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Influenza neuraminidase assembly: Evolution of domain cooperativity
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. (Robert Daniels)
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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 facilitates 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 examined 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 appropriate 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

Abstract [sv]

Influensa är en av de mest smittsamma sjukdomarna som drabbar människor 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 motsvarar ”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 lösningar på problemet med att behöva bilda en tetramer. När vi gjorde ändringar i en TMD som vanligtvis gick ihop till en tetramer, och därmed förhindrade detta, så noterade vi att huvuddomänens funktion påverkades vilket ledde till att influensaviruset hade svårt att spridas. Vidare så har våra pågående 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 influensaforskningen, utan även potentiellt för framställandet av nya influensavacciner/-mediciner.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2016. , 64 p.
Keyword [en]
Influenza, neuraminidase, assembly, transmembrane domain, evolution
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-134470ISBN: 978-91-7649-553-7ISBN: 978-91-7649-554-4OAI: oai:DiVA.org:su-134470DiVA: diva2:1033451
Public defence
2016-11-25, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.

Available from: 2016-11-01 Created: 2016-10-06 Last updated: 2016-10-25Bibliographically approved
List of papers
1. Assembly of Subtype 1 Influenza Neuraminidase Is Driven by Both the Transmembrane and Head Domains
Open this publication in new window or tab >>Assembly of Subtype 1 Influenza Neuraminidase Is Driven by Both the Transmembrane and Head Domains
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2013 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, no 1, 644-653 p.Article in journal (Refereed) Published
Abstract [en]

Neuraminidase (NA) is one of the two major influenza surface antigens and the main influenza drug target. Although NA has been well characterized and thought to function as a tetramer, the role of the transmembrane domain (TMD) in promoting proper NA assembly has not been systematically studied. Here, we demonstrate that in the absence of the TMD, NA is synthesized and transported in a predominantly inactive state. Substantial activity was rescued by progressive truncations of the stalk domain, suggesting the TMD contributes to NA maturation by tethering the stalk to the membrane. To analyze how the TMD supports NA assembly, the TMD was examined by itself. The NA TMD formed a homotetramer and efficiently trafficked to the plasma membrane, indicating the TMD and enzymatic head domain drive assembly together through matching oligomeric states. In support of this, an unrelated strong oligomeric TMD rescued almost full NA activity, whereas the weak oligomeric mutant of this TMD restored only half of wild type activity. These data illustrate that a large soluble domain can force assembly with a poorly compatible TMD; however, optimal assembly requires coordinated oligomerization between the TMD and the soluble domain.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-87689 (URN)10.1074/jbc.M112.424150 (DOI)000313197200065 ()
Note

AuthorCount:5;

Available from: 2013-02-15 Created: 2013-02-14 Last updated: 2016-10-06Bibliographically approved
2. Polar Residues and Their Positional Context Dictate the Transmembrane Domain Interactions of Influenza A Neuraminidases
Open this publication in new window or tab >>Polar Residues and Their Positional Context Dictate the Transmembrane Domain Interactions of Influenza A Neuraminidases
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2013 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, no 15, 10652-10660 p.Article in journal (Refereed) Published
Abstract [en]

Interactions that facilitate transmembrane domain (TMD) dimerization have been identified mainly using synthetic TMDs. Here, we investigated how inherent properties within natural TMDs modulate their interaction strength by exploiting the sequence variation in the nine neuraminidase subtypes (N1-N9) and the prior knowledge that a N1 TMD oligomerizes. Initially, consensus TMDs were created from the influenza A virus database, and their interaction strengths were measured in a biological membrane system. The TMD interactions increased with respect to decreasing hydrophobicity across the subtypes (N1-N9) and within the human N1 subtype where the N1 TMDs from the pandemic H1N1 strain of swine origin were found to be significantly less hydrophobic. The hydrophobicity correlation was attributed to the conserved amphipathicity within the TMDs as the interactions were abolished by mutating residues on the polar faces that are unfavorably positioned in the membrane. Similarly, local changes enhanced the interactions only when a larger polar residue existed on the appropriate face in an unfavorable membrane position. Together, the analysis of this unique natural TMD data set demonstrates how polar-mediated TMD interactions from bitopic proteins depend on which polar residues are involved and their positioning with respect to the helix and the membrane bilayer.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-90186 (URN)10.1074/jbc.M112.440230 (DOI)000317565000043 ()
Funder
Swedish Research Council
Note

AuthorCount:5;

Available from: 2013-05-28 Created: 2013-05-28 Last updated: 2016-10-06Bibliographically approved
3. The Influenza Virus Neuraminidase Protein Transmembrane and Head Domains Have Coevolved
Open this publication in new window or tab >>The Influenza Virus Neuraminidase Protein Transmembrane and Head Domains Have Coevolved
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2015 (English)In: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 89, no 2, 1094-1104 p.Article in journal (Refereed) Published
Abstract [en]

Transmembrane domains (TMDs) from single-spanning membrane proteins are commonly viewed as membrane anchors for functional domains. Influenza virus neuraminidase (NA) exemplifies this concept, as it retains enzymatic function upon proteolytic release from the membrane. However, the subtype 1 NA TMDs have become increasingly more polar in human strains since 1918, which suggests that selection pressure exists on this domain. Here, we investigated the N1 TMD-head domain relationship by exchanging a prototypical old TMD (1933) with a recent (2009), more polar TMD and an engineered hydrophobic TMD. Each exchange altered the TMD association, decreased the NA folding efficiency, and significantly reduced viral budding and replication at 37 degrees C compared to at 33 degrees C, at which NA folds more efficiently. Passaging the chimera viruses at 37 degrees C restored the NA folding efficiency, viral budding, and infectivity by selecting for NA TMD mutations that correspond with their polar or hydrophobic assembly properties. These results demonstrate that single-spanning membrane protein TMDs can influence distal domain folding, as well as membrane-related processes, and suggest the NA TMD in H1N1 viruses has become more polar to maintain compatibility with the evolving enzymatic head domain. IMPORTANCE The neuranainidase (NA) protein from influenza A viruses (IAVs) functions to promote viral release and is one of the major surface antigens. The receptor-destroying activity in NA resides in the distal head domain that is linked to the viral membrane by an N-terminal hydrophobic transmembrane domain (TMD). Over the last century, the subtype 1 NA TMDs (N1) in human H1N1 viruses have become increasingly more polar, and the head domains have changed to alter their antigenicity. Here, we provide the first evidence that an old N1 head domain from 1933 is incompatible with a recent (2009), more polar N1 TMD sequence and that, during viral replication, the head domain drives the selection of TMD mutations. These mutations modify the intrinsic TMD assembly to restore the head domain folding compatibility and the resultant budding deficiency. This likely explains why the N1 TMDs have become more polar and suggests the N1 TMD and head domain have coevolved.

National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-113554 (URN)10.1128/JVI02005-14 (DOI)000347178900018 ()
Note

AuthorCount:6;

Available from: 2015-02-06 Created: 2015-02-04 Last updated: 2016-10-06Bibliographically approved
4. Assembly co-cooperativity between the influenza 1 NA stalk and 2 transmembrane domain defines the insertion deletion boundary
Open this publication in new window or tab >>Assembly co-cooperativity between the influenza 1 NA stalk and 2 transmembrane domain defines the insertion deletion boundary
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-134372 (URN)
Available from: 2016-10-05 Created: 2016-10-05 Last updated: 2016-10-06Bibliographically approved

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