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Overexpression of CIN85 suppresses the growth of herpes simplex virus in HeLa cells
Experimental Cell Research 311 (2005) 265 – 271 www.elsevier.com/locate/yexcr
Research Article
Overexpression of CIN85 suppresses the growth of herpes simplex virus in HeLa cells Tadashi Naritaa, Akikazu Andob, Yuzuru Mikamic, Tadayoshi Taniyamaa,* a
Laboratory of Bacterial Infection and Immunity, Department of Immunology, National Institute of Infectious Diseases, 1-23-1, Toyama, Shinjuku, Tokyo 162-8640, Japan b Graduate School of Science and Technology, Chiba University, 648 Matsudo, Matsudo-city, Chiba 271-8510, Japan c Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, 1-81-1, Inohana, Chuo-ku, Chiba 260-8673, Japan Received 21 June 2005, revised version received 12 September 2005, accepted 14 September 2005 Available online 11 October 2005
Abstract The adaptor protein CIN85 is widely distributed in different tissues and has three Src homology 3 (SH3) domains, a proline-rich region (PRR), and a coiled-coil domain. During studies on the function of CIN85, it was reported to form a complex with herpes simplex virus 1 (HSV-1) infected cell protein 0 (ICP0), which plays a key role in enabling viral replication. Here, we demonstrate that plaque formation by HSV-1 is reduced on HeLa cells expressing CIN85 ectopically. The PRR of CIN85 was found to be essential for the inhibition of virus growth, whereas the three SH3 domains were not required. CIN85 also suppressed HSV-1 growth in Chinese hamster ovary (CHO) cells expressing the receptor for herpes simplex virus entry (herpes virus entry mediator A; HVEM). However, immunoprecipitation experiments showed that CIN85 did not interact with HVEM directly, indicating that CIN85 is not involved in the HSV-1 cell-entry pathway, but rather in another downstream pathway. Collectively, our data indicate that CIN85 might play an important role in HSV-1 infection. D 2005 Elsevier Inc. All rights reserved. Keywords: CIN85; Herpes simplex virus, HSV; Herpes virus entry mediator A, HVEM; Proline-rich region; SH3KBP1; SH3 domain
Introduction Cbl-interacting protein of 85 kDa (CIN85) is an adaptor protein containing three Src homology 3 (SH3) domains, a proline-rich region (PRR), and a coiled-coil domain [1]. We identified this protein independently as SH3 domain kinase binding-protein 1 (SH3KBP1) and reported that its gene maps to the human X chromosome region p22.1 –p21.3 [2]. CIN85 and its rat homolog, Ruk/SETA [3,4], are widely expressed in a variety of tissues and form complexes with many signaling proteins via its PRR [3– 7] or SH3 domains [8– 11]. These complexes play a role in various biological functions, including control of receptor tyrosine kinase (RTK) signaling [6,7,10 – 12], rearrangement of the actin
* Corresponding author. Fax: +81 3 5285 1150. E-mail address: [email protected] (T. Taniyama). 0014-4827/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2005.09.007
cytoskeleton [13], and apoptosis in neuronal cells [3,14]. Interestingly, CIN85 forms a complex of Cbl – CIN85 – endophilins. This complex associates with RTKs, such as epidermal growth factor (EGF), hepatocyte growth factor (HGF), and platelet-derived growth factor (PDGF) receptors, and regulates the internalization of activated RTKs through clathrin-coated pits. We have shown that CIN85 associates with tumor necrosis factor (TNF) receptor 1 that does not have a tyrosine kinase domain through Src, and enhances TNF-a-induced apoptosis in T cells [15]. Recently, Roizman and his group demonstrated that a protein from herpes simplex virus 1 (HSV-1), infected cell protein 0 (ICP0), interacts with CIN85 [16]. ICP0 is expressed immediately after infection and is not essential for virus replication. ICP0 plays an important role in enabling vial replication [17]. Therefore, it is of great interest whether CIN85 might influence HSV infection. Here, we show that overexpression of CIN85 suppresses
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HSV-1 growth in HeLa cells and that the PRR in CIN85 was required to achieve this effect.
Materials and methods Materials The cell culture media and antibiotics used were purchased from the following sources: Dulbecco’s modified Eagle’s medium (DMEM) was from Sigma; Eagle’s MEM medium (MEM) was from Nissui Seiyaku (Tokyo, Japan); F-12 nutrient mixture medium (HAM) and fetal bovine serum (FBS) were from GIBCO BRL; blasticidin S (BS) was from Funakoshi (Tokyo, Japan); geneticin (GN) was from GIBCO BRL; and zeocin was from Invitrogen. Polyclonal anti-CIN85 antiserum was produced by immunizing a rabbit with a KLH-conjugated peptide, CKKIRLRLQMEVNDIKKALQSK (amino acids 645 – 665 from CIN85). IgG antibodies were affinity purified on a Sepharose column (Amersham Biosciences) to which the same peptide was bound. Anti-herpes virus entry mediator A (HVEM) antibody was obtained from Santa Cruz Biotechnology. Horseradish peroxidase (HRP)-conjugated anti-V5 antibody and HRP-conjugated anti-FLAG antibody were purchased from Invitrogen and Sigma, respectively. The expression vectors pEF6/V5-His C and pEF1/ V5-His C were from Invitrogen, and pFLAG-CMV-4 was from Sigma. The V5-tagged herpes virus entry mediator A (HVEM) expression vector (pcDNA3.1/GS/V5-HVEM, clone ID#, RG000784) was obtained from Invitrogen. Cells and virus Human cervix adenocarcinoma HeLa 229 and African green monkey kidney fibroblast Vero cells were obtained from the Dainippon Pharmaceutical Co., Ltd. (Osaka, Japan) and maintained in DMEM containing 10% FBS, at 37-C in a 5% CO2 incubator. Chinese hamster ovary (CHO-K1) cells were obtained from the Health Science Research Resources Bank (HSRRB, Osaka, Japan) and maintained in HAM containing 10% FBS. HSV-1 (HF strain, ATCC VR-260) was obtained from the American Type Culture Collection and grown in Vero cells in MEM with 2% FBS. It was harvested by three freeze-thaw cycles at concentrations of 3.4 107 TCID50 units/ml, where TCID50 (50% tissue-culture infectious dose) is the quantity of virus
