Viral Apoptotic Mimicry Is an Immune Evasion Viral Apoptotic Mimicry Mechanism Used by Hepatitis B Virus (HBV)

PROGRESS

(REF. 13), when they hypothesized that viral apoptotic mimicry is an immune evasion Viral apoptotic mimicry mechanism used by hepatitis B virus (HBV). The premise being that during chronic HBV Ali Amara and Jason Mercer infection, large-scale production of non- infectious subviral particles, composed of Abstract | As opportunistic pathogens, viruses have evolved many elegant phosphatidylserine-rich host membranes strategies to manipulate host cells for infectious entry and replication. Viral and viral membrane proteins, would be able apoptotic mimicry, defined by the exposure of phosphatidylserine — a marker for to suppress adaptive immunity through apoptosis — on the pathogen surface, is emerging as a common theme used by interactions between phosphatidylserine and cellular apoptotic clearance receptors. enveloped viruses to promote infection. Focusing on the four best described Since Vanlandschoot and Leroux-Roels’ examples (vaccinia virus, dengue virus, Ebola virus and pseudotyped lentivirus), we prediction more than a decade ago, viral summarize our current understanding of apoptotic mimicry as a mechanism for apoptotic mimicry has been experimentally virus entry, binding and immune evasion. We also describe recent examples of confirmed for several enveloped viruses. non-enveloped viruses that use this mimicry strategy, and discuss future directions These include alphaviruses, flaviviruses, filoviruses, some arenaviruses, baculoviruses, and how viral apoptotic mimicry could be targeted therapeutically. poxviruses and rhabdoviruses14–21 (TABLE 1). By containing phosphatidylserine within Through millions of years of coexistence of an anti-inflammatory response. The their membranes, these viruses effectively with their hosts, viruses have evolved to externalization of phosphatidylserine on mimic apoptotic cells, thereby subverting exploit the essential functions of the cells apoptotic cells was recently shown to depend apoptotic clearance mechanisms to facilitate that they invade, such as endocytosis, on caspase-mediated cleavage of the phos- virus entry or infection. For many viruses secretion, cell division and apoptosis. Viral pholipid flippase ATP11C5. When external- that use apoptotic mimicry, their cellular pathogens use many mechanisms to evade ized, phosphatidylserine can be recognized phosphatidylserine receptors have been or subvert apoptosis, including inactivation by subsets of receptors on the surface of identified, and the role of this strategy in of apoptotic sensors, molecular mimicry of both professional and non-professional virus infection has been characterized apoptotic regulators and perturbation of phagocytes (reviewed in REF. 6). These spe- (reviewed in REF. 22). caspase cleavage (reviewed in REF. 1). It was cialized receptors perform two functions: Interestingly, accumulating evidence sug- recently shown that viruses also hijack apop- they trigger the signalling cascades and gests that apoptotic mimicry can facilitate totic recognition and clearance mechanisms actin rearrangements that are required for infection by several different mechanisms. In for their own means. By eliminating abnor- the endocytic engulfment, trafficking and this Progress article, we discuss recent stud- mal or harmful cells, dampening immune degradation of apoptotic debris, and they ies indicating that viral apoptotic mimicry responses and assuring normal tissue and concomitantly initiate the production of promotes virus entry, enhances virus bind- organ formation during development, the anti-inflammatory cytokines while suppres ing to host cells or dampens host immune clearance of apoptotic cells serves an essen- sing the transcription of pro-inflammatory responses, depending on the virus and the tial function in all multicellular organisms cytokines7–9 (BOX 1). phosphatidylserine receptor or receptors that (reviewed in REF. 2). Not surprisingly, this Given these properties, it is easy to envi- are engaged. Recent findings suggest that process is highly conserved and, importantly, sion why a virus would evolve to use a strat- both enveloped and non-enveloped viruses actively anti-inflammatory3. egy of apoptotic mimicry. By masquerading can use this strategy, so we introduce the To distinguish dead and dying cells as apoptotic debris, viruses could directly terms classic and non-classic apoptotic from their healthy counterparts, phosphati- engage the apoptotic clearance machinery. mimicry, respectively. Focusing on the dylserine, which is a negatively charged This would in turn trigger uptake by endo- best described examples to date, we review phospholipid, is exposed on the external cytosis and simultaneously dampen the host phosphatidylserine-mediated entry of vac- leaflet of the plasma membrane as a hall- immune response. As many (perhaps all) cinia virus (VACV), Ebola virus (EBOV) mark of programmed cell death4. There cell types are capable of clearing apoptotic and dengue virus (DENV), phosphatidyl- are five key steps involved in the clearance cells10–12, an apoptotic mimicry strategy serine-enhanced binding of lentivirus vec- of apoptotic debris: phosphatidylserine may enable a virus to expand its cell type tors and the dampening of innate immune exposure on the apoptotic cell; engage- tropism without the need to encode specific responses by phosphatidylserine-containing ment of phosphatidylserine receptors on receptor-binding proteins. The idea that pseudotyped lentiviral particles. We also a phagocyte; endocytic engulfment of the viruses might subvert cellular apoptotic discuss how a non-enveloped virus, simian apoptotic cell; intracellular trafficking of the clearance machinery was first proposed by virus 40 (SV40), engages phosphatidylser- phagosome for degradation; and induction Vanlandschoot and Leroux-Roels in 2003 ine receptors for internalization. As all of

