Tetherin and its viral antagonists.

Abstract

Restriction factors comprise an important layer of host defense to fight against viral infection. Some restriction factors are constitutively expressed whereas the majority is induced by interferon to elicit innate immunity. In addition to a number of well-characterized interferon-inducible antiviral factors such as RNaseL/OAS, ISG15, Mx, PKR, and ADAR, tetherin (BST-2/CD317/HM1.24) was recently discovered to block the release of enveloped viruses from the cell surface, which is regarded as a novel antiviral mechanism induced by interferon. Here, we briefly review the history of tetherin discovery, discuss how tetherin blocks virus production, and highlight the viral countermeasures to evade tetherin restriction.

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INVITED REVIEW
Tetherin and Its Viral Antagonists
Björn D. Kuhl & Vicky Cheng & Mark A. Wainberg &
Chen Liang
Received: 2 November 2010 /Accepted: 27 December 2010 /Published online: 11 January 2011
# The Author(s) 2011. This article is published with open access at Springerlink.com
Abstract Restriction factors comprise an important layer
of host defense to fight against viral infection. Some
restriction factors are constitutively expressed whereas the
majority is induced by interferon to elicit innate immunity.
In addition to a number of well-characterized interferon-
inducible antiviral factors such as RNaseL/OAS, ISG15,
Mx, PKR, and ADAR, tetherin (BST-2/CD317/HM1.24)
was recently discovered to block the release of enveloped
viruses from the cell surface, which is regarded as a novel
antiviral mechanism induced by interferon. Here, we briefly
review the history of tetherin discovery, discuss how
tetherin blocks virus production, and highlight the viral
countermeasures to evade tetherin restriction.
Keywords HIV-1.Tetherin.Vpu.Virus release.Interferon
Introduction
Interferon triggers innate immunity to protect cells from
viral infection. Production of interferon is initiated with
recognition of viral components, mainly viral nucleic acids,
by cellular pathogen recognition receptors that are located
on cellular membranes or within the cytosol (reviewed in
Kawai and Akira 2006; Douville and Hiscott 2010). For
example, membrane-associated Toll-like receptor 3 (TLR3)
recognizes double-stranded viral RNA, recruits TIR
domain-containing adaptor inducing IFN-β, and activates
the interferon regulatory factor 3 (IRF3) and nuclear factor
kappa-light-chain-enhancer of activated B cells (NF-κB)
pathways to induce type I interferon production. TLR7 and
TLR9 are both located in endosomes. After recognizing
single-stranded viral RNA or double-stranded viral DNA,
they recruit myeloid differentiation primary response gene
88 and activate the IRF7 and NF-κB pathways. The
cytosolic retinoic-acid-inducible gene I and melanoma
differentiation-associated gene 5 recognize either double-
stranded viral RNA or 5′-triphospho viral RNA and activate
the MAVS (also known as IPS-1, VISA, and Cardif)
signaling transduction pathway, which leads to interferon
production.
Interferon binds to its receptor on the cell surface and
activates tyrosine kinase 2 and Janus kinase 1 that in turn
phosphorylates signal transducer and activator of transcrip-
tion 1 (STAT1) and STAT2. The phosphorylated STAT1/
STAT2 then translocates into the nucleus and forms the
interferon-stimulated gene factor 3 (ISGF3) complex
together with IRF9. The ISGF3 complex activates the
expression of a wide spectrum of genes, collectively named
interferon-stimulated genes (ISGs) that together establish an
antiviral state. Among hundreds of ISGs, those with well-
defined antiviral activities include RNaseL/OAS (2′,5′-
oligoadenylate synthetase), ISG15, myxovirus resistance
(Mx), protein kinase R (PKR), and adenosine deaminase,
RNA-specific (ADAR) that inhibit virus infection by
different mechanisms (reviewed in Sadler and Williams
2008). Upon binding to double-stranded RNA, OAS
Björn D. Kuhl and Vicky Cheng contributed equally to this work.
