, Magnus Essand, Jorma Hinkula, Göran Akusjärvi and Leif Andersson. PNAS. 2018 Apr 17;115(16): E3808-E3816. doi: 10.1073/pnas.1722333115

IV. Influenza A Virus Cell Entry, Replication, Virion Assembly and Movement. Dou D, Revol R, Östbye H, Wang H, Daniels R. Front Immunol. 2018 Jul 20;9:1581. doi: 10.3389/fimmu.2018.01581

Abstract

Influenza A viruses (IAVs) are one of the most common human respiratory pathogens and are largely responsible for the seasonal influenza epidemics that cause mild to severe disease. The two IAV glycoproteins, hemagglutinin (HA or H) and neuraminidase (NA or N), serve as the major surface antigens and also are the main determinants of infectivity, pathogenicity and transmis-sibility. Due to the high abundance in the IAV envelope and its defined func-tions of mediating cell binding and viral entry, current influenza vaccines have primarily been developed based on HA. The less abundant NA is a receptor-destroying enzyme that facilitates virion release from the infected cell and the escape from decoy receptors during the entry process. Despite these important roles for infection, NA has been largely neglected in vaccines because of its low abundance and labile properties.

The work in this thesis involves several studies that have primarily focused

on establishing a general overview of NA maturation and providing a bio-chemical assessment of the enzymatic properties in the NAs from circulating H1N1 IAVs. The results from these studies show that the membrane integra-tion of a class of NAs is dependent on the synthesis of its long C-terminus, NA tetramerization is coordinated by its N-terminal transmembrane domain (TMD) and the distal enzymatic head domain, NA stability changes are related to intrinsic and extrinsic determinants, and that the N-linked glycosylation sites on the NA head domain contribute to viral incorporation. In addition, we demonstrated that NA oligomeric structure possesses sufficient plasticity to allow the formation of heterotetramers, which increases the tolerance for suboptimal substitutions and contributes to the diversification of its enzymatic properties.

Together, these results provide new insights into the NA maturation pro-

cess and the biochemical mechanisms that are responsible for the NA property differences that are observed in circulating H1N1 IAVs.

Contents

Influenza A virus ............................................................................... 1 IAV subtypes ......................................................................................... 2 IAV life cycle ........................................................................................ 2

IAV Neuraminidase ........................................................................... 7 Structure ................................................................................................ 8 Synthesis and Maturation ..................................................................... 11 Functions ............................................................................................. 14 NA as antiviral target and a vaccine antigen ......................................... 17

Results summary ............................................................................. 21

Conclusions and future perspectives ................................................ 25

Sammanfattning på svenska ............................................................. 29

Acknowledgments ........................................................................... 31

References ....................................................................................... 33

Abbreviations

ADCC antibody-dependent cell-mediated cytotoxicity ADPC antibody-dependent phagocytosis CRM1 chromosomal maintenance 1 cRNA complementary RNA DANA 2,3-dehydro-2-deoxy-N-acetylneuraminic acid ER endoplasmic reticulum HA hemagglutinin IAV Influenza A virus M1 matrix protein 1 M2 matrix protein 2 MAbs monoclonal antibodies NA neuraminidase NAIs neuraminidase inhibitors NK natural killer NLS nuclear localization sequence NP nucleoprotein OST oligosaccharyltransferase PA polymerase acidic protein PB1 polymerase basic protein 1 PB2 polymerase basic protein 2 SA sialic acid SRP signal recognition particle SS ER signal sequence TMD transmembrane domain vRNP viral ribonucleoprotein vRNA viral RNA

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Influenza A virus

IAVs are one of the most common human respiratory pathogens in humans

and are partly responsible for the seasonal influenza epidemics [1]. Aquatic birds are considered the natural reservoir for IAVs [2], but IAVs can also in-fect many other species, such as swine, horses and humans [3, 4]. To enable cross-species replication and transmission, IAVs generally need to obtain adaptions in the replication machinery, which often involves substitutions in the receptor binding domain [5]. The introduction of new IAV strains from avian or/and swine to human population can lead to sporadic influenza pan-demics, which subsequently become seasonal epidemic strains following the years [6]. In the past 100 years, four IAV’s pandemic have been occurred, the 1918 H1N1, the 1957 H2N2, 1968 H3N2 and the recent one 2009 H1N1 [7].

IAVs are enveloped viruses that contain eight negative-sense, single-

stranded viral RNAs (vRNAs) that encode for one or more viral proteins [Fig-ure 1A] [8]. The viral envelope is derived from the infected cell plasma mem-brane and decorated by three membrane proteins with different ratios: hemag-glutinin (HA) is the most abundant, followed by neuraminidase (NA) and the matrix protein 2 (M2) [9-11]. Matrix protein 1 (M1) is located under the en-velope, where it contributes to the support and shape of the lipid bilayer. Within the core of the virus the eight vRNAs exist as individual viral ribonu-cleoprotein (vRNP) complexes [12]. Each vRNP is comprised of a vRNA coiled around multiple copies of the viral nucleoprotein (NP) and a single copy of the heterotrimeric RNA-dependent RNA polymerase (polymerase basic protein 1 [PB1], polymerase basic protein 2 [PB2], and polymerase acidic pro-tein [PA]), which is bound to the short helical hairpin formed by the vRNA terminus [Figure 1B] [13, 14].

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Figure 1. IAV and vRNP diagram. A. Diagram of an IAV particle containing eight gene segments. Viral structural proteins HA, NA, M1, M2 and the viral ribonucleo-proteins (vRNPs). B. A vRNP is comprised of a single-stranded viral RNA (vRNA) coiled around multiple nucleoprotein (NP) copies. The terminal 5’ and 3’ regions of vRNA form a short helical hairpin, which is bound by the viral RNA-dependent RNA polymerase comprised of PB1, PB2, and PA. The illustration is modified from Dou D., et al. Front Immunol. 2018 [15].

IAV subtypes

The subtype categorization of IAVs is based on the diverse antigenicity of the two surface antigens, HA and NA [10, 15]. Within IAVs from aquatic birds, 16 HA subtypes (H1-H16) and 9 NA subtypes (N1-N9) have been iden-tified [3]. Two additional HA (H17 and H18) and NA (N10 and N11) subtypes have been sequenced from bats [16, 17]. In humans only two IAV subtype combinations (H1N1 and H3N2) currently contribute to seasonal influenza epidemics [18].

IAV life cycle

The IAV infectious life cycle can be summarized into the following steps: i- virus attachment and penetration; ii- viral genome transcription and replica-tion; iii- viral protein translation and viral component trafficking; and iv- virus assembly, budding and release [Figure 2] [15].

