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Advances in Anatomy, Embryology and Cell Biology
Klaus Osterrieder Editor
Cell Biology
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Klaus Osterrieder
Editor
Cell Biology of
Herpes Viruses
Editor
Klaus Osterrieder
Institut für Virologie
Freie Universität Berlin
Berlin, Germany
ISSN 0301-5556 ISSN 2192-7065 (electronic)

Advances in Anatomy, Embryology and Cell Biology
ISBN 978-3-319-53167-0 ISBN 978-3-319-53168-7 (eBook)
DOI 10.1007/978-3-319-53168-7
Library of Congress Control Number: 2017941254
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Preface
Viruses belonging to the order Herpesvirales are found in many species and all
around the world. It is commonly surmised that herpesviruses are species specific
and have a relatively long history of coevolution with their respective hosts
(Davison 2002). In addition, there are numerous examples where different herpes-
viruses have been found in one species. For example, in humans we know nine
different herpesviruses that are members of the Alpha- (3), Beta- (4), or
Gammaherpesvirinae (2) subfamilies, respectively, in the Herpesviridae family
(Davison et al. 2009; Roizman 1996). Although the dogma of species specificity
is regularly challenged and has become more and more questionable as we retrieve
and analyze more and more virus sequences from animals of different taxa, the
nomenclature of the Herpesvirales remains organized such that it reflects the host
species from which it is isolated and which is considered the definitive host
(Davison et al. 2009).
Regardless of nomenclature and species specificities, all herpesviruses share
fundamental biological principles. The signature lifestyle of herpesviruses includes
phases of lytic replication with the production of fully infectious progeny that are
clearly distinct from phases of quiescence, commonly referred to as latency (Arvin
et al. 2007). The herpesvirus lifestyle means that “herpesviruses are forever”: once
a host is infected, the virus stays with it for prolonged periods of time, yes, we
believe for the lifetime of the infected individual (Field et al. 2006; Preston and
Efstathiou 2007). While formally this is hard to prove and also challenged from
time to time, it is clear that primary infection, latency, and reactivation or recru-
descence from the latent state are central to herpesvirus epidemiology and mainte-
nance in populations (Bennett and Gilden 1996; Grinde 2013).
In this issue of Advances in Anatomy and Embryology, experts in the herpesvi-
rus field describe and shed new light on the cellular processes that herpesviruses
employ to manipulate host cells for their quest of survival. Being enveloped viruses,
herpesviruses attach to the plasma membrane and then enter target cells by fusion
(discussed in Chaps. 1 and 2). The next encounter is with the host cell’s innate
defenses (Chap. 3), before the nucleocapsid travels to nuclear pores and releases its
genome into the nucleus. Here, replication occurs after co-opting and partially
v
vi Preface
disarming antiviral functions in the nucleus (Chap. 4), and new nucleocapsids are
formed (Chap. 6), before newly produced virions have to leave the nucleus, which
they do by fusion with the inner nuclear membrane (Chap. 7). After
de-envelopment at the outer nuclear membrane and loss of the “primary” envelope,
the viruses make their way out of the infected cells (Chap. 8), which then allows
them to spread to new individuals (Chap. 9). However, as outlined before, in some
instances and dependent on the cell type, latency is established after DNA is
released into the nucleus, a condition characterized by the presence and mainte-
nance of viral genomes but without lytic viral gene expression (Chap. 5).
In summary, experts in the herpesvirus field here report on the recent advances in
knowledge of interaction between herpesviruses and their target cells. The authors
describe their work and that of others and how cell biology, biochemistry, and
genetics help us to continuously increase and refine our understanding of the very
peculiar pathogen–host interactions.
Berlin, Germany Klaus Osterrieder
References
Arvin A, Campadelli-Fiume G, Mocarski E, Moore PS, Roizman B, Whitley R, Yamanishi K

(2007) Human Herpesviruses: biology, therapy, and immunoprophylaxis. Cambridge
University Press, Cambridge, UK
Bennett JL, Gilden DH (1996) The molecular genetics of herpes simplex virus latency and
pathogenesis: a puzzle with many pieces still missing. J Neurovirol 2:225–229
Davison AJ (2002) Evolution of the herpesviruses. Vet Microbiol 86:69–88
Davison AJ, Eberle R, Ehlers B, Hayward GS, McGeoch DJ, Minson AC, Pellett PE, Roizman B,
Studdert MJ, Thiry E (2009) The order Herpesvirales. Arch Virol 154:171–177
Field HJ, Biswas S, Mohammad IT (2006) Herpesvirus latency and therapy – from a veterinary
perspective. Antiviral Res 71:127–133
Grinde B (2013) Herpesviruses: latency and reactivation - viral strategies and host response. J Oral
Microbiol 5
Preston CM, Efstathiou S (2007) Molecular basis of HSV latency and reactivation. In: Arvin A,
Campadelli-Fiume G, Mocarski E, Moore PS, Roizman B, Whitley R, Yamanishi K (eds)
Human Herpesviruses: biology, therapy, and immunoprophylaxis. Cambridge University
Press, Cambridge, UK
Roizman B (1996) Herpesviridae. In: Field BN, Knipe DM, Howley PM, Channock RM, Melnick
JL, Monath TP, Roizman B, Straus SE (eds) Virology, 3rd edn. Lippincott-Raven,
Philadelphia, NY
Contents
1 Initial Contact: The First Steps in Herpesvirus Entry . . . . . . . . . . . 1

Walid Azab and Klaus Osterrieder
2 Herpes simplex virus Membrane Fusion . . . . . . . . . . . . . . . . . . . . . . 29
Darin J. Weed and Anthony V. Nicola
3 Innate Immune Mechanisms and Herpes Simplex Virus
Infection and Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Evelyn A. Kurt-Jones, Megan H. Orzalli, and David M. Knipe
4 The Human CMV IE1 Protein: An Offender of PML
Nuclear Bodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Myriam Scherer, Eva-Maria Schilling, and Thomas Stamminger
5 Herpesvirus Latency: On the Importance of Positioning
Oneself . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Patrick Lomonte
6 Herpesvirus Capsid Assembly and DNA Packaging . . . . . . . . . . . . . 119
Jason D. Heming, James F. Conway, and Fred L. Homa
7 Herpesvirus Nuclear Egress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
Richard J. Roller and Joel D. Baines
8 Assembly and Egress of an Alphaherpesvirus Clockwork . . . . . . . . . 171
Gregory A. Smith
9 Interindividual Spread of Herpesviruses . . . . . . . . . . . . . . . . . . . . . . 195
Keith W. Jarosinski
vii
Chapter 1
Initial Contact: The First Steps
in Herpesvirus Entry
Walid Azab and Klaus Osterrieder
1.1 Introduction
Herpesviruses constitute a large family of DNA viruses that can infect a wide variety
of species of at least two animal phyla, the Chordata (mammals, birds, fishes,
reptiles, and amphibians) and the Mollusca (oysters) (Pellet and Roizman 2007).
Herpesviruses not only infect a large diversity of hosts, they also enter and replicate
in a broad spectrum of cell types within the same host. Hence, the viruses have
evolved successful approaches to enter and manipulate different cell types. It is
surprising, therefore, that herpesviruses tend to be closely associated with a specific
host species. However, this concept is challenged recently as some herpesviruses
seem to be endemic in more than one species, observations that go beyond the well-
known species jumps and often fatal diseases in non-definitive hosts (Abdelgawad
et al. 2014; Greenwood et al. 2012; Wohlsein et al. 2011; Huff and Barry 2003).
Based on their biological properties and genome sequences, the family Herpesviridae
is divided into three subfamilies (Alphaherpesvirinae, Betaherpesvirinae, and
Gammaherpesvirinae) (Davison et al. 2009). The Alphaherpesvirinae subfamily con-
tains five recognized genera featuring 37 different species, of which three virus species
routinely infect humans: herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) and
varicella-zoster virus (VZV). The remaining virus species (in total 34) infect different
animals and can cause mild to severe disease, which are often associated with economic
losses. A characteristic feature common to all herpesvirus infections is the establishment
of latent infections, a state from which virus can be reactivated and result in recurring
disease. The diseases associated with alphaherpesviruses differ, but usually range from
mild skin lesions, respiratory and reproductive disorders, and neurological disorders to
W. Azab (*) • K. Osterrieder

Institut für Virologie, Robert von Ostertag-Haus, Zentrum für Infektionsmedizin, Freie
Universität Berlin, Robert-von-Ostertag-Str. 7-13, 14163 Berlin, Germany
e-mail: wfazab@zedat.fu-berlin.de
© Springer International Publishing AG 2017 1

K. Osterrieder (ed.), Cell Biology of Herpes Viruses, Advances in Anatomy,
Embryology and Cell Biology 223, DOI 10.1007/978-3-319-53168-7_1
2 W. Azab and K. Osterrieder
even tumors and death. Since virus infection starts with virus binding and entry into
target cells, we will focus, in this chapter, on the mechanisms and signaling cascades
associated with the entry of alphaherpesviruses into different cells.
To set the stage, the entry of herpesviruses into target cells is complex and,
obviously, requires synergisms between viral and cellular molecules. In contrast to
small enveloped viruses that have one or two glycoproteins to mediate entry,
herpesviruses encode for more than a dozen glycoproteins, several of which play
a role in entry. This multipartite nature of the entry machinery, which has been
evolved over long periods of time, endowed the virus with many opportunities and
choices that are “ready” and “capable” to ascertain successful entry according to
cell type. Herpesvirus entry is a coordinated process that involves a sequence of
events and can be separated into (1) attachment or tethering to the cell surface,
(2) binding to specific cell receptors, (3) intracellular signaling, and (4) fusion of the
viral envelope with cellular membranes (Campadelli-Fiume and Menotti 2007;
Connolly et al. 2011; Spear and Longnecker 2003). More details on the fusion
process are provided in Chap. 2 of this series: Herpes Simplex Virus Membrane
Fusion by Darin J. Weed and Anthony Nicola.
1.2 Attachment to Cells
Herpesvirus attachment to the cell surface is a charge-based and a relatively