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Box 1 | Apoptotic clearance obtain a phosphatidylserine-rich membrane. Several enveloped viruses that use apoptotic In a healthy adult, approximately 50 billion cells undergo apoptosis on a daily basis69. Given these mimicry, such as EBOV and Marburg virus vast numbers, it is not surprising that defects in the clearance of apoptotic cells can result in (MARV), have been suggested to bud from various diseases. The major hallmark of apoptosis is exposure of phosphatidylserine on the cell plasma membrane microdomains called surface70. This negatively charged phospholipid is found on the inner leaflet of the plasma membrane in healthy cells26, but during apoptosis, phosphatidylserine is exposed on the external lipid rafts, which are highly enriched for 29,30 leaflet of the plasma membrane. This change in phosphatidylserine localization is mediated by the phosphatidylserine in the external leaflet . inactivation of lipid flippases in combination with activation of calcium-dependent phospholipid Infection by many viruses triggers apoptosis scramblases. of the target cell; thus, it is likely that these Clearance of apoptotic cells is initiated when these phosphatidylserine-enriched membranes viruses acquire phosphatidylserine by bud- engage phosphatidylserine receptors. Two types of phosphatidylserine receptor have been ding from the surface of apoptotic cells, described: those that bind the phospholipid directly and those that use bridging molecules to as has been suggested for the arenavirus associate with it. Direct phosphatidylserine-binding receptors include T cell immunoglobulin and Pichinde virus (PICV)31. In lieu of inducing mucin receptor (TIM) proteins (TIM1, TIM3 and TIM4); the CD300 family members CD300a and apoptosis, cell surface phosphatidylserine CD300f (also known as CLM1); and the seven-transmembrane spanning receptors brain-specific exposure may be the consequence of a virus- angiogenesis inhibitor 1 (BAI1), stabilin 1 and receptor for advanced glycosylation end products induced increase in intracellular calcium (RAGE) (reviewed in REFS 2,71,72). The phosphatidylserine-bridging molecule MFGE8 is used for 32 apoptotic clearance through αvβ3 and αvβ5 integrins73, which are indirect phosphatidylserine concentrations . This rise in calcium levels receptors. Similarly, GAS6 and protein S (PROS) are the bridging molecules that link the indirect can inactivate lipid flippases — proteins phosphatidylserine receptors of the tyrosine protein kinase receptor 3 (TYRO3)–AXL–MER (TAM) that translocate phospholipids between family to phosphatidylserine to mediate apoptotic clearance72. membrane leaflets and thus maintain phos- The mechanism used for the engulfment of apoptotic cells by phagocytes most closely resembles phatidylserine asymmetry — and result in macropinocytosis74. This endocytic mechanism is largely actin driven and regulated by the RHO phosphatidylserine externalization33. It is GTPases RAC1 and CDC42 (REF. 75). Hallmarks of macropinocytosis include vigorous membrane likely that enveloped and non-enveloped 36 ruffling, uptake into loose-fitting fluid-filled vacuoles and bystander endocytosis . Although, the viruses alike would be able to exploit such a molecular details remain incomplete76, genetic screens in Caenorhabditis elegans have identified mechanism; however, as there is no literature components of the signalling pathways that drive RHO GTPase activation and the subsequent cytoskeletal rearrangements needed for clearance64. Once engulfment is complete, the apoptotic to support this theory, it remains to be deter- corpse is digested; akin to classic endosomes, the vacuole containing the apoptotic cell undergoes mined whether viruses use this strategy to stepwise maturation involving a switch of RAB GTPases, exchange of phosphoinositides, acquire phosphatidylserine. acidification and movement towards the nucleus (reviewed in REF. 76). These steps occur in preparation for fusion with lysosomes, which contain the lytic enzymes, peptidases and DNases Host cell entry via apoptotic mimicry needed to break down the incoming apoptotic debris. Many of the viruses that have been reported In addition, binding of apoptotic cells by professional phagocytes initiates the production of the to use apoptotic mimicry do so to facilitate 7,77 anti-inflammatory cytokines transforming growth factor‑β (TGFβ) and interleukin‑10 (IL‑10) . The host cell entry (TABLE 1). As mentioned above, interaction of apoptotic cells and phagocytes also modulates the inflammatory response at the divergent mechanisms of apoptotic mimicry transcriptional level by suppressing the transcription of genes encoding pro-inflammatory have been described, so we introduce the cytokines, including IL‑12 (REFS 8,9,78). Both professional and non-professional phagocytes produce a subset of cell survival and growth factors following apoptotic clearance3. Collectively, terms classic and non-classic apoptotic mim- apoptotic clearance prevents inflammation, shapes the immune response and maintains tissue icry. Classic apoptotic mimicry refers to an homeostasis. enveloped virus that exposes phosphatidylserine on the external leaflet of its membrane to engage phosphatidylserine receptors for these mechanisms rely on ligand binding to intracellular organelles or the plasma uptake and/or immunomodulatory pur- phosphatidylserine receptors, a comprehen- membrane23 (FIG. 1). DENV and other flavi poses. Conversely, non-classic apoptotic sive understanding of the various apoptotic viruses derive their membrane from the mimicry refers to a non-enveloped virus mimicry strategies used by these viruses may endoplasmic reticulum (ER) via budding24. that, through the use of mimicry or host aid the development of new broad-spectrum The luminal leaflet of the ER membrane is membrane hijacking, engages apoptotic antiviral agents. enriched for phosphatidylserine25,26, which clearance receptors to infect cells. suggests a potential mechanism by which Phosphatidylserine acquisition by virions flaviviruses acquire this phospholipid. It has Classic apoptotic mimicry. Viruses hijack During apoptosis, the asymmetrical dis- been reported that the membrane of VACV various endocytic mechanisms of a host tribution of phospholipids in the plasma is derived from membrane sheets that are cell for internalization (reviewed in REF. 34). membrane is disrupted, which results in generated by the rupture of ER cisternae27. Clathrin-mediated endocytosis (CME) and the externalization of phosphatidylserine A recent study of a VACV mutant arrested macropinocytosis (reviewed in REF. 35) are to the outer leaflet5. As phosphatidylserine during membrane formation shows that the two most common host mechanisms exposure is essential for apoptotic clearance4, viral membranes were contiguous with that are subverted for virus entry. CME is a the acquisition of this membrane phospho- the ER and oriented such that the external constitutive process driven by the formation lipid during viral assembly is critical for leaflet of the viral membrane faced the ER of clathrin-coated vesicles with a diameter viral apoptotic mimicry. The mechanisms lumen28. These findings further indicate that of 100 nm. During macropinocytosis, which by which viruses acquire phosphatidylserine the phosphatidylserine-rich luminal leaflet is not constitutive and requires induction, are poorly understood, but possible strate- of the ER becomes the external leaflet of the a large volume of extracellular material is gies include hijacking phosphatidylserine- viral membrane. Such a mechanism would endocytosed in macropinosomes, which are rich cellular membranes by budding from explain how VACV and other poxviruses vesicles with a diameter of up to 10 μm that

