The Great Escape: Viral Strategies to Counter BST-2/Tetherin
et al. (2010) The Great Escape: Viral Strategies to Counter BST-2/Tetherin. PLoS
Pathog 6(5): e1000913. doi:10.1371/journal.ppat.1000913
The Great Escape: Viral Strategies to Counter BST-2/ Tetherin
Janet L. Douglas 0
Jean K. Gustin 0
Kasinath Viswanathan 0
Mandana Mansouri 0
Ashlee V. Moses 0
Klaus Fru h 0
Glenn F. Rall, The Fox Chase Cancer Center, United States of America
0 Vaccine and Gene Therapy Institute, Oregon Health & Science University , Beaverton, Oregon , United States of America
The interferon-induced BST-2 protein has the unique ability to restrict the egress of HIV-1, Kaposi's sarcoma-associated herpesvirus (KSHV), Ebola virus, and other enveloped viruses. The observation that virions remain attached to the surface of BST-2-expressing cells led to the renaming of BST-2 as ''tetherin''. However, viral proteins such as HIV-1 Vpu, simian immunodeficiency virus Nef, and KSHV K5 counteract BST-2, thereby allowing mature virions to readily escape from infected cells. Since the anti-viral function of BST-2 was discovered, there has been an explosion of research into several aspects of this intriguing interplay between host and virus. This review focuses on recent work addressing the molecular mechanisms involved in BST-2 restriction of viral egress and the species-specific countermeasures employed by various viruses.
BST-2 (CD317/HM1.24) was initially identified by two
independent groups searching for novel surface markers of
terminally differentiated normal and neoplastic B cells [1,2]. In
a proteomics screen, our group subsequently identified BST-2 as a
novel target for the viral ubiquitin-ligase K5 of Kaposis sarcoma
associated herpesvirus (KSHV) . However, the function of
BST2 remained unknown until it was identified as an intrinsic
antiviral factor that restricts the egress of HIV-1 by tethering mature
virions to the host cell surface . Coincident with this discovery,
BST-2 was identified as a target of the HIV-1 accessory protein
Vpu, providing a plausible mechanism for the well-established, but
ill-defined, virus release function of Vpu . Work by other
investigators showing that Vpu downregulates BST-2 from the cell
surface [3,5] suggested a mechanism for Vpu antagonism of
BST2. These discoveries have stimulated an active area of research
that explores several intriguing aspects of BST-2 function,
including its role as a general inhibitor of enveloped virus release,
the mechanisms underlying its neutralization by viral
immunomodulators, and the possibility that additional activities for this
enigmatic protein remain to be identified. In addition to providing
a critical overview of recent discoveries in the field, the intent of
this review is to summarize the history of BST-2, its anti-viral
activities, and potential modes of action. We focus primarily on
human BST-2 and HIV-1 to describe the molecular characteristics
of BST-2, countermeasures employed by HIV-1 Vpu, and the
genetic and mechanistic aspects of the hostvirus interaction. To
put the significance of BST-2/HIV-1 into a larger perspective, we
also address species specificity and discuss other viruses restricted
by BST-2, and the means, if any, utilized by these viruses to
overcome BST-2. While much remains to be clarified regarding
the nature and significance of BST-2 function, its role as an
intrinsic mediator of anti-viral resistance provides unique insight
into the complexity of hostvirus relationships and reminds us of
the potential to exploit these relationships for therapeutic benefit.
Molecular Characteristics of BST-2
Membrane Topology of BST-2
Human, rat, and mouse BST-2 have been independently identified
and subsequently cloned by several groups [2,68]. This work and
that of others  revealed that bst-2 encodes a 20-kDa, single pass,
type II glycosylated membrane protein that localizes to lipid rafts via
its COOH-terminal glycosylphosphatidylinisotol (GPI) anchor
(Figure 1A). While BST-2 migrates as a heterogenous smear of
approximately 3036k Da in reducing SDS-PAGE, the protein
migrates as a larger dimer under non-reducing conditions,
presumably due to the formation of disulfide bonds among the three
conserved cysteine residues in the extracellular domain. Among
known proteins, this topology is relatively unique, as it has only been
observed for one variant of the prion protein .
Sub-Cellular Localization and Transport of BST-2
BST-2 localizes not only to the plasma membrane but also to
internal membranes, particularly the trans-Golgi network (TGN)
and recycling endosomes . Unlike other GPI-anchored proteins,
BST-2 is endocytosed from the cell surface in a clathrin-dependent
manner. This appears to depend upon an interaction between an
AP-2 subunit and a non-canonical, dual tyrosine motif within the
BST-2 cytosolic domain [9,11] (Figure 1A). Recent studies also
show that BST-2 expressed at the apical surface of polarized
epithelial cells is linked to the actin cytoskeleton through a series of
ezrin-radixin-moesin (ERM)-binding and adapter proteins:
RICH2, EBP50, and ezrin. Furthermore, siRNA knockdown of
BST-2 in these cells resulted in a re-organization of the actin
cytoskeleton in a Rac- and Rho-dependent manner . While
the implications of these interactions for the anti-viral function of
BST-2 have not yet been evaluated, BST-2 appears to locate to
subcellular sites frequently used for viral egress.
Transcriptional Regulation of BST-2
Within the bst-2 promoter region is a tandem repeat containing
interferon (IFN) response elements and three STAT3 binding sites
that are activated in response to interleukin (IL)-6 . Indeed,
BST-2 is upregulated in most mouse and human cell types upon
type I and type II IFN treatment [4,8], consistent with an
evolutionarily conserved innate immune function. Interestingly,
BST-2 can also inhibit the production of IFN and
proinflammatory cytokines, such as IL-6 and tumor necrosis factor
(TNF)-a by human plasmacytoid dendritic cells (pDCs) . This
inhibition is accomplished by BST-2 binding to the orphan
receptor immunoglobulin-like transcript 7 (ILT7), which is
expressed exclusively on pDCs. This interaction establishes a
negative feedback loop in which IFN-induced BST-2 binds to the
ILT7-FceRIc complex, thereby signaling the inhibition of IFN
and proinflammatory cytokines . In addition to the
wellstudied anti-viral function described below, BST-2 might also play
a role in regulating innate immune cells.
