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Type II transmembrane domain hydrophobicity dictates the cotranslational dependence for inversion
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.ORCID iD: 0000-0002-5864-8489
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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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.

Place, publisher, year, edition, pages
2014. Vol. 25, no 21, p. 3363-3374
National Category
Biophysics Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-110182DOI: 10.1091/mbc.E14-04-0874ISI: 000344236100019OAI: oai:DiVA.org:su-110182DiVA, id: diva2:787511
Note

AuthorCount:5;

Available from: 2015-02-10 Created: 2014-12-08 Last updated: 2019-12-12Bibliographically approved
In thesis
1. Influenza A Virus: Spatial analysis of influenza genome trafficking and the evolution of the neuraminidase protein
Open this publication in new window or tab >>Influenza A Virus: Spatial analysis of influenza genome trafficking and the evolution of the neuraminidase protein
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
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:nbn:se:su:diva-175202 (URN)978-91-7797-885-5 (ISBN)978-91-7797-886-2 (ISBN)
Public defence
2019-12-02, Vivi Täckholmsalen (Q-salen), NPQ-huset, Svante Arrhenius väg 20, Stockholm, 10:00 (English)
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Available from: 2019-11-07 Created: 2019-10-15 Last updated: 2019-10-28Bibliographically approved

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