that caused cytopathic effects (CPE) in 50% of Vero cells; this was measured using confluent monolayers of Vero cells in 96-well tissue-culture dishes, at eight dilutions (the 10-fold serial dilutions) of each sample, with five replicate wells at each dilution. The CPE was calculated using the method of Reed and Muench [18]. The solution of HSV-1 contained 2.2 107 plaque-forming units (PFU)/ml assayed using Vero cells and this quantity of virus was used to calculate of multiplicity of infection (MOI). Construction of stable transformants of HeLa and CHO-K1 cells The expression vectors used for CIN85 and its deletion mutants (Fig. 1) were as described previously and the same method was used to ligate a cDNA fragment of CIN85 into the BamHI/EcoRI site of pEF6/V5-His C (pEF6/V5-HisCCIN85) [15]. HeLa cells (1 107 cells) were transfected with 10 Ag of the CIN85 expression vector or its deletion derivatives by electroporation. After 48 h, transfectants were selected with 2 Ag/ml BS, for pEF6/V5-His C, or 0.8 mg/ml GN, for pEF1/V5-His C and pFLAG-CMV-4, for 2– 3 weeks and stable transformed clones were obtained. The BS selected cell lines were designated to as HeLa/V5-CIN85BS and HeLa/control-BS, while the GN selected cell lines to HeLa/V5-CIN85-GN and HeLa/control-GN. Expression of V5-CIN85, V5-CIN85-dSH3dPRR, FLAG-CIN85SH3ABC or FLAG-CIN85-PRR was verified by Western blot analysis, as described previously [15]. Control HeLa cells were established by introducing vector DNA without an insert into the cells using the same method. CHO-K1 cells were transfected with pcDNA3.1/GS/V5HVEM as described above, stable transformants were selected with 1 mg/ml zeocin and stable transformed clones were obtained. After verifying V5-HVEM expression by Western blot analysis, one clone that was sensitive to HSV-1 infection was selected for further experiments. This clone was transfected with the CIN85 expression vector, pEF1/V5-His C, as described above, stable transformants were selected in 1 mg/ml zeocin and 0.8 mg/ml GN and cloned. Control CHOK1 cells were obtained by introducing vector DNA without an insert into CHO-K1 cells using the same method. Plaque formation assay Confluent monolayers of HeLa transformants in 12-well tissue-culture dishes were rinsed with MEM containing 2%
Fig. 1. Schematic diagram of products expressed by CIN85 and its derived deletion vectors, shown on the right.
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FBS and infected with HSV-1 (6 102 TCID50/0.2 ml/ well). After incubation for 1 h at 37-C, the virus inocula were removed from the monolayers and replaced with 2 ml MEM containing 2% FBS and 0.6% methylcellulose. After 3 days incubation, the medium was removed and plaques were counted by staining the monolayers with 0.02% crystal violet containing 2% formaldehyde. All experiments were performed in triplicate. For CHO-K1 mutant cells, HSV-1 was inoculated at 1.2 104 TCID50/0.2 ml/well and then replaced with 2 ml HAM containing 1% FBS and 0.6% methylcellulose. After 4 days, the medium was removed and plaques were counted as described above.
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poly-vinylidene fluoride membranes (Millipore) and incubated with the appropriate antibodies. Bands were visualized using a chemiluminescent substrate (Pierce) and exposure to X-ray film. Measurement of cellular growth HeLa transformants were distributed into 96-well tissueculture dishes at 2 103 cells per well in DMEM containing 10% FBS. After incubation for 1, 3, and 5 days at 37-C, the number of viable cells was assessed using the WST-1 reagent (Roche). All experiments were performed in triplicate.
Measurement of infectious efficiency Statistical analysis The cells of HeLa transformants were seeded in 96-well tissue-culture dishes at 2.5 104 cells/well and incubated for 16 h at 37-C. After the monolayers were rinsed with MEM containing 2% FBS, the 10-fold serial dilutions of HSV-1 in MEM containing 2% FBS were added to the wells at 2.5 104 PFU/well (MOI = 1) to 2.5 10 3 PFU/well (MOI = 1 10 7) with 5 replicate wells for each dilution. After incubation for 1 h at 37-C, the virus inocula were replaced with 0.1 ml MEM containing 2% FBS. After 4 days incubation, the medium was removed and the monolayers were stained with 0.02% crystal violet containing 2% formaldehyde to visualize the CPE. Three times of experiments were performed. The infectious efficiency was determined as the percentage of wells detecting a CPE over five wells. HSV-1-replication assay Confluent monolayers of HeLa transformants in 24-well tissue-culture dishes (3 105 cells/well) were rinsed with MEM containing 2% FBS and infected with HSV-1 at 1 102 TCID50/0.1 ml/well. After incubation for 1 h at 37-C, virus inocula were removed and replaced with 1 ml MEM containing 2% FBS. After incubation for between 1 and 5 days, the virus was harvested by three freeze-thaw cycles. Experiments were performed in triplicate. The amount of virus in each suspension was expressed as TCID50 values, measured using confluent Vero cells. Western blot analysis and immunoprecipitation Western blot analysis and immunoprecipitation were carried out as described previously [15]. Briefly, cells were lysed with M-PER (Pierce) containing a protease inhibitor cocktail (Roche). One-milligram aliquots of the cleared extracts were incubated overnight at 4-C with anti-HVEM antibody and the immune complexes were precipitated by rotating the samples with 100 Al protein-G Sepharose (50%), at 4-C for 4 h. After the beads were washed, the immunoprecipitated proteins were separated by SDS-PAGE under reducing conditions. Proteins were transferred to
Significant differences between experimental groups were analyzed by Dunnett’s t test using Statistical Analysis System (SAS) software (SAS Institute Inc.). P values <0.05 were considered statistically significant.
