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• W2049212648 abstract "Development of strategies to prevent herpes simplex virus (HSV) infection requires knowledge of cellular pathways harnessed by the virus for invasion. This study demonstrates that HSV induces rapid phosphorylation of focal adhesion kinase (FAK) in several human target cells and that phosphorylation is important for entry post-binding. Nuclear transport of the viral tegument protein VP16, transport of viral capsids to the nuclear pore, and downstream events (including expression of immediate-early genes and viral plaque formation) were substantially reduced in cells transfected with dominant-negative mutants of FAK or small interfering RNA designed to inhibit FAK expression. These observations were substantiated using mouse embryonic fibroblast cells derived from embryonic FAK-deficient mice. Infection was reduced by >90% in knockout cells relative to control cells and was further reduced if the knockout cells were transfected with small interfering RNA targeting proline-rich tyrosine kinase-2, which was also phosphorylated in response to HSV. The knockout cells were permissive for viral binding, and virus triggered an intracellular calcium response, but nuclear transport was inhibited. Together, these results support a novel model for invasion that implicates FAK phosphorylation as important for delivery of viral capsids to the nuclear pore. Development of strategies to prevent herpes simplex virus (HSV) infection requires knowledge of cellular pathways harnessed by the virus for invasion. This study demonstrates that HSV induces rapid phosphorylation of focal adhesion kinase (FAK) in several human target cells and that phosphorylation is important for entry post-binding. Nuclear transport of the viral tegument protein VP16, transport of viral capsids to the nuclear pore, and downstream events (including expression of immediate-early genes and viral plaque formation) were substantially reduced in cells transfected with dominant-negative mutants of FAK or small interfering RNA designed to inhibit FAK expression. These observations were substantiated using mouse embryonic fibroblast cells derived from embryonic FAK-deficient mice. Infection was reduced by >90% in knockout cells relative to control cells and was further reduced if the knockout cells were transfected with small interfering RNA targeting proline-rich tyrosine kinase-2, which was also phosphorylated in response to HSV. The knockout cells were permissive for viral binding, and virus triggered an intracellular calcium response, but nuclear transport was inhibited. Together, these results support a novel model for invasion that implicates FAK phosphorylation as important for delivery of viral capsids to the nuclear pore. Herpes simplex virus (HSV) 1The abbreviations used are: HSV, herpes simplex virus; HIV, human immunodeficiency virus; ER, endoplasmic reticulum; FAK, focal adhesion kinase; siRNA, small interfering RNA; MEF, mouse embryonic fibroblast; GFP, green fluorescent protein; mAb, monoclonal antibody; PBS, phosphate-buffered saline; m.o.i., multiplicity of infection; pfu, plaque-forming units; HHV-8, human herpesvirus 8. type 2 is a major global health problem, the leading cause of genital herpes and neonatal herpes encephalitis, and a major cofactor for human immunodeficiency virus (HIV) infection. Novel strategies to prevent HSV-2 and other sexually transmitted infections are urgently needed. Development of new approaches requires an understanding of the molecular and cellular events critical for the establishment of infection. Previous work demonstrates that HSV entry is a complex process requiring the following steps: (i) binding to heparan sulfate receptors; (ii) engagement of glycoprotein D coreceptors; (iii) penetration, which, for human epithelial cells, is mediated primarily by fusion of the viral envelope with the cell membrane; (iv) transport of the viral tegument protein VP16 (trans-inducing factor (αTIF)) to the nucleus; and (v) transport of viral capsids to the nuclear pore with release of viral DNA into the nucleoplasm (1Sodeik B. Ebersold M.W. Helenius A. J. Cell Biol. 1997; 136: 1007-1021Crossref PubMed Scopus (552) Google Scholar, 2Spear P.G. Longnecker R. J. Virol. 