Journal of General Virology (2013), 94, 193–208
DOI 10.1099/vir.0.047423-0
Correlation between structure, protein composition,
morphogenesis and cytopathology of Glossina
pallidipes salivary gland hypertrophy virus
Henry M. Kariithi,1,2 Jan
W. M. van. Lent,1 Sjef Boeren,3
.
Adly M. M. Abd-Alla,2 Ikbal Agah Ince,4,5 Monique M. van Oers1
and Just M. Vlak1
Correspondence
1
Monique M. van Oers
2
Laboratory of Virology, Wageningen University, 6708 PB Wageningen, The Netherlands
Insect Pest Control Laboratory, International Atomic Energy Agency, A-1400 Vienna, Austria
monique.vanoers@wur.nl
3
Laboratory of Biochemistry, Wageningen University, 6703 HA Wageningen, The Netherlands
4
Department of Genetics and Bioengineering, Yeditepe University, 34755, Istanbul, Turkey
5
Department of Biosystems Engineering, Faculty of Engineering, Giresun University, 28100,
Giresun, Turkey
Received 22 August 2012
Accepted 8 October 2012
The Glossina pallidipes salivary gland hypertrophy virus (GpSGHV) is a dsDNA virus with rodshaped, enveloped virions. Its 190 kb genome contains 160 putative protein-coding ORFs. Here,
the structural components, protein composition and associated aspects of GpSGHV
morphogenesis and cytopathology were investigated. Four morphologically distinct structures: the
nucleocapsid, tegument, envelope and helical surface projections, were observed in purified
GpSGHV virions by electron microscopy. Nucleocapsids were present in virogenic stroma within
the nuclei of infected salivary gland cells, whereas enveloped virions were located in the
cytoplasm. The cytoplasm of infected cells appeared disordered and the plasma membranes
disintegrated. Treatment of virions with 1 % NP-40 efficiently partitioned the virions into envelope
and nucleocapsid fractions. The fractions were separated by SDS-PAGE followed by in-gel
trypsin digestion and analysis of the tryptic peptides by liquid chromatography coupled to
electrospray and tandem mass spectrometry. Using the MaxQuant program with Andromeda as a
database search engine, a total of 45 viral proteins were identified. Of these, ten and 15 were
associated with the envelope and the nucleocapsid fractions, respectively, whilst 20 were
detected in both fractions, most likely representing tegument proteins. In addition, 51 host-derived
proteins were identified in the proteome of the virus particle, 13 of which were verified to be
incorporated into the mature virion using a proteinase K protection assay. This study provides
important information about GpSGHV biology and suggests options for the development of future
anti-GpSGHV strategies by interfering with virus–host interactions.
INTRODUCTION
The Glossina pallidipes salivary gland hypertrophy virus
(GpSGHV) is a rod-shaped, enveloped virus measuring
50 nm in width and 1000 nm in length (Garcia-Maruniak
et al., 2009). The virus has a circular dsDNA genome
of 190 032 bp and contains 160 putative protein-coding
ORFs (Abd-Alla et al., 2010a). GpSGHV is a member
of the newly established family Hytrosaviridae, genus
Glossinavirus and species Glossina hytrosavirus (Abd-Alla
et al., 2009). To date, hytrosaviruses (SGHVs) have been
identified that infect the tsetse fly Glossina pallidipes
(GpSGHV) (Jaenson, 1978), the housefly Musca domestica
A supplementary table is available with the online version of this paper.
047423 G 2013 SGM
Printed in Great Britain
(MdSGHV) (Coler et al., 1993) and probably the narcissus
bulb fly Merodon equestris Fabricius (Amargier et al., 1979).
Recently, a similar virus has been reported in the accessory
gland filaments of the braconid wasp Diachasmimorpha
longicuadata (Luo & Zeng, 2010). GpSGHV and MdSGHV
induce similar gross pathology in their hosts, most notably
the characteristic hypertrophy of salivary glands of the
adult insects and a reduction in reproductive fitness (AbdAlla et al., 2010b; Lietze et al., 2007). Whilst MdSGHV
causes symptomatic infections in the housefly (Lietze et al.,
2011b, 2012), tsetse flies infected by GpSGHV exhibit both
asymptomatic and symptomatic infections, with the
asymptomatic state being the most widespread in the fly
colonies (Abd-Alla et al., 2007).
193
H. M. Kariithi and others
GpSGHV negatively affects laboratory colonies of G.
pallidipes, often leading to colony collapse (Abd-Alla et al.,
2007, 2010b). Maintenance of healthy, productive fly
colonies is vital to tsetse fly and trypanosomiasis eradication campaigns through the sterile insect technique, thus
creating an urgent need to develop antiviral strategies to
manage GpSGHV infections. During membrane feeding,
one viraemic fly deposits up to 107 viral genome copies in
the form of virus particles into a blood meal, which are
infectious per os to healthy G. pallidipes (Abd-Alla et al.,
2010b). Although it is unknown how the virus gets into
salivary glands, it is assumed that ingested virions enter via
the midgut, transverse the haemolymph-filled haemocoel
to reach the glands where they replicate (Garcia-Maruniak
et al., 2009) and reside until transmission to new tsetse
hosts.
The proteome of the GpSGHV particle consists of 61 virally
encoded proteins (Kariithi et al., 2010). However, the
localization and function of these proteins and their
respective contribution to the virus ultrastructure and
infection process are unknown. It is particularly important
to determine GpSGHV envelope proteins (likely to be
involved in the virus entry process) and to know which
(a)
virion proteins contribute to cytoplasmic trafficking of the
virus, as these proteins are possible targets for the
development of antiviral strategies against GpSGHV.
Enveloped viruses often contain numerous host-derived
proteins, some of which are specifically incorporated into
mature virions (reviewed by Cantin et al., 2005). One
would reason that GpSGHV virions also contain proteins
of cellular origin, which reflect the morphogenesis and
egress processes. In this paper, the structure of GpSGHV
virions is detailed and a comprehensive repertoire of viral
and cellular proteins with their localization within the
virion is presented.
