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Influenza A Virus: Spatial analysis of influenza genome trafficking and the evolution of the neuraminidase protein
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Influenza A viruses (IAVs) are a common infectious agent that seasonally circulates within the human population that causes mild to severe acute respiratory infections. The severity of the infection is often related to how the virus has evolved with respect to the pre-existing immunity in the population. For IAVs, the most common mechanisms to avoid the immune response are to vary the surface antigens, hemagglutinin (HA) and neuraminidase (NA), by processes known as antigenic drift and shift.

Antigenic drift refers to point mutations that accumulate in HA and NA as a result of the antibody-mediated selection pressure that exists in the population. The majority of the changes attributed to antigenic drift localize to HA and NA surface exposed regions, however this does not exclude that drift can also result in the selection of residues that are not exposed. One region where non-exposed residues have potentially been selected for is the NA transmembrane domain (TMD) of human H1N1 IAVs, where a temporal bias exists for the accumulation of polar residues. By examining these sequence changes in the NA TMD, we found that the polar residues contribute to the amphipathic characteristic of the NA TMD, which mediates the oligomerization of the N-terminus. As more polar residues became incorporated, the strength of the TMD-TMD interaction increased, presumably to benefit the NA head domain assembly into a functional tetramer. We determined that the amphiphilic drift in the NA TMD is able to bypass the strict hydrophobicity required for membrane insertion at the endoplasmic reticulum because it can utilize the co-translational translocation process to facilitate the insertion and inversion of its non-ideal TMD. The contribution of the TMD to proper NA assembly was traced to the formation of the Ca2+ binding pocket that is located at the center of the tetrameric assembly, as this pocket lies above the stalk linker regions and must be occupied for NA to function.

In addition to antigenic drift, NA and HA can also undergo antigenic shift. Antigenic shift occurs when either of the gene segments encoding NA or HA are exchanged with ones from another IAV encoding another subtype of NA or HA. Different from antigenic drift, antigenic shift can only occur when a cell is co-infected and most investigations on the process of reassortment have been made at the protein level due to the methodological issues for labeling the RNA genome in situ. To overcome these technical limitations, we developed an in situ RNA labeling approach that provides highly specific spatial resolution of the IAV genome throughout the infection process. By applying this approach to temporally analyze the co-infection process, we found that the entry of a second IAV is stalled in the cytoplasm if another IAV has begun to replicate. Together, these results provide insight into the low frequency of antigenic shift in nature and provide evidence that non-exposed residues may make an underappreciated contribution to NA antigenic drift in human H1N1 viruses.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2019. , p. 40
Keywords [en]
Influenza A virus, IAV, neuraminidase, NA, IAV genome trafficking, viral entry, viral replication, co-infection, antigenic drift, antigenic shift, NA assembly, transmembrane domain, evolution
National Category
Microbiology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-175202ISBN: 978-91-7797-885-5 (print)ISBN: 978-91-7797-886-2 (electronic)OAI: oai:DiVA.org:su-175202DiVA, id: diva2:1361347
Public defence
2019-12-02, Vivi Täckholmsalen (Q-salen), NPQ-huset, Svante Arrhenius väg 20, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2019-11-07 Created: 2019-10-15 Last updated: 2019-10-28Bibliographically approved
List of papers
1. 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: 2019-10-15Bibliographically approved
2. Type II transmembrane domain hydrophobicity dictates the cotranslational dependence for inversion
Open this publication in new window or tab >>Type II transmembrane domain hydrophobicity dictates the cotranslational dependence for inversion
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2014 (English)In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 25, no 21, p. 3363-3374Article in journal (Refereed) Published
Abstract [en]

