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NA transmembrane domain: Amphiphilic drift to accommodate two functions
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. (Robert Daniels)ORCID iD: 0000-0002-5864-8489
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Neuraminidase (NA) is one of two major antigens on the surface of influenza A viruses. It is comprised of a single N-terminal transmembrane domain (TMD), a stalk domain, and a C-terminal enzymatic head domain that cleaves sialic acid, most notably to release new particles from the host cell surface. NA is only enzymatically active as a homo-tetramer. However, it is not known which properties facilitate the oligomerization of NA during assembly. Our results show that, apart from anchoring the protein to the membrane, the NA TMD also contributes to the assembly process by keeping the stalk in a tetrameric conformation. The ability of the TMD to oligomerize is shown to be dependent on its amphiphilic characteristics that was largely conserved across the nine NA subtypes (N1-N9). Over time the NA TMDs in human H1N1 viruses were found to have become more amphiphilic, which correlated with stronger oligomerization. An old H1N1 virus with a more recent N1 TMD had impaired growth, but readily acquired compensatory mutations in the TMD to restore growth, by reverting the TMD oligomerization strength back to that of the old TMD, demonstrating a biological role of the TMD in folding and assembly. NA and the other viral proteins are spatially and temporally coordinated to achieve optimal viral production. By using a co-transfection analysis, the high AU-content in the NA and HA ER-targeting sequence coding regions (for NA TMD as well as the HA signal sequence) were found to inhibit their expression. The inhibition was alleviated by the early expressed influenza RNA-binding protein NS1, which promoted translation and showed enriched foci at the endoplasmic reticulum (ER). NS1, which expresses early during infection, is therefore likely the regulator of NA and HA to prevent premature expression. These results show that the NA TMD is under substantial selection pressure at both the nucleotide and amino acid level to accommodate its roles in ER-targeting, protein folding, and post-transcriptional regulation.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2017.
Keywords [en]
influenza, IAV, neuraminidase, NA, transmembrane domain, TMD, secretory protein, ER-targeting sequence, ER-targeting sequence coding region, protein regulation, NS1, GALLEX
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-142051ISBN: 978-91-7649-825-5 (print)ISBN: 978-91-7649-826-2 (electronic)OAI: oai:DiVA.org:su-142051DiVA, id: diva2:1090243
Public defence
2017-06-05, Magnelisalen, 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: Accepted.

Available from: 2017-05-11 Created: 2017-04-24 Last updated: 2017-05-12Bibliographically 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, p. 644-653Article 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: 2017-12-06Bibliographically 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, p. 10652-10660Article 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: 2017-04-28Bibliographically 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, p. 1094-1104Article 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/JVI.02005-14 (DOI)000347178900018 ()
Note

AuthorCount:6;

Available from: 2015-02-06 Created: 2015-02-04 Last updated: 2017-12-04Bibliographically approved
4. Translational regulation of viral secretory proteins by 5’ coding regions and a viral RNA-binding protein
Open this publication in new window or tab >>Translational regulation of viral secretory proteins by 5’ coding regions and a viral RNA-binding protein
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(English)In: Journal of Cell Biology, ISSN 0021-9525, E-ISSN 1540-8140Article in journal (Refereed) Accepted
Abstract [en]

A primary function of 5’ regions in many secretory protein mRNAs is to encode an endoplasmic reticulum (ER) targeting sequence. Here we show the regions coding for the ER-targeting sequences of the influenza proteins NA and HA also function as translational regulatory elements, which are controlled by the viral RNA-binding protein NS1. The translational increase depends on the nucleotide composition of the NA and HA ER-targeting sequences, their 5’ positioning, and is facilitated by the NS1 RNA-binding domain, which can associate with ER membranes. Inserting the ER-targeting sequence coding region of NA into different 5’UTRs confirmed that NS1 can promote the translation of secretory protein mRNAs based on the nucleotides within this region rather than the resulting amino acids. By analysing human protein mRNA sequences we found evidence that this mechanism of using 5’ coding regions and particular RNA-binding proteins to achieve gene-specific regulation may extend to human secreted proteins.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-141847 (URN)
Available from: 2017-04-21 Created: 2017-04-21 Last updated: 2017-04-28

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