IAV

NS

M

GenesegmentsPB2PB1

NS1

PAHANPNA

M1M2

NS2

NA

HA

M2

M1

vRNPs splic

ed

gene

s

vRNP

PB2

PB1

vRNA NP

PA 3’

5’

A B

3

Figure 2. Diagram of IAV life cycle. (i) IAVs attach to the cell surface by binding to sialic acid (SA) receptors that facilitate entry into the cell by endocytosis. The endo-cytic vesicle matures into an endosome which possess low pH. The drop in pH triggers the exposure of the HA fusion peptide that forms the fusion pore between the viral and endosomal membranes resulting in vRNPs release into the cytosol. (ii) vRNPs are transported into the nucleus through the nuclear pore complex (NPC). mRNAs and cRNAs are transcribed from incoming vRNPs by the attached RNA-dependent RNA polymerase. cRNAs assemble to cRNPs together with newly synthesized viral proteins. cRNPs are used to transcribed progeny vRNAs that assemble into progeny vRNPs together with newly synthesized viral proteins. (iii) The viral mRNAs are exported to

Golgi

(i)

IAV

SA

Cell

ER

Nucleus

(ii)IncomingvRNP(-)

ProgenyvRNP(-) x8

cRNP(+) x8

A(n)mRNA(+) x 8

NPC

A(n)

Ribosome

PAPB2PB1

NP

Endosome

NAHAM2

(iii)

(iv)

SA

IAV

4

the cytoplasm. Newly synthesized NP, PA, PB1 and PB2 are translated on cytosolic ribosome and imported back into the nucleus. The viral membrane proteins, HA, NA and M2 are translated at the ER and trafficked to the plasma membrane through the Golgi. Progeny vRNPs are exported through NPC and traffick towards the plasma membrane. (iv) After maturation, HA, NA and M2 together with the vRNPs are as-sembled into progeny viruses at budding site. The progeny viruses are released by the receptor-destroying function of NA. The illustration is modified from Dou D., et al. Front Immunol. 2018 [15].

IAVs require the proteolytic cleavage of the HA molecules on the viral sur-face into two subunits, HA1 and HA2, to be infectious [19-21]. The receptor binding sites within HA1 facilitate the attachment of IAVs to the potential host cell surface by binding to terminal sialic acid (SA) residues of glycoconjugates that reside on the cell plasma membrane [20, 22]. Once bound, the sialidase function of NA helps the virus move and identify the proper receptor for in-ternalization by cleaving the local SAs [23]. Upon binding to the proper re-ceptor, the virus enters into the cell by triggering either clathrin-dependent or -independent endocytosis [24].

Following entry into the cell, the virus traffics to the endosome where the

vacuolar-type proton ATPase creates an acidic pH environment. The low en-dosomal pH causes a conformational change in HA that exposes the fusion peptide located in HA2 [25] and it also causes the viral M2 ion channel to open resulting in the dissociation of the vRNPs from M1 [26, 27]. The exposed fusion peptide then interacts with the endosomal membrane, bringing the viral and endosomal membrane close enough to eventually form a fusion pore [28, 29], which facilitate the transfer of the vRNPs to the cytosol.

Within the cytosol, the vRNP complexes use the host cell machinery and

trafficking pathways to reach the nucleus. The primary mechanism involves an interaction of the exposed nuclear localization sequence (NLS) on the viral NP with importin-a, which is a component of the nuclear import machinery [30-32]. Subsequently, importin-a recruits importin-b and together these two proteins deliver the vRNP cargo to the nuclear pore complex (NPC) where it is transported into the nucleoplasm [33-35].

Transcription of the viral mRNA initiates almost immediately upon entry

into the nucleus, as the vRNP-associated polymerases can use the bound vRNA as a template and is aided by an association with the host cell RNA Polymerase II [36, 37]. The primers obtain using a mechanism called cap-

5

snatching, which involves the association of the PB2 cap-binding domain with the 5’ host mRNA cap followed by a cleavage of the mRNA by the PA endo-nuclease domain and the repositioning of the mRNA fragment into the cata-lytic center of PB1 where it pairs with 3’ end of the vRNA [37-40]. After elongation, a polyadenylated tail is carried out at the 3’ end of mRNA via reiterative stuttering process [41]. The polyadenylated mRNA is subsequently exported from the nucleus for translation [42]. Different from other segments, the M and NS mRNA contain splicing sites which can recruit the cellular spliceosome resulting in the production of two distinct mRNAs [43-45].

Viral protein translation also depends on the host cell machinery. The viral

envelope proteins (HA, NA and M2) are translated and folded on ER mem-brane-bound ribosomes and traffic through cellular secretory pathway to the plasma membrane for progeny virus assembling. The soluble proteins (PB2, PB1, PA, NP, M1, NS1 and NS2) are synthesized by cytosolic ribosomes and many are imported back into the nucleus for different purposes [15]. PB2, PB1, PA and NP are used for additional transcription and progeny vRNP for-mation [46, 47]. NS1 helps to inhibit interferon activation and also may en-gage in viral mRNA nuclear export [48]. M1 and NS2 have been speculated to facilitate the nuclear export of the progeny vRNPs by recruiting the cellular chromosomal maintenance 1 (CRM1) dependent nuclear export pathway [49, 50].

The progeny vRNA replication is a primer-independent process where the

viral polymerase uses the negative-sense vRNA as a template to transcribe the complementary positive-sense RNA (cRNA) [51-53]. The resulting cRNA acts as a template for progeny vRNA transcription [51, 54]. The progeny vRNA then associate with the newly synthesized NP molecules and a single copy of the viral polymerase complex to assemble into the progeny vRNP which is used either for further replication, or is exported from the nucleus to the plasma membrane for packaging into the progeny virus [46, 47, 51].

Once the vRNP reaches the plasma membrane, the it is thought to be pack-

aged into the virion through an interaction with M1 molecules that are associ-ated with HA and NA that are located at the viral budding site [55, 56]. The resulting progeny virion only contains a single copy of each of the eight gene segments and this may be attributed to the unique packaging signals on each segment [57, 58].

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Despite the different locations in synthesis, all the viral components need to be delivered to the host cell membrane to assemble into an infectious virion. Progeny virus assembly and budding is one of the least defined aspects of the IAV life cycle, but it has been indicated to occur at lipid raft domains on the apical membrane region of an infected cell, which are enriched in cholesterol and sphingolipids [59, 60]. The transmembrane domains of HA and NA in-trinsically interact with lipid rafts and M1 may be recruited to this site by as-sociating with the cytoplasmic tails of HA and NA [61]. However, the recruit-ment of M2 is not clear and has been proposed to involve an association with M1 and HA [62, 63]. Clustering of HA and NA may contribute to bud for-mation by altering the curvature of the lipid raft domain and M1 may addi-tionally contribute by functioning like a membrane-bending protein on the in-ner leaflet of the bilayer [64, 65]. During bud formation M2 has been shown to accumulate at the neck of the budding site where it is proposed to facilitate the membrane scission process [66, 67].