Membrane insertion by the Sec61 translocon in the endoplasmic reticulum (ER) is highly dependent on hydrophobicity. This places stringent hydrophobicity requirements on transmembrane domains (TMDs) from single-spanning membrane proteins. On examining the single-spanning influenza A membrane proteins, we found that the strict hydrophobicity requirement applies to the N-out-C-in HA and M2 TMDs but not the N-in-C-out TMDs from the type II membrane protein neuraminidase (NA). To investigate this discrepancy, we analyzed NA TMDs of varying hydrophobicity, followed by increasing polypeptide lengths, in mammalian cells and ER microsomes. Our results show that the marginally hydrophobic NA TMDs (Delta G(app) > 0 kcal/mol) require the cotranslational insertion process for facilitating their inversion during translocation and a positively charged N-terminal flanking residue and that NA inversion enhances its plasma membrane localization. Overall the cotranslational inversion of marginally hydrophobic NA TMDs initiates once similar to 70 amino acids past the TMD are synthesized, and the efficiency reaches 50% by similar to 100 amino acids, consistent with the positioning of this TMD class in type II human membrane proteins. Inversion of the M2 TMD, achieved by elongating its C-terminus, underscores the contribution of cotranslational synthesis to TMD inversion.

National Category
Biophysics Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-110182 (URN)10.1091/mbc.E14-04-0874 (DOI)000344236100019 ()
Note

AuthorCount:5;

Available from: 2015-02-10 Created: 2014-12-08 Last updated: 2019-10-15Bibliographically approved
3. Structural restrictions for influenza neuraminidase activity promote adaptation and diversification
Open this publication in new window or tab >>Structural restrictions for influenza neuraminidase activity promote adaptation and diversification
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2019 (English)In: Nature Microbiology, E-ISSN 2058-5276, Vol. 4, p. 2565-2577Article in journal (Refereed) Published
Abstract [en]

Influenza neuraminidase (NA) is a sialidase that contributes to viral mobility by removing the extracellular receptors for the haemagglutinin (HA) glycoprotein. However, it remains unclear why influenza NAs evolved to function as Ca2+-dependent tetramers that display variable stability. Here, we show that the Ca2+ ion located at the centre of the NA tetramer is a major stability determinant, as this Ca2+ ion is required for catalysis and its binding affinity varies between NAs. By examining NAs from 2009 pandemic-like H1N1 viruses, we traced the affinity variation to local substitutions that cause residues in the central Ca2+-binding pocket to reposition. A temporal analysis revealed that these local substitutions predictably alter the stability of the 2009 pandemic-like NAs and contribute to the tendency for the stability to vary up and down over time. In addition to the changes in stability, the structural plasticity of NA was also shown to support the formation of heterotetramers, which creates a mechanism for NA to obtain hybrid properties and propagate suboptimal mutants. Together, these results demonstrate how the structural restrictions for activity provide influenza NA with several mechanisms for adaptation and diversification.

National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-175186 (URN)10.1038/s41564-019-0537-z (DOI)
Available from: 2019-10-15 Created: 2019-10-15 Last updated: 2019-12-08Bibliographically approved
4. Analysis of IAV Replication and Co-infection Dynamics by a Versatile RNA Viral Genome Labeling Method
Open this publication in new window or tab >>Analysis of IAV Replication and Co-infection Dynamics by a Versatile RNA Viral Genome Labeling Method
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2017 (English)In: Cell reports, ISSN 2211-1247, E-ISSN 2211-1247, Vol. 20, no 1, p. 251-263Article in journal (Refereed) Published
Abstract [en]

Genome delivery to the proper cellular compartment for transcription and replication is a primary goal of viruses. However, methods for analyzing viral genome localization and differentiating genomes with high identity are lacking, making it difficult to investigate entry-related processes and co-examine heterogeneous RNA viral populations. Here, we present an RNA labeling approach for single-cell analysis of RNA viral replication and co-infection dynamics in situ, which uses the versatility of padlock probes. We applied this method to identify influenza A virus (IAV) infections in cells and lung tissue with single-nucleotide specificity and to classify entry and replication stages by gene segment localization. Extending the classification strategy to co-infections of IAVs with single-nucleotide variations, we found that the dependence on intracellular trafficking places a time restriction on secondary co-infections necessary for genome reassortment. Altogether, these data demonstrate how RNA viral genome labeling can help dissect entry and co-infections.

Keywords
RNA viruses, viral genome labeling and localization, influenza A virus, RNA labeling, vRNAs, IAV entry, IAV cell co-infections, IAV replication cycle, single-cell IAV genome trafficking, single-nucleotide specificity
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-146349 (URN)10.1016/j.celrep.2017.06.021 (DOI)000404899700022 ()
Available from: 2017-08-29 Created: 2017-08-29 Last updated: 2019-10-15Bibliographically approved

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