B. D. Kuhl:V. Cheng:M. A. Wainberg:C. Liang (*)
McGill AIDS Centre,
Lady Davis Institute-Jewish General Hospital,
Montréal, Quebec, Canada H3T 1E2
e-mail: chen.liang@mcgill.ca
B. D. Kuhl:M. A. Wainberg:C. Liang
Department of Medicine, McGill University,
Montréal, Quebec, Canada H3A 2B4
V. Cheng:M. A. Wainberg:C. Liang
Department of Microbiology and Immunology, McGill University,
Montréal, Quebec, Canada H3A 2B4
J Neuroimmune Pharmacol (2011) 6:188–201
DOI 10.1007/s11481-010-9256-1

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oligomerizes and synthesizes 2′,5′-oligoadenylates that bind
to and activate RNaseL. The activated RNaseL degrades
both cellular and viral RNA. ISG15 is conjugated to
cellular and viral proteins to modulate cellular response
and inhibit virus infection. This event is termed ISGylation
that is catalyzed sequentially by three enzymes called
ubiquitin E1 enzyme, E2-conjugating enzymes (such as
UBCH8), and E3 ligase enzymes (such as HERC5,
homologous to the E6-associated C terminus domain and
RCC1-like-domain-containing protein 5). Mx proteins are
dynamin-like large GTPases. They inhibit the infection of a
wide range of RNA and DNA viruses by binding to viral
nucleocapsids or other viral components and subsequently
trapping viral complexes as a result of Mx oligomerization.
PKR is a kinase that is dormant in its monomeric form.
Upon binding to viral double-stranded RNA, the kinase
activity is activated, which leads to phosphorylation and
dimerization of PKR. The activated PKR then phosphor-
ylates EIF2α and thus inhibits translation of cellular and
viral proteins. ADAR is an adenosine deaminase that binds
double-stranded RNA and converts adenosine to inosine. In
addition to these antiviral effectors, recent studies discov-
ered that an ISG named tetherin (also called BST-2/CD317/
HM1.24, bone marrow stromal cell antigen 2) inhibits viral
infection via a novel mechanism by blocking the release of
nascent enveloped viral particles from the cell surface.
Discovery of tetherin as an antiviral protein
BST-2 (or HM1.24) was first reported as a protein that
expresses at the surface of human plasma cell lines, bone
marrow stromal cell lines and B cells, and is thus
speculated to have a role in B-cell development (Goto et
al. 1994; Ishikawa et al. 1995). Its potential role as a
component of the host antiviral defense mechanism was
suggested by its being a target of the K5 protein (also
named MIR2) of Kaposi’s sarcoma-associated herpesvi-
rus (Bartee et al. 2006). The antiviral activity of BST-2
was subsequently demonstrated for its ability to inhibit the
release of HIV-1 that is deficient in Vpu and was therefore
given the name tetherin (Neil et al. 2008; Van Damme et
al. 2008).
In addition to structural and enzymatic proteins including
Gag, Pol, and Env, HIV-1 also encodes two regulatory
proteins Tat and Rev that are essential for viral gene
expression, as well as four accessory proteins named Vpr,
Vif, Vpu, and Nef that play important roles in HIV-1
pathogenesis (reviewed in Anderson and Hope 2004; Malim
and Emerman 2008). For example, Vif recognizes a cellular
cytidine deaminase named apolipoprotein B mRNA editing
enzyme, catalytic polypeptide 3 G (APOBEC3G), triggers
polyubiquitination of APOBEC3G through recruiting the
Cullin5-containing E3 ligase, and sends APOBEC3G to
proteasomes for degradation. In the absence of Vif,
APOBEC3G associates with HIV-1 RNP complex, gets
packaged into virus particles, and induces hypermutation of
viral cDNA by converting cytidines into uridines during
reverse transcription (reviewed in Henriet et al. 2009;
Niewiadomska and Yu 2009). It has been known for quite
a long time that Vpu is required for efficient HIV-1
production in certain cell lines such as HeLa and primary
T cells but not in cells such as HEK293 and COS-7 (Strebel
et al. 1989; Göttlinger et al. 1993). The Vpu-deficient HIV-
1 particles encounter a difficulty of leaving the surface of
Vpu-non-permissive cells such as HeLa. A dominant
cellular factor was speculated to underlie this restriction
activity because the restriction phenotype was recreated in
the HeLa/COS-7 heterokaryotic cells (Varthakavi et al.
