Table Of ContentHERPESVIRUS DNA
DEVELOPMENTS IN MOLECULAR VIROLOGY
VOLUME 1
Yechiel Becker, series editor
Series ISBN 90-247-2444-9
Herpesvirus DNA
Recent studies on the organization of viral genomes, mRNA
transcription, DNA replication, defective DNA, and viral DNA sequences
in transformed cells and bacterial plasmids
Edited by
YECHIEL BECKER
The Hebrew University of Jerusalem, Israel
I I
1981
SPRINGER-SCIENCE+BUSINESS MEDIA, B.V.
ISBN 978-94-015-6899-9 ISBN 978-94-015-6897-5 (eBook)
DOI 10.1007/978-94-015-6897-5
Copyright © 1981 by Springer Science+Business Media Dordrecht
Ursprünglich erschienen bei Martinus Nijhoff Publishers, The Hague 1981
Sollcoverreprint ofthe hardcoverist edition 1981
All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or
transmitted in any form or by any means, mechanical, photocopying, recording, or otherwise,
without written permission of the publisher, Springcr-Scicnce+Business Media, B. V.
v
PREFACE
Herpesviruses, classified in the family Herpesviridae, are
important human and animal pathogens that can cause primary,
latent or recurrent infections and even cancer. The major
interest in research on herpesviruses today focuses on
understanding the organization of the DNA genome, as well
as on characterizing the viral genes in regard to their
control and function. Modern techniques have allowed the
viral DNA to become a molecular tool in the study of gene
function, since it is now possible to implant the DNA into
eukaryotic cells.
This book contains original studies on the structure
and organization of the DNA of human and animal herpes
viruses. The various chapters acquaint the reader with the
organization of the viral DNA, the mRNA transcripts, the
replicative intermediates of the viral DNA, defective DNA
genomes and their mode of synthesis, analyses of the viral
DNA sequences in transformed cells, and the relationship
between the presence of viral DNA fragments in the cancer
cells and the transformed state of the cells.
An attempt has been made to provide insight into the
present experimental approach and to update the reader on
the use of the most advanced molecular techniques like
restriction enzyme analysis, blotting of viral nucleic
acids, cloning of viral DNA fragments in bacterial plasmids,
and transfection of cells with both cloned and uncloned
viral DNA fragments. These studies will give the reader a
clear perspective of the philosophy and technology of
herpesvirus DNA research. The book will thus be of great
value to all who are interested in understanding the mole
cular aspects of herpesvirus DNA and the biological proper
ties of the viral genes.
I wish to thank all the contributors to this volume
VI
which, I hope, will serve as a stimulus to scientists
engaged in research on herpesviruses. To my regret, cer
tain chapters which were promised did not arrive. The
assistance of Dr. Julia Hadar with editing and indexing
of the manuscripts and the excellent secretarial help of
Mrs. Esther Herskovics are much appreciated.
Yechiel Becker
Jerusalem, January 1981
VII
CONTENTS
Preface v
list of Contributors xii
1. Introduction: Current trends in herpesvirus DNA research (a review)
Y. Becker
Herpesvirus genomes have unique features 1
Restriction enzyme cleavage patterns in parental and recombinant DNA 3
Replication of herpesvirus DNA 5
Herpesvirus DNA sequences in transformed cells 7
Studies using cloned herpesvirus DNA sequences 7
HUMAN HERPESVIRUSES
Herpes simplex virus DNA structure
2. Structure ofHSV-1 DNA at the joint regions
M.J. Wagner and W.C. Summers
Introduction 11
Experimental 12
Discussion 21
Transcription of the viral DNA
3. Immediate-early transcription ofHSV-1 and HSV-2
J .B. Clements, A.J. Easton and F .J. Rixon
The immediate early (IE) replication phase 27
Genome map locations, orientations, sizes and polypeptides encoded
by IE mRNA's 28
Fine structure analysis ofHSV IE mRNA's 30
Overall conclusions 42
4. Isolation and characterization ofHSV-1 mRNA
E.K. Wagner, K.P. Anderson, R.E. Costa, G.B. Devi,
B.H. Gaylord, L.E. Holland, J .R. Stringer and L.L. Tribble
Summary/Introduction 45
Experimental approach 46
Populations ofHSV-1 mRNA abundant at different stages of infection 48
