**4.1.2 CD46 is a cellular receptor for HHV6 and measles**

CD46 also designated as membrane cofactor protein (MCP) is a member of a family of glycoproteins acting as regulators of complement activation, but has subsequently been shown to link innate immunity to adaptative immunity (Seya et al., 1986). CD46 mediates the inactivation of its natural complement ligands C3b and C4b and acts as a cofactor for serine protease factor I (Lublin et al., 1988). CD46 thus protect host cells from homologous complement attack which could result in extensive cytolysis and widespread tissue damage. Another function of CD46 is to promote T cell differentiation from a helper Th1 to a regulatory Tr1 phenotype, depending on IL-2 concentrations (Cardone et al., 2010). Human CD46 is alternatively spliced into several isoforms, resulting in a varying number of extracellular domains and two different cytoplasmic tails, Cyt1 and Cyt2 (Seya et al., 1999). CD46 seems to be constitutively recycled from the cell surface via clathrin-coated pits and transported to perinuclear multivesicular bodies (Crimeen-Irwin et al., 2003). The importance of CD46 in the homeostasis of the organism is highlighted by its ubiquitous expression on the surface of all nucleated human cells (Liszewski et al., 1991).

Human CD46 is a cellular receptor for Human Herpesvirus 6 (HHV-6) (Santoro et al., 1999) and MV (Dorig et al., 1993). The authors demonstrated that both acute infection and cell to cell fusion mediated by HHV-6 were specifically inhibited by a monoclonal antibody to CD46; fusion was also blocked by soluble CD46. Interestingly, they showed that nonhuman cells resistant to HHV-6 fusion and entry became susceptible upon expression of recombinant human CD46. Concerning the study of MV infection in the CNS, a murine model of MV-induced pathology was developed from several lines of transgenic mice expressing human CD46 molecule with Cyt1 or Cyt2 (Evlashev et al., 2000). The results of their experiments showed that all transgenic mice expressing CD46 protein in the brain were highly sensitive to intracerebral infection by the MV Edmonston strain, in comparison to nontransgenic controls. In addition, the two isoforms of CD46, Cyt1 and 2, are functionally similar in mediating MV entry into the brain.

#### **4.1.3 Ephrins are cellular receptors for Hendra virus (HeV) and Nipah virus (Niv)**

The Eph (erythropoietin-producing hepatocellular) receptors are the largest family of tyrosine kinase receptors (TRKs). They interact with cell surface bound ligands, the ephrins (Eph receptor interacting proteins) that are a highly conserved class of proteins with many homologous members. Based on structural differences, two classes of ephrins can be classified: ephrins-A (A1 to A6) are attached to the plasma membrane via glycophosphatidyl inositol (GPI) moiety and ephrins B (B1 to B3) crossing the plasma membrane and possessing a short cytoplasmic tail. Eph receptors have diverse functions, such as widespread effects on the actin cytoskeleton, cell-substrate adhesion, intercellular junctions, cell shape, and cell movement (Egea and Klein, 2007; Himanen et al., 2007; Pasquale, 2005; Pasquale, 2008).

KO and double KO mice. The results indicated that nectin-1 deficient mice showed no signs of disease after intracranial inoculation, and no HSV antigens were detectable in the brain parenchyma. However, HSV antigens were detected in non-parenchymal cells lining the ventricules. In the double KO mice, the results showed an absence of disease and no detectable expression of viral antigens even in non-parenchymal cells, indicating that infection of these cells in the nectin-1 KO mice was dependent on the expression of HveM.