RESULTS AND DISCUSSION
Signature features of GpSGHV virions in infected
host cells
Electron microscopy of cryo-sections of G. pallidipes
hypertrophied salivary glands showed nucleocapsids
embedded in a chromatin-like network of electron-dense
filaments (or virogenic stroma) within the nuclei of
infected cells (Fig. 1a, panel i), presumably induced by
(b)
(i)
(ii)
(i)
(ii)
(iii)
(iv)
Vs
Nc
V
(v)
(iii)
Lum
Nc
Env
Cy
Tg
Sp
Fig. 1. Electron micrographs of infected salivary glands and GpSGHV virions. (a) Cryo-sections through hypertrophied salivary
gland cells. Nucleocapsids (Nc) were embedded in virogenic stroma (Vs) present in the nucleus (i), whilst enveloped virions (V)
were observed in the cytoplasm (Cy) (ii) and in the lumen (Lum) of the glands (iii). Note that the infected cytoplasm appeared
jumbled, the plasma membrane appeared disintegrated (indicated by arrows) and the lumen was full of enveloped virions. Bars,
200 nm (i, ii); 1 mm (iii). (b) Electron micrographs of negatively stained GpSGHV enveloped virions. The surface of a mature
virion consists of regular helically arranged surface projections (i). The top, middle and bottom views of the virion particle shown
in (i) are also shown (ii–iv, respectively). A cryo-section through a group of virions in an infected cell cytoplasm is shown (v). The
inset shows a high-magnification image of a cross-section through a virion particle, revealing the nucleocapsid, tegument (Tg),
envelope (Env) and surface projections (Sp). Bars, 100 nm (i–iv); 200 nm (v).
194
Journal of General Virology 94
Structure and virion components of GpSGHV
GpSGHV infection. This is similar to the cytopathology
of baculoviruses, where studies have demonstrated that
packaging of virus particles occurs in the virogenic stroma,
where empty capsids assemble in the pockets between
chromatin-like filaments and the capsids then fill with DNA
acquired from the stroma (Fraser, 1986; Young et al., 1993).
Another question is how GpSGHV acquires its envelope.
Enveloped viruses have been shown to acquire their
envelopes through various mechanisms. For instance,
white spot syndrome virus (WSSV, family Nimaviridae)
acquires its envelope within the nucleus (Xie et al., 2006),
whilst herpesviruses are enveloped by budding either
through the nuclear membrane or into trans-Golgi
membranes (Johnson & Baines, 2011). Other herpesviruses
are enveloped entirely in the cytoplasm (Tandon &
Mocarski, 2011). Many enveloped vertebrate (e.g. orthoand paramyxoviruses and retroviruses) and invertebrate
viruses (baculovirus budded virus) acquire their envelopes
by budding through the plasma membrane. Our results
showed that naked GpSGHV nucleocapsids were most
abundant in the nucleus, whilst enveloped virions were
restricted to the cytoplasm (Fig. 1a, panel ii), suggesting a
cytoplasmic envelopment process, possibly via the endoplasmic reticulum–Golgi system. This is in agreement with
recent evidence that nucleocapsids of the related MdSGHV
egress from the nucleus via a nucleopore complex for
cytoplasmic envelopment (Boucias et al., 2012).
The lumen of infected salivary glands was filled with closely
packed arrays of rod-shaped, enveloped virions (Fig. 1a,
panel iii). Our previous reports have demonstrated that
virions shed into the saliva are infectious per os to healthy
flies during membrane feeding in tsetse colonies (Abd-Alla
et al., 2010b; Kariithi et al., 2011). The infected cells
appeared to be in disarray and extended into the adjoining
lumen, with the plasma membranes disintegrated (Fig. 1a,
panel iii). None of the examined sections showed evidence
of virus budding through the plasma membrane into the
salivary gland lumen. These observations suggest that
GpSGHV virions egress from the infected cell via
disintegration of the plasma membrane. This is remarkably
different from MdSGHV, where recent studies have
demonstrated that the particles migrate to and bud out
of the plasma membrane bordering the salivary gland
lumen (Boucias et al., 2012; Lietze et al., 2011a).
Negative-staining electron microscopy and electron tomography of enveloped virions extracted from freshly excised
glands showed a helical arrangement of elongated surface
projections, ~13 nm in length and with a periodicity of
15 nm (Fig. 1b, panels i–iv). Surface projections have
also been reported in vesicular stomatitis virus (VSV)
(Cartwright et al., 1969) and in several poxviruses
(Hiramatsu et al., 1999). In VSV, the surface projections
are composed of cellular material and virus-specific
antigens, and enzymic removal of these substructures
prevents virus attachment to susceptible cells (Cartwright
et al., 1969). De Giuli et al. (1975) suggested that the surface
http://vir.sgmjournals.org
projections in some strains of Rous sarcoma virus are
essential for the interaction with cellular receptors to permit
initiation of the infectious process. It is likely that the surface
projections observed in GpSGHV are made up of polymeric
structures of viral proteins, for which the 44 kDa proteins
encoded by ORF96 and ORF97, two of the most abundant
viral proteins (Table 1; see also Fig. 4), are possible
candidates. Additionally, host-derived proteins (see below)
may also be present in the GpSGHV surface projections.
The GpSGHV virion contains an internal core with a mean
diameter of 40 nm, which is separated from the envelope
by an electron-dense proteinaceous matrix of ~10 nm
thick (Fig. 1b, panel v). We propose to call this amorphous
structure the GpSGHV tegument. Almost 50 % of the
identified GpSGHV structural proteins are found in the
tegument (Table 1), similar to the situation in human
cytomegalovirus (Varnum et al., 2004). The GpSGHV
nucleocapsid core consists of a thin dense layer surrounding a central, higher-density area, suggesting that the core
is not hollow. Assuming an equal distribution of the
superhelical DNA in the nucleocapsid, the superhelicity of
GpSGHV DNA (190 kb, 900 nm long nucleocapsid) is
approximately half that of the average baculovirus (130 kb,
300 nm long nucleocapsid) (Jehle et al., 2006).
