Interaction between Core protein of classical swine fever virus with cellular IQGAP1 protein appears essential for virulence in swine.
ABSTRACT Here we show that IQGAP1, a cellular protein that plays a pivotal role as a regulator of the cytoskeleton interacts with Classical Swine Fever Virus (CSFV) Core protein. Sequence analyses identified residues within CSFV Core protein (designated as areas I, II, III and IV) that maintain homology to regions within the matrix protein of Moloney Murine Leukemia Virus (MMLV) that mediate binding to IQGAP1 [EMBO J, 2006 25:2155]. Alanine-substitution within Core regions I, II, III and IV identified residues that specifically mediate the Core-IQGAP1 interaction. Recombinant CSFV viruses harboring alanine substitutions at residues (207)ATI(209) (I), (210)VVE(212) (II), (213)GVK(215) (III), or (232)GLYHN(236) (IV) have defective growth in primary swine macrophage cultures. In vivo, substitutions of residues in areas I and III yielded viruses that were completely attenuated in swine. These data shows that the interaction of Core with an integral component of cytoskeletal regulation plays a role in the CSFV cycle.
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ABSTRACT: Heme oxygenase 1 (HO-1) is an inducible enzyme that exerts potent antioxidant and anti-inflammatory effects, which also plays a critical role in host defenses against microbial, and particularly viral, infections. In our previous study, up-regulation of HO-1 was observed in peripheral blood leukocytes (PBLs) by genomic expression profiling, following infection of pigs with virulent classical swine fever virus (CSFV), the causative agent of a highly contagious disease threatening global pig industry (Shi et al., 2009). To study the potential involvement of HO-1 in CSFV proliferation, the role of its down-regulation in CSFV-infected PK-15 cells was further investigated. Results showed that infection with virulent CSFV strain Shimen significantly up-regulated the expression of HO-1 and that its down-regulation by small interfering RNA (siRNA) could inhibit CSFV proliferation as measured by genomic replication and production of infectious virus. The study revealed the involvement of HO-1 in CSFV proliferation, indicating that HO-1 is a potential target for inhibition of CSFV replication.Virus Research 01/2013; · 2.75 Impact Factor
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ABSTRACT: The capsid (C) protein of the Flaviviridae family members is involved in nucleocapsid formation and virion assembly. However, the influence of C protein-interacting partners on the outcome of pestivirus infections is poorly defined. In this study, the hemoglobin subunit beta (HB) was identified as a C protein-binding protein using glutathione S-transferase (GST) pulldown and subsequent mass spectrometry analysis of PK-15 cells, which are permissive cells of classical swine fever virus (CSFV). Coimmunoprecipitation and confocal microscopy confirmed that HB interacts and colocalizes with the C protein in cytoplasm. Silencing of HB with small interfering RNAs promoted CSFV growth and replication, whereas overexpression of HB suppressed CSFV replication and growth. Interestingly, HB interacts with retinoic acid inducible gene I (RIG-I) and increases its expression, resulting in increased production of type I interferon (IFN). Overall, our results suggest that cellular HB antagonizes CSFV growth and genomic replication probably by triggering IFN signaling and might represent a novel antiviral restriction factor. This study reports for the first time the novel role of HB in innate immunity.Journal of Virology 03/2013; · 5.08 Impact Factor
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ABSTRACT: We have identified host IQGAP1 as an interacting partner for EBOV VP40, and its expression is required for EBOV VP40 VLP budding. IQGAP1 is involved in actin cytoskeletal remodeling during cell migration and formation of filopodia. The physical interaction and functional requirement for IQGAP1 in EBOV VP40 VLP egress links virus budding to the cytoskeletal remodeling machinery. Consequently, this interaction represents a novel target for development of therapeutics to block budding and transmission of filoviruses.Journal of Virology 05/2013; · 5.08 Impact Factor
Interaction between Core protein of classical swine fever virus with cellular IQGAP1
protein appears essential for virulence in swine
D.P. Gladuea, L.G. Holinkaa, I.J. Fernandez-Sainza, M.V. Prarata, V. O'Donnella,b, N.G. Vepkhvadzea, Z. Luc,
G.R. Risattib, M.V. Borcaa,⁎
aPlum Island Animal Disease Center, ARS, USDA, Greenport, NY 11944, USA
bDepartment of Pathobiology and Veterinary Science, University of Connecticut, Storrs, CT 06269, USA
cPlum Island Animal Disease Center, DHS, Greenport, NY 11944, USA
a b s t r a c ta r t i c l ei n f o
Received 17 September 2010
Returned to author for revision
28 December 2010
Accepted 30 December 2010
Available online 23 January 2011
Classical swine fever virus
Here we show that IQGAP1, a cellular protein that plays a pivotal role as a regulator of the cytoskeleton
interacts with Classical Swine Fever Virus (CSFV) Core protein. Sequence analyses identified residues within
CSFV Core protein (designated as areas I, II, III and IV) that maintain homology to regions within the matrix
protein of Moloney Murine Leukemia Virus (MMLV) that mediate binding to IQGAP1 [EMBO J, 2006 25:2155].
Alanine-substitution within Core regions I, II, III and IV identified residues that specifically mediate the Core-
IQGAP1 interaction. Recombinant CSFV viruses harboring alanine substitutions at residues
210VVE212(II),213GVK215(III), or232GLYHN236(IV) have defective growth in primary swine macrophage
cultures. In vivo, substitutions of residues in areas I and III yielded viruses that were completely attenuated in
swine. These data shows that the interaction of Core with an integral component of cytoskeletal regulation
plays a role in the CSFV cycle.
Published by Elsevier Inc.