Results and discussion Effect of overexpressing CIN85 in HeLa cells on HSV-1 plaque formation Two stable transformants of HeLa cells (HeLa/V5CIN85-BS cl. 1 and cl. 2) were constructed using pEF6/ V5-His C-CIN85 and BS selection. The effects of CIN85 on HSV-1 infection were determined using two HeLa cell clones, which, compared to the endogenous level in control cells, expressed 16- and 2-times the level of CIN85, respectively (Fig. 2A). When these clones were infected with HSV-1, plaque formation was reduced on HeLa/V5CIN85-BS cl. 1 cells but not on HeLa/V5-CIN85-BS cl. 2 cells (Fig. 2B). The plaque count on HeLa/V5-CIN85-BS cl. 1 was less than half of control. Furthermore, we compared sensitivity of these clones to HSV-1 infection. As shown in Fig. 2C, an infectious efficiency of 50% in HeLa/V5CIN85-BS cl. 1 and HeLa/control-BS cells was achieved when the amounts of inoculating HSV-1 were 2 10 3 and 3 10 5 MOI (PFU/cell), respectively. Thus, HeLa/V5CIN85-BS cl. 1 cells were approximately two orders of magnitude less sensitive to HSV-1 infection than HeLa/ control-BS cells. Since the condition of host cells can influence viral replication, we tested the growth of HeLa/ V5-CIN85-BS cl. 1 and HeLa/control-BS cells. As shown in Fig. 2D, the overexpression of V5-CIN85 did not affect cellular growth. We also tested HSV-1 infection in two additional stable HeLa transformants that were constructed using pEF1/V5-His C-CIN85 and GN selection and designated HeLa/V5-CIN85-GN cl. 1 and HeLa/V5CIN85-GN cl. 2 (Fig. 2E). Fig. 2F indicates that, in both clones, plaque formation with HSV-1 infection was reduced, as shown for HeLa/V5-CIN85-BS cl. 1. Moreover, protein
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Fig. 2. Inhibition of HSV-1 plaque formation as a result of the overexpression of CIN85 in HeLa cells. (A) Expression of V5-CIN85 in HeLa/V5-CIN85-BS cells (top) and endogenous or total CIN85 in each cell line (bottom) in Western blots. A 20-Ag sample of each cell lysate was applied to each lane. The ratio of the proteins shown below each track was determined by densitometry (ATTO, Tokyo, Japan). (B) Plaque formation assays were performed as described in the Materials and methods section. The numbers of plaques obtained with HSV-1 are expressed as the % of control plaques. Data are the means T standard deviations (SDs; n = 3). The number of plaques on HeLa/control cells was 84.7 T 13.3. Significant differences between HeLa/V5-CIN85-BS cells and HeLa/ control cells are indicated by asterisks (*P < 0.05; ns, not significant). (C) The sensitivity of HeLa/V5-CIN85-BS cells to HSV-1 infection was determined using confluent monolayers of HeLa transformants, originally seeded at 2.5 104 cells/well in 96-well tissue-culture dishes. Monolayers were infected with HSV-1 at the MOI, indicated on the x axis, with 5 replicate wells for each dilution. After 1 h at 37-C, the virus inocula were replaced with 0.1 ml MEM containing 2% FBS. After incubation for 4 days, the medium was removed and the monolayers were stained with 0.02% crystal violet containing 2% formaldehyde to visualize the CPE. The infectious efficiency ( y axis) was determined as the percentage of wells detecting a CPE over five wells. Data are the means T SDs (n = 3). (D) Growth of HeLa/V5-CIN85-BS and HeLa/control-BS cells. Cells were plated into 96-well tissue-culture dishes at 2 103 cells/100 Al in DMEM containing 10% FBS. After incubation for 1, 3, and 5 days, 10 Al WST-1 was added in each well and incubated for 2 h, and absorbance at 450 nm was measured. (E) Expression of V5-CIN85 in HeLa/V5-CIN85-BS and HeLa/V5-CIN85-GN cells, and expression of V5-CIN85-dSH3dPRR in HeLa/V5CIN85-dSH3dPRR cells, in Western blots. A 20-Ag sample of each cell lysate was applied to each lane. (F) Effects of V5-CIN85 and V5-CIN85-dSH3dPRR expression of HSV-1 plaque formation. Plaque formation assays were performed as described in the Materials and methods section. The numbers of plaques obtained with HSV-1 are expressed as the % of control plaques. Data are the means T SDs (n = 3). The numbers of plaques on HeLa/control-BS and HeLa/ control-GN cells were 50.0 T 12.3 and 59.3 T 4.9, respectively. Significant differences between HeLa/V5-CIN85-BS, HeLa/V5-CIN85-GN or HeLa/V5CIN85-dSH3dPRR cells and HeLa/control cells are indicated by asterisks (*P < 0.05; **P < 0.01; ns, not significant).
overexpression is a potential source of stress on cells and this could plausibly, in turn, cause HSV-1 growth suppression. However, we excluded this possible explanation by showing that HeLa/V5-CIN85-dSH3dPRR cells did not suppress HSV-1 infection (Fig. 2F). Thus, the overexpression of CIN85 was indeed associated with the suppression of HSV-1 growth in HeLa cells. The PRR in CIN85 is important for the inhibition of HSV infections The adapter functions of CIN85 are abolished in V5CIN85-dSH3dPRR, as it lacks both SH3 domains and the PRR. We therefore investigated whether the overexpression
of V5-CIN85-dSH3dPRR in HeLa cells inhibited HSV-1 infection. Three clones expressing V5-CIN85-dSH3dPRR ectopically were constructed (Fig. 2E). These clones did not inhibit plaque formation by HSV-1 (Fig. 2F), indicating that the SH3 domains and/or the PRR in CIN85 were important for its inhibition of HSV-1 growth. Our next experiment aimed to determine whether the three SH3 domains or the PRR in CIN85 were required to inhibit HSV infections. Two HeLa cell lines were constructed, one expressing FLAG-tagged CIN85-SH3ABC (HeLa/FLAGCIN85-SH3ABC) and the other expressing FLAG-tagged CIN85-PRR (HeLa/FLAG-CIN85-PRR). Three clones were obtained for each construct and infected with HSV-1 (Fig. 3A). When HeLa/FLAG-CIN85-SH3ABC cells were
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approximately two orders of magnitude less sensitive to HSV-1 infection than HeLa/FLAG-control cells (Fig. 3C). The growth rates of these cell lines were similar (data not shown). These results show that the PRR in CIN85 is essential for the inhibition of HSV-1 growth in HeLa cells. The CIN85 PRR binds to the SH3 domain of many other proteins. For example, we previously used a domain array to show that SH3 domains from the Src family kinases, interleukin-2-inducible T cell kinase, peroxisomal membrane protein pex13, Abl, and phospholipase Cg 1 strongly bind to the CIN85 PRR [15]. These cellular proteins might be involved in a number of HSV-1 infection processes. HSV-1 replication is inhibited by overexpression of CIN85 Although we had established that CIN85 overexpression inhibited plaque formation by HSV-1, it was unclear whether it also suppressed HSV-1 replication. In order to resolve this issue, therefore, we examined the yields of virus in various HeLa cell clones at day 1 to day 5 after inoculation of HSV-1. MOI of this experiment was used at 0.0002 PFU/cell. The yields of virus were calculated as TCID50 values. As shown in Fig. 4A, the quantity of HSV-1 in HeLa/V5-CIN85-BS was decreased at day 2 to day 5 compared to that of control cells. Similarly, in HeLa/FLAGCIN85-PRR cells, the quantity of virus was also decreased at day 2 to day 5 (Fig. 4B). Therefore, the replication of HSV-1 in the CIN85 overexpressing cells was suppressed, and it was approximately two orders of magnitude lower than that of control cells. These suppression ratios were similar the results in Figs. 2C or 3C. Thus, these results indicate that the overexpression of CIN85 containing a PRR inhibits not only plaque formation, but also the replication of HSV-1 in HeLa cells.