nonspecific process, by which viral envelope glycoproteins (mainly gC and gB in
the case of alphaherpesviruses) associate reversibly with cell surface heparan
sulfate proteoglycans (HSPG) and chondroitin sulfate proteoglycans (CSPG)
(Banfield et al. 1995; Spear and Longnecker 2003). In dendritic cells (DC),
C-type lectin (DC-SIGN) can bind to both gB and gC and facilitate initial HSV-1
attachment (de Jong et al. 2008). Similar to HSV-1, binding to DC-SIGN was also
detected in the case of HSV-2; however, there was no direct assay to confirm the
interaction between HSV-2 gB and/or gC and DC-SIGN (de Jong et al. 2008). This
charge-based contact serves the concentration of virus particles at the cell surface,
but is not sufficient to trigger virus entry.
In the case of HSV-1 and HSV-2 (Herold et al. 1991; WuDunn and Spear 1989),
VZV (Zhu et al. 1995; Jacquet et al. 1998), pseudorabies virus (PRV) (Mettenleiter
et al. 1990), bovine herpesvirus 1 (BHV-1) (Okazaki et al. 1991), equine herpesvirus
1 (EHV-1), and EHV-4 (Osterrieder 1999; Azab et al. 2010) virions tether to cells
after interaction with heparan sulfate through either gC or gB. Several studies
showed that (1) binding to cell surfaces devoid of heparan sulfate is significantly
reduced (Gruenheid et al. 1993; Herold et al. 1994; Mettenleiter et al. 1990; Okazaki
et al. 1991; Osterrieder 1999; Shieh et al. 1992; WuDunn and Spear 1989; Azab et al.
2010), (2) heparin inhibits virion binding to cells (Herold and Spear 1994; Herold
et al. 1991; Lycke et al. 1991; Okazaki et al. 1991; WuDunn and Spear 1989), and
(3) deletion of the gC encoding genes results in virions that are impaired in their
ability to bind to cells (Liang et al. 1991; Osterrieder 1999; Schreurs et al. 1988). In
1 Initial Contact: The First Steps in Herpesvirus Entry 3
the case of HSV-2, gB is the key glycoprotein in viral binding with HSPG (Gerber
et al. 1995). Commensurate with the obvious redundancy in the system and although
gC plays the predominant role in viral attachment during the early steps of infection,
gC is dispensable for virus growth. Generally, gC-negative mutants are still infec-
tious, but exhibit decreased efficiency of virus binding and infectivity compared to
wild-type viruses (Herold et al. 1991; Spear 2004).
Heparan sulfate (HS) is among the most highly negatively charged biopolymers
in nature and is abundantly expressed on the cell surface of almost all cell types
(Sarrazin et al. 2011). Both gB and gC have been shown to have several positively
charged amino acid residues that enable electrostatic interaction with the negatively
charged HS moieties. However, the relative contributions of viral proteins to HSPG
binding are difficult to assess. It was reported that HSV-1 gB contains a polylysine
region (68-KPKKNKKPK-76) that is responsible for HS binding (Laquerre et al.
1998). HSV-1 and HSV-2 gBs exhibit an 85% identity, with the most variability
seen in this lysine-rich sequence. This might explain the differences between the
two strains in the key glycoprotein required for HS binding (Herold et al. 1996). On
the other hand, a cluster of hydrophobic arginine residues localized at the cysteine
127-cysteine 144 loop were shown to be required for HSPG interaction with the
most active amino acids situated near the C-terminal region of the two cysteines
(Mardberg et al. 2001). This is consistent with the concept of electrostatic attrac-
tion: negatively charged heparan sulfate disaccharides bind virions to the plasma
membrane via their positively charged basic residues. Interestingly, it seems that
gB and gC do not bind the same receptor unit and that gC from different viruses
attach to distinct surface molecules (Trybala et al. 2000).
Besides providing attachment sites for herpesviruses, HS can act also as a
mediator of virus transport along filopodia. It was shown that filopodia express
HS that can interact with HSV-1 gB and transport the virions in a surfing-like
phenomenon toward cell bodies, where nectin-1 is mainly expressed, for subse-
quent entry events, be it directly at the plasma membrane or from endosomes. This
process is facilitated by the actin cytoskeleton and transient activation of small
GTPases. Filopodia also form bridges between cells providing carriageways to help
transfer of extracellular HSV-1 virions from infected to uninfected cells (Oh et al.
2010; Dixit et al. 2008; Lehmann et al. 2005). The involvement of HS in this
process suggests the intriguing probability that virions in general may exploit
interactions with cellular HS for targeted transport to either the cell body or
neighboring uninfected cells.
1.3 Receptor Binding
Recently, our understanding of the entry process of alphaherpesviruses has

increased due to an ever-growing body of work contributed by many laboratories.
From the virus side, there are a handful of glycoproteins that are responsible for all
entry events, starting from receptor binding and ending by nucleocapsid release into
the cytoplasm. Either all or some of the glycoproteins, depending on virus species,
show diversity with regard to binding to multiple cellular receptors that is depen-
dent on cell type. However, in all cases, this diversity leads to a conserved fusion
process that takes place either on the plasma membrane or from within endosomes
(Table 1.1).
1.3.1 gD-Binding Receptors
With the exception of VZV, which lacks the gD homolog in its genome (Davison
and Scott 1986), and Marek’s disease virus (MDV), which does not express gD in
cell culture (Tan et al. 2001), gD is conserved among the alphaherpesviruses and is
essential for virus entry (Campadelli-Fiume and Menotti 2007; Heldwein and
Krummenacher 2008; Krummenacher et al. 2013; Spear 1993). However, one
exception of this rule was reported recently for herpes B virus (Macacine herpes-
virus 1) (Perelygina et al. 2015). Although gD is functionally active and mediates
herpes B virus entry, it is not essential for entry into target epithelial and fibroblast
cells. In other words, B virus seems to have two entry strategies, a gD-dependent
and gD-independent entry pathway. However, the viral glycoprotein that mediates
the latter pathway is not identified yet (Perelygina et al. 2015).
1.3.1.1 Herpes Simplex Viruses
In HSV-1 and HSV-2, four gD receptors have been identified: a member of the
tumor necrosis factor (TNF) receptor family (HVEM); the poliovirus receptor
family, nectin-1 and nectin-2, which belong to the immunoglobulin superfamily;
and a modified form of heparan sulfate (3-O-sulfated heparan sulfate, 3-O-HS)
(Geraghty et al. 1998; Montgomery et al. 1996; Shukla et al. 1999; Warner et al.
1998). However, the preference of gD of either virus to each receptor is different.
HVEM and nectin-1 are used by both viruses, regardless of the clinical strains or the
origin of the virus (Krummenacher et al. 2004). Nectin-2, on the other hand, has
been shown to mediate entry for HSV-2 and only some HSV-1 mutants (Rid1
mutant) that do not exhibit gD-mediated restriction of entry (Warner et al. 1998).
Finally, 3-O-HS can only be utilized by HSV-1 (Shukla et al. 1999).
HVEM was the first gD receptor to be identified and is expressed on the surface
of different cell types, including T and B lymphocytes, epithelial cells, and fibro-
blasts present in human tissues of the lung, liver, and kidney (Montgomery et al.
1996; Spear 2004). The gD core comprises a V-like immunoglobulin fold that
flanked by an N-terminal hairpin loop and a long C-terminal extension. While the
N-terminal residues contain all gD receptor contact sites, the C-terminal extension
anchors the protein to the viral envelope and carries the profusion domain. The
HVEM binding site is limited to a very narrow region (aa 7–32) within the gD
N-terminus. On the HVEM side, the first two cysteine-rich domains (CRD1
Table 1.1 Glycoproteins of alphaherpesviruses and their cellular receptors

Virus Glycoprotein Function Host receptors
HSV-1 gC • Attachment • Heparan sulfate
• DC-SIGN
gD • Binds cells • HVEM
• Triggers fusion • Nectin-1
• Nectin-2
• 3-OS HS
• ZF-3-OS HS
gH/gL • Regulates fusion • αVβ3 integrin
• Activates gB • αVβ6 integrin
• αVβ8 integrin
gB • Attachment • Heparan sulfate
• Binds cells • DC-SIGN
• Catalyzes membrane fusion • PILRα
• MAG
• NMMHC-IIA
• NMMHC-IIB
HSV-2 gC • Attachment • Heparan sulfate
gD • Binds cells • HVEM
• Nectin-2
gB • Catalyzes membrane fusion • Heparan sulfate
VZV gE • Binds cells • IDE
• MPRci
gH • Binds cells • MPRci
• Binds cells • MAG
• Catalyzes membrane fusion • MPRci
gI • Binds cells • MPRci
EHV-1 gC • Attachment • Heparan sulfate
gD • Binds cells • MHC-I
• Triggers fusion
gH/gL • Triggers or regulates fusion • α4β1 integrin
• Catalyzes membrane fusion
EHV-4 gC • Attachment • Heparan sulfate
gD • Binds cells • MHC-I
• Triggers fusion
• Catalyzes membrane fusion
PRV gC • Attachment • Heparan sulfate
gD • Binds cells • Nectin-1
• PVR (CD155)
gB • Binds cells • PILRα
• Catalyzes membrane fusion • NMMHC-IIA
BHV-1 gC • Attachment • Heparan sulfate
gD • Binds cells • Nectin-1
BHV-5 gD • Binds cells • Nectin-1
Herpes B virus gD • Binds cells • Nectin-1
CeHV-2 gD • Binds cells • Nectin-1
residues 27–29 and 35–37 and CRD2 residues 74–76) are necessary and sufficient
for gD binding. The binding hot spot is mainly centered around the prominent
HVEM tyrosine (Y23) that protrudes into a pocket on the surface of gD (Carfi et al.
2001; Connolly et al. 2002, 2003).
Nectin-1 is broadly expressed in virtually all human tissues, including the central
nervous system, ganglia, and skin, but also in cell lines, including epithelial cells,
fibroblasts, endothelial cells, and keratinocytes (Campadelli-Fiume et al. 2000;
Linehan et al. 2004; Spear et al. 2000). The important domain of gD that interact
with nectin-1 (aa 35–38, 215, and 222–223) is topologically distinct from that
interacting with HVEM, with Y38 residue being the most critical position for
binding (Di Giovine et al. 2011). It was also shown that gD contacts exclusively
with one β-sheet of nectin-1 V-domain C00 C0 CFG residues. In particular, a promi-
nent interaction involves phenylalanine (F129), at the tip of the FG loop, which
protrudes into a pocket of gD (Di Giovine et al. 2011).
Nectin-2, which is also expressed on a wide variety of human tissues and cell
lines (Campadelli-Fiume et al. 2000), is considered a weak receptor for HSV-2 and
inactive for HSV-1 wild type (Krummenacher et al. 2004; Lopez et al. 2000;
Warner et al. 1998). A single amino acid substitution in gD rendered the
so-called Rid1 HSV-1 mutant capable of binding to nectin-2 without affecting its
ability to use nectin-1 but not HVEM (Connolly et al. 2003; Yoon et al. 2003).
Nectin-1, nectin-2, and the poliovirus receptor (PVR; CD155) also serve as
receptors for entry of PRV mediated by gD (Geraghty et al. 1998; Lopez et al.
2000; Warner et al. 1998; Connolly et al. 2001). Furthermore, nectin-1 was shown
to interact with BHV-1 and BHV-5 gD to facilitate virus entry (Geraghty et al.
1998; Gabev et al. 2010; Alves Dummer et al. 2014). However, BHV-5 gD seems to
interact with a wide range of cellular receptors compared to BHV-1 (Alves
Dummer et al. 2014). A recent study reported that productive infection of two
primate herpesviruses, herpes B virus and cercopithecine herpesvirus 2 (CeHV-2),
can be established only with nectin-1 (but not HVEM)-expressing cells, indicating
that nectin-1 serves as a receptor for these two viruses (Fan et al. 2012; Fan and
Longnecker 2012).
3-OS HS is a highly sulfated modified form of HS, which is present on the
surface of cells of different origins. HSV-1 gD binds to 3-OS, which can initiate
virus entry (Shukla et al. 1999; Shukla and Spear 2001). In addition, 3-OS HS
seems to play an important role in HSV-1 entry into primary corneal fibroblast
cultures (Tiwari et al. 2006). The sites on HS recognized by gD are generated by a
number of heparan sulfate d-glucosaminyl 3-O-sulfotransferase (3-OST) isoforms,
which can produce 3-OS HS with unique functions that depend on the modification
(O’Donnell and Shukla 2008). The gD crystal structure revealed a positively
charged pocket proximal to the N-terminus that seems to have a role in 3-OS HS
binding. Mutating this region significantly reduced binding of gD to 3-OS HS and
HVEM receptors, indicating that at least one 3-OS HS binding region of gD may
overlap with HVEM binding sites (Yoon et al. 2003). It was shown recently that
HSV-1 can infect nectin-1- and HVEM-deficient murine dermal fibroblasts,
although it was severely delayed. These results suggest the presence of a second
more inefficient receptor, possibly 3-OS HS, which can mediate virus entry into
these fibroblasts (Petermann et al. 2015a).
Recent studies provided evidence that some isoforms of zebrafish (ZF)-encoded
3-OSTs can modify HS, which can bind to HSV-1 gD and mediate virus entry and
spread (Antoine et al. 2014; Baldwin et al. 2013; Hubbard et al. 2010). Although the
amino acid sequences of ZF-3-OST enzymes show various degrees of similarity to
human 3-OST isoforms, the catalytic residues and the substrate-binding sites are
highly conserved (reached up to 100%) (Yakoub et al. 2014). This can explain the
ability of ZF-3-OSTs to mediate HSV-1 entry since both the catalytic residues and
the substrate-binding sites have been shown to be important for HSV-1 entry-
mediating activity of 3-OSTs (Shukla et al. 1999; Yakoub et al. 2014).
Inevitably the question arises what the receptor of choice would be if a cell is
co-expressing multiple receptors on its surface. The answer would be difficult since
there is limited information on this topic. The differential usage of receptors may
point to a tissue-specific usage that may play an important role in virus pathogenesis
in infected hosts. Recent studies suggest that nectin-1 acts as the sole receptor to
mediate HSV-1 uptake, while HVEM plays a minor role when both receptors are
equally expressed on murine dermal fibroblasts or epidermal keratinocytes
(Petermann et al. 2015a, b). It is worthwhile to mention that gD has the same affinity
to bind to nectin-1 and HVEM (Krummenacher et al. 1998). Studies in mice have
shown that the efficiency of HSV-2 infection of the vaginal epithelium was reduced
in the absence of nectin-1 expression, whereas the absence of HVEM expression
appeared to have no effect, indicating that nectin-1 is the primary receptor respon-
sible for infection (Taylor et al. 2007). However, nectin-1 is not the sole receptor
essential for HSV-2 spread from the vaginal epithelium to the nervous system. In
another study, after HSV-2 inoculation through the intracranial route, expression of
nectin-1, but not HVEM, was shown to be crucial for HSV-2 neuronal spread and
development of encephalitis (Kopp et al. 2009). However, when both receptors are
absent, infection was completely inhibited. In the ocular model, HSV-1 infection
was reported to be dependent on both HVEM and nectin-1, suggesting that receptor
requirements depends on the route of infection and/or serotype (Karaba et al. 2011).
Furthermore, it was shown that herpes B virus uses only nectin-1 to gain entry into
the cells (Fan et al. 2012). These findings support the idea that nectin-1 is the main
entry receptor for herpes simplex viruses and possibly related viruses.
1.3.1.2 Equine Herpesviruses
EHV-1 and EHV-4, members of the genus Varicellovirus, have significant genetic
and antigenic similarity (Davison et al. 2009; Roizman 1996). EHV-1 gD was
shown to utilize unique entry receptors that differ from those used by other
alphaherpesviruses (Frampton et al. 2005, 2007). The differential use of receptors
apparently allows EHV-1 to enter into a wide range of different cell types, including
those resistant to HSV-1. By contrast, EHV-4 was shown to be restricted mainly to
primary equine cells (Azab and Osterrieder 2012). Recently, equine major
histocompatibility (MHC) class I molecules were shown to serve as entry receptors

