Article |
Address correspondence to Betsy C. Herold, Dept. of Pediatrics and Microbiology, Mount Sinai School of Medicine, One Gustave L. Levy Place, Box 1657, New York, NY 10029. Tel.: (212) 241-5272. Fax: (212) 426-4813. email:betsy.herold{at}mssm.edu
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: focal adhesion kinase; viral entry; membrane fusion; IP3; tyrosine phosphorylation
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
For most cell types, HSV entry (defined as fusion of the viral envelope with the cell plasma membrane and delivery of the viral capsid into the cell cytoplasm) is resistant to agents such as amantadine, chloroquine, and trifluoperazine, whose actions are known to alter endocytic pathways (Wittels and Spear, 1991; Nicola et al., 2003). Fusion requires the concerted action of gD, oligomers of gB, and heterodimers of glycoproteins H and L (gHgL; Spear and Longnecker, 2003). Cells transiently or permanently expressing gD, gB, and gHgL (Turner et al., 1998; Muggeridge, 2000) can induce fusion. Deletion of any one of these glycoproteins results in loss of penetration (Cai et al., 1987; Johnson and Ligas, 1988; Ligas and Johnson, 1988; Forrester et al., 1992; Hutchinson et al., 1992). However, the mechanism by which interactions between these viral glycoproteins and the cell surface trigger fusion is not defined. No specific fusion domains in HSV envelope glycoproteins have been identified.
We observed that HSV entry is associated with tyrosine phosphorylation of cellular proteins (Qie et al., 1999). Using immunoprecipitation and Western blotting, we found that several host cell proteins become tyrosine phosphorylated within 510 min after exposure to either HSV-1 or HSV-2. However, no phosphorylation was detected when cells were exposed to a virus deleted in gL that binds but fails to penetrate. Phosphorylation was restored when the gL deletion virus was grown on complementing gL-expressing cells. Viral infection and gene expression were inhibited by tyrphostin B46, a protein tyrosine kinase inhibitor that prevents the phosphorylation of cellular proteins (Qie et al., 1999). These analyses suggest that phosphorylation pathways are activated in response to initiation of fusion and may be required for infection.
Several reports have demonstrated that phosphorylation may occur in conjunction with Ca2+ signaling (Sayeski, et al., 2000; Trinkaus-Randall et al., 2000). The ER is the main storage site for intracellular Ca2+, and mobilization of Ca2+ from this store is an essential triggering signal for downstream events, including activation of phosphorylation pathways. Moreover, increases in intracellular calcium concentration ([Ca2+]i) have been associated with membrane fusion for a variety of biological membranes, including some enveloped viruses. For example, binding of HIV-1 gp120 to CD4 and engagement of chemokine coreceptors results in activation of Ca2+-signaling pathways (Davis et al., 1997; Alfano et al., 1999; Liu et al., 2000). Ca2+ signaling also may play a role in human cytomegalovirus (HCMV) entry (Keay et al., 1995). Specifically, exposure of human fibroblasts to HCMV mediates an increase in inositol-1,4,5-triphosphate (IP3), leading to mobilization of internal Ca2+ stores. We hypothesize that exposure to HSV might also activate the IP3 pathway leading to an increase in [Ca2+]i, which, in turn, may activate phosphorylation pathways. Activation of these signaling pathways may facilitate viral penetration and/or the transport of incoming viral capsids to the nucleus.