Purification and fractionation of GpSGHV virions
For a comprehensive analysis of the full set of virion
proteins, the purity and integrity of the virus preparations
are critical. Initial purification of GpSGHV particles using
a sucrose gradient resulted in total loss of the virus
envelope, leading to the erroneous conclusion that
GpSGHV is a non-enveloped DNA virus consisting of 12
polypeptides (Odindo et al., 1986). Although purification
of the virus was later improved by the use of Nycodenz
gradient centrifugation (Abd-Alla et al., 2007; Kariithi et al.,
2010), the integrity of the virus particles was still
compromised. In the current study, an improved
GpSGHV purification protocol was developed using the
high-molecular-mass sugar Ficoll, prepared in an organic
buffer (HEPES) and supplemented with protease inhibitors
at high pH (pH 8.0) (Kariithi et al., 2012). This protocol
resulted in preservation of the rod shape of the GpSGHV
virions with intact envelope surrounding the virus particles
(Fig. 2a). Treatment of these particles with 1 % NP-40/
137 mM NaCl buffer resulted in efficient fractionation of
the envelope and nucleocapsid components (Fig. 2b, c).
Silver staining of SDS-PAGE gels of NP-40-treated virions
showed four dominant bands in the nucleocapsid fraction
(130, 55, 43 and 30 kDa) and two in the envelope fraction
of ~40 and 26 kDa (Fig. 2d, indicated with asterisks).
Several other protein bands observed in the intact virions
were associated with either the nucleocapsid or the
envelope fraction with varying intensities. A high-molecular-mass smear was observed close to the top of the
resolving gel in the envelope fraction (Fig. 2d) and may be
an indication of covalently modified glycoproteins.
195
Phosphorylated proteins are indicated with asterisks (see text for details). EPV, entomopoxvirus; ezrA, septation ring formation regulator ezrA; GBD-FH3, Rho GTPase-binding/formin homology 3
domain; GV, granulovirus; HDAC, histone deacetylase; NLS-BP, nuclear localization signal binding protein; NPV, nuclear polyhedrosis virus; PPase-tensin, pyrophosphatase tensin-type domain
profile; PUM, Pumilio RNA-binding repeat profile; RGD, Arg-Gly-Asp/cell-attachment sequence; SCG, serine-cysteine-glycine; SP, signal peptide; NUMOD3, nuclease-associated modular DNAbinding domain 3; TM, transmembrane domain.
Journal of General Virology 94
Classification
ORF no.
UniProt ID
Molecular mass Coverage (%)
(kDa)
No. peptides Homology to cellular
identified
proteins and/or
proteins in other
viruses
Envelope
components
1*
B0YLF6
81.4
11.8
7
88
102*
B0YLP2
B0YLQ6
77.8
76.1
8.7
15.5
6
8
41
B0YLJ5
48.8
13.6
5
53*
B0YLK7
40.2
14.2
4
39
B0YLJ3
37.7
9.6
3
36
B0YLJ0
13.8
18.3
3
68
72
B0YLM2
B0YLM6
12.7
31.8
32.4
22.3
4
6
Signature domain(s)/motifs
(amino acid regions harbouring
the motif/domain)
Functional annotation
(references)
PIF-0 (P74),
Spodoptera
pectinicornis NPV
SP; isoleucine-rich activation motif
(aa 648–686)
Oral infection (Peng et al.,
2010); virus replication
(Chazal & Gerlier, 2003);
tissue-specific gene
expression (Attardi & Tjian,
1993; Kuzio et al., 1989)
PIF-1, Neodiprion
abietis NPV
TM, SP; epidermal growth factor-like
domain; multiple tyrosine kinase
phosphorylation sites (aa 248–254,
273–280, 418–489 and 593–599)
Oral infection (Peng et al.,
2010); virus replication
(Chazal & Gerlier, 2003);
MAPK pathway/growth or
differentiation signalling
(Afonso et al., 2000; Alroy &
Yarden, 1997)
ORF MSV214,
Melanoplus
sanguinipes EPV
PIF-2, Gryllus
bimaculatus
nudivirus
SP; SCG gene family protein
ORF67, WSSV
SP
SP; threonine-rich regions
(aa 123–197) interrupted by
proline/serine residues (aa 167–179)
SP; thymidylate synthase
TM; ERV/ALR thiol oxidase domain
(aa 157–254)
Oral infection (Peng et al.,
2010); VIRUS replication
(Chazal & Gerlier, 2003)
Heavily O-glycosylated
protein – cellular binding
(McGeoch et al., 1993)
Protection of viral genome
from host DNases (Forterre
et al., 2004)
Disulfide-bond formation
during cytoplasmic virus
assembly (Hakim et al.,
2011; Hakim & Fass, 2009;
Senkevich et al., 2000a, b)
H. M. Kariithi and others
196
Table 1. Structural GpSGHV proteins identified by LC-MS/MS
http://vir.sgmjournals.org
Table 1. cont.
Classification
Nucleocapsid
components
UniProt ID
Molecular mass Coverage (%)
(kDa)
No. peptides Homology to cellular
identified
proteins and/or
proteins in other
viruses
197
45*
B0YLJ9
201.1
10.2
15
62*
B0YLL6
512.1
9.1
30
83
B0YLN7
81.6
16.6
9
104
108*
B0YLQ8
B0YLR2
77.9
63.9
7.6
19.4
4
8
107*
B0YLR1
59.6
35.9
16
61
B0YLL5
57.4
22.5
8
106*
B0YLR0
55.1
29.2
9
70
44
154
B0YLM4
B0YLJ8
B0YLV8
50.9
42.8
40.1
21.3
16.7
11.8
5
5
4
Signature domain(s)/motifs
(amino acid regions harbouring
the motif/domain)
Functional annotation
(references)
PPase-tensin (aa 299–603); coiled-coil Site-specific host–virus
region (aa 533–542); t-SNAREs
interactions and packaging
(aa 466–528); SF3 helicase (aa 1411– (James et al., 2003)
1581); asparagine-rich region (aa 417–
454)
ORF147, Trichoplusia SP; NLS-BP (aa 4357–4373); nebulin Nuclear targeting (Robbins
ni ascovirus-2c
repeats (aa 2541–2561 and 4207–4231); et al., 1991); actin-zipper
GBD-FH3 (aa 856–1247; 1492–1885; (Labeit & Kolmerer, 1995);
2138–2557); leucine zipper (aa 1148– homing endonuclease
2093); ezrA (aa 964–1584); NUMOD3 (Sitbon & Pietrokovski,
motifs (aa 850–863, 3416–3429, 3465– 2003); transcription
3478); spectrin repeats (aa 976–1066, regulation (Groves et al.,
1162–1299, 1546–1649); t-SNAREs
2001);
(aa 1677–1705); Ag332 (aa 2804–2869)
ORF AMV214 AmNLS-BP
sacta moorei EPV
TM; coiled-coil region
Cell-division protein SP; AAA-ATPase central domain
Molecular chaperone/
48, lymphocystis
protein (aa 240–384); PAN
remodelling of
disease virus (China (aa 50–463)
macromolecules (Iyer et al.,
isolate)
2004); DNA unwinding and/
or packaging (Gorbalenya &
Koonin, 1989)
Cell-division protein SP; P-loop/AA-ATPase central domain Molecular chaperone/
48, lymphocystis
protein (aa 212–388); PAN (aa 27–
remodelling of
disease virus (China 430)
macromolecules (Iyer et al.,
isolate)
2004); DNA unwinding and/
or packaging (Gorbalenya &
Koonin, 1989)
Serine/threonine/glutamine-rich
stretches (aa 25–106)
Glutamine-rich region (aa 265–286)
Sequence-specific
transcription activation
(Asković & Baumann, 1997;
Biggin et al., 1988)
Structure and virion components of GpSGHV
Nucleocapsid
components
ORF no.