2003; 77: 10179-10185Crossref PubMed Scopus (467) Google Scholar). Glycoprotein B plays the major role in mediating binding of HSV-2 to heparan sulfate receptors, whereas glycoprotein C plays a more dominant role in mediating binding of HSV-1 (3Cheshenko N. Herold B.C. J. Gen. Virol. 2002; 83: 2247-2255Crossref PubMed Scopus (72) Google Scholar, 4Herold B.C. WuDunn D. Soltys N. Spear P.G. J. Virol. 1991; 65: 1090-1098Crossref PubMed Google Scholar). For both serotypes, glycoproteins B, D, and H-L are required for penetration, but their precise role and the cellular pathways important for viral invasion have not been fully elucidated. The observations that heparan sulfate is the major attachment receptor for HSV and that heparan sulfate moieties also interact with gp120 of HIV fostered the development of sulfated or sulfonated polymers as candidate topical microbicides to prevent viral transmission. These compounds prevent genital herpes in the mouse and simian immunodeficiency virus in the macaque and are currently being evaluated in Phase II/III clinical trials (5Keller M.J. Klotman M.E. Herold B.C. J. Antimicrob. Chemother. 2003; 51: 1099-1102Crossref PubMed Scopus (21) Google Scholar, 6Keller M.J. Tuyama A. Carlucci M.J. Herold B.C. J. Antimicrobial. Chemother. 2005; 55: 420-423Crossref PubMed Scopus (43) Google Scholar). However, competitive antagonists of viral binding are not likely to be sufficient to fully protect against sexual transmission. Experience with systemic therapy indicates that combination therapies that target more than one step in the viral life cycle are optimal. Thus, elucidating the cellular pathways required for viral entry may provide novel targets for microbicide development. We reported previously that both serotypes of HSV trigger inositol triphosphate-mediated release of endoplasmic reticulum (ER) Ca2+ stores and that Ca2+ signaling plays a critical role in viral entry (7Cheshenko N. Del Rosario B. Woda C. Marcellino D. Satlin L.M. Herold B.C. J. Cell Biol. 2003; 163: 283-293Crossref PubMed Scopus (118) Google Scholar). Pharmacological inhibition of ER Ca2+ release or chelation of intracellular Ca2+ reduces nuclear transport of the viral tegument protein VP16, a surrogate marker for capsid transport, and the downstream events in the viral life cycle, including expression of viral immediate-early genes and viral plaque formation. Activation of Ca2+ signaling is associated with triggering of phosphorylation signaling pathways. Specifically, we observed previously that HSV-1 and HSV-2 trigger the phosphorylation of focal adhesion kinase (FAK) within 5-10 min following exposure of Vero (monkey kidney) or CaSki (human cervical epithelial) cells to virus. FAK phosphorylation appears to occur downstream of release of ER Ca2+ stores, as treatment of cells with 2-aminoethoxydiphenyl borate, an inositol triphosphate receptor antagonist that prevents release of ER Ca2+, or chelation of intracellular Ca2+ blocks the virus-induced phosphorylation of FAK. Activation of both pathways (Ca2+ and FAK phosphorylation) requires exposure to entry-competent virus, as HSV-1 deleted in the glycoproteins essential for penetration (glycoproteins B, D, and H-L) fails to induce Ca2+ release or to trigger FAK phosphorylation, whereas Ca2+ release and phosphorylation are preserved following exposure to the repaired viruses and to inactivated virus, which retain the ability to penetrate cells. These findings led to the proposal of a new paradigm for HSV entry in which HSV triggers inositol triphosphate-mediated release of ER Ca2+ stores, which may facilitate