We tested this hypothesis for HSV-1 and HSV-2 using two different epithelial cell lines. Although the two serotypes are similar in many respects, differences in epidemiology, cell tropism, and spectrum of clinical disease are well known. For example, HSV-1 commonly infects oral mucosa, whereas HSV-2 preferentially infects genital mucosa. With respect to viral entry, serotype differences in the relative contribution of gC and gB toward heparan sulfate binding and in use of gD coreceptors have been described previously (Herold et al., 1991, 1994; Spear et al., 2000). Thus, we explored the role of Ca2+-signaling pathways in viral infection for both serotypes.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
To explore the possibility that Ca2+ influx across voltage-operated channels also contributes to the response to HSV, cells were pretreated with 10-µM concentrations of verapamil or nifedipine. Minimal change in the amplitude of [Ca2+]i response to virus was observed. The [Ca2+]i in response to HSV-1 in nifedipine- or verapamil-treated Vero cells was 163 ± 65 (n = 11) and 251 ± 120 (n = 12), respectively (Fig. 2, A, D, and E). However, the response to virus was modified in cells treated with these agents as reflected in the loss of the shoulder (Fig. 2, F and G). Similar results were obtained for HSV-2 and with CaSki cells (unpublished data). Together, these results suggest that the peak response to virus reflects release of ER Ca2+ stores, whereas the shoulder may represent influx of Ca2+ across voltage-operated channels. The results obtained are consistent with the notion that plasma membrane Ca2+ channels may be linked to the IP3 receptors. Similar results have been described in several other systems (Berridge, 1995; Parekh and Penner, 1997; Uhlen et al., 2000; Bakowski et al., 2001; Straube and Parekh, 2001; Valencia et al., 2001).
Effects of Ca2+ inhibitors on viral infection
To determine whether the Ca2+ response is important for viral infection, synchronized infectivity assays were conducted in which 100 µM 2-APB, 10 µM nifedipine, or 10 µM verapamil were added at the time of viral penetration for 1 h. HSV attachment, which occurs at 4°C, can be differentiated from penetration, which occurs after a shift to 37°C. The effects of these pharmacological agents on cell viability were determined in parallel. Although Tg does not significantly reduce cell viability at a concentration of 10 µM, it is known to have pleiotropic effects on cell function, including inhibition of protein synthesis at concentrations as low as 30 nM (Soboloff and Berger, 2002), and for this reason, Tg was not included. Results for Vero and CaSki cells are summarized in Fig. 3. HSV-1 and HSV-2 but not vesicular stomatitis virus (VSV) infections were reduced after treatment of cells with 2-APB (P < 0.001, ANOVA). In contrast, the Ca2+ channel blockers had little or no effect on viral infection (Fig. 3). These results suggest that release of IP3-sensitive ER stores is required for HSV infection. To further assess the importance of [Ca2+]i on HSV infection, the cells were pretreated for 2 h with 50 µM BAPTA-AM, a cell-permeable cytosolic Ca2+ chelator, or with 0.5 mM EGTA, a concentration that chelates extracellular Ca2+ and thereby prevents entrance of extracellular Ca2+ (Zwick et al., 1999, Bouchard et al., 2001), and were then infected with virus. Cell viability was monitored in parallel. Pretreatment of cells with BAPTA-AM (but not EGTA) reduced HSV infection to a similar extent as that observed with 2-APB (Fig. 3, P < 0.001, ANOVA).
|
|
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Our analyses suggest that binding of virus to heparan sulfate proteoglycan receptors and engagement of gD coreceptors are not sufficient to trigger the cellular signaling pathways. Similarly, interactions between gHgL and the cell, in the absence of gD or gB, is not adequate. This follows from the observations that viruses deleted in gB, gD, or gL fail to induce the signaling events. In contrast, HI virus, which retains the ability to enter cells, induces FAK phosphorylation. The requirement for the full complement of essential glycoproteins (gB, gD, and gHgL) to trigger the signaling events suggests that the pathway is activated in response to initiation of the fusion process or subsequent early events in invasion. These results differ from those obtained with the related ß-herpes virus, HCMV. Activation of a cellular receptor for HCMV gHgL (a 92.5-kD cellular protein) directly or indirectly by anti-idiotypic antibodies mediates transmembrane signaling events resulting in increases in [Ca2+]i (Keay et al., 1995; Milne et al., 1998; Baldwin et al., 2000). No cellular receptor for the gHgL complex of HSV-1 or HSV-2 has yet been identified. Possibly, the gHgL complex for HSV (in the presence of gB and gD) also engages a cellular receptor that directly or indirectly activates PLC. Recent experiments have shown that phosphoinositide-specific PLC
may be activated by phosphatidylinositol-3-kinase, which can act upstream of PLC
(Bierne et al., 2000).