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IAV Neuraminidase

Influenza NA is a sialidase that catalyzes the hydrolysis of terminal sialic

acid residues found on various glycoconjugates. Sialidases are also commonly found in animals or microorganisms where they are involved in many regula-tory processes by modifying the surface sialic acid profiles on cells [68]. In animals, sialidases can regulate the immune system, the half-lives of circulat-ing cells and apoptosis [69-71]. In some bacterium, sialidases act as virulence factors that aid in pathogenesis and/or nutrient acquisition [72, 73]. Therefore, pathogenesis related sialidases are considered to be viable anti-bacterial or anti-viral drug targets [73, 74].

Structural studies imply that the catalytic domain of neuraminidases form

a similar 6-bladed b-propeller fold [75-78]. Currently, three different classes of sialidases have been categized based on the substrate specificity, for exam-ple a2-3-linked sialic acid versus a2-6-linked sialic acid, and the catalytic mechanisms [79]. Hydrolytic sialidases have better tolerance for differences in sialic acid linkage [80]. Trans-sialidases prefer a2-3-linked substrates and the hydrolytic activity is slower than the trans-sialylation activity resulting in the transfer of the cleaved sialic acid from one glycoconjugate to another [81]. Intramolecular trans-sialidases have a stringent linkage specificity, as they have been shown to cleave a2-3-linked sialic aid and release 2,7-anhydro- Neu5Ac [82, 83].

The NA from IAVs is a hydrolytic sialidase that displays a receptor-de-

stroying activity which opposes the function of the sialic acid binding protein HA [22, 74, 84, 85]. Phylogenetic analysis of NA sequences from different subtypes (N1-N9) suggest that NAs can be divided into two distinct groups: N1, N4, N5 and N8 in Group 1 and N2, N3, N6, N7 and N9 in Group 2 [Figure 3A] [86, 87]. Group 1 is structurally differentiated from group 2 by an addi-tional cavity near the catalytic site that is created by the movement of the ‘150 loop’ (residues 147-152) [Figure 3B] [87]. The ‘150 loop’ forms a corner of

8

the catalytic site and Russell, R.J., et al. showed that the cavity on N1, N4 and N8 can take two conformations based on the binding and release of the sialic acid substrate [87]. Recent studies on NA from a H1N1 2009 pandeimc like IAV suggest that the cavity on these NAs only have one conformation and this may also exist in avian N2s and earlier human N2s [88, 89]. In support of the ‘150 loop’ contributing to catalysis, Rudrawar et al. found that NA activity can be inhibited by locking of the ‘150 loop’ into a certain conformation [90].

Figure 3. IAV NA groups and the ‘150 loop’ cavity. A. Phylogenetic tree showing the nine NA subtypes from IAVs into two distinct groups: Group 1 (N1, N4, N5 and N8) and Group 2 (N2, N3, N6, N7 and N9). B. Comparison of the NA catalytic cavities from the two distinct groups. Left panel is N1 from H1N1 (A/Brevig Mission/1/1918; Blue; PDB ID: 3BEQ) [91]. Right panel is N2 from H2N2 (A/Tokyo/3/67; Red; PDB ID: 1NN2) [92]. The 150-cavity is pointed by white arrow. Images were generated using PyMOL. The illustrations are modified from Russell, R.J. et al. Nature 2006 [87] and Li, Q. et al. Nat Struct Mol Biol. 2010 [88].

Structure

Unlike bacterial sialidases which can function in varied oligomeric states, NAs from IAVs strictly function as a homotetramer [Figure 4A] [76, 92-97]. Each NA tetramer forms a mushroom like shape and is comprised of four do-mains: a globular enzymatic head domain, a stalk region with varied length, a transmembrane domain (TMD) and a short cytosolic N-terminal domain [Fig-ure 4A] [98].

N1 N2

N4

N5

N8

N3

N7

N9

N6

Group 1 Group 2

A BN1 N2

150-cavity

9

The 6-bladed propeller structure is located in the enzymatic head domain of each monomer where each blade contains four antiparallel b-sheets that are connected by loops and stabilized by intermolecular disulfide bonds [78, 99]. The catalytic site is located in the center of each in each NA monomer and is tilted towards the side of the mushroom, which may facilitate substrate acqui-sition [10, 100, 101]. The deep active site cavity is lined by eight highly con-served charged and polar amino acid residues which interact with the sialic acid substrate [Figure 4B] [102]. The cavity is further stabilized by six outer conserved residues, which form a “second shell” [91, 100, 101].

A second sialic acid binding site in the head domain has been identified on

N6 and N9. It has also recently been shown to exist on 2009 pandemic-like N1s where it contributes to cleaving sialic acid from complex N-linked gly-cans [103-106]. This site also commonly appears in avian NAs subtypes and is constructed by three surface loops [107, 108]. Recent observations indicate that the second binding site contributes to NA catalytic efficiency and cross-species transmission, as this site increase the valency of NA, which ultimately increases the affinity for the sialic acid substrate [106, 109, 110]. The ‘150 loop’ is also an important feature on the head domain of some NA subtypes and has been discussed above.

Each NA tetramer has also been shown to bind up to five calcium ions de-

pending on the strain and subtype. One calcium ion in each monomer and is located close to the catalytic site, whereas another one is positioned at the cen-ter of the fourfold axis of symmetry [Figure 4B] [78, 87, 91, 111]. In different NAs these calcium binding sites have been shown to be essential for enzyme activity, thermostability and to promote NA adaptation and diversification [111-113]. In early N1 and N4 structures an additionally putative calcium-binding site has been observed in each monomer, but the contributions of this site has not been examined [Figure 4B] [91].

NA co-translationally receives N-linked glycosylation modifications [114].

The N-linked glycans are discretely distributed at the top, lateral and bottom side of head domain and the amount vary among and within different species and subtypes [115, 116]. N-linked glycans generally provide benefits for pro-tein folding by recruiting the chaperones to reduce the folding energy barrier and contribute to sorting the protein to the appropriate destination [117-119]. In addition, N-linked glycans can also alter the antibody binding to an antigen

10

by masking the epitopes, consequently promoting immune evasion for the vi-rus [120, 121]. Removal of the glycosylation sites from the A/WSN/33 IAV increased the viral neurovirulence in mice model [122]. However, the function and influence of the N-linked glycans on NA still lack a comprehensive inves-tigation.

The NA stalk region generally contains approximate 50 amino acids with

length variation which can be created by deletions of coding regions corre-sponding to up to18 amino acids [123]. The length variation of the stalk can further impact virus biological properties, for instance the shorter stalk re-duces the efficiency of the viral elution from the chicken erythrocytes without changing NA activity, and an elongated stalk can lower the viral titer [123-125]. These suggest alterations of the NA stalk length may hinder the acces-sibility of NA to the sialic acid receptor and break the HA and NA spatial balance [126]. However, the clustering of NA on the virion suggests that al-tering the stalk region can impact the substrate affinity or enzymatic activity [9, 127-129]. The NA stalk region also contains at least one cysteine residue that contributes to NA dimerization by forming an intermolecular disulfide bond with a neighboring NA monomer assisting for the further tetramerization [Figure 4A] [130, 131]. The glycosylation sites in the stalk region have also been shown to change over the years, but these functional impact of these changes has also not been thoroughly investigated [116]. Virulence studies on the NA stalk region have also shown that it contributes to the virulence, path-ogenicity and transmission of influenza viruses [132-136].