CD46 also designated as membrane cofactor protein (MCP) is a member of a family of glycoproteins acting as regulators of complement activation, but has subsequently been shown to link innate immunity to adaptative immunity (Seya et al., 1986). CD46 mediates the inactivation of its natural complement ligands C3b and C4b and acts as a cofactor for serine protease factor I (Lublin et al., 1988). CD46 thus protect host cells from homologous complement attack which could result in extensive cytolysis and widespread tissue damage. Another function of CD46 is to promote T cell differentiation from a helper Th1 to a regulatory Tr1 phenotype, depending on IL-2 concentrations (Cardone et al., 2010). Human CD46 is alternatively spliced into several isoforms, resulting in a varying number of extracellular domains and two different cytoplasmic tails, Cyt1 and Cyt2 (Seya et al., 1999). CD46 seems to be constitutively recycled from the cell surface via clathrin-coated pits and transported to perinuclear multivesicular bodies (Crimeen-Irwin et al., 2003). The importance of CD46 in the homeostasis of the organism is highlighted by its ubiquitous

Human CD46 is a cellular receptor for Human Herpesvirus 6 (HHV-6) (Santoro et al., 1999) and MV (Dorig et al., 1993). The authors demonstrated that both acute infection and cell to cell fusion mediated by HHV-6 were specifically inhibited by a monoclonal antibody to CD46; fusion was also blocked by soluble CD46. Interestingly, they showed that nonhuman cells resistant to HHV-6 fusion and entry became susceptible upon expression of recombinant human CD46. Concerning the study of MV infection in the CNS, a murine model of MV-induced pathology was developed from several lines of transgenic mice expressing human CD46 molecule with Cyt1 or Cyt2 (Evlashev et al., 2000). The results of their experiments showed that all transgenic mice expressing CD46 protein in the brain were highly sensitive to intracerebral infection by the MV Edmonston strain, in comparison to nontransgenic controls. In addition, the two isoforms of CD46, Cyt1 and 2, are

**4.1.3 Ephrins are cellular receptors for Hendra virus (HeV) and Nipah virus (Niv)**  The Eph (erythropoietin-producing hepatocellular) receptors are the largest family of tyrosine kinase receptors (TRKs). They interact with cell surface bound ligands, the ephrins (Eph receptor interacting proteins) that are a highly conserved class of proteins with many homologous members. Based on structural differences, two classes of ephrins can be classified: ephrins-A (A1 to A6) are attached to the plasma membrane via glycophosphatidyl inositol (GPI) moiety and ephrins B (B1 to B3) crossing the plasma membrane and possessing a short cytoplasmic tail. Eph receptors have diverse functions, such as widespread effects on the actin cytoskeleton, cell-substrate adhesion, intercellular junctions, cell shape, and cell movement (Egea and Klein, 2007; Himanen et al., 2007; Pasquale, 2005;

expression on the surface of all nucleated human cells (Liszewski et al., 1991).

**4.1.2 CD46 is a cellular receptor for HHV6 and measles** 

functionally similar in mediating MV entry into the brain.

Pasquale, 2008).

Two independent studies identified Ephrin B2 (EFNB2) as the receptor for HeV and NiV, which are emergent paramyxovirus. Of the ten potential receptor-encoding plasmids they used to transfect non-permissive HeLa cells, only the human EFNB2 plasmid confers HeV fusion permissiveness. They further demonstrated that soluble EFNB2 blocks both viruses fusion in human EFNB2-transfected HeLa cells. They also showed that both HeV and NiV glycoprotein G and EFNB2 soluble protein could be specifically and reciprocally captured by ELISA. In addition, both viruses glycoprotein G were efficiently coprecipitated by EFNB2/Fc. Another study identified EFNB2 as an entry receptor for NiV (Negrete et al., 2005). The authors also reported that EFNB2 binds to the NiV glycoprotein G. They observed that soluble Fc-fusion proteins of EFNB2 effectively blocked NiV fusion and entry into permissive cell types. Moreover, transfection of EFNB2 into non-permissive cells renders them permissive for NiV fusion and entry. Interestingly they found that soluble EFNB2 inhibited NiV-envelope-mediated infection of microvascular endothelial cells and primary cortical rat neurons. EFNB3 was later discovered as an additional receptor for NiV using the Chinese hamster ovary cell line (CHO-pgsA745) that does not express ephrins endogenously (Negrete et al., 2006). Interestingly, the authors characterized the important conserved Leu-Trp residues in the G-H loop of EFNB2 and B3 that are critical for their binding with NiV glycoprotein G.

