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

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

Herpes simplex viruses (HSV) harness cellular calcium signaling pathways to facilitate viral entry. As a result, they become more stable and competent for nuclear egress. I think it may boil down to where is the virus going to hit so what disease occurs. The nature of the process by which dissociation of the scaffold is accomplished is unknown. Despite having the same rigidity, the scaffold-containing B capsids broke at significantly lower forces than A and C capsids. Quantitative analyses of levels of luciferase mRNA revealed that differential expression of luciferase was controlled at the transcriptional level. In contrast, no fusion or intracellular delivery of viral capsids was observed in CHOK1 cells, which are permissive for HSV binding only because the cells lack gD coreceptors (Montgomery et al., 1996 ).

To further validate the confocal model, studies were conducted with viral variants deleted in specific envelope glycoproteins. CaSki cells were infected with DiD-labeled purified parental virus [HSV-1(KOS) or HSV-2(G)] or viruses deleted in HSV-2 gB (GgB−), HSV-1 gD (KOSgD-1−), or HSV-1 gH (KOSgH-1−) in a synchronized infection assay. Deshmukh’s expertise is in neuron survival and death. Representative images obtained 1 h after entry are shown in A, bottom. Consistent with the results obtained with the dually labeled KVP26GFP virus, fusion of the envelope (red) with the membrane (blue) and intracellular delivery of VP5 (green) was readily detected after infection of CaSki cells with KOS. analyzed data; and W.H.R., K.R., B.S., and G.J.L.W. In contrast, no cell-associated virus was detected after exposure to HSV-2(G)gB− virus, consistent with previous studies demonstrating that gB plays a critical role in HSV-2 binding (Cheshenko and Herold, 2002 ).

Cell-bound virus was detected after infection with HSV-1 variants deleted in either gD-1 or gH-1, but no delivery of VP5 was observed. These findings are consistent with the essential roles played by gD and gH in mediating entry for both serotypes after binding. To examine the Ca2+ response during the entry process, CaSki cells were loaded with Calcium Green and mock infected or infected with an equivalent number of viral particles each of the purified DiD-labeled viruses in a synchronized assay. For presentation of these images, the plasma membranes are red and viral envelopes are blue (B). Live confocal images were acquired every 2 s immediately after the temperature reached 37°C. Temperature–kinetic studies demonstrated that viral entry occurs before the temperature reaches 37°C. That is, the histones associated with viral DNA did not undergo demethylation – a process that allows tightly packaged DNA to become more open so that gene expression can occur, including HSV gene expression, which was precisely what the virus needed in order to be reactivated.

At various times posttemperature shift, nonpenetrated virus was inactivated with a low pH buffer (or pH 7.4 buffer as a control), and infection was monitored by counting plaques 48 h pi (Gerber et al., 1995 ). Entry is most efficient at 37°C, with 50% of HSV-2(G) entering within 7 min. At room temperature, 50% of virus is internalized in 20 min and at 18°C, 25% of virus enters cells within 40 min (data not shown). These findings are consistent with the confocal images (B). Images acquired immediately after HSV-1(KOS)–infected cells reached 37°C already demonstrated an increase in membrane Ca2+. The microscopic techniques used in this study do not allow the definitive localization of the membrane Ca2+ signal. Within 16 s after reaching 37°C, an increase in global intracellular Ca2+ was also observed, which persisted for 5–10 min.

In contrast, no change in membrane or global Ca2+ was observed at any time after exposure to the HSV-2(G)gB− virus, which is impaired in binding and entry. Bound virus was detected following exposure to gD− virus, but the virus failed to elicit any Ca2+ response. In contrast, some increase in membrane Ca2+ was observed after exposure to the gH− virus, but no global intracellular Ca2+ response was detected. Together, these findings indicate that there are two distinct Ca2+ stores activated in response to HSV. Increases in Ca2+ at the membrane require viral binding (gB) and engagement of coreceptors (gD), whereas release of global intracellular stores also requires gH–gL. HSV enters CaSki, but not CHO-K1, cells, and it elicits an increase in both membrane and global intracellular Ca2+. (A) CaSki or CHO-K1 cells were exposed to DiD-envelope–labeled HSV-1(KV26GFP) virus (moi = 10 pfu/cell) in a synchronized assay, …