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Table 1 | Viruses using apoptotic mimicry Virus genus* Phosphatidylserine function Phosphatidylserine Form of apoptotic Refs in viral life cycle receptors mimicry

Alphavirus (CHIKV, EEEV, RRV and SINV) Binding, endocytosis and AXL, CD300a, Classic 15,19,20,52 infection MFGE8‑binding integrins, TIM1 and TIM4 Arenavirus (AMAV, LASV, LCMV, PICV and TCRV) Binding, endocytosis and AXL, TIM1 and TYRO3 Classic 15,21,31,48 infection Alphabaculovirus (AcMNPV) Binding, endocytosis and AXL and TIM1 Classic 19,52 infection Ebolavirus and Marburgvirus (EBOV and MARV) Binding, endocytosis, infection AXL, TIM1, TIM4 and Classic 14–16,19, and immune evasion TYRO3 44,79 Flavivirus (DENV, WNV and YFV) Binding, endocytosis, infection AXL, TIM1, TIM3, Classic 14,15,17 and immune evasion TIM4 and TYRO3 Orthopoxvirus (VACV; both mature virions and Signalling, endocytosis and AXL Classic 18,20, extracellular virions) infection 39,40 Vesiculovirus (VSV) Binding, endocytosis and AXL and TIM1 Classic 19,52 infection Enterovirus (PV) Infection Unknown Non-classic 56 (autophagosome-like vesicle hijacking) Hepatovirus (HAV) Unknown TIM1 Non-classic 54,80,81 (budding into MVBs) Polyomavirus (SV40) Binding, endocytosis and AXL Non-classic 53 infection (GAS6 mimicry) AcMNPV, Autographa californica multicapsid nucleopolyhedrovirus; AMAV, Amapari virus; CHIKV, Chikungunya virus; DENV, dengue virus; EBOV, Ebola virus; EEEV, eastern equine encephalitis virus; HAV, hepatitis A virus; LASV, Lassa virus; LCMV, lymphocytic choriomeningitis virus; MARV, Marburg virus; MBVs, multivesicular bodies; PICV, Pichinde virus; PV, poliovirus; RRV, Ross River virus; SINV, Sindbis virus; SV40, simian virus 40; TCRV, Tacaribe virus; TIM, T cell immunoglobulin and mucin receptor; TYRO3, tyrosine protein kinase receptor 3; VACV, vaccinia virus; VSV, vesicular stomatitis virus; WNV, West Nile virus; YFV, yellow fever virus. *Listed are the virus genera (and viruses in parenthesis) that have been shown to use apoptotic mimicry. The respective viral families represented by these genera are Togoviridae (Alphavirus), Baculoviridae (Alphabaculovirus), Filoviridae (Ebolavirus and Marburgvirus), Flaviviridae (Flavivirus), Poxviridae (Orthopoxvirus), Rhabdoviridae (Vesiculovirus), Picornaviridae (Enterovirus and Hepatovirus) and Polyomaviridae (Polyomavirus). form without a coat35,36. After endocytosis, phosphatidylserine exposed on the virion macropinocytosis and, thus, for productive clathrin-coated vesicles feed into the clas- membrane initiates viral uptake by binding infection18,36. A subsequent study confirmed sic endosome trafficking pathway, whereas to direct phosphatidylserine receptors, such that phosphatidylserine is the only biologi- macropinosomes traffic and mature in paral- as T cell immunoglobulin and mucin recep- cally relevant, naturally occurring lipid con- lel. In both cases, maturation of the vesicular tor 1 (TIM1; also known as HAVCR1) and stituent of VACV mature virions that can compartments provide the virus with a other members of the TIM family, or, via rescue infection39, and further work impli- plethora of cues that facilitate infection, bridging molecules, to indirect phosphati- cated AXL as the phosphatidylserine recep- including decreasing pH and the presence of dylserine receptors, such as the receptor tor for these viruses40 (FIG. 1). Overexpression proteases and inorganic ions. However, there tyrosine kinase AXL (also known as UFO) of the TAM ligand GAS6, which acts as a are differences between classic endosomes and other members of the TYRO3–AXL– bridging molecule between phosphatidyl- and macropinosomes, including intralu- MER (TAM) family (BOX 1). serine and AXL (BOX 1), was also reported minal vesicle formation, and the presence The first experimental evidence of a to enhance infection of VACV extracel- of RAB effectors, major histocompatibility virus using apoptotic mimicry for entry lular virions20, a second form of infectious complex (MHC) class II molecules and was provided in 2008 for VACV mature VACV that is produced when mature virions Toll-like receptors (TLRs)37. Together, these virions18. Previous studies had indicated acquire additional membranes from host two pathways facilitate the internalization of that the VACV membrane is phosphati- cell organelles. Although it has not been most viruses, regardless of their varying size dylserine rich and that phosphatidylserine demonstrated that the membranes of extra- and intracellular trafficking requirements. can reactivate VACV virions that have cellular virions contain exposed phosphati- Apoptotic cells are phagocytosed by a been delipidated38. More recently, detailed dylserine (as the mature-virion membrane mechanism that closely resembles macro- analysis of the entry mechanism of VACV does), these virions do enter host cells using pinocytosis. Some viruses that use classic mature virions showed that the virus enters macropinocytosis41, suggesting that they too apoptotic mimicry, such as poxviruses and host cells by macropinocytosis. Masking rely on apoptotic mimicry for infection. filoviruses, have been shown to enter cells by phosphatidylserine using annexin V (also Similarly to VACV and apoptotic cells, inducing this endocytic pathway. However, known as annexin A5; a protein that binds EBOV uses macropinocytosis for host cell as we describe below, other viruses, includ- to phosphatidylserine with very high affin- entry42,43. The finding that ectopic expression ing flaviviruses and alphaviruses, enter ity) indicated that phosphatidylserine on the of the phosphatidylserine receptors tyrosine host cells using CME (FIG. 1). In either case, viral surface is required for the induction of protein kinase receptor 3 (TYRO3), AXL or