2003). Subsequent studies revealed that this putative
restriction factor is interferon-inducible (Neil et al. 2007).
A long quest for this mysterious factor by several labs led
to the discovery of its identity as tetherin, named after its
function to tether nascent HIV-1 particles to the cell surface
(Neil et al. 2008; Van Damme et al. 2008). After this
finding, a variety of enveloped viruses were also shown to
be subject to the restriction of tetherin. These include HIV-2
(Le Tortorec and Neil 2009), simian immunodeficiency
virus (SIV) (Jouvenet et al. 2009), alpharetrovirus (Rous
sarcoma virus) (Jouvenet et al. 2009), betaretrovirus
(Mason-Pfizer monkey virus) (Jouvenet et al. 2009),
gammaretrovirus (human T-cell leukemia virus type 1
(HTLV-1)) (Jouvenet et al. 2009), filoviruses (Marburg
virus (Jouvenet et al. 2009; Sakuma et al. 2009) and Ebola
virus (Jouvenet et al. 2009; Kaletsky et al. 2009)),
arenaviruses (Lassa virus (Sakuma et al. 2009)), and
herpesvirus (Kaposi’s sarcoma-associated herpesvirus
(KSHV) (Mansouri et al. 2009)). The two forthcoming
questions are how tetherin retains enveloped viral particles
to the cell surface and how viruses evade this restriction.
What is tetherin?
Human tetherin is encoded by a single copy of the bst-2
gene on chromosome 19p13.2 that is split into four exons
(Ishikawa et al. 1995). This gene is relatively non-
polymorphic with only one nonsynonymous single-
nucleotide polymorphism that is listed in the human
genome database and located in the coding region for the
ectodomain of tetherin. Tetherin orthologs are present in the
genomes of mammals but with significant sequence
divergence (McNatt et al. 2009).
Tetherin is constitutively expressed in mature B cells,
some cancer cell lines, bone marrow stromal cells,
monocyte-derived macrophages, and plasmacytoid dendrit-
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ic cells (pDCs) (Ishikawa et al. 1995; Vidal-Laliena et al.
2005; Blasius et al. 2006; Mitchell et al. 2009; Miyagi et al.
2009). Interferon strongly stimulates tetherin expression
due to the presence of interferon response elements and a
binding site for STAT3 in the promoter region of bst-2 gene
(Blasius et al. 2006). However, the level of tetherin
expression as part of the interferon response appears to be
cell-type-dependent (Miyagi et al. 2009). Regarding cancer
T-cell lines, which are commonly used in laboratories to
study HIV infection, the basal expression levels of tetherin
vary but are all interferon-inducible (Neil et al. 2008; Van
Damme et al. 2008; Rong et al. 2009).
When first described in B cells, tetherin was suggested to
be a marker for B-cell maturation (Ishikawa et al. 1995). Its
subsequent detection on multiple myeloma cells suggested
a tetherin-based therapeutic strategy to induce cell-mediated
cytotoxicity with tetherin antibody (Ozaki et al. 1997; Ono
et al. 1999). In an epithelial cell line, tetherin interacts and
modulates the subapical actin cytoskeleton, indirectly, via
RICH2, EBP50, and ezrin (Rollason et al. 2009). It remains
undetermined whether the cytoskeleton-modulating activity
is involved in tetherin’s function of restricting viral release.
Tetherin is also a ligand for immunoglobulin-like transcript
7 (ILT7) on pDCs (Cao et al. 2009). Since ILT7-mediated
signaling strongly inhibits production of interferon and
other cytokines in pDCs (Brown et al. 2004; Cao et al.
