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Cip1 as a molecular sensor for BoHV-4 replication
Journal of Virological Methods 161 (2009) 308–311
Contents lists available at ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Short communication
p21Waf1/Cip1 as a molecular sensor for BoHV-4 replication A. Capocefalo a , V. Franceschi a , C.B.A. Whitelaw b , D.B. Vasey b , S.G. Lillico b , S. Cavirani a , G. Donofrio a,∗ a b
Facoltà di Medicina Veterinaria, Dipartimento di Salute Animale, Sezione di Malattie Infettive degli Animali, via del Taglio 10, 43100 Parma, Italy Division of Developmental Biology, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin, Midlothian EH25 9PS, Scotland, UK
a b s t r a c t Article history: Received 9 March 2009 Received in revised form 27 May 2009 Accepted 10 June 2009 Available online 18 June 2009 Keywords: BoHV-4 p21Waf1/Cip1 Cellular damage
BoHV-4 replication cycle is dependent on the S-phase of the cell-cycle at the stage of viral DNA synthesis. Because p21 is a rate-limiting regulator of the G1/S-phase transition and up-regulated by DNA-damaging agents, in this study p21 expression in BoHV-4 infected cells was investigated. The p21 promoter was found to be highly activated in a dose- and time-dependent manner following BoHV-4 infection only in cells which are permissive for BoHV-4 replication. Thus p21 expression reports on BoHV-4 replication and could represent a host cell defensive response to infection-associated cellular damage. © 2009 Elsevier B.V. All rights reserved.
Bovine herpesvirus 4 (BoHV-4) is a herpesvirus and members of the Gammaherpesvirinae subfamily. BoHV-4 was first isolated in Europe from animals with respiratory and ocular diseases by Bartha et al. (1966) and later in the United States by Mohanty et al. (1971). BoHV-4 has been isolated from a variety of samples and cells (Egyed, 1998) from healthy cattle and from cattle with abortion, metritis, pneumonia, diarrhea, respiratory infection, and mammary pustular dermatitis (Bartha et al., 1966; Thiry et al., 1989). Although BoHV4 has been demonstrated in many tissues, accumulated evidence suggests that one site of persistence in both the natural and experimental host is cells of the monocyte/macrophage lineage (Osorio et al., 1985). However, the pathogenic role of BoHV-4 remains unclear; the direct correlation between particular strains of BoHV-4 with variable disease conditions is a delicate question, unsolved even through experimental infection. BoHV-4 causes cytopathic effect (CPE) and replicates in a variety of cell lines and primary cultures of various bovine and other animal species in culture (Peterson and Goyal, 1988). In contrast, several other gammaherpesviruses establish a latent infection in vitro. Viral gene expression is restricted to a specific subset of genes and the cells survive and replicate. The viral genome is maintained as a circular episome (Ceserman et al., 1995) and the origin of replication for the circular viral genome, oriP, distinct from the origin of replication used during lytic viral replication, oriLyt, have been identified (Zimmermann et al., 2001). The outcome of BoHV-4 infection depends on the cell type: (i) permissive infection, characterized by strong cytopathic effect and an abundant viral replication and high level of infectious virus production. (ii) Un-permissive
∗ Corresponding author. Tel.: +39 0521032677; fax: +39 0521032672. E-mail address: [email protected] (G. Donofrio). 0166-0934/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2009.06.012
infection, where BoHV-4 infected cells produced low levels of early and late viral RNAs, but caused no cytopathic effect. (iii) Persistent infection, which could follow a permissive infection, surviving and replicating cells were found to be persistently infected, maintaining the viral genome over many passages and producing low levels of infectious virus (Donofrio et al., 2000; Donofrio and van Santen, 2001). In contrast to other viruses, replication of most herpesviruses is generally assumed to be dependent of the stage of the host cell in the cell-cycle (Knipe, 1990; Shadan et al., 1994). The possibility that BoHV-4 replication could be dependent on the cell-cycle was investigated by Vanderplasschen et al. (1995), who showed that the replication in vitro of the BoHV-4 is restricted by its DNA synthesis dependence on the S-phase of the cell-cycle and the following assumptions were stated: (i) cell transition through the S-phase quantitatively increased the rate of BoHV-4 DNA replication. (ii) BoHV-4 DNA synthesis could not be detected in cells arrested in G0. (iii) Drug synchronization of cells before infection increased the percentage of cells expressing L proteins. (iv) Infection of cells arrested in G0 led to few BoHV-4 positive cells. Because the CKI p21Waf1/Cip1 (p21) is a rate-limiting regulator of the G1/S-phase transition (Bartek and Lukas, 2000), its transcriptional regulation in permissive and unpermissive BoHV-4 infected cells was investigated in the present communication. Initially, a bovine macrophage cell line (BoMac, from V. van Santen, Auburn University, USA) susceptible to BoHV-4 infection (Donofrio and van Santen, 2001) was transfected with a reporter construct characterized by a 4 kb murine p21 promoter driving the -galactosidase reporter gene, pX3W (Vasey et al., 2008) (Fig. 1A). BoMac cells from a confluent 25-cm2 flask maintained as a monolayer with growth medium containing 90% DMEM, 10% FBS, 2 mM l-glutamine, 100 IU/ml penicillin and 10 g/ml streptomycin and
A. Capocefalo et al. / Journal of Virological Methods 161 (2009) 308–311
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Fig. 1. (A) Diagram showing the pX3W reporter construct, containing the murine p21 promoter in front of the -galactosidase ORF. (B) Representative phase contrast image of BoHV-4 treated cells expressing -galactosidase and the untreated control. (C) The experiment was repeated three times giving always the same result.
