Conformational Change in Herpes Simplex Virus Entry Glycoproteins Detected by Dot Blot

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M

E T H O D S I N

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O L E C U L A R

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Series Editor John M. Walker

School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, UK

For further volumes:

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For over 35 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimedMethods in Molecular Biology series. The series was the first to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by- step fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitute the key ingredient in each and every volume of theMethods in Molecular Biology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexed in PubMed.

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Herpes Simplex Virus

Methods and Protocols

Second Edition

Edited by

Russell J. Diefenbach

Department of Biomedical Sciences, Macquarie University, Sydney, NSW, Australia

Cornel Fraefel

Institute of Virology, University of Zurich, Zürich, Switzerland

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Editors

Russell J. Diefenbach

Department of Biomedical Sciences Macquarie University

Sydney, NSW, Australia

Cornel Fraefel Institute of Virology University of Zurich Zu¨rich, Switzerland

ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology

ISBN 978-1-4939-9813-5 ISBN 978-1-4939-9814-2 (eBook) https://doi.org/10.1007/978-1-4939-9814-2

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed.

The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use.

The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made.

The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Cover caption: HSV-1 virions in the extracellular space of Vero cells prepared for electron microscopy by high-pressure freezing, freeze-substitution, embedding in epon and ultrathin sectioning showing core, capsid, tegument, envelope and glycoproteins. The size difference is due to the section plane. Bar¼ 100 nm. Elisabeth M. Schraner, Institute of Virology, University of Zurich, Zu¨rich, Switzerland.

This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer Nature.

The registered company address is: 233 Spring Street, New York, NY 10013, U.S.A.

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Preface

Herpes simplex viruses type 1 and 2 (HSV-1, HSV-2) are important human pathogens.

HSV-1, for example, has a worldwide seroprevalence of more than 80% in adults. The virus typically enters orofacial mucosal epithelial cells, where productive infection takes place, but it can also infect genital mucosa epithelial cells. Productive replication in epithelial cells leads to release of progeny virus at the site of host entry, from where the virus can access neurons of the trigeminal ganglia to establish lifelong latency and to create a reservoir for periodic reactivation. In immunocompromised patients, HSV-1 can cause severe meningoencephali- tis or keratoconjunctivitis that can lead to permanent neurological damage and death or blindness, respectively, if not treated. The herpes simplex viruses have been the prototype viruses of theAlphaherpesvirinae subfamily and have been extensively studied for decades on all aspects of infection, replication, and pathogenesis. HSV-1 and HSV-2 have also become important tools to study cell biology and immunology, and for the development of innova- tive vaccines and vectors for gene- and tumor therapy.

It would be impossible to cover all aspects of methodology related to the investigation of herpes simplex viruses in one book. We hope in this second edition that we have again successfully encapsulated a significant breath of relevant methodology but also incorporated new rapidly developing technologies such as next-generation sequencing, CRISPR/Cas9 engineering, and the use of BioID to identify protein–protein interactions. The chapters contained within will be of interest to immunologists as well as molecular and cell biologists.

It will appeal to those researchers who wish to initiate molecular- and/or cellular-based approaches to investigate HSV. Many of the techniques can be readily translated to other closely related herpesviruses.

The first two chapters of this book include comprehensive reviews on HSV-1 biology and life cycle and the current state of play in antiviral and vaccine development. These are followed by a wide collection of protocols, including basic protocols on growing viruses in cell culture and manipulating viral DNA. Other chapters describe approaches to design and application of HSV-1 vectors for cancer- and gene therapy, or to study specific aspects of HSV-1 biology such as latency, intracellular transport, and protein–protein interaction using a number of cell culture and animal models. Rapidly developing areas such as the topic of extracellular vesicles, in the context of HSV-1, have also been included.

Procedures for structural analyses, microscopy, proteomics, and testing of antivirals are included as well. The methods provided are intended to aid new researchers in the field of herpes virology as well as those experienced investigators wishing to embark on new techniques.