Classicalswinefever virus (CSFV)isa small,envelopedvirus witha
positive, single-stranded RNA genome that causes classical swine
fever (CSF), a highly contagious disease of swine. CSFV, along with
Bovine Viral Diarrhea Virus (BVDV) and Border Disease Virus (BDV),
are members of the genus Pestivirus within the family Flaviviridae
(Fauquet et al., 2005). The CSFV genome is approximately 12.3 kb and
contains a single open reading frame encoding a polyprotein of 3898-
amino-acids. Co- and post-translational processing of the polyprotein
by cellular and viral proteases ultimately yields 11 to 12 final cleavage
NS5B-COOH) (Rice, 1996). Structural components of the CSFV virion
include the glycoproteins Erns, E1, E2, and a nucleocapsid of unknown
symmetry, the Core protein.
Within the Flaviviridae family, Core is encoded as the second
product of the polyprotein. The Core protein of pestiviruses is a small,
highly basic polypeptide that is cleaved at its N-terminus by Npro, an
event critical for infectious particle production. The Core protein of
BVDV has additionally been characterized as lacking significant
secondary structures (Murray et al., 2008). It is known that the C-
terminal of Core is cleaved by signal peptide peptidase. (Heimann
et al., 2006; Rümenapf et al., 1998; Meyers et al., 1989), and it has
been suggested that the CSFV Core protein influences regulation of
cellular transcription (Liu et al., 1998) and also interacts with host
SUMOylation proteins (Gladue et al., 2010).
Analysis of the Core protein of Hepatitis C Virus (HCV), another
member of the Flaviviridae family, provides further insight into the
possible functions of the Core protein of CSFV. HCV Core self-
assembles into nucleocapsid-like particles in the presence of nucleic
acids (Kunkel et al., 2001) and can directly interact with HCV RNA
(Fan et al., 1999; Shimoike et al., 1999; Tanaka et al., 2000). Core can
bind other HCV proteins such as NS5A and E1 (Goh et al., 2001; Lo
et al., 1996; Masaki et al., 2008) and interacts with host cellular
proteins (Jin et al., 2000; Mamiya and Worman., 1999; Otsuka et al.,
2000; Yoshida et al., 2002; You et al., 1999), influencing HCV
pathogenesis by modulation of signaling pathways, cell transforma-
tion and proliferation, regulation of cellular and viral gene expression,
apoptosis, and alteration of lipid metabolism. (Giannini and Brechot,
2003; Levrero, 2006; Tellinghuisen and Rice, 2002; Lai and Ware.,
2000; McLauchlan, 2000; Ray and Ray, 2001). HCV Core protein is
capable of impairing the host's immune response, by interacting with
cell molecules that results in suppression of IL-12 synthesis in human
macrophages (Eisen-Vandervelde et al., 2004), T cell dysfunction (Yao
Virology 412 (2011) 68–74
⁎ Corresponding author. Plum Island Animal Disease Center, USDA/ARS/NAA, P.O.
Box 848, Greenport, NY 11944-0848, USA. Fax: +1 631 323 3006.
E-mail addresses: email@example.com (D.P. Gladue),
firstname.lastname@example.org (L.G. Holinka), email@example.com
(I.J. Fernandez-Sainz), firstname.lastname@example.org (M.V. Prarat),
vivian.odonnell.@ars.usda.gov (V. O'Donnell), email@example.com
(N.G. Vepkhvadze), firstname.lastname@example.org (Z. Lu), email@example.com
(G.R. Risatti), firstname.lastname@example.org (M.V. Borca).
0042-6822/$ – see front matter. Published by Elsevier Inc.
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/yviro
et al., 2007), and inhibition of T-lymphocyte activation and prolifer-
ation (Chen et al., 1994; Kittlesen et al., 2000; Yao et al., 2001).
Although the role of CSFV structural glycoproteins in virus vir-
ulence has been studied in detail (Meyers et al., 1999; Risatti et al.,
2005a, 2005b, 2006, 2007a, 2007b; van Rijn et al., 1994; van Gennip
et al., 2004), knowledge about the role of Core protein on the outcome
of CSFV infection in swine is limited. Recently, we have shown that
CSFV Core protein interacts with proteins of the cellular SUMOylation
pathway, SUMO-1 (small ubiquitin-like modifier) and UBC9, a SUMO-
1 conjugating enzyme (Gladue et al., 2010). Substitution of Core
residues in CSFV involved with binding to SUMO1 and UBC9 resulted
in virus attenuation in swine. To further elucidate the role of Core
protein in CSFV virulence, we expanded this previous study to identify
additional host factors interacting with the Core protein during virus
infection. Here we report that the cellular IQGAP1 protein interacts
specifically with the CSFV Core protein. IQGAP1 is involved in a
diverse set of protein–protein interactions and is a prominent reg-
ulator of the cytoskeleton (for a review, see Noritake et al., 2005;
Brandt and Grosse, 2007). This protein binds monomeric G proteins:
Cdc42 and Rac, likely mediating their effects in reorganizing the
cytoskeleton (Fukata et al., 2002; Watanabe et al., 2004).
Specific interaction of IQGAP1 with the Moloney murine leukemia
virus (MMLV) matrix (M) protein suggests its involvement in in-
tracellular trafficking of the virus, an interaction that is essential for
virus replication (Leung et al., 2006). Mutational studies performed in
that report defined residues of MMLV M protein, critical for its
interaction with IQGAP1. Based on sites within the MMLV M protein
that recognize IQGAP1, four areas (I–IV) within the CSFV Core protein
that potentially recognize IQGAP1 were identified. Substitution of
Core native amino acid residues with alanine in areas I and III
completely abolished the Core–IQGAP1 protein–protein binding
whereas substitutions in areas II and IV only partially affected the
interaction. CSFV mutants harboring substitutions in these four areas
were developed to assess the importance of the Core–IQGAP1 protein
interaction for virulence in swine. Remarkably, substitutions in areas I
and III of the Core protein, significantly affecting IQGAP1 recognition,
correlate with complete absence of virulence in swine. Therefore, the
ability of CSFV Core protein to bind cellular IQGAP1 protein during
infection plays a critical role in virus virulence within the swine host.