Fig. 3. The PRR, and not the SH3 domains, of CIN85 suppressed plaque formation by HSV-1 in HeLa cells. (A) Expressions of FLAG-CIN85SH3ABC (left) and FLAG-CIN85-PRR (right), in Western blots. A 20-Ag sample of each cell lysate was applied to each lane. (B) Plaque formation assays were performed as described in the Materials and methods section. The numbers of plaques obtained with HSV-1 are expressed as the % of control plaques. Data shown are the means T SDs (n = 3). The number of plaques on HeLa/FLAG-control was 73.3 T 10.4. Significant differences between HeLa/FLAG-CIN85-SH3ABC or HeLa/FLAG-CIN85-PRR cells and HeLa/FLAG-control cells are indicated by asterisks (*P < 0.05; **P < 0.01; ns, not significant). (C) The sensitivity of HeLa/FLAG-CIN85-PRR and HeLa/FLAG-CIN85-SH3ABC cells to HSV-1 infection. Experiments were performed as described in the legend of Fig. 2C.
infected with HSV-1, the extent of plaque formation was almost identical to (or greater than) HeLa/FLAG-control cells (Fig. 3B). However, when HeLa/FLAG-CIN85-PRR cells were infected with HSV-1, plaque formation was reduced compared to control cells. Similarly, HeLa/FLAG-CIN85PRR cells (but not HeLa/FLAG-CIN85-SH3ABC cells) were
Fig. 4. Inhibition of HSV-1-replication by CIN85 expression in HeLa cells. An HSV-1-replication assay was performed as described in the Materials and methods section. The quantities of HSV-1 were indicated as TCID50 values ( y axis). Solid lines indicate HSV-1 quantity in HeLa/V5-CIN85-BS cells (A) and HeLa/FLAG-CIN85-PRR cells (B). Dotted lines indicate HSV-1 quantity in each of the HeLa/control cells. Data are the means T SDs (n = 3).
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Overexpression of CIN85 inhibits HSV-1 growth in CHO-K1 cells expressing HVEM, but CIN85 does not interact with HVEM Although CHO-K1 cells are naturally resistant to HSV entry, expression of HVEM sensitizes them, allowing virus penetration [19]. This system is therefore useful for examining the process of HSV cell entry. Stably transformed CHO-K1 cells expressing V5-tagged HVEM (V5HVEM) were constructed and one clone that was sensitive to HSV-1 infection was isolated. Next, a V5-CIN85 expression vector was introduced into these cells and stable transformants expressing V5-CIN85 were obtained. The expression of both V5-HVEM and V5-CIN85 is shown in Fig. 5B (lower panel). This cell line allowed us to determine whether the overexpression of CIN85 in HSV-infectable CHO-K1 cells expressing HVEM suppressed HSV growth after HVEM had permitted virus entry. Inoculation with HSV-1 confirmed that CHO-K1/V5HVEM cells were sensitive to infection, as plaque formation occurred. CHO-K1/V5-HVEM/V5-CIN85 cells were also sensitive to HSV-1 infection, although the numbers of plaques formed were lower than on CHO-K1/V5-HVEM cells (Fig. 5A). As expected, no HSV-1 plaques formed on CHO-K1/control or CHO-K1/V5-CIN85 cells. CIN85 interacts with TNF receptor-associated factor 1 (TRAF1) via Src [15], and TRAF2 and TRAF5 bind to the cytoplasmic tail of HVEM [20,21]. Therefore, we investigated whether CIN85 interacted directly with the HVEM cytoplasmic domain. Cell lysates were prepared from the CHO-K1 mutants and immunoprecipitated with an antiHVEM antibody. The immunoprecipitates were then subjected to Western blotting and probed with an anti-V5 antibody. This demonstrated that V5-CIN85 did not coprecipitate with V5-HVEM (Fig. 5B) and indicated that CIN85 does not interact directly with the HVEM cytoplasmic domain. Thus, CIN85 does not participate directly in the HSV-1 cell-entry pathway, but acts further downstream in the infection process. The precise mechanisms by which CIN85 suppresses the growth of HSV-1 are still unknown. Recently, Roizman and his group reported that ICP0 from HSV-1, which plays a key role in enabling viral replication, forms a complex with CIN85 and Cbl. This complex mediates EGF receptor degradation. ICP0 contains several SH3 binding sites and binds to CIN85 SH3 domain [16]. Herpes simplex virus 2 (HSV-2) ribonucleotide reductase (ICP10), which has Ser/Thr protein kinase activity, has been shown to prevent apoptosis in infected cells [22,23]. It is interesting to note that the ICP10 protein kinase has SH3 binding sites and is known to bind to Grb2 [24]. The PRR of CIN85 also binds to Grb2 [3 –5]. Thus, CIN85 might form complexes with the other HSV-1 molecules in addition to ICP0. Although further work is required to determine the mechanisms by which CIN85 suppresses the growth of HSV-1, our results strongly suggest a previously
Fig. 5. Inhibition of HSV-1 growth by CIN85 in CHO-K1 cells expressing V5-HVEM does not involve a direct interaction between CIN85 and HVEM. (A) Plaque formation assays were performed as described in the Materials and methods section. Two clones from each transfection were tested. HSV-1 was inoculated at 1.2 104 TCID50/well and incubated for 4 days. Data shown are the means T SDs (n = 3). Significant differences between CHO-K1/V5-HVEM/V5-CIN85 and CHO-K1/V5-HVEM cells are indicated by asterisks (**P < 0.01). (B) One-milligram aliquots of cell lysates from each CHO-K1 transformant were immunoprecipitated with 10 Ag anti-HVEM antibody. Western blots of the immunoprecipitates were probed with anti-V5 antibody (top panel). To determine the expression of V5-HVEM and V5-CIN85 in each cell line, a 20-Ag sample of each cell lysate was applied on each lane (bottom).
unknown role for CIN85, involving its PRR, in HSV-1 infection.