2006), tetherin may negatively regulate interferon and
cytokine production (Cao et al. 2009).
Tetherin localizes tothe plasma membrane, the trans-Golgi
network (TGN) and the early and recycling endosomes, and
cycles between these membrane compartments (Kupzig et al.
2003; Habermann et al. 2010). Endocytosis of tetherin from
the plasma membrane is clathrin-dependent and involves an
interaction of tetherin with the adaptor proteins AP-1 and
AP-2 (Rollason et al. 2007; Masuyama et al. 2009). In an
epithelial cell line, tetherin localizes to the apical membrane
following cellular polarization (Rollason et al. 2009). At the
cell surface, tetherin localizes to lipid rafts (Kupzig et al.
2003; Rollason et al. 2007, 2009; Goffinet et al. 2009).
Immunofluorescence microscopy reveals tetherin as punctual
clusters at the cell surface (Hinz et al. 2010). HIV-1 Gag
assembly and virus budding also occur at these membrane
microdomains (Waheed and Freed 2009). In addition, lipid
rafts are also involved in forming virological synapses and
promoting cell-to-cell transmission of viruses (Popik et al.
2002; Piguet and Sattentau 2004; Popik and Alce 2004; Jolly
and Sattentau 2005).
How does tetherin block virus release?
Tetherin retains nascent viral particles at the cell surface by
linking them to the cellular membrane and to each other
(Fig. 1a). Electron microscopy shows accumulation of viral
particles at the surface of tetherin-expressing cells (Neil et
al. 2008; Perez-Caballero et al. 2009; Fitzpatrick et al.
2010). Tetherin is colocalized with viral Gag at the cell
surface (Neil et al. 2008; Van Damme et al. 2008; Perez-
Caballero et al. 2009) and is detected in the membrane of
viruses that are released from the cell surface either
spontaneously (Fitzpatrick et al. 2010; Habermann et al.
2010) or by shearing force (such as vortexing) (Miyagi et
al. 2009). Results of immunoelectron microscopy further
revealed that tetherin is located between the tethered viral
particles and between the tethered virus particle and the
plasma membrane, which indicates a direct role of tetherin
in blocking virus release. This conclusion is further
supported by two lines of evidence. First, human tetherin,
when expressed in non-human cells, can restrict release of
budding virions (Sato et al. 2009). Second and more
importantly, the antiviral function of tetherin is, to a certain
degree, independent of its sequence. An antiviral competent
artificial tetherin has been reported, which has its trans-
membrane domain derived from transferrin receptor, coiled-
coil domain from dystrophia myotonica protein kinase, and
glycophosphoinisitol (GPI) anchor from urokinase plasmin-
ogen activator receptor (Perez-Caballero et al. 2009). These
results suggest that the antiviral activity of tetherin unlikely
requires a co-factor.
Structural determinants of tetherin’s antiviral function Te-
therin is a type II transmembrane protein (Ishikawa et al.
1995; Kupzig et al. 2003). It bears a short cytoplasmic N-
terminal region, a transmembrane region, an ectodomain,
and a C-terminal GPI anchor (Kupzig et al. 2003). This
topology is rare and only shared with an isoform of the
prion protein (Hegde et al. 1998; Stewart et al. 2001).
Tetherin forms disulfide-linked dimers. It is heterogeneous-
ly glycosylated and thus migrates as 30–36-kDa proteins on
a sodium dodecyl sulfate–polyacrylamide gel. These struc-
tural features together determine tetherin’s antiviral function
and also regulate its sensitivity to different viral antagonists.
The short cytoplasmic domain (at amino acid positions 1
to 21) contains a conserved dual tyrosine motif (YXY6–8)
that is crucial for clathrin-mediated endocytosis through
recruiting AP-1 and AP-2 adaptor proteins (Rollason et al.