Classification
Tegument
components
ORF no.
UniProt ID
Molecular mass Coverage (%)
(kDa)
No. peptides Homology to cellular
identified
proteins and/or
proteins in other
viruses
Journal of General Virology 94
52
B0YLK6
36.7
5.6
2
31
B0YLI5
33.6
16.8
5
113*
B0YLR7
33.1
30.6
8
98*
101*
38*
B0YLQ2
B0YLQ5
B0YLJ2
13.5
12.3
136.7
44.3
46.2
46.3
7
6
47
10
B0YLG4
127.0
68.4
81
71
86
64
B0YLM5
B0YLP0
B0YLL8
72.0
70.2
70.0
4.1
43.8
47.4
2
25
29
46
B0YLK0
61.5
41.7
18
47
B0YLK1
47.2
38.1
17
97*
B0YLQ1
44.4
55.1
24
Signature domain(s)/motifs
(amino acid regions harbouring
the motif/domain)
Functional annotation
(references)
Cellular protein,
Plasmodium
falciparum
Cellular protein
PY00593, Plasmodium
yoelli strain 17XNL
Leucine/isoleucine-rich regions (aa 4– Export of macromolecules
30); N-myristoylation sites (aa 48–53); from nucleus to cytoplasm
HDAC-interaction like domain protein (Görlich & Kutay, 1999);
Gene regulation (Gwack
et al., 2001; Kuo & Allis,
1998; Zhu et al., 1999)
SP
TM
TM; SP
SP; RGD motif (aa 914–916)
ORF MSV156, M.
sanguinipes EPV
ORF AMV130 A.
moorei EPV
Virus–host cell interactions
during viral entry (Bai et al.,
1993; Belin & Boulanger,
1993; Roivainen et al., 1991;
Ruoslahti, 1996;
Shayakhmetov et al., 2005)
Potential N-glycosylation sites (aa 157, Nuclear targeting (Robbins
313, 327 and 342); multiple serine/
et al., 1991); recruitment of
threonine/tyrosine-rich regions;
proteins in signalling (Kay
proline-rich profile (aa 292–357);
et al., 2000); chromatin
bipartite NLS (aa 232–249);
regulation of virus infection
bromodomain-2 profile (aa 662–732); (Lieberman, 2006;
PPase-tensin (aa 801–1013)
Nicewonger et al., 2004)
ATP-binding cassette transporter;
PUM (aa 1–14)
PPase (inorganic pyrophosphatase)
(aa 438–444)
Cellular protein
(CBG22662);
Caenorhabditis
briggsae
TM; SP
Regulation of cholesterol
efflux (Mujawar et al., 2006)
Recruitment of proteins in
signalling (Kay et al., 2000)
H. M. Kariithi and others
198
Table 1. cont.
TM; SP
TM
6
9
24.0
61.1
B0YLR6
B0YLJ7
112
43
19.1
16.9
Matrixin peptidase,
TM; SP; zinc-dependent metalloSpodoptera litura GV protease
19
12
18
18
20
10
2
55.0
44.7
66.7
62.9
62.6
38.4
11.4
B0YLP7
B0YLK4
B0YLP8
B0YLM1
B0YLM3
B0YLN9
B0YLR4
93*
50
94
67*
69*
85
110
http://vir.sgmjournals.org
Tegument
components
B0YLQ0
B0YLG1
B0YLF7
38.5
32.7
32.7
31.0
30.9
30.1
23.6
Identification and characterization of GpSGHV
virion structural proteins
96*
7
2
43.5
41.0
38.7
66.7
7.9
46.8
28
3
17
TM
Signature domain(s)/motifs
(amino acid regions harbouring
the motif/domain)
No. peptides Homology to cellular
identified
proteins and/or
proteins in other
viruses
Molecular mass Coverage (%)
(kDa)
UniProt ID
ORF no.
Classification
Table 1. cont.
Western blots using polyclonal antibody directed against
the product of ORF10 showed multiple bands in both the
nucleocapsid and envelope fractions, the most prominent
of which were present in the nucleocapsid fraction (Fig.
3a). Western blot analysis confirmed the presence of the
marker for the GpSGHV envelope (the P74 protein) in the
envelope fraction (Fig. 3b).
Involved in ectodomain
shedding (Dolnik et al.,
2004)
Functional annotation
(references)
Structure and virion components of GpSGHV
A total of 45 virion proteins were identified by liquid
chromatography coupled to electrospray and tandem mass
spectrometry (LC-MS/MS) analysis of the envelope and
nucleocapsid fractions (see Table 1 for details). Of the 45
GpSGHV proteins, ten were found only in the envelope
fraction, 15 in the nucleocapsid fraction only and 20 were
measurably present in both fractions. Nine of the identified
proteins had potential transmembrane domains (TMs) and
15 had predicted signal peptides (SPs), of which seven were
identified in the envelope fraction. The giant viral protein
encoded by ORF62 (4373 aa) was the least abundant
protein measured. Of the 20 proteins associated with both
fractions, five (encoded by ORF50, -10, -94, -46 and -86)
were found to be much more abundant in the nucleocapsid
fraction than in the envelope fraction (Fig. 4). Similarly,
five other proteins (encoded by ORF96, -97, -69, -64 and
-112) were much more abundant in the envelope than in
the nucleocapsid fraction.