In summary, these analyses demonstrate that Ca2+ signaling pathways play a key role in facilitating early events in HSV-1 and HSV-2 invasion. Because the signaling responses occur within minutes after exposure to virus, it seems likely that they are associated with fusion of the viral envelope and cell membrane and/or an immediate post-entry event. Activation and release of IP3-sensitive Ca2+ stores and subsequent phosphorylation of FAK are dependent on the presence of the full complement of essential envelope glycoproteins. Defining the specific cellular pathways activated during HSV entry and precisely how the virus triggers these pathways is key to understanding the process of viral invasion, and should facilitate development of novel strategies to prevent HSV entry and infection.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Purification and quantification of viruses
Viruses were purified from Vero or complementing cells on dextran gradients as described previously (Herold et al., 1991, 1994). Titers of the purified virus were determined by plaque assays. Relative viral particle numbers were determined by comparing the amounts of gB or gD by optical densitometry after Western blotting with mAb 1123 (anti-gB) or mAb1103 (anti-gD) (Goodwin Institute, Plantation, FL), as described previously (Tal-Singer et al., 1995; Qie et al., 1999; Cheshenko and Herold, 2002).
Reagents
Verapamil, nifedipine, 2-APB, Tg, EGTA, EGTA-AM, and ionomycin were purchased from Calbiochem and diluted in DMSO or PBS per manufacturer's instructions. The acetoxymethyl ester of Fura-2 (Fura-2/AM) and the cell-permeable cytosolic Ca2+ chelator, BAPTA-AM, were purchased from Molecular Probes, Inc. Heparin was purchased from Sigma-Aldrich.
Measurement of [Ca2+]i
Cells were loaded with 25 µM Fura-2/AM prepared in PBS for 3060 min, rinsed with PBS for 30 min at 37°C, and then exposed sequentially to buffer or pharmacological inhibitors and purified HSV diluted in PBS at 37°C. The viral inoculum was equivalent to an moi equal to 15 pfu/cell (based on titer on complementing cells) or an equivalent number of viral particles for noninfectious virus. Using an inverted epifluorescence microscope (Eclipse TE300; Nikon) linked to a cooled CCD camera (Pentamax; Princeton Instruments) interfaced with a digital imaging system (MetaFluor; Universal Imaging Corp.), [Ca2+]i was measured in individually identified Fura-2loaded cells visualized using an S Fluor 40x objective (NA 0.9, WD 0.3; Nikon) as described previously (Woda et al., 2002). Cells were alternately excited at 340 and 380 nm, and the images were digitized for subsequent analysis. Images were acquired every 210 s. An intracellular calibration was performed at the conclusion of each experiment according to previously described techniques (Grynkiewicz et al., 1985; Woda et al., 2002). The 340/380-nm fluorescence ratio was determined initially in the presence of a Ca2+-free bath plus 10 µM EGTA-AM (Rmin), and then in a 2-mM Ca2+ bath containing 10 µM ionomycin (Rmax). The equation used to calculate experimental values of [Ca2+]i was: [Kd(R-Rmin)/Rmax-R)](Sf2/Sb2), where R is the observed ratio of emitted light, Kd is the dissociation constant for Fura-2 and Ca2+ (assumed to be 224 nM), and Sf2 and Sb2 are the fluorescence signals of free and bound dye at 380 nm, respectively (Grynkiewicz et al., 1985). 715 cells were monitored for each experiment.