for EHV-1 and EHV-4 (Kurtz et al. 2010; Sasaki et al. 2011; Azab et al. 2014).
Equine MHC-I acts as a gD-binding receptor for EHV-1 and EHV-4 entry into
different equine cells, including fibroblasts, epithelial cells, and endothelial cells.
Although MHC-I molecules are expressed on virtually all somatic cells, they seem
an unlikely choice as a viral entry receptor: they are present on all tissues, which
would restrict tissue specificity, and at the same time, they are among the most
polymorphic of all mammalian proteins. Some MHC loci have many variable
alleles that can differ by 10–25% of their amino acid composition (Tallmadge
et al. 2010; Gilcrease 2007). Yet, other non-equine cells can still be infected with
EHV-1 and EHV-4 independently of equine MHC-I (Sasaki et al. 2011; Azab et al.
2014) indicating that these viruses utilize different receptors to gain entry into these
cells. We further provided evidence that alanine at position 173 (A173) within the
α2 domain of equine MHC-I is necessary, but not sufficient, for gD-MHC-I
interaction. However, amino acids and domains outside of A173 have a major
influence on virus binding (Azab et al. 2014). Horse MHC-I molecules are encoded
by a diverse set of genes in the horse MHC haplotypes that include genes at
polymorphic classical MHC-I loci (Tallmadge et al. 2005, 2010). Certain classical
loci have alleles that encode alanine at position 173 and permit EHV entry (Azab
et al. 2014). Each MHC haplotype also carries a variable number of other
nonclassical MHC-I genes that lack polymorphism and other structural features
and carry alleles with amino acids other than alanine at position 173 (Ellis et al.
1995). We showed that these genes do not support EHV entry into equine cells
(Azab et al. 2014). From the whole body of literature, we concluded that EHV-1
and EHV-4 target a domain of classical polymorphic equine MHC-I molecules that
include A173 and other as yet unidentified structures. All MHC-I molecules that do
not specify A173 are highly resistant to EHV-1 and EHV-4 entry.
The crystal structures of EHV-1 or EHV-4 gD are not yet resolved, and the gD
domain(s) mediating binding to MHC-I is unknown. In an attempt to determine the
gD-binding residue(s) responsible for binding to MHC-I and different cellular
receptors, we constructed a panel of gD mutants and based the constructs on
previous studies with HSV-1 and HSV-2 (Carfi et al. 2001; Connolly et al. 2003,
2005). We next determined the abilities of the generated gD virus mutants to bind
and enter different (equine and non-equine) cell lines. Our data showed that:
1. Deletion of amino acids 7–32 following the signal peptide cleavage site, which is
between R35 and A36, completely inhibited virus entry and infection of equine
dermal (ED) cells as well as other non-equine cells, indicating that this domain is
essential for gD-receptor interaction.
2. Mutating the conserved tyrosine at position 38 to alanine (Y38A) did not alter
virus entry into equine and non-equine cells.
3. The N-terminal region of EHV-1 gD or EHV-4 gD (amino acids 1–348) is
required for functional interaction with MHC-I and other non-MHC-I cellular
receptors (Fig. 1.1) (Azab and Osterrieder, unpublished data).
Fig. 1.1 Schematic diagram of the generated gD mutants and their ability to infect cells. EHV-1
tropism, infects the same cells as EHV-1; EHV-4 tropism, infects the same cells as EHV-4;
gD-1_gD4N, EHV-1 gD with the N-terminal domain of EHV-4 gD; gD-4_gD1N, EHV-4 gD
with the N-terminal domain of EHV-1 gD
In summary, our data indicates that both EHV-1 and EHV-4 gDs seem to have the
same MHC-I-binding domain, which is located in the N-terminal region of the
protein. We previously showed that EHV-1, but not EHV-4, can efficiently enter
and replicate in CHO-A, CHO-B, and CHO-C cells (Azab and Osterrieder 2012);
however, EHV-1 cannot use the equine HVEM homolog as an entry receptor (Azab
and Osterrieder, unpublished data). This means that EHV gDs might have different
binding residues from those of HSV-1 and HSV-2 and use a different set of
receptors to enter non-equine cells. Swapping the N-terminal region between
EHV-1 gD and EHV-4 gD changed the tropism of EHV-1 and EHV-4 in a manner
similar to swapping the entire genes (Azab and Osterrieder 2012). Replacing amino
acids 40–312 of EHV-1 gD with the corresponding EHV-4 gD sequence resulted in
a virus, which has a significant replication deficit and can only infect ED cells. The
data indicates that gD seems to have different binding domains for equine MHC-I
and cellular receptors of non-equine cells.
1.3.2 gB-Binding Receptors
gB is the most conserved glycoprotein of the cell entry machinery of herpesviruses

