Virion Glycoprotein-Mediated Immune Evasion by Human Cytomegalovirus: a Sticky Virus Makes a Slick Getaway.

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Virion Glycoprotein-Mediated Immune Evasion by Human Cytomegalovirus: a Sticky Virus Makes a Slick Getaway Thomas J. Gardner, Domenico Tortorella Icahn School of Medicine at Mount Sinai, Department of Microbiology, New York, New York, USA

SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .663 INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .663 GLYCOPROTEIN COMPONENTS OF THE CMV VIRION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .664 gB, a Multifunctional Viral Glycoprotein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .665 gH/gL Complexes, Critical Determinants of Tropism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .667 gM/gN Complex, a Diverse and Highly Expressed Protein Pair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .667 IMMUNE EVASION MECHANISMS OF CMV VIRION GLYCOPROTEINS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .667 Strain Polymorphism Contributes to Immune Evasion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .668 Epitope Competition Misleads the Humoral Immune Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .669 Fc Receptor-Mediated Endocytosis and the MSL-109 Effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .670 Glycan Shielding Protects Essential Viral Epitopes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .671 Cell-to-Cell Spread Avoids Antibody-Mediated Inhibition. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .671 CONCLUDING REMARKS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .672 ACKNOWLEDGMENTS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .672 REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .672

SUMMARY

The prototypic herpesvirus human cytomegalovirus (CMV) exhibits the extraordinary ability to establish latency and maintain a chronic infection throughout the life of its human host. This is even more remarkable considering the robust adaptive immune response elicited by infection and reactivation from latency. In addition to the ability of CMV to exist in a quiescent latent state, its persistence is enabled by a large repertoire of viral proteins that subvert immune defense mechanisms, such as NK cell activation and major histocompatibility complex antigen presentation, within the cell. However, dissemination outside the cell presents a unique existential challenge to the CMV virion, which is studded with antigenic glycoprotein complexes targeted by a potent neutralizing antibody response. The CMV virion envelope proteins, which are critical mediators of cell attachment and entry, possess various characteristics that can mitigate the humoral immune response and prevent viral clearance. Here we review the CMV glycoprotein complexes crucial for cell attachment and entry and propose inherent properties of these proteins involved in evading the CMV humoral immune response. These include viral glycoprotein polymorphism, epitope competition, Fc receptor-mediated endocytosis, glycan shielding, and cell-to-cell spread. The consequences of CMV virion glycoprotein-mediated immune evasion have a major impact on persistence of the virus in the population, and a comprehensive understanding of these evasion strategies will assist in designing effective CMV biologics and vaccines to limit CMV-associated disease.

The prototypic betaherpesvirus, human cytomegalovirus (CMV), is particularly adept at dissemination and chronic infection. The examination of how CMV is able to persist in its human host despite a potent humoral immune response has been a particularly active area of research over the last few decades. In fact, CMV has long served as a lens through which microbiologists have studied molecular mechanisms of infection and immune evasion. Recent estimates place CMV seroconversion at more than 60% worldwide by the age of 50 years, and the likelihood of infection increases with age (2). Following primary infection, which is usually benign in immunocompetent individuals, CMV can establish latency in myeloid and endothelial cells and can periodically reactivate to an active infection (3). CMV disease is a major cause of morbidity and mortality in immunosuppressed patients, especially recipients of solid organ or bone marrow transplants, neonates, AIDS patients, cardiovascular disease patients, and the elderly (4–6). CMV disease is a major medical problem projected to cost a total of $4.4 billion/year (7). Reactivated CMV disease is observed in a striking 8 to 39% of solid organ transplants (SOT) and hematopoietic stem cell transplants (HSCT), of which there are ⬎45,000 procedures in the United States annually (8–10). Furthermore, CMV infections remain the most common congenital viral infection in the United States (0.2 to 2.5% of all births) and cost ⬃$300,000 per congenitally infected child (7, 11). Congenitally infected neonates can develop extensive central nervous sys-

Published 15 June 2016

INTRODUCTION

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erpesviruses have coexisted with their hosts for millions of years (1). Infection typically occurs at a young age, and once infected, a healthy human host will likely remain chronically infected for the remainder of his or her life. Successful immune evasion thus presents a unique challenge to this family of viruses.

September 2016 Volume 80 Number 3

Citation Gardner TJ, Tortorella D. 2016. Virion glycoprotein-mediated immune evasion by human cytomegalovirus: a sticky virus makes a slick getaway. Microbiol Mol Biol Rev 80:663– 677. doi:10.1128/MMBR.00018-16. Address correspondence to Domenico Tortorella, [email protected]. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Microbiology and Molecular Biology Reviews

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Gardner and Tortorella

FIG 1 Schematic of the CMV virion. The CMV virion consists of an ⬃235-kb double-stranded DNA genome contained within an icosahedral protein capsid. A tegument layer (blue zone) consisting of many phosphoproteins lies between the capsid and the outer membrane. The outer lipid bilayer membrane is studded with multiple glycoprotein complexes.