The N-terminal transmembrane domain (TMD) anchors NA to the viral en-

velope [137, 138]. Within and between subtypes the TMD shows variability in amino acid sequence, but in general is predicted to form an a-helix [130, 139]. Beside the anchor function, the TMD has also been suggested to associ-ate with lipid rafts, directing NA to the site of viral budding [140, 141], and substitutions within TMD have been shown impact NA activity and reduce viral growth [142]. More recent work has shown that the NA TMDs from hu-man H1N1 IAVs have become more polar over the last 100 years and that these polar residues interact between adjacent monomers to aid in NA tetram-erization [143, 144]. Further studies demonstrated that not all TMD and head domains are compatible, supporting previous observation showing that NA assembly is a concerted process between the enzymatic head domain and the TMD [145]. Following the TMD is the cytoplasmic tail, which contain six

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highly conserved amino acids ‘MNPNQK’ in all NA subtypes [130]. The cy-toplasmic tail has been proposed to associate with M1 to facilitate viral as-sembly [61, 137, 146], as viruses containing NA without the tail show altered morphology and reduced budding efficiency [61, 147-149].

Figure 4. Structure of NA and the enzymatic head domain. A. Diagrams of the NA homotetramers from H1N1 depicting the enzymatic head domain, the disulfide bond between cysteine residues (red) in the stalk region and the TMD. B. The top view of NA enzymatic head domains from H1N1 consisting of 6-bladed propeller structure and each blade contains four anti-parallel β-sheets connected by loops and stabilized by intermolecular disulfide bonds. The substrate (zanamivir, red stick) located in the NA catalytic site (right magnification) and the nine Ca2+ ions (black spheres) are dis-played in the respective binding sites. Images were generated using PyMOL (PDB ID: 3B7E) [91].

Synthesis and Maturation Influenza NA is a type II membrane protein that utilizes the secretory path-

way of the host cell for synthesis, maturation and transport to the plasma mem-brane where progeny virion assembly occurs.

During synthesis NA is co-translationally targeted to the endoplasmic re-

ticulum (ER) by the signal recognition particle (SRP) [138]. SRP in the GTP-bound state recognizes the N-terminal TMD of NA once it emerges from the ribosomal exit tunnel and directs the ribosome-nascent chain complex to the GTP-bound SRP receptor that is located next to the Sec61 protein-conducting channel (translocon) in the ER membrane [Figure 5(i)] [150-152]. The asso-ciation of SRP with the SRP receptor causes GTP hydrolysis which results in

A B

N-terminal

Enzymatic head domain

Stalk domain

Transmembrane domain (TMD) TM

DTM

D

TMD

TMD

Cytoplasm

Extracellular

Top view

12

the transfer of the ribosome-nascent chain complex to the Sec61 translocon [Figure 5(ii)] [153].

Figure 5. NA synthesis process. (i) The GTP-bound signal recognition particle (SRP) recognizes the newly synthesized N-terminal TMD of NA. (ii) The SRP-ribosome-nas-cent chain complex is guided to the ER membrane and associates with the GTP-bound SRP receptor (SR) next to the translocon. (iii) The GTPs are hydrolyzed resulting in SRP release from the ribosome and translation is resumed at the translocon. The il-lustration is modified from Dou, D., et al. Mol Biol Cell, 2014 [154] and Dou D., et al. Front Immunol. 2018 [15].

Once NA synthesis resumes at the ER membrane, the N-terminus inverts

and the TMD passes through the lateral gate of the Sec61 translocon and inte-grates into the ER membrane while the C-terminal portion of NA is translo-cated into the ER lumen [155]. This ER membrane integration of NA TMD is a complexed process, TMD stalling in the translocon may accommodate for the TMD to orientate and properly interact with the lateral gate [156]. The opening of the lateral gate is triggered by the hydrophobic property of the TMD and the loss of hydrophobicity that is displayed by the NA TMD from H1N1 viruses is predicted to significantly hinder the insertion process [144, 154, 157-159]. However, NA likely has evolved to compensate for the lack of insertion efficiency by another mechanism that includes the sequence compo-sition that is downstream from the TMD [160] .

(ii)

Cell

ER lumen

A(n)TMD

SRP(i)GTP

SR

TransloconN

N

C

(iii)

NA mRNARibosome

Cytoplasm

13

Upon entry into the ER lumen, the NA stalk and the enzymatic head domain both receive multiple N-linked glycans. The N-linked glycans are transferred to the Asn residues of the glycosylation consensus sequence Asn–X–Ser/Thr by the oligosaccharyltransferase (OST) in the ER lumen [161]. Following ad-dition, the core glycan structures are immediately trimmed by glucosidases I and II. This trimming creates monoglucosylated glycans that are substrates for the lectin chaperones calnexin and calreticulin and the associated protein di-sulfide isomerase ERp57, which are part of the ER quality control machinery in the cell. Previous work has shown that NA is bound by the lectin chaperones in the ER and that the association with the head N-linked glycans promotes maturation by preventing aggregation [114, 162-164]. Another ER chaperone, GRP78-BiP, can also transiently associate with NA in the early stage of the synthesis till the NA oligomerization begin [165, 166].

Oligomerization of NA starts with co-translational dimerization that in-

volves the formation of an intermolecular disulfide bond between the cysteine residues in the stalk region [114]. Surprisingly, this process occurs before the synthesis of the second monomer is completed, indicating that the second copy of NA may template on the first copy. Following one additional conforma-tional change, the dimers then post-translationally assemble into the enzymat-ically active tetrameric structure [114, 167]. Previous studies found that NA TMD and the enzymatic head domain can oligomerize independently and that these two regions cooperate during maturation to increase the folding effi-ciency of NA [143]. The main driving force for the TMD oligomerization was shown to be polar residues that are positioned to one side of the TMD in order to facilitate interactions with a TMD from another copy of NA [144]. The folding link between these two distal regions partially explains the observed hydrophobicity changes in the NA TMDs from H1N1 IAVs [145].

Oligomerization is essential for NA’s sialidase function, even though each

individual subunit contains the active site structural information [95, 100, 168]. This restriction has been observed for decades, but still lacks a clear explanation. A unique property of influenza NA is the central calcium binding site, which is formed by residues from each monomer and NA has been shown to be a Ca2+-dependent enzyme. These observations suggest that the NA te-trameric dependent activity could be related to the requirement of Ca2+ ion in the central pocket. However, the central Ca2+ ion is not been seen in the crystal structures of all NAs, indicating that the relationship of the central calcium

14

binding site and NA tetrameric depending activity may not be so straightfor-ward [87, 169].