Three of the GpSGHV envelope proteins were homologues
of the baculovirus occlusion-derived virus envelope
proteins P74, PIF-1 and PIF-2, which in baculoviruses
are essential in the initial stages of oral infection of the host
(reviewed by Slack & Arif, 2006). They are highly likely to
have similar roles in oral infections of GpSGHV in tsetse
midguts. After ingestion, hytrosaviruses find their way to
the salivary glands, thereby causing distinct hypertrophy of
the gland tissues (Garcia-Maruniak et al., 2009). It is not
known how these viruses induce hyperplasia of the infected
glands. However, it is noteworthy that the GpSGHV PIF-1
sequence contains an epidermal growth factor (EGF)-like
domain, SP, TMs and multiple tyrosine kinase phosphorylation sites (Table 1). EGF-like domains are known to
initiate a tyrosine kinase-mediated signalling cascade that
culminates in recruitment of the evolutionally conserved
mitogen-activated protein kinase (MAPK) pathway and
results in growth/differentiation signals (reviewed by Alroy
& Yarden, 1997). Interestingly, it has been demonstrated
that the fowlpox virus ORF FPV211 product, which
contains these structural features, contributes to the
hyperplasia of fowlpox virus-infected tissues (Afonso et al.,
2000). Whether PIF-1 plays a role in tsetse salivary gland
hyperplasia remains to be investigated.
Motif analyses using ExPASy revealed several features in
the virion protein sequences, among which were an
arginine-glycine-aspartate (RGD) motif/cell-attachment
sequence, bipartite nuclear localization signals, a P-loop
199
H. M. Kariithi and others
(a)
(b)
(c)
(a)
M
GpSGHV
Nucleocapsid
Envelope
kDa
130
95
72
55
(d)
M
GpSGHV
Envelope
Nucleocapsid
kDa
170
130
*
(b)
M
kDa
95
95
72
72
55
43
*
*
*
GpSGHV
Envelope
Nucleocapsid
55
Fig. 3. Western blot analysis of GpSGHV virion proteins using
anti-rabbit serum against proteins encoded by GpSGHV ORF10
(a) and ORF1 (P74) (b). Lane M, marker (kDa).
34
*
26
*
17
Fig. 2. (a) Electron microscopy micrographs of high-quality
GpSGHV virions after purification using 10–40 % Ficoll gradient
centrifugation. (b, c) Treatment of the purified virions with 1 % NP40/137 mM NaCl buffer resulted in efficient removal of the
envelope (b) from the nucleocapsids (c). Bars, 100 nm. (d)
Protein profiles of intact virions, envelope and nucleocapsid
components of purified virions. Dominant bands in the envelope
and nucleocapsid fractions are indicated by asterisks. Lane M,
marker (kDa).
nucleotide-binding motif (ATP/GTP-A2), nebulin repeats
and regions enriched in specific amino acids such as
proline, serine, isoleucine/leucine, asparagine and glutamine. In addition to the signature domains and predicted
structures, 14 of the identified viral proteins had homologues in other viruses, whilst four showed similarity to
known cellular proteins (Table 1).
Analysis of the phosphorylation status of GpSGHV
structural proteins by Western blotting showed six major
signals in the intact virus sample (marked with asterisks in
Fig. 5; two signals .170 kDa, and signals of approximately
43, 38, 30 and 15 kDa. Of these, four signals were also
200
visualized in the nucleocapsid fraction (Fig. 5). Several
minor bands were also observed in the intact virion
preparation, some of which were also observed in the
nucleocapsid fraction. Only two signals were observed in
the envelope fraction (~44 kDa). Bioinformatics analysis of
the GpSGHV proteins in the structural components
indicated that at least six GpSGHV tegument proteins
(encoded by ORF38, -67, -69, -93, -96 and -97) are likely to
be phosphorylated (data not shown). Based on the
molecular sizes of the proteins identified by LC-MS/MS,
phosphorylation of these proteins was confirmed by
Western blot analysis using mouse anti-phosphoserine/
-threonine/-tyrosine mAbs (marked with asterisks in Table
1; Fig. 5). In conclusion, the majority of the phosphorylated viral proteins were localized in the tegument.
Although no common sequences/motifs have been identified to direct proteins into the viral tegument, it has been
suggested for herpesviruses that phosphorylation facilitates
incorporation of proteins into the viral tegument (Kalejta,
2008), with the majority of tegument proteins being
phosphorylated (Shenk & Stinski, 2008). Viral phosphoproteins may have a significant influence on the assembly
(Sen & Todaro, 1977; Sen et al., 1977) and uncoating of
virions (Lackmann et al., 1987; Witt et al., 1981), on the
interaction between viral proteins and host DNA
(Scheidtmann et al., 1984) and on transcriptional regulation (Hsu & Kingsbury, 1985; Hsu et al., 1982; Kingsford
& Emerson, 1980). Antiviral interventions could be
Journal of General Virology 94
Structure and virion components of GpSGHV
7
7
96
6
6
43
93
97
50
5
Log total iBAQ peak intensity
69
47
10
5
38
67
64
4
4
94
112
46
86
3
3
101
110
107
98
2
113
108
106
61
44
83 7
154
1
–2
2
85
72
71
45
52
70
Nucleocapsid
–3
P74
2
31
–1
88 41
104
39
62
68
PIF1
1
36
Envelope
PIF2
0
1
2
0
Log protein abundance ratio (envelope/capsid)
Fig. 4. Abundance distribution of GpSGHV-encoded proteins (&) and virion-associated host-derived cellular proteins (m)
identified by LC-MS/MS. Large ovals enclose the proteins that were measured only in the envelope or nucleocapsid fraction,
whilst the rest were detectable in both fractions. Numbers correspond to the GpSGHV ORFs. IBAQ, Intensity-based absolute
quantification. See Table S1A for the identities of the cellular proteins detected in the envelope and nucleocapsid fractions.
directed against incorporation of these proteins into the
tegument, which may arrest virus morphogenesis.
