MINIREVIEW Viral entry mechanisms: cellular and viral mediators of herpes simplex virus entry Jihan Akhtar 1 and Deepak Shukla 1,2 1 Department of Ophthalmology and Visual Sciences, College of Medicine, University of Illinois at Chicago, IL, USA 2 Department of Microbiology and Immunology, College of Medicine, University of Illinois at Chicago, IL, USA Introduction The herpesvirus family consists of over 100 double- stranded DNA viruses divided into a, b and c subgroups [1]. Only eight herpesviruses are known to commonly infect humans and the remainder are animal herpesviruses infecting a wide variety of animal species. All members of the herpesvirus family cause lifelong latent infections and, structurally, all have a linear, double-stranded DNA genome packaged into an icosahedral capsid (Fig. 1) [1]. This capsid, in turn, is enclosed by the tegument, a layer of proteins. The tegument is then covered by a bilayer lipid membrane with embedded proteins and glycoproteins. The pro- tected DNA genome is essential for viral infectivity. Closely-related herpes simplex type-1 (HSV-1) and type-2 (HSV-2) viruses are members of the alphaher- pesvirus subfamily and are responsible for highly pre- valent infections among humans [2], although a number of common experimental animals also demon- strate susceptibility to HSV infections. Symptomatic disease caused by HSV-1 is typically limited to cold sores of the mouth and keratitis in the eyes. HSV-2, in contrast, is mostly responsible for genital lesions. However, both viruses are capable of causing lesions on identical body sites and both can cause life-threat- ening diseases in immunocompromised individuals, including newborns, patients with HIV or patients undergoing immunosuppressive treatment [1,2]. Trans- mission among humans requires physical contact and often occurs during kissing (HSV-1) or sexual inter- course (HSV-2). The area of the lesions depends on Keywords cell-to-cell spread; endocytosis; entry; filopodia; glycoproteins; herpes simplex virus; herpes viruses; phagocytosis; viral surfing Correspondence D. Shukla, 1855 W. Taylor Street (M ⁄ C 648), Chicago, IL 60612, USA Fax: +1 312 996 7773 Tel: +1 312 355 0908 E-mail: dshukla@uic.edu (Received 18 June 2009, revised 9 September 2009, accepted 18 September 2009) doi:10.1111/j.1742-4658.2009.07402.x Herpes simplex virus type-1 and type-2 are highly prevalent human patho- gens causing life-long infections. The process of infection begins when the virions bind heparan sulfate moieties present on host cell surfaces. This ini- tial attachment then triggers a cascade of molecular interactions involving multiple viral and host cell proteins and receptors, leading to penetration of the viral nucleocapsid and tegument proteins into the cytoplasm. The nucleocapsid is then transported to the nuclear membrane and the viral DNA is released for replication in the nucleus. Recent studies have revealed that herpes simplex virus entry or penetration into cells may be a highly complex process and the mechanism of entry may demonstrate unique cell-type specificities. Although specificities clearly exist, past and ongoing studies demonstrate that herpes simplex virus may share certain common receptors and pathways that are also used by many other human viruses. This minireview helps to shed light on recent revelations on the herpes simplex virus entry process. Abbreviations HS, heparan sulfate; HSPG, heparan sulfate proteoglycan; HSV-1, herpes simplex virus type-1; HSV-2, herpes simplex virus type-2; HVEM, herpesvirus entry mediator; 3-OST, 3-O sulfotransferase; PILR, paired immunoglobulin-like receptor. 7228 FEBS Journal 276 (2009) 7228–7236 ª 2009 The Authors Journal compilation ª 2009 FEBS the inoculation site; therefore, sores are most com- monly found on the mouth or genital areas. Medical professionals and others not wearing surgical gloves could also acquire lesions on their fingers from the virions shed from their patients’ vaginal mucosa and ⁄ or mouth. This is commonly known as finger herpes or herpetic whitlow. After initial infection, the virus remains latent in neurons, a key feature of alphaherpesviruses [1]. Dur- ing this period, hosts are still capable of spreading infection to other humans via asymptomatic shedding of the virions. Reactivation of the virus can be a result of a variety of environmental triggers, including