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Type II transmembrane domain hydrophobicity dictates the cotranslational dependence for inversion
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.ORCID-id: 0000-0002-5864-8489
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
Vise andre og tillknytning
2014 (engelsk)Inngår i: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 25, nr 21, s. 3363-3374Artikkel i tidsskrift (Fagfellevurdert) 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.

sted, utgiver, år, opplag, sider
2014. Vol. 25, nr 21, s. 3363-3374
HSV kategori
Forskningsprogram
biokemi
Identifikatorer
URN: urn:nbn:se:su:diva-110182DOI: 10.1091/mbc.E14-04-0874ISI: 000344236100019OAI: oai:DiVA.org:su-110182DiVA, id: diva2:787511
Merknad

AuthorCount:5;

Tilgjengelig fra: 2015-02-10 Laget: 2014-12-08 Sist oppdatert: 2022-03-23bibliografisk kontrollert
Inngår i avhandling
1. Influenza A Virus: Spatial analysis of influenza genome trafficking and the evolution of the neuraminidase protein
Åpne denne publikasjonen i ny fane eller vindu >>Influenza A Virus: Spatial analysis of influenza genome trafficking and the evolution of the neuraminidase protein
2019 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
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.

sted, utgiver, år, opplag, sider
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2019. s. 40
Emneord
Influenza A virus, IAV, neuraminidase, NA, IAV genome trafficking, viral entry, viral replication, co-infection, antigenic drift, antigenic shift, NA assembly, transmembrane domain, evolution
HSV kategori
Forskningsprogram
biokemi
Identifikatorer
urn:nbn:se:su:diva-175202 (URN)978-91-7797-885-5 (ISBN)978-91-7797-886-2 (ISBN)
Disputas
2019-12-02, Vivi Täckholmsalen (Q-salen), NPQ-huset, Svante Arrhenius väg 20, Stockholm, 10:00 (engelsk)
Opponent
Veileder
Tilgjengelig fra: 2019-11-07 Laget: 2019-10-15 Sist oppdatert: 2022-02-26bibliografisk kontrollert
2. Influenza Neuraminidase: Novel mechanisms of influenza NA that enable adaptation and promote diversification
Åpne denne publikasjonen i ny fane eller vindu >>Influenza Neuraminidase: Novel mechanisms of influenza NA that enable adaptation and promote diversification
2020 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

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

sted, utgiver, år, opplag, sider
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2020. s. 50
Emneord
Influenza, IAV, neuraminidase, transmembrane domain, the central Ca2+ binding site, heterotetrameric formation, viral incorporation, evolution, adaptation, diversification
HSV kategori
Forskningsprogram
biokemi
Identifikatorer
urn:nbn:se:su:diva-182612 (URN)978-91-7911-214-1 (ISBN)978-91-7911-215-8 (ISBN)
Disputas
2020-09-29, via Zoom. A link will be published on https://www.dbb.su.se/, Stockholm, 14:00 (engelsk)
Opponent
Veileder
Tilgjengelig fra: 2020-09-04 Laget: 2020-08-13 Sist oppdatert: 2022-02-26bibliografisk kontrollert

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