Acknowledgments We thank K. Nashimoto and K. Nakazawa of SSP Co., Ltd. for technical assistance in the culture of HSV-1.
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Research Article
Overexpression of CIN85 suppresses the growth of herpes simplex virus in HeLa cells Tadashi Naritaa, Akikazu Andob, Yuzuru Mikamic, Tadayoshi Taniyamaa,* a
Laboratory of Bacterial Infection and Immunity, Department of Immunology, National Institute of Infectious Diseases, 1-23-1, Toyama, Shinjuku, Tokyo 162-8640, Japan b Graduate School of Science and Technology, Chiba University, 648 Matsudo, Matsudo-city, Chiba 271-8510, Japan c Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, 1-81-1, Inohana, Chuo-ku, Chiba 260-8673, Japan Received 21 June 2005, revised version received 12 September 2005, accepted 14 September 2005 Available online 11 October 2005
Abstract The adaptor protein CIN85 is widely distributed in different tissues and has three Src homology 3 (SH3) domains, a proline-rich region (PRR), and a coiled-coil domain. During studies on the function of CIN85, it was reported to form a complex with herpes simplex virus 1 (HSV-1) infected cell protein 0 (ICP0), which plays a key role in enabling viral replication. Here, we demonstrate that plaque formation by HSV-1 is reduced on HeLa cells expressing CIN85 ectopically. The PRR of CIN85 was found to be essential for the inhibition of virus growth, whereas the three SH3 domains were not required. CIN85 also suppressed HSV-1 growth in Chinese hamster ovary (CHO) cells expressing the receptor for herpes simplex virus entry (herpes virus entry mediator A; HVEM). However, immunoprecipitation experiments showed that CIN85 did not interact with HVEM directly, indicating that CIN85 is not involved in the HSV-1 cell-entry pathway, but rather in another downstream pathway. Collectively, our data indicate that CIN85 might play an important role in HSV-1 infection. D 2005 Elsevier Inc. All rights reserved. Keywords: CIN85; Herpes simplex virus, HSV; Herpes virus entry mediator A, HVEM; Proline-rich region; SH3KBP1; SH3 domain
Introduction Cbl-interacting protein of 85 kDa (CIN85) is an adaptor protein containing three Src homology 3 (SH3) domains, a proline-rich region (PRR), and a coiled-coil domain [1]. We identified this protein independently as SH3 domain kinase binding-protein 1 (SH3KBP1) and reported that its gene maps to the human X chromosome region p22.1 –p21.3 [2]. CIN85 and its rat homolog, Ruk/SETA [3,4], are widely expressed in a variety of tissues and form complexes with many signaling proteins via its PRR [3– 7] or SH3 domains [8– 11]. These complexes play a role in various biological functions, including control of receptor tyrosine kinase (RTK) signaling [6,7,10 – 12], rearrangement of the actin
* Corresponding author. Fax: +81 3 5285 1150. E-mail address: [email protected] (T. Taniyama). 0014-4827/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2005.09.007
cytoskeleton [13], and apoptosis in neuronal cells [3,14]. Interestingly, CIN85 forms a complex of Cbl – CIN85 – endophilins. This complex associates with RTKs, such as epidermal growth factor (EGF), hepatocyte growth factor (HGF), and platelet-derived growth factor (PDGF) receptors, and regulates the internalization of activated RTKs through clathrin-coated pits. We have shown that CIN85 associates with tumor necrosis factor (TNF) receptor 1 that does not have a tyrosine kinase domain through Src, and enhances TNF-a-induced apoptosis in T cells [15]. Recently, Roizman and his group demonstrated that a protein from herpes simplex virus 1 (HSV-1), infected cell protein 0 (ICP0), interacts with CIN85 [16]. ICP0 is expressed immediately after infection and is not essential for virus replication. ICP0 plays an important role in enabling vial replication [17]. Therefore, it is of great interest whether CIN85 might influence HSV infection. Here, we show that overexpression of CIN85 suppresses
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HSV-1 growth in HeLa cells and that the PRR in CIN85 was required to achieve this effect.
Materials and methods Materials The cell culture media and antibiotics used were purchased from the following sources: Dulbecco’s modified Eagle’s medium (DMEM) was from Sigma; Eagle’s MEM medium (MEM) was from Nissui Seiyaku (Tokyo, Japan); F-12 nutrient mixture medium (HAM) and fetal bovine serum (FBS) were from GIBCO BRL; blasticidin S (BS) was from Funakoshi (Tokyo, Japan); geneticin (GN) was from GIBCO BRL; and zeocin was from Invitrogen. Polyclonal anti-CIN85 antiserum was produced by immunizing a rabbit with a KLH-conjugated peptide, CKKIRLRLQMEVNDIKKALQSK (amino acids 645 – 665 from CIN85). IgG antibodies were affinity purified on a Sepharose column (Amersham Biosciences) to which the same peptide was bound. Anti-herpes virus entry mediator A (HVEM) antibody was obtained from Santa Cruz Biotechnology. Horseradish peroxidase (HRP)-conjugated anti-V5 antibody and HRP-conjugated anti-FLAG antibody were purchased from Invitrogen and Sigma, respectively. The expression vectors pEF6/V5-His C and pEF1/ V5-His C were from Invitrogen, and pFLAG-CMV-4 was from Sigma. The V5-tagged herpes virus entry mediator A (HVEM) expression vector (pcDNA3.1/GS/V5-HVEM, clone ID#, RG000784) was obtained from Invitrogen. Cells and virus Human cervix adenocarcinoma HeLa 229 and African green monkey kidney fibroblast Vero cells were obtained from the Dainippon Pharmaceutical Co., Ltd. (Osaka, Japan) and maintained in DMEM containing 10% FBS, at 37-C in a 5% CO2 incubator. Chinese hamster ovary (CHO-K1) cells were obtained from the Health Science Research Resources Bank (HSRRB, Osaka, Japan) and maintained in HAM containing 10% FBS. HSV-1 (HF strain, ATCC VR-260) was obtained from the American Type Culture Collection and grown in Vero cells in MEM with 2% FBS. It was harvested by three freeze-thaw cycles at concentrations of 3.4 107 TCID50 units/ml, where TCID50 (50% tissue-culture infectious dose) is the quantity of virus