emo- tional or physical stress, which subsequently leads to virus replication in epithelial cells and a lifetime of intermittent mucocutaneous lesions [2]. The ability of the virus to avoid immune detection and establish latency in a significant patient population (up to 80% human adults for HSV-1 and approximately 40% for HSV-2) is facilitated by its unique ability to produc- tively enter cells of the epithelia for viral gene expres- sion, replication and eventual spread from cell-to-cell to innervating nerves and, ultimately, to trigeminal (HSV-1) or sacral (HSV-2) ganglia for the estab- lishment of latency. Thus, HSV entry into host cells marks the first and possibly most critical step in viral pathogenesis. Five viral glycoproteins have been implicated in the viral entry process: gB, gC, gD, gH and gL [3,4]. All but gC are essential for entry. The initial interaction, or binding to cells, is mediated via interactions of gC and ⁄ or gB with heparan sulfate proteoglycans (HSPGs). F-actin-rich membrane protrusions called filopodia may facilitate attachment by providing HSPG-rich sites for the initial binding (Fig. 1). Although gC is not essential for viral entry, its absence decreases the overall viral binding to cell surfaces [1]. After the initial attachment to cells, the process of pen- etration begins. The latter, depending on the host cell type and the mode of entry [4,5], may require fusion of the virion envelope with the plasma membrane or with the membrane of an intracellular vesicle (Fig. 1) [5]. In either case, the membrane fusion requires essen- tial participation from viral glycoproteins gB, gD, gH and gL. Although gD is not considered to be a fusogen, other essential glycoproteins, more impor- tantly, gB and gH, demonstrate many characteristics of viral fusion proteins [4,6]. Similar to attachment, membrane fusion also requires participation from cellular receptors. A number of unrelated receptors for gD have been discovered. These include nectin-1 and -2, herpesvirus entry mediator (HVEM) and 3-O sul- fated heparan sulfate (3-O HS) [3,7]. The current, widely accepted model for membrane fusion suggests that binding of gD to one of its cognate receptors induces conformational changes in gD that mobilize a fusion active multi-glycoprotein complex involving gB, gD, gH and gL (Fig. 2) [8]. Fusion of viral envelope HSV surfing Glycoproteins Tegument DNA HSV surfing Nucleocapsid Envelope HSPG gD receptor I II Cytoplasm I II Nucleus Fig. 1. HSV virion and its two major modes of entry into cells. Structural components of a typical HSV virion are shown (box). HSV virions can enter into cells via a pH-independent fusion of viral envelope with the plasma membrane (I) or, alternatively, via an endocytic pathway that may be phagocytosis-like (II) in terms of the viral uptake. In both pathways, HSV particles may initially associate with filopodia-like mem- brane protrusions via HSPG. Unidirectional transport of extracellular particles bound to filopodia (HSV surfing) then brings the particles closer to the cell body for entry via interactions with the cellular receptors, including gD receptor and possibly gB receptor. Fusion at the plasma membrane results in the release of the naked viral nucleocapsid in the cytoplasm for transport to the nucleus. Similarly, endocytosis also requires fusion of the enveloped particles with the vesicular membrane for the release of the viral nucleocapsid proximal to the nucleus. J. Akhtar and D. Shukla Cellular and viral mediators of HSV entry FEBS Journal 276 (2009) 7228–7236 ª 2009 The Authors Journal compilation ª 2009 FEBS 7229 with a cellular membrane results in content mixing and the eventual release of the viral nucleocapsid and tegu- ment proteins into the host cytoplasm (Figs 1 and 2). Subsequently, symbolizing post-entry steps, HSV nucleocapsids dissociate with tegument proteins and bind a microtubule-dependent, minus end-directed motor, dynein [9]. Although most of the tegument proteins are required for activation and modulation of viral gene expression and shut-off of host protein syn- thesis, some may participate in dynein-propelled trans- port of the nucleocapsids along microtubules toward the nuclear membrane for uncoating and the release of viral DNA into the nucleus. Transcription, replication of viral DNA and assembly of progeny capsids take place within the host nucleus. The