(Connolly et al. 2011). In alphaherpesviruses, gB was shown to mediate virus entry
process through attaching to cell surface HS, binding to specific receptors, and
catalyzing membrane fusion (Campadelli-Fiume and Menotti 2007; Connolly et al.
2011; Spear and Longnecker 2003; Karasneh and Shukla 2011). The crystal
structure of gB showed that this glycoprotein has five distinct domains with domain
IV being fully exposed, carrying neutralizing epitopes and possibly interacting with
cellular receptors (Heldwein et al. 2006).
Paired immunoglobulin-like type 2 receptor-α (PILR-α) is one of the paired
inhibitory receptors that likely acts as an immune system regulator. It is expressed
mainly on the surface of immune cells, including monocytes, macrophages, and
dendritic cells, and can tune the inflammatory response through delivering either
activating or inhibitory signals (Mousseau et al. 2000; Yamada and McVicar 2008).
HSV-1 was shown to use the inhibitory PILR-α to gain entry into HSV-1-resistant
cells (CHO-K1) that are transduced with PILR-α as well as human CD14-positive
PBMC (Satoh et al. 2008; Arii et al. 2009). HSV-1 infection of human PBMC was
blocked by either anti-PILR-α or anti-HVEM antibodies, suggesting the need for
both receptors (Arii et al. 2009). Interestingly, PILR-α has been shown to mediate
the entry of PRV, another alphaherpesvirus, but not HSV-2 (Arii et al. 2009).
Mutational analysis showed that gB-PILR-α interaction is dependent on sialylated
O-glycans on gB with two main threonine residues (T53 and T480) being essential
for this interaction (Wang et al. 2009; Arii et al. 2010b).
Non-muscle myosin II (NM-II), a member of the motor protein myosin super-
family, is an actin-binding protein and widely expressed in different tissues
(Hartman and Spudich 2012; Ivanov et al. 2007). NM-II is a heterohexamer
composed of two heavy (H) chains, two essential light (L) chains, and two regula-
tory (L) chains. Three different isoforms, A, B, and C, of the heavy (H) chain have
been reported to have different roles in the control of cell adhesion, intracellular
vesicular traffic, cell migration, and tissue architecture (Lu et al. 2008; Vicente-
Manzanares et al. 2009). Non-muscle myosin heavy chain IIA (NMHC-IIA) and
NMHC-IIB were shown to physically bind to HSV-1 gB, facilitate virus entry, and
mediate virus infectivity in vivo (Arii et al. 2010a, 2015). Overexpression of
NMHC-IIA in human promyelocytic HL60 cells also enhanced PRV infection,
which was inhibited by anti-NMHC-IIA antibodies (Arii et al. 2010a). In mamma-
lian cells, NMHC-IIA and NMHC-IIB are expressed endogenously and were shown
to function mainly in the cytoplasm, but not on the cell surface (Vicente-
Manzanares et al. 2009). However, after exposure of cells to HSV-1, NMHC-IIA
and NMHC-IIB expression was shown to be redistributed and markedly enriched at
the plasma membrane. These findings indicated that HSV-1 universally upregulated
cell surface expression of both molecules after adsorption to facilitate entry. Of
note, cells used in these studies were naturally permissive to HSV-1 infection,
indicating that NMHC-IIA and NMHC-IIB might have additional, yet unidentified,
role(s) during virus entry (Arii et al. 2010a, 2015). As mentioned above, HSV-1 can
surf along filopodia toward cell bodies to meet its entry receptors. Actin cytoskel-
eton organization and gB were shown to be critical for this process (Clement et al.
2006; Oh et al. 2010). It was speculated that, besides heparan sulfate, NMHC-IIA
and NMHC-IIB aid in virus surfing after binding to gB. Although gB was shown not
to be involved in the Rho-mediated induction of filopodia, activation of signaling
cascades can be induced by other viral glycoproteins, perhaps gD, after binding to
its cognate receptors (Van den Broeke and Favoreel 2011). Further studies will be
needed to address the role of these receptors in virus surfing.
Myelin-associated glycoprotein (MAG, Siglec-4), a member of the sialic-acid-
binding Ig-like lectin (Siglec) family, is a cell surface glycoprotein that expressed
specifically in myelin-forming cells (oligodendrocytes and Schwann cells in the
CNS and PNS, respectively). It plays important roles in regulating axonal growth,
maintenance of myelinated axons, and promoting the differentiation, maintenance,
and survival of oligodendrocytes (Quarles 2007). Expression of MAG on the
surface of promyelocytes and oligodendroglia cells confers susceptibility of these
cells to HSV-1 and VZV infection, respectively, after binding to gB (Suenaga et al.
2010). Furthermore, sialic acids (SA) on VZV gB were shown to be required for
MAG interaction. Mutating the conserved SA-binding residue (R118) on MAG
abrogated MAG-gB interaction and reduced VZV entry. In addition, gB with
mutations in the sialylated N-glycosylation sites (N557 and N686) cannot bind to
MAG and impaired cell-to-cell fusion (Suenaga et al. 2015). However, the possi-
bility cannot be excluded that conformational changes of gB caused by these point
mutations influence the fusion process. Since MAG is mainly expressed in neural
tissues, it might be involved in VZV and HSV-1 infection of glia cells with
subsequent induction of neurological disorders. After all, the point mutations in
gB (Arii et al. 2010a; Suenaga et al. 2015) were mostly in domains III and V,
indicating that the exposed nature of domain IV is not important to mediate the
interaction with cellular receptors as it was previously proposed. However, solving
the crystal structure of gB in combination with its receptor(s) will give more insight
into this important gB-cell interaction.
1.3.3 gH/gL-Binding Receptors
The heterodimeric gH/gL complex is a unique and stable complex that does not
resemble any other viral fusion protein identified to date. The resolved crystal
structure showed that HSV-2 and PRV gH have distinct architectures with three/
four domains (Chowdary et al. 2010; Backovic et al. 2010). A recent study
confirmed, at least in two alphaherpesviruses, the specificity of the N-terminal
domain I of gH for gL binding, which is also required for correct folding and
trafficking of gH. Furthermore, the C-terminal domain III is important for interac-
tion with gB (Bohm et al. 2015). It is now believed that gH/gL has no fusion
activity; instead, they act as fusion regulators to activate gB, probably through
direct binding (Chowdary et al. 2010).
There are few cellular gH/gL receptors detected so far; most of them are from the
integrin family and they are nonessential for virus entry. Nevertheless, they determine
the route of entry and entry efficiency (Azab et al. 2013; Gianni et al. 2010). Among
alphaherpesviruses, only HSV-1, HSV-2, and EHV-1-gHs were shown to have
integrin-binding motifs, RGD and SDI, respectively (Azab et al. 2012; Gianni et al.
2010). The RGD motif is the minimal peptide region of many proteins known to
interact with cell surface integrins, such as αVβ3, αVβ5, and α3β1 (Takada et al. 2007).
On the other hand, the LDV and SDI motifs are the primary binding site for α4β1
integrins in fibronectin (Graham et al. 2005; Komoriya et al. 1991; Davis et al. 1997).
Among gammaherpesviruses, Epstein-Barr virus (EBV) gH was shown to interact with
αVβ6 and αVβ8 (Hutt-Fletcher and Chesnokova 2010), while Kaposi’s sarcoma-
associated virus (KSHV) gH was reported to bind to heparan sulfate and ephrin
receptor A2 (EphA2) tyrosine kinase (Hahn and Desrosiers 2014; Veettil et al. 2014).
Recent studies have shown that αVβ3 integrins are important for routing the entry
pathway of HSV-1 into cells. In the absence of αVβ3 integrin, HSV-1 enters
CHO-nectin-1, J-nectin-1, and 293 T cells through a pathway independent of
cholesterol-rich rafts and dynamin-II and at neutral pH. Overexpressing αVβ3
integrin on the surface of these cells directs the entry of HSV-1 through a pathway
dependent on lipid rafts, dynamin-II, and acidic pH that is independent of caveolin-1
(Gianni et al. 2010). In an artificial model where 293 T cells overexpressed αVβ3
integrins and nectin-1, αVβ3 integrin was shown to relocalize nectin-1 receptor to
lipid rafts independently of virus binding (Gianni and Campadelli-Fiume 2012).
This may explain routing HSV-1 entry to a lipid raft-dependent pathway. The
mechanism of how αVβ3 integrins relocalize nectin-1 to the lipid raft and whether
this is also the case in cells naturally expressing αVβ3 integrin and nectin-1 still to be
investigated.
Not only αVβ3 integrins but also other members of the αV integrins, namely,
αVβ6 and αVβ8 integrins, can serve as receptors for HSV-1 entry into experimental
models of keratinocytes and other epithelial and neuronal cells (Gianni et al. 2013).
Both integrins can bind to gH/gL with high affinity and mediate the entry of HSV-1,
independently of each other, to acidic endosomes. Only αVβ8, but not αVβ6
integrins, route the virus through a lipid microdomain that requires dynamin-II.
HSV-1 infection of K562 (myelocytic cell line expresses a very limited number of
integrins, but not αV) increases only after transgenic expression of αVβ6 integrin
on its surface. However, J cells expressing either αVβ6 or αVβ8 integrin alone, in
the absence of nectin-1, were not susceptible to HSV-1 infection, indicating that
neither of the two integrins can promote HSV-1 entry in cells negative for gD
receptors. Interestingly, interaction of gH/gL with these integrins resulted in gL
dissociation and its release into the medium. gL dissociation was shown to require
all of the actors of the entry scene, meaning receptor-bound gD and gB (Gianni
et al. 2015). It seems that gL acts as a gH regulator and prevents its activation until
it binds to cellular integrins, which results in conformational changes finally
resulting in gL dissociation.
Recently, our laboratory investigated the entry pathway of EHV-1 and EHV-4,
for which the cellular and viral determinants routing virus entry were unknown. We
found that the interaction between EHV-1 gH and α4β1 integrins mediated fusion
of the viral envelope with the plasma membrane. Disrupting this interaction
rerouted the virus to an endocytic pathway, which is dependent on dynamin-II,
cholesterol, caveolin-1, and tyrosine kinase activity. Exchanging EHV-1 gH with
its counterpart in EHV-4, which has no integrin-binding motif, mutating the EHV-1
gH SDI motif, or blocking the interaction with α4β1 integrins, by means of using
blocking antibodies or soluble α4β1 integrins, rerouted EHV-1 to the endocytic
pathway. The molecular mechanisms underlying the change of the virus entry
pathway will be discussed later. Cumulatively, we could confirm that the decision
which of the two pathways will be taken is mainly dependent on gH and its ability to
interact with α4β1 integrins (Azab et al. 2013).
1.3.4 gE-Binding Receptors
In contrast to most members of the Alphaherpesvirinae, VZV does not express gD,
which is the main receptor-binding glycoprotein (Cole and Grose 2003). It was
shown that VZV gE is essential for virus infection and is involved in virus entry and
cell-to-cell spread (Mo et al. 2002). Besides MAG, the gB-binding receptor,
insulin-degrading enzyme (IDE) was shown to interact with gE through its extra-
cellular domain (Li et al. 2006). Blocking or downregulating cellular IDE inhibited
virus infection. Furthermore, overexpression of IDE in resistant cells mediated
VZV entry and enhanced virus infection not only for cell-free but also for cell-
associated virus.
Cation-independent mannose 6-phosphate receptor (MPRci) has been shown to
facilitate entry of cell-free VZV, probably by interacting with viral glycoproteins
(gB, gH, gI, and gE) that contain phosphorylated N-linked complex oligosaccha-
rides with mannose 6-phosphate (Man 6-P) groups. However, soluble MPRci did not
bind to viral glycoproteins in a ligand-blotting assay, even though similar blots
detected the interaction of the soluble MPRci with lysosomal enzymes (Zhu et al.
1995). Adding Man 6-P to the medium blocked infection with cell-free VZV. In
addition, stable cell lines deficient in MPRci were resistant to infection with cell-free
VZV, but not cell-associated virus (Chen et al. 2004). It seems likely that MPRci is a
cellular receptor for cell-free VZV but not for cell-to-cell spread of the virus.
1.4 Signaling Triggered by Alphaherpesvirus Binding

to Cellular Receptors
Viruses have to overcome formidable hurdles before they can invade cells and be
able to deliver their genomes into cells. Studies of the last decade showed that
viruses are very well adapted and have evolved different strategies to manipulate
cellular responses. Cellular signaling is one of the important events through which
cells respond to any intruder including viruses. It is now clear that many viruses,
including alphaherpesviruses, can not only counteract cellular signaling but also
make use of them to optimize infection (Greber 2002). In the coming section, the
most common signaling events associated with alphaherpesvirus entry will be
discussed.
1.5 Calcium-Signaling Pathways
Ca2+ is one of the most prominent and universal carriers of signals, which acts as a
second messenger and can modulate a number of steps during virus entry (Zhou
et al. 2009; Hay 2007). Free cytosolic Ca2+ has a concentration in the 100 nM range.
The concentration of stored Ca2+, particularly in the endoplasmic reticulum (ER), is
maintained at several hundred μM, whereas extracellular Ca2+ concentrations can
reach the mM range (Zhou et al. 2009). Due to this big difference between
intracellular and extracellular levels, cells have to tightly control Ca2+ homeostasis
to avoid acute gigantic fluctuations (Berridge et al. 2003). The increase of cytosolic
Ca2+ is usually triggered by specific ligand-receptor interactions on the cell surface
that often converge on phospholipase C (PLC) activation (Mikoshiba and Hattori
2000; Zhou et al. 2009). Among the cellular receptors involved in Ca2+-mediated
signaling are integrins. The engagement of α4β1 integrin activated PLC and
resulted in an increase of cytosolic Ca2+ (Kanner et al. 1993; Van Seventer et al.
1992). Activation of PLC leads to the hydrolysis of phosphatidylinositol
4,5-bisphosphate (PIP2) to generate two intracellular molecules: inositol 1,4,5-
triphosphate (IP3), which can trigger release of Ca2+ from intracellular stores, and
diacylglycerol (DAG), which is responsible for the activation of different down-
stream proteins, for example, protein kinase C (PKC) (Rhee 2001; Sekiya et al.
2004). IP3 can bind to the IP3 receptor (IP3R) localized on the cytoplasmic side of
the ER, which in turn mobilizes ER-resident Ca2+ (Rhee 2001; Berridge 1993). As
mentioned above, viruses have adopted different strategies to hijack Ca2+-mediated
signaling events to promote their entry and ensure virus replication (Zhou et al.
2009).
Previous studies have shown that exposure of cells to either HSV-1 or HSV-2
triggers a rapid and transient increase in cytosolic Ca2+. This process requires activation
of PLC and subsequent IP3-IP3R interaction, which in turn results in Ca2+ release from
ER stores (Cheshenko et al. 2003). Inhibiting IP3R or chelating intracellular Ca2+
inhibited HSV entry and VP16 transport. Interestingly, these studies showed that the
full set of entry glycoproteins (gB, gD, gH/gL) is needed to activate this pathway. The
absence of any of the glycoproteins inhibited Ca2+ release and prevented virus entry,
suggesting that this pathway might be activated in response to initiation of the fusion
process or other early events during entry (Cheshenko et al. 2003, 2007). Recently, the
same group showed that HSV binding to cellular receptors triggered Akt translocation
to microdomains of the outer leaflet of the plasma membrane where it can interact with
gB (Cheshenko et al. 2013). Akt is then phosphorylated resulting in increased expres-
sion of αVβ3 integrins on the cell surface, which may in turn facilitate interaction with
gH. This sequence of events is required for the significant increase of cytosolic Ca2+
and efficient virus entry (Cheshenko et al. 2014). The entry process is aborted when Akt
is silenced, Akt phosphorylation is blocked, or gH-αVβ3 integrin interaction is
interrupted. Moreover, gH-αVβ3 integrin interaction leads to the activation of the
focal adhesion molecule (FAK), which, together with proline-rich kinase 2 (Pyk2),
can promote the transport of viral capsids and tegument proteins to the nuclear pore
through reorganization of the actin cytoskeleton (Cheshenko et al. 2005, 2014). Still,
the signal cascade that triggered FAK phosphorylation remains to be identified.
A similar but distinct mechanism is active in the case of EHV-1. We showed that
binding of EHV-1 to equine epithelial cells induces release of Ca2+ from intracel-
lular stores, which may facilitate virus fusion with the plasma membrane. Our
results suggest that the increase of cytosolic Ca2+ is initiated by the interaction
between gH and α4β1 integrin, which activates PLC and the subsequent IP3-IP3R
pathway. Mutants in which integrin-binding motif was disrupted (EHV-1gHS440A)
(Azab et al. 2012) were not able to induce a significant increase in cytosolic Ca2+.
Furthermore, blocking the interaction between EHV-1 gH and integrins, by means
of either using α4β1 integrin-blocking antibodies or soluble α4β1 integrins, also
inhibited the increase in cytosolic Ca2+. Interestingly, Ca2+ release activated phos-
pholipid scramblases, which cause a rapid collapse of lipid asymmetry, ultimately
leading to phosphatidylserine (PS) exposure on the plasma membrane. Treating
cells with calcium or scramblase inhibitors prevented PS exposure on cell surface.
MHC-I (EHV-1 entry receptor) levels or distribution was not affected by the
increased exposure of PS, indicating that PS may facilitate lipid mixing. Addition-
ally, we found that most of the exposed PS were colocalized with virus particles;
however, it is not clear if or how any of the viral glycoproteins (particularly, the
fusogenic protein gB) interact with PS (DuBois et al. 2013; Heldwein et al. 2006). It
is important to mention that not only direct interaction of PS with viral glycopro-
teins can help virus entry but also the elevated level of PS on the plasma membrane
may influence virus entry through differential distribution of lipids, modifying
membrane fluidity, or promoting local changes in the bilayer phase (Rawat et al.
2003). However, the exact mechanism is not yet identified and will require detailed
biophysical studies. We currently exclude that reorganization of the actin cytoskel-
eton through intracellular signaling facilitates virus internalization (Iyengar et al.
1998; Pontow et al. 2004), as we were unable to find changes in actin rearrangement
after EHV-1 binding and infection. In contrast to HSV, blocking increased cytosolic
Ca2+ levels did not inhibit EHV-1 entry, rather it redirected the virus to a caveolin-
dependent endocytic pathway, indicating that virus fusion at the plasma membrane
may be enhanced in response to Ca2+ and subsequent exposure of PS (Azab et al.
2015). Only blocking endocytic pathways together with Ca2+ release from ER
significantly reduced virus infection.
1.6 Small GTPases
The role of GTPase-signaling cascade during alphaherpesvirus entry was reported

