Survival of infection with Ebola virus (EBOV) depends on the ability of the host to
mount early and strong immune responses [1], [2]. However, given that EBOV cases are
associated with 40%–90% human mortality, EBOV has developed intricate solutions to
human immunological defenses. Enveloped viruses, like EBOV, must deposit their genetic
material within a cell to ensure their propagation. The roles of viral envelope glycoproteins
in mediating virus attachment to host cells and catalyzing the subsequent fusion of
the viral and host plasma membranes have been well described (reviewed in [3]). Given
the limited number of genes in EBOV and other viruses, it stands to reason that these
conformationally labile glycoproteins are also involved in more than just the initial
steps of a productive infection. There is strong evidence that viral entry glycoproteins
(GP) are modulators of host antiviral defenses (Table 1). In this article, we discuss
our current structural understanding of the functions of envelope entry glycoproteins
in immune evasion using EBOV as our example.
10.1371/journal.ppat.1003258.t001
Table 1
Viral entry glycoprotein-mediated immune evasion strategies in other viral families.
Viral Family
Immune Evasion Mechanism
Examples/Comments
Ref
Arenaviridae
Glycoprotein shedding/secretion
Lassa virus: shed GP1-mediated immune evasion has been attributed to differential
glycosylation of the shed and transmembrane glycoprotein complex.
[37], [38]
Coronaviridae
Direct humoral antagonism
SARS CoV: spike protein acts as a ligand for phenotypic conversion of B cells into
macrophage-like cells.
[39]
Filoviridae
see article
Flaviviridae
Glycan shielding
The hepatitis C virus E1/E2 glycoprotein escapes neutralizing antibodies in a glycoprotein-dependent
manner.
[40], [41]
NK and innate immune antagonism
The hepatitis C virus E2 glycoprotein binds CD81 and blocks natural killer cell activation.
[42]
Herpesviridae
Glycan shielding
Bovine herpes virus gp180 O-linked glycans shield against humoral assault and are
conserved across all gammaherpesvirus gp350 homologs.
[43]
Antigen presentation antagonism
Epstein Barr virus sgp42 binds MHC class II, thereby interfering with antigen presentation
to CD4+ T-cells.
[44]
Orthomyxoviridae
Glycan shielding
Glycans present on the Influenza A virus HA glycoprotein protect temporally diverse
pandemic strains in a conserved manner.
[45], [46]
Paramyxoviridae
Glycoprotein shedding/secretion
Respiratory syncytial virus G glycoprotein acts both as a decoy for host antibodies
and can modulate immunity via immune receptor interactions.
[47]
Glycan shielding
Nipah virus F protein contains N-linked glycans that offer a protective role against
the host antibody response.
[48]
Retroviridae
Glycoprotein shedding/secretion
HIV-1 gp120 shedding competes with the gp160 complex for host antibodies.
[49]
Immunosuppressive structural motif
Peptides derived from HIV-1 gp41 inhibit T-cell activation.
[19]
Immunosuppressive structural motif
HTLV-1 gp21 immunomodulatory region inhibits IgG response by ∼40 fold when compared
to mutant recombinant protein.
[50]
Glycan shielding
HIV-1 gp120 glycan shield protects otherwise neutralizing epitopes from humoral antagonism
and directs antibodies towards variable loops.
[7], [51]
Direct innate immunity antagonism
HIV-2 env-encoded glycoprotein counteracts BST-2-mediated viral tethering.
[32]
Antigen presentation antagonism
HIV-1 gp41 can interrupt TCR-CD3 interactions to modulate T-cell proliferation.
[52]
Antigen presentation antagonism
HIV-1 gp41 interacts with HLA-associated invariant chain and may have a role in MHC-directed
antagonism.
[28], [53]
How Does Glycosylation of Ebola Virus Envelope Proteins Facilitate Immune Evasion?