Mechanisms of BST-2 Anti-Viral Restriction and
The HIV-1 accessory protein Vpu is a small NH2-terminally
anchored TM protein that mediates the degradation of CD4 
via interaction with the beta transducin repeat-containing protein
(bTrCP), a subunit of the Skp1-Cullin-F-box (SCF) E3 ubiquitin
ligase complex  (Figure 1B). In addition, Vpu enhances the
release of progeny virions from certain cell types (Vpu
responsive cells), a phenomenon that was discovered 20 years
ago . However, the mechanism of this enhancement remained
obscure. The recent identification of BST-2 as a putative viral
restriction factor subject to Vpu antagonism has answered a
longstanding question regarding Vpus virion release function.
However, it has also stimulated many intriguing new questions
about the evolution and function of both of these proteins.
Evidence for a BST-2 Anti-Viral Tethering Function
Electron micrographs of Vpu-responsive cells infected with
DVpu HIV-1 reveal the presence of viral particles accumulated at
the cell surface in what appear to be tethered chains . In two
seminal papers it was shown that the expression of BST-2 confers
the Vpu-responsive phenotype, and that in cells lacking BST-2
expression, there is a marked reduction in tethered DVpu
virions [4,5]. To reflect this unique activity, BST-2 was renamed
tetherin . Tethered, cell-associated virions appear to be fully
mature, based on the presence of both electron dense cores and
the functional reverse transcriptase activity of particles that have
been physically dislodged from the infected cell surface . The
virions can also be released by protease treatment, which Neil et
al. present as evidence for a protein-based tether, as opposed to a
budding defect that prevents membrane separation [4,17].
However, this protease sensitivity does not rule out a potential
role for other host proteins besides BST-2 in restricting virion
release or alternative hypotheses to tethering as the mechanism of
viral restriction. A recent report has identified BCA2 as a
BST-2interacting factor, which is thought to supplement the BST-2 viral
restriction by enhancing the internalization and degradation of
tethered virions from the cell surface . Because BST-2 can
restrict a large number of enveloped viruses (see Table 1), it is
unlikely that it interacts with a specific viral protein to induce
tethering. Neil et al. hypothesized that because BST-2 forms
dimers and higher order multimers, perhaps BST-2 is
incorporated into virions, thereby allowing for tethering between
virusand cell-associated BST-2 . Perez et al. tested this hypothesis
and found that they could only detect BST-2 in DVpu HIV-1
particles when BST-2 was functionally inactivated via deletion of
either its transmembrane (TM) domain or GPI anchor .
Wildtype BST-2 could only be detected in over-expressed Gag viral-like
particles (VLPs). Interestingly, only the DTM mutant was
incorporated into wild-type HIV-1 virions, suggesting that Vpu
limits BST-2 incorporation into viral particles via the TM domain.
Several other reports have confirmed the incorporation of BST-2
into HIV virions, although discrepancies remain. For example,
Hammonds et al.  were able to detect IFN-induced BST-2 in
DVpu virions, but not wild-type HIV virions, while Fitzpatrick et
al.  and Habermann et al.  detected endogenous BST-2 in
both wild-type and DVpu HIV. In contrast to these studies, Miyagi
et al. were able to detect endogenous BST-2 in DVpu, but not
wild-type HIV virions released via vortexing from infected cells
. However, they also detected BST-2 in control preparations
of vesicles isolated from uninfected cells, and therefore concluded
that BST-2 is not specifically incorporated into viral particles. Neil
et al.  went on to hypothesize that if BST-2 were incorporated
into viral particles, a tethering mechanism might depend upon
homo-dimeric/oligomeric interactions between cell- and
virusassociated BST-2 molecules. This has been tested by several
groups. Treatment of cell surface-tethered HIV and Ebola VLPs
 or wild-type HIV  with reducing agents did not induce
particle release, suggesting that tethering does not involve disulfide
linkage of BST-2 dimers or oligomers. Similarly, treatment of
tethered virions with the GPI anchor-cleaving enzyme Pi-PLC did
not effectively release the virions . Thus, while it is now clearly
established that Vpu-responsiveness is caused by BST-2,
additional studies are required to further elucidate the
BST-2dependent tethering mechanism and to determine whether there is
a functional role for virion-associated BST-2.
BST-2 Domains Important for Restricting Virus Release
To date, the majority of BST-2 mapping studies have revealed
species-specific residues important for virus-mediated antagonism
of BST-2, but not for the anti-viral function of BST-2. The
original studies identifying BST-2 as a viral release restriction
factor suggested that the COOH-terminal GPI anchor is necessary
for the anti-viral function of human BST-2, as an NH2-terminally
hemagglutinin (HA)-tagged mutant missing the GPI anchor and
downstream sequences was unable to restrict HIV release . This
same group later showed that along with the GPI anchor, both the
Other lentiviruses (EIAV, FIV); other
retroviruses (RSV, MPMV, HTLV-1,PFV)
Filoviruses (Ebola, Marburg, Lassa)
it expresses Vpu)
? not evaluated
Cell surface downregulation/degradation
Presumably same as HIV-1 Vpu
cell surface downregulation/sequestration
Cell surface downregulation/not degradation No
Species Specificity of
See Table 3
TM domain and the predicted internal coiled-coil (CC) domain
are also important for the BST-2 tethering function .