fusion of the viral envelope and cell membrane, allowing delivery of capsids to the cytoplasm (7Cheshenko N. Del Rosario B. Woda C. Marcellino D. Satlin L.M. Herold B.C. J. Cell Biol. 2003; 163: 283-293Crossref PubMed Scopus (118) Google Scholar). In response to activation of the Ca2+ pathways, we hypothesize that FAK and related signaling pathways are activated, which may promote transport of the viral capsids to the nuclear pores. This study was designed to test this hypothesis and to evaluate whether HSV exploits FAK signaling pathways to promote its nuclear transport. We used complementary molecular techniques, including dominant-negative variants, small interfering RNA (siRNA), and knockout cells, to explore the role played by FAK in HSV invasion. We tested the hypothesis with both serotypes using human and murine cells. Our findings indicate that FAK phosphorylation plays a central role in promoting transport of viral capsids. Cells and Viruses—CaSki (human cervical epithelial), CaCo-2 (human colonic epithelial), and SK-N-SH (human neuroblastoma) cells were obtained from American Type Culture Collection and passaged in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Immortalized human endocervical and ectocervical cells were a generous gift from R. N. Fichorova and D. J. Anderson (8Fichorova R.N. Rheinwald J.G. Anderson D.J. Biol. Reprod. 1997; 57: 847-855Crossref PubMed Scopus (262) Google Scholar). Mouse embryonic fibroblast (MEF) cells derived from embryonic FAK-deficient mice (FAK-/-) and control MEF cells (FAK+/+) were originally described by Ilic et al. (9Ilic D. Furuta Y. Kanazawa S. Takeda N. Sobue K. Nakatsuji N. Nomura S. Fujimoto J. Okada M. Yamamoto T. Nature. 1995; 377: 539-544Crossref PubMed Scopus (1587) Google Scholar) and were a generous gift from I. H. Gelman (Mount Sinai School of Medicine). The FAK+/+ cells were derived from the same age embryos (day 8). Cells were cultured overnight in serum-free medium to minimize background tyrosine phosphorylation due to growth factors in serum. HSV-2(G) (provided by B. Roizman, University of Chicago, Chicago, IL) and vesicular stomatitis virus-Indiana (provided by P. Palese, Mount Sinai School of Medicine) were grown on Vero cells. HSV-1 containing a green fluorescent protein (GFP)-VP26 fusion protein (designated K26GFP) was a generous gift from P. Desai (The Johns Hopkins University) (10Desai P. Person S. J. Virol. 1998; 72: 7563-7568Crossref PubMed Google Scholar). Heat-inactivated virus was prepared by heating to 56 °C for 30 min. Purification and Quantification of Viruses—For binding and Ca2+ studies, dextran-purified viruses were used as described previously (4Herold B.C. WuDunn D. Soltys N. Spear P.G. J. Virol. 1991; 65: 1090-1098Crossref PubMed Google Scholar, 11Herold B.C. Visalli R.J. Susmarski N. Brandt C.R. Spear P.G. J. Gen. Virol. 1994; 75: 1211-1222Crossref PubMed Scopus (295) Google Scholar). Titers of the purified virus were determined by plaque assays. Relative viral particle numbers were determined by comparing the amounts of glycoprotein D by optical densitometry after Western blotting with anti-glycoprotein D monoclonal antibody (mAb) 1103 (Rumbaugh-Goodwin Institute, Plantation, FL) as described previously (3Cheshenko N. Herold B.C. J. Gen. Virol. 2002; 83: 2247-2255Crossref PubMed Scopus (72) Google Scholar, 12Qie L. Marcellino D. Herold B.C. Virology. 1999; 256: 220-227Crossref PubMed Scopus (43) Google Scholar). Reagents—EGTA and ionomycin were purchased from Calbiochem and diluted in Me2SO or phosphate-buffered saline (PBS) following the manufacturer's instructions. Fura-2 acetoxymethyl ester was purchased from Molecular Probes, Inc. (Eugene, OR). Heparin and cycloheximide were purchased from Sigma. Plasmids and Transfections—pKH3-FAK carrying the gene for wild-type FAK and pKH3-Y397 carrying the FAK gene with a Y397F mutation were gifts from J.