Building from these observations, we next focused on the cellular components that contribute to the signaling response. The initial contact of HSV with epithelial cells is binding to heparan sulfate chains of cell surface proteoglycans, but the core proteins involved have not been identified. Transmembrane heparan sulfate proteoglycans include glypican, cerebroglycan, betaglycan, CD44, and members of the syndecan family. Recent studies indicate that the predominant proteoglycans on human genital epithelial cell surfaces are syndecan-1 and syndecan-2 (Bobardt et al., 2007 ). We confirmed these findings by examining CaSki cells for the expression of syndecan-1, syndecan-2, and glypican by Western blotting and confocal microscopy. Syndecan-2 predominated with no glypican detected (data not shown). Transfection of CaSki cells with syndecan-2 sequence-specific siRNA markedly reduced syndecan-2 expression 48 h after transfection as illustrated by scanning of confocal microscopy images or Western blots, whereas transfection with the unrelated HVEM sequence-specific siRNA had no effect on syndecan-2 expression (A).

The effect of syndecan gene silencing on infection was monitored by microscopy using K26GFP or plaque assays. CaSki cells were transfected with nonspecific or syndecan-2 siRNA, and then 48 h later, they were infected with K26GFP (5 pfu/cell). One and 4 h pi, the cells stained with EZ-Link SuLfo-NHS-Biotin to detect plasma membranes, and then the cells were fixed and stained with DAPI to detect nuclei and viewed by confocal microscopy. Viral GFP is detected in the cytoplasm of CaSki cells as early as 1 h pi, and it increases in intensity by 4 h, consistent with a productive infection (B). In contrast, no intracellular GFP is detected in cells transfected with syndecan-2–specific siRNA. Parallel studies demonstrate that transfection with syndecan-specific siRNA reduced viral binding by >90% (C) and inhibited viral plaque formation by 98% (D). Transfection with syndecan-1–specific siRNA had more modest effects and reduced infection by 82%, suggesting that syndecan-1 also plays a role as a core proteolycan for viral binding (D).

Together, these findings demonstrate for the first time a role for syndecans as core proteoglycan proteins important for HSV infection of human cervical epithelial cells. The next step in viral entry is binding of gD to a coreceptor. Several studies suggest that nectin-1 is the major coreceptor on human epithelial cells (Linehan et al., 2004 ; Galen et al., 2006 ). Transfection of cells with nectin-1 sequence-specific, but not HVEM-specific, siRNA reduced its expression by at least 90% (based on scanning of confocal microscopy images and Western blots) (A). Binding of virus was not impaired (B), but no viral entry was detected in cells transfected with nectin siRNA by confocal microscopy using K26GFP virus (C). Moreover, nuclear transport of VP 16, a viral tegument protein, was also reduced by 98% (D), and viral plaque formation was reduced by 90% (E). These findings confirm an important role for nectin-1 in facilitating HSV infection of human epithelial cells.

The heterodimeric complex of gH–gL is also required for viral entry. Although no gH–gL receptor has been definitively identified, several studies suggest a possible role for integrins. For example, a soluble form of HSV-1 gH–gL bound to Vero cells and mutation of a potential integrin-binding motif, Arg-Gly-Asp (RGD), in gH abolished the binding (Parry et al., 2005 ). Consistent with the possible role of integrins in HSV entry is the observation that binding of many ligands to integrins triggers Ca2+ signaling pathways (Giancotti and Ruoslahti, 1999 ). To explore the possible role of αv integrins in HSV entry, CaSki cells were transfected with integrinαv siRNA, which reduced integrin expression by 63–83% (A). Controls included cells transfected with HVEM siRNA, which had no impact on integrinαv expression (data not shown). Confocal microscopy studies with purified dually labeled K26GFP virus demonstrated that the viral envelope (blue) merged with the cell membrane (red), but the virus was retained within the membrane and failed to release viral capsids (green); in contrast, release of capsids was readily detected in cells transfected with nonspecific siRNA, and by 4 h pi, intracellular GFP was readily detected (B).
Multiple Receptor Interactions Trigger Release of Membrane and Intracellular Calcium Stores Critical for Herpes Simplex Virus Entry