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Cell membrane Exposed phosphatidylserine Phosphatidylserine- bridging molecule Lipid raft AXL or TYRO3

Budding from lipid rafts TIM1 or EBOV TIM4 Phosphatidylserine acquisition from ER Phosphatidylserine sheets (?)

Macropinocytic virus entry and signalling AXL ER Viral glycoprotein Lysis VACV

Budding from the ER lumen AXL or TYRO3 Clathrin

TIM1, TIM3 or TIM4 DENV Clathrin-mediated Flippase virus entry and Modulation of signalling lipid ﬂippases (?)

Figure 1 | Classic apoptotic mimicry. During classic apoptotic mimicry, evidence indicates that phosphatidylserine exposed on the viral surface a virus acquires host cell phosphatidylserine and incorporates it into the binds to both direct phosphatidylserine receptors,Nature Reviews such as T| Microbiologycell immuno- viral membrane. Exposed phosphatidylserine on the viral surface binds globulin and mucin receptor (TIM) proteins, and indirect phosphatidylserine directly or indirectly to phosphatidylserine receptors, which facilitate virus receptors, such as AXL and tyrosine protein kinase receptor 3 (TYRO3), entry or infection. Shown are several potential strategies that viruses may which require phosphatidylserine-bridging molecules. Both EBOV and use to acquire phosphatidylserine in their membranes during assembly. DENV have been shown to use both direct and indirect phosphatidylserine Ebola virus (EBOV) has been shown to bud from plasma membrane micro- receptors, whereas VACV has only been shown to use the indirect receptor domains, or lipid rafts, that are highly enriched for phosphatidylserine in AXL. Whether EBOV and DENV can engage these various receptors simul- the external leaflet. Furthermore, it has been proposed that vaccinia virus taneously or whether VACV can use other phosphatidylserine receptors (VACV), which acquires its membrane within the host cytoplasm and exits has not been determined. For some viruses, such as EBOV and VACV, host cells by inducing cell lysis, derives its membrane from endoplasmic engagement of phosphatidylserine receptors triggers their internalization reticulum (ER) sheets generated by the rupture of ER cisternae. Finally, by macropinocytosis. For other viruses, including DENV, binding of dengue virus (DENV) and other flaviviruses derive their membrane via ER phosphatidylserine to receptors on the host cell surface induces clathrin- budding. Although these examples cover a range of mechanisms, it is also mediated uptake, which is an alternative mechanism of endocytosis. After possible that phosphatidylserine enrichment is facilitated by the viral internalization, downstream signalling cascades promote additional steps modulation of lipid flippases or of apoptosis (not illustrated). Recent of infection.

TIM1 enhanced macropinocytic virus entry the phosphatidylserine receptor-mediated enveloped RNA viruses that cause various provided the first link between apoptotic enhancement of infection. Consistent medically relevant human diseases, includ- mimicry and EBOV16,44,45. It was originally with this, phosphatidylserine-containing ing haemorrhagic fever and encephalitis46. It proposed that EBOV glycoproteins medi- liposomes and annexin V can both inhibit was recently shown that ectopic expression ate the association with TIM1 (REF. 16). TIM1‑mediated enhancement of EBOV of either TIM receptors (TIM1, TIM3 (also However, recent studies showed that expo- infection, as they compete for phosphatidyl- known as HAVR2) and TIM4 (also known sure of phosphatidylserine on viral particles serine receptors and mask phosphatidylserine, as TIMD4)) or TAM receptors (AXL and is essential for EBOV binding to TIM1 and respectively15,19. TYRO3) enhanced flavivirus endocytosis subsequent virus entry15,19 (FIG. 1), dem- Members of the Flavivirus genus, such as through CME (A.A., unpublished observa- onstrating that phosphatidylserine within DENV, West Nile virus (WNV) and yellow tions) (FIG. 1) and that subsequent infection the EBOV membrane was responsible for fever virus (YFV), are mosquito-borne by all DENV serotypes, WNV and YFV was