CSFV structural Core protein binds swine IQGAP1 protein
To identify host cellular proteins that interact with CSFV Core
protein, we constructed an N-terminal fusion of the Gal4 protein
binding domain to the Core protein as ‘bait’ for the yeast two-hybrid
system. Approximately 1×107independent yeast colonies derived
from a swine primary macrophage cDNA library containing 3×106
independent clones were screened. These colonies were selected for
growth using -Leu/-Trp/-His/-Ade media. Plasmids were isolated from
positive colonies and sequenced. In-frame proteins were retested for
specificity to the Core protein. As a negative control proteins were
tested for binding the Lam-BD protein. Several proteins were
identified as specific binding partners for the CSFV Core protein
(data not shown). One of these proteins, IQGAP1, was selected for
further study since IQGAP1 functions as a prominent regulator of the
cytoskeleton which likely plays a role in viral pathogenesis. IQGAP1
specifically bound Core protein when compared to binding of Lam-BD
protein (Fig. 1).
Mapping areas of the CSFV Core protein critical for IQGAP1 recognition
Previous studies have described the binding of Moloney murine
leukemia virus (MMLV) matrix (M) protein with IQGAP1 protein
(Leung et al., 2006). Mutational studies defined residues of the MMLV
M protein critical for interaction with IQGAP1, revealing a correlation
between binding and virus replication. Based on the sites of MMLV M
protein that recognize IQGAP1 (Leung et al., 2006), four (I–IV)
homologous areas (Fig. 2) were identified in CSFV Core protein using
harboring alanine substitutions in place of native amino acid residues
within these four areas were developed to assess whether these
the following mutant proteins containing Ala substitutions within the
(213GVK215), and CoreΔIQ.IV (232GLYHN236) (Table 1). These CoreΔIQ
proteinswere testedin the yeast two-hybrid system against theswine
IQGAP1 protein. Interestingly, substitutions within areas I and III
resulted in loss of ability to bind IQGAP1 (Fig. 1). In contrast,
substitutions in areas II and IV only caused a decrease in the ability
of the Core protein to bind the swine IQGAP1 protein. All CoreΔIQ
mutatedproteinsmaintainedtheir abilityto bind swineclathrin in the
yeast two-hybrid at similar levels (data not shown), indicating that
these mutated areas within the CoreΔIQ mutated proteins are areas
specific for IQGAP1 binding.
Sequence analysis of the 100 amino acid residues of Core protein
from geographically and temporally different CSFV isolates revealed a
high degree of sequence similarity and conservancy at putative
IQGAP1 targetsites (Fig. 2), suggesting these sites playa critical rolein
the biology of CSFV. Additionally, the areas predicted in CSFV to bind
IQGAP1 are highly conserved in BVDV and BDV, further suggesting a
critical role for these residues in other pestiviruses (data not shown).
Replication of CoreΔIQ mutant viruses in vitro
To further evaluate the role of CSFV Core IQGAP1 binding sites in
the biology of the virus, recombinant viruses based on virulent strain
Brescia (BICv) were constructed, containing alanine substitutions in
the previously described four critical IQGAP1 binding sites of the Core
protein. Mutant viruses referred to as CoreΔIQ.Iv, CoreΔIQ.IIv,
CoreΔIQ.IIIv, and CoreΔIQ.IVv represent each of the four putative
Viruses were rescued from transfected cells by 4 dpi (days post-
identical to parental DNA plasmids, confirming that only mutations at
predicted mutated sites were reflected in rescued viruses.
In vitro growth characteristics of these mutant viruses were eval-
uated relative to parental BICv in a single-step growth curve. Primary
swine macrophage cell cultures were infected at a multiplicity of
infection (MOI) of 0.01 TCID50per cell. Virus was adsorbed for 1 h
(time zero), and samples were collected at 72 h post-infection (hpi).
All mutant viruses exhibited about one log10 decrease in titer when
compared with parental BICv (Fig. 3), suggesting that all mutant
Effect of Core–IQGAP interactions on the cell cytoskeleton
The effects of viral infection on γ-tubulin and vimentin were
examined to compare the distribution of microtubules and interme-
diate filaments in cells infected with BICv, CoreΔIQ.Iv, CoreΔIQ.IIv,
CoreΔIQ.IIIv, CoreΔIQ.IVv, to that seen in uninfected cells (Fig. 5). In
uninfected cells, the microtubules (visualized with antibodies recog-
nizing γ-tubulin) as well as the intermediate filaments (visualized
network running throughout the cytoplasm. In BICv-infected cells, γ-
tubulin was rearranged into a ring surrounding the nucleus. When
infected cells. Examining the appearance of γ-tubulin and vimentin in
infected cells also revealed differences between the effects of BICv and
CoreIQ viruses. Infection with CoreΔIQ.IIv and CoreΔIQ.IVv led to a
rearrangement of both markers, γ-tubulin and vimentin, into a ring
D.P. Gladue et al. / Virology 412 (2011) 68–74
surrounding the nucleus similar to the one observed with BICv. In
contrast, when CoreΔIQ.Iv and CoreΔIQ.IIIv were studied, both
proteins, γ-tubulin and vimentin, retained a radial pattern as seen in
uninfected cells (Fig. 5).
Evaluation of the role of CSFV Core IQGAP1 binding sites in CSFV
virulence in swine
with IQGAP1 protein, all four CoreΔIQv mutants were intranasally (IN)
inoculated into naïve swine, at doses of 105TCID50. After inoculation,
survival of pigs inoculated with mutant viruses was assessed relative to
weremonitoreddailyfor clinicaldisease.BICv exhibiteda characteristic
virulent phenotype (Table 2); none of the control pigs survived the
infection, dying or beingeuthanized around 8 dpi. Interestingly, viruses
CoreΔIQ.Iv and CoreΔIQ.IIIv were completely attenuated in swine.