#### **4.1.4 Scavenger receptor class B member 2 (SCARB2) and EV71**

The concept of scavenger receptors (SCAR) was first described by their ability to bind modified low-density lipoproteins (Goldstein et al., 1979). SCAR can bind a variety of natural/endogenous, modified host, microbial (bacterial, viral, fungal, and parasitic), environmental, soluble or particulates ligands by endocytosis and phagocytosis (reviewed in (Mukhopadhyay and Gordon, 2004)). SCARB2 (also known as lysosomal integral membrane protein II or CD36b like-2) belongs to the scavenger receptor class B subfamily, which also includes SCARB1 and CD36 (Calvo et al., 1995; Eskelinen et al., 2003). SCARB2 is a type III double-transmembrane protein with N- as well as C-terminal cytoplasmic tails and is located primarily in lysosomes and endosomes. SCARB2 participates in membrane transportation and the reorganization of the endosomal-lysosomal compartment (Kuronita et al., 2002). Deficiency of SCARB2 in mice causes ureteric pelvic junction obstruction, deafness and peripheral neuropathy (Gamp et al., 2003). SCARB2 is widely expressed in lysosomes and late endosomes (reviewed in (Eskelinen et al., 2003).

Since its identification in California in 1969 (Schmidt et al., 1974), enterovirus 71 (EV71) has been recognized as a frequent cause of epidemics of hand-foot-and-mouth disease (HFMD) associated with severe neurological sequelae in some cases (Blomberg et al., 1974). EV71 infections can progress to aseptic meningitis, acute flaccid paralysis and fatal encephalitis (McMinn, 2002). A recent paper demonstrated that SCARB2 is a receptor for EV71 (Yamayoshi et al., 2009). The authors showed that EV71 binds soluble SCARB2 or cells expressing SCARB2, and the binding is inhibited by an antibody to SCARB2. Furthermore, expression of human SCARB2 enables normally unsusceptible cell lines to support EV71 propagation and develop cytopathic effects. Yet, the detail of the interaction between EV71 and SCARB2 at the cellular level, resulting in CNS infection has not been identified.

#### **4.1.5 CD4 as the primary receptor for HIV**

Human CD4 exhibits a poly-Ig-like domain structure which is homologous to an increasingly large number of recognition molecules, and consists in four tandem variable-

Virus-Induced Encephalitis and

1996); fusin was thus renamed CXCR4.

2003).

2004; Kroemer et al., 2007).

Innate Immune Responses – A Focus on Emerging or Re-Emerging Viruses 69

terminus. The two major classes are the CXC chemokines (CXCR1 to CXCR5) in which the first two cysteines are separated by a single residue, and the CC chemokines (CCR1 to CCR9) in which the first two cysteines are adjacent. CXCR4 (also termed fusin, HUMSTSR, LESTR, HM89, LCR1, NPYR, D2S201E) and CCR5 (also known as CKR5, CC CKR5, ChemR13, CMKBR5) are membrane-bound co-receptors for HIV entry. CXCR4 is expressed on neutrophils, myeloid cells (as well as microglia), and particularly on T lymphocytes (Loetscher et al., 1994). CCR5 mRNA expression was detected in lymphoid organs such as the thymus and spleen, and in peripheral T lymphocytes and macrophages (Raport et al., 1996). The first HIV-1 co-receptor was identified with the use of a recombinant vaccinia virus-based construct in which fusion between the HIV gp120 envelope glycoprotein (Env) expressing cells and CD4-expressing cells would lead to the activation of a reporter gene *Escherichia coli* LacZ (Feng et al., 1996). The screening of a HeLa cDNA plasmid library and subsequent sequence analysis of the insert revealed that the protein was a member of the G protein-coupled receptor superfamily and was designated "fusin". In addition, loss-offunction experiments showed that anti-fusin antibodies blocked cell fusion and infection of primary human CD4+ T lymphocytes. Interestingly, both experiments stressed that fusin functioned preferentially for T cell line (TCL)-tropic rather than for macrophage (M)-tropic HIV-1 isolates. The discovery of fusin provided an impetus for the identification of the Mtropic HIV isolates coreceptor. Several independent reports demonstrated that CCR5 is the major coreceptor for M-tropic HIV-1 strains (Alkhatib et al., 1996; Choe et al., 1996; Deng et al., 1996; Doranz et al., 1996; Dragic et al., 1996). The evidence was based on both gain-offunction studies using recombinant CCR5, and loss-of-function studies with CCR5 chemokines ligands (MCP-1, MIP-1, MIP-1 and RANTES) as blocking agents. After the discovery of CCR5, it was demonstrated that fusin is a chemokine receptor that specifically recognize the CXC chemokines ligands SDF-1 and SDF-1 (Bleul et al., 1996; Oberlin et al.,