tem (CNS) disorders, in the form of encephalitis, deafness, upper motor neuron disorders, psychomotor retardation, myopathy, and choroidoretinitis (12). Toxicity, drug-drug interactions, and antiviral resistance are common limitations of existing antivirals that necessitate the development of more CMV therapeutics that are both safe and effective (13, 14). Passive immunotherapy and, ultimately, an effective vaccine are promising treatment options that will avoid the unwanted side effects observed with small-molecule antiviral inhibitors. Indeed, recent interest in the development of neutralizing monoclonal antibodies (MAbs) and the investigation of hyperimmune globulin therapies for treatment of CMV reveals a great potential for antibody-mediated CMV therapeutics (15). Consideration of the potential immune evasion strategies that permit the CMV virion glycoproteins to escape the humoral immune response will aid in selecting the most suitable antigen candidates for these endeavors. Furthermore, CMV-based vaccine vectors show promise in administering protection from HIV (16), and the recent FDA approval of the first-ever engineered oncolytic virus, based on the herpes simplex virus 1 (HSV-1) genome (TVEC), has ushered in a new class of viral oncolytics (17). Thus, a comprehensive understanding of the humoral response to herpesvirus glycoproteins is vital in advancing these promising avenues. CMV is a true behemoth; its large double-stranded DNA genome is ⬃235 kb long and contains between 192 and up to 751 open reading frames (ORFs), including those for as many as 65 unique glycoproteins (18–21). The extents of expression and functions of the majority of these glycoproteins are unknown (22). Approximately 20 viral proteins, including glycoproteins, are expressed in the virus envelope (23), and additional glycoproteins may aid in viral dissemination or alter cell proliferation (24– 27). While viral membrane envelope proteins are known to play critical roles in the virus life cycle by permitting cellular attachment and fusion with the host membrane, many CMV glycopro-

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teins perform additional roles by serving critical functions in immune evasion, both within and outside the cellular environment. We and others previously reviewed the elegant methods by which CMV “immune evasin” proteins dampen the anti-CMV immune response (3, 28–30). Therefore, in the sections below, we aim to highlight the critical and diverse roles of CMV-encoded virion envelope proteins and their “innate” ability to evade the immune response. GLYCOPROTEIN COMPONENTS OF THE CMV VIRION

The CMV virion consists of a proteinaceous capsid containing an ⬃235-kb double-stranded DNA genome surrounded by a tegument layer, composed of ⬎14 bona fide viral proteins, and an outer envelope that contains several glycoprotein complexes necessary for entry into various cell types (Fig. 1) (31–36). Upon viral egress, viral capsids undergo primary envelopment at the inner nuclear membrane followed by deenvelopment at the outer nuclear membrane. Cytoplasmic viral capsids then undergo a second envelopment step by budding into a nearby assembly complex. The CMV assembly complex is the result of a dramatic reorganization of the secretory pathway that is essential for the recruitment of tegument and structural proteins for viral envelopment. Following attachment of tegument proteins, the virion is enveloped with a lipid bilayer containing virion glycoprotein complexes consisting of soluble and membrane proteins (37, 38). These envelope proteins represent a significant structural component of the mature CMV virion, comprising up to ⬃13% of the total protein material as determined by mass spectrometry studies (36). The surface of the CMV virion is studded with numerous complexes composed of heterogeneous assemblies of various glycoproteins. These complexes were previously designated glycoprotein complexes (GCs) I to III based on sedimentation and reactivity to neutralizing monoclonal antibodies following ratezonal centrifugation (39). CMV glycoprotein complexes include



Immune Evasion by CMV Envelope Glycoproteins

FIG 2 CMV glycoprotein complexes. The essential CMV glycoprotein complexes required for cell attachment and entry are depicted. The glycoprotein complex (GC) designations are indicated. The pentameric complex (PC) is illustrated on the right and includes the UL128, UL130, and UL131a proteins. Parallel dashed lines represent disulfide linkages between individual envelope proteins.

the gB oligomer (GCI), the gM/gN dimer (GCII), the gH/gL/gO trimer (GCIII), and the gH/gL/UL128/UL130/UL131a pentamer complex (PC) (Fig. 2 and Table 1). While the precise functional roles of the glycoprotein complexes on the CMV virion are not fully understood, it is clear that the envelope glycoproteins play critical roles in the virus life cycle and that distinct glycoprotein complexes permit attachment and entry into various cell types (Fig. 3). More specifically, gB, gH, and gL, which are conserved in all herpesviruses, make up the core fusion machinery and are required for attachment and entry steps (40, 41). CMV entry likely requires various cellular receptors (Fig. 3A) and involves distinct cell-type-dependent mechanisms (Fig. 3B). Entry into fibroblasts occurs in a pH-independent manner via fusion at the cell surface through gB and the gH/gL/gO complex (42) and/or through macropinocytosis (43). In contrast, entry into epithelial, endothelial, dendritic, and monocytic cells requires gB, gH/gL/gO, and the PC and occurs through endocytosis or macropinocytosis followed by a pH-dependent fusion event (Fig. 3B) (31–33, 35, 44–49). The stark difference in CMV entry pathways underscores the involvement of fundamentally distinct entry mechanisms that likely extend beyond mere differences in cell-type-specific receptors. A more detailed overview of the major CMV glycoprotein complexes and their roles in entry follows. gB, a Multifunctional Viral Glycoprotein

gB (UL55) is an essential component of the CMV virion glycoprotein repertoire (Fig. 2). The nascent gB polypeptide is synthesized

as a 160-kDa monomer that is cleaved in the trans-Golgi network by a furin endoprotease to generate two polypeptides (gp58 and gp116) covalently linked by a disulfide bond. These polypeptides form a heterodimer that consists of an ectodomain surface subunit (gp116) and a smaller polypeptide resembling a type I membrane protein (gp58) (50–52). While the multifunctional nature of gB continues to be explored, it is required for cell entry and cell-to-cell spread (53). gB interacts with heparan sulfate proteoglycans in the initial tethering step of CMV cell attachment (Fig. 3A) (54). Interaction of gB with cell surface proteins, such as integrins (55–58), epithelial growth factor receptor (EGFR) (58–60), and platelet-derived growth factor receptor alpha (PDGFR␣) (61, 62), may activate cellular signaling pathways that facilitate entry, although reports about the role of EGFR as a CMV receptor were subsequently challenged (63). The involvement of integrins in Kaposi’s sarcomaassociated herpesvirus (KSHV), Epstein-Barr virus (EBV), and herpes simplex virus (HSV) bolsters the evidence for involvement of these proteins in CMV infection (64–68). Recently, THY-1 (CD90) was identified as a putative entry factor that may form complexes with CMV gB and gH to link viral entry with signaling events that permit infection (69). A recent study by Wille et al. demonstrated that CMV gB expressed on the cell surface could successfully complement infection by virions lacking the gB protein, arguing that gB is involved in fusion and is not required in a receptor-binding role in fibroblasts (70).