2007). The two lysine residues (at positions 18 and 21) are
ubiquitinized by the RING-CH K5 ligase of KSHV, which
leads to tetherin degradation (Mansouri et al. 2009; Pardieu
et al. 2010). Human tetherin lacks the 14-D/GDIWK-18
motif that constitutes the interaction site of Nef and is thus
resistant to Nef-mediated antagonism (Jia et al. 2009;
Zhang et al. 2009). Additionally, the cytoplasmic domain
of tetherin has been described to indirectly modulate
subapical actin cytoskeleton in polarized endothelial cells
via RICH2, EBP50, and ezrin (Rollason et al. 2009).
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The transmembrane region (at amino acid positions 22 to
43) consists of a single membrane-spanning helix. In the
case of HIV-1, the transmembrane region renders human
tetherin sensitive to Vpu-mediated downmodulation from
the cell surface and subsequent degradation (Gupta et al.
2009a; McNatt et al. 2009; Rong et al. 2009). Several
amino acid residues across the transmembrane domain of
tetherin have been reported to regulate the sensitivity to
Vpu and tetherin/Vpu interaction; these include L22, L23,
G25, I26, V30, I33, I36, and T45 (Fig. 2) (Gupta et al.
2009a; McNatt et al. 2009; Rong et al. 2009). Interestingly,
results of computer modeling of the structure of tetherin
transmembrane domain show that these amino acid residues
are positioned at one side of the helix, suggesting that they
may together form the contact site with Vpu (Fig. 2).
The ectodomain (amino acids 44 to 160) assumes a long
single α-helix as shown by the results of X-ray crystallog-
raphy (Fig. 3) (Hinz et al. 2010; Schubert et al. 2010; Yang
et al. 2010a). The complete ectodomain extends to a length
of 150 to 170Å which includes the 90-Å C-terminal coiled-
coil domain (Hinz et al. 2010). Two tetherin molecules
dimerize via this parallel disulfide-linked coiled-coil struc-
ture that is mainly stabilized by interactions throughout the
two-third C-terminal portion of the ectodomain (Fig. 3).
The N-terminal portion of ectodomain appears to be
relatively flexible at two hinges (positions A88 and G109)
and mediates the tetramerization of two tetherin dimers by
forming an antiparallel four-helix bundle (Fig. 3) (Hinz et
al. 2010; Schubert et al. 2010). Although two tetherin
dimers form a tetramer in crystals, mutants that are
deficient in tetramerization retain most of the antiviral
activity (Schubert et al. 2010; Yang et al. 2010a). The
length of ectodomain is crucial for tetherin to block virus
release, which suggests a molecular ruler function to keep
the two membrane-spanning termini at a distance that is
required for maximal antiviral activity (Hinz et al. 2010;
Yang et al. 2010a).
The ectodomain of tetherin also contains three cysteines
(C53C63C91) that form disulfide bonds and therefore
stabilize tetherin homodimers. In addition, there exist two
sites for post-translational N-linked glycosylation (N65N92)
(Ohtomo et al. 1999; Andrew et al. 2009). While
glycosylation was shown to be dispensable for the antiviral
function, formation of at least one disulfide bond is crucial
for tetherin to block virus release (Andrew et al. 2009;
Perez-Caballero et al. 2009; Hinz et al. 2010).
The C-terminal region of tetherin contains a signal for
the addition of a GPI anchor. The nascent protein is
predictably cleaved at a serine residue (position 160) in
the endoplasmic reticulum (ER) followed by the attachment
Fig. 1 Antiviral mechanism of
tetherin. a Tetherin localizes to
the virus budding site and
incorporates into the viral
membrane. Tetherin exerts its
antiviral action by tethering viral
particles to the cell and to each
other. Further, tetherin inhibits
protease-mediated viral
maturation of released viral
particles. b, c Models of
molecular tethering mechanism.
Tetherin homodimers are likely
oriented in parallel. Tetherin
links viral particles to cells by
insertion of one terminus into
the virion membrane, while the
other is located in the cellular
membrane (b), or tetherin may
link viral particles and cells via
dimerization of the ectodomains
of monomers with both termini
inserted in either the virion
membrane or the cellular
membrane
J Neuroimmune Pharmacol (2011) 6:188–201 191

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of the prefabricated GPI anchor (Kupzig et al. 2003).