intricate details of the steps required for entry are discussed below. Viral binding to filopodia Although the virus binding to cells results from the interaction of gC and ⁄ or gB with heparan sulfate (HS), the location on the cell where this binding can occur the earliest has only recently been elucidated. In human conjunctival epithelial cells, virions were observed attaching to filopodia-like membrane protrusions [10]. It was also observed that virus attachment to filopodia was followed by unilateral movement of the virions towards the cell body. Staining of many natural target cells with anti-HSV receptor antibodies indicated the expression of HS but not other entry receptors. Nectin- 1 expression, for example, was limited to cell bodies with no detectable expression on filopodia (M. J. Oh & D. Shukla, unpublished results). The phenomenon of extracellular HSV-1 moving unilaterally towards the cell body on filopodia has also been observed in retinal pigment epithelial (RPE) and P19N neural cells [11,12]. A similar phenomenon for the transport of extracellular virus particles, termed ‘viral surfing’, has been observed with other viruses, such as retroviruses and human pap- illomavirus type-16 [13,14]. Surfing is also shared by many additional herpesviruses, including cytomegalo- virus and human herpesvirus-8 (V. Tiwari & D. Shukla, unpublished results). Exposure of HSV-1 to cells can induce the forma- tion of filopodia [5]. This presumably enhances the effi- cacy of viral infection by targeted delivery (via surfing) of virus particles to cell bodies for subsequent fusion with plasma membrane or endocytosis [11]. Based on functional analogy with retroviral surfing, it is quite likely that myosin-dependent F-actin retrograde flow is responsible for the HSV movements along filopodia [13]. Ongoing studies demonstrate that filopodial bridges formed between two cells can help transfer extracellular HSV-1 virions from an infected to an uninfected cell (V. Tiwari & D. Shukla, unpublished results). Although viral trafficking on filopodia has not yet been observed or studied in HSV-2, because of strong similarities between HSV-1 and HSV-2 viral entry mechanisms, including the use of HS as an attachment receptor, it is quite possible that this phe- nomenon also plays a role in HSV-2 entry. It is also worth noting that filopodia are not essential for virus attachment, with the latter occuring at virtually any place on the plasma membrane as long as receptors such as HS are present. However, because of the pres- ence of filopodia at the leading edges of tissue layers (e.g. in vivo during wound healing), they may provide easy ‘roadways’ for HSV to reach cell bodies for infection. Fusion at cell and vesicular membranes Fusion at the plasma membrane is a pH-independent process that requires gB, gD, gH and gL (Fig. 2) [3]. Cellular receptors such as nectin-1, HVEM or 3-OS HS are also required. The process is triggered by gC HSPG Membrane fusion Attachment HSV gB + gB gD gH-gL gD receptor HSPG g g Fusion complex gH-gL gD gB Lipid mixing Content mixing + gD receptor Fig. 2. Molecular interactions that facilitate HSV entry. Initial attachment to cells is mediated by interaction between HSPGs with HSV glycoproteins gC and ⁄ or gB. Membrane fusion is required for penetration of the viral nucleocapsid and tegument into the cytoplasm. Interaction between gD, gH-gL and a gD receptor may be sufficient to bring conformational changes within gD to trig- ger merging of viral and cellular membranes or lipid mixing. How- ever, a fourth glycoprotein, gB, is also required for complete fusion and content mixing, which essentially results in the release of the tegument and nucleocapsid into the cytoplasm. A receptor for gB, PILR-a, is also expected to play a role during the fusion process. Its precise role is still emerging and therefore it is not shown here. Cellular and viral mediators of HSV entry J. Akhtar and D. Shukla 7230 FEBS Journal 276 (2009) 7228–7236 ª 2009 The Authors Journal compilation ª 2009 FEBS conformational changes in gD that occur upon recep- tor binding [15]. Alteration in gD conformation then mobilizes gH and gL, a heterodimer, and gB to initiate the fusion process at the cell surface. Multiprotein complexes involving gD ⁄ gB and gD ⁄ gH ⁄ gL have been detected previously [9,16] and the entire complex colo- calizes at the