Functions

The primary function of influenza NA is to facilitate viral mobility by removing the sialic acid receptors that are necessary for HA binding [170]. How NA catalyzes the hydrolysis of the glycosidic bond that attaches the sialic acid residue has not been fully determined and the mechanistic insight has primarily been provided from structural studies [101, 171]. The catalytic pro-cess begins with substrate binding via interactions between the sialyl group and the conserved functional residues in the NA active site, which include ionic bonds between the carboxyl groups on the sialic acid and the three pos-itively charged arginine residues in the NA active site [101]. Upon binding, the chair conformation of sialic acid is changed to a boat, creating a positively charged oxocarbenium ion at the C2 atom of the sialic acid [Figure 6(i)] [172, 173]. This conformation change in sialic acid is considered a key step in the reaction as it creates a transition state that is susceptible to nucleophilic attack by the nearby deprotonated hydroxyl group of Tyr 402 (N1 numbering). Fol-lowing the nucleophilic attack, the glycosidic bond attaching the sialic acid residue is broken and the underlying sugar (aglycone) molecule leaves the NA active site and is protonated by the solvent [Figure 6(ii)] [171]. The sialic acid residue bound to Tyr 402 is then released following a nucleophilic attack on the C2 atom by a solvent hydroxide ion [Figure 6(iii)], resulting in the hydrox-ylation of the sialic acid residue and the recharging of Tyr 402 [Figure 6(iv)] [102, 171, 174].

15

Figure 6. The catalytic mechanism of NA and substrate reaction. (i) Substrate bind-ing into the NA activity site causes conformational change of sialic acid from chair to boat and creates a positively charged oxocarbenium ion (red) at the C2 atom. (ii) The deprotonated hydroxyl group of Tyr 402 (N1 numbering) performs a nucleophilic at-tack (blue arrow) on the C2 atom and breaks the glycosidic bond attaching the sialic acid residue. The underlying sugar is protonated by the solvent (red). (iii) The hy-droxide ion (red) from the solvent then perform a nucleophilic attack (blue arrow) on the C2 atom and (iv) Tyr 402 is recharged. Consequently, the sialic acid residue is hydroxylated (red) and released. The illustration is modified from Shtyrya, et al. Acta Naturae, 2009 [102].

During the infection process, influenza viruses are exposed to sialylated

glycoconjugates in the mucin, at the cell surface, and even on the NA and HA antigens of neighboring virions [84, 175, 176]. As a result of the ubiquitous nature of these critical residues, the sialidase function of NA has been linked to several functions that promote the infection process [Figure 7]. One of the main roles of NA is to help the virus reach the respiratory epithelium by re-moving the sialic acid residues from the dense population of glycoproteins (mucins) in the mucus layer that can act as decoy receptors for HA [Figure 7B] [177, 178]. NA also facilitates the release of the viral progeny by remov-ing the local sialic acid receptors from the infected cell surface during the bud-ding process [Figure 7C] [179, 180], and prevents binding between viruses by

OO O-

OHHN

O

OHHOHO

2

34

5

6 OR

Tyr 402

OH

O O

O-HN

O

OHHO

HO

Tyr 402

O-

2345

6

OR

Substrate transition state Enzyme - substrate intermediate

HOR

O O

O-HN

O

OHHO

HO

Tyr 402

O

O

OHH

O O

O-HN

OHHO

HO

Tyr 402

O

Sialic acid release

OO O-

OHHN

O

OHHOHO

2

34

5

6 OH

Tyr 402

OH

Substrate binding

Enzyme recharge

+(i) (ii)

(iii)

(vi)

16

removing the sialic acid residues from the N-linked glycans on the viral gly-coproteins HA and NA [Figure 7D] [176].

Recent studies have shown evidence that NA can also aid in entry as well

by removing local sialic acid residues on the cell surface, enabling the virus to more efficiently scan for the proper receptor [Figure 7E] [23, 181]. In strains possessing inefficient NAs, the inactive catalytic site has been proposed to aid more directly in viral internalization by acting as a receptor binding protein [182-184]. However, it is unclear if this function occurs outside of the in vitro environments that have been examined. Although not all functions that have been associated with NA are well established, the many that have been are crucial for efficient IAV replication and likely contribute to transmission [185].

Figure 7. NA functions during infection. A. Diagram of the sialylated glycoconju-gate receptor and the opposing functions of HA and NA. B. To reach the target in-fected cell, IAVs need penetrate the protective mucus layer containing a high density of mucin decoy receptors. NA facilitates the penetration of IAVs by removing the de-coy receptors for HA. C. NA promotes progeny virus releasing by removing the local sialic acid receptors during the viral budding process. D. NA prevents HA-mediated

Cellendocytosis

Viral movement on cell surface

IAV 1

NA

IAV 1

IAV 2IAV 2

Mucin secreted cell

Mucin

Ciliated epithelial cell

Infected cell

GlcNAc

Mannose

GalactoseSialic acid

Sialylated glycoconjugate Infected cell

HA bind receptor

Infected cell

NA cleave receptor

Mucin

NAInfected cell

Progeny virion Release progeny virion

Infected cell

IAV

NA

A

C

ED

B

17

viral aggregation by cleaving off the sialic acid from the N-linked glycans of HA and NA. E. NA and HA create viral movement by removing and binding the located sialic acid receptors on the cell surface resulting in efficiently scanning for the proper re-ceptor. The illustration is modified from Dou D., et al. Front Immunol. 2018 [15].

NA as antiviral target and a vaccine antigen NA has a rigid and conserved active site that is important for a productive

influenza infection. These properties formed the premise for the development of several competitive inhibitors that target the NA active site. The three ma-jor NA inhibitors (NAIs) that have been globally licensed for the treatment and prevention of influenza infection are zanamivir (Relenza), oseltamivir (Tamiflu), and peramivir (Rapivab). An additional long-acting NAI, la-ninamivir (Inavir), has also been approved for influenza treatment in Japan [186]. All of the current NAIs are designed based on modifying the structure of 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA) which is a transi-tion state analogue of sialic acid. For improving the binding affinity of zanamivir, the hydroxyl on the C4 of DANA was substituted with a 4-guani-dino group and oseltamivir was designed by using a cyclohexene ring with two additional substitutions, an amino group on C4 and a hydrophobic pentyl ether side chain [Figure 8] [187-192]. Although, zanamivir and oseltamivir are both effective NA inhibitors, resistant influenza strains were isolated shortly after the drugs were introduced, likely due to the high mutation rate of the influenza virus polymerase and the plasticity in NA [104, 186].

Figure 8. Chemical structures of sialic acid and NA inhibitors. DANA is a transition state analogue of sialic acid bound to NA. Zanamivir and Oseltamivir are designed base on the principle of DANA with modification to improve the binding affinity. The red highlights the substitution in DANA that differ from sialic acid and the blue high-lights the substitutions in zanamivir and oseltamivir that differ from DANA. The illus-tration is modified from the Jennifer L. et al. Influenza Other Respir Viruses, 2013 [186].