incubated at 37 ◦ C in a humidified atmosphere of 95% air–5% CO2 were electroporated with pX3W (5 g) in 500 l DMEM without serum (Equibio apparatus, 186 V, 960 F, 4-mm gap cuvettes). To monitor the efficiency of transfection a parallel electroporation was performed with pEGFP-C1 (Invitrogen, Milan, Italy). Twenty-four hours post-electroporation, cells were infected with 5 multiplicity of infection of BoHV-4EGFPTK, a recombinant BoHV-4 delivering an enhanced green fluorescent protein (EGFP) expression cassette under the control of human cytomegalovirus (CMV) promoter, inserted into the thymidine kinase (TK) locus of BoHV-4 genome (DN599 strain, ATCC VR631) (Donofrio et al., 2002). This recombinant BoHV-4 allowed a direct monitoring of the infection through EGFP expression. Twenty-four hours post-infection, cells were fixed with 0.2% of glutaraldehyde and stained with X-gal for -galactosidase expression (Vasey et al., 2008). BoHV-4EGFPTK infected cells expressed -galactosidase as shown by blue cells (Fig. 1B), in contrast no blue cells appeared in the uninfected control (Fig. 1C). To be able to quantify better the p21 promoter activity in transfected cells, a luciferase reporter construct was developed by sub-cloning the p21 promoter in front of the luciferase open read-
ing frame (ORF) reporter gene contained into the pGL3-Basic vector (Promega, Milan, Italy), to generate p21-Luc reporter construct, facilitated by generating a new EcoRI site in the pGL3-Basic vector multi-cloning site. A 1 kb kanamycin amplicon obtained by PCR using primers (Sense, 5 -aaacccggtaccgaattcccggaattgccagctggg3 ; Antisense, 5 -gggcccctcgaggaaattgtaagcgttaataat-3 ) containing KpnI and EcoRI sites to the 5 -end and XhoI to the 3 end, was subcloned into KpnI/XhoI cut pGL3-Basic vector. Then, the kanamycin amplicon was removed with EcoRI/XhoI restriction digestion and the 4 kb p21 promoter, excised-out from pX3W with EcoRI/XhoI was ligated into EcoRI/XhoI linearized pGL3-Basic vector to get p21-Luc (Fig. 2A). BoMac cells were transfected by electroporation with p21-Luc and infected with BoHV-4EGFPTK as described above. Cells were harvested at different times after infection (6, 12 and 24 h postinfection) and luciferase activity measured. Luciferase reporter assay was performed with a Dual Luciferase Reporter Assay System kit (Promega, Milan, Italy) with minor modifications. Following treatments, cells were washed with PBS, lysed with 100 l of lysis passive buffer by freeze–thawing at −80 ◦ C. 20 l of the cell lysate was added to 50 l of LAR and luciferase activity was determined
Fig. 2. (A) Diagram showing the p21-luc reporter construct, containing the p21 promoter in front of the luciferase ORF. (B) p21 transiently transfected BoMac cells, BoHV-4 treated (black box) or untreated (gray box) and analyzed for luciferase expression at different time points (6, 12 and 24 h). The fluorescence microscope image showing green fluorescent cells, represent the infection level monitored through EGFP expression conferred by the recombinant BoHV-4 delivering an EGFP expression cassette. (C) p21 transiently transfected BoMac cells, treated with different dilutions and compared with the untreated control cells. The data represents the mean relative luciferase units. Each reaction was done in quadruplicate, and each point represents the average ± standard deviation from three experiments. * P < 0.001, as calculated by Student’s t-test or one-way ANOVA. (For interpretation of the references to color in this figure caption, the reader is referred to the web version of the article.)