**4.2.2 Phosphatidylserine (PS), apoptotic blebs and virus spreading from cells to cells**  PS is a phospholipid located on the inner leaflet of the plasma membrane (Williamson and Schlegel, 1994; Zwaal and Schroit, 1997). Its internal positioning is maintained by translocases that catalyze aminophospholipid transport from the external to the internal leaflet of the plasma membrane. The loss of PS asymmetry can occur during several cellular events including cell injury, cell activation or apoptosis (Balasubramanian and Schroit,

Two apoptotic pathways can be identified; (i) the extrinsic apoptotic pathway is initiated by members of the Tumour necrosis factor (TNF) family of death ligands that bind to TNFR death receptor family members, Tumour Necrosis Factor Receptor –related apoptosis inducing signal ligand (TRAIL) and Fas ligand (FASL). This results in the formation of the Death Inducing Signal Complex DISC at the cell membrane. DISC subsequently interacts with Fas-associated death domain protein (FADD) in caspase 8, pro caspase 3 and this activates caspase 3, resulting in host cell death (Wilson et al., 2009); (ii) The intrinsic apoptosis pathway is dependent upon the activation of host proteins such as tumour suppressor protein P53 by virus infection. This in turn activates BH-3 domain only members of the B cell lymphoma 2 (Bcl-2) family, e.g the pro-apoptotic proteins Bax and Bak. The activation of these two proteins produces pores in the outer membrane of mitochondria with the release of cytochrome C activating caspase 9 and cell death (Danial and Korsmeyer,

joining (VJ)-like domains (Maddon et al., 1987). The Ig-like domains of CD4 are anchored to the membrane by a hydrophobic segment, and followed by a short cytoplasmic region. CD4 is expressed on the surface of T lymphocytes, monocytes, macrophages, and brain microglia (resident macrophages) (Wyatt and Sodroski, 1998).

HIV invades the CNS early following viral infection but provokes brain disease only years later. During the periods of progressive immunosuppression, neurological disease occurs in 15 to 20% of infected individuals which develop cognitive and motor impairments referred to as the HIV type 1 (HIV-1)-associated dementia complex also termed as HIV-associated dementia or HIV encephalopathy (Janssen et al., 1991; Price et al., 1988). Neurological disease is often associated with a marked depletion of CD4+ T lymphocytes (Navia et al., 1986). It is now know that HIV encephalitis is a common pathological manifestation characterized by monocyte infiltration into the brain, the formation of macrophage-derived multinucleated giant cells, microglial nodules, and demyelination (Sharer et al., 1985).