In EBOV, four variants of the envelope glycoprotein are synthesized as a result of
transcriptional stuttering or post-translational processing (Figure 1A). About 25%
of transcripts from the GP gene produce the virion-attached or envelope spike GP that
is important for entry. The surface of the envelope GP is covered with N- and O-linked
glycans. Depending on the EBOV species, the envelope GP contains 11–18 N-linked glycan
sites. Furthermore, EBOV GP contains an unstructured ∼150-residue mucin-like domain
that is heavily modified with O-linked glycans (∼80 sites) [4]. The N-linked glycans
are a heterogeneous mixture of ∼60 different species of high-mannose, hybrid, and
complex oligosaccharides, while the O-linked glycans consist of primarily smaller
trisaccharide structures (core 2) that contain varying amounts of sialic acids [5].
10.1371/journal.ppat.1003258.g001
Figure 1
Ebola virus glycoproteins.
(A) Processing of EBOV glycoproteins. The EBOV genome contains seven genes (3′-NP-VP35-VP40-GP-VP30-VP24-L-5′),
but nine proteins are produced due to editing events in the GP gene. The GP gene primary
transcript encodes for a ∼110 kDa, dimeric secreted GP (pre-sGP). Furin cleavage of
pre-sGP produces mature sGP and a secreted Δ-peptide. Transcriptional stuttering results
in the production of the envelope-attached GP and a small, secreted GP (ssGP). The
GP is the only virally encoded protein on the EBOV surface and is cleaved by furin
to form a disulfide-linked GP1-GP2 heterodimer, which then assembles as trimers on
the virus surface. GP1 contains the receptor-binding site for host cell attachment,
while GP2 contains a helical heptad-repeat (HR) region, transmembrane anchor (TM),
and a 4-residue cytoplasmic tail. A cleavage at the membrane-proximal external region
by the tumor necrosis factor-α converting enzyme (TACE) releases the shed GP. The
first 295 residues of ssGP, sGP, and GP are common, but each protein has a different
C-terminus, leading to different functions. (B) Epitope masking by EBOV glycoproteins.
Molecular surface of EBOV GP subunits (PDB code: 3CSY) are shown in green (GP1) and
yellow (GP2). Complex-type N-linked glycans are modeled onto the EBOV GP surface as
red/white spheres to reveal a heavy glycan layer that buries much of the GP surface,
including the receptor-binding site; only a small patch at the base of the GP is accessible
(KZ52/16F6 antibody-binding site). The O-linked glycosylated mucin-like domain (blue)
is modeled onto EBOV GP, and thought to form an extended structure that provides another
glycan layer of protection to the virus. EBOV GP is estimated to be ∼150 Å in height.
Given the size and shape of EBOV GP, smaller cellular surface proteins, such as MHC
class I and β-integrins (∼70 Å in height), may be sterically blocked.
Epitope masking is a major mechanism of viral immune evasion. Modeling of the EBOV
GP core structure reveals a surface covered in oligosaccharides (Figure 1B). The dense
clustering of glycans creates an unfavorable environment for the interaction of otherwise
neutralizing antibodies. Moreover, critical regions on EBOV GP, such as the receptor-binding
site, are hidden under layers of glycan. No antibodies have been identified to target
the receptor-binding site, however a number of neutralizing antibodies have been generated
against the more variable mucin-like domain [6]. The mucin-like domain is not necessary
for EBOV entry [4]. Essentially, the EBOV GP glycans direct the immune system to produce
antibodies against highly variable or dispensable regions on the viral surface. This
also occurs in hosts infected with HIV-1; nonbroadly neutralizing antibodies are generated
against the variable V1/V2/V3 loops [7]. In mice, removal of the mucin-like domain
of the EBOV GP leads to the production of cross-species antibodies directed at the
conserved glycoprotein core structure [8]. A small area near the base of the EBOV
GP core is available to immune surveillance (Figure 1B). This nonglycosylated patch
on GP is conserved in both Zaire and Sudan EBOV species, and the neutralizing antibodies
KZ52 and 16F6-1 bind to this hotspot [9]. However, given its close proximity to the
viral membrane and the density of GP spikes on the surface, it is not clear how accessible
this epitope is.