Cellular proteins in GpSGHV virions
Fifty-one host (cellular) proteins were identified in the
GpSGHV virion proteome. Of these, eight were measurable
only in the nucleocapsid fraction, including several 26S/60S
ribosomal proteins, histone H3-II, phage terminase
(Sodalis glossinidius strain morsitans) and vesicle coat
complex COPI-e (see Table S1, available in JGV Online,
and Fig. 4). Similarly, five of the cellular proteins were
measurable only in the envelope fraction, including cargo
transport protein EMp24, a major outer-membrane
lipoprotein (Sodalis glossinidius strain morsitans), F0F1type ATP synthase-b and an uncharacterized membranetrafficking protein. Other cellular proteins were detected in
both the envelope and nucleocapsid fractions with varying
abundances (see Fig. 4). Furthermore, enzymic codes could
be assigned to 22 of the 51 cellular proteins. The identified
cellular proteins could be divided into nine categories by
their (putative) functions (Table S1).
http://vir.sgmjournals.org
Treatment of purified GpSGHV particles with proteinase K
(PK) removed many of the proteins associated with the
virus (Fig. 6a). After passing the PK-treated sample
through a 20 % Ficoll cushion followed by LC-MS/MS,
none of the ten virion envelope proteins was detectable, in
contrast to the majority of the tegument proteins. Based on
the molecular masses, the major viral proteins that
disappeared after PK treatment (marked with asterisks in
Fig. 6a, lane 2) included proteins encoded by ORF45
(201 kDa), ORF38 (137 kDa), ORF107 (60 kDa), ORF47
and -97 (~50 kDa), ORF-69 and -85 (~30 kDa), and ORF68 and -101 (~12 kDa). When observed by transmission
electron microscopy, the PK-treated virus particles were
devoid of intact envelopes (compare Fig. 6b and c).
Thirteen host proteins could still be identified in the
sample that was passed through the 20 % Ficoll cushion. Of
these proteins, six (heat-shock cognate-70 family proteins,
histone H2A, myosin, tubulin, actin and glyceraldehyde-3phosphate dehydrogenase) have been demonstrated to be
incorporated in viruses (Table 2 and references therein).
Western blot analysis of the PK-treated samples with
antibodies against cellular host proteins produced a clear
201
H. M. Kariithi and others
M
kDa
170
130
95
72
GpSGHV
Nucleocapsid
*
*
*
Envelope
55
43
34
26
*
*
*
*
*
Fig. 5. Western blot analysis of the phosphorylation status of GpSGHV structural proteins
in intact virions and the nucleocapsid and
envelope fractions. Major phosphorylated proteins in the intact virus sample and the
nucleocapsid fraction are indicated by asterisks, whilst minor ones are indicated by arrows.
Lane M, marker (kDa).
17
*
*
signal with anti-myosin IgG (Fig. 6d). Western blot
analysis using antibodies against the other tested cellular
host proteins showed either a negative signal (actin) or a
(a)
weak signal (ubiquitin and tubulin). Limitations of virus
quantities, however, precluded optimization of the protocol for detection of these host proteins.
(b)
M
(– PK)
(+ PK)
(c)
(+ PK + purity)
(+ PK + purity)
(+ PK)
kDa
170
130
95
*
*
*
72
55
43
34
**
**
(d)
kDa
M
(– PK)
(+ PK)
(+ PK + purity)
95
26
72
*
17
Fig. 6. PK protection assay of purified GpSGHV virions. (a) Silver-stained SDS-polyacrylamide gel of non-treated (”PK), PKtreated (+PK) and PK-treated samples that were passed through a 20 % Ficoll cushion to remove PK and free-floating
peptides (+PK+purity). Viral proteins that disappeared after PK treatment are indicated by asterisks in lane 2. (b, c) PK
treatment followed by 20 % Ficoll purification resulted in naked nucleocapsids, as shown by comparison of (b) and (c). (d)
Western blotting with rabbit polyclonal anti-myosin antibody detected myosin in the (+PK+purity) fraction (see Table 2). Bars,
200 nm (b, c). Lane M, marker (kDa).
202
Journal of General Virology 94
http://vir.sgmjournals.org
Table 2. Verification of incorporation of cellular proteins in mature GpSGHV virions
Purified virions were either treated with 0.08 mg PK per 1 mg virion protein or treated and purified through a 20 % Ficoll cushion followed by LC-MS/MS analysis. HIV-1, Human
immunodeficiency virus 1; Mo-MuLV, Moloney murine leukemia virus; SV-40, simian virus 40; WNV, West Nile virus.
Classification
Protein synthesis
Host molecule
Mass (kDa) Unique peptides
Incorporated in
other viruses
Virus (reference)
50.4
94.5
6
5
No
No
HIV-1 (Cimarelli & Luban, 1999), WNV
(Blackwell & Brinton, 1997)
Protein processing
machinery
Hsc70-4
Hsc70-3
D3TPL1
D3TRH2
60.8
72.6
4
10
Yes
Yes
HIV-1 (Gurer et al., 2002)
Transcription regulation
Histone H2A
D3TPW0
15.0
2
Yes
SV-40 (Chen et al., 1979)
Cytoskeleton
Myosin (heavy chain)
Tubulin-a
Actin 5C
D3TQ00
D3TQG7
D3TQK0
87.3
49.9
41.8
30
11
3
Yes
Yes
Yes
HIV-1 (Ott et al., 1996, 2000b)
Mo-MuLV (Wang et al., 2003)
Mo-MuLV (Nermut et al., 1999; Wang
et al., 2003)
Tubulin-b
D3TR30
50.2
7
Glyceraldehyde-3-phosphate
dehydrogenase
Endoplasmic reticulum glucoseregulated protein
Protein disulfide isomerase
D3TRU0
35.7
2
Yes
HIV-1 (Ott et al., 2000a)
D3TS03
82.1
16
D3TRE3
57.4
3
No
HIV-1 (Ott 2002)
Porin
D3TRY2
30.4
5
Ion transport
203
Structure and virion components of GpSGHV
Elongation factor 2
D3TNV8
D3TP87
Cellular metabolism
Elongation factor 1-a
UniProt protein
ID
H. M. Kariithi and others
Implications of finding host proteins in GpSGHV
The results presented in this study clearly showed that
GpSGHV virions can contain numerous cellular proteins.
Cellular host proteins incorporated into or onto virus
particles have been demonstrated to play specific or
supplementary roles in virus life cycles. Actin and myosin
have been reported to be incorporated, for instance, into
Mo-MuLV (Nermut et al., 1999) and HIV-1 (Ott et al.,
1996, 2000b). In HIV-1, inhibition of the interaction
between the Gag protein and actin and myosin markedly
reduced the amount of virus released from infected cells
(Ott, 1997). Glyceraladehyde-3-phosphate dehydrogenase
has been reported to be co-incorporated with actin inside
mature HIV-1 virions, where it play roles in the
enhancement of gene expression (Ott et al., 2000b).
