Interaction-mediating sequences within class I viral fusion glycoproteins : their roles in viral infection and in applications
Author: Zhang, Simin
Date: 2015-11-13
Location: Room 4Z, Nobel's Alle 8, Karolinska University Hospital, Huddinge
Time: 09.00
Department: Inst för laboratoriemedicin / Dept of Laboratory Medicine
View/ Open:
Thesis (3.323Mb)
Abstract
Class I viral fusion glycoproteins facilitate fusion of the viral envelope with cell membranes and entry of the virus into the cell, through extensive short sequence-specific interactions. Regions mediating these interactions include the N-terminal hydrophobic fusion peptide, a pair of extended 4,3-hydrophobic heptad repeats (HRs), a membrane-active membrane proximal external region (MPER), a hydrophobic transmembrane domain and the cytoplasmic tail region. In particular, the anti-parallel binding of the C-terminal HR to the central N-HR trimeric coiled-coil forms the 6-helix bundle fusion core. These interaction-mediating sequences are generally well preserved sequentially and structurally, allowing their peptidyl analogues to be developed as antiviral therapeutics and/or research reagents (e.g. HR-derived peptides). Novel targets for the development of antiviral drugs and viral detection reagents are required when facing drug-resistant viral strains, viral pathogens without effective and/or economical treatment, and newly emerging viral pathogens. This thesis focuses on the systematic identification of novel interaction-mediating sequences within Class I viral fusion glycoproteins, and the investigation of their involvements in viral replication as well as their potential applications in diagnosis and anti-viral interventions.
In Paper I, peptide array scanning identified 34 spike (S) protein-derived peptides that bound to the S protein of severe acute respiratory syndrome-associated coronavirus (SARS-CoV). These putative self-binding peptides contain five core octapeptide consensus sequences, among which the octapeptide GINITNFR was predicted to form β-zipper-mediated amyloid-like fibrils. The peptide C6 containing this sequence was subsequently shown to oligomerize and form amyloid-like fibrils. The potential of C6 to conduct β-zipper-mediated interactions was further applied to detect the S protein expression by immunofluorescence staining. The peptide array scanning in Paper I used the S protein ectodomain without the MPER and beyond. Using chemical crosslinking and immunofluorescence staining, in Paper II we could show that the S protein MPER could oligomerize and further heteromerize with the N-terminal internal fusion peptide (IFP). The MPER-derived peptides also inhibited the coronavirus entry in a dose-dependent manner, potentially through disrupting the MPER-mediated interactions.
The ability of peptides derived from the MPER in inhibiting viral entry and infection was subsequently studied in Paper III, in the context of HIV-1. The antiviral activities of the HIV-1 Env MPER-derived peptides were abrogated upon Ala substitution of the Trp residues or deletion of the C-terminal cholesterol-interacting motif. Unexpectedly, Ala substitutions of the Trp residues within HIV-1 Env significantly elevated the biosynthesis of another viral structural protein, the p55/Gag, which led to enhanced viral particle release. In Paper IV, besides the MPER we identified the signal sequence of HIV-1 Env as another region that could negatively regulate the expression of p55/Gag. The HIV-1 Env signal sequence, which mediates the co-translational translocalization of nascent Env polypeptide into the endoplasmic reticulum, inhibited the viral protein expression and production, probably at a post-ER-targeting stage. N-terminal truncations of the Env signal sequence significantly elevated the intracellular and intraviral levels of late viral proteins and the proviral genome transcription in a time- and dose-dependent fashion. Moreover, the truncations suppressed the HIV-1 promotor (LTR)-driven expression of a reporter protein, suggesting that the Env signal sequence inhibited viral genome transcription through LTR-dependent interactions.
This thesis focused on three novel interaction-mediating sequences within two Class I viral fusion glycoproteins, which could regulate the viral infectivity, at both viral entry and assembly, through protein-protein, protein-lipid, and/or protein-nucleic acids interactions. These sequences and the interactions that they are meditating could be further targeted by their peptidyl analogues for viral detection and/or inhibition.
In Paper I, peptide array scanning identified 34 spike (S) protein-derived peptides that bound to the S protein of severe acute respiratory syndrome-associated coronavirus (SARS-CoV). These putative self-binding peptides contain five core octapeptide consensus sequences, among which the octapeptide GINITNFR was predicted to form β-zipper-mediated amyloid-like fibrils. The peptide C6 containing this sequence was subsequently shown to oligomerize and form amyloid-like fibrils. The potential of C6 to conduct β-zipper-mediated interactions was further applied to detect the S protein expression by immunofluorescence staining. The peptide array scanning in Paper I used the S protein ectodomain without the MPER and beyond. Using chemical crosslinking and immunofluorescence staining, in Paper II we could show that the S protein MPER could oligomerize and further heteromerize with the N-terminal internal fusion peptide (IFP). The MPER-derived peptides also inhibited the coronavirus entry in a dose-dependent manner, potentially through disrupting the MPER-mediated interactions.