TABLE 1 Glycoprotein complexes of the CMV virion Glycoprotein

ORF

Topology

No. of potential N-linked glycosylation sites

Complex(es)a

gB (gp58/116) gH gL gM gN gO UL128 UL130 UL131a

UL55 UL75 UL115 UL100 UL73 UL74 UL128 UL130 UL131

Transmembrane type I Transmembrane type I Soluble Transmembrane type III Transmembrane type I Soluble Soluble Soluble Soluble

18 6 1 1 2 18 0 3 1

Oligomer, gB:gH/gL gH/gL, gH/gL/gO, PC, gB:gH/gL gH/gL, gH/gL/gO, PC, gB:gH/gL gM/gN gM/gN gH/gL/gO PC PC PC

a

gB:gH/gL refers to a noncovalent interaction.



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FIG 3 Model of CMV attachment and entry. (A) CMV entry receptors. (1) CMV initially tethers to cells through interactions between heparan sulfate proteoglycans (HSPGs) and the gB and gM/gN complexes. (2) gB then interacts with cell surface receptors, which may include integrins, epidermal growth factor receptor (EGFR), and platelet-derived growth factor receptor alpha (PDGFR␣). gH complexes may interact with integrins and additional cell surface receptors. Receptor binding results in signal transduction that likely primes the fusion event. (3) gH/gL complexes and gB then act together to initiate membrane fusion. (B) Cell entry pathways. CMV entry into fibroblast cells occurs at the cell surface and/or in macropinocytic vacuoles and requires gB and the gH/gL/gO complex in a pH-independent fusion event. Entry into epithelial, endothelial, dendritic, and monocytic cells occurs via endocytosis and/or macropinocytosis followed by a pH-dependent fusion event and requires gB, gH/gL/gO, and the PC.

This contrasts with existing mechanistic knowledge of herpesvirus cell attachment that implicates a receptor-binding role by gB. In fact, HSV gB may interact with cellular receptors associated with lipid rafts, and KSHV utilizes gB to bind to an integrin receptor (66, 71, 72). Recently, EBV gB was found to directly interact with neuropilin 1 (NRP1) on the cell surface to promote infection through activation of receptor tyrosine kinase (RTK) signaling (73).

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Regardless of its precise function in receptor binding, gB is essential for the CMV viral fusion process. Electron microscopy data indicate that CMV gB exists as a trimer with a tertiary structure similar to that of the HSV-1 and EBV gB proteins (74). HSV and EBV gB also likely induce the viral fusion process when triggered by gH/gL complexes (40, 75–77). Indeed, the recently solved crystal structure of the CMV gB ectodomain reveals that it closely resembles the postfusion structures of the HSV-1 and EBV ho-




mologs, establishing it as an additional class III fusion protein (74, 78–81). These data support the paradigm that gB is a critical member of the CMV glycoprotein repertoire for virus entry. gH/gL Complexes, Critical Determinants of Tropism

The type I membrane glycoprotein gH (UL75) is a critical component of the viral binding and fusion machinery (Fig. 2 and 3). It exists in multiple glycoprotein complexes on the surface of the CMV virion, paired through a disulfide bond with the soluble glycoprotein gL (UL115). The dimerization of gH and gL in the endoplasmic reticulum (ER) increases the export of both proteins and enables their formation of the mature entry complexes required by CMV to infect its full range of target cells (82–85). gO (UL74) is a highly glycosylated, soluble protein that interacts with gH/gL via a disulfide bond to gL (86, 87). gO is a critical component of the gH/gL/gO complex, and its association with gH/gL promotes export of the trimer from the ER (48, 83, 88). Numerous studies have demonstrated that the gH/gL/gO complex is essential for entry into fibroblasts via surface membrane fusion as well as for entry into epithelial cells (49, 84, 87, 89). An additional virion surface complex is formed between gH/gL and the soluble UL128, UL130, and UL131a proteins to form the gH/gL/UL128/UL130/UL131a pentameric complex (PC) (Fig. 2). These proteins interact with the gH/gL heterodimer via a disulfide bond between gL and UL128. A recent report demonstrates that gO and UL128 bind to the same site on gH/gL, competing for binding and generating a disulfide bond with gL-Cys-144 (86). An additional ER-resident viral factor, UL148, may regulate the ratio of gH/gL/gO to PC, and thus the subsequent tropism of nascent virions (90). The PC permits entry into epithelial, endothelial, monocytic, and some dendritic cells via a pH-dependent, endocytic route of entry (Fig. 3B) (31, 35, 44, 47, 91, 92). Interestingly, the ratio of gH/gL/gO to PC can vary significantly between CMV strains (88). Extensive passaging of laboratory strains of CMV, such as AD169, has led to the selection of viruses which lack an intact PC (32, 33, 93). Mutants lacking PC replicate in fibroblasts as well as or better than wild-type virus strains (33, 35, 91), while mutants lacking gH/gL/gO display severe replication defects despite containing elevated amounts of PC (48, 94, 95). Strains lacking an intact PC do not infect epithelial cells, endothelial cells, monocytes, or macrophages (32, 35, 47). Infection levels of both fibroblasts and epithelial cells correlate with the abundance of the gH/gL/gO complex on the virus surface, suggesting that only the trimer complex can impart the conserved herpesvirus gH/gL entry function, while the trimer and PC can both impart a receptor-binding function (49). gH complexes may interact with ␣v␤3 integrin during the binding step of CMV entry (56). This is supported by evidence that integrins can serve as specific receptors for EBV and HSV gH/gL and may provide the viral fusion trigger or promote viral endocytosis (64, 96–99). Competition studies have revealed that cell surface expression of the PC, but not the gH/gL complex, the UL128/ UL130/UL131a complex, or gB, on the surfaces of epithelial cells can prevent CMV infection, implying that the PC may be involved in receptor binding by the virus (34) (Fig. 3A). EGFR does not enhance infection by the clinical strain TR in any cell type (100, 101), while PDGFR␣ enhances CMV entry in epithelial and endothelial cells and increases viral entry into cells that normally cannot be infected by CMV (100). Intriguingly, this enhancement of epithelial/endothelial cell infection does not require the PC, and