membrane during fusion [9]. Although it is expected that members of fusion active complex undergo additional changes in conformation, no clear information is available because of the lack of 3D structures of any fusion active glycoprotein complex. It is, however, known that gD bound to its receptor dem- onstrates significant changes in its conformation [15]. The HSV-induced membrane fusion is accomplished by initially bringing the membranes of both the host cell and virus into close contact by receptor ⁄ glycopro- tein interactions followed by mixing of the membranes or lipids to create an intermediary state, sometimes referred as ‘hemifusion intermediate’ [17]. Subse- quently, a fusion pore is formed that allows mixing of the cytoplasmic contents with viral contents, which, in the case of HSV entry, essnetially implies the delivery of viral tegument proteins and the nucleocapsid into the cytoplasm. Although gD and the gH ⁄ gL hetero- dimer may be sufficient to initiate the lipid mixing, a full-scale fusion resulting in the mixing of the contents requires the presence of gB as well (Fig. 2) [17]. It has been suggested that fusion may require a sequential action by the glycoproteins [17], although a recent study did not find any pressing evidence for this [16]. Recent studies have implicated gB and gH as having multiple fusogenic domains [4,6,18]. gB is highly con- served among herpesviruses; however, this glycoprotein demonstrates a unique characteristic in HSV-1 and HSV-2 because it remains uncleaved, whereas many other herpesvirus gBs are post-translationally cleaved. Interestingly, both uncleaved and cleaved forms of gBs share mixed features of class I and class II viral fusion proteins, and thereby define a new, hybrid class of viral fusogens [6]. Class I proteins contain hairpin trimers with N-terminal hydrophobic membrane-penetrating peptides and centrally located a-helical coiled-coils. By contrast, class II proteins contain b-structures with internally located fusion domains. Similar to class I, the gB trimer also contains a central a-helical core but the fusion loops, similar to class II, are part of the elongated b-hairpins. The gB trimer shows strong resemblance to fusogenic glycoprotein G of vesicular stomatitis virus [6]. Similar to gB, gH was also reported to contain two heptad repeat regions that were found to be necessary for fusion induction [4]. Peptides that corresponded to these regions effectively prevented HSV-1 infection, suggesting the importance of these regions. The exact significance of gL is still unclear, although it is likely to play an important role by stabi- lizing or regulating gH conformations. The role of gD and its receptor in the fusion process is likely that of a catalyst [3,15]. Although gD does not contain any fusion peptides or domains, binding to its receptor is required for the initiation of fusion, unless a poorly understood gD-dependent but gD- receptor-independent pathway is initiated [19]. It is worth noting that gD homologs are rare and, among human herpesviruses, only HSV-1 and HSV-2 appear to express gD [1]. It is unclear why HSV virions have evolved with a gD-based fusion trigger mechanism, whereas many other herpesviruses can do without it. It is conceivable that a gD-based mechanism may pro- vide some explanation for the differences in tissue tro- pism demonstrated by HSV-1 and HSV-2. It is also possible that the involvement of gD, which is capable of interacting with at least three distinct classes of entry receptors, may enhance the host range for HSV because a vast majority of cultured cells of human or animal origin are susceptible to HSV entry. Other human herpesviruses demonstrate more restrictive host ranges in terms of the cell lines that they infect [1]. Although fusion at the plasma membrane was origi- nally considered to be the only route of viral entry for HSV, recent studies have supported the existence of an alternate entry mechanism utilizing endocytosis [4,5]. This atypical endocytosis resembles phagocytosis in the virus uptake mechanism (Fig. 1) [5]. Under this pro- cess, the endocytosed enveloped particles subsequently fuse with a vesicular membrane. Similar interactions between viral gB, gD, gH, gL and host cell receptors are expectedly mirrored within an intracellular vesicle such as an endosome to facilitate the fusion. The gD receptor has been colocalized