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Tytuł pozycji:

Insights into the Organization of the Poxvirus Multicomponent Entry-Fusion Complex from Proximity Analyses in Living Infected Cells.

Tytuł:
Insights into the Organization of the Poxvirus Multicomponent Entry-Fusion Complex from Proximity Analyses in Living Infected Cells.
Autorzy:
Schin AM; Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
Diesterbeck US; Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
Moss B; Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
Źródło:
Journal of virology [J Virol] 2021 Jul 26; Vol. 95 (16), pp. e0085221. Date of Electronic Publication: 2021 Jul 26.
Typ publikacji:
Journal Article; Research Support, N.I.H., Intramural
Język:
English
Imprint Name(s):
Publication: Washington Dc : American Society For Microbiology
Original Publication: Baltimore, American Society for Microbiology.
MeSH Terms:
Virus Internalization*
Poxviridae/*physiology
Viral Fusion Proteins/*metabolism
Animals ; Cell Line ; Cytoplasm/metabolism ; Green Fluorescent Proteins/genetics ; Green Fluorescent Proteins/metabolism ; Protein Binding ; Vaccinia virus/genetics ; Vaccinia virus/metabolism ; Vaccinia virus/physiology ; Viral Fusion Proteins/genetics
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Grant Information:
Division of Intramural Research, National Institute of Allergy and Infectious Diseases (DIR, NIAID)
Contributed Indexing:
Keywords: green fluorescent protein; membrane proteins; multiprotein complex; proximity analysis; vaccinia virus; virus entry
Substance Nomenclature:
0 (Viral Fusion Proteins)
147336-22-9 (Green Fluorescent Proteins)
Entry Date(s):
Date Created: 20210602 Date Completed: 20211011 Latest Revision: 20220127
Update Code:
20240104
PubMed Central ID:
PMC8312868
DOI:
10.1128/JVI.00852-21
PMID:
34076488
Czasopismo naukowe
Poxviruses are exceptional in having a complex entry-fusion complex (EFC) that is comprised of 11 conserved proteins embedded in the membrane of mature virions. However, the detailed architecture is unknown and only a few bimolecular protein interactions have been demonstrated by coimmunoprecipitation from detergent-treated lysates and by cross-linking. Here, we adapted the tripartite split green fluorescent protein (GFP) complementation system in order to analyze EFC protein contacts within living cells. This system employs a detector fragment called GFP1-9 comprised of nine GFP β-strands. To achieve fluorescence, two additional 20-amino-acid fragments called GFP10 and GFP11 attached to interacting proteins are needed, providing the basis for identification of the latter. We constructed a novel recombinant vaccinia virus (VACV-GFP1-9) expressing GFP1-9 under a viral early/late promoter and plasmids with VACV late promoters regulating each of the EFC proteins with GFP10 or GFP11 attached to their ectodomains. GFP fluorescence was detected by confocal microscopy at sites of virion assembly in cells infected with VACV-GFP1-9 and cotransfected with plasmids expressing one EFC-GFP10 and one EFC-GFP11 interacting protein. Flow cytometry provided a quantitative way to determine the interaction of each EFC-GFP10 protein with every other EFC-GFP11 protein in the context of a normal infection in which all viral proteins are synthesized and assembled. Previous EFC protein interactions were confirmed, and new ones were discovered and corroborated by additional methods. Most remarkable was the finding that the small, hydrophobic O3 protein interacted with each of the other EFC proteins. IMPORTANCE Poxviruses are enveloped viruses with a DNA-containing core that enters cells following fusion of viral and host membranes. This essential step is a target for vaccines and therapeutics. The entry-fusion complex (EFC) of poxviruses is unusually complex and comprised of 11 conserved viral proteins. Determination of the structure of the EFC is a prerequisite for understanding the fusion mechanism. Here, we used a tripartite split green fluorescent protein assay to determine the proximity of individual EFC proteins in living cells. A network connecting components of the EFC was derived.

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