Surprisingly, they discovered that the amino acid sequence of
these domains was not important for tethering function. A
molecule consisting of structurally similar domains from three
unrelated proteins (TM from transferrin receptor, CC from
dystrophia myotonica protein kinase [DMPK], and GPI anchor
from urokinase plasminogen activator receptor) was able to restrict
viral release as efficiently as BST-2. To investigate whether dimer
formation plays a role in the BST-2 release function, Andrew et al.
and Perez-Caballero et al. both constructed mutants substituted
for the putative disulfide-linked cysteines located within the BST-2
extracellular domain. Each group found that when all three
extracellular cysteine residues were mutated (C53A, C63A, and
C91A), both dimer formation and BST-2 function were prevented,
while single and double substitutions had no effect [19,25],
suggesting that promiscuous dimer formation is important for
BST-2 anti-viral activity. Conversely, both groups made
substitutions for the two putative N-linked glycosylation sites (N65 and
N92) and obtained conflicting results. Andrew et al. found that
substituting both of these Asn residues with Gln affected
glycosylation, but they had no impact upon either BST-2 function
or sensitivity to Vpu . In contrast, Perez-Caballero et al.
replaced both Asn residues with Ala, which resulted in a
nonfunctional BST-2 . However, because this latter mutant was
not efficiently expressed at the cell surface, it is likely that in
addition to affecting BST-2 glycosylation, this particular mutation
impacted intracellular transport. In summary, the sequence
requirements for BST-2 tethering seem to be extraordinarily
flexible as long as overall topology and intracellular transport are
Molecular Mechanisms of Vpu-Dependent BST-2
Earlier attempts to map the Vpu domains necessary for
enhanced virus release were inconclusive. One group found that
the two phosphorylation sites within the Vpu C-terminus that are
essential for binding to bTrCP were dispensable for virus release
, while another group showed an approximate 50% reduction
in virus release when substitutions were made for these serines
[27,28]. A role for the Vpu TM domain in viral egress was first
noted when a Vpu TM mutant that was functional with respect to
CD4 downregulation failed to enhance viral release . While all
of these studies were performed prior to the discovery that BST-2
inhibits viral egress, recent studies (detailed below) have confirmed
a role for Vpus bTrCP-binding and TM domains in
The bTrCP-Binding Domain of Vpu Is Important for BST-2
Our group and others have determined that the bTrCP-binding
site located within the Vpu cytoplasmic domain is necessary for the
downregulation of BST-2  (Figure 1B). This was
demonstrated by showing that a Vpu bTrCP-binding mutant
did not induce downregulation or degradation of BST-2. In
addition, both a dominant negative bTrCP mutant and an siRNA
directed against bTrCP effectively block Vpus ability to
downregulate BST-2. While the bTrCP-binding domain is
necessary for counteracting BST-2, it does not appear to be
necessary for direct interaction between the proteins, as both
wildtype Vpu and the bTrCP-binding mutant co-immunoprecipitate
and co-localize with BST-2 [30,33]. These results suggested that
another region(s) within Vpu mediates BST-2 binding.
The Vpu Transmembrane Domain May Mediate BST-2
One candidate region for a putative BST-2 binding site is the
Vpu TM domain (Figure 1B). While recent data suggest that the
Vpu TM domain interacts with BST-2 [32,35] and is important
for Vpus ability to downregulate BST-2 , no comprehensive
mapping has been reported thus far.
The Transmembrane Proximal Region Affects the
Subcellular Localization of Vpu
Varthakavi et al. have suggested that the localization of Vpu to a
specific pericentriolar compartment of the TGN is necessary for its
ability to enhance virion release . The domain responsible for this
TGN localization was later mapped to the Vpu TM proximal region,
which contains two overlapping putative sorting signals
(tyrosinebased YXXW and di-leucine based (D/E)XXXL(L/I)) 
(Figure 1B). This region was first identified in Vpu C, where it was
shown to be involved in both the plasma membrane localization of
Vpu C and viral egress . Mutagenesis of this region in Vpu B was
also shown to reduce viral release .
Degradation of BST-2 in the Presence of Vpu
Flow cytometry analyses from many studies clearly indicate that
the levels of endogenous BST-2 at the cell surface of HeLa cells are
markedly diminished in the presence of Vpu [5,23,30,31,34]. This
decrease in cell surface expression could be caused by either
BST2 degradation or BST-2 sequestration within an intracellular
compartment (see Figure 2). Due to conflicting data that has likely
arisen from the different methodologies employed (see Table 2),
distinguishing between these mechanisms has not been
straightforward. However, immunoblot analyses from the majority of
studies have demonstrated a decrease in total cellular BST-2 levels
in the presence of Vpu, which would favor a degradation
mechanism [3,23,3033]. Table 3 provides a compilation of the
reagents and techniques used, as well as the results obtained from
each of these mechanistic studies.
Vpu-Mediated Degradation Pathways of BST-2
While it remains to be determined whether BST-2 is directly
ubiquitinated upon interaction with Vpu and bTrCP, support for
a ubiquitin-dependent mechanism was provided by experiments in
which the Vpu-mediated downregulation of BST-2 was
significantly inhibited by concanamycin A , bafilomycin A1 ,
and long-term MG132 treatment ($12 h) [31,33,34].
Concanamycin A and bafilomycin A1 are both vacuolar H(+)-ATPase
inhibitors that block endosomal maturation and thus lysosomal
degradation. In contrast, MG132 is a proteasome inhibitor that,
when used for extended periods, prevents cellular ubiquitin
recycling. Since the resulting ubiquitin depletion can affect
ubiquitin-mediated endocytosis and other ubiquitin-dependent
pathways, MG132-treatment does not necessarily implicate
proteasomal degradation. Depending on the drugs used, opposing
conclusions have been reached, in which Vpu-mediated
degradation of BST-2 occurs via either the lysosome [30,32,34] or the
proteasome [31,33] (see Table 3 and Figure 2). Another possible
cause for these conflicting results may be the BST-2 expression
systems utilized (see Table 2). In general, data supporting a
lysosomal degradation mechanism have come from studies of
endogenously expressed BST-2, while data supporting a
proteasome-dependent pathway have arisen from the use of exogenously
expressed, epitope-tagged BST-2, which often results in the
accumulation of immature BST-2 within the endoplasmic
reticulum (ER) .
Viral Egress Assays
HIV reporter assay
N Physiologically relevant
N 100% of cells express protein
N No need for expression vectors
N Correct modifications and localization
N Only measures infectious particles
N Reliable quantitation
Table 2. Advantages and Disadvantages of Various Expression and Assay Systems.