Animals survived the infection and remained clinically normal
throughout the observation period (21 days) with only one animal
out of five infected with CoreΔIQ.IIIv presenting a transient rise in body
Fig. 1. Reactivity of CSFV wild-type and mutant Core proteins with IQGAP1 protein or Clathrin protein (positive control) in the yeast two-hybrid system. Wild-type Core and mutants
I, II, III and IV proteins were tested for their ability to bind IQGAP1 protein. Yeast growth on selective SD-Ade-His-Leu-Trp media (A), and growth on non-selective SD-Leu-Trp media
(B). Schematic representation of putative IQGAP1 binding motifs found in CSFV Core protein (C).
Fig. 2. Multiple alignments of CSFV Core proteins revealed the presence of highly conserved putative IQGAP1 sites I, II, III, and IV (bold underlined). Shaded are Lys residues involved
with Core-SUMO-1 and Core-UBC9 protein–protein interactions as described by Gladue et al. (2010). Shown here is a comparison between geographically and temporally separated
D.P. Gladue et al. / Virology 412 (2011) 68–74
temperature. In contrast, animals infected with mutant CoreΔIQ.IIv or
CoreΔIQ.IVv, although presenting less severe CSF symptoms than those
infected with BICv, died around 12 and 14 dpi, respectively (Table 2).
Therefore, there appears to be a close correlation between disrupting
Core–IQGAP1 protein–protein binding as detected in the yeast two-
hybrid system and the induction of virus attenuation.
Virus shedding (detected in tonsil scrapings and nasal swabs) and
viremia in CoreΔIQ.Iv and CoreΔIQ.IIIv-inoculated animals were almost
sampled (Fig. 4). Interestingly, high virus loads in blood, nasal swabs,
the infection when compared to BICv-infected pigs (Fig. 4). Similarly, a
delayed onset of disease wasobserved in animals inoculatedwith these
mutant viruses relative to wild-type infected animals.
Animals that survive inoculation with CoreΔIQ.Iv and CoreΔIQ.IIIv
when challenged with virulent BICv by 28 dpi were not protected,
against E2 and Ernswere not detected in these animals by the time of
challenge as measured by ELISA tests (CSF SERO ELISA and CHEKIT-
CSF-MARKER Kits, Idexx Laboratories, Westbrook, ME, USA).
Here we describe a specific interaction between IQGAP1 protein, a
major cytoskeleton regulator, with CSFV Core protein. Substitution of
residues207ATI209or213GVK215within Core, disrupting Core–IQGAP1
protein–protein interaction completely, correlates with the induction
of virus attenuation in vivo. Partial disruption of Core–IQGAP1 binding
by substituting Core residues210VVE212or232GLYHN236resulted in
viruses inducing a delayed onset of CSF, suggesting that the degree of
attenuation closely correlates with the ability of the Core protein to
interact with IQGAP1, and that this swine–host protein interaction
plays an important role in CSFV pathogenesis.
CoreΔIQ viruses showed a slight growth defect in cultured primary
swine macrophages. Impaired replication observed in vitro usually
correlates with virus attenuation in vivo, with animals overcoming the
infection, likely because of poor virus replication in vivo and a rapid
clearance of mutant viruses mediated by the host immune response.
In fact, CoreΔIQ.Iv and CoreΔIQ.IIIv mutants, presenting the highest
extent of attenuation, demonstrated decreased replication during
infection in animals. However, CoreΔIQ.IIv and CoreΔIQ.IVv mutants
exhibited more limited replication than parental BICv in cultured
swine macrophages, but replicated almost as efficiently in vivo
inducing disease similar to wild-type virus, although with a delayed
progression. These two recombinant viruses likely do not contain
sufficient mutations within the Core–IQGAP1 interaction to limit CSFV
tropism in vivo, with viruses retaining the ability to replicate in
multiple tissues and cause disease.
The observed interaction of Core with the cell's cytoskeleton may
inhibit in vivo host immune cell migration, hampering viral CSFV
clearance. Salmonella, a facultative intracellular pathogen, releases a
bacterial effector protein, SseI (also known as or SrfH), that is able to
mechanism involves the interaction of SseI with IQGAP1, reducing
dendritic cell (DC) migration in vivo that in turn correlates with a
reduction in the number of DC and CD4+ T cells in spleens of
Salmonella-infected mice. Salmonella invasion promotes the interac-
tion of IQGAP1 with Rho GTPases Rac1 and Cdc42 to induce actin
polymerization (Brown et al., 2007). In vitro, knockdown of IQGAP1
significantly reduces Salmonella invasion and abrogates activation of
Cdc42 and Rac1 by Salmonella. Similarly, Ibe, an effector protein of
enteropathogenic and enterohemorrhagic Escherichia coli interacts
E. coli-induced pedestals and actin-rich membrane ruffles. Interest-
ingly, these pathogens use common effector mechanisms to increase
alterations in the distribution of the cytoskeleton of cells infected with
since mutations introduced in CoreΔIQ.Iv and CoreΔIQ.IIIv appear to
disrupt the induction of those cytoskeleton alterations (Fig. 5).
with the cellular SUMOylation pathway proteins, SUMO-1 (small
ubiquitin-like modifier) and UBC9, a SUMO-1 conjugating enzyme. In
important for CSFV pathogenesis since mutant viruses harboring
substitutions at those positions were attenuated in swine. Interactions
of viral proteins with the SUMOylation pathway seems to be important
for viral infectivity, as shown for Ebola Virus Zaire VP35, Adenovirus
CELO Gam1, Dengue Virus envelope protein, Human Herpesvirus 6 IE2,
and Human Cytomegalovirus IE2, either preventing or inducing SUMO
conjugation of target proteins (Ahn et al., 2001; Chang et al., 2009;
Chiocca, 2007; Tomoiu et al., 2006). Here we extended the analysis of
host factors interacting with CSFV proteins, specifically identifying
IQGAP1 as a swine host protein binding partner for the CSFV Core
protein. We also observed that mutations in the binding site in Core
protein can completely abrogate CSFV virulence, demonstrating that
acquisition of attenuation correlates with loss of binding to IQGAP1.