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also enhanced14,15,17. Moreover, it was shown that these viruses use an entry mechanism also been observed52. In addition, transduc- that phosphatidylserine is incorporated into that requires interactions with both phos- tion of a baculovirus was enhanced by the the membrane of flavivirus particles and phatidylserine receptors and virus-specific expression of TIM1 in non-permissive cell is essential for infection of TIM- or TAM- entry receptors. Infectivity of the arenavirus lines, and this enhancement was shown to expressing cells, suggesting that flaviviruses Lassa virus (LASV) is also enhanced by occur in a phosphatidylserine-dependent use apoptotic mimicry to invade host cells. phosphatidylserine48. LASV binds to well- manner19. Both baculoviruses and lenti- Characterization of the interactions revealed described extracellular (α‑dystroglycan) viruses are used as viral vectors for gene that virions bind directly to the TIM recep- and intracellular (lysosomal-associated delivery. The fact that these unrelated viral tors TIM1 and TIM4 and require the metal membrane protein 1 (LAMP1)) receptors vectors were found to use apoptotic mimicry ion-dependent ligand-binding site in these on host cells to facilitate entry49–51, but it for enhanced transduction suggests that it is receptors. By contrast, binding of TAM is not known whether these receptors are important to consider the consequences of receptors occurred indirectly via the bridg- required for phosphatidylserine-mediated phosphatidylserine-mediated residual trans- ing molecules GAS6 or protein S (PROS). enhancement of LASV infection or whether duction before the use of these vectors for These data strongly argue for a tripartite phosphatidylserine receptors are sufficient gene delivery. model, whereby TAM ligands bind to phos- for infection in the absence of these virus- phatidylserine exposed on DENV particles specific receptors. It is possible that, in some Non-classic apoptotic mimicry. Recent and bridge virions to TAM receptors. cases, phosphatidylserine receptors act as a findings indicate that even non-enveloped Collectively, these studies suggest that scaffold, recruiting additional virus-specific viruses have evolved strategies to engage viruses which use classic apoptotic mimicry receptors or cell factors that are required to apoptotic clearance receptors for internali- to enter cells undergo macropinocytosis or assemble an entry complex for viral endo- zation (FIG. 2), underscoring the adaptive CME. Currently, no clear pattern linking the cytosis. However, these proposed mecha- advantage offered by apoptotic mimicry choice of endocytic mechanism to receptor nisms of apoptotic mimicry have yet to be to a wide range of viruses. Non-enveloped preference can be delineated, nor can alter- experimentally tested. viruses do not have a membrane in which native endocytic mechanisms be ruled out. Classic apoptotic mimicry may also to incorporate phosphatidylserine and For viruses using CME, many of the studies enhance virus binding to host cells. The so are not able to engage in classic apop- are gain‑of‑function assays performed in first link between cellular phosphatidyl- totic mimicry. In the most divergent viral non-permissive cell lines; therefore, addi- serine receptors and enhanced virus bind- apoptotic mimicry mechanism, SV40, a tional experimental evidence is needed ing was provided by a study investigating non-enveloped polyomavirus, displays to determine whether phosphatidylserine the mechanism of residual transduction its own ligand for AXL in lieu of a bridg- receptor engagement by enveloped viruses of pseudotyped lentiviruses in which the ing molecule, thereby circumventing the is required for CME. Thus, it may be impor- natural envelope proteins had been substi- requirement for phosphatidylserine53. An tant to reinvestigate the endocytic mecha- tuted to ablate receptor-binding activity20. elegant predictive approach was taken to nism of virus internalization in the context Although the endocytic pathway used by identify this SV40 receptor, based on the of apoptotic mimicry. these pseudotyped lentiviruses when engag- principle that pathogens have evolved to The molecular mechanisms by which ing phosphatidylserine receptors was not exploit the cellular machinery of target cells phosphatidylserine receptors mediate virus determined, the authors showed that the and on the resultant hypothesis that pro- entry are poorly understood. It remains to be TAM ligands GAS6 and PROS enhanced teins from pathogens mimic the structure clarified whether phosphatidylserine recep- lentiviral binding to target cells expressing of host proteins which are recognized by tors mediate virus internalization directly AXL and TYRO3, thus leading to increased cellular receptors. In silico ligand–receptor or act in concert with unknown molecules transduction. Staining of pseudovirus par- modelling of the SV40 major capsid protein to coordinate viral endocytosis. In addi- ticles with the phosphatidylserine-binding VP1 against a library of known receptor tion, as these receptors are used by various protein annexin V showed that the lentivi- structures suggested that VP1 structurally viruses with different entry mechanisms, it is ral vectors contained phosphatidylserine mimics the bridging molecule GAS6 to plausible that the receptors are exploited for regardless of the viral envelope protein bind TAM receptors. This prediction was different purposes, depending on the virus. used for pseudotyping. This suggests that experimentally confirmed53 by binding For instance, several enveloped viruses enter phosphatidylserine acquisition, and thus studies between SV40 and both TYRO3–Fc host cells by receptor-mediated endocytosis apoptotic mimicry, is an intrinsic feature and AXL–Fc soluble recombinant proteins, and deliver their genome into the host of lentiviruses. Overexpression of the TAM and also by infection experiments in which cytosol through low-pH‑triggered fusion of receptors TYRO3 and AXL in a non-per- SV40 colocalized with AXL-expressing the viral and endosomal membranes47. For missive cell line enhanced GAS6‑mediated cells immediately after viral adsorption. flaviviruses and alphaviruses, this membrane transduction20. The link between lentiviral Apoptotic mimicry seems to be required fusion reaction is exclusively dependent on phosphatidylserine, GAS6 and AXL was for SV40 infectivity, as AXL knockdown in endosome acidification. The fact that phos- confirmed by showing that either mutation SV40‑permissive cells inhibits infection53. phatidylserine receptors are not required for of the phosphatidylserine-binding domain of The fact that SV40 usurps the apoptotic this step suggests that they act as bona fide GAS6 or incubation with AXL-specific anti- clearance pathway by mimicking the phos- entry receptors that are necessary and suf- bodies could abrogate viral transduction20. phatidylserine-bridging molecule GAS6 ficient to promote infection. Conversely, Apoptotic mimicry by lentiviruses does not suggests that the use of phosphatidylserine for the filoviruses MARV and EBOV, TIM1 seem to be limited to the use of TAM recep- receptors and the concept of viral apoptotic overexpression does not enhance virus entry tors, as bridging of viral phosphatidylserine mimicry is not restricted to enveloped in cells lacking the cognate entry receptor to integrin by the phosphatidylserine recep- viruses harbouring exposed phosphatidyl- for these viruses, NPC1 (REF. 15), suggesting tor MFGE8 (also known as lactadherin) has serine on their membranes.