EBOV GP also has the unique ability to mask the function of host cellular proteins
important in response to viral pathogens. Transient expression of EBOV GP results
in low detectable levels of various cell surface proteins such as major histocompatibility
complex (MHC) class I proteins and several members of the β-integrin family [10],
[11]. Initially, it was thought that EBOV GP downregulated expression or degraded
these proteins from the cell surface. However, MHC class I and β-integrins are not
removed from the cell surface. Rather, the mucin-like domain of EBOV GP provides a
“glycan umbrella” that shields surface epitopes and inhibits surface protein recognition
[11]–[13] (Figure 1B). This represents a novel mechanism of disrupting immune function
that does not involve downregulation or degradation of surface proteins.
What Roles Do Shed Viral Glycoproteins Play in Immune Evasion?
The shedding or secretion of soluble viral glycoproteins exemplifies another viral
strategy of humoral misdirection. Many enveloped viruses, including EBOV, Lassa, respiratory
syncytial, herpes simplex, and HIV-1, generate free glycoproteins that act as either
“antibody sinks” or decoys of host immunity (Table 1). EBOV-infected cells secrete
two glycoproteins (secreted GP and shed GP) into an infected person's sera [14], [15].
Most of the transcripts (70%) for the GP gene encode the 110-kDa, dimeric, secreted
GP (sGP) (Figure 1A). A cleavage at the membrane-proximal external region by the tumor
necrosis factor-α converting enzyme (TACE) releases the trimeric glycoprotein, termed
shed GP. In 5% of the transcripts, insertion of two adenosines produces a small 298-residue
secreted GP (ssGP), of unknown function. The first 295 amino acids of sGP are common
with the envelope GP, but due to transcriptional stuttering, the sGP C-terminus forms
different disulfide linkages leading to a homodimeric rather than a trimeric assembly
(Figure 1A). As a result, sGP lacks regions found in GP that have been shown to be
important in the neutralization of the virus [9]. sGP and shed GP likely compete with
virion-attached GP for antibody binding [16]. Most of the antibodies derived from
EBOV survivors or macaques are directed towards sGP rather than the virion-attached
GP [17], [18]. Antibodies that bind to sGP or shed GP are likely nonneutralizing,
and those neutralizing antibodies that cross-react between sGP and GP may be absorbed
by the much more abundant sGP. In a guinea pig model of EBOV infection, shed GP inhibits
the neutralizing activity of EBOV antibodies [15].
How Do Viral Glycoproteins Actively Suppress Host Immunity?
In a seminal paper, Cianciolo et al. described immunomodulation by a synthetic peptide
derived from the fusion subunit of the HIV-1 envelope glycoprotein [19]. The immunomodulatory
region (IR) is comprised of a disulfide-linked loop situated between the heptad-repeat
regions of the fusion subunit, and similar structures have been identified in numerous
retroviruses and filoviruses (Figure 2A). Point mutations introduced into the IR of
HIV-1 gp41 abrogate the modulation of host cytokine expression in vitro and increase
antibody responses in rats immunized with mutant protein [20]. Synthetic peptides
derived from the IR regions of GP2 of Ebola and Marburg viruses inhibit the expression
of IFN-γ, IL-2, and IL-10, lower CD4+ and CD8+ cell activation, and increase immune
cell apoptosis [21]. The fusion domain from Moloney murine leukemia virus expressed
on various tumor cell lines facilitates xenograph immune evasion and natural killer
cell antagonism [22]. Interestingly, the human endogenous retrovirus-derived syncytin-2
retains the immunomodulatory activity associated with the viral envelope glycoproteins,
but the closely related syncytin-1 differs in the IR region, ablating this function
[23]. These retrovirus-derived syncytin proteins are implicated in both cell–cell
fusion during placental development and in maternal–fetal tolerance, clearly pointing
to a role in immune evasion [24]. Available structures of the post-fusion glycoprotein
subunit show that the disulfide-bonded immunomodulatory motif exists as a conformationally
conserved region at the apex of the fusion subunit, with residues identified by mutagenesis
as important for immunosuppression displayed outwards (Figure 2B). Interestingly,
in the SIV fusion subunit the same region is not found at the apex but rather on the
central helical heptad-repeat region. One possible target of the HIV-1 gp41 IR is
CD74, a type II single-pass transmembrane protein that, among other functions, chaperones
MHC class II dimers from the endoplasmic reticulum to the MHC class II compartment
(MIIC) for antigen loading [25], [26], and is also implicated in MHC class I cross-presentation
[27]. Recently, it was determined that the ectodomain of human CD74 binds to residues
corresponding to a region adjacent to the conserved HIV-1 gp41 IR. When peripheral
blood mononuclear cells (PBMCs) were incubated with recombinant post-fusion HIV-1
gp41, an increase in phosphorylated ERK occurred. Furthermore, this activation was
inhibited in a dose-dependent manner by treatment with soluble recombinant CD74 ectodomain
[28]. The activation of the ERK/MAPK pathway via high levels of the CC-chemokine RANTES
(or other exogenous signals) is responsible for increased infectivity of HIV-1 [29].