mainly for HSV-1 and PRV (Akhtar and Shukla 2009; Favoreel et al. 2007). The
early contact between herpesviruses and cells leads to cytoskeletal rearrangements,
which induce filopodia formation and membrane fusion. Following uptake by host
cells, viral capsids travel to the nucleus through microtubules-based system
(Dohner et al. 2005). Different alphaherpesviruses, including HSV-1, PRV,
EHV-1, and EHV-4, were shown to utilize microtubules for transport from the
cell surface toward the nucleus (Azab et al. 2013; Frampton et al. 2010; Lyman and
Enquist 2009).
Before being transported along microtubules, HSV-1 seems to rely on the actin
cytoskeleton to facilitate virus entry. As mentioned above, HSV-1 can induce cell
cytoskeletal rearrangements leading to the formation of filopodia, which in turn
direct virus transport to the cell body for subsequent entry. During entry into
primary cultures of human corneal fibroblast and nectin-1-overexpressing CHO
cells, HSV-1 virions were associated with cellular protrusions followed by phago-
cytic uptake, which involves specific arrangement of actin cytoskeleton, internal-
ization of virions into large phagosome-like vesicles, and activation of Rho
GTPases (Rho-A and Cdc42) (Clement et al. 2006). Furthermore, activation of
Rac1/Cdc42 signaling facilitated HSV-1 entry into MDCKII epithelial cells, but not
keratinocytes (Hoppe et al. 2006; Petermann et al. 2009). One downstream signal-
ing pathway for filopodia formation is phosphoinositide 3 kinase (PI3K), which can
regulate the phosphorylation of multiple cellular kinases (Greber 2002). In addition,
PI3K signaling was shown to regulate RhoA activation, actin networks, and
filopodia formation in HSV-1-infected cells (Tiwari and Shukla 2010). Possibly,
a role of gD-nectin-1 interaction is important for actin modification during entry
(Mizoguchi et al. 2002; Sakisaka et al. 2001; Ch’ng and Enquist 2005). Nectins can
regulate Ras and Rho GTPase activation with subsequent modifications in actin
cytoskeleton reorganization (Sakisaka et al. 2007). Binding of PRV gD to nectin-1
induced the formation of “synaptic buttons” during entry into sensory neurons of
the trigeminal ganglia through Cdc42-based remodeling of actin cytoskeleton
(De Regge et al. 2006).
The need of specific signaling cascades during herpesvirus entry may differ
depending on cell types and the route of entry. However, it seems likely that there is
a common pathway shared by many herpesviruses, for example, Ca2+ release and
actin rearrangements at the time of entry, although not dramatic (in some cases), but
significant to assure successful entry of the virus.
1.7 Model of Entry: Refinement
We now have a better understanding of virus entry and draw a more detailed picture
of the entry process of alphaherpesviruses (Fig. 1.2):
1. Attachment: tethering of the virions to cell surface occurs upon interaction of
viral gC/gB to heparan sulfate (sometimes DC-SIGN), which can also help virus
“surfing” to the cell body where entry can take place.
2. Receptor binding: this step is considered the trigger for the entire entry process.
Many players (viral and cellular) are involved depending on cell type and virus
species. Receptor-bound gD can send activation signal to gH/gL complex and
initiate cellular signaling cascades through PI3k and Rho GTPases to modulate
the actin cytoskeleton and prepare cell membranes for fusion and virion uptake.
3. Co-receptor binding: this step mainly is through gH/gL and cellular integrins.
The signal that the gH/gL heterodimer receives from gD and from integrin
receptor leads to the dissociation of gL from the heterodimer. At this point, gH
(activated form) signals to gB inducing gB activation.
Fig. 1.2 Entry of alphaherpesviruses. (a) Virus particles associate with HS on the surface of
filopodia to facilitate virus transport to the cell body where it can bind to entry receptors. Signaling
cascades through PI3k and Rho GTPases can modulate actin cytoskeleton and prepare cell
membrane for virion uptake. (b) Virus entry proceeds either by direct fusion with the plasma
membrane or through endocytic route. Attachment first takes place through gC/gB-heparan sulfate
interaction. Receptor binding with either gD or gB triggers the entry process. gH/gL-integrin
interaction signals inside the cell and results in Ca+2 release from ER through a PLC-IP3R
pathway. Scramblase activation with subsequent exposure of PS may help virus fusion. Phosphor-
ylation of Akt resulted in increased expression of αVβ3 integrins on the cell surface, which may
facilitate the interaction with gH. Dissociation of gL from the gH/gL heterodimer promotes gH to
induce gB activation, which subsequently executes the fusion process. Finally, activation of FAK
and Pyk2 facilitates capsid transport within the cells after actin reorganization
4. Signaling cascades: gH-integrin interaction initiates signaling that results in Ca+2