We would like to thank all who have contributed to the completion of this book, in particular the authors of the chapters. We would also like to thank the editor of theMethods in Molecular Biology series, John Walker, for his constant support during the preparation of this volume. We gladly accepted John’s invitation to co-edit a second edition of a HSV protocols book largely based on our prior experience with the first edition and how well it

v

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has been received. Finally, we hope that our book will help many researchers in the herpes virus field in their pursuit of understanding the complex interactions between herpes virus and host. Still, much remains to be discovered!

Sydney, NSW, Australia Russell J. Diefenbach

Zu¨rich, Switzerland Cornel Fraefel

vi Preface

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Contributors

RAQUELBELLO-MORALES " Departamento de Biologı´a Molecular, Universidad Aut!onoma de Madrid, Madrid, Spain; Centro de Biologı´a Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain

KIRSTIEBERTRAM " Centre for Virus Research, The Westmead Institute for Medical Research, Westmead, NSW, Australia; Sydney Medical School, The University of Sydney, Westmead, NSW, Australia

ROSSA. BOADLE " Westmead Research Hub, Westmead, NSW, Australia

KATHRINBOHN-WIPPERT " Department of Bioengineering, University of Illinois at Urbana- Champaign, Urbana, IL, USA

GABRIELLACAMPADELLI-FIUME " Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy

MUJEEBR. CHEERATHODI _" Department of Biomedical Sciences, Florida State University College of Medicine, Tallahassee, FL, USA

CAMILLECOHEN " Univ Lyon, Universite´ Claude Bernard Lyon 1, CNRS UMR 5310, INSERM U 1217, LabEx DEVweCAN, Institut NeuroMyoGe`ne (INMG), Team Chromatin Assembly, Nuclear Domains, Virus, Lyon, France

JUSTUSB. COHEN " Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

REBECCAS. COOPER _" Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA, USA

ARMELLECORPET " Univ Lyon, Universite´ Claude Bernard Lyon 1, CNRS UMR 5310, INSERM U 1217, LabEx DEVweCAN, Institut NeuroMyoGe`ne (INMG), Team Chromatin Assembly, Nuclear Domains, Virus, Lyon, France

ANTHONYL. CUNNINGHAM " Centre for Virus Research, The Westmead Institute for Medical Research, Westmead, NSW, Australia; Sydney Medical School, The University of Sydney, Westmead, NSW, Australia

KEVINDANASTAS " Centre for Virus Research, The Westmead Institute for Medical Research, Westmead, NSW, Australia; The University of Sydney, Westmead, NSW, Australia

CHRISTOPHERE. DENES " Centre for Virus Research, The Westmead Institute for Medical Research, The University of Sydney, Westmead, NSW, Australia

RUSSELLJ. DIEFENBACH " Centre for Virus Research, The Westmead Institute for Medical Research, The University of Sydney, Westmead, NSW, Australia; Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Macquarie University, Sydney, NSW, Australia

LICHUNDONG " Department of Medicine, University of Washington, Seattle, WA, USA NABIL ELBILALI " Department of Pathology and Cell Biology, University of Montreal,

Montreal, QC, Canada

ALBERTOL. EPSTEIN _" UMR INSERM U1179, University of Versailles Saint Quentin en Yvelines (UVSQ), Montigny le Bretonneux, France

ROGERD. EVERETT " MRC-University of Glasgow Centre for Virus Research, Glasgow, Scotland, UK

CORNELFRAEFEL " Institute of Virology, University of Zurich, Zu¨rich, Switzerland

xi

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VALENTINAGATTA " Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy

TATIANAGIANNI " Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy

KATRINAA. GIANOPULOS " Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, Pullman, WA, USA; School of

Molecular Biosciences, College of Veterinary Medicine, Washington State University, Pullman, WA, USA

JOSEPH C. GLORIOSO " Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

WILLIAMF. GOINS " Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

ANJALIGOWRIPALAN " John Curtin School of Medical Research, The Australian National University, Canberra, ACT, Australia

BONNIEHALL " Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

EKATERINAE. HELDWEIN " Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA, USA

HUI-LANHU _" Department of Biochemistry and Molecular Pharmacology, New York

University School of Medicine, New York, NY, USA

SHAOHUAHUANG " Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

TONYT. HUANG " Department of Biochemistry and Molecular Pharmacology, New York

University School of Medicine, New York, NY, USA

FRANCELINEJUILLARD _" Univ Lyon, Universite´ Claude Bernard Lyon 1, CNRS UMR 5310, INSERM U 1217, LabEx DEVweCAN, Institut NeuroMyoGe`ne (INMG), Team