The ability of peptides derived from the MPER in inhibiting viral entry and infection was subsequently studied in Paper III, in the context of HIV-1. The antiviral activities of the HIV-1 Env MPER-derived peptides were abrogated upon Ala substitution of the Trp residues or deletion of the C-terminal cholesterol-interacting motif. Unexpectedly, Ala substitutions of the Trp residues within HIV-1 Env significantly elevated the biosynthesis of another viral structural protein, the p55/Gag, which led to enhanced viral particle release. In Paper IV, besides the MPER we identified the signal sequence of HIV-1 Env as another region that could negatively regulate the expression of p55/Gag. The HIV-1 Env signal sequence, which mediates the co-translational translocalization of nascent Env polypeptide into the endoplasmic reticulum, inhibited the viral protein expression and production, probably at a post-ER-targeting stage. N-terminal truncations of the Env signal sequence significantly elevated the intracellular and intraviral levels of late viral proteins and the proviral genome transcription in a time- and dose-dependent fashion. Moreover, the truncations suppressed the HIV-1 promotor (LTR)-driven expression of a reporter protein, suggesting that the Env signal sequence inhibited viral genome transcription through LTR-dependent interactions.
This thesis focused on three novel interaction-mediating sequences within two Class I viral fusion glycoproteins, which could regulate the viral infectivity, at both viral entry and assembly, through protein-protein, protein-lipid, and/or protein-nucleic acids interactions. These sequences and the interactions that they are meditating could be further targeted by their peptidyl analogues for viral detection and/or inhibition.
List of papers:
I. Si Min Zhang, Ying Liao, Tuan Ling Neo, Yanning Lu, Ding Xiang Liu, Anders Vahlne, James P. Tam. Identification and applications of self-binding zipper-like sequences in SARS-CoV spike protein. [Manuscript]
II. Ying Liao, Si Min Zhang, Tuan Ling Neo, James P. Tam. Tryptophan-dependent membrane interaction and heteromerization with internal fusion peptide by membrane proximal external region of SARS-CoV spike protein. Biochemistry. 2015; 54 (9): 1819−1830.
Fulltext (DOI)
Pubmed
View record in Web of Science®
III. Si Min Zhang, Alenka Jejcic, James P. Tam, Anders Vahlne. Membrane-active Sequences within gp41 Membrane Proximal External Region (MPER) Modulate MPER-containing Peptidyl Fusion Inhibitor Activity and the Biosynthesis of HIV-1 Structural Proteins. PLoS ONE. 2015; 10(7): e0134851.
Fulltext (DOI)
Pubmed
View record in Web of Science®
IV. Si Min Zhang, Anders Vahlne, Alenka Jejcic. The gp160 Signal Sequence Suppresses HIV-1 Replication in an LTR-Dependent Manner. [Manuscript]
I. Si Min Zhang, Ying Liao, Tuan Ling Neo, Yanning Lu, Ding Xiang Liu, Anders Vahlne, James P. Tam. Identification and applications of self-binding zipper-like sequences in SARS-CoV spike protein. [Manuscript]
II. Ying Liao, Si Min Zhang, Tuan Ling Neo, James P. Tam. Tryptophan-dependent membrane interaction and heteromerization with internal fusion peptide by membrane proximal external region of SARS-CoV spike protein. Biochemistry. 2015; 54 (9): 1819−1830.
Fulltext (DOI)
Pubmed
View record in Web of Science®
III. Si Min Zhang, Alenka Jejcic, James P. Tam, Anders Vahlne. Membrane-active Sequences within gp41 Membrane Proximal External Region (MPER) Modulate MPER-containing Peptidyl Fusion Inhibitor Activity and the Biosynthesis of HIV-1 Structural Proteins. PLoS ONE. 2015; 10(7): e0134851.
Fulltext (DOI)
Pubmed
View record in Web of Science®
IV. Si Min Zhang, Anders Vahlne, Alenka Jejcic. The gp160 Signal Sequence Suppresses HIV-1 Replication in an LTR-Dependent Manner. [Manuscript]
Institution: Karolinska Institutet
Supervisor: Vahlne, Anders
Issue date: 2015-10-23
Rights:
Publication year: 2015
ISBN: 978-91-7676-114-4
Statistics
Total Visits
Views | |
---|---|
Interaction-mediating ...(legacy) | 370 |
Interaction-mediating ... | 282 |
Total Visits Per Month
September 2023 | October 2023 | November 2023 | December 2023 | January 2024 | February 2024 | March 2024 | |
---|---|---|---|---|---|---|---|
Interaction-mediating ... | 0 | 2 | 2 | 2 | 2 | 1 | 0 |
File Visits
Views | |
---|---|
Thesis_Zhang_Simin.pdf | 303 |
Thesis_Zhang_Simin.pdf(legacy) | 302 |
null(legacy) | 1 |
Top country views
Views | |
---|---|
United States | 170 |
Germany | 104 |
Sweden | 76 |
China | 47 |
South Korea | 31 |
Poland | 14 |
Singapore | 11 |
Japan | 10 |
Canada | 9 |
Ireland | 8 |
Top cities views
Views | |
---|---|
Ashburn | 77 |
Seoul | 27 |
Kiez | 26 |
Lansing | 26 |
Beijing | 19 |
Stockholm | 10 |
Kraków | 9 |
Singapore | 9 |
Wilmington | 9 |
Dublin | 7 |