Plaque assays
Synchronized plaque assays in which pharmacological inhibitors were added during viral penetration were conducted as described previously (Herold et al., 2002). In brief, cells in 6-well dishes were precooled and exposed to HSV or VSV at 4°C for 2 h to allow binding. The moi was selected to yield 200500 pfu/well (e.g., moi
0.005 pfu/cell) on control wells. Unbound virus was removed and the cells were washed three times with PBS and then treated with 10 µM verapamil or nifedipine, 100 µM 2-APB, 10 µM Tg, or buffer (DMSO or PBS). Cells were immediately transferred to 37°C to allow viral penetration for 1 h. Unpenetrated virus was inactivated by washing the cell monolayer with a low pH citrate buffer (50 mM sodium citrate and 4 mM KCl, adjusted to pH 3.0) for 2 min, and then by washing three times with PBS. The cells were then overlaid with medium containing 0.5% methylcellulose for 48 h. For HSV-1(KOS) and HSV-2(G), plaques were counted by immunoassay (black-plaque; Herold et al., 1991). For VSV, cells were overlaid with 0.1% methylcellulose, fixed after 24 h with methanol, and stained with Giemsa.
VP16 and ICP4 time-course assays
To examine which steps in HSV infection are inhibited by the pharmacological agents, the time of introduction of the drug was varied and infection was monitored by examining transport of the tegument protein VP16 to the nucleus or expression of immediate early gene product, ICP4. Synchronized infectivity assays were conducted as described above, except the moi was equivalent to 1 pfu/cell and the penetration time (time from temperature shift to citrate treatment) was reduced to 15 min. The pharmacological agents or control buffers were added during the 4°C binding period, at the time the cells were transferred to 37°C (penetration), or immediately after the citrate treatment for 1 h. The cells were then overlaid with medium and viral infection was monitored by examining transport of VP16 or expression of ICP4 as described below.
To examine transport of VP16 to the nucleus, nuclear extracts were prepared 4 h after infection as described previously (Melchjorsen et al., 2002). The nuclear proteins were separated on an 8.5% SDS-polyacrylamide gel and transferred to a PVDF membrane (PerkinElmer) using a Trans-Blot system (Bio-Rad Laboratories) and blocked overnight in 5% milk-TBS. Membranes were incubated with mouse anti-VP16 (1:500; Santa Cruz Biotechnology, Inc.), diluted in 5% milk-TBS for 2 h, and rinsed extensively in TBST (50 mM Tris-Cl, pH 7.4, 150 mM NaCl, and 0.05% Tween 20). The membranes were then incubated with HRP-conjugated goat antimouse IgG (1:1,000; Calbiochem) in 5% milk-PBS for 2 h. After rinsing, the membranes were developed using the Western Lightning Chemiluminescence Reagent Plus (PerkinElmer). Blots were scanned and analyzed using the Gel Doc 2000 system (Bio-Rad Laboratories).
To examine ICP4 expression, infected cells were harvested 5 h after infection in lysis buffer (1% SDS, 50 mM Tris-Cl, pH 7.4, 55 mM EDTA, and I mM DTT), which was supplemented with complete protease inhibitors (Roche). The supernatant (soluble fraction) and pellet (insoluble fraction) were separated by centrifugation at 16,000 g at 4°C, and the protein concentrations in the supernatants were determined using the DC Protein Assay (Bio-Rad Laboratories). The supernatants were suspended in SDS sample buffer (100 mM Tris-Cl, pH 6.8, 1% ß-mercaptoethanol, 4% SDS, 0.2% bromophenol blue, and 20% glycerol) boiled for 4 min. The proteins were separated on an 8.5% SDS-polyacrylamide gel and transferred to a PVDF membrane (PerkinElmer) using a Trans-Blot system (Bio-Rad Laboratories) and blocked overnight in 5% milk-TBS. Membranes were incubated with mouse anti-ICP4 1101 (Goodwin Institute, Plantation, FL) or anti-ß-actin A5441 (Sigma-Aldrich) diluted in 5% milk-TBS for 2 h, then rinsed extensively in TBST (50 mM Tris-Cl, pH 7.4, 150 mM NaCl, and 0.05% Tween 20). The membranes were then incubated with HRP-conjugated goat antimouse IgG (1:1,000; Calbiochem) in 5% milk-PBS for 2 h. After rinsing, the membranes were developed using the Western Lightning Chemiluminescence Reagent Plus (PerkinElmer). Blots were scanned and analyzed using the Gel Doc 2000 system.