CSFV by elucidating what appear to be multiple roles for Core in the
to control virus infection in swine.
Materials and methods
Viruses and cells
Swine kidney cells (SK6) (Terpstra et al., 1990), free of BVDV, were
cultured in Dulbecco's Minimal Essential Media (DMEM) (Gibco, Grand
Island, NY) with 10% fetal calf serum (FCS) (Atlas Biologicals, Fort
Set of CSFV CoreΔIQ mutant viruses constructed in this study.
Core wild type sequence
aCore mutant sequence Mutant virus
aAmino acid position relative to CSFV BICv polyprotein.
Fig. 3. In vitro growth characteristics of CoreΔIQv mutants and parental BICv. Primary
swine macrophage cell cultures were infected (MOI=0.01) with each of the mutants or
BICv and virus yield titrated at 72 h post-infection in SK6 cells. Data represent means
and standard deviations from two independent experiments. Sensitivity of virus
detection: ≥log10 1.8 TCID50/ml.
D.P. Gladue et al. / Virology 412 (2011) 68–74
Collins, CO). Virulent CSFV Brescia strain was propagated in SK6 cells
et al., 2005a). Growth kinetics was assessed on primary swine
macrophage cell cultures prepared as described (Zsak et al., 1996).
96-well plates (Costar, Cambridge, MA). Viral infectivity was detected,
after 4 days in culture, by an immunoperoxidase assay using the CSFV
monoclonal antibody WH303 (Edwards et al., 1991) and the Vectastain
ABC kit (Vector Laboratories, Burlingame, CA). Titers were calcu-
lated using the method of Reed and Muench (1938) and expressed as
TCID50/ml. As performed, test sensitivity was ≥1.8 TCID50/ml.
Construction of CSFV CoreΔIQGAP (CΔIQ) mutants
Full-length pBIC was used as a template in which putative IQGAP1
binding sites in the Core protein were mutated. IQGAP1 binding sites
were predicted using the Clustal W Analysis Program (http://www.
ebi.ac.uk/Tools/clustalw2/index.html) using regions of the MMLV M
protein that bind IQGAP1 as a template (Leung et al., 2006). Amino
acids in the predicted binding regions were substituted with alanine,
introduced by site-directed mutagenesis using the QuickChange XL
Site-Directed Mutagenesis kit (Stratagene, Cedar Creek, TX) per-
formed per manufacturer's instructions. Primers were designed using
the Stratagene Primer Mutagenesis program.
In vitro rescue of CSFV Brescia and CΔIQ mutants
Full-length genomic clones were linearized with SrfI and in vitro
transcribed using the T7 MEGAscript system (Ambion, Austin, TX).
RNA was precipitated with lithium chloride and transfected into
SK6 cells by electroporation at 500 V, 720 Ω, 100 W with a BTX 630
electroporator (BTX, San Diego, CA). Cells were seeded in 12-well
plates (Costar, Cambridge, MA) and incubated for 4 days at 37 °C and
5% CO2. Virus was detected by immunoperoxidase staining as
described above, and stocks of rescued viruses were stored at −70 °C.
DNA sequencing and analysis
Full-length clones and in vitro rescued viruses were completely
sequenced with CSFV-specific primers by the dideoxynucleotide chain-
termination method (Sanger et al., 1977). Sequencing reactions were
prepared with the Dye Terminator Cycle Sequencing Kit (Applied
Biosystems, Foster City, CA). Reaction products were sequenced on an
ABI PRISM 3730xl automated DNA sequencer (Applied Biosystems,
Foster City, CA). The final DNA consensus sequence represented an
average five-fold redundancy at each base position. Sequence compar-
isons were conducted using BioEdit software (http://www.mbio.ncsu.
Development of the cDNA library
A porcine primary macrophage cDNA expression library was
constructed (Clontech, Mountain View, CA) using monocytes/macro-
phages obtained from healthy CSFV-free swine exactly as previously
described (Gladue et al., 2010). Macrophage cultures were prepared
cells using an RNeasy Mini kit (Qiagen, Valencia, CA). Contaminant
genomic DNAwas removed byDNase treatmentusingTURBODNA-free
(Ambion, Austin, TX). After DNase treatment, genomic DNA contami-
nation of RNA stocks was assessed by real-time PCR amplification
targetingtheporcineβ-actingene.RNA qualitywasassessed usingRNA
Swine survival and fever response following infection with CSFV IQGAP1 mutants and parental BICv.
Virus No. of survivors/total no.Mean time to death
No. of days to onset
Duration no. of days
Max daily temperature (±SD)
aOnly one animal had a transient raised body temperature.
468 1214 21
Fig. 4. Virus titers in clinical samples (blood, tonsil scrapings, and nasal swabs) from
pigs infected with CoreΔIQ mutants and parental BICv. Each point represents the mean
log10 TCID50/ml and standard deviations from at least two animals. Sensitivity of virus
detection: ≥log 10 1.8 TCID50/ml.
D.P. Gladue et al. / Virology 412 (2011) 68–74
Clara,CA).Cellularproteinswere expressed asGAL4-ADfusionproteins
while CSFV Core was expressed as GAL4-BD fusion proteins.