Chromatin Assembly, Nuclear Domains, Virus, Lyon, France

BITAKHADIVJAM " Department of Pathology and Cell Biology, University of Montreal, Montreal, QC, Canada

DAVID M. KOELLE " Department of Medicine, University of Washington, Seattle, WA, USA;

Department of Laboratory Medicine, University of Washington, Seattle, WA, USA;

Department of Global Health, University of Washington, Seattle, WA, USA; Vaccine and Infectious Diseases Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA;

Benaroya Research Institute, Seattle, WA, USA

ANDREAS. LAIMBACHER " Musculoskeletal Research Unit (MSRU), Vetsuisse Faculty,

University of Zurich, Zu¨rich, Switzerland; Center for Applied Biotechnology and Molecular Medicine (CABMM), University of Zurich, Zu¨rich, Switzerland

JENNIFERH. LAVAIL " Department of Anatomy, University of California San Francisco, San Francisco, CA, USA

VALERIO LEONI " Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy

ROGERLIPPE´ " Department of Pathology and Cell Biology, University of Montreal, Montreal, QC, Canada

PATRICKLOMONTE " Univ Lyon, Universite´ Claude Bernard Lyon 1, CNRS UMR 5310, INSERM U 1217, LabEx DEVweCAN, Institut NeuroMyoGe`ne (INMG), Team Chromatin Assembly, Nuclear Domains, Virus, Lyon, France

xii Contributors

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JOSE´ ANTONIOLO´ PEZ-GUERRERO " Departamento de Biologı´a Molecular, Universidad Aut!onoma de Madrid, Madrid, Spain; Centro de Biologı´a Molecular Severo Ochoa, CSIC- UAM, Madrid, Spain

PEGGY MARCONI " Department of Chemical and Pharmaceutical Sciences (DipSCF), University of Ferrara, Ferrara, Italy

MOHAMEDALIMAROUI _" Univ Lyon, Universite´ Claude Bernard Lyon 1, CNRS UMR 5310, INSERM U 1217, LabEx DEVweCAN, Institut NeuroMyoGe`ne (INMG), Team Chromatin Assembly, Nuclear Domains, Virus, Lyon, France

JOSHUA O. MARSHAK " Department of Medicine, University of Washington, Seattle, WA, USA

MARCOMARZULLI " Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

DAVIDG. MECKESJR. " Department of Biomedical Sciences, Florida State University College of Medicine, Tallahassee, FL, USA

ANITAF. MEIER " Institute of Virology, Vetsuisse Faculty, University of Zurich, Zu¨rich,

Switzerland

LAURAMENOTTI " Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy

MONICAMIRANDA-SAKSENA _" Centre for Virus Research, The Westmead Institute for Medical Research, Westmead, NSW, Australia; The University of Sydney, Westmead, NSW,

Australia

IANMOHR " Department of Microbiology, New York University School of Medicine, New York,

NY, USA

ANTHONYV. NICOLA " Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, Pullman, WA, USA; School of Molecular Biosciences, College of Veterinary Medicine, Washington State University, Pullman, WA, USA

SIMONAPEPE " Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy

BILJANA PETROVIC " Nouscom Srl, Rome, Italy

MOLLYM. RATHBUN " Department of Biochemistry and Molecular Biology, Center for Infectious Disease Dynamics, The Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, PA, USA

KERRIEJ. SANDGREN " Centre for Virus Research, The Westmead Institute for Medical Research, Westmead, NSW, Australia; Sydney Medical School, The University of Sydney, Westmead, NSW, Australia

TRIKOMALASARI " Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, Pullman, WA, USA

ANDREASSAUERBREI _" Section of Experimental Virology, Institute for Medical Microbiology, Jena University Hospital, Jena, Germany

NANCYM. SAWTELL " Division of Infectious Diseases, Department of Pediatrics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA

MICHAELSEYFFERT " Institute of Virology, University of Zurich, Zu¨rich, Switzerland MACKENZIEM. SHIPLEY " Department of Biochemistry and Molecular Biology, Center for

Infectious Disease Dynamics, The Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, PA, USA

Contributors xiii

(13)

JACINTAB. SMITH " Centre for Virus Research, The Westmead Institute for Medical Research, Westmead, NSW, Australia; Sydney Medical School, The University of Sydney, Westmead, NSW, Australia

KALANGHADPUTHANKALAMSRINIVAS " Department of Microbiology, New York University School of Medicine, New York, NY, USA

SAMUELD. STAMPFER _" Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA, USA

SEREINAO. SUTTER " Institute of Virology, Vetsuisse Faculty, University of Zurich, Zu¨rich, Switzerland

MORIAHL. SZPARA " Department of Biochemistry and Molecular Biology, Center for

Infectious Disease Dynamics, The Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, PA, USA

RICHARDL. THOMPSON " Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Cincinnati, OH, USA

NAOMIR. TRUONG " Centre for Virus Research, The Westmead Institute for Medical Research, Westmead, NSW, Australia; Sydney Medical School, The University of Sydney, Westmead, NSW, Australia

DAVID C. TSCHARKE " John Curtin School of Medical Research, The Australian National University, Canberra, ACT, Australia

ANDREAVANNINI " Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy

THILAGAVELUSAMY " John Curtin School of Medical Research, The Australian National University, Canberra, ACT, Australia

ELLEN M. WHITE " Department of Molecular Biology and Microbiology, Tufts University

School of Medicine, Boston, MA, USA

ANGUSC. WILSON _" Department of Microbiology, New York University School of Medicine, New York, NY, USA

xiv Contributors

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Metadata of the chapter that will be visualized online

Chapter Title Conformational Change in Herpes Simplex Virus Entry Glycoproteins Detected by Dot Blot

Copyright Year 2020

Copyright Holder Springer Science+Business Media, LLC, part of Springer Nature

Author Family Name Sari

Particle

Given Name Tri Komala Suffix

Division Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine

Organization Washington State University Address Pullman, WA, USA

Author Family Name Gianopulos

Particle

Given Name Katrina A.

Suffix

Division School of Molecular Biosciences, College of Veterinary Medicine

Corresponding Author Family Name Nicola Particle

Given Name Anthony V.

Suffix

Division School of Molecular Biosciences, College of Veterinary Medicine

Email [email protected] Abstract

BookID 467481_2_En__ChapID 18_Proof# 1 - 16/7/19

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Conformational changes in viral membrane proteins drive membrane fusion, a critical step in virus entry and infection. Here we describe a simple and rapid virus blotting immunoassay to define conformational changes with a panel of monoclonal antibodies to distinct sites across a viral glycoprotein. This dot blot technique has been utilized to define low pH-triggered changes in the prefusion form of the herpesviral fusogen gB. At pH of <6.2 there are specific changes in herpes simplex virus 1 gB domains I and V. This corresponds broadly to host cell endosomal pH. Many of the identified changes are at least partially reversible. This method can be adapted to document changes in viral proteins that are not fusion proteins, including those induced by alternate triggers such as receptor-binding or protease cleavage.

Keywords

(separated by ‘-’)

Immunoassay - Dot blot - Antibodies - Nitrocellulose membrane - Virus entry - Glycoproteins - Herpesvirus - Herpes simplex virus - Conformational change - Membrane fusion - Low pH

BookID 467481_2_En__ChapID 18_Proof# 1 - 16/7/19

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Chapter 18

1

Conformational Change in Herpes Simplex Virus Entry

2

Glycoproteins Detected by Dot Blot

3

4 AU1

Tri Komala Sari, Katrina A. Gianopulos, and Anthony V. Nicola

Abstract 5

Conformational changes in viral membrane proteins drive membrane fusion, a critical step in virus entry and 6

infection. Here we describe a simple and rapid virus blotting immunoassay to define conformational 7

changes with a panel of monoclonal antibodies to distinct sites across a viral glycoprotein. This dot blot 8

technique has been utilized to define low pH-triggered changes in the prefusion form of the herpesviral 9

fusogen gB. At pH of <6.2 there are specific changes in herpes simplex virus 1 gB domains I and V. This 10

corresponds broadly to host cell endosomal pH. Many of the identified changes are at least partially 11

reversible. This method can be adapted to document changes in viral proteins that are not fusion proteins, 12

including those induced by alternate triggers such as receptor-binding or protease cleavage. 13