Detection of FAK phosphorylation by immunoblot
Nearly confluent CaSki or Vero monolayers were preincubated with serum-free medium for 24 h before infection, and were then exposed to HSV viruses at an moi of 10 pfu/cell for 5, 10, 20, or 30 min. Cell lysates were prepared as described above. Proteins were separated by SDS-PAGE and immunoblots prepared as described above. Membranes were incubated with a 1:1,000 dilution of rabbit anti-FAK[pY397], which recognizes the autophosphorylation site of FAK (Biosource International). The membranes were then stripped and reincubated with a 1:1,000 dilution of mouse anti-FAK mAb (F-15020; Transduction Laboratories), which recognizes total FAK.
Effects of BAPTA-AM or EGTA on viral infection and FAK phosphorylation
CaSki or Vero cells were pretreated for 2 h at 37°C with 10 or 50 µM BAPTA-AM or with 0.5 mM EGTA, washed three times, and exposed to HSV or VSV as described above. Infection was monitored using plaque assays, transport of VP16 to the nucleus, or expression of ICP4. To examine the effects of the chelators on FAK phosphorylation, cells were serum starved for 24 h, treated with the chelators for 2 h, and then infected. At various times after infection, cell lysates were prepared as described above. Proteins were separated by SDS-PAGE and immunoblots were incubated with antibodies directed against FAK[pY397] or ICP4.
Cell viability assay
The effects of the pharmacological agents on cell viability were determined by the metabolic reduction of the tetrazolium salt MTS (CellTiter 96®; Promega). Half-confluent Vero or CaSki cells were exposed to various concentrations of each drug for the indicated times, washed three times, and overlaid with fresh medium. Cell viability was examined at 24 h. Controls included cells exposed to media in the absence of any drugs and cells exposed to 0.1% nonoxynol-9 (Sigma-Aldrich), a detergent cytotoxic to cells in culture (Herold et al., 1999).
![]() |
Acknowledgments |
---|
Submitted: 22 January 2003
Accepted: 5 September 2003
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alfano, M., H. Schmidtmayerova, C.A. Amella, T. Pushkarsky, and M. Bukrinsky. 1999. The B-oligomer of pertussis toxin deactivates CC chemokine receptor 5 and blocks entry of M-tropic HIV-1 strains. J. Exp. Med. 190:597605.
Bakowski, D., M.D. Glitsch, and A.B. Parekh. 2001. An examination of the secretion-like coupling model for the activation of the Ca2+ release-activated Ca2+ current I(CRAC) in RBL-1 cells. J. Physiol. 532:5571.
Baldwin, B.R., C.O. Zhang, and S. Keay. 2000. Cloning and epitope mapping of a functional partial fusion receptor for human cytomegalovirus gH. J. Gen. Virol. 81:2735.
Berridge, M.J. 1995. Capacitative calcium entry. Biochem. J. 312:111.[Medline]
Bierne, H., S. Dramsi, M.P. Gratacap, C. Randriamampita, G. Carpenter, B. Payrastre, and P. Cossart. 2000. The invasion protein InIB from Listeria monocytogenes activates PLC-gamma1 downstream from PI 3-kinase. Cell. Microbiol. 2:465476.[CrossRef][Medline]
Bouchard, M., L.-H. Wang, and R.J. Schneider. 2001. Calcium signaling by HBx protein in hepatitis B virus DNA replication. Science. 294:23762378.