-L. Guan (Cornell University, Ithaca, NY). All transfections were done with Effectene transfection reagent (Qiagen Inc.) following the manufacturer's protocol. CaSki, FAK+/+, and FAK-/- cells were grown in 12-well plates and transfected with siRNA for FAK and Pyk2 and with control nonspecific siRNA using siRNA/ siAB™ assay kits (Upstate Biotechnology Inc.) at a concentration of 100 pmol/well. 24 h post-transfection, the cells were infected with virus and analyzed for expression of phosphorylated FAK/FAK and phosphorylated Pyk2/Pyk2 by preparing cell lysates and performing Western blotting as detailed below. Measurement of [Ca2+]i—Cells were loaded with 25 mm fura-2 acetoxymethyl ester prepared in PBS for 30-60 min, rinsed with PBS for 30 min at 37 °C, and then exposed sequentially to PBS buffer or HSV diluted in PBS at 37 °C. The viral inoculum was equivalent to a multiplicity of infection (m.o.i.) equal to 1-5 plaque-forming units (pfu)/cell. Using a Nikon Eclipse TE300 inverted epifluorescence microscope linked to a cooled Pentamax CCD camera (Princeton Instruments) interfaced with a digital imaging system (MetaFluor, Molecular Devices, Downingtown, PA), [Ca2+]i was measured in individually identified fura-2-loaded cells visualized using a Nikon S Fluor ×40 objective (numerical aperture of 0.9 and window of 0.3) as described previously (7Cheshenko N. Del Rosario B. Woda C. Marcellino D. Satlin L.M. Herold B.C. J. Cell Biol. 2003; 163: 283-293Crossref PubMed Scopus (118) Google Scholar). Cells were alternately excited at 340 and 380 nm, and the images were digitized for subsequent analysis. Images were acquired every 2-10 s. An intracellular calibration was performed at the conclusion of each experiment as described previously (7Cheshenko N. Del Rosario B. Woda C. Marcellino D. Satlin L.M. Herold B.C. J. Cell Biol. 2003; 163: 283-293Crossref PubMed Scopus (118) Google Scholar). 8-15 cells were monitored for each experiment. Plaque Assays—Briefly, cells in 6-well dishes were exposed to virus for 2 h at 37 °C; the inoculum was removed; and cells were washed 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) (4Herold B.C. WuDunn D. Soltys N. Spear P.G. J. Virol. 1991; 65: 1090-1098Crossref PubMed Google Scholar). For vesicular stomatitis virus, cells were overlaid with 1% methylcellulose, fixed after 24 h with methanol, and stained with Giemsa stain. Unless indicated otherwise, the m.o.i. was selected to yield ∼200-500 pfu/well (e.g. m.o.i. ∼ 0.005 pfu/cell) on control wells. Viral Binding and VP16 Time Course Assays—To examine which steps in HSV infection are blocked in transfected or knockout cells, viral binding and VP16 transport to the nucleus were compared. For binding studies, cells were exposed to serial 2-fold dilutions of purified HSV-2(G) for 5 h at 4 °C. Unbound virus was removed by washing, and cell-bound virus was analyzed by preparing Western blots of cell lysates and probing with anti-glycoprotein D mAb 1103. To examine transport of VP16 to the nucleus, synchronized infection assays were conducted as described previously (7Cheshenko N. Del Rosario B. Woda C. Marcellino D. Satlin L.M. Herold B.C. J. Cell Biol. 2003; 163: 283-293Crossref PubMed Scopus (118) Google Scholar). Cells were precooled and exposed to HSV at 4 °C for 2 h at a m.o.i. of 0.1, 1, or 10 pfu/cell to allow binding. Unbound virus was removed, and the cells were washed three times with PBS and then transferred to 37 °C for 15 min to allow penetration. Unpenetrated virus was inactivated by washing the cell monolayer with a low pH citrate buffer adjusted to pH 3.0 for 2 min and then by washing three times with PBS. The cells were overlaid with fresh medium, and nuclear extracts were prepared 1 h after citrate treatment. The nuclear proteins were separated on a 10% SDS-polyacrylamide gel, transferred to a polyvinylidene difluoride membrane (PerkinElmer Life Sciences) using a Trans-Blot system (Bio-Rad), and blocked overnight in 5% milk-containing Tris-buffered saline. Membranes