OO

O

NH2

NH

O

OseltamivirZanamivir

OH

O

HNO

O

NH2

HN

HO

HO

OH

H

HN

OH

O

HOHN

O

O

HO

HO

OH

HOH

Sialic acid

OH

O

HOHN

O

O

HO

HO

OH

H

DANA

18

In addition to the competitive inhibitors, NA antibodies have received more and more consideration as an alternative or compensatory option for prevent-ing and treating influenza infections [193]. This is not a new idea as NA spe-cific antibodies have been shown to provide protection against influenza in-fections in humans several decades ago [194-196]. However, recent studies have begun to provide further evidence between the correlation of anti-NA antibodies and protection during influenza viruses challenge, which is inde-pendent of the more prevalent HA-based immunity [197-199]. Moreover, NA specific antibodies displayed the ability to cross-react with different NAs in-dicating that NA may act as a broadly protective antigen against virus infec-tion [200-203].

Influenza infected cells are generally considered to be cleared through an-

tibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent phag-ocytosis (ADPC), which have been broadly studied using anti-HA antibodies [204-208]. ADCC is mediated by NK cells and neutrophils, whereas ADPC is mediated by macrophages. Monoclonal antibodies (MAbs) against NA have also been shown to cause an ADCC effect in vitro and in a protective virus challenge in mouse models [209], and NA is known to contain a number of CD4+ and CD8+ T-cells epitopes [210, 211]. However, the mechanism of protection provided by anti-NA antibodies requires further investigations.

The currently established mechanism of action for NA antibodies is the

ability to neutralize the infection by preventing virus budding and egress from the infected cell. The neutralizing effects of NA antibodies is generally attrib-ute to the inhibition of enzymatic activity which is achieved by blocking the active site or by sterically hindering the access of large branched glycoconju-gates [103, 212-214]. Recent studies have also indicated that some neutraliz-ing MAbs that protect against lethal virus infection also recognize epitopes on the lateral surface of NA [215, 216]. Although it is equally plausible that these antibodies could also trap virions in the mucus, inhibiting entry, or promoting virus-virus binding, these aspects have not been thoroughly examined [217].

Similar to the competitive inhibitors, viral strains can also acquire re-

sistance against therapeutic NA antibodies. Often this is achieved by surface point mutations or by the addition of N-linked glycans. For N2, Wan et al showed that a recently introduced N-linked glycosylation site at residue 245

19

(N2 numbering) significantly inhibited the ability of neutralizing MAbs to bind [120, 201]. However, other studies have shown that the cross-reactivity of MAbs against N1 is likely due to the presence of conserved epitopes [120, 201]. Currently, it remains difficult to discern how many NA antibodies pro-vide protection, due to the technical problems associated with crystalizing NA-antibody Fab complexes, but this problem is likely to be overcome by combining previous studies with technology advancements. A good example of this was shown by Zhu et al. where the authors systematically defined N9 antibody epitopes using cryo-electron microscopy of a recombinant N9 that was stabilized by a single substitution in the central-calcium binding site [113, 218].

Vaccination is recommended by WHO as the optimums method for pre-

vention and control of influenza infection [219]. Three classes of seasonal vaccines have been licensed currently, which are inactivated, live attenuated, and recombinant HA vaccine, and all licensed vaccines are standardized based on the content of HA [219, 220]. However, the effectiveness and performance of current vaccines is unsatisfactory and new vaccine formulations or strate-gies are desired [221-223]. Despite the therapeutic benefit of NA antibodies mentioned above, the low abundancy on the viral envelope and its lability re-sult in inconsistent quantities and qualities of NA in current vaccines [194, 198, 202, 224, 225]. The benefits provided by NA antigens strongly suggest that a comprehensive and systematic investigation of NA’s fundamental prop-erties to effectively integrate and standardize NA in IAV vaccines.

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21

Results summary

Paper I. Type II Transmembrane Domain Hydrophobicity Dictates the Co-translational Dependence for Inversion.

Hydrophobicity is an essential characteristic of single-spanning membrane proteins that insert into the ER membrane via the Sec61 translocon during synthesis. Influenza viruses contain three single-spanning membrane proteins, HA, NA and M2. Predictive analysis shows that the hydrophobicity rule strictly applies to the TMDs of the type I membrane proteins (Nout-Cin) HA and M2, but not to the TMDs from the type II membrane protein (Nin-Cout) NA. The marginal hydrophobicity of the NAs TMDs from human H1N1 IAVs, was shown to be sufficient for targeting the ribosome-nascent NA chain complex to the Sec61 translocon. However, the correct orientation of NA was only achieved when a substantial portion of the C-terminus was present, indi-cating that the elongation of the C-terminus facilitates the inversion and inte-gration of the marginally hydrophobic NA TMDs. We verified the contribu-tion of the co-translational synthesis to inversion by demonstrating that adding residues after another TMD can also facilitate its inversion. Furthermore, we performed a predictive analysis of human type II single-spanning membrane proteins with marginally hydrophobic TMDs and observed a clear preference for these proteins possessing a long C-terminus. Together our results show that in addition to flanking positive charge residues, the position of the TMD with respect to the C-terminus can also influence its topology.

Paper II. The Influenza Virus Neuraminidase Protein Transmembrane and Head Domains Have Coevolved.

Influenza NAs require tetramerization to be enzymatically active. Our pre-

vious studies showed that the interaction between NA TMDs contribute to the proper folding of the enzymatic head domain and that this interaction is driven by polar residues within the TMD [143, 144]. We also demonstrated that the hydrophobicity of the NA TMD in human H1N1 IAVs has been decreasing

22

over the years, resulting in a stronger TMD association [144], indicating that the TMD is under some selection pressure. Unlike the head domain that is exposed to different environments, the TMD resides in the viral envelope, which is a relatively stable environment. Therefore, we hypothesized that the NA TMDs and head domains communicate with each other during the folding process. To investigate this question, we generated two viruses, WSN wild type (WT) virus and recombinant WSN virus that contained a chimeric NA where the TMD from WSN was replaced with a recent more polar TMD. A comparison of the two viruses revealed a decrease in the folding efficiency of NA and a temperature dependent growth defect for the virus containing the NA chimera with the more recent TMD. Rescue mutations were identified following the passaging of the virus at 37˚C and all the substitutions were in the TMD. Analysis of the mutant TMDs showed that the substitutions de-creased the association to a level that matched the original TMD, demonstrat-ing that the NA TMDs and head domains have coevolved to maintain compat-ibility during the assembly process.

Paper III. Structural restrictions for influenza neuraminidase activity promote adaptation and diversification.