with endosomal markers and electron micrographs show the fusion and exit of nuclecapsids from the endosomes [5]. Interestingly, unlike other bacterial and viral entry mechanisms, HSV-1 endocytosis does not appear to be mediated by clathrin-coated pits or caveolae. Furthermore, although endosomes provide an acidic background that may augment viral infectivity in certain cell types, this pH dependency is not required in all cell lines [20–22]. The choice between endocytosis and fusion at the plasma membrane appears to depend on individual cell types. In Vero and Hep2 cells, fusion at the plasma membrane is the mechanism of choice; however, in cell types such as CHO, HeLa, RPE, human epidermal keratinocytes and human conjunctival epithelial cells, evidence of endocytosis of virions has been observed [11,21,22]. Interestingly, gD was shown to down-regu- late nectin-1 in cells utilizing endocytosis in HSV-1 J. Akhtar and D. Shukla Cellular and viral mediators of HSV entry FEBS Journal 276 (2009) 7228–7236 ª 2009 The Authors Journal compilation ª 2009 FEBS 7231 entry, such as HeLa cells [23]. By contrast, this down- regulation was not seen in cells where HSV fuses at the plasma membrane, such as in Vero cells. This suggests that the gD ⁄ nectin-1 interaction is a possible factor in the cell-type specific mode of HSV-1 internalization. Cellular receptors for gD The main receptors that gD utilizes for cell entry are nectin-1, nectin-2, HVEM and 3-O HS. HSV-1 and HSV-2 differ in their preference of gD receptor types. HVEM and nectin-1 are utilized by both virus types; however, 3-O HS can only be used by HSV-1. Simi- larly, nectin-2 has not been shown to allow substantial wild-type HSV-1 entry and it may have a greater effect on HSV-2 entry [3]. Various cell types rely on different gD receptors for HSV-1 entry: T lymphocytes and trabecular meshwork cells utilize HVEM, whereas neuronal and epithelial cells require nectin-1 [3,24]. The dependence of HSV-2 on nectin-1 and HVEM for entry and spread in vivo has been successfully demonstrated using single and double knockout mice [25]. 3-O HS, which is not a receptor for HSV-2, appears to play a major role in HSV-1 entry into primary cultures of corneal fibro- blasts [26]. Because 3-O HS is the most recently dis- covered and perhaps least understood gD receptor, a relatively detailed analysis of 3-O HS is presented below. HS essentially represents a structurally diverse family of polysaccharides sharing a common backbone structure with varying degrees of additional modifica- tions and functions [27]. Demonstrating its structural complexity, HS, which is essentially a polymer of repeating disaccharide units containing a glucosamine and a glucuronic acid residue (Fig. 3), is expressed in a variety of chain lengths with varying degrees of addi- tional modifications on the cell surfaces and extracellu- lar matrices of almost all cell types [27]. It is an important attachment receptor shared by many patho- genic viruses, including all human herpesviruses except Epstein–Barr virus [1]. The significance of HS is partic- ularly higher for HSV-1 because, in addition to attach- ment, it can also mediate membrane penetration by HSV-1 virions [7]. Although a relatively less modified backbone HS chain can facilitate HSV attachment, a highly modified version, 3-OS HS, is required for interaction with gD and for independently triggering membrane fusion during entry and cell-to-cell spread processes [7,28,29]. This fusion triggering 3-OS HS is generated after numerous modifications, including 2-O-, 6-O- and 3-O sulfations and epimerization (Fig. 3) [7,27]. The final step of parent chain sulfation to create 3-O HS is performed by a number of 3-O sulfotrans- ferase (3-OST) isoforms: 3-OST-1, -2, -3A, -3B, -4, -5 and -6 [1,30]. Each isoform may demonstrate cell-type specific expression patterns and may produce a unique 3-OS HS chain with uniquely different functions. 