The GAL4-based yeast two-hybrid system was used for this study
(Chien et al., 1991; Fields and Song, 1989). The ‘bait’ protein, CSFV
Brescia Core protein (amino acid residues 168–268 of the CSFV
polyprotein), was expressed with an N-terminus fusion to the GAL4
Binding Domain (BD). As ‘prey’, the previously described swine
macrophage cDNA library containing proteins fused to the GAL4
Activation Domain (AD) was used. Screening was done as previously
contained amino acids (502–704) of homo sapiens IQGAP1 (NCBI
Infection of cells and confocal microscopy
Sub-confluent monolayers of SK6 cells grown on 12 mm glass
coverslips in 24-well tissue culture dishes, were infected with BICv,
CoreΔIQ.Iv, CoreΔIQ.IIv, CoreΔIQ.IIIv, CoreΔIQ.IVv at a multiplicity of
infection (MOI) of 1–5 TCID50/cell, or mock infected for 48 h in
Dulbecco's minimum essential medium (DMEM, Invitrogen, CA) con-
taining 1% heat inactivated fetal bovine serum and 1% antibiotics. At
forty-eight hours after infection the cells were fixed with 4% parafor-
maldehyde (EMS, Hatfield, PA) and analyzed by confocal microscopy.
Immunofluorescence and confocal microscopy using the antibodies
listed below were performed as previously described (O'Donnell et al.,
2005). Monoclonal antibodies against γ-tubulin (Sigma) and vimentin
(Sigma), at a 1/100 dilution, were used as markers to identify
microtubules and intermediate filaments respectively.
phenotypein swinerelative to thevirulent Brescia strain.Swineused in
these studies were 10 to 12 weeks old, forty-pound commercial breed
pigs inoculated intranasally with 105TCID50of either mutant or wild-
type parental virus (BICv). Clinical signs (anorexia, depression, purple
skin discoloration, staggering gait, diarrhea and cough) and changes in
body temperature were recorded daily throughout the 21-day exper-
iment. Total and differential white blood cell and platelet counts were
obtained using a Beckman Coulter ACT (Beckman, Coulter, CA).
We thank the Plum Island Animal Disease Center animal care unit
staff for their excellent technical assistance. This work was partially
supported by the National Pork Board grant # 09-111 and USDA
Agricultural and Food Research Initiative (AFRI) grant # 2009-01614.
Ahn,J.H., Xu, Y., Jang, W.J., Matunis, M.J., Hayward, G.S., 2001. Evaluation of interactions of
like modifiers and their conjugation enzyme Ubc9. J. Virol. 75 (8), 3859–3872.
Brandt, D.T., Grosse, R., 2007. Get to grips: steering local actin dynamics with IQGAPs.
EMBO Rep. 8 (11), 1019–1023.
Brown, M.D., Bry, L., Li, Z., Sacks, D.B., 2007. IQGAP1 regulates Salmonella invasion
through interactions with actin, Rac1, and Cdc42. J. Biol. Chem. 282 (41),
Buss, C., Muller, D., Ruter, C., Heusipp, G., Schmidt, M.A., 2009. Identification and
characterization of Ibe, a novel type III effector protein of A/E pathogens targeting
human IQGAP1. Cell. Microbiol. 11 (4), 661–677.
Chang, T.H., Kubota, T., Matsuoka, M., Jones, S., Bradfute, S.B., Bray, M., Ozato, K., 2009.
Ebola Zaire virus blocks type I interferon production by exploiting the host SUMO
modification machinery. PLoS Pathog. 5 (6), e1000493.
Chen, C.H., Sheu, J.C., Wang, J.T., Huang, G.T., Yang, P.M., Lee, H.S., Lee, C.Z., Chen, D.S.,
1994. Genotypes of hepatitis C virus in chronic liver disease in Taiwan. J. Med. Virol.
44 (3), 234–236.
Chien, C.T., Bartel, P.L., Sternglanz, R., Fields, S., 1991. The two-hybrid system: a method
to identify and clone genes for proteins that interact with a protein of interest. Proc.
Natl Acad. Sci. USA 88 (21), 9578–9582.
Chiocca, S., 2007. Viral control of the SUMO pathway: Gam1, a model system. Biochem.
Soc. Trans. 35 (Pt 6), 1419–1421.
Edwards, S., Moennig, V., Wensvoort, G., 1991. The development of an international
reference panel of monoclonal antibodies for the differentiation of hog cholera
virus from other pestiviruses. Vet. Microbiol. 29 (2), 101–108.
Eisen-Vandervelde, A.L., Waggoner, S.N., Yao, Z.Q., Cale, E.M., Hahn, C.S., Hahn, Y.S.,
2004. Hepatitis C virus core selectively suppresses interleukin-12 synthesis in
human macrophages by interfering with AP-1 activation. J. Biol. Chem. 279 (42),
Fan, Z., Yang, Q.R., Twu, J.S., Sherker, A.H., 1999. Specific invitro association between the
hepatitis C viral genome and core protein. J. Med. Virol. 59 (2), 131–134.
Fauquet, C. M., M.A. Mayo, J. Maniloff, U. Desselberger, and L.A. Ball, Ed. (2005). VIRUS
TAXONOMY: VIIIth Report of the International Committee on Taxonomy of Viruses.
Edited by M. A. M. C.M. Fauquet, J. Maniloff, U. Desselberger, and L.A. Ball (Elsevier
Academic Press): ICTV.
Fields, S., Song, O., 1989. A novel genetic system to detect protein–protein interactions.
Nature 340 (6230), 245–246.
Fukata, M., Watanabe, T., Noritake, J., Nakagawa, M., Yamaga, M., Kuroda, S., Matsuura,
Y., Iwamatsu, A., Perez, F., Kaibuchi, K., 2002. Rac1 and Cdc42 capture microtubules
through IQGAP1 and CLIP-170. Cell 109 (7), 873–885.
Giannini, C., Brechot, C., 2003. Hepatitis C virus biology. Cell Death Differ. 10 (Suppl 1),
Gladue, D.P., Holinka, L.G., Fernandez-Sainz, I.J., Prarat, M.V., O'Donell, V., Vepkhvadzea,
N., Lu, Z., Rogers, K., Risatti, G.R., Borca, M.V., 2010. Effects of the interactions of
Classical Swine Fever Virus Core protein with proteins of the SUMOylation pathway
on virulence in swine. Virology 407 (1), 129–136.