Therefore, the mature tetherin is a heterogeneously glycosy-
lated 160-amino-acid single pass transmembrane protein with
a C-terminal GPI anchor incorporated into lipid rafts. The GPI
anchor-mediated localization to lipid rafts promotes the
clathrin-mediatedendocytosis(Rollasonetal.2007). Removal
of GPI anchor abolishes the antiviral activity of tetherin (Neil
et al. 2008; Perez-Caballero et al. 2009).
Models of “tethering” Several models have been proposed
to illustrate how tetherin retains viral particles on the cell
surface. These models are conceived on the basis of two
key features of tetherin, dimerization across the ectodomain
and insertion of the two termini into either the cellular
plasma membrane or the membrane of viral particle. Since
not only does tetherin act as a link to retain a virus particle
to the cell surface but also tethers the viral particles to each
other, it is believed that the N and the C termini of tetherin
do not distinguish between the cellular and the viral
membranes for insertion, which is not surprising given that
viral membrane is derived from the cellular plasma
membrane (Fig. 1b, c) (Neil et al. 2008; Perez-Caballero
et al. 2009). Theoretically, a tetherin dimer may consist of
two monomers that are positioned either in a parallel or in
an antiparallel orientation. These two possibilities are now
resolved by the crystal structures of tetherin ectodomains
that are shown to form parallel dimers. Tetherin may thus
function as a parallel homodimer to bridge cellular and viral
membrane directly by insertion of one terminus into the
cellular membrane and the other into the viral membrane
(Fig. 1b). However, there is no conclusive evidence to rule
out the possibility that tetherin dimers work by inserting the
two termini of the same tetherin molecule into the same
membrane (Fig. 1c).
Fate of tethered viral particles The tethered virions may be
internalized and sent to late endosomes and lysosomes for
degradation(Neiletal.2007; Van Damme et al. 2008; Sato et
al. 2009). It was recently shown that internalization and
lysosomal degradation are promoted by the interaction of the
cytosolic domain of tetherin with breast cancer associated
gene 2 (Rabring7, ZNF364, RNF115) (Miyakawa et al.
2009). However, the molecular mechanisms behind this
remain unknown. Furthermore, it is unclear whether inter-
nalized virions can cycle back to the cell surface if not
degraded.
C
C
N
N
T45
L22
L23
V30
G25
I26
I33
I36
A14
A18
W22
Tetherin HIV-1 Vpu
Fig. 2 Models of tetherin and Vpu transmembrane domains. The
transmembrane domains of human tetherin (left) and HIV-1 Vpu
(right) are illustrated in regard to their orientation in the membrane.
Amino acids of the tetherin transmembrane known to impact on the
interaction without abruption of tetherin function are highlighted in
blue and indicated left to the model; amino acids are selected
according to Gupta et al. (2009a), McNatt et al. (2009), and Rong et
al. (2009). Amino acids that are involved in tetherin downmodulation
from the cell surface are highlighted in yellow on the Vpu
transmembrane model (Vigan and Neil 2010). Models were created
with PyMol software on the basis of the sequences of human tetherin
(GenBank ID NP_004326.1) and HIV-1 Vpu from viral clone pNL_4-3
(GenBank ID AAK08488.1)
Fig. 3 Crystal structure of tetherin ectodomain. Shown are the crystal
structures of a tetherin dimer (top) and tetramer (bottom). The
disulfide bond building cysteine residues C53C63C91are highlighted
in blue (top and bottom); N-glycosylation sites N65N92are presented
in purple (top). Structures are based on the X-ray crystallography data
from Schubert et al. (2010) (PDB ID: 3NWH) and were created using
PyMol software. Structures of the ectodomains were created using
PyMol software
192J Neuroimmune Pharmacol (2011) 6:188–201