Cai, W.Z., S. Person, S.C. Warner, J.H. Zhou, and N.A. DeLuca. 1987. Linker-insertion nonsense and restriction-site deletion mutations of the gB glycoprotein gene of herpes simplex virus type 1. J. Virol. 61:714721.[Medline]
Cheshenko, N., and B.C. Herold. 2002. Glycoprotein B plays predominant role in mediating herpes simplex virus type 2 attachment and is required for entry and cell-cell spread. J. Gen. Virol. 83:22472252.
Cicala, C., J. Arthos, M. Ruiz, M. Vaccarezza, A. Rubbert, A. Riva, K. Wildt, O. Cohen, and A.S. Fauci. 1999. Induction of phosphorylation and intracellular association of CC chemokine receptor 5 and focal adhesion kinase in primary human CD4+ T cells by macrophage-tropic HIV envelope. J. Immunol. 163:420426.
Davis, C.B., I. Dikic, D. Unutmaz, C.M. Hill, J. Arthos, M.A. Siani, D.A. Thompson, J. Schlessinger, and D.R. Littman. 1997. Signal transduction due to HIV-1 envelope interactions with chemokine receptors CXCR4 or CCR5. J. Exp. Med. 186:17931798.
Dean, H.J., S.S. Terhune, M.T. Shieh, N. Susmarski, and P.G. Spear. 1994. Single amino acid substitutions in gD of herpes simplex virus 1 confer resistance to gD-mediated interference and cause cell-type dependent alterations in infectivity. Virology. 199:6780.[CrossRef][Medline]
Forrester, A., H. Farrell, G. Wilkinson, J. Kaye, N. Davis-Poynter, and T. Minson. 1992. Construction and properties of a mutant of herpes simplex virus type 1 with glycoprotein H coding sequences deleted. J. Virol. 66:341348.[Abstract]
Grynkiewicz, G., M. Poenie, and R.Y. Tsien. 1985. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260:34403450.[Abstract]
Herold, B.C., D. WuDunn, N. Soltys, and P.G. Spear. 1991. Glycoprotein C of herpes simplex virus type 1 plays a principal role in the adsorption of virus to cells and in infectivity. J. Virol. 65:10901098.[Medline]
Herold, B.C., R.J. Visalli, N. Susmarski, C.R. Brandt, and P.G. Spear. 1994. Glycoprotein C-independent binding of herpes simplex virus to cells requires cell surface heparan sulphate and glycoprotein B. J. Gen. Virol. 75:12111222.[Abstract]
Herold, B.C., R. Kirkpatrick, D. Marcellino, A. Travelstead, V. Pilipenko, H. Krasa, J. Bremer, L.J. Dong, and M.D. Cooper. 1999. Bile salts: natural detergents for the prevention of sexually transmitted diseases. Antimicrob. Agents Chemother. 43:745751.
Herold, B.C., I. Scordi-Bello, N. Cheshenko, D. Marcellino, M. Dzuzelewski, F. Francois, R. Morin, V.M. Casullo, R.A. Anderson, C. Chany, II, et al. 2002. Mandelic acid condensation polymer: novel candidate microbicide for prevention of human immunodeficiency virus and herpes simplex virus entry. J. Virol. 76:1123611244.
