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      Mutations in the Leucine Zipper-Like Motif of the Human Parainfluenza Virus 3 Fusion Protein Impair Fusion Activity

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          Abstract

          Objective

          To investigate the effect of the leucine zipper-like motif between HRA and HRB of the human parainfluenza virus 3 fusion protein on fusion activity.

          Methods

          Site-directed mutagenesis was utilized to substitute the heptadic residues at 257, 264, 271, 278, 285, 292, and 299 in this motif with alanine. Additionally, 3 middle heptadic leucine residues at 271, 278, and 285 were replaced with alanine singly or in combination. A vaccinia virus-T7 RNA polymerase transient expression system was employed to express the wild-type or mutated fusion (F) proteins. Three different types of membrane fusion assays were performed to analyze the fusogenic activity, fluorescence-activated cell sorting (FACS) analysis was executed to examine the cell surface expression level, and a coimmunoprecipitation assay was conducted to probe the hemagglutinin-neuraminidase (HN)-F interaction at the cell surface.

          Results

          All of the substitutions in this motif exhibited diminished or even lost fusion activity in all stages of fusion, although they all had no effect on cell surface expression. In the coimmunoprecipitation assay, all mutants resulted in decreased detection of the HN-F complexes compared with that of the wild-type F protein.

          Conclusions

          This motif has an important influence on fusion activity, and its integrality is indispensable for membrane fusion.

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          Most cited references44

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          The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins

          S. McKnight, P. Johnson, W. Landschulz (1988)
          A 30-amino-acid segment of C/EBP, a newly discovered enhancer binding protein, shares notable sequence similarity with a segment of the cellular Myc transforming protein. Display of these respective amino acid sequences on an idealized alpha helix revealed a periodic repetition of leucine residues at every seventh position over a distance covering eight helical turns. The periodic array of at least four leucines was also noted in the sequences of the Fos and Jun transforming proteins, as well as that of the yeast gene regulatory protein, GCN4. The polypeptide segments containing these periodic arrays of leucine residues are proposed to exist in an alpha-helical conformation, and the leucine side chains extending from one alpha helix interdigitate with those displayed from a similar alpha helix of a second polypeptide, facilitating dimerization. This hypothetical structure is referred to as the "leucine zipper," and it may represent a characteristic property of a new category of DNA binding proteins.
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            Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase.

            Edward Niles, T. Fuerst, Richard Moss … (1986)
            DNA coding for bacteriophage T7 RNA polymerase was ligated to a vaccinia virus transcriptional promoter and integrated within the vaccinia virus genome. The recombinant vaccinia virus retained infectivity and stably expressed T7 RNA polymerase in mammalian cells. Target genes were constructed by inserting DNA segments that code for beta-galactosidase or chloramphenicol acetyltransferase into a plasmid with bacteriophage T7 promoter and terminator regions. When cells were infected with the recombinant vaccinia virus and transfected with plasmids containing the target genes, the latter were expressed at high levels. Chloramphenicol acetyltransferase activity was 400-600 times greater than that observed with conventional mammalian transient-expression systems regulated either by the enhancer and promoter regions of the Rous sarcoma virus long terminal repeat or by the simian virus 40 early region. The vaccinia/T7 hybrid virus forms the basis of a simple, rapid, widely applicable, and efficient mammalian expression system.
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              Lipid-anchored influenza hemagglutinin promotes hemifusion, not complete fusion.

              Lisa J White, T Danieli, G Kemble (1994)
              It has been proposed that membrane fusion events such as virus-cell fusion proceed through a hemifusion intermediate, a state where lipids but not contents of the fusing compartments mix. We engineered the influenza hemagglutinin (HA) such that it would be anchored in membranes via a glycosylphosphatidylinositol (GPI) tail. GPI-anchored HA forms a trimer that can bind red blood cells (RBCs) and change conformation under fusion-inducing conditions. Using RBCs labeled with fluorescent lipid or fluorescent soluble content probes, we found that GPI-anchored HA mediated lipid mixing with similar time course and efficiency as wt-HA, yet did not mediate transfer of soluble contents. Hence, GPI-anchored HA appears to initiate, but not complete, a fusion reaction. We interpret our results as evidence for uncoupling a physiological fusion reaction, for trapping a hemifusion intermediate, and for assigning a role to a transmembrane domain in a fusion event.
              Article 0 comments Cited 110 times – based on 0 reviews     Review now
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                Author and article information

                Journal
                Journal ID (nlm-ta): Intervirology
                Journal ID (iso-abbrev): Intervirology
                Journal ID (publisher-id): INT
                Title: Intervirology
                Publisher: S. Karger AG (Allschwilerstrasse 10, P.O. Box · Postfach · Case postale, CH–4009, Basel, Switzerland · Schweiz · Suisse, Phone: +41 61 306 11 11, Fax: +41 61 306 12 34, karger@karger.com )
                ISSN (Print): 0300-5526
                ISSN (Electronic): 1423-0100
                Publication date (Print): February 2016
                Publication date (Electronic): 23 December 2015
                Volume: 58
                Issue: 5
                Pages: 297-309
                Affiliations
                [1] aDepartment of Virology, School of Public Health
                [2] bInstitute of Biochemistry and Molecular Biology, School of Medicine
                [3] cKey Laboratory for Experimental Teratology of the Ministry of Education, Shandong University
                [4] dDepartment of Laboratory Medicine, Provincial Hospital Affiliated to Shandong University
                [5] eDepartment of Neurosurgery, Qilu Hospital of Shandong University
                [6] fDepartment of Laboratory Medicine, Shandong Tumor Hospital and Institute
                [7] gShandong Center for Disease Control and Prevention, Jinan, PR China
                Author notes
                *Zhiyu Wang, Department of Virology, School of Public Health, Shandong University, 44 Wenhua Western Road, Jinan, Shandong 250012 (PR China), E-Mail zhiyu.wang@ 123456sdu.edu.cn
                Article
                Publisher ID: int-0058-0297
                DOI: 10.1159/000441978
                PMC ID: 7179560
                PubMed ID: 26694747
                SO-VID: 273eb9bf-cd35-42b0-b983-6b1cfb89eb04
                Copyright © Copyright © 2015 by S. Karger AG, Basel
                License:

                This article is made available via the PMC Open Access Subset for unrestricted re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the COVID-19 pandemic or until permissions are revoked in writing. Upon expiration of these permissions, PMC is granted a perpetual license to make this article available via PMC and Europe PMC, consistent with existing copyright protections.

                History
                Date received : 13 July 2015
                Date accepted : 24 October 2015
                Date: 2015
                Page count
                Figures: 8, References: 42, Pages: 13
                Categories
                Subject: Original Paper

                Keywords: human parainfluenza virus 3,fusion protein,leucine zipper-like motif,mutations,membrane fusion
                Data availability:
                Keywords: human parainfluenza virus 3, fusion protein, leucine zipper-like motif, mutations, membrane fusion

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