that caused cytopathic effects (CPE) in 50% of Vero cells; this was measured using confluent monolayers of Vero cells in 96-well tissue-culture dishes, at eight dilutions (the 10-fold serial dilutions) of each sample, with five replicate wells at each dilution. The CPE was calculated using the method of Reed and Muench [18]. The solution of HSV-1 contained 2.2 107 plaque-forming units (PFU)/ml assayed using Vero cells and this quantity of virus was used to calculate of multiplicity of infection (MOI). Construction of stable transformants of HeLa and CHO-K1 cells The expression vectors used for CIN85 and its deletion mutants (Fig. 1) were as described previously and the same method was used to ligate a cDNA fragment of CIN85 into the BamHI/EcoRI site of pEF6/V5-His C (pEF6/V5-HisCCIN85) [15]. HeLa cells (1 107 cells) were transfected with 10 Ag of the CIN85 expression vector or its deletion derivatives by electroporation. After 48 h, transfectants were selected with 2 Ag/ml BS, for pEF6/V5-His C, or 0.8 mg/ml GN, for pEF1/V5-His C and pFLAG-CMV-4, for 2– 3 weeks and stable transformed clones were obtained. The BS selected cell lines were designated to as HeLa/V5-CIN85BS and HeLa/control-BS, while the GN selected cell lines to HeLa/V5-CIN85-GN and HeLa/control-GN. Expression of V5-CIN85, V5-CIN85-dSH3dPRR, FLAG-CIN85SH3ABC or FLAG-CIN85-PRR was verified by Western blot analysis, as described previously [15]. Control HeLa cells were established by introducing vector DNA without an insert into the cells using the same method. CHO-K1 cells were transfected with pcDNA3.1/GS/V5HVEM as described above, stable transformants were selected with 1 mg/ml zeocin and stable transformed clones were obtained. After verifying V5-HVEM expression by Western blot analysis, one clone that was sensitive to HSV-1 infection was selected for further experiments. This clone was transfected with the CIN85 expression vector, pEF1/V5-His C, as described above, stable transformants were selected in 1 mg/ml zeocin and 0.8 mg/ml GN and cloned. Control CHOK1 cells were obtained by introducing vector DNA without an insert into CHO-K1 cells using the same method. Plaque formation assay Confluent monolayers of HeLa transformants in 12-well tissue-culture dishes were rinsed with MEM containing 2%
Fig. 1. Schematic diagram of products expressed by CIN85 and its derived deletion vectors, shown on the right.
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FBS and infected with HSV-1 (6 102 TCID50/0.2 ml/ well). After incubation for 1 h at 37-C, the virus inocula were removed from the monolayers and replaced with 2 ml MEM containing 2% FBS and 0.6% methylcellulose. After 3 days incubation, the medium was removed and plaques were counted by staining the monolayers with 0.02% crystal violet containing 2% formaldehyde. All experiments were performed in triplicate. For CHO-K1 mutant cells, HSV-1 was inoculated at 1.2 104 TCID50/0.2 ml/well and then replaced with 2 ml HAM containing 1% FBS and 0.6% methylcellulose. After 4 days, the medium was removed and plaques were counted as described above.
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poly-vinylidene fluoride membranes (Millipore) and incubated with the appropriate antibodies. Bands were visualized using a chemiluminescent substrate (Pierce) and exposure to X-ray film. Measurement of cellular growth HeLa transformants were distributed into 96-well tissueculture dishes at 2 103 cells per well in DMEM containing 10% FBS. After incubation for 1, 3, and 5 days at 37-C, the number of viable cells was assessed using the WST-1 reagent (Roche). All experiments were performed in triplicate.
Measurement of infectious efficiency Statistical analysis The cells of HeLa transformants were seeded in 96-well tissue-culture dishes at 2.5 104 cells/well and incubated for 16 h at 37-C. After the monolayers were rinsed with MEM containing 2% FBS, the 10-fold serial dilutions of HSV-1 in MEM containing 2% FBS were added to the wells at 2.5 104 PFU/well (MOI = 1) to 2.5 10 3 PFU/well (MOI = 1 10 7) with 5 replicate wells for each dilution. After incubation for 1 h at 37-C, the virus inocula were replaced with 0.1 ml MEM containing 2% FBS. After 4 days incubation, the medium was removed and the monolayers were stained with 0.02% crystal violet containing 2% formaldehyde to visualize the CPE. Three times of experiments were performed. The infectious efficiency was determined as the percentage of wells detecting a CPE over five wells. HSV-1-replication assay Confluent monolayers of HeLa transformants in 24-well tissue-culture dishes (3 105 cells/well) were rinsed with MEM containing 2% FBS and infected with HSV-1 at 1 102 TCID50/0.1 ml/well. After incubation for 1 h at 37-C, virus inocula were removed and replaced with 1 ml MEM containing 2% FBS. After incubation for between 1 and 5 days, the virus was harvested by three freeze-thaw cycles. Experiments were performed in triplicate. The amount of virus in each suspension was expressed as TCID50 values, measured using confluent Vero cells. Western blot analysis and immunoprecipitation Western blot analysis and immunoprecipitation were carried out as described previously [15]. Briefly, cells were lysed with M-PER (Pierce) containing a protease inhibitor cocktail (Roche). One-milligram aliquots of the cleared extracts were incubated overnight at 4-C with anti-HVEM antibody and the immune complexes were precipitated by rotating the samples with 100 Al protein-G Sepharose (50%), at 4-C for 4 h. After the beads were washed, the immunoprecipitated proteins were separated by SDS-PAGE under reducing conditions. Proteins were transferred to
Significant differences between experimental groups were analyzed by Dunnett’s t test using Statistical Analysis System (SAS) software (SAS Institute Inc.). P values <0.05 were considered statistically significant.