3-OST-1 creates 3-O HS with anti-thrombin binding sites and no gD binding activity, whereas other iso- forms such as 3-OST-2, -3, -4 and -6, create forms of 3-O HS that can act as gD receptors [1,7,30]. Their 6-O S 6-O S 6-O S 6-O S 6-O S 6-O S 6-O 3-O sulfotransferase 6-O sulfotransferase 2-O sulfotransferase N-deacetylase, N-sulfotransferase, epimerase S 6-O S 3-O S S N S N S N SS 2-O S 2-O S N S 2-O S N S N S 2-O S N S 2-O S N S N multiple copies = Glucose = Galactose = Xylose = Serine = α-linkage = β-linkage = Glucuronic acid = Iduronic acid S 2-O S N S 2-O S 2-O N S N Fig. 3. Outline of HS maturation. A number of listed enzymes participate in the modifi- cation of the parent chain HS, which is a polymer of repeating disaccharide units containing a glucosamine and a glucuronic acid residue. All possible modifications and their preferred sequences (arrows) are shown. Cellular and viral mediators of HSV entry J. Akhtar and D. Shukla 7232 FEBS Journal 276 (2009) 7228–7236 ª 2009 The Authors Journal compilation ª 2009 FEBS individual physiological functions are yet not well known. A 3D structure for 3-O HS interaction with gD has been suggested [15]. The crystal structure of gD contains a positively-charged deep pocket and a flat region with clusters of basic amino acid residues. Mutagenesis stud- ies indicate that the pocket proximal to N-terminus may have a role in 3-OS HS binding because mutations in this region affect 3-OS HS usage by HSV-1 [31]. Inter- estingly, the same mutations also affect HVEM usage but not nectin-1. Thus, it is quite possible that at least one 3-OS HS binding region of gD may overlap with HVEM binding sites [15,31]. Efforts have also been made to identify the structural specificity within HS that is required for gD binding. Apart from a 3-O sulfated glucosamine, an upstream 2-O sulfated iduronic acid appears to be required as well [7]. To define the mini- mum size requirement for this interaction, a 3-O sulfated octasaccharide was generated [32]. This octasaccharide binds gD and demonstrates an inherent ability to block gD-triggered cell-to-cell fusion. Because the fusion was blocked in cells that may or may not use 3-OS HS as the major receptor for entry, it again suggests that 3-OS HS binding sites may overlap with other receptor (HVEM and possibly nectin-1) binding sites on gD. Another interesting property of 3-OS HS is that HSV-2 gD fails to use it as a receptor for entry [7]. Indeed, even for the attachment process, HSV-2 demonstrates many dif- ferences in the HS structure recognition than HSV-1 [33]. It remains to be determined whether the inability to use 3-OS HS and additional differences in HS binding may have a role in the tissue tropism shown by the two HSV serotypes. Paired immunoglobulin-like receptor (PILR)-a: a new receptor for HSV-1 gB Although viral attachment to cells shows strong depen- dence on the interactions of gB and gC with HS, stud- ies have also shown that cells without HS are still susceptible to low-efficiency HSV-1 infection and retain the ability to bind soluble gB [34,35]. The sug- gestion that there may be other receptors interacting with gB was upheld by a recent finding regarding the ability of gB to act as a ligand for PILR-a, one of the paired inhibitory receptors found on monocytes, mac- rophages and dendritic cells [19]. CHO-K1 cells, natu- rally resistant to HSV-1 infection, demonstrate susceptibility to infection after transfection with PILR- a plasmid. Human cell lines expressing both HVEM and PILR-a experienced decreased viral entry when antibodies to either PILR-a or HVEM were used, supporting a separate need for both receptors. Additionally, PILR-a contains a tyrosine-based motif in its cytoplasmic domain that delivers inhibitory signals to the host cell. Thus, by interacting with this inhibitory receptor, gB may allow HSV-1 to escape host immune system recognition via suppression at the same time exploiting the receptor for viral entry. The most current notion with respect to HSV-induced membrane fusion may include PILR-a as an important and a balancing component of the fusion machinery [36]. Interestingly, HSV-2 entry may not be so strongly dependent on PILR-a as a co-receptor, as shown for RPE cells [37] and further confirmed by additional findings [38]. HSV-1 binding mediated by PILR-a also appears to mediate cell–virus fusion at the plasma membrane. Susceptible CHO cells utilize only endocytosis as an HSV entry mechanism. However, after inhibiting endo- cytosis and transfecting CHO cells with PILR-a, HSV-1 entry was still possible, presumably at the plasma membrane [38]. Therefore, PILR-a may be responsible for an alternate, but poorly understood, plasma membrane fusion mechanism of entry (Fig. 2). It is likely that PILR-a mediated fusion is a gD-depen- dent process that may