In support of these findings, siRNA knockdown of CD74 effectively curbs HIV-1 infectivity
[30]. Although these works are stimulating, more extensive research is required to
generate a complete description of viral fusion glycoprotein-associated immunosuppression.
Like HIV-1, the host targets of the immunomodulatory motif found in other species
of virus remain poorly defined and await further studies.
10.1371/journal.ppat.1003258.g002
Figure 2
Structural conservation of the viral glycoprotein immunomodulatory region.
The immunomodulatory region is approximately 20 amino acids long and is found within
numerous retroviruses and filoviruses. In each case presented here, the experimentally
defined immunomodulatory region is rendered in yellow and residues that are necessary
for the observed immunomodulatory activity are depicted as spheres. (A) Head-on view
of viral glycoproteins exhibiting a conserved three-fold pinwheel structure. (B) Side-view
illustrating the differences in possible interaction faces between the lentiviral
gp41 immunomodulatory region and a representative retrovirus, HTLV-1. The outward
positioning of the important immunomodulatory residues shown for HTLV-1 gp21 can be
observed in all available retroviral and filoviral post-fusion glycoprotein structures
except SIV gp41. The PDB codes for the fusion glycoproteins are as follows: EBOV GP2,
2EBO; MARV GP2, 4G2K; HTLV-1 gp21, 1MG1; syncytin-2, 1Y4M; SIV gp41, 2EZO.
What Are the Innate Restriction Strategies Targeted toward Viral Glycoproteins?
Host strategies for viral restriction are not limited to the humoral arm of the immune
system. The interferon-α-induced innate viral restriction factor BST-2 (also called
tetherin and CD317) is a common target of viral glycoprotein modulation [31]. As viruses
bud from the cell surface, they are coated with a membrane derived from the host cell.
As a result, host BST-2 is incorporated in the membrane of the nascent virion and
forms a protein tether to prevent viral release. This nonspecific restriction factor
potentially plays a protective role against infections due to retroviruses, filoviruses,
arenaviruses, flaviviruses, rhabdoviruses, and orthomyxoviruses (Table 1).
EBOV and HIV-2 both downregulate BST-2 by interactions mediated through their respective
viral glycoproteins [32], whereas HIV-1 makes use of the accessory protein Vpu to
achieve this same outcome. Some viruses degrade BST-2 or sequester it in intracellular
compartments. For example, the HIV-2 envelope glycoprotein appears to sequester the
constitutively endocytosed BST-2 in transferrin-positive endosomes [33]. Recent studies
have shown that EBOV GP does not remove BST-2 from the cellular surface [34] or sequester
it in intracellular sites or lipid rafts [35]. EBOV GP interferes with BST-2-mediated
virion capture independently of the mucin-like domain, and neither an engineered form
of GP lacking the transmembrane domain nor the dimeric sGP antagonize BST-2 restriction
[32]. HIV-1 Vpu interacts with BST-2 via a helical interface found within the transmembrane
domains of the two proteins [36]. Accordingly, it may be worthwhile to explore the
role of the transmembrane domain of EBOV GP in BST-2 antagonism.
Perspectives
Viruses have developed remarkable mechanisms to inhibit the adaptive and innate immune
systems of their hosts. Clearly, viral entry glycoproteins play critical roles in
these activities. However, many of these roles and biological pathways are poorly
defined. With new infectious diseases emerging and classical viral diseases reemerging,
closer examination of viral entry glycoproteins as targets for preventative or therapeutic
strategies is warranted.