were incubated with mouse anti-VP16 antibody (1:500 dilution; Santa Cruz Biotechnology, Inc.), diluted in 5% milk-containing Tris-buffered saline for 2 h, and rinsed extensively with 50 mm Tris-Cl (pH 7.4), 150 mm NaCl, and 0.05% Tween 20. The membranes were then incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (1:1000 dilution; Calbiochem) in 5% milk-containing PBS for 2 h. After rinsing, the membranes were developed using Immuno-Star-AP chemiluminescent kit (Bio-Rad). Blots were scanned and analyzed using the GelDoc 2000 system. Detection of FAK or Pyk2 Phosphorylation by Immunoblotting—Nearly confluent cell monolayers were preincubated with serum-free medium for 12 h before infection and then exposed to HSVs at a m.o.i. of 1 pfu/cell for the indicated times (5-120 min). Cell lysates were prepared as described above, and proteins were separated by SDS-PAGE and immunoblotting. Membranes were incubated with a 1:500 dilution of rabbit anti-phospho-Tyr397 FAK antibody, which recognizes the tyrosine autophosphorylation site at position 397 of FAK (catalog no. 44-624G, BIOSOURCE, Camarillo, CA), or with a 1:500 dilution of rabbit anti-phospho-Tyr402 Pyk2, which recognizes the tyrosine autophosphorylation site at position 402 (catalog no. 44-618G, BIOSOURCE). The membranes were then stripped and reincubated with a 1:1000 dilution of mouse anti-FAK mAb (catalog no. F15020, Transduction Laboratories), which recognizes total FAK, or with a 1:500 dilution of rabbit anti-Pyk2 antibody, which recognizes total Pyk2 (catalog no. 07-437, Upstate Biotechnology Inc.). Confocal Microscopy—CaSki, FAK+/+, and FAK-/- cells were grown on glass coverslips in 12-well plates and, if indicated, transfected with siRNA as described above and then infected 24 h post-transfection with K26GFP at the indicated m.o.i. (range of 0.1-10 pfu/cell). At the indicated times post-infection, the infected cell monolayers were washed three times with PBS. To label plasma membranes, the cells were stained for 30 min with EZ-Link sulfosuccinimidobiotin reagent (1:1000 dilution; Pierce), which reacts with primary amines on cell-surface proteins prior to fixation (13Foster T.P. Melancon J.M. Olivier T.L. Kousoulas K.G. J. Virol. 2004; 78: 13262-13277Crossref PubMed Scopus (61) Google Scholar), and the biotinylated cells were reacted with Alexa Fluor 647-conjugated streptavidin antibody (1:1000 dilution; Molecular Probes, Inc.). Microtubules were labeled with anti-α-tubulin mAb (1:200 dilution; catalog no. A1126, Molecular Probes, Inc.) and Alexa Fluor 647-conjugated goat anti-mouse secondary antibody (1:200 dilution; Molecular Probes, Inc.). Nuclei were detected by staining with 4′,6-diamidino-2-phenylindole nucleic acid stain (Molecular Probes, Inc.). Images were examined using a Zeiss LSM 510 Meta confocal microscope fitted with ×100 objective. Image analysis and subcellular co-localization were generated and analyzed using the LSM confocal software package. Quantification of amounts of cell-associated GFP was performed with NIH Image densitometric software. In addition, to determine the fraction of cells expressing nuclear GFP 8 h post-infection, 100 cells were analyzed and counted. We previously showed that HSV induces FAK phosphorylation in CaSki or Vero cells and that phosphorylation requires fusion-competent virus (7Cheshenko N. Del Rosario B. Woda C. Marcellino D. Satlin L.M. Herold B.C. J. Cell Biol. 2003; 163: 283-293Crossref PubMed Scopus (118) Google Scholar). This study was expanded to include multiple cell types known to be susceptible to HSV-2. Cells were serum-starved overnight and exposed to HSV-2 or mock-infected. At the indicated times post-infection, cell lysates were prepared and examined for expression of total and phosphorylated FAKs by Western blotting. HSV-2 induced an increase in phosphorylated FAK within 5 min in CaSki cells (Fig. 1). Similar results were obtained with immortalized human ectocervical cells (Ecto1E6/E7), colonic cells (CaCo-2), neuronal cells (SH-N-SK), and macrophages (Fig. 1). To determine whether phosphorylated FAK contributes to HSV infection, we used several different strategies. First, we exposed MEF cells derived from embryonic FAK-deficient mice (FAK-/-) (9Ilic D. Furuta Y. Kanazawa S. Takeda N. Sobue K. Nakatsuji N. Nomura S. Fujimoto J. Okada M. Yamamoto T. Nature. 