Key words Immunoassay, Dot blot, Antibodies, Nitrocellulose membrane, Virus entry, Glycopro- 14

teins, Herpesvirus, Herpes simplex virus, Conformational change, Membrane fusion, Low pH 15

1 Introduction 16

Virus entry is a key process in the viral replication cycle by which the 17

incoming viral particle gains initial access to the host cell. All 18

enveloped viruses must fuse with the target cell membrane to 19

initiate successful entry [1]. The energetically unfavorable merging 20

of two stable lipid bilayer membranes is thought to be driven by the 21

conformational transition from prefusion to postfusion forms of 22

the viral fusion glycoprotein. This major structural rearrangement, 23

or conformational change, results in exposure of hydrophobic 24

fusion peptide sequences. The exposed hydrophobic regions then 25

interact with lipids of the target membrane, which is an essential 26

destabilizing step in the fusion reaction [2]. Conformational 27

changes in viral fusion proteins can be triggered by host cell cues, 28

the most common of which is the low pH environment of an 29

endosomal compartment. Enfuvirtide, a HIV antiviral, blocks a 30

critical conformational change in the viral fusion protein gp41 [3]. 31

Russell J. Diefenbach and Cornel Fraefel (eds.), Herpes Simplex Virus: Methods and Protocols, Methods in Molecular Biology, vol. 2060,https://doi.org/10.1007/978-1-4939-9814-2_18,© Springer Science+Business Media, LLC, part of Springer Nature 2020

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There are biochemical, biophysical, and structural approaches 32

to detecting and evaluating protein conformational change. The 33

binding of a specific monoclonal antibody, for example, can depend 34

on the primary, secondary, or quaternary structure of the antigen. 35

Thus antigenicity as defined by antibody reactivity can be employed 36

to distinguish different conformations of a single protein. Antibo- 37

dy–antigen interactions can be assessed by many immunological 38

techniques, including flow cytometry, enzyme-linked immunosor- 39

bent assay (ELISA), Western blotting, and dot blotting. 40

The Herpesviridae family of large, enveloped DNA viruses 41

causes significant morbidity and mortality. Many different virus- 42

encoded membrane proteins, glycosylated and nonglycosylated, 43

decorate the surface of herpesvirions. They comprise the multicom- 44

ponent machinery that orchestrates receptor-binding, membrane 45

fusion, and entry. Glycoprotein B (gB) is the core fusion protein, 46

but it typically requires two or more additional viral membrane 47

proteins to execute fusion. Glycoprotein B is thought to undergo 48

major structural rearrangements during membrane fusion. The 49

other required glycoproteins play critical triggering and regulatory 50

roles and may also assume multiple conformations, particularly 51

when comparing prefusion and postfusion states. In addition to 52

gB, the gH/gL heterodimer and gD are necessary for herpes 53

simplex virus (HSV) entry and membrane fusion [4–8]. 54

Here we describe a rapid virus blotting (dot blot) immunoassay 55

to define conformational changes with a panel of monoclonal anti- 56

bodies to distinct sites across a viral glycoprotein. Scientists have 57

taken experimental advantage of the ability of proteins to bind to 58

nitrocellulose membranes for many years. In the simplest example 59

of a dot blot, a protein is added to the membrane substrate and 60

passively binds. The dot blot method (also known as slot blot) 61

mirrors Western blotting (or immunoblotting), except that pro- 62

teins are not first separated by electrophoresis. Thus dot blot may 63

allow proteins to be analyzed in a more native state. 64

The prefusion form of HSV-1 gB undergoes low pH-triggered 65

conformational changes that are thought to be fusion-associated 66

[9–12]. These changes are at least partially reversible. In addition to 67

the dot blot approach, gB conformation changes have been 68

detected by polyacrylamide gel electrophoresis, tryptophan fluores- 69

cence spectroscopy, and immunofluorescence microscopy 70

[13–17]. The dot blot approach described here can be adapted to 71

document changes in viral proteins that are not fusion proteins, 72

including those induced by alternate triggers such as receptor- 73

binding or protease cleavage. 74

Tri Komala Sari et al.