Hutchinson, L., H. Browne, V. Wargent, N. Davis-Poynter, S. Primorac, K. Goldsmith, A.C. Minson, and D.C. Johnson. 1992. A novel herpes simplex virus glycoprotein, gL, forms a complex with glycoprotein H (gH) and affects normal folding and surface expression of gH. J. Virol. 66:22402250.[Abstract]
Johnson, D.C., and M.W. Ligas. 1988. Herpes simplex viruses lacking glycoprotein D are unable to inhibit virus penetration: quantitative evidence for virus-specific cell surface receptors. J. Virol. 62:46054612.[Medline]
Keay, S., and B.R. Baldwin. 1996. Evidence for the role of cell protein phosphorylation in human cytomegalovirus/host cell fusion. J. Gen. Virol. 77:25972604.[Abstract]
Keay, S., B.R. Baldwin, M.W. Smith, S.S. Wasserman, and W.F. Goldman. 1995. Increases in [Ca2+]i mediated by the 92.5-kDa putative cell membrane receptor for HCMV gp86. Am. J. Physiol. 269:C11C21.[Medline]
Li, E., D.G. Stupack, S.L. Brown, R. Klemke, D.D. Schlaepfer, and G.R. Nemerow. 2000. Association of p130CAS with phosphatidylinositol-3-OH kinase mediates adenovirus cell entry. J. Biol. Chem. 275:1472914735.
Ligas, M.W., and D.C. Johnson. 1988. A herpes simplex virus mutant in which glycoprotein D sequences are replaced by beta-galactosidase sequences binds to but is unable to penetrate into cells. J. Virol. 62:14861494.[Medline]
Liu, Q.H., D.A. Williams, C. McManus, F. Baribaud, R.W. Doms, D. Schols, E. De Clercq, M.I. Kotlikoff, R.G. Collman, and B.D. Freedman. 2000. HIV-1 gp120 and chemokines activate ion channels in primary macrophages through CCR5 and CXCR4 stimulation. Proc. Natl. Acad. Sci. USA. 97:48324837.
Melchjorsen, J., F.S. Pedersen, S.C. Mogensen, and S.R. Paludan. 2002. Herpes simplex virus selectively induces expression of the CC chemokine RANTES/CCL5 in macrophages through a mechanism dependent on PKR and ICP0. J. Virol. 76:27802788.
Milne, R.S., D.A. Paterson, and J.C. Booth. 1998. Human cytomegalovirus glycoprotein H/glycoprotein L complex modulates fusion-from-without. J. Gen. Virol. 79:855865.[Abstract]
Montgomery, R.I., M.S. Warner, B.J. Lum, and P.G. Spear. 1996. Herpes simplex virus-1 entry into cells mediated by a novel member of the TNF/NGF receptor family. Cell. 87:427436.[Medline]
Morgan, C., H.M. Rose, and B. Mednis. 1968. Electron microscopy of herpes simplex virus. I. Entry. J. Virol. 2:507516.[Medline]
Moriuchi, M., H. Moriuchi, R. Williams, and S.E. Straus. 2000. Herpes simplex virus infection induces replication of human immunodeficiency virus type 1. Virology. 278:534540.[CrossRef][Medline]
Muggeridge, M.I. 2000. Characterization of cell-cell fusion mediated by herpes simplex virus 2 glycoproteins gB, gD, gH and gL in transfected cells. J. Gen. Virol. 81:20172027.
Nicola, A.V., A.M. McEvoy, and S.E. Straus. 2003. Roles for endocytosis and low pH in herpes simplex virus entry into HeLa and Chinese hamster ovary cells. J. Virol. 77:53245332.
Novotny, M.J., M.L. Parish, and P.G. Spear. 1996. Variability of herpes simplex virus 1 gL and anti-gL antibodies that inhibit cell fusion but not viral infectivity. Virology. 221:113.[CrossRef][Medline]
Parekh, A.B., and R. Penner. 1997. Store depletion and calcium influx. Physiol. Rev. 77:901930.
Qie, L., D. Marcellino, and B.C. Herold. 1999. Herpes simplex virus entry is associated with tyrosine phosphorylation of cellular proteins. Virology. 256:220227.[CrossRef][Medline]
Roop, C., L. Hutchinson, and D.C. Johnson. 1993. A mutant herpes simplex virus type 1 unable to express glycoprotein L cannot enter cells, and its particles lack glycoprotein H. J. Virol. 67:22852297.[Abstract]
Sayeski, P.P., M.S. Ali, and K.E. Bernstein. 2000. The role of Ca2+ mobilization and heterotrimeric G protein activation in mediating tyrosine phosphorylation signaling patterns in vascular smooth muscle cells. Mol. Cell. Biochem. 212:9198.[CrossRef][Medline]
Soboloff, J., and S.A. Berger. 2002. Sustained ER Ca2+ depletion suppresses protein synthesis and induces activation-enhanced cell death in mast cells. J. Biol. Chem. 277:1381213820.