Influenza NAs display diverse properties among and within the different subtypes. For instance, changes are commonly observed in the number of gly-cosylation sites, the substrate specificity, substrate binding affinity, catalytic activity and stability. However, the mechanisms that allow NA to accommo-date so many variations remain unclear. In this study we investigated why NAs possess distinct differences in stability, a property that previously has been shown to be influenced by the presence of Ca2+ [112, 226]. Initially, we confirmed that the stability varied within the N1 subtype and that it is impacted by intrinsic properties as well as the environmental Ca2+ concentration. Through a mutational analysis we traced the calcium stability dependence to the central Ca2+-binding site, which is assembled from a portion of each of the four NA monomers. Substitutions that altered the central Ca2+-binding affinity also caused a decrease in substrate binding, tetramer dissociation, and im-pacted viral growth. By analyzing N1 sequences from circulating strains, we identified substitutions surrounding the central Ca2+-binding site, which varied NA stability and the calcium binding affinity, indicating the importance of this calcium binding site in nature. Complementation experiments demonstrated the requirement of the central-calcium for NA activity and the plasticity in the

23

NA structure, suggesting that the oligomerization dependence may also func-tion as a mechanism for NA to rescue deficient mutants during error prone transcription. Together these findings illustrate how NA utilizes its structural restrictions for activity to increase mutational tolerance and to promote its ad-aptation and diversification. Paper IV. Conserved N-linked glycans on the influenza NA head domain con-tribute to viral incorporation but are not essential for H1N1 replication.

Influenza NAs possess a variable number of N-linked glycosylation sites on the head domains. However, it is not known if the N-linked glycan sites aid in NA maturation or primarily function to mask antigenic determinants. Se-quence-based analysis shows there are three well-conserved glycosylation sites on N1 head domain and one additional site on N2. Variable sites were primarily found on the NA head domain from human H1N1 and H3N2 IAVs. Experimental analysis showed that the removal of the conserved N-linked gly-can sites decreased IAV incorporation and replication efficiency in cells and eggs for N1. Interestingly, the variable sites dramatically influenced the ther-mostability of N1. Thus, in addition to masking antigenic epitopes, the N-linked glycan sites on the NA head domain can also affect the enzymatic prop-erties and the incorporation of NA into virions.

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25

Conclusions and future perspectives

Many efforts have been made to investigate the influenza antigens from structural, functional, immunological and evolutionary aspects. However, compared to the HA, NA has been neglected despite its contributions to mul-tiple steps during the infection process. One of the likely reasons for the ex-clusion is the discovery of NA inhibitors. The works presented here focused on understanding the biological features of NA, which involve NA synthesis and maturation, the relationship between the different domains, the variability in its enzymatic properties and the N-linked glycosylation sites [Figure 9].

Paper I demonstrated an alternative mechanism for ER membrane integra-

tion of NA with marginally hydrophobic TMDs. Our results show that the co-translational process and the length of the C-terminus are both critical for the proper insertion and inversion of the NA TMD into the ER membrane. The co-translational process extends the time for the hydrophobic side of the am-phipathic a-helix to interact with the lateral gate of translocon while the con-tinuous translation can potentially provide a driving force for the inversion process prior to the integration into the ER membrane. This mechanism pro-vides an advantage for NA TMDs as it alleviates the strict hydrophobicity re-quirements for insertion, which allows the TMDs more sequence space for evolving, as evidence by the recent trend towards becoming more marginal hydrophobic. Previously we showed that the incorporation of polar residues in the TMD contributes to oligomerization and NA folding [143, 144]. These findings indicate that the TMDs possess the ability to adapt by altering the hydrophobic property and that this is mainly possible due to the positioning within the protein. This type of approach could be applied to improve NA expression in different environmental conditions without alternating the enzy-matic domains. However, the increasing TMD interactions indicate this region is possibly subject to selective pressure by the distal enzymatic head domain.

Paper II investigated the hypothesis discussed above and revealed a com-

patibility requirement between the NA TMDs and the enzymatic head do-mains in N1. The compatibility mismatch profoundly affects the biological

26

function of NA by decreasing the folding efficiency. The defective function of NA accordingly influences the viral budding and replication processes. In line with this premise, the rescue mutations that appeared during the viral rep-lication process were located in the TMD region and restore the compatibility with the head domain. These results further indicate that the NA TMD can compensate for suboptimal mutations in other domains to maintain the com-patibility that is necessary for efficient NA folding. This study introduces that a relationship exists between the different domains in NA and emphasizes the importance of them working together. This presumably places some limita-tions on the diversity and evolution of NAs.

NA also only functions as a tetramer, a unique property among sialidases

that has been observed decades ago. In Paper III we asked if this unique oli-gomeric requirement contributes or limits NA diversification. Tetramerization of NA results in the formation of a cavity at the axis of symmetry, which is created by residues from each monomer. Recent structural studies resolved a Ca2+ ion in the cavity and defined it as the central Ca2+-binding site [91, 101, 227]. Ca2+ ions have previously been linked to NA stability, but the Ca2+-bind-ing sites involved had not been defined yet [112, 226]. Paper III specifically pointed out that the variation of NA stability primarily depends on the struc-ture around the Ca2+-binding site and the Ca2+ ion binding affinity. Experi-mental analyses showed that in addition to stability, the central Ca2+-binding site and the Ca2+ ion are involved in multiple fundamental properties of NA, catalysis and oligomerization efficiency, and that these are reversible by re-moving and re-adding calcium. These observations give a rational explanation for the tetrameric dependent activity of NA, as it requires the presence of the central Ca2+ ion.

Viruses containing NAs with variable central Ca2+-binding affinities also

displayed extracellular Ca2+ level dependent release and replication, especially when decoy receptors were present in the environment. Bioinformatic analysis of NAs from pandemic 2009 H1N1 viruses showed the temporal cycling of N1 stability involves substitutions around the central Ca2+-binding site in na-ture. These indicate that IAVs may utilize the central Ca2+-binding site of NA to regulate and optimize the functional balance between NA and HA.

In addition to adaptation, the Ca2+-dependent tetramer requirement also of-

fers NA more structural plasticity as it allows for heterotetrameric formation. This property enhances the tolerance of NA for suboptimal mutations that

27

arise from the error-prone transcriptional process as complementation can mask deleterious mutations expanding the breadth of potential viral qua-sispecies, leading to greater property diversification. This study highlights a novel mechanism of NA adaptive regulation and defines a potential target for disrupting NA enzymatic activity. It also provides new approaches for rescu-ing labile NA expression and introduces new considerations for how NA is evolving.

NAs also display variable numbers of N-linked glycosylation sites on the

enzymatic head domains among and within the different subtypes. Paper IV shows that the highly conserved N-linked glycosylation sites are required for the proper folding of NA which is necessary to have efficient NA virion in-corporation and viral replication in cells and eggs. Two N-linked glycosylation sites (Asn146 and Asn434, N1 numbering) demonstrated an influence on N1 stability and this property may be attributed to the positioning being close to the central Ca2+-binding site. This study provides original insight into the N-linked glycosylation sites on NA and how they can influence enzymatic prop-erties and the NA-HA ratio in the virion.

In all, these studies found a series of new regulatory mechanisms of the NA

from IAVs and contribute to the fundamental understanding of NA synthesis at the ER, the cooperativity between the different NA domains, regulation of NA enzymatic properties and adaptation. These valuable insights should help to improve IAV vaccine development by potentially increasing the quality and quantity of NA in the viral particles from which many vaccines are derived.