Considerable amount of data identified CD4 as the main entry receptor for HIV. Previous studies on the two AIDS retroviruses, human T-lymphotropic virus type III (HTLV-III) and lymphadenopathy-associated virus (LAV) helped to characterize the primary cellular receptor involved in HIV infection (Dalgleish et al., 1984; Klatzmann et al., 1984). Dalgleish and colleagues identified receptor-positive cells by assessing induction of multinucleated syncytia and by testing the susceptibility of various cell types to pseudotypes of vesicular stomatitis virus (VSV) carrying envelope antigens of HTLV strains. Their results showed that both VSV (HTLV-I) and VSV (HTLV-II) pseudotypes plated on a variety of cell types whereas the VSV (HTLV-III) was more specific to the two human T-cell lines (JM and CEM) tested. In addition, only cells expressing the CD4 antigen were sensitive to syncytium induction by HTLV-III and infection by VSV (HTLV-III) pseudotypes. After the screening of 155 monoclonal antibodies to T-cell surface antigens for their ability to inhibit the syncytia formation induced by HTLV-III, they demonstrated that pre-incubation of cells with all anti-CD4 antibodies blocked the cell fusion induced by HTLV-III. In another paper published the same year, Klatzman *et al*. (Klatzmann et al., 1984) investigated the relationship between the CD4+ tropism of LAV and the presence of CD4 on T lymphocytes. Similarly to the previous paper on HTLV-III, they reported that pre-incubation of CD4+ T lymphocytes with three monoclonal antibodies directed against the CD4 glycoprotein specifically blocked cell infection by LAV.

It is well documented that efficient entry of HIV-1 into target cells is dependent upon binding of the viral exterior envelope glycoprotein, gp120, to the CD4-amino-terminal domain (McDougal et al., 1986). After the virus binding, the HIV-1 envelope glycoproteins mediate the fusion of viral and host cell membranes to complete the fusion process. The formation of syncytia results in the membrane fusion directed by HIV-1 envelope glycoproteins expressed on the infected cell surface with uninfected CD4-positive cells. Although CD4 expression on a target cell seems to be sufficient for HIV attachment, the fusion process appeared to be more complex.

#### **4.2 Secondary receptors of virus entry**

#### **4.2.1 Chemokine receptors CXCR4 and CCR5 as HIV infection cofactors**

Chemokine receptors are part of the serpentine GTP-binding protein (G protein)-coupled receptors superfamily that include receptors for hormones, neurotransmitters, paracrine substance, inflammatory mediators, certain proteinases, taste and odorant molecules. All known human chemokines fit within four classes based on the cysteine motifs near the N-

joining (VJ)-like domains (Maddon et al., 1987). The Ig-like domains of CD4 are anchored to the membrane by a hydrophobic segment, and followed by a short cytoplasmic region. CD4 is expressed on the surface of T lymphocytes, monocytes, macrophages, and brain microglia

HIV invades the CNS early following viral infection but provokes brain disease only years later. During the periods of progressive immunosuppression, neurological disease occurs in 15 to 20% of infected individuals which develop cognitive and motor impairments referred to as the HIV type 1 (HIV-1)-associated dementia complex also termed as HIV-associated dementia or HIV encephalopathy (Janssen et al., 1991; Price et al., 1988). Neurological disease is often associated with a marked depletion of CD4+ T lymphocytes (Navia et al., 1986). It is now know that HIV encephalitis is a common pathological manifestation characterized by monocyte infiltration into the brain, the formation of macrophage-derived multinucleated giant cells, microglial nodules, and demyelination (Sharer et al., 1985). Considerable amount of data identified CD4 as the main entry receptor for HIV. Previous studies on the two AIDS retroviruses, human T-lymphotropic virus type III (HTLV-III) and lymphadenopathy-associated virus (LAV) helped to characterize the primary cellular receptor involved in HIV infection (Dalgleish et al., 1984; Klatzmann et al., 1984). Dalgleish and colleagues identified receptor-positive cells by assessing induction of multinucleated syncytia and by testing the susceptibility of various cell types to pseudotypes of vesicular stomatitis virus (VSV) carrying envelope antigens of HTLV strains. Their results showed that both VSV (HTLV-I) and VSV (HTLV-II) pseudotypes plated on a variety of cell types whereas the VSV (HTLV-III) was more specific to the two human T-cell lines (JM and CEM) tested. In addition, only cells expressing the CD4 antigen were sensitive to syncytium induction by HTLV-III and infection by VSV (HTLV-III) pseudotypes. After the screening of 155 monoclonal antibodies to T-cell surface antigens for their ability to inhibit the syncytia formation induced by HTLV-III, they demonstrated that pre-incubation of cells with all anti-CD4 antibodies blocked the cell fusion induced by HTLV-III. In another paper published the same year, Klatzman *et al*. (Klatzmann et al., 1984) investigated the relationship between the CD4+ tropism of LAV and the presence of CD4 on T lymphocytes. Similarly to the previous paper on HTLV-III, they reported that pre-incubation of CD4+ T lymphocytes with three monoclonal antibodies directed against the CD4 glycoprotein specifically blocked cell