BST-2 Expression Systems Advantages
Long-term viral replication
N Most physiologically relevant
To further investigate a role for ubiquitination of BST-2, two
groups have mutated the cytoplasmic lysine residues of BST-2,
which are the most likely targets for ubiquitin addition (Figure 1B).
Both groups found that the double lysine mutant retained the
ability to restrict viral egress and was still downregulated by Vpu.
These data suggest that if BST-2 ubiquitination is required for its
viral restriction function or necessary for Vpu-mediated
downregulation, then residues other than the cytoplasmic lysines must be
the ubiquitin target [33,34]. A definitive mechanism for the
Vpumediated degradation of BST-2 awaits a more extensive analysis of
the role that ubiquitin plays in this process.
The Role of BST-2 Endocytosis in the Vpu-Mediated
Downregulation of BST-2
Mitchell et al. presented data that indicates a role for the
endosomal adapter protein complex member AP-2 (m2) in the
Vpudependent downregulation of BST-2 . However, Vpu did not
appear to enhance the rate of BST-2 internalization, leading the
authors to conclude that Vpu acts after BST-2 is naturally
endocytosed. In contrast, Iwabu et al. mutated a dual-tyrosine site
in BST-2 (Y6/8A) (Figure 1B) involved in clathrin-dependent
endocytosis and found that Vpu was still able to induce BST-2
downregulation, suggesting that natural BST-2 endocytosis is not
required for this process . The interpretation of any effects Vpu
might have on BST-2 endocytosis are complicated by the conflicting
reports regarding which AP-2 subunit, either m2  or a-adaptin
, is involved in the natural BST-2 endocytosis pathway.
Species-Specific Lentiviral Countermeasures
The retroviral restriction factor TRIM5a was identified in
studies designed to identify host factors responsible for HIV-1
N Well-characterized, efficient antibodies to tag
N Could adversely affect processing, localization and function
N More time consuming to generate stable cell lines
N Potential loss of BST-2 expression over time
N Over-expression anomalies
N (processing/secretion defects)
N Variable transfection efficiency (cell-type dependent)
N Must perform dose-response
N Need efficient antibodies to molecule
N Measures all particle release including non-infectious
N Quantitation of immunoblots
N Viral entry, reverse transcription, and integration must not be affected
restriction in Old World monkeys . A number of recent
publications (described below) suggest a similar species specificity
in the abilities of primate lentiviruses to overcome BST-2
restriction by their respective hosts.
Non-Human BST-2 Proteins Restrict HIV-1
Several studies have found that HIV-1 egress is inhibited by
BST-2 proteins from a wide selection of mammalian species. This
list includes Old World monkeys, such as rhesus macaques
[40,41], African green monkeys (AGMs) [40,42,43], and
Mustached monkeys , as well as both mice and rats [31,40]. Thus
far, the only primate BST-2 shown not to restrict HIV-1 was
found in a species of New World owl monkey (Aotus lemurinus
griseimembra) . However, when the sequence of this defective
BST-2 was compared to that of closely related owl monkeys
encoding functional BST-2 proteins, the defect mapped to residue
181 (I 181 T) within the predicted COOH-terminal GPI-anchor
signal peptide. This mutation altered normal BST-2 glycosylation,
which leads to the inactivation or mistargeting of the protein in
this owl monkey species. Taken together, these data suggest that as
long as BST-2 is able to mature properly, BST-2 restriction of
HIV-1 is remarkably species independent. This generalization was
extended further by Sato et al., who showed that when transfected
into a variety of mammalian and bird cell lines, human BST-2 can
still restrict HIV-1 release. This suggests that BST-2 function
requires no species-specific cofactors .
HIV-1 Vpu Does Not Counteract Non-Hominid BST-2
Another interesting aspect of the aforementioned studies was the
consistent observation that HIV-1 Vpu counteracts human and
chimpanzee (cpz) BST-2, but not BST-2 proteins encoded by
nonhominids [31,4045]. These findings explain the previous
observation that regardless of Vpu expression, COS-7 cells
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Further support for the species specificity of Vpu-mediated
antagonism of BST-2 came from studies demonstrating that Vpu
encoded by SIVmus (which infects Moustached monkeys
[Cercopithecus cephus]) could antagonize the C. cephus and the closely
related AGM BST-2s, but not human BST-2. This phenomenon
has recently been extended to include numerous other
Vpuexpressing simian immunodeficiency virus (SIV) isolates (SIVgsn/
mus/mon) [48,49]. Surprisingly, the Vpus encoded by SIVcpz and
SIVgor were inactive against all BST-2s tested. Instead, these SIV
strains appear to utilize Nef for this purpose (see below) [48,49].
Thus, while BST-2s ability to restrict viral egress appears to be
pleiotropic, there appears to be a clear adaptation of viral Vpu
proteins to their respective host species, with the notable exception
BST-2 Domains Required for Sensitivity to Vpu
The species specificity of BST-2 antagonism has provided the
unique opportunity to map residues within human BST-2 that
are required for Vpu-mediated downregulation. Swapping the
cytoplasmic, TM, and extracellular domains between human
and mouse BST-2 showed that important determinants are
present in each human domain that are required for Vpu
downregulation . Other studies showed that rhesus BST-2
was downregulated by Vpu when the TM was replaced with
that of human BST-2 . Conversely, replacing the TM of
human BST-2 with that of rhesus BST-2 rendered the chimeric
protein resistant to Vpu. In an alternative approach, a
comparison of primate BST-2 nucleotide sequences suggested
that the ratios of non-synonymous substitutions (nucleotide
changes that affect the protein sequence) to synonymous
substitutions were higher in the cytoplasmic and TM domains
compared to those in the extracellular domain [40,42]. Focusing
on these regions led to the identification of residues within the
TM of human BST-2 that influenced Vpu-mediated
downregulation (Figure 3) [40,42,43]. However, due to the wide
variation in both BST-2 expression and maturation presented
in these studies, no clear consensus has emerged.