1995; 377: 539-544Crossref PubMed Scopus (1587) Google Scholar) and control MEF cells (FAK+/+) to HSV-2 and examined FAK response and susceptibility to infection. HSV-2 induced an ∼3-fold increase in the level of phosphorylated FAK as a percentage of total FAK compared with mock-infected cells (Fig. 2A, left panel); the response persisted for ∼1 h. In contrast, no FAK or phosphorylated FAK was detected in FAK-/- cells (Fig. 2A, right panel). Moreover, FAK-/- cells showed reduced susceptibility to infection by either serotype (Fig. 2B). Viral titers were reduced by 93 and 96% in FAK-/- cells relative to FAK+/+ cells for HSV-1 and HSV-2, respectively. Notably, no reduction in susceptibility to vesicular stomatitis virus was observed for FAK-/- cells relative to FAK+/+ cells. Susceptibility to HSV infection was partially restored if cells were transfected with the wild-type FAK gene (data not shown). To more carefully explore which steps in viral infection are impaired in FAK-/- cells, we compared viral binding and nuclear transport of the viral tegument protein VP16 in FAK-/- and FAK+/+ cells. The results demonstrate that there was no reduction in binding of virus to cells in FAK-/- cells relative to FAK+/+ cells (Fig. 3A). However, nuclear transport of VP16 1 h after citrate treatment was substantially reduced in FAK-/- cells compared with FAK+/+ cells (Fig. 3B). The blots were scanned, and optical densitometry corrected for differences in loading. At a m.o.i. of 10 pfu/cell, there was an ∼93% reduction in the relative amounts of VP16 detected in the nuclei of FAK-/- cells compared with FAK+/+ cells. The amount of nuclear VP16 detected was comparable with that observed if FAK+/+ cells were infected with 10-fold less virus (m.o.i. = 1 pfu/cell). To ensure that nuclear VP16 reflects transported and not newly synthesized protein, experiments were also conducted in the presence of cycloheximide, an inhibitor of protein synthesis (Fig. 3C). Comparable amounts of nuclear VP16 were detected 1 h after citrate treatment in FAK+/+ cells independent of cycloheximide treatment and accounted for ∼73% of total cell-associated VP16. As anticipated, a substantial reduction in viral ICP4 expression was also observed in FAK-/- cells compared with FAK+/+ cells, consistent with impaired delivery of viral DNA to the cell nucleus and thus viral gene expression (data not shown). Taken together, these results suggest that, in the absence of FAK, viral invasion is blocked at a step post-binding, leading to reduced nuclear transport of VP16 and inhibition of downstream events, including expression of immediate-early gene products and plaque formation. Because the embryonic knockout cells are murine and because our goal is to identify the signaling pathways required for HSV infection of human target cells, we adopted a complementary strategy to assess the role of FAK and its phosphorylation in human cervical epithelial cells. CaSki cells were transfected with siRNA specific for FAK, a dominant-negative variant of FAK (Y397F) in which the major autophosphorylation site has been mutated (a gift from J.-L. Guan) (14Zhao J. Pestell R. Guan J.