antibodies targeting PDGFR␣ do not inhibit CMV entry into fibroblasts or epithelial or endothelial cells. This suggests that CMV does not directly interact with PDGFR␣ but may utilize it in an alternative macropinocytosis entry pathway that requires pH-dependent fusion (100) (Fig. 3B). Importantly, gH/gL complexes likely contribute to more than just cell attachment, as the PC serves as a ligand for receptor-mediated signaling events that lead to internalization of CMV into monocytes, while the gH/gL/gO complex triggers Src-mediated signaling to permit fusion at the cell membrane (45). While details of the specific mechanisms are still being elucidated, gH/gL complexes clearly carry out important functions in viral attachment and fusion. gM/gN Complex, a Diverse and Highly Expressed Protein Pair

Despite being the most abundant glycoprotein complex on the surface of the CMV virion envelope (36), the gM/gN heterodimer is the least-characterized glycoprotein complex. gM (UL100), a type III membrane glycoprotein, contains 8 membrane-spanning segments and is associated, through a disulfide bridge, with the glycosylated type I gN protein (UL73) (Fig. 2) (102, 103). The gM/gN complex is among the few envelope proteins that are conserved among herpesviruses, likely indicating an important role for viral pathogenesis (104). While mutagenesis studies of gM and gN indicate that they are essential for virus replication (105, 106), and while the gM/gN complex is capable of binding to heparan sulfate proteoglycans in the first step in viral cell attachment prior to entry (Fig. 3A), the functions of gM and gN as virion glycoproteins are largely unknown (107, 108). Although no role in specific viral receptor attachment or membrane fusion has been reported for the gM/gN complex, antibodies targeting the complex are capable of neutralizing infection to an extent comparable to neutralization by anti-gB antibodies (105, 109–111). Thus, targeting the gM/gN complex may be an effective strategy to limit CMV infection and dissemination. IMMUNE EVASION MECHANISMS OF CMV VIRION GLYCOPROTEINS

Many CMV proteins are adept at manipulating the cellular machinery that modulates cellular metabolism and major histocompatibility complex (MHC) class I and class II antigen presentation (3, 112–114). As with all herpesviruses, CMV possesses the ability to maintain its viral genome within a cell without generating infectious virions (115). Latent virus, however, can periodically reactivate as a productive infection, with the capacity to replicate and disseminate virus. In fact, a significant clinical threat from CMV arises from cases of reactivated CMV infection, typically brought on by immunosuppression (116). While CMV latency is an effective and elegant method to avoid elimination by the immune system, host-to-host transmission requires that the virus exist, at least for a time, outside a cellular environment, where it is subject to a hostile immune system. Healthy humans exhibit a potent and well-documented humoral response to the CMV virion (117). How can CMV disseminate within the host in the presence of circulating anti-CMV antibodies? As we describe below, the CMV virion glycoproteins possess “innate” characteristics that support their ability to evade clearance by neutralizing antibodies through diverse mechanisms (Table 2). These strategies allow the CMV virion to disseminate freely and to infect new


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TABLE 2 CMV evasion strategies against humoral immunity and potential therapeutic countermeasures Immune evasion mechanism Strain polymorphism Epitope competition

Advantage(s) to virus

Therapeutic countermeasure(s)

Superinfection by multiple viral strains, increased diversity of strains within the host Protection of susceptible envelope protein regions from neutralizing antibodies

Subunit vaccines that elicit antibody response to conserved epitopes, therapeutic antibodies that target conserved epitopes Immunization with chimeric or truncated proteins (subunit vaccine or mutant virus) to direct the immune response to vulnerable neutralizing epitopes, therapeutic antibodies that target vulnerable neutralization regions, neutralizing antibodies isolated from hyperimmune globulin sera Fab fragments to avoid endocytosis-mediated uptake of virusantibody immune complexes, antibody-drug/toxin conjugates to eliminate virus-infected cells Immunization using chimeric or truncated proteins (subunit vaccine or mutant virus) to direct the immune response to vulnerable neutralizing epitopes, therapeutic antibodies that target vulnerable neutralization regions of low glycan density Combined use of antibody therapy directed to cell surface-expressed envelope proteins with pharmaceutical inhibitors, nanoantibodies (e.g., camelid antibodies) to envelope proteins

Fc receptor-mediated endocytosis

Enhancement of viral entry in diverse cell types, transplacental transmission

Glycan shielding

Protection of susceptible glycoprotein regions surrounded by high glycan density

Cell-to-cell spread

Avoidance of extracellular immune surveillance, viral dissemination

cells, ultimately permitting spread within the host and transmission to additional individuals (Fig. 4). Strain Polymorphism Contributes to Immune Evasion