Sodeik, B., M.W. Ebersold, and A. Helenius. 1997. Microtubule-mediated transport of incoming herpes simplex virus 1 capsids to the nucleus. J. Cell Biol. 136:10071021.
Spear, P.G., and R. Longnecker. 2003. Herpesvirus entry: an update. J. Virol. 77:1017910185.
Spear, P.G., R.J. Eisenberg, and G.H. Cohen. 2000. Three classes of cell surface receptors for alphaherpesvirus entry. Virology. 275:18.[CrossRef][Medline]
Straube, S., and A.B. Parekh. 2001. Effects of phosphatidylinositol kinase inhibitors on the activation of the store-operated calcium current ICRAC in RBL-1 cells. Pflugers Arch. 442:391395.[CrossRef][Medline]
Tal-Singer, R., C. Peng, M. Ponce De Leon, W.R. Abrams, B.W. Banfield, F. Tufaro, G.H. Cohen, and R.J. Eisenberg. 1995. Interaction of herpes simplex virus glycoprotein gC with mammalian cell surface molecules. J. Virol. 69:44714483.[Abstract]
Thastrup, O., P.J. Cullen, B.K. Drobak, M.R. Hanley, and A.P. Dawson. 1990. Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2+-ATPase. Proc. Natl. Acad. Sci. USA. 87:24662470.[Abstract]
Trinkaus-Randall, V., R. Kewalramani, J. Payne, and A. Cornell-Bell. 2000. Calcium signaling induced by adhesion mediates protein tyrosine phosphorylation and is independent of pHi. J. Cell. Physiol. 184:385399.[CrossRef][Medline]
Turner, A., B. Bruun, T. Minson, and H. Browne. 1998. Glycoproteins gB, gD, and gHgL of herpes simplex virus type 1 are necessary and sufficient to mediate membrane fusion in a Cos cell transfection system. J. Virol. 72:873875.
Uhlen, P., A. Laestadiu, T. Jahnukainen, T. Soderblom, F. Backhed, G. Celsi, H. Brismar, S. Normark, A. Aperia, and A. Richter-Dahlfors. 2000. Alpha-haemolysin of uropathogenic E. coli induces Ca2+ oscillations in renal epithelial cells. Nature. 405:694697.[CrossRef][Medline]
Valencia, L., M. Bidet, S. Martial, E. Sanchez, E. Melendez, M. Tauc, C. Poujeol, D. Martin, M.D. Namorado, J.L. Reyes, and P. Poujeol. 2001. Nifedipine-activated Ca2+ permeability in newborn rat cortical collecting duct cells in primary culture. Am. J. Physiol. Cell Physiol. 280:C1193C1203.
Wittels, M., and P.G. Spear. 1991. Penetration of cells by herpes simplex virus does not require a low pH-dependent endocytic pathway. Virus Res. 18:271290.[CrossRef][Medline]
Woda, C.B., M. Leite, Jr., R. Rohatgi, and L.M. Satlin. 2002. Effects of luminal flow and nucleotides on [Ca2+]i in rabbit cortical collecting duct. Am. J. Physiol. Renal Physiol. 283:F437F446.
Zwick, E., C. Wallasch, H. Daub, and A. Ullrich. 1999. Distinct calcium-dependent pathways of epidermal growth factor receptor transactivation and PYK2 tyrosine phosphorylation in PC12 cells. J. Biol. Chem. 274:2098920996.