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Cell membrane Mimicry of AXL phosphatidylserine- bridging molecule

Lysis SV40

VP1 (GAS6 mimic)

TIM1

Exosome-like HAV Budding into MVBs Phosphatidylserine

Indirect phosphatidylserine receptor (?) Phosphatidylserine- bridging molecule (?) PV receptor

Direct Autophagosome-like PV-containing phosphatidylserine double-membrane phosphatydylserine receptor (?) capture vesicle

Figure 2 | Non-classic apoptotic mimicry. In non-classic apoptotic MVBs fuse with the plasma membrane, the HAV particles cloaked in the mimicry, non-enveloped viruses use alternative means to engage phos- cell-derived envelope are released in a processNature Reviews thought | toMicrobiology be akin to phatidylserine receptors. For instance, the non-enveloped polyomavirus exosome egress. The phosphatidylserine-enriched exosome-like parti- simian virus 40 (SV40), which exits cells by lysis, mimics the phosphati- cles bind to T cell immunoglobulin and mucin receptor 1 (TIM1) on target dylserine-bridging molecule GAS6 to engage tyrosine protein kinase cells. To facilitate bulk virus transfer, poliovirus (PV) virions are captured receptor 3 (TYRO3)–AXL–MER (TAM) family receptors. By sharing struc- by autophagosome-like double-membrane vesicles. The outer mem- tural homology with GAS6, the SV40 major structural protein VP1 brane of these vesicles fuses with the cell surface to release phosphati- engages the indirect phosphatidylserine receptor AXL to initiate inter- dylserine-rich vesicles containing multiple PV virions. Both PV receptors nalization. Another non-enveloped virus, hepatitis A virus (HAV), prob- and phosphatidylserine in these vesicles are required for subsequent ably hijacks phosphatidylserine-containing membranes by budding into infection. However, the phosphatidylserine receptors required remain cellular organelles known as multivesicular bodies (MVBs). When the undefined.

Hepatitis A virus (HAV), which is a into MVBs may exit cells as exosome-like extracellular PV‑containing vesicles with member of the Picornaviridae family, uses membrane vesicles55. It is therefore plau- phosphatidylserine exposed on their outer TIM1 as an entry receptor54, suggesting that sible that phosphatidylserine within this leaflet. Blocking experiments using annexin this virus uses apoptotic mimicry for infec- acquired membrane engages TIM1 directly, V indicated that, in addition to the PV recep- tion (FIG. 2). As HAV is a non-enveloped thereby facilitating an apoptotic mimicry tors, phosphatidylserine on these vesicles virus and thus has no membrane, it is pos- mechanism. was required for infection56. Although the sible that it exploits TIM1 by mechanisms Another recent example of non-classic phosphatidylserine receptors and endocytic other than phosphatidylserine binding. apoptotic mimicry posits that the spread of mechanism exploited for entry remain to be However, it has been proposed that HAV enteroviruses, including poliovirus (PV), is determined, infection by PV within phos- particles become enveloped in host cell facilitated by bulk transfer of virions within phatidylserine vesicles was more efficient membrane when they bud into host phosphatidylserine vesicles56. After assem- than infection by free PV, emphasizing the cell organelles known as multivesicular bod- bly, non-enveloped cytoplasmic PV virions advantage of this non-classic apoptotic ies (MVBs)55. These organelles are thought are captured by large double-membrane mimicry strategy. to release vesicles known as exosomes into autophagosome-like vesicles. These vesi- To date, viruses from at least nine dif- the extracellular space on fusion with the cles then bud from the cell, leaving behind ferent families have been shown to use an plasma membrane. HAV particles that bud the outermost membrane, resulting in apoptotic mimicry strategy to engage host