Other Viruses Restricted by BST-2 and Their
BST-2 has been shown to inhibit the release of viral or viral-like
particles from a variety of enveloped viruses (Table 1 and
references therein). Many of these viruses share little or no
homology with one another, thus highlighting BST-2s intrinsic
anti-viral function. Because viruses co-evolve with their hosts, it
was perhaps not surprising to find that HIV-1 encodes a BST-2
countermeasure in the form of Vpu. Therefore, by extension, one
might suspect that other viruses have also developed mechanisms
to deal with BST-2. The following section explores the manner in
which viruses other than HIV-1 antagonize BST-2.
In contrast to HIV-1, HIV-2 does not encode a Vpu protein.
Regardless, some strains of HIV-2 have been shown to exhibit an
enhanced release phenotype in Vpu-restrictive cells [50,51].
Interestingly, this HIV-2 release function maps to the envelope
(Env) protein. Attempts to map those residues that contribute to
this phenotype have revealed both a single amino acid (T598)
within the gp41 ectodomain  and a GYXXh endocytic-sorting
motif within the cytoplasmic tail . Although these studies were
performed prior to the identification of BST-2, recent data
confirms that egress-competent HIV-2 strains can downregulate
cell surface BST-2, and that both residue T598 [30,41] and the
GYXXh motif  may be involved in this process. Mutations
that prevent envelope processing are also defective for both egress
 and BST-2 antagonism . Mechanistically, it has been
shown that, like Vpu, HIV-2 Env co-immunprecipitates with
BST2 . However, unlike Vpu, no evidence for HIV-2
Envdependent BST-2 degradation has been shown. In one study,
BST-2 was found to accumulate in the TGN in the presence of
HIV-2 Env, suggesting that BST-2 sequestration may be the
mechanism whereby HIV-2 Env enhances viral egress  (see
Figure 2). Of note, HIV-2 Env was also able to antagonize rhesus
BST-2 , suggesting that HIV-2 Env functions in a
Simian Immunodeficiency Viruses
Like HIV-2, most SIV strains do not encode a Vpu homolog.
However, in contrast to HIV-2, two recent studies have shown
that deleting the SIV env gene does not significantly inhibit
SIVmac release from cells expressing rhesus BST-2 [41,55].
Instead, these studies revealed that SIV Nef counteracts BST-2.
This inhibition appears to be species specific; while Nef proteins
from various SIV strains effectively antagonize BST-2 from their
respective hosts, they are inactive against human BST-2 . New
evidence suggests that this is also the case for SIVgor and SIVcpz
even though they express Vpu . Interestingly, both HIV-1 and
HIV-2 Nef appear to have lost much of this functionality, as they
do not antagonize human BST-2 [41,55]. However, they have
maintained some detectable activity against the rhesus BST-2 .
Using chimeras between human and rhesus BST-2, the region
necessary for sensitivity to antagonism by SIVmac Nef was
mapped to five amino acids (GDIWK) within the cytoplasmic
domain of rhesus BST-2, which are missing in human BST-2
[41,55] (Figure 3). Mutational analyses have shown that both the
Nef myristoylation site [41,55] and cholesterol recognition motif
 are important for Nefs ability to counteract rhesus BST-2,
thus highlighting the importance of Nef membrane localization
(Figure 1C). Nef mutations that abolish downregulation of CD4
and CD28, but not MHC-I, also prevented BST-2 antagonism,
suggesting potential mechanistic similarities between Nef-mediated
downregulation of both CD4 and BST-2  (see Figure 2).
However, aside from the observation that SIV Nef induced cell
surface downregulation of rhesus BST-2 , no other
mechanistic studies have been performed to date. Interestingly, the use of
Nef to counteract BST-2 may not be universal among SIV strains.
One group has found that, like HIV-2, the SIVagmTan Env
downregulates cell surface BST-2 in a species-independent
manner . However, this study relied exclusively on
exogenously expressed SIVagm Env; env deletion viruses were not tested,
and control experiments to determine Nefs role were not
performed. Further complicating these conclusions, Lim et al.
observed only a modest antagonism of AGM BST-2 by wild-type
SIVagmTan. They hypothesize that this particular SIV strain may
not require a BST-2 antagonist because it does not induce a robust
IFN response in vivo . More systematic, comparative studies
will be necessary to a) confirm which strains of SIV have evolved
BST-2 countermeasures and b) clarify the contributions that Vpu,
Nef, and/or Env make towards SIV egress.
The inhibition of Ebola VLP release provided the first
demonstration that BST-2 limits the egress of a non-retrovirus
. Kaletsky et al. screened four Ebola proteins that are known
to impact viral egress for their ability to overcome BST-2 .
Only the glycoprotein (GP) restored VLP release in cells
expressing BST-2. In contrast to Vpu, Ebola GP was found to
counteract both murine and human BST-2, suggesting a lack of
species specificity. While a direct interaction between GP and
BST-2 was inferred from their co-localization and
co-immunoprecipitation, no degradation or obvious mislocalization of BST-2
was observed, leaving the mechanism of antagonism by GP
Kaposis SarcomaAssociated Herpesvirus
Although KSHV is the only DNA virus currently known to
counteract BST-2, our studies of KSHV-encoded
immunomodulators established the first viral connection for BST-2 . In a
proteomics screen for new host targets of the viral TM ubiquitin
ligase K5, we observed that BST-2 levels were reduced in the
presence of K5 . More recently, we demonstrated that, similar
to other K5 targets, BST-2 is ubiquitinated by K5, resulting in
ubiquitin-mediated endocytosis and lysosomal destruction .
Ubiquitination occurred at lysines in the cytoplasmic domain of
BST-2 (Figure 1A) and removal of the two lysines rendered BST-2
resistant to K5. In contrast, lysine-less BST-2 is still degraded by
Vpu , indicating that either alternative residues act as
ubiquitin substrates or BST-2 is not a direct target of ubiquitin
ligases in HIV-1-infected cells. Further analyses revealed that upon
knockdown of K5, BST-2 reduced the release of KSHV from
HeLa cells . While this result indicates that BST-2 interferes
with KSHV egress, further studies will be needed to determine
how this interference is achieved since, unlike retroviral budding,
herpesviral egress occurs by vesicular transport. Nevertheless,
these studies indicate that the anti-viral function of BST-2 acts
across an exceptionally wide spectrum of viruses.