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2 Materials 75

1. Dot blot apparatus (such as Minifold I Dot-Blot System, 76

Schleicher and Schuell). 77

2. Nitrocellulose membrane (such as Protran Nitrocellulose Blot- 78

ting Membrane, 0.45 μM pore size). 79

3. Filter paper (such as Whatman 3 MMChromatography Paper). 80 AU2

4. Vacuum source (central or pump) (see Note 1). 81

5. Source of virus antigen (such as extracellular HSV-1 strain KOS 82

virions, ~10⁵–10⁶PFU per dot). 83

6. Mildly acidic pH medium: Dulbecco’s modified Eagle’s 84

medium (DMEM) (bicarbonate-free) solid, 0.2% bovine 85

serum albumin (BSA), 5 mM succinate, 5 mM 2-(N-morpho- 86

lino)ethanesulfonic acid (MES), 5 mM HEPES. Make up to 87

1 L with sterile, cell-culture grade water. Filter and store at 88

4 ^!C. 89

7. Laboratory vortex mixer. 90

8. Laboratory rocker. 91

9. Primary antibody (monoclonal or polyclonal) to viral envelope 92

protein. Mouse monoclonal antibodies to HSV gB, H126, and 93

H1817 (Virusys) are examples used here. 94

10. Horseradish peroxidase (HRP)-conjugated secondary anti- 95

body (e.g., goat anti-mouse HRP). 96

11. Phosphate buffered saline (PBS). 97

12. PBS-T wash buffer: 0.2% Tween 20 in PBS. 98

13. Blocking buffer: 5% nonfat dry milk prepared in PBS-T. Pre- 99

pare just prior to use. 100

14. Distilled water. 101

15. 0.05 N HCl. 102

16. 0.05 N NaOH. 103

17. Water bath, 37^!C. 104

18. Chemiluminescence substrate for HRP. 105

19. Film cassette. 106

20. X-ray film. 107

21. Plastic transparency sheets, tweezer, cutting board, and 108

low-lint, delicate task wipes. 109

22. Automatic film developer in darkroom. 110

Dot Blot Detection of Conformational Change

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3 Methods 111

3.1 Assembly of Dot Blot Apparatus

1. Measure and cut nitrocellulose membrane to the desired size. 112

Handle membrane with forceps and gloved hands. 113

2. Place nitrocellulose membrane in a clean dish, and add PBS to 114

activate. 115

3. Rock membrane for 20 min at room temperature. 116

4. Wet filter paper with PBS. 117

5. Attach filtration plate to vacuum plenum (Fig. 1). Back the 118

nitrocellulose membrane with filter paper and place on top of 119

the filtration plate. Place sample well plate with silicone O-rings 120

facing down on top of the membrane. Clamp the sandwich in 121

place with the adjustable latches (see Note 2). 122

6. Attach vacuum hose to the vacuum plenum on the assembled 123

dot blot apparatus (see Note 3). 124

7. Turn vacuum on for 2 min to test. Turn off vacuum. Remove 125

the sample plate, and verify that there are uniform O-ring 126

indentations on the membrane. This is an indication that 127

proper filtration is achieved. 128

8. Turn vacuum off until samples are ready to be applied. 129

130

Fig. 1 Schematic of assembly for dot blot immunoassay of conformational change of viral entry glycoproteins Tri Komala Sari et al.