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cell phosphatidylserine receptors (TABLE 1). Apoptotic cell However, it should be noted that conflict- Exposed Enveloped virus ing results have been observed in the phosphatidylserine Phosphatidylserine- case of the Old World arenaviruses LASV bridging molecule and lymphocytic choriomeningitis virus (LCMV). Whether these discrepancies are Indirect IL-10 phosphatidylserine due to experimental approaches (differ- IL-10 receptor TGFβ receptor ences in infection readouts, cell types and IFNAR virus production protocols) remains to be TLR clarified15,19,21,48. The most striking similar- ity between all these viruses is their broad in vitro and in vivo cell type specificity. This STATs broad tropism is possibly facilitated by the expression of phosphatidylserine receptors on various different host cell types. SOCS1 and SOCS3 Viral immune evasion Experimental evidence suggests that in addition to subverting host cell apoptotic Increased Dampened anti-inﬂammatory innate immune clearance machinery for the promotion of response response virus binding and uptake, viruses subvert this machinery for other purposes. For filo Cytokines SOCS1 and SOCS3 viruses as well as some arenaviruses and some flaviviruses, TAM receptor kinase activity has been implicated in the enhancement of infection14,17,21,57. Deletion of the Figure 3 | Viral apoptotic mimicry and immune evasion. The clearance of apoptotic cells and debris Nature Reviews | Microbiology cytoplasmic tail of AXL or mutation of its induces an anti-inflammatory response. Binding of apoptotic cells to phosphatidylserine receptors ATP-binding site — a site that is essential and the subsequent engulfment of these cells by phagocytes initiates the production of anti-inflam- for AXL kinase activity — inhibits DENV matory cytokines such as interleukin‑10 (IL‑10) and transforming growth factor-β (TGFβ). This initiates and WNV infection without having an a feed-forward suppression of the innate immune response at the level of transcription, and this suppression is dependent on prolonged signalling through signal transducer and activator of transcription effect on endocytosis14,17. Although the (STAT) family proteins. Akin to apoptotic cells, enveloped viruses, including pseudotyped lentivirus underlying mechanism remains unknown, vectors and West Nile virus, are thought to use apoptotic mimicry to dampen innate immune these results suggest that activation of AXL responses. Binding of an enveloped virus complexed to bridging molecules (such as GAS6) promotes facilitates a post-entry step of flavivirus the activation of tyrosine protein kinase receptor 3 (TYRO3)–AXL–MER (TAM) family receptors, which infection. Interestingly, apoptotic clearance heterodimerize with type I interferon receptor (IFNAR) to induce suppressor of cytokine signalling 1 is intimately linked with a dampening of (SOCS1) and SOCS3 expression, and this in turn inhibits IFNAR and Toll-like receptor (TLR) signalling. inflammatory responses (BOX 1; FIG. 3). On Although phosphatidylserine receptors are often studied individually, it is likely that viruses using formation of a tripartite complex with phos- apoptotic mimicry can simultaneously engage different phosphatidylserine receptors to modulate phatidylserine and GAS6, TAM receptors various innate immune and anti-inflammatory pathways and thus promote immune evasion. Of note, have been shown to heterodimerize with T cell immunoglobulin and mucin receptor (TIM) family phosphatidylserine receptors are not included in the figure owing to a lack of evidence for their participation in the dampening of immune response type I interferon receptor (IFNAR). This by viruses. leads to activation of IFNAR signalling, which triggers nuclear translocation of signal transducer and activator of transcription 1 in bone marrow-derived dendritic cells Interestingly, expression of CD300a (STAT1) and the subsequent transcription of (BMDCs) from knockout mice lacking all (also known as CLM8), a recently identi- suppressor of cytokine signalling 1 (SOCS1) three TAM receptors showed that pseudo fied direct phosphatidylserine receptor and SOCS3, which encode proteins that typed lentiviral particles containing expressed mainly on myeloid and mast contribute to the pleiotropic inhibition of phosphatidylserine on the viral surface cells59,60, has been shown to increase the inflammatory cytokines and of TLR signal- produced a strong antiviral response, with binding of pseudotyped lentiviral particles ling58. The major question that arises from high levels of Ifna4, Ifnb and Socs1 mRNAs to host cells, but without any change in these findings is whether virus-mediated compared with levels in infected BMDCs transduction efficiency52. Of note, engage- activation of phosphatidylserine receptors from wild-type mice. Importantly, inclusion ment of CD300a by apoptotic cells does not also dampens the innate immune response of antibodies against IFNα and IFNβ pro- promote their clearance; instead, on bind- to potentiate infection. teins restored the level of infection in TAM ing to phosphatidylserine, the cytoplasmic A recent study suggests that TAM ligands triple-knockout BMDCs from the reduced immunoreceptor tyrosine-based inhibi- complexed to pseudotyped lentiviral par- level caused by the antiviral response to that tion motif (ITIM) of CD300a recruits the ticles are ‘super TAM agonists’ that disable seen in wild-type cells. Therefore, these data phosphatase SHP1 (also known as PTPN6), host immune responses and facilitate virus indicate that inhibition of the antiviral type I which blocks the release of inflammatory spread14. It was shown that TAM-mediated IFN response is the primary mechanism of cytokines and chemokines61. Thus, it is inhibition of type I interferon (IFN) signal- TAM-mediated enhancement of lentiviral plausible that binding of a virus to CD300a ling enhances viral infection14. Experiments infection (FIG. 3). modulates the inflammatory and innate