release from ER through PLC-IP3R and FAK activation. The increase of cyto-
solic Ca+2 results in scramblase activation with subsequent exposure of
PS. Activation of FAK facilitates capsid transport within the cells after actin
reorganization.
5. Fusion: this process is mainly executed by gB after activation by gH and perhaps
by gB receptors. Fusion can take place either with the plasma membrane or from
within endosomes. The decision of which route the virus will take depends on
viral gH and/or cellular integrins.
1.8 Conclusion and Remarks
Alphaherpesviruses are associated with a variety of diseases that can affect a wide
range of hosts. This big difference in host (tissue) tropism may well be associated
with differences in gene products (envelope glycoproteins) involved in virus entry,
which translates into different abilities in entering cells. Alphaherpesviruses have
shown considerable flexibility with respect to the use of receptors and entry
pathways, including direct fusion at the plasma membrane or from within
endosomes, either at neutral or acidic pH, depending on the cell type and available
receptor(s) (Geraghty et al. 1998; Hutchinson et al. 1992; Mercer et al. 2010;
Shukla et al. 1999). However, with this clear difference in the elements included
in the entry process, alphaherpesviruses still share many features during entry,
which include the glycoprotein set involved, the concerted series of events, signal-
ing cascades, and fusion with cellular membranes. The identification of cellular
receptors and co-receptors, crystal structures of glycoproteins, the different signal-
ing cascades, and, to some extent, the interaction between viral glycoproteins has
provided a better understanding of the entry process. Still, however, many aspects
need to be resolved, mainly those associated with the receptor of choice to be
utilized by the virus during entry. In addition, the role of different cellular mole-
cules and signaling events during entry is still unknown.
The main question still is as to how the community benefits from this all?
Definitely, developing rational vaccines or potent antiviral drugs would be worth-
while goals. Alphaherpesviruses have a high prevalence in human and animal
populations worldwide and cause significant disease and economic losses. Cur-
rently, no antiviral drugs targeting herpesvirus entry are available. A clear under-
standing of the entry mechanisms may provide clues to developing a new
generation of therapeutics that target virus entry: glycoprotein-receptor interaction,
glycoprotein-glycoprotein interaction, or the fusion process itself. Since virus entry
is a decisive process and shares common features among all alphaherpesviruses,
effective therapeutics can control different virus infections resulting in less virus
replication and reduced deaths.
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Another random document with
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has mixed up some tradition of this ancient work in Nubia. At any
rate, whatever be the truth about Lake Mœris, his account proves
beyond all question that the idea of a Reservoir was familiar to the
ancient Egyptians.
The tradition of a Reservoir somewhere on the upper waters of
the Nile lingered long in Egypt. There is a curious reference to it in a
book of travel by F. Vansleb, a Dutchman who visited Egypt about
the year 1670. The fertility of the Nile flood is caused, he says, by a
fall of dew, which usually takes place on June 17, just after the
appearance of the ‘green’ water. This dew purifies the foul water, and
makes it swell by fermentation. ‘Some of the country,’ however, he
proceeds, ‘that are ignorant of the true causes of this increase,
imagine that it proceeds from a large pond in Ethiopia in the river
itself, which the Abyssins begin to open about June 12, and let the
water out by degrees, more and more till September 14, by which
time they begin to shut it again. But this is a foolish fancy of the
Copties.’
We have seen how the tradition of Lake Mœris fascinated
Mehemet Ali; but the methods of Haroun-al-Raschid were not suited
to solid engineering works, as the history of the Barrage too plainly
shows. None of his descendants, with the exception of the Khedive
Ismail, had the wit to conceive or the ability to execute such an
undertaking, and Ismail’s fantastic imagination was fully occupied in
other directions. Fortunately for Egypt, the project had to wait until
the success of the Barrage made the time ripe for its execution, and
until skilful brains and strong hands were ready to plan and carry it
out in the most efficient manner possible.
There were three problems to be faced: first, Where could such a
Reservoir be erected? second, What arrangements could be devised
to avoid the danger of large silt deposits, which would soon seriously
diminish the capacity of the Reservoir, and, if allowed to accumulate,
render it in no long time entirely useless? third, Supposing that the
difficulties of site and design could be overcome, where was the
money to be found? During the first years of the British occupation,
while Egypt was still painfully struggling upwards from the abyss of
bankruptcy into which she had been cast by the mad whirlwind of
extravagance in Ismail’s reign, it was no time for the inception of
original works on a grand scale. But in 1890 the matter became an
affair of practical politics, and was at last seriously taken in hand.
Meantime discussion had been raging as to the best locality for the
Reservoir. An American gentleman, Mr. Cope Whitehouse, took up
the case of the Wadi Rayan, a depression in the desert to the south-
west of the Fayoum. This, he maintained, was the real site of the
ancient Lake Mœris, and here the Reservoir ought to be. He had no
professional knowledge, and he was utterly wrong in his ideas; but
his vehement method of controversy kept the subject thoroughly
alive. The whole land was filled with his clamour, and every expert
was forced to give his own views in self-defence. The debate served
a useful purpose. Gradually it came to be recognised that the river-
bed itself was the proper place for storing the water by means of a
Dam. Authorities differ as to whom belongs the credit of first making
this suggestion, or, rather, of first reviving the tradition of the past;
but it seems pretty clear that Sir Samuel Baker suggested the
construction of a Dam at the first cataract at Assouan as far back as
1867.
However that may be, in 1890 the Government took the matter
up, and charged Mr. W. Willcocks with the task of examining the river
north of Wadi Halfa, reporting upon the best available site for the
Dam, and preparing a design for it. After a careful survey, his plans
were completed in 1894, and his design for a Reservoir at Assouan
was then submitted to an International Committee of Engineers,
consisting of Sir Benjamin Baker, M. Boulé, and Signor Torricelli. Mr.
Willcocks’ plans were, with some modifications, accepted by a
majority of the Commission, and to him belongs the honour of having
designed the Dam. The selection of the Assouan site solved the first
of the three difficulties. There is at this point an extensive outcrop of
granite clean across the valley of the Nile, which it was thought
would give sound rock everywhere at a very convenient level for the
foundations of the Dam. Moreover, the trough of the river above the
cataract and a long way south of it is exceptionally deep, and this
makes it possible for a greater amount of water to be stored up
behind the Dam. But the prime necessity was for a solid foundation.
Elsewhere in Egypt the bed of the Nile is composed of shifting
sands, on which it would have been impossible to build a Dam
capable of holding up so great a head of water.
Mr. Willcocks’ design solved the second difficulty, the problem of
constructing a Dam strong enough for the purpose, and yet of
avoiding the danger of filling up the Reservoir by too great
accumulations of silt. The other great Dams in the world, as, for
instance, that built by Sir Arthur Cotton on the Godavery River, in
India, are solid throughout. They are planned so that the rising flood
shall pass freely over the top of them. But the Assouan Dam is of a
type previously unknown, and its success ought to stimulate
perennial irrigation in many parts of the world where such projects
have hitherto proved failures. Its principle is that even the highest
flood shall pass, not over it, but through it. To this end it is pierced
with 180 openings, which are like tunnels in the great mass of
masonry. The openings are controlled by powerful sliding-gates
worked from above. During the months of the flood every gate will be
up, and the ‘red’ water, carrying all its heavy burden of silt, will pass
through without impediment. Later in the year, about the end of
November, when the flood has subsided and very much less matter
is carried in suspension, the sluice-gates begin to be gradually
closed, and by the end of February the Reservoir is full, without
having affected the normal discharge of the river in any appreciable
degree. From April to July the water thus stored up is let out by
degrees for employment, according to the state of the river and the
requirement of the crops. By the time the next flood begins to come
down all the stored water will have passed out, and every sluice will
be once more open to give free passage to the rising stream.
Although the Nile in December and January carries an insignificant
amount of sediment compared to that brought down in August and
September, it yet brings down a very considerable quantity, far
greater than most other rivers at any time, and quite enough to go a
long way towards silting up the bed of the Reservoir, if it was allowed
to remain. But for this the river provides its own remedy: every year
the force of the flood will act like a gigantic broom, sweeping the floor
of the Reservoir. The sluices, arranged in sets of five, are distributed
at different levels, according to the formation of the river-bed on the
upstream side, so as to facilitate this process to the utmost. During
the months of the inundation the Nile at Assouan pours down for
weeks together a volume of 10,000 tons of water per second, and
sometimes as much as 14,000 or 15,000 tons per second. The rush
of this stupendous mass is sufficient to assure us that there will be
no silting up of the Reservoir.
Two of the difficulties had been thus overcome, when, from a new
and unexpected quarter, a storm sprang up, which very nearly
brought to a standstill the rising fabric of Egyptian prosperity. The
project of the Reservoir would have raised the level of the water, and
held up the river above the Dam to a head of 100 feet; this would
have involved the temporary submersion every year of the island of
Philæ, with its famous Temple of Isis, Pharaoh’s Bed, and other
monuments. A terrific hubbub arose. Archæological and antiquarian
societies, which until then had sometimes belittled the monuments of
Philæ as belonging to an inferior period, poured in their protests.
People who had never heard of Philæ before, but who were none the
less influential for that, joined in the outcry. Diplomatists, whose one
desire was to embarrass our progress in Egypt, took up the cause of
Art with a will. These champions of humanity at large forgot the poor
fellaheen, to whom the extra water means all the difference between
misery and happiness; nothing would satisfy them but the complete
abandonment of the project. The engineers fought stoutly in the
interests of Egypt; they offered to raise the whole of the monuments
bodily, or to transport them to the neighbouring island of Bigeh; but,
though they saved the Dam, the original design was lost, and the
Dam to-day is 33 feet lower than it ought to have been. The
foundations of Philæ have been underpinned and strengthened, the
island will only be partially submerged, and the injury to Egypt can
only be faintly estimated.
Was the sacrifice worth it? The value of Philæ lies in its beauty
more than in its antiquarian interest. No one who has witnessed
night after night the glorious sunsets on the Nile, the mysterious
charm of the changing waters, the dark belt of palms reflected in the
river below and standing out in strong contrast against the sky, the
limestone cliffs of the desert clear-cut in the dry air, and flushing pink
in the radiance of the indescribable after-glow, no one who has seen
the Temples of Karnak, could hesitate to make so small a sacrifice,
in comparison, for the sake of the river-side people. Moreover, Egypt
is rich in treasures of the past, as yet undiscovered, and wanting
only money for their development, which the Reservoir would in time
supply. And how few people visit Philæ at all! Surely, even in a
country a thousand times poorer than Egypt in artistic and
archæological interests, the well-being of the living and of the unborn
should have prevailed.
If all those who joined to swell the uproar had been really
disinterested lovers of the beautiful, there would have been small
reason to complain of their insistence. Enthusiasts can hardly be
expected to listen to the voice of reason, and Philæ has charms to
soften the heart of the most savage utilitarian. Its fate is a mournful
necessity, but it is a necessity, for the question of the Reservoir had
come to be a question of existence for Egypt. Even the advantage
gained by the opposition in lowering the height of the Dam is only a
delay. On the pylon of the Temple of Isis at Philæ is carved a huge
representation of the Pharaoh of the day, one of the most
degenerate of the Ptolemies, catching his defeated enemies by the
hair of their heads with one hand, an uplifted sword in the other. The
whole is a copy of the work of his warlike ancestors, and even as a
copy it is a delusion and a sham; for he won no victories, defeated
no enemies, and, indeed, scarcely ventured outside the walls of his
harem. The apparent victory of these lovers of Philæ, to call them by
their more honourable title, was not less delusive. Philæ is doomed.
Between half drowned and wholly drowned there is not much
difference in the case of an island, certainly not a difference worth
fighting for, and the Dam will be raised to its full height, perhaps as
soon as Egypt is ready for the extra water.
The financial difficulty remained. In spite of the prosperity of
Egypt, she is, as everyone acquainted with her history is aware,
bound hand and foot by international fetters in matters financial. The
Caisse de la Dette, founded to protect Egyptian creditors against the
dangers of bad administration, has remained to be an obstacle to
any improvements that must benefit these interests. The practical
outcome of the system is that, if the Caisse be hostile—and hostile it
has often been—no public work like the Nile Reservoir can be
carried out without the imposition of extra taxation to the amount of
double the annual expenditure required.
Time passed, the need became more pressing, but the prospects
of the Reservoir seemed further off than ever. Besides the regulation
of her water-supply, Egypt had on her hands the question of the
Soudan. From every point of view the reconquest of that province
and the upper waters of the Nile was a prime necessity; no one
could tell how long the war might last, or how great the expense
might be. It seemed impossible that she could bear the cost of two
such enterprises simultaneously, and under such circumstances her
credit would not have been sufficient to raise the capital sum
required on anything like reasonable terms. Not only so, but by the
peculiar constitution of Egyptian finance it was illegal for her to raise
a loan without the consent of the Caisse, a consent which it was
impossible to obtain.
But, fortunately for Egypt, there were a few men with clearer
vision and more faith in the future, and chief among these was Lord
Cromer. The statesman who had controlled the tangled destinies of
Egypt through so many dark years, and baffled so many tortuous
intriguers, as well as more open foes, was not the man to despair in
such a situation. In 1897 the first negotiations were quietly opened
with Sir E. Cassel. Then came the vote of the majority of the Caisse
to grant £500,000 towards the Soudan Railway, and the successful
action taken in the Courts against that vote. Everyone knows how
this seeming defeat was turned to overwhelming victory by the
decision of the English Government to grant £750,000 for the railway
on certain conditions. The enemies of England in Egypt received a
staggering blow.
But the story was not yet complete. In April, 1898, Sir E. Cassel
arrived at Cairo; in one day the details of the arrangements to
finance the Dam were settled; all that night the lawyers drafted the
necessary documents; a Council of Ministers was hastily called in
the morning, and the contracts were signed. Sir E. Cassel was to
provide the necessary funds for the execution of the work,
£2,000,000; repayment by the Egyptian Government was to be
deferred altogether for five years, and then to be spread over a
period of thirty years. The first payment of about £78,000 is included
in the Budget for 1903.
Looking back now after five years of prosperity, when Egyptian
securities have actually increased in value, while Consols
themselves have so greatly declined, it is easy to see that the
statesman and the financier were justified in their faith. But in those
days it needed a clear vision and a stout heart to calculate thirty
years ahead—nay, even five—in a country so much the sport of
international politics. The Soudan Campaign was not yet ended;
behind the dervishes there loomed vague possibilities of worse
complications. The Egyptian Government made a good bargain then,
though it would doubtless make a better now. But it was then, and
not now, that the business had to be settled.
CHAPTER VIII
THE DAM AND THE NEW BARRAGES
Once the financial difficulty was settled, no time was lost in setting to
work. As soon as the flood of 1898 began to subside, Messrs. Aird
and Co., the contractors, were busy with the foundations of the Dam.
Five years was the period allowed by the contract, but a succession
of low Niles gave unusual facilities for the work, and everything was
completed before the flood of 1902, a year before the specified time.
From its vast proportions, the Dam is infinitely more impressive to
the imagination than any other of the irrigation works in Egypt. But
from an engineering point of view its construction was a plain,
straightforward business compared with the difficulties of building a
Barrage, where the river-bed offered no more solid foundation than
shifting sands. Still, there was a moment, on the first uncovering of
the river-bed, when its fate seemed to hang in the balance. The
Assouan site had been selected principally because the outcrop of
granite, there running clean across the valley, would give, it was
thought, solid foundation at a convenient level. It was found that in
some places the rock was rotten to a depth of 40 feet. It was an
anxious moment, both from an engineering and a financial point of
view. Every foot of rotten rock meant a considerable addition to the
calculated expense, besides modifying the building plan. Once more
Lord Cromer’s strong will saved the situation. On the financial side
he stood on firm ground, and he proved as good an engineer as he
had been a financier. Solid rock was reached, and the work went
steadily forward. Ten thousand men was the usual sum of those
employed, and of these 800 were Italian stone-cutters specially
brought over to deal with the tough granite of which the Dam is built.
Granite and Portland cement are the two great materials used for
welding the fetters of the Nile.
The Dam is about one mile and a quarter in length, and at its
deepest point it is 126 feet high. Sixty-five feet of water can be held
up when the reservoir is full, and it is capable of storing about
1,200,000,000 cubic metres of water—that is to say, about the same
amount of water as passes through Assouan in a single day when
the flood is at its height. The face of the wall is a slope on the
downstream side, and its width at the bottom corresponds
approximately to its height. Seven hundred and eighty thousand
cubic yards of masonry have been used. On the western side a
ladder of four locks gives passage to boat and steamer traffic at all
seasons.
All parts of Egypt are to benefit in a greater or less degree from
the extra summer supply. The original calculation assumed an
amount to be distributed of 1,065,000,000 cubic metres. It was
allotted as follows:
Cubic Metres.
South of Assiout 170,000,000
Assiout to Cairo with the Fayoum 510,000,000
Gizeh Province 85,000,000
Lower Egypt 300,000,000
This division meant that 52,000 acres could be reclaimed to