While it is widely held that DNA viruses are more genetically stable than RNA viruses, a number of studies have revealed that considerable genetic diversity exists within the CMV genomes in infected hosts. A recent analysis of the genomic variability of CMVs isolated from congenitally infected neonates indicates that CMV exists within its host as a mixture of genetically diverse genomes (118–120). Recent efforts have employed high-throughput sequencing to study the extent of genome-wide variability in clinical isolates (121). Remarkably, intrahost CMV populations were found to be as variable as some RNA virus quasispecies, and that CMV diversity spanned the entire length of the genome, including the genes for the envelope proteins present on the CMV membrane (118). Mixed populations of CMV genomes within an infected individual have been observed by analyzing gB genotypes (122–130) as well as the genotypes for additional virion envelope proteins: gN, gO, gH, and gL (127–129). These differences permitted the categorization of 4 polymorphic gB genotypes (131), 7 polymorphic gO genotypes, and 4 polymorphic gN genotypes (132, 133). Protein alignment of CMV strain VR1814 to 10 CMV strains isolated from geographically distinct regions demonstrated diversity in all of the CMV glycoproteins, including the UL128, UL130, and UL131a proteins, with the greatest diversity appearing in gN and gO (Table 3). The natural humoral immune response to CMV primarily induces antibodies directed against gB; however, antibodies targeting gM/gN and gH/gL have also been identified (109, 134, 135). Recent work also revealed the exquisite potency of human-derived monoclonal antibodies (MAbs) that target the PC in blocking infection of epithelial, endothelial, and monocytic cells (111, 136, 137). Numerous neutralizing epitopes exist across the CMV virion glycoprotein repertoire, and thus only a fraction of neutralizing antibodies are likely to be affected by the existence of any one polymorphism. The threshold for maintaining neutralizing potency may be influenced by polymorphisms in a specific region that significantly limit the efficacy of the existing humoral

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immune response for controlling infection when the host is exposed to a viral strain with a different genotype (Fig. 4A). The existence of such strain-specific epitopes leads to the intriguing concept that although CMV-specific antibodies may exhibit equal neutralizing capacities in cell culture assays, some epitopes may represent more important targets in vivo. Consideration of these factors may improve strategies for the development of therapeutic antibodies or vaccine antigens (Table 2). gB, a well-characterized CMV virion glycoprotein, plays an important role in infectivity and cell-to-cell spread. There is a robust antibody response to gB in the human host (135, 138, 139). Five antigenic domains, AD-1 to AD-5, have been described for gB, among which four appear to elicit a neutralizing antibody response (140). The linear epitopes AD-1 and AD-2 contained within the gB protein are major targets for neutralizing antibodies (20). However, amino acid changes in AD-1 and AD-2 have been identified between clinical strains (141–143), and binding of monoclonal antibodies and human serum to the AD-2 segment differs between clinical and laboratory strains (144). Variations in this region can be generated by homologous recombination in patients infected with multiple strains of CMV (145). Given that these immunodominant regions can exchange genetic sequences, homologous recombination in the gene encoding this critical glycoprotein likely expands the repertoire of neutralizing antibodies required to suppress CMV infection and thus functions as a mechanism by which the virus can increase the number of CMV-specific antibodies required to achieve a neutralizing antibody threshold. Neutralization of CMV by human sera has revealed strainspecific anti-gH antibodies (146). Additionally, a specific domain within the amino-terminal portion of gH that is capable of inducing strain-specific neutralizing antibodies has been identified (133). Furthermore, studies of renal transplant recipients demonstrated an increased occurrence of adverse events in seropositive patients whose gH antibodies were mismatched with the gH from their seropositive organ donor (147). The potential for emergence of gH escape mutants was also demonstrated by use of the human-derived antibody MSL-




FIG 4 Strategies of CMV envelope protein-mediated immune evasion. (A) Strain polymorphism. Initial CMV infection elicits an antibody response that may not effectively neutralize infection by alternate strains due to variability in the CMV virion envelope proteins. (B) Epitope competition. Antibodies targeting regions of a CMV envelope protein that do not participate in critical attachment or entry processes may sterically block the binding of neutralizing antibodies to critical epitopes. (C) Fc receptor-mediated endocytosis. A CMV virion that is bound by CMV-specific antibodies may cross-link Fc receptors on the cell surface and induce endocytosis of the virion-IgG complex. Viral entry then occurs when the virus fuses with the endosomal membrane within the cell. (D) Glycan shielding. Highly glycosylated surface proteins, such as CMV gB, gO, and gN (Table 1), may prevent antibody binding by limiting access to susceptible epitopes within the glycoprotein or glycoprotein complex. (E) Cell-to-cell fusion. Surface viral envelope proteins may induce fusion between an infected cell and a bystander cell, permitting transfer of infectious material while avoiding exposure to the host neutralizing antibody response.

109 following continuous passaging of infected epithelial cells in the presence of the antibody (148). Finally, CMV-seropositive individuals also display strain-specific neutralizing antibodies to gN, suggesting that a broad gNneutralizing activity may be required to impart protection when an individual is infected with multiple CMV strains (147, 149). Indeed, CMV-seropositive sera from various donors displayed varied neutralizing activities against four recombinant viruses that differed only in their gN genotype, thus demonstrating that gN


polymorphism may contribute to antibody evasion and facilitate superinfection of CMV-positive individuals (150). Epitope Competition Misleads the Humoral Immune Response

Variability among CMV strains present in an infected host can provide a population-based strategy of immune evasion. However, an individual strain within an infected host must possess


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TABLE 3 Alignment of glycoproteins from geographically diverse CMV strains

Strain

Origin

Collection date (yr)

VR1814 3301 BE/19/2011 CZ/1/2011 Davis Han Han30 Merlin PAV7 TR UKNEQAS2

Italy UK Belgium Czech Republic USA China Germany UK Italy USA Australia

1996 2001 2011 2011 1957 2007 2006 1999 2007 1996 2013

a

Identity (%)a Accession no.