Mapping HSV-1 mRNA abundant at different stages of infection 50
Isolation and characterization of specific HSV-1 mRNA species 57
Discussion 63
Replication of viral DNA
5. Properties of the replicating HSV DNA
I. Hirsch
Introduction 69
Single-stranded regions in replicating HSV DNA molecules 70
The processing of HSV DNA in infected cells 73
Topology of the replicating HSV DNA molecule 74
Concluding remarks 79
VIII
6. Electron microscopy of branched HSV DNA molecules: Possible
recombination intermediates
A. Friedmann, S. Rabkin andY. Becker
Introduction 85
Experiment& 86
Branched dsDNA molecules 86
dsDNA molecules connected by a single-stranded component 87
Discussion 89
7. Replication ofHSV-1 DNA: Isolation of a subnuclear DNA
synthesizing fraction
S. Rabkin, Y. Shtram andY. Becker
Summary/Introduction 95
Experiment& 96
Isolation of a subnuclear fraction from HSV-1 infected nuclei 98
Isolation of DNA synthesizing subnuclear fraction in sucrose layers 100
Properties of the subnuclear fraction from nuclei synthesizing
defective DNA 100
Discussion 103
Defective viral DNA
8. Tandem repeat defective DNA from the L segment of the HSV genome
D.M. Cuifo and G.S. Hayward
Summary 107
Introduction 108
Serial passaging to generate defective virus 109
Characterization of the MP&8 repeat unit 112
The MPal8 repeat unit originates from the L component of the parent
genome 115
Cleavage maps of the MPal8 repeat unit 117
Packaged defective DNA contains an exactly integrru number of
repeat units 118
Homology between the L segment repeat unit and sequences in the
S segment of the parent genome 120
Discussion 121
9. Structure and physic& mapping of different classes of defective HSV-1
ANGDNA
H.C. Kaerner
Summary 129
Introduction 130
Experiment& approach 130
The DNA of the parent& virus strain HSV-1 ANG 132
Locruization of the nucleotide sequences of defective HSV-1 ANG DNA
on the parental viral genome 133
The structural organization of defective HSV-1 ANG DNA 141
Discussion 144
10. Structure and expression of Class I and II defective interfering HSV
genomes
N. Frenkel, H. Locker and D. Vlazny
Introduction 149
Derivation of the serially passaged virus stocks 150
The structure of the variant genomes 151
Studies of defective genome replication 158
IX
Models for the replication of defective HSV genomes 163
Models for the evolution of defective genomes 164
The expression of defective HSV genomes 167
Biologic properties of virus populations containing Class I defective
virus genomes 169
Biologic properties of serially passaged virus populations containing
Class II defective genomes 172
General aspects of the biologic properties of serially passaged HSV
populations 176
Aspects of ICP overproduction 177
The mechanism of interference 178
11. Biosynthesis of defective HSV DNA
S. Rabkin and Y. Becker
Summary /Introduction/Experimental 185
Defective viral DNA 187
Electron microscopy of defective DNA 189
Fate of prelabeled viral DNA during defective DNA biosynthesis 191
Discussion 193
HSV DNA fragments in herpesvirus transformed cells
12. Mapping of the HSV sequences in transformed cells
J.M. Leiden and N. Frenkel
Introduction 197
Characterization of the mapping approach 200
Mapping of the HSV-1 and HSV-2 tk genes 203
Comparison of the HSV DNA sequences present in stable and unstable
HSV-1 tk+ cells 206
Comparison of the HSV DNA sequences present in an unstable
HSV-1 tk+ cell line with those present in tk" revertant and tk+
rerevertant derivative cell lines 210
Mapping of the viral DNA sequences present in hamster cells
morphologically transformed with the Xbai F fragment of HSV-1 DNA 210
Discussion 212
13. Transfection with HSV DNA fragments and DNA from HSV trans
formed cells
G. Darby, K.F. Bastow and A.C. Minson
General introduction 223
The generation of 'biochemically' transformed cells 226
Properties of 'biochemically' transformed cells 227
Isolation of revertant cells with TK-phenotype 241
Transformed cell DNA as a TK gene donor 246
Summary and conclusions 249
14. Viral genes in HSV transformed cells as detected genetically
J.C.M. Macnab
Summary and introduction 255
Experimental approach and results 256
Discussion 267