Fig. 5. Effect of BICv and CoreΔIQ viruses infection on host cell microtubules and intermediate filaments. Monolayers of SK6 cells were infected with BIC and CoreΔIQ viruses or mock
infected, fixed with paraformaldehyde at 48 h post-infection, and processed for IF staining and confocal microscopy as described in Materials and methods. The microtubule was
immunolabeled with mouse anti-γ-tubulin (a–f), and intermediate filaments with mouse-anti-vimentin (g–l). AlexaFluor 594-conjugate antibodies were used as secondary
D.P. Gladue et al. / Virology 412 (2011) 68–74
Goh, P.Y., Tan, Y.J., Lim, S.P., Lim, S.G., Tan, Y.H., Hong, W.J., 2001. The hepatitis C virus
core protein interacts with NS5A and activates its caspase-mediated proteolytic
cleavage. Virology 290 (2), 224–236.
Heimann, M., Roman-Sosa, G., Martoglio, B., Thiel, H-J., Rümenapf, T., 2006. Core protein
of pestiviruses is processed at the C terminus by signal peptide peptidase. J. Virol.
80 (4), 1915–1921.
Jin, D.Y., Wang, H.L., Zhou, Y., Chun, A.C., Kibler, K.V., Hou, Y.D., Kung, H., Jeang, K.T.,
2000. Hepatitis C virus core protein-induced loss of LZIP function correlates with
cellular transformation. EMBO J. 19 (4), 729–740.
Kittlesen, D.J., Chianese-Bullock, K.A., Yao, Z.Q., Braciale, T.J., Hahn, Y.S., 2000.
Interaction between complement receptor gC1qR and hepatitis C virus core
protein inhibits T-lymphocyte proliferation. J. Clin. Invest. 106 (10), 1239–1249.
Kunkel, M., Lorinczi, M., Rijnbrand, R., Lemon, S.M., Watowich, S.J., 2001. Self-assembly
of nucleocapsid-like particles from recombinant hepatitis C virus core protein.
J. Virol. 75 (5), 2119–2129.
Lai, M.M., Ware, C.F., 2000. Hepatitis C virus core protein: possible roles in viral
pathogenesis. Curr. Top. Microbiol. Immunol. 242, 117–134.
Leung, J., Yueh, A., Appah Jr., F.S., Yuan, B., de los Santos, K., Goff, S.P., 2006. Interaction of
Moloney murine leukemia virus matrix protein with IQGAP. EMBO J. 25 (10),
Levrero, M., 2006. Viral hepatitis and liver cancer: the case of hepatitis C. Oncogene 25
Liu, J.J., Wong, M.L., Chang, T.J., 1998. The recombinant nucleocapsid protein of classical
swine fever virus can act as a transcriptional regulator. Virus Res. 53 (1), 75–80.
Lo, S.Y., Selby, M.J., Ou, J.H., 1996. Interaction between hepatitis C virus core protein and
E1 envelope protein. J. Virol. 70 (8), 5177–5182.
Mamiya, N., Worman, H.J., 1999. Hepatitis C virus core protein binds to a DEAD box RNA
helicase. J. Biol. Chem. 274 (22), 15751–15756.
Masaki, T., Suzuki, R., Murakami, K., Aizaki, H., Ishii, K., Murayama,A., Date, T., Matsuura,
Y., Miyamura, T., Wakita, T., Suzuki, T., 2008. Interaction of hepatitis C virus
nonstructural protein 5A with core protein is critical for the production of
infectious virus particles. J. Virol. 82 (16), 7964–7976.
McLauchlan, J., 2000. Properties of the hepatitis C virus core protein: a structural
protein that modulates cellular processes. J. Viral Hepat. 7 (1), 2–14.
McLaughlin, L.M., Govoni, G.R., Gerke, C., Gopinath, S., Peng, K., Laidlaw, G., Chien, Y.H.,
Jeong, H.W., Li, Z., Brown, M.D., Sacks, D.B., Monack, D., 2009. The Salmonella SPI2
effector SseI mediates long-term systemic infection by modulating host cell
migration. PLoS Pathog. 5 (11), e1000671.
Meyers, G., Rumenapf, T., Thiel, H.J., 1989. Molecular cloning and nucleotide sequence
of the genome of hog cholera virus. Virology 171 (2), 555–567.
Meyers, G., Saalmuller, A., Buttner, M., 1999. Mutations abrogating the RNase activity in
glycoprotein E(rns) of the pestivirus classical swine fever virus lead to virus
attenuation. J. Virol. 73 (12), 10224–10235.
Murray, C.L., Marcotrigiano, J., Rice, C.M., 2008. Bovine viral diarrhea virus core is an
intrinsically disordered protein that binds RNA. J. Virol. 82 (3), 1294–1304.
Noritake, J., Watanabe, T., Sato, K., Wang, S., Kaibuchi, K., 2005. IQGAP1: a key regulator
of adhesion and migration. J. Cell Sci. 118 (Pt 10), 2085–2092.
O'Donnell, V., LaRocco, M., Duque, H., Baxt, B., 2005. Analysis of foot-and-mouth disease
virus internalization events in cultured cells. J. Virol. 79 (13), 8506–8518.
Otsuka, M., Kato, N., Lan, K., Yoshida, H., Kato, J., Goto, T., Shiratori, Y., Omata, M., 2000.
Hepatitis C virus core protein enhances p53 function through augmentation of DNA
binding affinity and transcriptional ability. J. Biol. Chem. 275 (44), 34122–34130.
Ray, R.B., Ray, R., 2001. Hepatitis C virus core protein: intriguing properties and
functional relevance. FEMS Microbiol. Lett. 202 (2), 149–156.