Results and discussion Effect of overexpressing CIN85 in HeLa cells on HSV-1 plaque formation Two stable transformants of HeLa cells (HeLa/V5CIN85-BS cl. 1 and cl. 2) were constructed using pEF6/ V5-His C-CIN85 and BS selection. The effects of CIN85 on HSV-1 infection were determined using two HeLa cell clones, which, compared to the endogenous level in control cells, expressed 16- and 2-times the level of CIN85, respectively (Fig. 2A). When these clones were infected with HSV-1, plaque formation was reduced on HeLa/V5CIN85-BS cl. 1 cells but not on HeLa/V5-CIN85-BS cl. 2 cells (Fig. 2B). The plaque count on HeLa/V5-CIN85-BS cl. 1 was less than half of control. Furthermore, we compared sensitivity of these clones to HSV-1 infection. As shown in Fig. 2C, an infectious efficiency of 50% in HeLa/V5CIN85-BS cl. 1 and HeLa/control-BS cells was achieved when the amounts of inoculating HSV-1 were 2 10 3 and 3 10 5 MOI (PFU/cell), respectively. Thus, HeLa/V5CIN85-BS cl. 1 cells were approximately two orders of magnitude less sensitive to HSV-1 infection than HeLa/ control-BS cells. Since the condition of host cells can influence viral replication, we tested the growth of HeLa/ V5-CIN85-BS cl. 1 and HeLa/control-BS cells. As shown in Fig. 2D, the overexpression of V5-CIN85 did not affect cellular growth. We also tested HSV-1 infection in two additional stable HeLa transformants that were constructed using pEF1/V5-His C-CIN85 and GN selection and designated HeLa/V5-CIN85-GN cl. 1 and HeLa/V5CIN85-GN cl. 2 (Fig. 2E). Fig. 2F indicates that, in both clones, plaque formation with HSV-1 infection was reduced, as shown for HeLa/V5-CIN85-BS cl. 1. Moreover, protein
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Fig. 2. Inhibition of HSV-1 plaque formation as a result of the overexpression of CIN85 in HeLa cells. (A) Expression of V5-CIN85 in HeLa/V5-CIN85-BS cells (top) and endogenous or total CIN85 in each cell line (bottom) in Western blots. A 20-Ag sample of each cell lysate was applied to each lane. The ratio of the proteins shown below each track was determined by densitometry (ATTO, Tokyo, Japan). (B) Plaque formation assays were performed as described in the Materials and methods section. The numbers of plaques obtained with HSV-1 are expressed as the % of control plaques. Data are the means T standard deviations (SDs; n = 3). The number of plaques on HeLa/control cells was 84.7 T 13.3. Significant differences between HeLa/V5-CIN85-BS cells and HeLa/ control cells are indicated by asterisks (*P < 0.05; ns, not significant). (C) The sensitivity of HeLa/V5-CIN85-BS cells to HSV-1 infection was determined using confluent monolayers of HeLa transformants, originally seeded at 2.5 104 cells/well in 96-well tissue-culture dishes. Monolayers were infected with HSV-1 at the MOI, indicated on the x axis, with 5 replicate wells for each dilution. After 1 h at 37-C, the virus inocula were replaced with 0.1 ml MEM containing 2% FBS. After incubation for 4 days, the medium was removed and the monolayers were stained with 0.02% crystal violet containing 2% formaldehyde to visualize the CPE. The infectious efficiency ( y axis) was determined as the percentage of wells detecting a CPE over five wells. Data are the means T SDs (n = 3). (D) Growth of HeLa/V5-CIN85-BS and HeLa/control-BS cells. Cells were plated into 96-well tissue-culture dishes at 2 103 cells/100 Al in DMEM containing 10% FBS. After incubation for 1, 3, and 5 days, 10 Al WST-1 was added in each well and incubated for 2 h, and absorbance at 450 nm was measured. (E) Expression of V5-CIN85 in HeLa/V5-CIN85-BS and HeLa/V5-CIN85-GN cells, and expression of V5-CIN85-dSH3dPRR in HeLa/V5CIN85-dSH3dPRR cells, in Western blots. A 20-Ag sample of each cell lysate was applied to each lane. (F) Effects of V5-CIN85 and V5-CIN85-dSH3dPRR expression of HSV-1 plaque formation. Plaque formation assays were performed as described in the Materials and methods section. The numbers of plaques obtained with HSV-1 are expressed as the % of control plaques. Data are the means T SDs (n = 3). The numbers of plaques on HeLa/control-BS and HeLa/ control-GN cells were 50.0 T 12.3 and 59.3 T 4.9, respectively. Significant differences between HeLa/V5-CIN85-BS, HeLa/V5-CIN85-GN or HeLa/V5CIN85-dSH3dPRR cells and HeLa/control cells are indicated by asterisks (*P < 0.05; **P < 0.01; ns, not significant).
overexpression is a potential source of stress on cells and this could plausibly, in turn, cause HSV-1 growth suppression. However, we excluded this possible explanation by showing that HeLa/V5-CIN85-dSH3dPRR cells did not suppress HSV-1 infection (Fig. 2F). Thus, the overexpression of CIN85 was indeed associated with the suppression of HSV-1 growth in HeLa cells. The PRR in CIN85 is important for the inhibition of HSV infections The adapter functions of CIN85 are abolished in V5CIN85-dSH3dPRR, as it lacks both SH3 domains and the PRR. We therefore investigated whether the overexpression
of V5-CIN85-dSH3dPRR in HeLa cells inhibited HSV-1 infection. Three clones expressing V5-CIN85-dSH3dPRR ectopically were constructed (Fig. 2E). These clones did not inhibit plaque formation by HSV-1 (Fig. 2F), indicating that the SH3 domains and/or the PRR in CIN85 were important for its inhibition of HSV-1 growth. Our next experiment aimed to determine whether the three SH3 domains or the PRR in CIN85 were required to inhibit HSV infections. Two HeLa cell lines were constructed, one expressing FLAG-tagged CIN85-SH3ABC (HeLa/FLAGCIN85-SH3ABC) and the other expressing FLAG-tagged CIN85-PRR (HeLa/FLAG-CIN85-PRR). Three clones were obtained for each construct and infected with HSV-1 (Fig. 3A). When HeLa/FLAG-CIN85-SH3ABC cells were
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approximately two orders of magnitude less sensitive to HSV-1 infection than HeLa/FLAG-control cells (Fig. 3C). The growth rates of these cell lines were similar (data not shown). These results show that the PRR in CIN85 is essential for the inhibition of HSV-1 growth in HeLa cells. The CIN85 PRR binds to the SH3 domain of many other proteins. For example, we previously used a domain array to show that SH3 domains from the Src family kinases, interleukin-2-inducible T cell kinase, peroxisomal membrane protein pex13, Abl, and phospholipase Cg 1 strongly bind to the CIN85 PRR [15]. These cellular proteins might be involved in a number of HSV-1 infection processes. HSV-1 replication is inhibited by overexpression of CIN85 Although we had established that CIN85 overexpression inhibited plaque formation by HSV-1, it was unclear whether it also suppressed HSV-1 replication. In order to resolve this issue, therefore, we examined the yields of virus in various HeLa cell clones at day 1 to day 5 after inoculation of HSV-1. MOI of this experiment was used at 0.0002 PFU/cell. The yields of virus were calculated as TCID50 values. As shown in Fig. 4A, the quantity of HSV-1 in HeLa/V5-CIN85-BS was decreased at day 2 to day 5 compared to that of control cells. Similarly, in HeLa/FLAGCIN85-PRR cells, the quantity of virus was also decreased at day 2 to day 5 (Fig. 4B). Therefore, the replication of HSV-1 in the CIN85 overexpressing cells was suppressed, and it was approximately two orders of magnitude lower than that of control cells. These suppression ratios were similar the results in Figs. 2C or 3C. Thus, these results indicate that the overexpression of CIN85 containing a PRR inhibits not only plaque formation, but also the replication of HSV-1 in HeLa cells.