-L. Mol. Biol. Cell. 2001; 12: 4066-4077Crossref PubMed Scopus (163) Google Scholar), or appropriate controls, and the impact on HSV infection was examined. Transfection with FAK sequence-specific siRNA (but not control nonspecific siRNA) reduced FAK protein expression by ∼85% (based on optical densitometry scanning of blots) and prevented HSV-induced FAK phosphorylation (Fig. 4A). Similarly, a substantial reduction (∼70%) in virus-triggered phosphorylated FAK was also observed in CaSki cells transfected with Y397F compared with cells transfected with wild-type FAK (Fig. 4B). Transfection with the FAK-specific siRNA or the Y397F FAK variant also reduced HSV-2 infection (Fig. 4C), supporting the results obtained for FAK-/- cells showing that FAK and its phosphorylation play important roles in HSV infection. To determine whether FAK inactivation prevents viral capsid transport, confocal microscopy studies were conducted. For these studies, we took advantage of the viral variant, K26GFP, an HSV-1 that expresses a GFP-VP26 fusion protein; VP26 is a major viral capsid protein. K26GFP grows comparably to wild-type virus in cell culture (10Desai P. Person S. J. Virol. 1998; 72: 7563-7568Crossref PubMed Google Scholar), and infection of FAK-/- cells was impaired to a similar extent for K26GFP compared with HSV-2(G) (Fig. 2B). To determine the optimal m.o.i. for confocal experiments, CaSki cells were first infected with K26GFP at a m.o.i. of 0.1, 1, or 10 pfu/cell; at the indicated times post-infection, fixed and stained with EZ-Link sulfosuccinimidobiotin to detect cellular plasma membranes and with 4′,6-diamidino-2-phenylindole to detect nuclei; and viewed by confocal microscopy. Viral GFP was detected in the cytoplasm of most of the cells infected with 10 pfu/cell as early as 1 h post-infection and in some cells infected with 1 pfu/cell (Fig. 5A). However, at a m.o.i. of 0.1 pfu/cell, GFP was not visible. Notably, 4 h post-infection, newly synthesized GFP appeared to accumulate in the nucleus in cells infected with 1 or 10 pfu/cell. To confirm that the observed nuclear GFP reflects newly synthesized protein, experiments were conducted in the presence or absence of cycloheximide (m.o.i. = 5 pfu/cell). Cycloheximide had no effect on delivery of viral capsids to the cytoplasm and its transport to the nuclear pore at 1 and 2 h post-infection, but blocked synthesis of GFP at 4 and 8 h post-infection (Fig. 5B). Next, to explore the role played by FAK in HSV entry, CaSki cells were transfected with nonspecific siRNA (Fig. 6A, upper panels) or FAK-specific siRNA (middle panels), infected with K26GFP (m.o.i. = 10 pfu/cell), and then fixed and stained 1, 2, 4, and 8 h post-infection as described above. Viral GFP was detected at the plasma membrane and in the cytoplasm of CaSki cells as early as 1 h post-infection independent of transfection. However, transport of viral capsids to the nuclear pore and newly synthesized GFP were rarely detected in cells transfected with FAK-specific siRNA 4 and 8 h post-infection. Parallel experiments were conducted with MEF cells (Fig. 6B). As reported previously (15Gelman I.H. Cell Biol. Int. 2003; 27: 507-510Crossref PubMed Scopus (29) Google Scholar), knockout of FAK alters the morphology of fibroblast cells with loss of typical elongated morphology. Intracellular GFP-VP26 was detected 1 h post-infection in both FAK+/+ and FAK-/- cells, but newly synthesized GFP was observed only in FAK+/+ cells. The relative amounts of intracellular and nuclear GFPs at 1 and 8 h post-infection, respectively, were determined by scanning ∼100 cells using NIH Image densitometric software (Fig. 6C). No measurable reduction in intracellular GFP was detected 1 h post-infection in the absence of FAK (FAK-/- cells or CaSki cells transfected with FAK-specific siRNA), suggesting no impediment to viral entry. In contrast, a substantial reduction in newly synthesized GFP was detected, consistent with a block at the level of capsid transport to the nuclear pore. To further explore the role of FAK in capsid transport, confocal experiments were modified; and instead of staining the plasma membrane, microtubules were immunostained using anti-α-tubulin mAb. In CaSki and FAK+/+ cells, viral capsids were readily detected associated with microtubule structures 1 and 2 h post-infection (Fig. 7, upper