Consistent with the block in infection observed by microscopy in B, nuclear transport of VP 16 was also reduced by 74% (A, left) and viral yields were reduced by 94% in a plaque assay (C). Together, these findings demonstrate that integrinαv plays a role in viral entry, possibly through engagement of gH. To address whether gH might be mediating the binding to integrinαv, cells were mock infected or infected with ∼100 particles/cell of purified labeled gD− or gH− virus. The cells were fixed 15 min posttemperature shift, and then they were stained with a mAb to integrinαv (). In the mock-infected cells, integrinαv is diffusely distributed near the plasma membrane. After exposure to the gD− virus, the viral envelope (blue) colocalizes with both the cell membrane (red) and integrinαv (green). In contrast, after infection with gH− virus, no colocalization with integrin is observed.

These findings suggest that gH interacts with integrinαv, even in the absence of gD. Notably, integrinαv seems to clump together with the viral envelope at the cell membrane after exposure to the gD−, but not the gH−, virus. We previously used calcium fluorometry and pharmacological agents to examine the Ca2+ response to HSV. Although fluorometry provides quantitative data on the intracellular Ca2+ concentration, the cellular source cannot be easily determined. Additionally, pharmacological agents are relatively nonspecific and have multiple effects. To overcome these limitations, we used confocal microscopy coupled with siRNA targeting of the IP3Rs to further define the role Ca2+ plays in viral infection. Activation of IP3Rs triggers Ca2+ release mostly from the ER, although some cells express IP3R isoforms in their plasma membrane (Tanimura et al., 2000 ).

Three different isoforms of the IP3R may be expressed. Transfection of CaSki cells with a combination of IP3R sequence-specific siRNAs directed at all three isoforms reduced IP3R expression by 70–80% (based on scanning of confocal microscopy images and Western blots) (A). The impact on viral entry was monitored by confocal microscopy with dually labeled virus. No internalized GFP was detected in cells transfected with IP3R-specific siRNA at any time pi (compare B with 4B, top). Rather, the viral envelope seemed to fuse with the cell membrane, but it remained trapped at the membrane. Consistent with the confocal images, nuclear transport of VP 16 was reduced by 81% (C) and viral yields by 89% (D). We previously reported that HSV preferentially infects the apical surface of polarized ECC-1 cells and that nectin-1 sorted to the apical surface of these cells (Galen et al., 2006 ).

We expanded this work by also examining the polarity of expression of syndecan-2, integrinαv, and IP3R on ECC-1 cells. Confocal images demonstrated that syndecan-2 preferentially sorted to the basal surface of cells, although the core proteoglycan was readily detected at both membranes. In contrast, nectin-1, integrinαv, and IP3R preferentially sorted to the apical membrane of cells (A). Moreover, when cells that had been grown on Transwells and loaded with Calcium Green were exposed to HSV-2(G) from the apical or basolateral membrane, Ca2+ release was observed almost exclusively after apical exposure (B). These observations are consistent with previous studies with polarized pancreatic acinar cells and enterocytes (Nathanson et al., 1994 ; Matovcik et al., 1996 ; Yamamoto-Hino et al., 1998 ), which demonstrate that IP3R are concentrated near the apical pole of the cell membrane and indicate that the preferential apical expression of nectin-1, integrinαv, and IP3R may contribute to the observation that HSV preferentially infects the apical surface of ECC-1 cells (Galen et al., 2006 ). Syndecans interact with cytoskeletal proteins including integrins to facilitate cell attachment and motility (Bishop et al., 2007 ). To explore the relationship between these cellular proteins, the subcellular localization of integrinαv after syndecan-2 siRNA knockdown, and conversely, the localization of syndecan-2 after siRNA integrinαv knockdown were evaluated.