It is well documented that efficient entry of HIV-1 into target cells is dependent upon binding of the viral exterior envelope glycoprotein, gp120, to the CD4-amino-terminal domain (McDougal et al., 1986). After the virus binding, the HIV-1 envelope glycoproteins mediate the fusion of viral and host cell membranes to complete the fusion process. The formation of syncytia results in the membrane fusion directed by HIV-1 envelope glycoproteins expressed on the infected cell surface with uninfected CD4-positive cells. Although CD4 expression on a target cell seems to be sufficient for HIV attachment, the

Chemokine receptors are part of the serpentine GTP-binding protein (G protein)-coupled receptors superfamily that include receptors for hormones, neurotransmitters, paracrine substance, inflammatory mediators, certain proteinases, taste and odorant molecules. All known human chemokines fit within four classes based on the cysteine motifs near the N-

**4.2.1 Chemokine receptors CXCR4 and CCR5 as HIV infection cofactors** 

(resident macrophages) (Wyatt and Sodroski, 1998).

infection by LAV.

fusion process appeared to be more complex.

**4.2 Secondary receptors of virus entry** 

terminus. The two major classes are the CXC chemokines (CXCR1 to CXCR5) in which the first two cysteines are separated by a single residue, and the CC chemokines (CCR1 to CCR9) in which the first two cysteines are adjacent. CXCR4 (also termed fusin, HUMSTSR, LESTR, HM89, LCR1, NPYR, D2S201E) and CCR5 (also known as CKR5, CC CKR5, ChemR13, CMKBR5) are membrane-bound co-receptors for HIV entry. CXCR4 is expressed on neutrophils, myeloid cells (as well as microglia), and particularly on T lymphocytes (Loetscher et al., 1994). CCR5 mRNA expression was detected in lymphoid organs such as the thymus and spleen, and in peripheral T lymphocytes and macrophages (Raport et al., 1996). The first HIV-1 co-receptor was identified with the use of a recombinant vaccinia virus-based construct in which fusion between the HIV gp120 envelope glycoprotein (Env) expressing cells and CD4-expressing cells would lead to the activation of a reporter gene *Escherichia coli* LacZ (Feng et al., 1996). The screening of a HeLa cDNA plasmid library and subsequent sequence analysis of the insert revealed that the protein was a member of the G protein-coupled receptor superfamily and was designated "fusin". In addition, loss-offunction experiments showed that anti-fusin antibodies blocked cell fusion and infection of primary human CD4+ T lymphocytes. Interestingly, both experiments stressed that fusin functioned preferentially for T cell line (TCL)-tropic rather than for macrophage (M)-tropic HIV-1 isolates. The discovery of fusin provided an impetus for the identification of the Mtropic HIV isolates coreceptor. Several independent reports demonstrated that CCR5 is the major coreceptor for M-tropic HIV-1 strains (Alkhatib et al., 1996; Choe et al., 1996; Deng et al., 1996; Doranz et al., 1996; Dragic et al., 1996). The evidence was based on both gain-offunction studies using recombinant CCR5, and loss-of-function studies with CCR5 chemokines ligands (MCP-1, MIP-1, MIP-1 and RANTES) as blocking agents. After the discovery of CCR5, it was demonstrated that fusin is a chemokine receptor that specifically recognize the CXC chemokines ligands SDF-1 and SDF-1 (Bleul et al., 1996; Oberlin et al., 1996); fusin was thus renamed CXCR4.