Aside from the mechanistic questions regarding both the
manner by which BST-2 inhibits viral egress and the means by
which various viruses neutralize this activity, still larger questions
remain. For example, how important is it for HIV to improve viral
release if the virus can easily spread via cell-to-cell fusion? In
longterm viral replication cultures, DVpu viruses show increased
syncytia formation and cell-to-cell spread , suggesting that
under these conditions, overall viral replication is not decreased,
even though particle release is significantly inhibited. Also, since
the majority of studies investigating the BST-2 viral restriction and
Vpu countermeasures have been performed in cell lines that are
not physiological targets of HIV, will the same conclusions be
reached when primary CD4+ T cells are evaluated? Regardless,
the very existence and current prevalence of Vpu among HIV-1
subtypes points to an evolutionary pressure to maintain this
molecule. This raises the possibilities that a) viral release plays a
much larger role in vivo, b) that the selection for the maintenance
of Vpu is a result of one of its other functions (i.e., CD4
downregulation), and c) that BST-2 has other important anti-viral
function(s) in addition to tethering virions. This latter hypothesis is
intriguing in light of the study showing that BST-2 activates the
ILT7 receptor on pDCs, leading to inhibition of IFN and
proinflammatory cytokine production . This result is
somewhat counterintuitive, as it suggests that HIV is promoting
immune activation. At the same time, if the goal of this activity is
the continued recruitment of T cells to sites of infection, then the
result of BST-2 downregulation might be expanded to include
both enhanced viral egress and dissemination. Further evidence
for an alternative BST-2 function(s) comes from the finding that an
entirely synthetic, functional tetherin can be assembled from
entirely non-BST-2 sequences . If structure trumps sequence
regarding tethering, compensatory mutations within BST-2 would
easily arise in response to viral countermeasures, such that there
would be little cross-species consensus among BST-2 sequences.
That this is not true suggests that BST-2 does indeed perform
other functions that require sequence fidelity, although these may
or may not serve an anti-viral purpose. While a great deal has been
accomplished in this emerging field, many loose ends remain, such
that it is too early to become tethered to any particular model
for either BST-2 function or antagonism.
1. Goto T , Kennel SJ , Abe M , Takishita M , Kosaka M , et al. ( 1994 ) A novel membrane antigen selectively expressed on terminally differentiated human B cells . Blood 84 : 1922 - 1930 .
2. Ishikawa J , Kaisho T , Tomizawa H , Lee BO , Kobune Y , et al. ( 1995 ) Molecular cloning and chromosomal mapping of a bone marrow stromal cell surface gene, BST2, that may be involved in pre-B-cell growth . Genomics 26 : 527 - 534 .
3. Bartee E , McCormack A , Fruh K ( 2006 ) Quantitative membrane proteomics reveals new cellular targets of viral immune modulators . PLoS Pathog 2 : e107. doi:10.1371/journal.ppat.0020107.
4. Neil SJ , Zang T , Bieniasz PD ( 2008 ) Tetherin inhibits retrovirus release and is antagonized by HIV-1 Vpu . Nature 451 : 425 - 430 .
5. Van Damme N , Goff D , Katsura C , Jorgenson RL , Mitchell R , et al. ( 2008 ) The interferon-induced protein BST-2 restricts HIV-1 release and is downregulated from the cell surface by the viral Vpu protein . Cell Host Microbe 3 : 245 - 252 .
6. Kupzig S , Korolchuk V , Rollason R , Sugden A , Wilde A , et al. ( 2003 ) Bst-2/ HM1.24 is a raft-associated apical membrane protein with an unusual topology . Traffic 4 : 694 - 709 .
7. Ohtomo T , Sugamata Y , Ozaki Y , Ono K , Yoshimura Y , et al. ( 1999 ) Molecular cloning and characterization of a surface antigen preferentially overexpressed on multiple myeloma cells . Biochem Biophys Res Commun 258 : 583 - 591 .
8. Blasius AL , Giurisato E , Cella M , Schreiber RD , Shaw AS , et al. ( 2006 ) Bone marrow stromal cell antigen 2 is a specific marker of type I IFN-producing cells in the naive mouse, but a promiscuous cell surface antigen following IFN stimulation . J Immunol 177 : 3260 - 3265 .
9. Masuyama N , Kuronita T , Tanaka R , Muto T , Hirota Y , et al. ( 2009 ) HM1.24 is Internalized from Lipid Rafts by Clathrin-Mediated Endocytosis through Interaction with -adaptin . Journal of Biological Chemistry . pp 1 - 27 .
10. Hegde RS , Mastrianni JA , Scott MR , DeFea KA , Tremblay P , et al. ( 1998 ) A transmembrane form of the prion protein in neurodegenerative disease . Science 279 : 827 - 834 .
11. Rollason R , Korolchuk V , Hamilton C , Schu P , Banting G ( 2007 ) Clathrinmediated endocytosis of a lipid-raft-associated protein is mediated through a dual tyrosine motif . J Cell Sci 120 : 3850 - 3858 .
12. Rollason R , Korolchuk V , Hamilton C , Jepson M , Banting G ( 2009 ) A CD317/ tetherin-RICH2 complex plays a critical role in the organization of the subapical actin cytoskeleton in polarized epithelial cells . J Cell Biol 184 : 721 - 736 .
13. Cao W , Bover L , Cho M , Wen X , Hanabuchi S , et al. ( 2009 ) Regulation of TLR7/9 responses in plasmacytoid dendritic cells by BST2 and ILT7 receptor interaction . Journal of Experimental Medicine 206 : 1603 - 1614 .
14. Willey RL , Maldarelli F , Martin MA , Strebel K ( 1992 ) Human immunodeficiency virus type 1 Vpu protein regulates the formation of intracellular gp160- CD4 complexes . J Virol 66 : 226 - 234 .
15. Margottin F , Bour SP , Durand H , Selig L , Benichou S , et al. ( 1998 ) A novel human WD protein, h-beta TrCp, that interacts with HIV-1 Vpu connects CD4 to the ER degradation pathway through an F-box motif . Mol Cell 1 : 565 - 574 .
