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326 | Coppo et al.

Thymidine kinase is a dispensable virulence factor in ILTV 2003a; Pavlova et al., 2010; Mundt et al., 2011), deletion mutants
that is not involved in viral replication or cell-to-cell spread in lacking this glycoprotein are attractive vaccine candidates in con-
vitro. Two TK deletion-mutants have been isolated and exam- trol strategies based on the differentiation between infected and
ined for in vitro growth characteristics and in vivo attenuation vaccinated animals (DIVA) (Pavlova et al., 2010).
(Schnitzlein et al., 1995; Han et al., 2002). ORFs UL27 (gB), The UL49.5 ORF, which encodes for gN, has already been
UL31, UL37, UL46 and UL48 have been investigated in regard described regarding its interactions with gM (see above). The
to protein expression, processing and/or intracellular localization UL50 ORF encodes a deoxyuridine triphosphatase (dUTPase),
and have been found to have functions consistent with those of dispensable for viral replication in vitro and in vivo, and its dele-
homologues in other herpesviruses (Poulsen and Keeler, 1997; tion had no effects on ILTV replication, cell-to-cell spread or
Helferich et al., 2007a) (Table 11.1). Characterization using poly- virulence (Fuchs et al., 2000).
clonal antibodies defined gB to be expressed in the endoplasmic Within the US region of the ILTV genome lay a cluster of five
reticulum (ER) as 110 kDa monomers that rapidly assemble into conserved alphaherpesvirus genes (US4–US8), which includes
homodimers composed of 100 kDa subunits. Immature homodi- members of the gD-family of herpesvirus proteins that comprises
mers are then transported to the trans-Golgi network where they US4 (glycoprotein G, gG), US6 (gD) and US7 (glycoprotein
are further modified by the addition of sugars (fucose) and con- I, gI) (McGeoch, 1990; Kongsuwan et al., 1993a; Thureen and
version of oligosaccharide side-chains to complex carbohydrates. Keeler, 2006), all of which contain conserved cysteine residues at
The modified gB dimers are then proteolytically cleaved to form the N-terminus of the translated polypeptide (McGeoch, 1990).
two disulphide-linked species, each with an apparent molecular Within the US region also lays UL47, which in ILTV has been
mass of 58 kDa (Poulsen and Keeler, 1997). translocated from the UL region, upstream to the US4 ORF
The UL44 gene encodes for gC, a conserved alphaherpesvirus (Kongsuwan et al., 1995; Wild et al., 1996; Ziemann et al., 1998a)
protein involved in viral cell attachment and entry, expressed as (Fig. 11.1). Except for US2, US3 (protein kinase) and US6 (gD),
a 60 kDa N-glycosylated product that is incorporated into the specific gene-deletion mutants have been isolated for each of
virion and expressed at the cell surface of infected cells (York the ORFs within the US region (Fuchs et al., 2005; Devlin et al.,
et al., 1990; Kingsley et al., 1994; Veits et al., 2003a; Mundt et 2006a,b; Helferich et al., 2007a,b; Pavlova et al., 2013). While
al., 2011). Monoclonal antibody (mAb) produced in differ- ILTV US2 has not been investigated in any capacity, sequence
ent laboratories have consistently detected a 60 kDa product and transcription analysis of the US3 ORF has found conserved
(York et al., 1987, 1990; Veits et al., 2003a; Mundt et al., 2011), domains characteristic of protein-serine/threonine kinases,
which originally was thought to represent gJ (Kongsuwan et al., which would be consistent with its function in other herpesvi-
1993b), but has been confirmed to represent ILTV gC (Veits et ruses (Kongsuwan et al., 1995). The kinase encoded by US3 was
al., 2003a). A deletion-mutant has been isolated (Pavlova et al., categorized as a late (γ1) gene by (Kongsuwan et al., 1995), but as
2010) and some functional analyses of this glycoprotein have an early III gene by (Mahmoudian et al., 2012).
been conducted (Kingsley et al., 1994; Kingsley and Keeler, 1999; Glycoprotein G is a highly characterized protein in ILTV.
Pavlova et al., 2010). Glycoprotein C is an abundant ILTV com- It is encoded by US4 and functions as a viral chemokine bind-
ponent that lacks a positively charged region in the ecto-domain ing protein (vCKBP) (Devlin et al., 2010), a function that is
at the N-terminal, which in other herpesviruses is responsible for conserved among other alphaherpesviruses (see ‘Pathogenesis
interactions with cell surface proteoglycans. Consequently, ILTV and immunity’) (Van De Walle et al., 2008). Glycoprotein G is a
does not interact with proteoglycans containing heparan- or secreted glycosylated protein (Kongsuwan et al., 1993a; Helferich
chondroitin-sulphate at the cell surface (Kingsley and Keeler, et al., 2007a,b; Devlin et al., 2010; Pavlova et al., 2013) that has
1999), which has justified hypotheses that ILTV gC may not be been implicated in cell-to-cell spread during infection of cultured
important in cell-surface viral attachment. Further in vitro stud- cells (Sun and Zhang, 2005). However, in vitro studies that meas-
ies using an ILTV gC-deletion mutant confirmed those findings, ured the growth and replication characteristics of two different
demonstrating that pre-adsorption of wild type or gC-deficient gG-deletion ILTV mutants determined that gG is not associated
ILTV with soluble heparin or chondroitin sulphate did not have with viral replication or cell-to-cell spread (Devlin et al., 2006b;
any effects on the capacity of the virus to infect cultured CEKs. Helferich et al., 2007b). Transcriptional analysis classified US4
Nevertheless, the gC-deletion mutant did exhibit slightly slower as a predominantly late (γ) gene; however, US4 transcription
entry kinetics in vitro and produced significantly smaller sized could also be observed under the influence of cycloheximide
plaques than the wild-type virus. These results suggest firstly, and phosphonoacetic acid, thus US4 also falling into the imme-
that the heparan or chondroitin binding capacity of gC has not diate early (α) and early categories (β) (Helferich et al., 2007a;
been adopted by other viral components and secondly, that gC Mahmoudian et al., 2012). Comparative analysis of viral gene
has a yet undefined role in viral entry (Pavlova et al., 2010). In transcription kinetics between infection with gG-deficient and
vivo studies determined that gC is also dispensable during in vivo gG-expressing ILTV determined that the absence of gG was
infection, that it is a virulence factor in ILTV and that it may be associated with changes in the transcription of a number of other
involved in immune-modulation strategies (see ‘Pathogenesis and viral genes in vitro and in vivo, thus indicating potential roles of
immunity’), which may help explain the attenuated phenotype gG in gene regulation (Mahmoudian et al., 2013), which would
of the gC-deletion mutant (Pavlova et al., 2010). Given the high be consistent with US4 transcription at earlier (α and β) phases of
level of immunogenicity of gC (York et al., 1987, 1990; Veits et al., the transcription cascade (Helferich et al., 2007a; Mahmoudian