#### **4.2.2 Phosphatidylserine (PS), apoptotic blebs and virus spreading from cells to cells**

PS is a phospholipid located on the inner leaflet of the plasma membrane (Williamson and Schlegel, 1994; Zwaal and Schroit, 1997). Its internal positioning is maintained by translocases that catalyze aminophospholipid transport from the external to the internal leaflet of the plasma membrane. The loss of PS asymmetry can occur during several cellular events including cell injury, cell activation or apoptosis (Balasubramanian and Schroit, 2003).

Two apoptotic pathways can be identified; (i) the extrinsic apoptotic pathway is initiated by members of the Tumour necrosis factor (TNF) family of death ligands that bind to TNFR death receptor family members, Tumour Necrosis Factor Receptor –related apoptosis inducing signal ligand (TRAIL) and Fas ligand (FASL). This results in the formation of the Death Inducing Signal Complex DISC at the cell membrane. DISC subsequently interacts with Fas-associated death domain protein (FADD) in caspase 8, pro caspase 3 and this activates caspase 3, resulting in host cell death (Wilson et al., 2009); (ii) The intrinsic apoptosis pathway is dependent upon the activation of host proteins such as tumour suppressor protein P53 by virus infection. This in turn activates BH-3 domain only members of the B cell lymphoma 2 (Bcl-2) family, e.g the pro-apoptotic proteins Bax and Bak. The activation of these two proteins produces pores in the outer membrane of mitochondria with the release of cytochrome C activating caspase 9 and cell death (Danial and Korsmeyer, 2004; Kroemer et al., 2007).

Virus-Induced Encephalitis and

infection.

numbers of receptor for HSV.

**4.2.4 Heparan sulfate proteoglycans (HSPGs) and HSV** 

Innate Immune Responses – A Focus on Emerging or Re-Emerging Viruses 71

expressed by monocytes/microglia and possesses a globular carboxy terminal sequence that binds to virus proteins containing mannose molecules. The transmembrane lectins include the Mannose Membrane Receptor (MMR also known as CD206) and the Dentritic Cell-Specific ICAM-3 Grabbing Non-integrin (DC-SIGN also known as CD209) receptor that recognizes "self " intercellular adhesion molecule 2 (ICAM2) and ICAM3 (van Kooyk and Geijtenbeek, 2003). MMR contains eight CRD (Weis et al., 1998). DC-SIGN is a type II transmembrane protein which possesses a single globular-structured CRD. DC-SIGN's CRD is separated from the transmembrane region by a neck domain involved in oligomerization and which regulates carbohydrate specificity. Finally, a cytoplasmic tail is present, which includes internalization motifs and an incomplete immunoreceptor tyrosine-based activation motif (ITAM). Microglia, perivascular macrophages and dendritic cells express MMR and DC-SIGN (Burudi et al., 1999; Linehan et al., 1999; Mukhtar et al., 2002; Sallusto et al., 1995; Schwartz et al., 2002). The expression of DC-SIGN on brain microvascular endothelial cells could be advantageous for viral entry into the CNS (Mukhtar et al., 2002). Two major lineages of WNV, L1 and L2, have been identified after the phylogenetic analyses of glycoprotein E (Lanciotti et al., 1999). L1 strains have a large distribution and have been found in Africa, Europe, the Middle-East, North and Central America whereas the L2 strain is mainly localized in Africa and Madagascar. WNV human infection is usually asymptomatic, however life-threatening neurological disease, including encephalitis or meningitis, generally occurs in older or immunocompromised individuals (Campbell et al., 2002; Granwehr et al., 2004). A recent study investigated the relative contribution of DC-SIGN in infection by glycosylated L1 and non-glycosylated L1 and L2 strains (Martina et al., 2008). The authors showed that in contrast to a non-glycosylated L1 strain, the glycosylated L1 strains use DC-SIGN as an attachment receptor on dendritic cells, leading to enhanced