Reed, L.J., Muench, H.A., 1938. A simple method of estimating fifty per cent endpoints.
Am. J. Hyg. 27, 493–497.
Rice, C.M., 1996. In: Knipe, B.N.F.D.M., Howley, P. (Eds.), Flaviviridae: The Viruses and
Their Replication, Third ed. : Fundamental Virology. Lippincott Raven, Philadelphia,
Risatti, G.R., Borca, M.V., Kutish, G.F., Lu, Z., Holinka, L.G., French, R.A., Tulman, E.R., Rock,
D.L., 2005a. The E2 glycoprotein of classical swine fever virus is a virulence
determinant in swine. J. Virol. 79 (6), 3787–3796.
Borca, M.V., 2005b. Mutation of E1 glycoprotein of classical swine fever virus affects
viral virulence in swine. Virology 343 (1), 116–127.
Risatti, G.R., Holinka, L.G., Carrillo, C., Kutish, G.F., Lu, Z., Tulman, E.R., Sainz, I.F., Borca,
M.V., 2006. Identification of a novel virulence determinant within the E2 structural
glycoprotein of classical swine fever virus. Virology 355 (1), 94–101.
Risatti, G.R., Holinka, L.G., Fernandez Sainz, I., Carrillo, C., Kutish, G.F., Lu, Z., Zhu, J., Rock,
D.L., Borca, M.V., 2007a. Mutations in the carboxyl terminal region of E2
glycoprotein of classical swine fever virus are responsible for viral attenuation in
swine. Virology 364 (2), 371–382.
Risatti, G.R., Holinka, L.G., Fernandez Sainz, I., Carrillo, C., Lu, Z., Borca, M.V., 2007b. N-
linked glycosylation status of classical swine fever virus strain Brescia E2
glycoprotein influences virulence in swine. J. Virol. 81 (2), 924–933.
Rümenapf, T., Stark, R., Heimann, M., Thiel, H-J., 1998. N-terminal protease of
pestiviruses: identification of putative catalytic residues by site-directed muta-
genesis. J. Virol. 72 (3), 2544–2547.
Sanger, F., Nicklen, S., Coulson, A.R., 1977. DNA sequencing with chain-terminating
inhibitors. Proc. Natl Acad. Sci. USA 74 (12), 5463–5467.
Shimoike, T., Mimori, S., Tani, H., Matsuura, Y., Miyamura, T., 1999. Interaction of
hepatitis C virus core protein with viral sense RNA and suppression of its
translation. J. Virol. 73 (12), 9718–9725.
Tanaka, Y., Shimoike, T., Ishii, K., Suzuki, R., Suzuki, T., Ushijima, H., Matsuura, Y.,
Miyamura, T., 2000. Selective binding of hepatitis C virus core protein to synthetic
oligonucleotides corresponding to the 5′ untranslated region of the viral genome.
Virology 270 (1), 229–236.
Tellinghuisen, T.L., Rice, C.M., 2002. Interaction between hepatitis C virus proteins and
host cell factors. Curr. Opin. Microbiol. 5 (4), 419–427.
Terpstra, C., Woortmeyer, R., Barteling, S.J., 1990. Development and properties of a cell
culture produced vaccine for hog cholera based on the Chinese strain. Dtsch
Tierärztl. Wochenschr. 97 (2), 77–79.
Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity
of progressive multiple sequence alignment through sequence weighting, position-
specific gap penalties and weight matrix choice. Nucleic Acids Res. 22 (22),
Tomoiu, A., Gravel, A., Tanguay, R.M., Flamand, L., 2006. Functional interaction between
human herpesvirus 6 immediate-early 2 protein and ubiquitin-conjugating
enzyme 9 in the absence of sumoylation. J. Virol. 80 (20), 10218–10228.
Van Gennip, H.G., Vlot, A.C., Hulst, M.M., De Smit, A.J., Moormann, R.J., 2004.
Determinants of virulence of classical swine fever virus strain Brescia. J. Virol. 78
van Rijn, P.A., Miedema, G.K., Wensvoort, G., van Gennip, H.G., Moormann, R.J., 1994.
Antigenic structure of envelope glycoprotein E1 of hog cholera virus. J. Virol. 68 (6),
Watanabe, T., Wang, S., Noritake, J., Sato, K., Fukata, M., Takefuji, M., Nakagawa, M.,
Izumi, N., Akiyama, T., Kaibuchi, K., 2004. Interaction with IQGAP1 links APC to
Rac1, Cdc42, and actin filaments during cell polarization and migration. Dev. Cell 7
Yao, Z.Q., Nguyen, D.T., Hiotellis, A.I., Hahn, Y.S., 2001. Hepatitis C virus core protein
inhibits human T lymphocyte responses by a complement-dependent regulatory
pathway. J. Immunol. 167 (9), 5264–5272.
Yao, Z.Q., King, E., Prayther, D., Yin, D., Moorman, J., 2007. T cell dysfunction by hepatitis
C virus core protein involves PD-1/PDL-1 signaling. Viral Immunol. 20 (2),
Yoshida, T., Hanada, T., Tokuhisa, T., Kosai, K., Sata, M., Kohara, M., Yoshimura, A., 2002.
Activation of STAT3 by the hepatitis C virus core protein leads to cellular
transformation. J. Exp. Med. 196 (5), 641–653.
You, L.R., Chen, C.M., Yeh, T.S., Tsai, T.Y., Mai, R.T., Lin, C.H., Lee, Y.H., 1999. Hepatitis C
virus core protein interacts with cellular putative RNA helicase. J. Virol. 73 (4),
Zsak, L., Lu, Z., Kutish, G.F., Neilan, J.G., Rock, D.L., 1996. An African swine fever virus
virulence-associated gene NL-S with similarity to the herpes simplex virus ICP34.5
gene. J. Virol. 70 (12), 8865–8871.
D.P. Gladue et al. / Virology 412 (2011) 68–74