Translation elongation factor-1a has been proposed to
target WNV RNA to a microenvironment for efficient virus
replication (Blackwell & Brinton, 1997) and play roles in
the packaging of HIV-1 into nascent virions (Cimarelli &
Luban, 1999). Heat-shock protein 70 family members have
been demonstrated to be bona fide proteins of the primate
lentiviral virions and have been proposed to play roles in
virus assembly and egress (Gurer et al., 2002). Finally,
virions of SV40 have been demonstrated to contain
biosynthetically active histone H2A protein (Chen et al.,
1979). Taken together, it seems likely that the cellular host
proteins in GpSGHV virions may have specific or auxiliary
roles in the virus life cycle. Further investigations are
needed to determine whether the incorporated cellular
proteins are distributed over infectious and non-infectious
GpSGHV virions. These data provide important leads
towards an understanding of the process of GpSGHV
assembly.
Conclusions
The GpSGHV virion has a rod-shaped protein nucleocapsid core surrounded by a proteinaceous tegument, an outer
envelope and helical surface projections. The tegument
proteins of GpSGHV comprise almost half of the total
virion proteins. In addition, the virus contains numerous
virion-associated cellular proteins, some of which appear
to be specifically incorporated into the mature virion. The
presence of cellular proteins in GpSGHV virions may be a
reflection of their requirement in the infection process or
may be remnants of interactions between the virus and
host proteins. The GpSGHV progeny nucleocapsids
translocate to the cytoplasm where envelopment is
orchestrated. Cytoplasmic assembly of the virus particles
induces cellular damage, which culminates in disintegration of the cell plasma membrane as the mature virions
egress from the infected cell. Finally, the data presented in
this study may offer new directions in antiviral strategies
based on virus–host interactions. Potential strategies have
been reviewed recently by Kariithi et al. (2012) and include
blocking of the initial attachment of GpSGHV to the tsetse
midgut receptors using either antibodies against envelope
204
proteins or competing peptides as reported for Autographa
californica multicapsid nucleopolyhedrovirus in Heliothis
virescens (Sparks et al., 2011), GpSGHV-specific gene
silencing using RNA interference, inhibition of GpSGHV
DNA polymerase (ORF79) by commercially available drugs
(Abd-Alla et al., 2011) and blocking of RGD-directed cell
adhesion as has been demonstrated for adenovirus (Bai
et al., 1993).
METHODS
Electron
microscopy
of
hypertrophied
salivary
glands.
Hypertrophied salivary glands were freshly dissected from adults of
a laboratory colony of G. pallidipes flies (IAEA Laboratories) and
immediately fixed (4 h at 4 uC) in 2 % paraformaldehyde/3 %
glutaraldehyde in 0.1 M phosphate/citrate buffer (pH 7.2). The
glands were washed, infiltrated with 2.3 M sucrose in 0.1 M
phosphate/citrate buffer (16 h at 4 uC) and cryo-fixed by plunging
into liquid ethane at 2160 uC using a Reichert KF80 plunger. Cryosections (80 nm in thickness) were cut at 2110 uC with a Leica
Ultracut S microtome equipped with an FCS cryo-system, mounted
on Formvar-coated copper grids (100 mesh), negatively stained with
3 % ammonium molybdate (pH 6.5) and air dried. Images were
recorded with a Gatan 4K CCD camera on a JEOL 2100 transmission
electron microscope equipped with a LaB6 filament operating at
200 kV.
Electron microscopy of virus particles. Salivary glands were
squashed gently in 1 : 1-diluted fixative and extracts incubated on
Formvar- and carbon-coated copper grids (100 mesh) and stained
with 1 % uranyl acetate (pH 3.7). Similar specimens were prepared
from purified virus suspensions (see below). For electron tomography, gold fiducial markers of 10 nm were included in the virus
extract and a series of 26-binned images was recorded with SerialEM
(Mastronarde, 2005) at tilt angles from 265 to +65u with increments
of 1u. The series of tilted projection images were converted into threedimensional tomograms using the IMOD program (Kremer et al.,
1996).
Virus purification. Three replicate extractions were conducted on 25
pairs each of hypertrophied salivary glands dissected from adult G.
pallidipes. The glands were disrupted immediately by two strokes of a
glass/Teflon homogenizer (on ice) in 1 ml homogenization buffer
[50 mM HEPES (pH 8.0), 10 mM Ficoll PM400 (GE Healthcare),
2 mM EDTA and protease inhibitors (Roche)]. The volumes were
brought to 2 ml and clarified by centrifugation three times (7500 g
for 10 min at 4 uC). The supernatants were pooled and layered onto
5 ml of a 10–40 % (w/v) Ficoll PM 400 discontinuous density
gradient and ultracentrifuged (25 000 g for 1 h at 4 uC). The virus
band was collected, resuspended in 50 mM buffer (pH 8.0) and
ultracentrifuged (60 000 g for 60 min at 4 uC). The resultant pellet
was allowed to dissociate into 1 ml 50 mM HEPES (pH 8.0)
overnight at 4 uC. The integrity of the purified virions was checked
by negative staining using a JEOL 2100 transmission electron
microscope (Kariithi et al., 2010).
Fractionation of virions into envelope and nucleocapsids.
Purified virions were incubated (30 min at room temperature) in
250 ml reaction volumes with lysis buffer [1 % NP-40, 50 mM Tris/
HCl (pH 8.0), 137 mM NaCl, 10 % glycerol and 2 mM EDTA]. The
NP-40-treated virions were layered onto a 5 ml 10–60 % (v/v)
glycerol discontinuous gradient and ultracentrifuged (110 000 g for
1 h at 4 uC). The envelope fraction was collected from the top 2 ml of
the gradient. To ensure complete removal of virion envelopes, the
Journal of General Virology 94
Structure and virion components of GpSGHV
pellet containing the nucleocapsids was resuspended and subjected to
another round of 1 % NP-40 extraction and glycerol ultracentrifugation. The purity of nucleocapsids was checked by negative-staining
transmission electron microscopy. The envelope fraction was
precipitated with trichloroacetic acid (overnight at 4 uC). The
precipitated proteins were recovered by centrifugation (20 000 g for
15 min) and the trichloroacetic acid was neutralized by three washes
with ice-cold acetone. The pellets were dried and resuspended in
10 mM Tris/HCl (pH 8.0).