Heparan sulfate proteoglycans (HSPGs) are secreted and membrane associated proteins covalently attached to unbranched glycosaminoglycan heparan sulfate (HS) molecules which are composed of linear polysaccharide chains (Esko and Selleck, 2002). HS molecules are synthesized on a variety of cell surface proteins but are found consistently on members of two major families of membrane-bound proteoglycans: the transmembrane core proteins syndecans and the GPI-linked glypicans (Bernfield et al., 1999). HS can influence cellenvironment interactions by binding to a heterogeneous group of growth factors, matrix ligands, and cell surface molecules. The possibility that HS could serve as an entry receptor for HSV type 1 and 2 was assessed on HEp-2 cells (WuDunn and Spear, 1989). They found that heparin blocked both virus adsorption. Different adsorption inhibitory agents including, heparin and poly-L-lysine (both bind to anions on the surface of virions and cells) and platelet factor 4 (which has a high affinity for heparin and heparan sulfate) was tested. After the incubation of these agents with HEp-2 cells either immediately before or during exposure with HSV-1 or HSV-2, an inhibition of virions adsorption by heparin was observed. Same results for poly-L-lysine and platelet factor 4 were obtained. A further study investigated the role of cell surface heparan sulfate in HSV infection using heparan sulfatedeficient mutants CHO cells (Shieh et al., 1992). They demonstrated that CHO mutants with defect in heparan sulfate biosynthesis are resistant to HSV infection and have reduced

Apoptosis following viral infection is a robust line defence mechanism, since the cell auto destruction appears one of the best ways to limit virus production and spreading (Griffin and Hardwick, 1997). Unequivocally, the expected purpose of this cell suicide in response to infection is to short-circuit the viral expansion and to promote the clearance of dead infected cells by professional phagocytes (Fujimoto et al., 2000; Hashimoto et al., 2007). However, most viruses deal with this defensive reaction and have evolved strategies to evade or delay apoptosis (Teodoro and Branton, 1997).

Under normal cellular conditions, exposure PS serves as a recognition signal for internalization of apoptotic cells or debris. PS-mediated apoptotic clearance is a highly conserved mechanism, occurring in both professional and nonprofessional phagocytes (Henson et al., 2001). Importantly, this process does not elicit an inflammatory response (Savill et al., 2002). "Apoptotic mimicry" or the exposure of PS on the surface of a pathogen provides a signal for virus uptake and may represent a mechanism for viruses to stunt the host inflammatory response and evade immune recognition. Some viruses like CMV or HSV-1 and HSV-2, which acquire their envelopes from intracellular organelles, have externalized PS (Pryzdial and Wright, 1994; Sutherland et al., 1997). A previous study investigated the early apoptotic events in real time in intact live ND7 neuron-like cells following HSV-1 infection (Gautier et al., 2003). The authors demonstrated that infection of differentiated ND7 cells by HSV-1 triggers detectable alterations including PS exposure indicative of physiological changes associated with early stages of apoptosis.

Furthermore, control of the apoptotic process by the viruses is key, either to establish a permanent infection when they are able to block apoptosis, or to facilitate their dissemination and prevent inflammatory and immune response when they are able to control and activate the late phase of apoptosis (Chen et al., 2006). Dissemination of viral particles through the production of apoptotic blebs from host cells could confer protection from the immune system (Chen et al., 2006; Everett and McFadden, 1999). Conversely, it has been proposed that phagocytosis of these pathogen (Sindbis for example) -enriched apoptotic blebs by antigen presenting cells (e.g. dendritic cells) could contribute to a robust and uncontrolled adaptive immune response leading to autoimmunity against self-antigens contained within the blebs (Rosen et al., 1995). In the case of CHIKV, it has recently been evidenced that completion of the apoptotic process is an important element for efficient virus propagation (Krejbich-Trotot et al., 2011). Indeed, CHIKV was revealed to exploit, may be in parallel to classical ways of entry and cell exit, an ingenious way of camouflage in apoptotic blebs to enhance viral spreading to neighbouring cells while being shielded from the immune system.