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3.2 Antigen Preparation

1. Adjust mildly acidic pH medium to the target pH by adding a 131

predetermined volume of 0.05 N HCl (see Note 4). 132

2. Add HSV-1 to adjusted pH medium to a final concentration of 133

~10⁵PFU per 200 μL sample to be dotted. Vortex briefly. 134

3. Incubate pH-treated virus in 37^!C water bath for 10 min. 135

4. To measure for reversibility of conformational changes, add 136

predetermined amount of 0.05 N NaOH (see Note 5), then 137

incubate virus for another 10 min in a 37 ^!C water bath. 138

Otherwise, virus is added to nitrocellulose membrane directly. 139 140

3.3 Antigen Blotting 1. Turn the vacuum on. Vortex samples briefly and apply sample ¹⁴¹ (200–500μL) to appropriate well. 142

2. Add 200 μL of sample per dot using a micropipette to allow 143

rapid and even dispersal of sample to the membrane. 144

3. Let sample sit with vacuum on while preparing to add blocking 145

buffer. 146

147

3.4 Membrane Blocking

1. Turn off vacuum. Disassemble dot blot apparatus. Release 148

latches, and remove the sample well plate. With forceps, trans- 149

fer nitrocellulose membrane to parafilm. Clean the apparatus 150

properly (see Note 6). 151

2. On a clean cutting board, use scalpel and ruler to trim mem- 152

brane as necessary. Mark with pencil to orient the position of 153

samples. 154

3. Transfer nitrocellulose to a clean dish. Add blocking buffer to 155

cover the surface of the membrane (see Note 7). 156

4. Place dish on a rocker, and rock for at least 20 min at room 157

temperature. 158

159

3.5 Addition of Primary and

Secondary Antibody

1. Prepare primary antibody dilution in blocking buffer. Dilution 160

(ascites or serum) is to be determined empirically, and can 161

range from 1:500 to 1:30,000. If using purified IgG, optimal 162

concentration must also be determined. 163

2. Replace blocking buffer with primary antibody solution. 164

3. Place dish on a rocker and rock overnight at room temperature. 165

4. Discard primary antibody. Perform 3–4 initial washes, by 166

quickly adding and replacing PBS-T wash buffer. 167

5. Wash the membrane by adding wash buffer to dish and rocking 168

for 10 min at room temperature. Remove buffer, and repeat 169

three times. 170

6. Prepare secondary (HRP-conjugated) antibody dilution in 171

blocking buffer. Dilutions typically range from 1:10,000 to 172

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1:30,000. Add sufficient volume to cover surface of nitrocellu- 173

lose membrane. 174

7. Rock for 20–30 min at room temperature. 175

8. Discard secondary antibody. Perform 3–4 initial washes, by 176

quickly adding and replacing PBS-T wash buffer. 177

9. Wash the membrane, by adding PBS-T wash buffer to dish and 178

rocking for 10 min at room temperature. Remove buffer, and 179

repeat three times. 180

181

3.6 Developing Blot 1. Immediately prior to use, prepare chemiluminescence HRP ¹⁸² substrate according to manufacturer’s instructions. Prepare 183

~20μL substrate per dot. 184

2. Discard wash buffer. Using forceps, gently blot membrane with 185

wipe to remove excess substrate. 186

3. Transfer membrane to a plastic transparency sheet. 187

4. Pipette substrate evenly across the surface of membrane. 188

5. Quickly place another transparency sheet on top, and use 189

gloved fingers to press across the membrane gently and thor- 190

oughly to allow even distribution of substrate. Avoid air 191

bubbles. 192

6. With forceps, transfer membrane to between two new trans- 193

parency sheets. Place in film cassette. 194

7. In darkroom, place film on top and seal cassette. Expose for 195

desired period of time. Develop film in automatic processor. 196

8. Completed dot blot experiments have indicated that HSV-1 gB 197

undergoes conformational changes in response to mildly acidic 198

pH (Fig. 2), and that these changes are partially reversible 199

(Fig. 3). 200

201

Fig. 2 Dot blots of HSV-1 strain KOS probed with monoclonal antibodies to gB. Virions were treated for 10 min at 37 ^!C with medium buffered to the indicated pHs and were blotted immediately to membrane. Blots were probed at neutral pH with the indicated gB-specific antibodies, followed by horseradish peroxidase-conjugated goat secondary antibody. Decreased reactivity of acid- treated HSV with H126 indicates conformational change (see Note 8) (Reproduced from ref. 9 with permission from the American Society for Microbiology)