Identification of GpSGHV structural proteins by LC-MS/MS.
described above. A portion of the PK-treated sample was subjected to
Western blotting using mouse anti-tubulin-a mAb (clone DM1A;
Sigma), mouse cytoplasmic actin mAb (clone 10-b3; Sigma), mouse
IgG anti-ubiquitin mAb (clone P4DI; Santa Cruz Biotechnology) or
rabbit anti-myosin polyclonal antibody (clone H-300; Santa Cruz
Biotechnology) as the primary antibody (diluted 1 : 3000). Alkaline
phosphatase-conjugated anti-mouse IgG (diluted 1 : 2000; Sigma) was
used as the secondary antibody using the conditions described above.
Functional and structural characterization of GpSGHV proteins. Identified viral proteins were annotated using Blast2GO
Portions of the envelope and nucleocapsid fractions were treated with
lysis buffer [8 M urea, 4 mM CaCl2, 0.2 M Tris/HCl (pH 8.0)] and
separated by SDS-PAGE (12 % acrylamide). The gel was stained with a
Colloidal Staining kit (Invitrogen). The middle sections of entire gel lanes
were excised and the gel sections cut into approximately 1 mm3 pieces.
In-gel trypsin digestions were performed and the resultant peptides were
analysed by LC-MS/MS (Kariithi et al., 2010). The data generated by LCMS/MS were analysed by the MaxQuant software package version 1.2.2.5
(Cox & Mann, 2008; Cox et al., 2011) with the following constructed
databases: a Glossina morsitans morsitans database (http://www.sanger.
ac.uk/resources/downloads/vectors/glossina-morsitans-morsitans.html),
a GpSGHV ORF database (http://www.uniprot.org/) and a contaminant database containing sequences of common contaminants
(BSA: NCBI protein accession no. P02769, bovine serum albumin
precursor; trypsin: P00760, bovine; trypsin: P00761, porcine;
keratin K22E: P35908, human; keratin K1C9: P35527, human;
keratin K2C1: P04264, human; keratin K1CI: P35527, human).
Proteins were identified with the MaxQuant software using default
settings for the Andromeda search engine (Cox et al., 2011),
except that extra variable modifications were set for deamidation of
N and Q. Peptides and proteins with a false discovery rate of ,1 %,
and proteins with at least two peptides of which at least one was
unique were accepted for further analyses. Student’s t-test of the
identified proteins was performed in the Perseus module version
1.2.0.17 on normalized peak abundances (Cox & Mann, 2011). The
normalized protein abundances were used to construct a scatter plot
to determine protein distribution in the GpSGHV structural
components.
software version 2.5.0 (Conesa et al., 2005), whilst the protein motifs
were analysed using the ExPASy PROSITE database (http://www.expasy.
org), and the numbers of TM helices were predicted by TMHMM
version 2.0. Signal peptide sequences were predicted using SignalP
3.0. The phosphorylation potential of the identified proteins was
predicted by the NetPhos 2.0 CBS Prediction server with a threshold
value set at 0.7. Confirmation of the phosphorylation statuses of
identified viral proteins was performed by Western blotting using
mouse anti-phospho-serine/threonine/tyrosine mAbs (diluted
1 : 1500; Thermo Scientific) and anti-mouse IgG alkaline phosphatase
(diluted 1 : 3000; Sigma) as the primary and secondary antibodies,
respectively, following the supplier’s instructions.
Localization of GpSGHV envelope and nucleocapsid proteins
by Western blotting. The envelope and nucleocapsid fractions of
Abd-Alla, A., Bossin, H., Cousserans, F., Parker, A., Bergoin, M. &
Robinson, A. (2007). Development of a non-destructive PCR method
purified GpSGHV virions were separated by SDS-PAGE (12 %
acrylamide) and transferred onto Immobilon-P (Millipore) membranes by semi-dry electrophoresis transfer. Membranes were blocked
by overnight incubation with 5 % non-fat milk powder and 0.05 %
Tween 20 in TBS buffer [10 mM Tris/HCl (pH 8), 150 mM NaCl]
(TBS-T) at 4 uC. The membranes were then incubated (1 h at room
temperature) with rabbit polyclonal antibodies against proteins
encoded by GpSGHV ORF10 or ORF1 generated in rabbits as the
primary antibodies (diluted 1 : 1000), washed three times with TBS-T
and further incubated (1 h at room temperature) with alkaline
phosphatase-conjugated polyclonal goat anti-rabbit IgG antibody
(diluted 1 : 2000; Sigma) as the secondary antibody. Blots were
developed with nitro-blue tetrazolium/BCIP (Sigma).
Verification of incorporation of cellular proteins into GpSGHV
virions. Purified GpSGHV virions were incubated for 30 min at
37 uC with 0.08 mg PK (Invitrogen) (mg total protein)21 (Moerdyk-
Schauwecker et al., 2009). PK activity was stopped by the addition of
PMSF to a final concentration of 5 mM, followed by incubation on
ice for 15 min. Contaminating vesicles that commonly co-purify with
enveloped viruses (Ott et al., 1996) were removed by passing a
portion of the PK-treated virions through a 5 ml 20 % Ficoll cushion
by ultracentrifugation (60 000 g for 1 h at 4 uC). The untreated and
PK-treated samples were separated by SDS-PAGE (12 % acrylamide),
followed by in-gel trypsin digestion and LC-MS/MS analysis as
http://vir.sgmjournals.org
ACKNOWLEDGEMENTS
This research was supported by Netherlands Fellowship Grant award
CF7548/2011 (http://www.nuffic.nl) and the FAO/IAEA Joint Program of Nuclear Techniques in Food and Agriculture (http://wwwnaweb.iaea.org/nafa/index.html). The authors acknowledge Biqualys
(http://www.biqualys.nl) and Wageningen Electron Microscopy
Centre (http://www.cat-agrofood.wur.nl/UK/Facilities/List+of+Facilities/
wageningen_electron_microscopy_centre.htm) for technical support.
The authors thank D. G. Boucias and M. Bergoin for thoughtful
review of the manuscript.
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