16. Klimkait T , Strebel K , Hoggan MD , Martin MA , Orenstein JM ( 1990 ) The human immunodeficiency virus type 1-specific protein vpu is required for efficient virus maturation and release . J Virol 64 : 621 - 629 .
17. Neil SJ , Eastman S , Jouvenet N , Bieniasz P ( 2006 ) HIV-1 Vpu promotes release and prevents endocytosis of nascent retrovirus particles from the plasma membrane . PLoS Pathog 2 : e39. doi:10.1371/journal.ppat.0020039.
18. Miyakawa K , Ryo A , Murakami T , Ohba K , Yamaoka S , et al. ( 2009 ) BCA2/ Rabring7 promotes tetherin-dependent HIV-1 restriction. PLoS Pathog 5: e1000700 . doi:10.1371/journal.ppat.1000700.
19. Perez-Caballero D , Zang T , Ebrahimi A , Mcnatt MW , Gregory DA , et al. ( 2009 ) Tetherin Inhibits HIV-1 Release by Directly Tethering Virions to Cells . Cell 139 : 499 - 511 .
20. Hammonds J , Wang JJ , Yi H , Spearman P ( 2010 ) Immunoelectron microscopic evidence for Tetherin/BST2 as the physical bridge between HIV-1 virions and the plasma membrane . PLoS Pathog 6 : e1000749. doi:10.1371/journal.ppat.1000749.
21. Fitzpatrick K , Skasko M , Deerinck TJ , Crum J , Ellisman MH , et al. ( 2010 ) Direct restriction of virus release and incorporation of the interferon-induced protein BST-2 into HIV-1 particles. PLoS Pathog 6: e1000701 . doi:10.1371/ journal.ppat.1000701.
22. Habermann A , Krijnse-Locker J , Oberwinkler H , Eckhardt M , Homann S , et al. ( 2010 ) CD317/tetherin is enriched in the HIV-1 envelope and downregulated from the plasma membrane upon virus infection . J Virol 84 : 4646 - 4658 .
23. Miyagi E , Andrew AJ , Kao S , Strebel K ( 2009 ) Vpu enhances HIV-1 virus release in the absence of Bst-2 cell surface down-modulation and intracellular depletion . Proc Natl Acad Sci U S A 106 : 2868 - 2873 .
24. Kaletsky RL , Francica JR , Agrawal-Gamse C , Bates P ( 2009 ) Tetherin-mediated restriction of filovirus budding is antagonized by the Ebola glycoprotein . Proc Natl Acad Sci U S A 106 : 2886 - 2891 .
25. Andrew AJ , Miyagi E , Kao S , Strebel K ( 2009 ) The formation of cysteine-linked dimers of BST-2/tetherin is important for inhibition of HIV-1 virus release but not for sensitivity to Vpu . Retrovirology 6: 80 .
26. Friborg J , Ladha A , Gottlinger H , Haseltine WA , Cohen EA ( 1995 ) Functional analysis of the phosphorylation sites on the human immunodeficiency virus type 1 Vpu protein . J Acquir Immune Defic Syndr Hum Retrovirol 8 : 10 - 22 .
27. Schubert U , Clouse KA , Strebel K ( 1995 ) Augmentation of virus secretion by the human immunodeficiency virus type 1 Vpu protein is cell type independent and occurs in cultured human primary macrophages and lymphocytes . J Virol 69 : 7699 - 7711 .
28. Schubert U , Strebel K ( 1994 ) Differential activities of the human immunodeficiency virus type 1-encoded Vpu protein are regulated by phosphorylation and occur in different cellular compartments . J Virol 68 : 2260 - 2271 .
29. Schubert U , Bour S , Ferrer-Montiel AV , Montal M , Maldarell F , et al. ( 1996 ) The two biological activities of human immunodeficiency virus type 1 Vpu protein involve two separable structural domains . J Virol 70 : 809 - 819 .
30. Douglas JL , Viswanathan K , Mccarroll MN , Gustin JK , Fruh K , et al. ( 2009 ) Vpu Directs the Degradation of the Human Immunodeficiency Virus Restriction Factor BST-2/Tetherin via a TrCP-Dependent Mechanism . J Virol 83 : 7931 - 7947 .
31. Goffinet C , Allespach I , Homann S , Tervo H , Habermann A , et al. ( 2009 ) HIV1 Antagonism of CD317 Is Species Specific and Involves Vpu-Mediated Proteasomal Degradation of the Restriction Factor . Cell Host and Microbe 5 : 285 - 297 .
32. Iwabu Y , Fujita H , Kinomoto M , Kaneko K , Ishizaka Y , et al. ( 2009 ) HIV-1 accessory protein Vpu internalizes cell-surface BST-2/tetherin through transmembrane interactions leading to lysosomes . Journal of Biological Chemistry . pp 1 - 22 .
33. Mangeat B , Gers-Huber G , Lehmann M , Zufferey M , Luban J , et al. ( 2009 ) HIV-1 Vpu Neutralizes the Antiviral Factor Tetherin/BST-2 by Binding It and Directing Its Beta-TrCP2-Dependent Degradation . PLoS Pathog 5 : e1000574. doi:10.1371/journal.ppat.1000574.
34. Mitchell R , Katsura C , Skasko M , Fitzpatrick K , Lau D , et al. ( 2009 ) Vpu Antagonizes BST-2-Mediated Restriction of HIV-1 Release via b-TrCP and Endo-Lysosomal Trafficking . PLoS Pathog 5 : e1000450. doi:10.1371/ journal.ppat.1000450.
35. Banning C , Votteler J , Hoffmann D , Koppensteiner H , Warmer M , et al. ( 2010 ) A flow cytometry-based FRET assay to identify and analyse protein-protein interactions in living cells . PLoS ONE 5: e9344. doi:10.1371/journal.- pone.0009344.
36. Varthakavi V , Smith R , Martin K , Derdowski A , Lapierre L , et al. ( 2006 ) The pericentriolar recycling endosome plays a key role in Vpu-mediated enhancement of HIV-1 particle release . Traffic 7 : 298 - 307 .