and middle panels, respectively). In contrast, no GFP was detected in microtubules in FAK-/- cells (Fig. 7, lower panels). Taken together, these results indicate that, in the absence of FAK, there is a restriction to capsid transport, leading to a reduction in the delivery of viral DNA to the nucleus and subsequent viral gene expression. Our previous study suggested that phosphorylation of FAK is triggered downstream of Ca2+ signaling because pharmacological agents that block the release of ER Ca2+ or chelation of intracellular Ca2+ prevent virus-induced phosphorylation of FAK (7Cheshenko N. Del Rosario B. Woda C. Marcellino D. Satlin L.M. Herold B.C. J. Cell Biol. 2003; 163: 283-293Crossref PubMed Scopus (118) Google Scholar). These observations suggest that Ca2+ signaling should be preserved in FAK-/- cells. To test this directly, we examined the virus-induced Ca2+ signaling response by excitation ratio fluorometry of cells loaded with the Ca2+ indicator fura-2. FAK+/+ and FAK-/- cells were exposed sequentially to PBS or HSV-2(G) at a m.o.i. of 5 pfu/cell. A rapid increase in the superfusate flow rate of PBS alone, simulating the conditions elicited by the addition of virus to the bathing medium, had no effect on resting [Ca2+]i. The resting [Ca2+]i of FAK+/+ and FAK-/- cells were comparable (∼100 nm) (Fig. 8). Exposure of both FAK+/+ and FAK-/- cells to HSV resulted in a significant rapid increase in [Ca2+]i, which peaked within 1 min and returned to the base line within ∼3 min (p < 0.001, analysis of variance) (Fig. 8), although the mean change in peak [Ca2+]i was greater for FAK+/+ cells compared with FAK-/- cells (181.4 ± 101.1 versus 70 ± 45 nm, respectively; mean ± S.E., n = 20 cells), possibly reflecting a reduction in virus-induced Ca2+ signaling in the absence of FAK or perturbations of other signal transduction pathways in the knockout cells. The ability of HSV to induce a significant Ca2+ response in FAK-/- cells supports the notion that FAK phosphorylation occurs downstream of virus-induced release of ER Ca2+. Pyk2 is a non-receptor tyrosine kinase related to FAK; is activated by various" @default.
• W2049212648 created "2016-06-24" @default.
• W2049212648 creator A5001969147 @default.
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• W2049212648 creator A5049888042 @default.
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• W2049212648 date "2005-09-01" @default.
• W2049212648 modified "2023-10-18" @default.
• W2049212648 title "Focal Adhesion Kinase Plays a Pivotal Role in Herpes Simplex Virus Entry" @default.
• W2049212648 cites W1572556994 @default.
• W2049212648 cites W1977990503 @default.
• W2049212648 cites W1985977887 @default.
• W2049212648 cites W1995964370 @default.
• W2049212648 cites W2016171668 @default.
• W2049212648 cites W2016347089 @default.
• W2049212648 cites W2028691987 @default.
• W2049212648 cites W2030604587 @default.
• W2049212648 cites W2036594578 @default.
• W2049212648 cites W2066888192 @default.
• W2049212648 cites W2089333797 @default.
• W2049212648 cites W2096039378 @default.
• W2049212648 cites W2106604059 @default.
• W2049212648 cites W2116403673 @default.
• W2049212648 cites W2118016056 @default.
• W2049212648 cites W2121768774 @default.
• W2049212648 cites W2125967276 @default.
• W2049212648 cites W2129251085 @default.
• W2049212648 cites W2131239923 @default.
• W2049212648 cites W2133298868 @default.
• W2049212648 cites W2134940342 @default.
• W2049212648 cites W2136242594 @default.
• W2049212648 cites W2142862392 @default.
• W2049212648 cites W2143783595 @default.
• W2049212648 cites W2147890616 @default.
• W2049212648 cites W2156423903 @default.
• W2049212648 cites W2168733588 @default.
• W2049212648 cites W2171667802 @default.
• W2049212648 doi "https://doi.org/10.1074/jbc.m503518200" @default.
• W2049212648 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/15994312" @default.
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