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immune responses of the host rather than As the field of viral apoptotic mimicry of preclinical development as cancer thera- facilitating viral uptake by endocytosis. is still young, many important questions peutics65,66. In light of the use of these recep- Although direct evidence for using apo- have not yet been answered. How many tors by highly pathogenic viruses, including ptotic mimicry to dampen the host immune and which viruses exploit apoptotic mim- EBOV and DENV, these compounds should response is thus far limited to pseudotyped icry? For many of the enveloped viruses be tested for their potential antiviral efficacy. lentiviruses, similarities have been noted described, we still do not know how they In support of this, a recent report showed between immunosuppression during apo- acquire phosphatidylserine in their mem- that AXL-specific antibodies increased ptotic cell clearance and that during in vivo brane nor whether this is an active pro- innate immune responses and could attenu- VACV infections; VACV infections lead cess. For viruses that bud from the plasma ate influenza virus and respiratory syncytial to the upregulation of anti-inflammatory membrane, timed induction of apoptosis virus (RSV) lethality in vivo67. Although cytokines, including transforming growth or modulation of cellular ATP-dependent these particular viruses do not use apop- factor‑β (TGFβ) and interleukin‑10 (IL‑10), phospholipid flippases5 could possibly totic mimicry, this study serves as a proof which prevent macrophage infiltration provide a means of enriching phosphati- of concept for the therapeutic potential of and inhibit T cell maturation62. It has been dylserine on the membrane before virion in vivo AXL targeting. Conversely, it was proposed that the suppression of innate exit from the cell. Once a virus displays demonstrated that TIM receptors on virus- immunity facilitates unchecked viral replica- phosphatidylserine, does the distribution producing cells can capture HIV‑1 virions tion, but whether this is due to the engage- or concentration of this phospholipid in the through phosphatidylserine exposed on the ment of phosphatidylserine receptors — and viral membrane dictate binding to a distinct HIV‑1 membrane, thus restricting virion hence to apoptotic mimicry — remains to be phosphatidylserine receptor and, by exten- release and spread of infection68. Perhaps determined. sion, the cell type specificity of the virus? To for some viruses, therapeutic overexpres- These studies do suggest that phosphati- better understand viral apoptotic mimicry, sion (rather than inhibition) of phosphatidylserine receptors are activated by viruses it may be important to elucidate why some dylserine receptors could be considered. during binding and/or entry to modulate enveloped viruses, including herpes simplex Collectively, these results suggest that future innate immunity, but additional studies are virus 1 (HSV‑1), severe acute respiratory efforts should be directed at exploiting viral required to determine the exact molecular syndrome coronavirus (SARS‑CoV), influ- apoptotic mimicry, phosphatidylserine mechanisms involved and to extend the enza A virus and some arenaviruses, do not exposure and phosphatidylserine receptors findings to other viral systems. In addition, use apoptotic mimicry for infection17,19 and for the development of antiviral therapies. the concept of viral apoptotic mimicry being why some phosphatidylserine receptors, such Ali Amara is at Institut National de la Santé et de la a means of immune evasion has to date been as brain-specific angiogenesis inhibitor 1 Recherche Médicale U944 and Centre National reported only in infection studies performed (BAI1) and receptor for advanced glycosyla- de la Recherche Scientifique UMR 7212, with mouse BMDCs and awaits confirmation tion end products (RAGE), do not medi- Laboratoire de Pathologie et Virologie Moléculaire, Institut Universitaire d’Hématologie, Université Paris 52 in additional cell types and, more impor- ate virus entry . Also, the possibility that Diderot, Sorbonne Paris Cité, Hôpital Saint-Louis, tantly, in vivo. Furthermore, as several viruses viruses using apoptotic mimicry simultane- 1 avenue Claude Vellefaux, 75010 Paris, France. can engage both direct and indirect phos- ously engage different phosphatidylserine Jason Mercer is at the Medical Research Council phatidylserine receptors, whether apoptotic receptors to promote virus entry and modu- Laboratory for Molecular Cell Biology, University mimicry for the purpose of immune evasion late the immune response at the same time College London, Gower Street, London WC1E 6BT, UK. can be extended to direct phosphatidylserine should be investigated. e-mails: [email protected]; receptors, such as TIMs, should be explored. In addition, the in vivo relevance of viral [email protected] apoptotic mimicry needs to be explored. doi:10.1038/nrmicro3469 Perspectives Established mouse pathogenesis models exist Published online 8 June 2015 Apoptotic cell death and clearance are for several of the viruses described in this arti- 1. Benedict, C. 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Hochreiter-Hufford, A. & Ravichandran, K. S. Clearing the external leaflet of viral membranes phosphatidylserine showed a protective the dead: apoptotic cell sensing, recognition, engulfment, and digestion. Cold Spring Harb. and is required for binding, endocytosis or effect against viral infection in vivo, indi- Perspect. Biol. 5, a008748 (2013). infectivity. The broad use of viral apoptotic cating that targeting of phosphatidylserine 7. Voll, R. E. et al. Immunosuppressive effects of apoptotic cells. Nature 390, 350–351 (1997). mimicry strategies to enhance entry and may hold promise as an effective antiviral 8. Cvetanovic, M. & Ucker, D. S. Innate immune infection suggests that it is a general mech- strategy31. Several small-compound inhibi- discrimination of apoptotic cells: repression of proinflammatory macrophage transcription is coupled anism that is exploited by many enveloped tors and TAM decoy receptors directed directly to specific recognition. J. 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Human CD300a binds to Competing interests statement 1–17 (2009). phosphatidylethanolamine and phosphatidylserine, The authors declare no competing interests.

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