cultivation in the Fayoum, and 120,000 acres in the Delta. Further,
south of Assiout 200,000 acres could be converted to perennial
irrigation by means of pumps upon the Nile banks. In Middle Egypt
458,000 acres could be converted to perennial irrigation, and in
Gizeh Province 106,000.
A low Nile on the average comes about once in five years, but
assuming that it happened every year, these results may be
expressed in terms of money on the basis that conversion to
perennial irrigation increases the yield per acre by £2 annually, and
that reclaimed land produces a yield valued at £5 annually. This
gives an increase of value in the annual yield:
£E
South of Assiout 420,000
Middle Egypt and Fayoum 1,176,000
Gizeh Province 212,000
Lower Egypt 600,000
Total £E2,408,000
while the direct annual gain to the State Exchequer in rental and
taxation would amount to £E378,400.
This does not exhaust the full extent of the benefits of the
Reservoir on which a money value can be placed. For some time
Egypt had been living beyond her real resources in the matter of
water. Encouraged by a series of good years, the acreage of cotton
had been greatly extended. The cotton-plant is very hardy, and can
retain its vitality for a certain period on a very scanty and irregular
supply of water. Cultivators, especially in the Delta, had taken
advantage of this quality up to the very hilt, and the annual crop had
reached an amount of 6,000,000 kantars.
Taking the not very high price of 175 piastres per kantar (1 kantar
= nearly 100 pounds), the annual value reached £10,000,000. A
season like that of 1889, when the summer supply was very low, and
not the lowest on record, would mean the loss of at least one-tenth
of this amount, and even more, in spite of the most successful
working of the Barrage, and the most careful system of rotations.
Against such a loss the Reservoir is a complete insurance, and, as a
low year cannot safely be reckoned as occurring less than once in
five years, the annual value of such insurance must be set down as
£200,000. The figures give some idea of the value of the new supply.
The estimate does not err on the side of exaggeration. It was framed
in the most cautious and conservative manner possible, and, in fact,
it would be by no means rash to put the total annual value a good
deal higher.
According to the financial arrangement, the first payment towards
defraying the cost of the works was included in the budget for 1903,
but no fresh taxation for the purpose was to be imposed till 1904,
and even then the full amount of direct benefit to the Exchequer will
not be realized till 1910. Time is thus given for the full effects of the
change to be felt, and the Reservoir will be paid for out of its own
profits. The alteration of the basin lands to the new system must take
some time, and their cultivators will thus be given full opportunity to
familiarize themselves with the new methods of agriculture which
they will have to employ.
The scheme of distribution allotted nearly 50 per cent. of the
Reservoir supply to the Fayoum and the province between Assiout
and Cairo. Just south of Assiout is the head of the great Ibrahimiyah
Canal, which not only supplies these provinces, but also feeds the
Bahr Yusuf, which waters the Fayoum. It was necessary, in view of
the increased discharge, to widen the upper reaches of this canal,
and to provide it with a new regulating head. But more than this was
required to insure its receiving the proper proportion of water
whenever the sluice-gates of the Dam were opened. Accordingly, at
the same time that the foundations of the Dam at Assouan were laid,
a new Barrage was begun at Assiout just downstream of the head of
the Ibrahimiyah Canal.
In principle the Assiout Barrage is exactly the same as that at the
point of the Delta, and the difficulties of construction were also
exactly similar, for in both cases the foundations had to be laid on
the same shifting sands, and as each section of the work was
undertaken a portion of the river had to be diverted from its course
by means of temporary earthen dams. The shifting nature of the
river-bed, the almost personal malignity of the water, constantly
bursting through in countless springs, each of which had to be
separately dealt with and suppressed, called forth the highest
exercise of engineering skill. But the experience gained in the long
struggle with the imperfections of the earlier Barrage infallibly told its
tale. Every difficulty was successfully encountered, and the Assiout
Barrage was completed by the summer of 1902, and was able to
hold up the 10 feet of water required of it, without any failure, at the
first attempt.
The visible part of this Barrage, which is just over half a mile in
length, consists of a viaduct or bridge with 111 archways, each 16
feet 5 inches in width, closed by strong iron gates, working in
grooves made in the supporting piers, and raised or lowered from
above. In contrast to the Delta Barrage, it is built throughout of stone,
and not of brick. At the western end is a lock, the largest in Egypt,
through which the largest of the boats that ply upon the Nile can
easily pass. Below the water lies the strength of the structure. The
viaduct rests upon a solid platform of granite and cement, 10 feet
deep and 87 feet wide, set at a suitable depth below the bed of the
river. As a further precaution against the action of the water, there
are also below the platform two continuous lines of iron sheet-piles,
with hermetically sealed joints. With such a series of obstacles to
encounter, the danger of the water forcing its way through
underneath the Barrage is small indeed. The whole amount of
masonry used in this Barrage is 220,000 cubic yards.
The prosecution of these great works in Upper Egypt by no
means exhausted the activity of the Irrigation Department; indeed, it
would almost seem that the building of Barrages has become part of
its ordinary routine, for a third remains to be chronicled. This is the
Zifta Barrage on the Damietta branch of the Nile, halfway between
the point of the Delta and the sea. Because it lies in a district
unvisited by tourists, though very important commercially, its
construction has not been heralded by any blowing of trumpets. Yet
it is a work of the very first class, and deserves to be reckoned
among the greatest of the triumphs of the department. Built on the
same plan as the Assiout Barrage, and, like it, capable of holding up
10 feet of water, it is designed to secure a better distribution of the
supply north of the Delta Barrage. As the area of the cultivated land
extended gradually northward, it became apparent that the canal
system taking off from above the original Barrage was becoming too
long to admit of the water in times of pressure reaching the
northernmost parts of the country. There were some who held that a
second Barrage, with a new system of canals taking off from it,
should have been erected on the Damietta branch, even in
preference to the new stone weirs, which have increased the
strength and distributing power of the old one. The dispute has been
happily settled by the adoption of both projects, and with the Zifta
Barrage completed in time for the summer of 1903, and the new
supply from Assouan, the reclamation of the northern lands will go
steadily forward.
It has been already pointed out that it is almost as important to get
the water off the land as to get it on, and the proper drainage of the
Delta lands has been the necessary complement of all the new
schemes. Though eclipsed by the splendour of the Reservoir and
other creations more taking to the eye, the performance of the
engineers in this direction during the last few years has been
sufficient to make them very memorable in irrigation annals. Since
1896 about 1,000 kilometres of new drains have been dug, and
nearly as great a length of existing drains remodelled. It has cost the
Egyptian Government close upon a million of money, but that this
expenditure has not been thrown away is proved by the great rise in
the value of all the lands affected.
The Zifta Barrage cost about £500,000, the Assouan Dam and the
Assiout Barrage £3,200,000. Apart from the ordinary expenditure on
maintenance and the suppression of the corvée, the twenty years
ended 1902 have seen an expenditure of £9,000,000 devoted to
irrigation and drainage. There could be no greater proof of the
wisdom of those who have directed Egyptian policy during that
period. However pressed they have been at different times by
demands for immediate expenditure on other objects when
resources were low, they have always adhered steadily to a policy of
liberality towards public works likely to be of a remunerative
character. In no other country do economic laws work out their
results with greater directness and certainty. Reproductive
expenditure is really worthy of its name, and brings its visible and
tangible fruit almost without a moment’s delay. As they have sown,
so have they reaped. There can be no comparison between this
expenditure and the benefits it has conferred upon Egypt. Three
salient examples may be given to point the force of these remarks.
First, the works undertaken by Colonel Ross to improve the
system of basin irrigation in Upper Egypt. 1877 was a year of very
low flood, and nearly 1,000,000 acres were sharaki—that is, entirely
exempted from taxation owing to absence of irrigation. In 1899, a
worse year—in fact, the lowest flood of the century—the sharaki
lands were no more than 250,000 acres, a result mainly due to the
successful reforms carried out by Colonel Ross in 1888-89. 1902
was a year very similar to 1899, and the sharaki acreage was no
more than 140,000 acres.
Second, the case of the Assiout Barrage. The work was finished a
year before the time named in the contract. On August 15, 1902, the
usual date of filling the basins, the flood was exceptionally low, and it
was decided to lower the gates of the Barrage. By so doing the
water-level of the canals was raised by 1½ metres, an increase
which was more than sufficient to avert the threatened disaster. The
money value of the crops thus secured to the land-owners of the
Fayoum and Middle Egypt is estimated at not less than £600,000.
The cost of the new works at Assiout, including the new regulator on
the Ibrahimiyah Canal, was about £875,000. Thus, in the first year of
their existence they nearly repaid their whole cost, and that, too, as it
were, by a sidewind; for the Barrage’s real function is to hold up the
river in the low summer season, and not in the flood.
Third, the case of the Assouan Reservoir itself. The summer
supply in 1903 has been the lowest on record; the discharge at
Assouan has fallen to 200 cubic metres per second. By means of the
Reservoir this supply has been actually doubled. Had the flood been
late in coming down, it would have been impossible to distribute the
water at so liberal a rate. The Soudan gauges gave warning that an
early flood was to be expected, and thus the authorities were able to
calculate with certainty, and open the sluice-gates with much greater
freedom. But the calculation would have been a useless exercise
unless there had been a store to draw upon. At the lowest
computation the loss avoided may be reckoned at a couple of
millions.
The administrators of Egypt have had many difficulties and
obstacles in their path, but to be able to point to such results is a
great compensation; their efforts need no formal monument.
CHAPTER IX
THE INAUGURATION OF THE RESERVOIR
December 10, 1902, was the official date of the inauguration of the
Reservoir, a memorable day in the history of Egypt, and worthy to be
marked with red even in the unchanging Mohammedan calendar.
The making of the Dam has been a great time for Assouan. The
town has thriven and prospered beyond all knowledge since the
days—not so very long ago—when two British battalions occupied
the barracks on the hill overlooking the river to the south. The
barracks are crumbling to pieces now, and only one or two
blockhouses remain as memorials of the past state of siege and fear
of dervish raids. The tide of war has rolled far away and spent itself
utterly in remote corners. The whole of the Soudan lies between
Assouan and the frontier of any possible enemy. Even the yellow fort
is untenanted save by a few policemen. True, for four years an army
of 10,000 men has been marshalled here, but it was an invasion of
the arts of peace, creative and not destructive. Possibly the
inhabitants would have liked their occupation to go on for ever; but
even the best of times must have an end, and it was a good
occasion for a holiday. In every Nile village flags and bunting were
flying. For once the fields were deserted, and everywhere the people
crowded to the bank in the hope of catching a glimpse of the
Khedive and his distinguished guests. Here and there the gaffirs, or
local policemen, lined the shore, standing stiffly to attention, or
saluting with their Remingtons in the regulation attitude of European
soldiers, which contrasted quaintly with their loose, flowing robes
and white turbans.
At Assouan the faithful subjects of the Khedive surpassed
themselves. Sunny Assouan lends itself readily to a festal garb.
Situated where the Nile broadens out after emerging from the rocky
defile of the cataract, the town has a most picturesque aspect at all
times; its embanked river-front makes it the neatest of all the cities of
Upper Egypt. Elephantine and the Sirdar’s Island rise green and
smiling out of the broad bosom of the river; the perpetual blue sky
makes everything doubly attractive to the Northern visitor. Dressed
for the festival, it was a charming scene. Triumphal arches of the
sacred yellow and brown, gorgeous hangings and many-coloured
festoons, bore testimony to the Oriental love of vivid hues; steamers
and dahabiehs, moored in line along the shore as well as along
Elephantine Island, vied with each other in their decorations, and
numerous feluccas were plying to and fro, half hidden by their
burdens of flags and palm-leaves. At night thousands of lamps
decorated shore and river alike, and the whole scene resembled
nothing so much as a Henley in Regatta Week, with its illuminations
unrestrained by doubts of weather. To the ear, however, the voices of
the night told a very different tale. The crooning song of the Nubian
boatmen, ‘Great is the Prophet, praise be to him!’ accompanying the
creaking of their clumsy oars with monotonous persistency, sounded
weird and barbaric over the twinkling waters. The bustle in the town,
too, was no mere ordinary festal murmur; for this was the month of
Ramadan, and the feast of lamps meant a great deal to all faithful
Moslems. All day long they have abstained from food or drink, and
the going down of the sun is keenly welcomed as the end of one day
more of fasting.
The secret of the prosperity of Assouan lies in its granite. It is the
granite bed of the river at this point that makes the Reservoir
possible; here are the granite quarries from which the Dam was built,
and from which every ruler of Egypt who wished to raise a
monument for all time has drawn his supplies. Nothing that I have
seen in this country brings the past so near as these quarries. Here
lies a rough-hewn obelisk, just ready to be rolled away; here an
enormous block of stone half hollowed into a bath for an Emperor, or
a sarcophagus for an Apis bull, designed by some mighty ruler who
‘thought in continents,’ and recked little of the lives and labours of
thousands provided he gratified his whim. But suddenly death or
some other fate intervened, and a feebler or more merciful
generation has never taken up the work. You may see the marks of
the wedges on some great face of rock, as fresh as if it was only
yesterday that Pharaoh’s workmen had driven them in and poured
water on the wood till it swelled and burst the stone; down below is
the fallen piece still waiting for the mason who never came back to it.
Perhaps some of the very stones cut by Cheops or Rameses have
been smoothed and planed and set in the Great Dam.
Passing along the raised causeway, down which so many great
monuments have been rolled slowly to the river, I came through a
long stretch of burning desert to a spur on the northern extremity of
the granite hills. Here, unexpectedly, I found myself overlooking the
lake formed by the filling of the Reservoir. Graceful lines of palm-
trees showed where the banks of the river had once been. Philæ
was but an insignificant speck on the blue waters, overpowered by
the fantastic piles of granite boulders that hem in the valley. In the far
distance rose some lofty hills, crowned by dazzling sand that might
easily have been mistaken for snow. The Dam builders have been
accused of vandalism, but they have created a standing pool in the
wilderness of surpassing beauty. The view of the lake was not the
only attraction of the spot; at my feet lay a colossal statue of Osiris,
destined for some temple, but never moved from the spot where it
was hewn.
Ancient Egypt may well look on with scornful wonder at our pride
in our achievements. The Great Pyramid at Gizeh contains three
times as much solid masonry as the Dam, cut from these same
quarries. Every one of those huge blocks had to be dragged to the
river, and carried down 600 miles, before it was hoisted into its place.
As an achievement of mechanical power the Great Dam cannot
compare with the Great Pyramid; but when at last I climbed a little
hill hard by the river, below the Reservoir itself, and saw the whole
length of the great stone rampart, stretching right across the valley,
the contrast between the world of Pharaoh and our own came strong
upon me.
Pharaoh, to whom time and life were nothing, out of the misery of
the forced labour of his subjects, raised a perfectly useless
monument of his own folly; yet he achieved his object, and made his
tomb one of the wonders of the world. We, with the free labour of
voluntary workers—paid, fed, and cared for, instead of being driven
by the whip—have dared to harness Nile himself. It is a work vital to
the interests of millions of dwellers by the river. Yet who can say that
the fame of the Pyramid will not endure the longer? Hundreds of
years after the time of Cheops a mighty Dam was built in Southern
Arabia for a like purpose of irrigation; it lasted for eight centuries or
more, and its bursting in 100 A.D. is mentioned in the Koran. Its ruins
remain to this day, and show that it was larger than the Nile Dam.
Eight hundred years is a long life for a reservoir, but if this one lasts
a quarter of that period it will have repaid its cost many hundred
times over.
Certainly it looks strong enough to last as long as the Nile itself.
Strength, and nothing but strength, shows in every stone of it.
Square, solid, and massive, it runs from shore to shore in an
absolutely straight line, without the slightest attempt at any trace of
ornament or decoration. Clearly, effect has been the last thing
thought of. Even the sluice-gates have absolutely plain rectangular
openings, and it detracts from the symmetry of the design that,
owing to engineering exigencies, they are not all of one height, but
run in sections of five, some of them lower than others. The top of
the wall runs in a simple, unbroken level; there is nothing to catch
the eye as it travels up the steep face of masonry except the
slightest change in the angle of the slope to a nearly complete
perpendicular. The wall simply leaves off because its builders
thought it high enough for the present. Along the broad surface of
the summit runs a tramway, with plenty of room for a man to walk on
either side, flanked by perfectly plain, solid parapets as high as the
waist, and more than a yard thick, of a piece with the masonry below.
On the eastern side the Dam is unostentatiously built into the
living rocks; no arch or pylon marks its start. On the western side it is
flanked by a ladder of four immense locks, set in a mountainous
embankment. The gallows-like arms of the draw-bridge, hideous in
appearance, but a marvel of mechanical ingenuity, over the upper
gate of the highest lock are the only break in the long, unrelieved
level. The hard gray colour of the granite strengthens the general
impression, though in time every part exposed to the action of the
water will be coated with the shining black varnish which the Nile
mud always lays on granite, and it will look exactly the same as the
natural bed of the stream.
How different all this is from the prettiness of the Delta Barrage,
with its brickwork, originally designed and built under French
influence, adorned with archways and towers, of which the lovely
garden, with its flowers and shrubs, its green lawns and leafy trees,
on the tongue of land which it crosses, seems a natural and
appropriate part! The Assouan Dam is the work of a practical,
unimaginative race. Its builders have had before them the problem of
harnessing the great river with a yoke that cannot be broken; they
had to hold up a reservoir containing 100,000,000 tons of water, so
that for 140 miles the river is turned back upon itself; and they have
succeeded.
In December all but a few of the sluice-gates are shut, for the
reservoir has to be filled. But imagine it at the height of the flood,
when the collected rainfall of half a continent is crashing past at the
rate of nearly 1,000,000 tons a minute, and through each of the 180
openings shoots a solid cube of dark water to dash thundering in
clouds of white foam on the rocks below, and rush tumultuously
down the swirling slopes of the cataract. Think of the huge bulk of
water held up when the reservoir is full. Then you will understand
something of the difficulties of the work. Solidity and strength could
not but be the first and overpowering idea in the minds of the
builders. The longer you look, the more you are impressed. The vast
dimensions of the Dam grow upon you from moment to moment.
There is, after all, a fierce beauty in those uncompromising features,
grimly set in the determination to hold the river in bondage. The
massive structure is in harmony with the forces of Nature. Feeble
and puny it may be compared with even the least of their handiwork,
but it is impossible not to feel that its builders have been inspired
with a spark of the same creative power. They drew their plans, and
dug and built and strove their best to control the great river. They
have succeeded because a portion of that spirit of Nature against
which they struggled has passed into their work.
Is it too much to hope that a scheme of decoration may yet be
found in consonance with these ideas? Some day the Dam will be
raised to the full height of the original design, thus doubling the
present capacity of the Reservoir. Then will be the time to finish the
work magnificently, and make of it a stately monument, to be the
glory of Egypt as well as the foundation of her material prosperity.
The subject is worthy of a great artist. Only a scheme conceived on
grand lines, perfectly simple and bold, can have the least chance of
success. Anything else would be as ridiculous as a proposal to place
a statue of ordinary dimensions on the top of the Great Pyramid. The
difficulties are great, and so would be the expense, so great, indeed,
that it would be far better to avoid any attempt at decoration, unless
the results are to be admirable beyond all question. Are there no
possible successors to the architects of Karnak?
The ceremony of inauguration was, like the masonry of the Dam
itself, sensible and solid, but it was not impressive. Those who
arranged its details had forgotten the vastness of the theatre in
which it was performed. From the purely spectacular point of view it
was a failure. Egypt has no money yet to spend on functions.
Perhaps a better stage management might have made a better
display without any greater expense. The material benefits of English
rule would be appreciated none the less gratefully for a little gilding.
But the Englishman in Egypt has had other things to think about than
the organization of what the native would call ‘fantasias.’ So he fell
back on the established custom of his own country. Wherever he
goes he carries with him law and order and equity and righteousness
and commonsense, and he also carries a peculiar kind of public
ceremonial routine. Everybody knows it. The invited guests arrive in
special trains, and perspire in top-hats and frock-coats for an
interminable time, until Royalty arrives full of gracious smiles amid a
cheering crowd. The distinguished persons pass into a pen carpeted
with red baize, where all the notables are assembled. Somebody
makes a perfectly inaudible speech, which receives a gracious and
inaudible reply. A button is pressed here, a lever turned there, and
several extraordinary things begin to happen in consequence. Then
a number of good men and true receive some well-earned
decorations, Royalty graciously departs, and everybody presses
home as best he can, while the band plays the National Anthem. The
whole is accompanied by the clicking of innumerable cameras, and