gB

gH

gL

gM

gN

gO

UL128

UL130

UL131a

GU179289 GQ466044 KP745654 KP745718.1 JX512198 KJ426589 KJ361953 AY446894 KJ361963 KF021605 KT634296

100 93.2 96.4 93.2 93.2 93.2 93.2 93.2 93.2 95.9 93.2

100 96.1 99.5 96.1 97 96 96 96 96.2 98.9 99.6

100 98.6 98.6 98.2 100 98.2 98.2 98.2 98.2 98.9 98.9

100 97.6 97.6 97.6 97.6 99.7 99.7 99.7 97.6 100 97.3

100 93.4 71 87.5 71.7 100 100 100 100 86.2 78.3

100 72.9 75.8 75.4 73.7 100 100 100 100 82.8 77.8

100 99.4 98.8 98.2 98.8 98.8 98.8 * 99.4 99.4 100

100 99.1 96.7 98.1 * 100 100 98.6 100 98.1 97.2

100 100 100 100 100 100 100 100 100 99.2 100

*, the gene is mutated or missing.

some means by which to avoid neutralizing antibodies for propagation. Such a mechanism may exist through epitope competition, whereby the antibody response targets juxtaposed or overlapping epitopes, some of which may block antibody binding and others of which may actually interfere with the ability of the neutralizing antibody to block an infection (Fig. 4B). The induction of both neutralizing and nonneutralizing antibodies that compete for the same antigenic region on a CMV virion has been described for gB (139). Peptide mapping with neutralizing monoclonal antibodies cloned from human peripheral blood lymphocytes revealed that the gp58 segment of gB possesses a high epitope density which elicits both neutralizing and nonneutralizing antibodies (151). Subsequent studies demonstrated that a large proportion of human polyclonal antibodies elicited against regions of the immunodominant AD-1 region of gB do not neutralize infection and may thus provide a mechanism by which CMV evades binding by neutralizing antibodies (139, 152). A recent comprehensive analysis of the human antibody response to gB revealed that over 90% of gB-specific antibodies secreted from memory B cells do not neutralize virus infection (140). It is possible that such nonneutralizing antibodies may limit infection through other mechanisms, such as antibody-dependent cell-mediated cytotoxicity (ADCC); however, this requires additional studies. Immunogenic regions of the gB protein may serve in part as a decoy to elicit a nonneutralizing immune response and potentially coat this critical entry factor in antibodies that do not affect its ability to bind and enter cells. Indeed, recent speculation suggests that the majority of gB proteins on the viral surface are in the postfusion conformation, thus providing an escape mechanism by eliciting an antibody response that does not impair fusion (81). Whether such a mechanism exists for additional envelope proteins remains to be determined; however, therapeutic antibody and vaccine antigen design efforts should consider whether to focus the neutralizing antibody response on protein domains that are not susceptible to epitope competition by nonneutralizing antibodies (Table 2). Fc Receptor-Mediated Endocytosis and the MSL-109 Effect

Canonically, neutralizing antibodies that engage with the surface of a viral glycoprotein can prevent infection either by blocking viral interactions with the host cell or by inhibiting the fusion process. While this is most often the case, several notable exceptions have been observed for CMV infections. In a report by Mai-

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dji et al., syncytiotrophoblasts from the placentas of mothers with high titers of CMV-neutralizing antibodies contained intracellular vesicles containing both IgG and gB (153). Additional villus explant experiments demonstrated that trypsin treatment and soluble protein A could block IgG-virion transcytosis, and polarized epithelial cells pretreated with IgG Fc fragments, but not F(ab=)2, were resistant to neonatal Fc receptor (FcRn)-mediated viral transcytosis. The FcRn transfers maternal antibodies across the placental barrier to the developing fetus. Thus, in the presence of CMV-specific antibodies, the CMV virion may coopt the syncytiotrophoblast IgG transport pathway in order to disseminate to the placenta and infect the fetus. This would permit an antibodycoated virion that would otherwise be unable to bind and infect a cell to enter through an endocytosis mechanism, subsequently fusing with the endosomal membrane to deliver its infectious material within the cell (Fig. 4C). This strategy of escape is a considerable concern, as hyperimmune globulin therapy is currently being investigated as a potential treatment for pregnant women with primary or reactivated CMV infection (154, 155) (Table 2). This example further highlights the complexities of therapeutically targeting envelope proteins of a highly evolved chronic human virus. CMV may possess additional, nongenetic mechanisms to escape neutralizing antibodies in the host through Fc receptor-mediated mechanisms. Manley et al. demonstrated that the humanderived anti-gH antibody MSL-109 could be incorporated into assembling virions within a CMV-infected cell (156). The nascent virus then utilized the Fc domain of gH-bound antibody to infect additional cells through clathrin-dependent endocytosis. This led to rapid resistance to MSL-109 (in as few as three rounds of infection in vitro). The process permitted infection only of fibroblasts that did not express classical Fc-␥ receptors, and virus propagated in the presence of MSL-109 lost the ability to infect epithelial and endothelial cells. It is interesting that this phenomenon resulted in narrowed tropism rather than enhanced tropism, as observed with antibody-dependent enhancement in dengue virus infection (157). Recently, Fouts et al. demonstrated that viral resistance to MSL-109 occurred following ⬎2 months of passaging in epithelial cells with suboptimal antibody concentrations and that the escape mutants exhibited attenuated cell entry, though this occurred through nonreversible genetic adaptation by the virus (148). Why was a rapid and nongenetic resistance to MSL-109 not observed? The use of primary epithelial cells may explain the difference in findings, as cell-type-specific Fc receptor expression may influ-




ence the emergence of nongenetic MSL-109 resistance. Note that Manley et al. also reported difficulties in raising escape mutants to an MSL-109 F(ab=)2 despite prolonged culture periods at 20⫻ the 50% inhibitory concentration (IC50), confirming the large barrier to genetic adaptation of CMV (148). CMV encodes its own Fc receptors (158); however, it remains to be determined whether they are involved in MSL-109-mediated uptake. The MSL-109 effect was not observed when virus was cultured in the presence of additional anti-gH neutralizing antibodies, suggesting that this mechanism of evasion may function only with specific domains of gH. Future work will likely investigate whether the process of nongenetic resistance can occur with neutralizing antibodies targeting alternate gH epitopes or additional CMV envelope proteins. Glycan Shielding Protects Essential Viral Epitopes