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4 Notes 202

1. A reliable, strong vacuum source is recommended for quality 203

dots that are well-defined and reproducible. 204

2. Adjust the tightness of the latches so proper tension is 205

achieved. Overtightening or undertightening will affect how 206

the vacuum is distributed to the sample well plate. 207

3. Before blotting samples, apply a volume of PBS to a control 208

well to verify that vacuum filtration is working properly. 209

4. Prior to experiment, determine the volume of 0.05 N HCl 210

necessary to adjust the medium to the pH to be tested. 211

5. Prior to experiment, determine the volume of 0.05 N NaOH 212

necessary to return the sample to the initial pH, typically 213

pH 7.2–7.6. 214

6. Rinse wells thoroughly with 70% ethanol using a squirt bottle. 215

Wash thoroughly with soapy water. Do not soak, as warping of 216

plates and O-rings may occur. Rinse thoroughly with distilled 217

water. Air-dry apparatus. 218

7. Use as small a container as possible to reduce the volume of 219

antibody solution needed. This will increase the liquid to 220

membrane surface ratio. 221

8. Immunoassays such as the dot blot benefit greatly from mono- 222

clonal antibody development efforts and extensive epitope 223

mapping studies (see ref.18). 224

Acknowledgments 225

This work was supported by Public Health Service grants 226

AI119159 and GM008336 from the National Institutes of Health. 227

Fig. 3 Dot blot illustrating partial reversibility of conformational change in HSV-1 gB. HSV-1 KOS virions were treated with medium buffered to pH 7.2 or 5.5. For the indicated samples, pH was neutralized back to 7.2 for 10 min at 37 ^!C.

Membranes were probed at neutral pH with the indicated antibodies, followed by horseradish peroxidase-conjugated secondary antibody (Reproduced from ref.9 with permission from the American Society for Microbiology)

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228 References

230 1. Nicola AV, Aguilar HC, Mercer J, Ryckman B,

231 Wiethoff CM (2013) Virus entry by endocyto-

232 sis. Adv Virol 2013:469538

233 2. White JM, Whittaker GR (2016) Fusion of

234 enveloped viruses in endosomes. Traffic 17

235 (6):593–614

236 3. Warnke D, Barreto J, Temesgen Z (2007)

237 Antiretroviral drugs. J Clin Pharmacol 47

238 (12):1570–1579

239 4. Cai WH, Gu B, Person S (1988) Role of glyco-

240 protein B of herpes simplex virus type 1 in viral

241 entry and cell fusion. J Virol 62(8):2596–2604

242 5. Forrester A et al (1992) Construction and

243 properties of a mutant of herpes simplex virus

244 type 1 with glycoprotein H coding sequences

245 deleted. J Virol 66(1):341–348

246 6. Johnson DC, Ligas MW (1988) Herpes sim-

247 plex viruses lacking glycoprotein D are unable

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249 dence for virus-specific cell surface receptors.

250 J Virol 62(12):4605–4612

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252 requirements for rapid endocytic entry of her-

253 pes simplex virus. J Virol 78(14):7508–7517

254 8. Weed DJ, Nicola AV (2017) Herpes simplex

255 virus membrane fusion. Adv Anat Embryol

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257 9. Dollery SJ, Delboy MG, Nicola AV (2010)

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260 84(8):3759–3766

261 10. Roller DG, Dollery SJ, Doyle JL, Nicola AV

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264 from-without activity. Virology 382

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triggers an irreversible conformational change 268

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mildly acidic pH. Virol J 7:352 279

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16. Wudiri GA, Schneider SM, Nicola AV (2017) 291

Herpes simplex virus 1 envelope cholesterol 292

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8:2383 294

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Conformational Change in Herpes Simplex Virus Entry Glycoproteins Detected by Dot Blot

M

M

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Herpes Simplex Virus

Methods and Protocols

Second Edition

Edited by

Russell J. Diefenbach

Department of Biomedical Sciences, Macquarie University, Sydney, NSW, Australia

Cornel Fraefel

Institute of Virology, University of Zurich, Zürich, Switzerland

Preface

Contents

Contributors

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Chapter 18

Conformational Change in Herpes Simplex Virus Entry

Glycoproteins Detected by Dot Blot

Tri Komala Sari, Katrina A. Gianopulos, and Anthony V. Nicola

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