not require a gD receptor as an essential component. Most recent studies appear to indicate that PILR-a plays a role both in binding and fusion; however, additional studies are needed to better elucidate the importance of this receptor. Additional receptors for HSV entry A few cell surface molecules have been implicated as putative HSV-1 gH receptors. These include B5 and avb3 integrins [39,40]. Expression cloning in porcine kidney cells resistant for HSV-1 entry has led to the discovery of B5, a type-2 membrane protein containing an extracellular heptad repeat potentially capable of forming an a-helix for coiled-coils [39]. Such structures may facilitate membrane fusion by interacting with viral proteins containing a-helices. A synthetic peptide identical to the heptad repeat region blocks HSV infec- tion of B5-expressing porcine cells and human HEp-2 cells, suggesting a possible role for B5 in HSV-1 entry. Because gH also contains an a-helix, it is speculated that it may be a ligand for B5 [39]. No direct interac- tion between B5 and gH has been demonstrated to date. By contrast, in a separate study, a soluble form of gH-gL heterodimer was found to specifically bind cells expressing avb3 integrins, although the effect of this interaction on HSV-1 entry remains elusive [40]. The binding appears to be highly specific because it can be prevented by mutating a potential integrin- binding motif, Arg-Gly-Asp (RGD), in gH. Again, J. Akhtar and D. Shukla Cellular and viral mediators of HSV entry FEBS Journal 276 (2009) 7228–7236 ª 2009 The Authors Journal compilation ª 2009 FEBS 7233 additional studies are needed to determine and estab- lish the significance of potential gH receptors. Cell-to-cell viral spread HSV-1 virus is also unique in that the spread of infec- tion is not dependent on a hematogenous or lymphatic route. Cell-to-cell contact is essential in HSV-1 and HSV-2 infection and elucidating the mechanism of cell-to-cell spread is important for fully understanding the overall viral infectious process. Viral spread from cell-to-cell depends on the same gD interaction with its receptor(s) as that observed when free virions initially infect a host cell [41]. Interestingly, gE and gI are glycoproteins that form a heterodimer required for cell-to-cell spread but are not required for the initial entry of free virus particles [42]. During HSV infection, the gE ⁄ gI heterodimers move from the trans-Golgi network to epithelial cell junctions along with other viral glycoproteins and virion particles. Removing the gene responsible for the early sorting through the TGN prevents subsequent cell-to-cell spread and viri- ons are observed to travel towards the apical surface instead of toward cell–cell junctions. Assortment through the TGN early in infection was found to be necessary for efficient cell-to-cell spread of HSV-1. In addition, gK has also been shown to play an essential role in cell-to-cell spread in corneal and trigeminal ganglia cells, comprising two cell lines that lead to the more devastating consequences of diseases such as blindness and meningitis [43]. Mice infected with mutated virus with the gene for gK deleted were dem- onstrated a significantly decreased corneal spread and also had decreased clinical signs of infection. Nectin-1 helps induce HSV-1 cell-to-cell spread. AF6, a multidomain protein that interacts with nectin- 1, leads to decreased cell-to-cell HSV-1 spread when knocked down [44]. Similar to nectin-1, this protein is also involved in cell–cell adhesion and is found at cell junctions. Interestingly, AF6 knockdown does not affect the nectin-1 clustering that occurs at junctions for cell-to-cell transmission. Therefore, nectin-1 recep- tor clustering appears to take place independently of expression of the gene for AF6 and the clustering is insufficient for cell-to-cell spread. Although 3-O HS has been shown to act as an gD entry receptor for free virions fusing at the plasma membrane, 3-O HS has also been shown to play a role in cell-to-cell fusion and spread [28]. Previously, HVEM and nectin-1 expression were considered to be required for cell-to-cell spread [41]. However, more recent studies imply that 3-O HS can also mediate cell- to-cell fusion, which is required during spread. Cells expressing gB, gD, gH and gL, and therefore mimick- ing the essential viral machinery for membrane fusion, were able