The N- or O-linked glycosylation state of proteins expressed on the surface of a virion can affect the potency of the host antibody response. Glycan modifications that function as a “shield” to protect viruses from antibody-mediated neutralization are a welldocumented characteristic for HIV and influenza virus infections (159, 160). A recent study examined a potential glycan-shielding role of CMV gN. gN is highly polymorphic, and thus far four genotypes have been defined based on sequence identity (161). Despite the large amount of variation in the surface domain of gN, there is a high level of conservation of potential N-linked glycosylation sites, indicating that a selective pressure to maintain the level of glycan density may exist. Kropff et al. generated viruses containing deletions in Ser/Thr-rich primary sequences of the gN ectodomain and a gN-truncated virus (103). Remarkably, while the modifications to gN had no influence on formation of the gM/gN complex or viral replication, the mutant viruses were significantly more susceptible to neutralizing antibodies targeting gN, gH, and gB. Immunization of mice with virions expressing truncated gN variants resulted in higher serum titers of neutralizing antibodies against the homologous strain, including increased titers of gH- and gB-neutralizing antibodies. It was hypothesized that this may have been due to increased induction of neutralizing antibodies that target epitopes that would otherwise be obscured by glycans located on gN. There is some evidence for a protective role of gO N-linked glycosylation in dampening antibody-mediated inhibition. gO contains a significant amount of carbohydrate modifications and sits in close juxtaposition to gH/gL (162). Cell-to-cell spread of a mutant virus lacking gO was more sensitive to neutralization by polyclonal sera than that of the parent virus (163). The glycoprotein cross-protection observed with gN and gO may represent an important mechanism through which CMV is able to avoid neutralization by antibodies targeting crucial glycoproteins and should be taken into consideration for the development of effective vaccines and therapeutic antibodies (Fig. 4D and Table 2). Unlike that of HSV or EBV, CMV gB is extensively glycosylated, with 18 predicted N-linked glycosylation sites (Table 1) (74). Recent structural data obtained through analysis of the CMV gB ectodomain revealed that epitopes of several neutralizing antibodies to the AD-5 region of the protein, which is expected to undergo dramatic refolding during fusion, are surrounded by N-linked glycans that may hinder antibody binding (80). In a manner similar to the glycan shielding of critical fusion subunits of HIV Env (164) and Ebola gp (165), glycans of the surrounding AD-1 and AD-2 domains of gB may protect the crucial AD-3 and AD-5


domains, which themselves cannot carry glycosylated residues due to their involvement in fusion. Thus, targeting of the most susceptible regions of the CMV fusion machinery may not be a simple feat. Cell-to-Cell Spread Avoids Antibody-Mediated Inhibition

A hallmark of CMV infection is its signature cytomegaly phenotype and its ability to promote cell-to-cell fusion. This indicates a significant morphological alteration of infected cells that may permit the intercellular passage of infectious material, thereby avoiding the exposure of virions to the host antibody response altogether (Fig. 4E) (166). In vivo, CMV most often displays a cell-associated method of dissemination. For example, in patients with acute CMV infection, virus is found in the white blood cell compartment but not in plasma or serum (167). Furthermore, low-passage-number clinical viruses display a highly cell-associated phenotype (168–170). A variety of experimental approaches propose a role for CMV virion glycoproteins in inducing cell-tocell fusion, thereby permitting the cytoplasmic transfer of infectious virus and limiting exposure to neutralizing antibodies. Gerna et al. observed microfusion events between endothelial cells and leukocytes that may allow the transfer of infectious particles by wild-type CMV strains (171). A virus lacking the UL99-encoded pp28 protein, which failed to assemble mature enveloped viral particles, could spread from cell to cell despite the absence of infectious progeny virus in the cell supernatant (172). Similarly, infection with a strain of AD169 with replacement of the UL16 gene with enhanced green fluorescent protein (EGFP) demonstrated the free diffusion of EGFP between fibroblasts (173). These studies provide strong support for the occurrence of cell-to-cell dissemination by CMV during infection. Various CMV virion glycoprotein complexes are likely involved in cell-to-cell fusion processes. Overexpression of CMV glycoproteins in direct cell-to-cell fusion assays has implicated both gB and gH/gL complexes in inducing cell surface fusion between an infected cell and a bystander cell. U373 cells engineered to constitutively express gB form multinucleate syncytia that are dependent on the density of gB expressed on the plasma membrane (174). Kinzler and Compton observed an inherent fusogenic activity of the gH/gL complex following retroviral delivery in CHO, HeLa, and immortalized fibroblast cells (175). Studies by Vanarsdall et al. involving adenoviral delivery of gB and gH/gL into distinct cell lines and subsequent mixing of both cell types resulted in substantial cell fusion in U373, MRC5, and ARPE-19 cells and human umbilical vein endothelial cells (HUVEC), indicating that gB and gH/gL form a “multicomponent viral fusion machine” that does not require expression of the complexes within the same bilayer (84, 101). Naturally, cell fusion data obtained from overexpression of viral proteins outside the context of an infection provide insight into protein function, yet viral gene delivery or transient transfection may lead to high levels of glycoproteins that do not mimic levels observed during infection. Furthermore, some cell types, such as CHO and U373 cells, have been demonstrated to fuse naturally (176). However, the mutual requirement of gB and gH/gL for fusion by additional herpesviruses is consistent with data indicating a requirement for both gB and gH/gL protein complexes in CMV fusion (177, 178). The fact that the CMV gB ectodomain closely resembles those of its HSV-1 and EBV homologs and the observation that CMV gH/gL displays a boot-shaped structure as observed for the HSV-2 gH/gL complex support the