Fig. 3. The PRR, and not the SH3 domains, of CIN85 suppressed plaque formation by HSV-1 in HeLa cells. (A) Expressions of FLAG-CIN85SH3ABC (left) and FLAG-CIN85-PRR (right), in Western blots. A 20-Ag sample of each cell lysate was applied to each lane. (B) Plaque formation assays were performed as described in the Materials and methods section. The numbers of plaques obtained with HSV-1 are expressed as the % of control plaques. Data shown are the means T SDs (n = 3). The number of plaques on HeLa/FLAG-control was 73.3 T 10.4. Significant differences between HeLa/FLAG-CIN85-SH3ABC or HeLa/FLAG-CIN85-PRR cells and HeLa/FLAG-control cells are indicated by asterisks (*P < 0.05; **P < 0.01; ns, not significant). (C) The sensitivity of HeLa/FLAG-CIN85-PRR and HeLa/FLAG-CIN85-SH3ABC cells to HSV-1 infection. Experiments were performed as described in the legend of Fig. 2C.
infected with HSV-1, the extent of plaque formation was almost identical to (or greater than) HeLa/FLAG-control cells (Fig. 3B). However, when HeLa/FLAG-CIN85-PRR cells were infected with HSV-1, plaque formation was reduced compared to control cells. Similarly, HeLa/FLAG-CIN85PRR cells (but not HeLa/FLAG-CIN85-SH3ABC cells) were
Fig. 4. Inhibition of HSV-1-replication by CIN85 expression in HeLa cells. An HSV-1-replication assay was performed as described in the Materials and methods section. The quantities of HSV-1 were indicated as TCID50 values ( y axis). Solid lines indicate HSV-1 quantity in HeLa/V5-CIN85-BS cells (A) and HeLa/FLAG-CIN85-PRR cells (B). Dotted lines indicate HSV-1 quantity in each of the HeLa/control cells. Data are the means T SDs (n = 3).
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Overexpression of CIN85 inhibits HSV-1 growth in CHO-K1 cells expressing HVEM, but CIN85 does not interact with HVEM Although CHO-K1 cells are naturally resistant to HSV entry, expression of HVEM sensitizes them, allowing virus penetration [19]. This system is therefore useful for examining the process of HSV cell entry. Stably transformed CHO-K1 cells expressing V5-tagged HVEM (V5HVEM) were constructed and one clone that was sensitive to HSV-1 infection was isolated. Next, a V5-CIN85 expression vector was introduced into these cells and stable transformants expressing V5-CIN85 were obtained. The expression of both V5-HVEM and V5-CIN85 is shown in Fig. 5B (lower panel). This cell line allowed us to determine whether the overexpression of CIN85 in HSV-infectable CHO-K1 cells expressing HVEM suppressed HSV growth after HVEM had permitted virus entry. Inoculation with HSV-1 confirmed that CHO-K1/V5HVEM cells were sensitive to infection, as plaque formation occurred. CHO-K1/V5-HVEM/V5-CIN85 cells were also sensitive to HSV-1 infection, although the numbers of plaques formed were lower than on CHO-K1/V5-HVEM cells (Fig. 5A). As expected, no HSV-1 plaques formed on CHO-K1/control or CHO-K1/V5-CIN85 cells. CIN85 interacts with TNF receptor-associated factor 1 (TRAF1) via Src [15], and TRAF2 and TRAF5 bind to the cytoplasmic tail of HVEM [20,21]. Therefore, we investigated whether CIN85 interacted directly with the HVEM cytoplasmic domain. Cell lysates were prepared from the CHO-K1 mutants and immunoprecipitated with an antiHVEM antibody. The immunoprecipitates were then subjected to Western blotting and probed with an anti-V5 antibody. This demonstrated that V5-CIN85 did not coprecipitate with V5-HVEM (Fig. 5B) and indicated that CIN85 does not interact directly with the HVEM cytoplasmic domain. Thus, CIN85 does not participate directly in the HSV-1 cell-entry pathway, but acts further downstream in the infection process. The precise mechanisms by which CIN85 suppresses the growth of HSV-1 are still unknown. Recently, Roizman and his group reported that ICP0 from HSV-1, which plays a key role in enabling viral replication, forms a complex with CIN85 and Cbl. This complex mediates EGF receptor degradation. ICP0 contains several SH3 binding sites and binds to CIN85 SH3 domain [16]. Herpes simplex virus 2 (HSV-2) ribonucleotide reductase (ICP10), which has Ser/Thr protein kinase activity, has been shown to prevent apoptosis in infected cells [22,23]. It is interesting to note that the ICP10 protein kinase has SH3 binding sites and is known to bind to Grb2 [24]. The PRR of CIN85 also binds to Grb2 [3 –5]. Thus, CIN85 might form complexes with the other HSV-1 molecules in addition to ICP0. Although further work is required to determine the mechanisms by which CIN85 suppresses the growth of HSV-1, our results strongly suggest a previously
Fig. 5. Inhibition of HSV-1 growth by CIN85 in CHO-K1 cells expressing V5-HVEM does not involve a direct interaction between CIN85 and HVEM. (A) Plaque formation assays were performed as described in the Materials and methods section. Two clones from each transfection were tested. HSV-1 was inoculated at 1.2 104 TCID50/well and incubated for 4 days. Data shown are the means T SDs (n = 3). Significant differences between CHO-K1/V5-HVEM/V5-CIN85 and CHO-K1/V5-HVEM cells are indicated by asterisks (**P < 0.01). (B) One-milligram aliquots of cell lysates from each CHO-K1 transformant were immunoprecipitated with 10 Ag anti-HVEM antibody. Western blots of the immunoprecipitates were probed with anti-V5 antibody (top panel). To determine the expression of V5-HVEM and V5-CIN85 in each cell line, a 20-Ag sample of each cell lysate was applied on each lane (bottom).
unknown role for CIN85, involving its PRR, in HSV-1 infection.
Acknowledgments We thank K. Nashimoto and K. Nakazawa of SSP Co., Ltd. for technical assistance in the culture of HSV-1.
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