Gene silencing of syndecan-2 resulted in a change in integrinαv subcellular localization with less integrin being detected at the plasma membrane. In contrast, silencing of integrinαv had little impact on syndecan-2 expression (). Knockdown of syndecan-2 also modified localization of nectin-1, whereas knockdown of nectin had little impact on syndecan-2 (data not shown). The dependence of integrin (and nectin) localization on syndecan protein expression is consistent with the known interactions between these proteins in other biological systems. For example, fibronectin binds simultaneously to heparan sulfate chains of syndecan and one or more integrins to induce cell spreading and focal adhesion formation (Couchman et al., 2001 ). Similarly, HSV may interact with heparan sulfate chains of syndecan, nectin, and integrins to form a functional complex that facilitates viral entry. Knockdown of syndecan-2 expression with siRNA inhibits viral binding and infection.

(A) Cells were transfected with nonspecific, HVEM-specific, or syndecan-2–specific siRNA. Forty-eight hours after transfection, they were stained with anti-syndecan … Blockade of nectin-1 by siRNA prevents viral entry after binding. (A) Cells were transfected with nonspecific or nectin-1–specific siRNA and 48 h after transfection, they were stained with a cocktail of anti-nectin-1 Abs (green) and examined by … Integrinαv interacts with virus to facilitate viral entry and infection. (A) Cells were transfected with nonspecific or integrinαv-specific siRNA, and 48 h after transfection, cells were stained with a mAb specific for integrinαv … Interactions with integrinαv requires viral gH.

Transfected CaSki cells were mock infected or infected with purified DiD-envelope labeled gD− or gH− virus (viral particle numbers equivalent to moi = 10 pfu/cell) in a synchronized … Blockade of IP3R with siRNA Prevents HSV Entry. (A) Cells were transfected with nonspecific or a combination of IP3R-specific siRNAs targeting all three isoforms, and 48 h after transfection, cells were stained with a mAb to the IP3Rs (green) and examined … Polarized expression of receptors on ECC-1 cells. (A) ECC-1 cells were grown on Transwells and stained with EZ-Link Sulfo-NHS-Biotin to detect cellular plasma membranes (red). Then, they were permeabilized, and the nuclei were detected by DAPI (blue). …

Gene silencing of syndecan-2 alters subcellular localization of integrinαv. CaSki cells were transfected with nonspecific and syndecan-2–specific siRNA and 48 h later, fixed, and stained for expression of integrinαv (green), plasma … Having established a role for these cellular proteins in viral entry, the impact of each on the Ca2+ responses was evaluated. No increase in membrane-associated or global Ca2+ was observed in cells transfected with syndecan-2 siRNA, consistent with the marked reduction in viral binding (A, top). Bound virus (blue) was observed in cells transfected with nectin siRNA, but, consistent with the results obtained with the gD− virus (B), in the absence of nectin, no membrane or global Ca2+ response was elicited. However, increases in membrane, but not global, Ca2+ were observed in cells transfected with siRNA targeting integrinαv or IP3R. The source of the changes in membrane Ca2+ could represent release from stores within or just beneath the membrane, but it is unlikely to reflect influx of extracellular Ca2+, because the cells were washed three times with Ca2+-free PBS before shifting the temperature to 37°C.

These findings are consistent with results obtained with the gH− virus, and they suggest that engagement of syndecan and nectin alone is sufficient to trigger membrane Ca2+ responses, but the global Ca2+ response requires signaling through integrinαv and activation of IP3R. Infection of cells transfected with nonspecific siRNA or an unrelated siRNA targeting HVEM (bottom) elicited both the membrane and global intracellular Ca2+ response. We also examined the viral-induced Ca2+ signaling response by excitation ratio fluorometry of cells loaded with Ca2+ indicator, fura-2. Exposure of cells transfected with nonspecific siRNA resulted in a significant rapid increase in [Ca2+]i, which peaked within 1 min (, B and C; p < 0.001). A blunted, albeit significant increase in [Ca2+]i, was also observed after transfection with either integrinαv or IP3R siRNA, reflecting the membrane Ca2+ response. No significant increases in [Ca2+]i were observed in cells transfected with either of the other siRNAs or in CHOK1 cells. HSV triggers membrane Ca2+ release if syndecan and nectin are present, but requires integrinαv and IP3R for the global Ca2+ response. (A) CaSki cell membranes were labeled with VYBRANT-DiI (red) and transfected with nonspecific or specific siRNAs ...

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