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existence of a CMV fusion mechanism that is similar to the fusion mechanisms of these herpesviruses (74, 78–81, 84, 86, 101, 179). Collectively, the studies demonstrate the complexity of CMV cellto-cell spread and the involvement of glycoprotein function therein. Mutant virus strains lacking critical glycoproteins have provided additional insight into the potential roles of gB and gH/gL in cell-to-cell fusion. For example, no cell-to-cell spread was observed when fibroblasts were infected with a gB-null virus (53). Wille et al. complemented gB-null or gH/gL-null virions with the corresponding protein(s) in normal human dermal fibroblasts (70). Infection with the mutant viruses revealed that gB expressed in trans could complement gB-null virions, but gH/gL could not complement gH/gL-null virions, arguing for a role for gB in fusion, whereas gH and gL may be important for receptor binding. Antibodies directed to CMV limit cell-to-cell spread in endothelial cells (180–182) and epithelial cells (183), as evidenced by a lack of focal spread in the presence of CMV-neutralizing antibodies, although virus strains may exhibit various degrees of sensitivity. Cell-to-cell spread in fibroblasts, in contrast, may proceed through transient microfusion events (173) and are largely insensitive to antibody-mediated inhibition (138, 163, 180, 183–185). Given the cell-type-specific mechanisms of CMV entry, it is not surprising that the virus exhibits cell-type-dependent mechanisms of cell-to-cell spread. A recent study by Scrivano et al. observed that virus spread in fibroblast cultures was predominantly supernatant driven, while spread in endothelial cell cultures was focal, likely due to the retention of endothelial cell-tropic virus by endothelial cells, while fibroblasts released both endothelial celltropic and non-endothelial cell-tropic viruses (180). The precise cause(s) for the various sensitivities to antibody-mediated inhibition of cell-to-cell spread is an important phenomenon for understanding how CMV proliferates in vivo, which remains largely unexplored. Regardless of cell type or virus strain, antibody inhibition of cell-to-cell spread will depend on the ability of an antibody to access regions of CMV glycoproteins that are poised to engage with a bystander cell and initiate fusion. Virus spread in polarized ARPE-19 cells occurs across lateral membranes where glycoproteins are unaffected by neutralizing antibodies (186). Thus, consideration of in vivo mechanisms of viral spread as well as the requirements for antibody access and sufficient suppression of cell-to-cell spread should be considered in the design of vaccines and therapeutic antibodies (Table 2). CONCLUDING REMARKS

Despite the numerous and multifaceted methods of immune evasion carried out by CMV, a competent immune response can effectively mitigate virus-associated diseases. On the other hand, rampant CMV propagation in individuals with compromised immune health is a true menace that results in severe disease complications. Unfortunately, each year, organ transplants, chronic inflammatory conditions, congenital CMV transmission, and elderly immune senescence all contribute to a substantial number of patients who are threatened by CMV-associated diseases. This necessitates the development of improved therapeutics to curb the devastating effects of unchecked CMV infection in these patient populations. Given that a healthy individual naturally controls CMV infection, it is reasonable to assume that utilization of antiCMV antibodies—whether through the use of recombinant biologics or the induction of a humoral immune response by vacci-

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nation—would be an effective strategy to prevent or control CMV infection. However, the requirements for an effective antibodymediated therapeutic strategy are not readily apparent. The processes of virion glycoprotein-mediated immune evasion described in this review highlight some of the difficulties in developing antibody-mediated CMV therapeutics that can provide substantial, long-lasting protection. The best strategies to minimize viral subversion of neutralizing antibodies will include careful selection of vaccine antigens and combinatorial therapeutic antibody treatment to target multiple components of the viral envelope repertoire. Adequate consideration of immune evasion mechanisms will undoubtedly expedite the development of therapeutic strategies against CMV-associated diseases that loom on the horizon. ACKNOWLEDGMENTS This work was supported in part by NIH grants AI112318 and AI101820. T.J.G. is a predoctoral trainee supported in part by an American Heart Association predoctoral fellowship.

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Cytomegalovirus immune evasion by perturbation of endosomal trafficking.

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CD8(+) T-cell Immune Evasion Enables Oncolytic Virus Immunotherapy.

Immune Response of Amebiasis and Immune Evasion by Entamoeba histolytica.

Immune recognition. Mls: makes a little sense.

Humoral immune response to human cytomegalovirus proteins: a brief review.

Lymphoma: immune evasion strategies.

Peroxisomes are platforms for cytomegalovirus' evasion from the cellular immune response.

The p36 isoform of murine cytomegalovirus m152 protein suffices for mediating innate and adaptive immune evasion.

Innate immune evasion by hepatitis B virus-mediated downregulation of TRIF.

Spirochetal Lipoproteins and Immune Evasion.

Signaling through RIG-I and type I interferon receptor: Immune activation by Newcastle disease virus in man versus immune evasion by Ebola virus (Review).

Immune evasion: See no evil.

A previously unidentified mechanism of immune evasion in glioblastoma.

More than just immune evasion: Hijacking complement by Plasmodium falciparum.

HIV suppression by host restriction factors and viral immune evasion.

Immune evasion by oncogenic proteins of acute myeloid leukemia.

Innate Immunity and Immune Evasion by Enterovirus 71.

Reactivation of herpes simplex virus type 2 from a quiescent state by human cytomegalovirus.

The circumsporozoite protein of Plasmodium: a mechanism of immune evasion by the malaria parasite?

Two novel human cytomegalovirus NK cell evasion functions target MICA for lysosomal degradation.

Activation of nucleotide oligomerization domain 2 (NOD2) by human cytomegalovirus initiates innate immune responses and restricts virus replication.