to fuse with 3-O HS-expressing cells [28]. Importantly, these cells expressed neither nectin-1 nor HVEM. When heparinase was used to eliminate the total HS, cell-to-cell fusion was dramatically decreased. In separate studies, it was found that 3-OS HS is cru- cial for the HSV-1-induced cell-to-cell fusion observed with primary cultures of corneal fibroblasts [45]. Therefore, the role of 3-O HS may be more extensive with respect to the process of viral entry and spread than previously thought. Conclusions HSV-1 and HSV-2 both utilize similar mechanisms of binding, fusion and subsequent cell-to-cell spread. Although fewer studies have been performed on HSV-2, the two viruses share 82% of their amino acid sequence and demonstrate high structural similarities [1]. Therefore, many of the concepts developed for HSV-1 are likely to be applicable for HSV-2, although further confirmation is needed. Different glycoproteins play unique key roles in the infectious cycle. Binding can be initiated on filopodia with the interaction of gC and gB; however, subsequent fusion and penetration utilize gB, gD, gH and gL. Entry can directly occur at the plasma membrane or via an intracellular vesicle. Fusion appears to be mediated by fusogenic regions of gB and gH triggered by the interaction of gD with its receptor. Finally, cell-to-cell spread utilizes gD recep- tors, all fusion essential glycoproteins, the gE ⁄ gI heterodimer and gK. Clearly, significant advances have been made in our quest to understand the mechanism of HSV entry, although many areas still remain poorly understood. For example, the role of cellular signaling pathways is poorly defined. Activation of Rho signaling has been demonstrated [5,46], as has the involvement of focal adhesion kinases [47], although a complete picture is yet to emerge. It is also unclear why the virus undergoes endocytosis when identical receptors can promote fusion at the plasma membrane. A better understanding of how the virus fuses with a vesicular membrane is also needed. For commonly spreading viruses such as HSV, such studies would have a high impact. In lieu of a protective vaccine [2], efficient microbicides or prophylactics can be designed by devel- oping a better understanding of virus entry mecha- nisms. Spread of the virus within a host can be contained by antiviral agents that target membrane fusion required for the spread. Similarly, targeting shared entry phenomena, such as surfing, can lead to the production of broad-spectrum antiviral agents. In Cellular and viral mediators of HSV entry J. Akhtar and D. Shukla 7234 FEBS Journal 276 (2009) 7228–7236 ª 2009 The Authors Journal compilation ª 2009 FEBS summary, viral entry is an important area of research with strong potential for identifying new strategies aim- ing to prevent epidemic and pandemic viral diseases caused by HSV, HIV, papilloma, influenza and many additional viruses. Continuing research in this area can lead to a greater understanding of the disease process and new treatments for viral diseases. Acknowledgements The authors thank Dr Beatrice Yue (UIC) for critically reading the manuscript. The work described here that was performed in the laboratory of D. Shukla was supported by a NIH RO1 grant AI057860 and a NIH core grant EY01792. D.S. is a recipient of Lew Wasserman Merit award from Research to Prevent Blindness, New York, NY, USA. References 1 Shukla D & Spear PG (2001) Herpesviruses and hepa- ran sulfate: an intimate relationship in aid of viral entry. J Clin Invest 108, 503–510. 2 Whitley RJ & Roizman B (2001) Herpes simplex virus infections. Lancet 357, 1513–1518. 3 Spear PG (2004) Herpes simplex virus: receptors and ligands for cell entry. Cell Microbiol 6, 401–410. 4 Campadelli-Fiume G, Amasio M, Avitabile E, Cerretani A, Forghieri C, Gianni T & Menotti L (2007) The mul- tipartite system that mediates entry of herpes simplex virus into the cell. 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Shukla 7236 FEBS Journal 276 (2009) 7228–7236 ª 2009 The Authors Journal compilation ª 2009 FEBS . MINIREVIEW Viral entry mechanisms: cellular and viral mediators of herpes simplex virus entry Jihan Akhtar 1 and Deepak Shukla 1,2 1 Department of Ophthalmology and. plays a pivotal role in herpes simplex virus entry. J Biol Chem 280, 31116–31125. Cellular and viral mediators of HSV entry J. Akhtar and D. Shukla 7236 FEBS