37. Dube M, Roy BB , Guiot-Guillain P , Mercier J , Binette J , et al. ( 2009 ) Suppression of Tetherin-restricting activity upon human immunodeficiency virus type 1 particle release correlates with localization of Vpu in the trans-Golgi network . J Virol 83 : 4574 - 4590 .
38. Ruiz A , Hill MS , Schmitt K , Guatelli J , Stephens EB ( 2008 ) Requirements of the membrane proximal tyrosine and dileucine-based sorting signals for efficient transport of the subtype C Vpu protein to the plasma membrane and in virus release . Virology 378 : 58 - 68 .
39. Stremlau M , Owens CM , Perron MJ , Kiessling M , Autissier P , et al. ( 2004 ) The cytoplasmic body component TRIM5alpha restricts HIV-1 infection in Old World monkeys . Nature 427 : 848 - 853 .
40. McNatt MW , Zang T , Hatziioannou T , Bartlett M , Fofana IB , et al. ( 2009 ) Species-specific activity of HIV-1 Vpu and positive selection of tetherin transmembrane domain variants . PLoS Pathog 5 : e1000300. doi:10.1371/ journal.ppat.1000300.
41. Jia B , Serra-Moreno R , Neidermyer W , Rahmberg A , Mackey J , et al. ( 2009 ) Species-Specific Activity of SIV Nef and HIV-1 Vpu in Overcoming Restriction by Tetherin/BST2. PLoS Pathog 5: e1000429 . doi:10.1371/journal.ppat.1000429.
42. Gupta R , Hue S , Schaller T , Verschoor E , Pillay D , et al. ( 2009 ) Mutation of a Single Residue Renders Human Tetherin Resistant to HIV-1 Vpu-Mediated Depletion . PLoS Pathog 5 : e1000443. doi:10.1371/journal.ppat.1000443.
43. Rong L , Zhang J , Lu J , Pan Q , Lorgeoux R , et al. ( 2009 ) The Transmembrane Domain of BST-2 Determines Its Sensitivity to Down-Modulation by HIV-1 Vpu . J Virol. pp 1 - 35 .
44. Lim E , Emerman M ( 2009 ) Simian immunodeficiency virus from African green monkeys (SIVAGM) does not antagonize endogenous levels of African green monkey tetherin/ BST-2. J Virol . pp 1 - 33 .
45. Wong SK , Connole M , Sullivan JS , Choe H , Carville A , et al. ( 2009 ) A New World primate deficient in tetherin-mediated restriction of human immunodeficiency virus type 1 . Journal of Virology 83 : 8771 - 8780 .
46. Sato K , Yamamoto S , Misawa N , Yoshida T , Miyazawa T , et al. ( 2009 ) Comparative study on the effect of human BST-2/Tetherin on HIV-1 release in cells of various species . Retrovirology 6 : 53 .
47. Neil SJ , Sandrin V , Sundquist WI , Bieniasz P ( 2007 ) An interferon-alphainduced tethering mechanism inhibits HIV-1 and Ebola virus particle release but is counteracted by the HIV-1 Vpu protein . Cell Host & Microbe 2 : 193 - 203 .
48. Sauter D , Schindler M , Specht A , Landford WN , Munch J , et al. ( 2009 ) Tetherin-Driven Adaptation of Vpu and Nef Function and the Evolution of Pandemic and Nonpandemic HIV-1 Strains . Cell Host and Microbe 6 : 409 - 421 .
49. Yang SJ , Lopez LA , Hauser H , Exline CM , Haworth KG , et al. ( 2010 ) Antitetherin activities in Vpu-expressing primate lentiviruses . Retrovirology 7 : 13 .
50. Bour S , Strebel K ( 1996 ) The human immunodeficiency virus (HIV) type 2 envelope protein is a functional complement to HIV type 1 Vpu that enhances particle release of heterologous retroviruses . J Virol 70 : 8285 - 8300 .
51. Ritter GD , Yamshchikov G , Cohen SJ , Mulligan MJ ( 1996 ) Human immunodeficiency virus type 2 glycoprotein enhancement of particle budding: role of the cytoplasmic domain . J Virol 70 : 2669 - 2673 .
52. Bour S , Akari H , Miyagi E , Strebel K ( 2003 ) Naturally occurring amino acid substitutions in the HIV-2 ROD envelope glycoprotein regulate its ability to augment viral particle release . Virology 309 : 85 - 98 .
53. Abada P , Noble B , Cannon PM ( 2005 ) Functional domains within the human immunodeficiency virus type 2 envelope protein required to enhance virus production . J Virol 79 : 3627 - 3638 .
54. Le Tortorec A , Neil SJD ( 2009 ) Antagonism and intracellular sequestration of human tetherin by the HIV-2 envelope glycoprotein . J Virol : 1 - 49 .
55. Zhang F , Wilson SJ , Landford WC , Virgen B , Gregory D , et al. ( 2009 ) Nef Proteins from Simian Immunodeficiency Viruses Are Tetherin Antagonists . Cell Host and Microbe : 1 - 14 .
56. Gupta R , Mlcochova P , Pelchen-Matthews A , Petit S , Mattiuzzo G , et al. ( 2009 ) Simian immunodeficiency virus envelope glycoprotein counteracts tetherin/ BST-2/CD317 by intracellular sequestration . Proc Natl Acad Sci USA.
57. Mansouri M , Viswanathan K , Douglas J , Hines J , Gustin J , et al. ( 2009 ) Molecular mechanism of BST2/Tetherin downregulation by K5/MIR2 of Kaposi's sarcoma herpesvirus . J Virol.
58. Gruber M , Soding J , Lupas AN ( 2005 ) REPPER-repeats and their periodicities in fibrous proteins . Nucleic Acids Res 33 : W239 - 243 .
59. Jouvenet N , Neil SJ , Zhadina M , Zang T , Kratovac Z , et al. ( 2009 ) Broadspectrum inhibition of retroviral and filoviral particle release by tetherin . J Virol 83 : 1837 - 1844 .