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Download textbook Cell Biology Of Herpes Viruses 1St Edition Klaus Osterrieder ebook all chapter pdf

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Cell Biology of Herpes Viruses 1st

Edition Klaus Osterrieder

Herpes Simplex Viruses First Edition Tomas Bergstrom

Molecular and Cellular Biology of Viruses 1st Edition

Neural cell biology 1st Edition Cheng Wang

Biology of the Fungal Cell Dirk Hoffmeister

Essential Cell Biology Fifth Edition Alberts

Cell biology : translational impact in cancer biology

Cell biology 3rd Edition Thomas D. Pollard

Essential Cell Biology 5th Edition Bruce Alberts

Klaus Osterrieder Editor

The series publishes volumes in two different formats:

Manuscripts should be addressed to

Prof. Dr. F. CLASCÁ, Department of Anatomy, Histology and Neurobiology

Prof. Dr. J.-P. TIMMERMANS, Department of Veterinary Sciences, University of Antwerpen,

More information about this series at

ISSN 0301-5556 ISSN 2192-7065 (electronic)

Library of Congress Control Number: 2017941254

© Springer International Publishing AG 2017

Printed on acid-free paper

This Springer imprint is published by Springer Nature

Berlin, Germany Klaus Osterrieder

Arvin A, Campadelli-Fiume G, Mocarski E, Moore PS, Roizman B, Whitley R, Yamanishi K

1 Initial Contact: The First Steps in Herpesvirus Entry . . . . . . . . . . . 1

Walid Azab and Klaus Osterrieder

W. Azab (*) • K. Osterrieder

© Springer International Publishing AG 2017 1

1.2 Attachment to Cells

Herpesvirus attachment to the cell surface is a charge-based and a relatively

1.3 Receptor Binding

Recently, our understanding of the entry process of alphaherpesviruses has

1.3.1 gD-Binding Receptors

1.3.1.1 Herpes Simplex Viruses

Table 1.1 Glycoproteins of alphaherpesviruses and their cellular receptors

1.3.1.2 Equine Herpesviruses

histocompatibility (MHC) class I molecules were shown to serve as entry receptors

1.3.2 gB-Binding Receptors

gB is the most conserved glycoprotein of the cell entry machinery of herpesviruses

1.3.3 gH/gL-Binding Receptors

1.3.4 gE-Binding Receptors

1.4 Signaling Triggered by Alphaherpesvirus Binding

1.5 Calcium-Signaling Pathways

1.6 Small GTPases

The role of GTPase-signaling cascade during alphaherpesvirus entry was reported

1.7 Model of Entry: Refinement

4. Signaling cascades: gH-integrin interaction initiates signaling that results in Ca+2

1.8 Conclusion and Remarks

Abdelgawad A, Azab W, Damiani AM, Baumgartner K, Will H, Osterrieder N, Greenwood AD

Berridge MJ (1993) Inositol trisphosphate and calcium signalling. Nature 361(6410):315–325.

THE DAM AND THE NEW BARRAGES

This division meant that 52,000 acres could be reclaimed to

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