Multiple Receptor Interactions Trigger Release of Membrane and Intracellular Calcium Stores Critical for Herpes Simplex Virus Entry

Abstract

Herpes simplex viruses (HSV) harness cellular calcium signaling pathways to facilitate viral entry. Confocal microscopy and small interfering RNA (siRNA) were used to identify the source of the calcium and to dissect the requisite viral-cell interactions. Binding of HSV to human epithelial cells induced no calcium response, but shifting the cells to temperatures permissive for penetration triggered increases in plasma membrane calcium followed by a global release of intracellular calcium. Transfection with siRNA targeting the proteoglycan syndecan-2 blocked viral binding and abrogated any calcium response. Transfection with siRNA targeting nectin-1, a glycoprotein D receptor, also prevented both membrane and intracellular calcium responses. In contrast, the membrane response was preserved after transfection with siRNA targeting integrin␣v, a novel glycoprotein H receptor. The membrane response, however, was not sufficient for viral entry, which required interactions with integrin␣v and release of inositol-triphosphate receptor-dependent intracellular calcium stores. Thus, calcium plays a critical, complex role in HSV entry. INTRODUCTIONHerpes simplex viruses (HSV) are a global health problem and the leading cause of genital ulcers, neonatal encephalitis, and a major cofactor for human immunodeficiency virus infection. Development of novel strategies to prevent infection requires elucidation of the molecular and cellular events critical for entry. Previous work demonstrates that viral entry is a complex process that requires multiple interactions at the cell surface involving four envelope glycoproteins, gD, gB, and heterodimers of gH-gL. The number of glycoproteins required illustrates the complexity of HSV entry compared with that observed with most other viruses, which typically require one or at most two glycoproteins. The cascade is initiated by binding of HSV to heparan sulfate proteoglycans at the cell surface; glycoprotein C plays the major role in mediating binding for HSV-1, whereas gB plays the dominant role in mediating binding for HSV-2 (Herold et al., 1991;Cheshenko and Herold, 2002). After engagement of this attachment receptor, gD interacts with one of several coreceptors, of which nectin-1 may play the predominant role on human epithelial cells (Linehan et al., 2004). Independently of its role in attachment, gB is also required for fusion as are heterodimers of gH-gL (Spear, 2004). Accumulating evidence points to gH as the executor of fusion, in part, because it exhibits structural-functional features typical of fusion proteins, including a fusion peptide and heptad repeat segments able to form coiled coils (Gianni et al., 2005(Gianni et al., , 2006. However, no receptor for gH has been definitively identified. Precisely how these interactions trigger viral entry is not clear. The concentration of intracellular free Ca 2ϩ ([Ca 2ϩ ] i ) regulates a variety of cellular processes, including sperm-egg fusion, contraction, secretion, cell growth, and apoptosis (Berridge, 2005). The specificity of Ca ...

Binding of herpes simplex virus glycoprotein D to nectin-1 exploits host cell adhesion

“…Despite that the nectin-1-mediated signalling pathway remains largely unknown, a previous study indeed shows an increase of the intracellular calcium level during HSV infection 33 . Th is change in calcium concentration requires the nectin-1 / gD interaction, and could in turn facilitate virus entry 34 . Further work is needed to investigate the cellular signalling pathway response to the binding of gD to nectin-1.…”

Herpes Simplex Virus Type 2 Glycoprotein H Interacts with Integrin αvβ3 To Facilitate Viral Entry and Calcium Signaling in Human Genital Tract Epithelial Cells

“…Entry into target cells by either serotype (HSV-1 or HSV-2) is complex, presumably reflecting the ability of virus to infect multiple cell types by either direct fusion or one of several endocytic pathways (2). Entry is initiated by attachment of HSV-1 glycoprotein C (gC) or HSV-2 gB to heparan sulfate moieties on syndecan proteoglycans (3)(4)(5)(6) followed by engagement of one of several gD coreceptors, most commonly nectin-1 on epithelial cells (7)(8)(9). Engagement of the gD coreceptor is followed by the translocation of Akt to microdomains on the outer leaflet of the plasma membrane, where interactions with gB lead to Akt phosphorylation and release of calcium (Ca 2ϩ ) near the plasma membrane (10).…”

“…Engagement of the gD coreceptor is followed by the translocation of Akt to microdomains on the outer leaflet of the plasma membrane, where interactions with gB lead to Akt phosphorylation and release of calcium (Ca 2ϩ ) near the plasma membrane (10). This initiates a signaling cascade that promotes the release of inositol-triphosphate receptor (IP 3 R)-dependent endoplasmic reticulum (ER) Ca 2ϩ stores, leading to entry of viral capsids and tegument proteins and their transport to the nuclear pore (5,11). The role played by gH in this Akt-Ca 2ϩ entry pathway has not yet been delineated.…”

Syndecan-1 and syndecan-2 play key roles in herpes simplex virus type-1 infection

“…These GAGs are predominantly heparan sulfated (Carey, 1997;Lopes et al, 2006) but syndecan-1 and syndecan-4 can also contain chondroitin sulfate (CS) GAGs in addition to HS-GAGs (Deepa et al, 2004;Shworak et al, 1994). HSV-1 commonly infects epithelial cells, which express detectable amounts of syndecan-1 and syndecan-2 (Bobardt et al, 2007;Cheshenko et al, 2007). In addition, syndecan-1 (CD138; NCBI reference sequence: NP_001006947) is expressed by many cell types including plasma cells.…”