Furthermore, these studies open up several new questions for future inves-

tigations on NAs. For instance, the NA stalk directly feeds into the base of each head domain around the axis of symmetry and it would be interesting to know whether the stalk could structurally support the assembly of the central Ca2+-binding site. The central Ca2+ ion is also not resolved in the crystalized structure from some NA subtypes and N2 displays less conservation compared to N1 around the central region. While our data indicates the central Ca2+-binding site likely exists in all N1 and N2, it is by no means exhaustive. Thus, it is possible that alternative regulatory mechanisms are implemented by other NA subtypes. It also is unclear if the requirements of the N-linked glycosyla-tion sites differ between NA subtypes and we did not examine those present on the stalk. The potential role of calcium in regulating NA also raises many questions which we did not have the time to examine such as how does the

28

central Ca2+-binding affinity affect viral replication and transmissibility in dif-ferent tissue and animal models. Undoubtedly future studies will investigate some of these questions and build on this work to create a comprehensive pic-ture of NA evolution in IAVs that is similar to the one which already exists for HA.

Figure 9. Diagram of the studies’ summary. This thesis works focus on understand-ing the biological features of NA, which involved NA synthesis (paper I); the compat-ibility requirement between the NA TMDs and the head domains (paper II); the cen-tral Ca2+-binding site and heterotetrameric formation diversified NA enzymatic prop-erties (paper III) and the N-linked glycosylation modification (paper IV). The illus-tration is modified from Dou, D., et al. Mol Biol Cell, 2014 [154] and Dou D., et al. Front Immunol. 2018 [15].

Summary

Synthesis (I) Relationship between the domains (II)

Enzymatic properties (III)Glycosylation (IV)

ER lumen

N

TMD

Head

29

Sammanfattning på svenska

Influensa A (IAV) är ett höljebärande virus som säsongsvis cirkulerar i människor vilket ger en mild till svår respiratorisk infektion. Höljet består av ett lipidmembran som omsluter genomet samt ger viruspartikeln sin form av en sfär eller ett filament. IAV innehåller åtta RNA-segment av negativ polari-tet som kodar för minst tio olika proteiner, och kan också koda för flera extra stamspecifika proteiner. Det finns tre integrala membranproteiner på virusets yta, matrixprotein 2 (M2), hemagglutinin (HA) och neuraminidas (NA). HA och NA är de främsta antigenerna och avgör infektiviteten, patogeniciteten, smittspridningsförmågan, och är huvudmålen för neutraliserande antikroppar. Det finns stora behov av vaccin mot IAV och mycket fokus har ägnats åt stu-dier på HA, vars proteinmängd nuvarande vaccin är standardiserade mot. NA däremot har negligerats på grund av dess instabilitet och vår begränsade för-ståelse av proteinet. Dock visar ackumulera forskning att NA spelar en viktig och multifunktionell roll under infektionsprocessen, och dessutom att vaccin innehållande funktionellt NA ger bättre skyddseffekt.

Denna avhandling fokuserar på att översiktligt förstå NA mognad från ett biokemiskt och virologiskt perspektiv. Upptäckterna kan summeras i fyra om-råden: I. Den ko-translationella processen och en viss längd på C-terminalen underlättar insättningen och inversionen av NA marginellt hydrofobiska trans-membrana domän (TMD) in till ER-membranet. II. NA TMD och avlägsna enzymatiska huvuddomän har ko-evolverat för att sammanfogas till en enzy-matisk aktiv form. III. NA centrala kalciumbindningsyta, samt kalciumjonen i sig, påverkar NA stabilitet och enzymatiska egenskaper. Tetrameriseringen erbjuder NA en strukturell plasticitet och ökad tolerans för suboptimala mu-tationer. Detta har främjat NA anpassningsförmåga samt NA diversifiering av egenskaper. IV. Det konserverade N-glykosyleringsstället i NA huvuddomän är viktigt för att NA effektivt ska inkorporeras i viruspartikeln samt för effek-tiv viral replikation.

30

Dessa resultat ger en högre förståelse för mekanismerna bakom NA syntes, oligomerisering, stabilitetsoptimering samt egenskapsdiversifiering. Tillsam-mans kan detta bidra till en tillverkningsprocess för vaccin innehållande NA.

31

Acknowledgments

First, I would like to express the gratitude to my supervisor, Robert Dan-iels, thank you for offering me an opportunity to work in your lab. You brought me to the field and trained me from a blank to who am I now. You are always helpful, patient, encouraging and motivating for my PhD study and life. Having you as my mentor is my best luck. I am really appreciating and thankless.

A special thanks to my co-supervisor, Jan-Willem de Gier, many thanks

for all the helps and suggestions you have given, especially during the difficult time of my PhD.

I would also like many thanks to Prof. Stefan Nordlund, Pia Ädelroth

and Martin Högbom for their time and efforts of organizing PhD programs and during all my PhD ‘check points’.

Sincerely thanks to Maria and Alex, you made my PhD life much warm

and convenience by taking care of all my problems. I wouldn’t focus on my research without your helps. Thank you!

Great thanks to the former and current lab members. You guys made my

PhD life much fun and amazing. Diogo (D), you were the ‘guide man’ for most of us in the lab. I am so grateful to have you as my lab teacher and good friend. You are always positive, full of energy, generous, responsibly and cover my back for no matter work or life! Johan (Big J), We were always like making fun of each other, competitive for everything and motivated to be the best as we could. I am really enjoyed. The other side, you were always helpful and generously sharing your knowledge, experiences and ideas with me. Of course, Mac man. Thank you, my good friend! Dan, 感谢你对我无论是工作

上还是生活上的无私包容和全力支持 ❤. Annika, many thanks for all the gen-erous helps and constructive suggestions for the paper. Henrik, thank you for

32

all the helps and objective opinions you were shared. Rebecca, it was nice work with you.

To the ‘lunch group’: Beata, Pedro, Cata and Chenge, thanks a lot, you

guys brought me a lot of the good moments, many laughs and helps. Zhe, Biao, Weihua, Fan, Huabin, Xin, many thanks to all the helps for

my projects. We had a lot of fun talk and great time after works. You made me homesick less.

Thanks to the previous and present lab neighbors at DBB: Claudio,

Kiavash, Patrick, Thomas, Alexandros, Regeia, Grant, Renuka, Felix, Daphne, Ane, Nir, Theresa, thank you for making the corridor such a great place.

Thanks to everyone in the DBB for creating a convenient working environ-

ment. My mother-in-law: 我今天的成就，离不开您的支持和帮助。感恩! My parents: 没有你们就没有我，感谢你们一直以来对我无私的奉献，疼爱

和教导，爱你们！

My family: 博士几年，家庭成员也增加了，你们是我的方向和前进的动

力，我会用尽一生去疼爱和保护你们！❤

33

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