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A. Capocefalo et al. / Journal of Virological Methods 161 (2009) 308–311
Fig. 3. (A) p21 transiently transfected HC11 cells, BoHV-4 treated (black box) or untreated (gray box) and analyzed for luciferase expression at different time points (6, 12 and 24 h). (B) p21 transiently transfected RD-4 cells, BoHV-4 treated (black box) or untreated (gray box) and analyzed for luciferase expression at different time points (6, 12 and 24 h). The data represents the mean relative luciferase units. Each reaction was done in quadruplicate, and each point represents the average ± standard deviation from three experiments. * P < 0.001, as calculated by Student’s t-test or one-way ANOVA.
with a PerkinElmer Victor3 Multilabel Counter, according to the manufacturer’s specifications. The p21 promoter was highly activated within 24 h postinfection (Fig. 2b) as observed for -galactosidase reporter system (Fig. 1B). However, further time post-infection was not possible to analyse due to the CPE and cell death induced by viral infection and replication. Hence, to confirm the specificity of this data, a dose dependant activation of p21 promoter by BoHV-4 infection was investigated. BoMac cells were transfected as before with p21Luc and infected with different dilution of BoHV-4EGFPTK and 24 h post-infection analyzed for luciferase activity. As expected, p21 promoter was activated in a dose dependent manner (Fig. 2c). Next, a similar experiment was performed on the HC11 mouse cell line (mouse mammary gland cells) permissive for BoHV-4 replication and on the RD-4 human cell line (human rhabdomyosarcoma cells, obtained from D. Derse, National Cancer Institute, Frederick, MD, USA) unpermissive to BoHV-4 replication. p21 promoter was activated only in BoHV-4 permissive HC11 cells which displayed a CPE (Fig. 3A) but not in RD-4 unpermissive cells which remain healthy (Fig. 3B). Therefore, p21 promoter activation correlated with BoHV4 replication. Viruses have evolved elegant approaches to use optimally limited genetic capacity to manipulate the intracellular environment. One common viral strategy is to usurp cellular signalling pathways to create an optimal environment for viral replication. Herpesvirus genomes are linear DNA molecules that circularize upon entry into the cell. The circular form of viral DNA conserved as a template for rolling-circle type of replication with viral genome concatamers are resolved by yet unidentified endonuclease(s). Replication of DNA is associated with increased levels of linear DNA molecules or abnormal DNA structures within the cell nucleus that could be recognized by cellular sensors of DNA damage and trigger DNA damage signalling. The mechanism of DNA damage signalling induction as well its physiological relevance for murine herpesvirus 68 (MHV68), a gamma herpesvirus highly related to BoHV-4, was provided by Tarakanova et al., 2007. p21 is a key mediator of the growth arrest induced by the tumor suppressor protein p53 in response to DNA damage (El-Deiry et al., 1993). p53 can stimulate transcription of the p21 gene by binding to the two p53-response elements located at positions −2281 to −2262 and −1395 to −1376 in the p21 promoter (El-Deiry et al., 1992; Resnick-Silverman et al., 1998). p21 was originally identified as an inhibitor of cyclin-dependent kinases (CDKs), including CDK2, CDK4 and CDC2 kinase complexes (Gu et al., 1993; Harper et al., 1993; Xiong et al., 1993). Moreover, it was shown that p21 can inhibit CDK3 and CDK6 kinases, and
that p21 is most effective towards G1/S cyclin-CDKs (Harper et al., 1995). Taking into account that BoHV-4 replication is dependent on the S-phase of the cell-cycle and p21 is a rate-limiting regulator of the G1/S-phase transition, p21 promoter activation reflects BoHV-4 replication and it could be hypothesized that p21 up-regulation is an attempt of the infected cells to block viral replication. In this brief communication the induction of p21 following BoHV-4 infection and replication in permissive cells has been shown. Although the viral and cellular factors required for p21 promoter transactivation were not investigated, a new door has been opened to a new insight into BoHV-4 and cellular interaction. Acknowledgement We would like to thank the Fondazione Cariparma (Cassa di Risparmio di Parma, Italy) for founding contribution to the project. References Bartha, A., Juhasz, M., Liebermann, H., 1966. Isolation of a bovine herpesvirus from calves with respiratory disease and keratoconjuntivitis. Acta Vet. Acad. Sci. Hung. 16, 357–358. Bartek, J., Lukas, J., 2000. Pathways governing G1/S transition and their response to DNA damage. FEBS Lett. 490, 117–122. Ceserman, E., Moore, P.S., Rao, P.H., Inghirami, G., Knowles, D.M., Chang, Y., 1995. In vitro establishment and characterization of two acquired immunodeficiency syndrome-related lymphoma cell lines (BC-1 and BC-2) containing Kaposi’s sarcoma-associated herpesvirus-like (KSHV) DNA sequences. Blood 86, 2708–2714. Donofrio, G., Cavirani, S., van Santen, V.L., 2000. Establishment of a cell line persistently infected with bovine herpesvirus-4 by use of a recombinant virus. J. Gen. Virol. 81, 1807–1814. Donofrio, G., van Santen, V.L., 2001. A bovine macrophage cell line supports bovine herpesvirus-4 persistent infection. J. Gen. Virol. 82, 1181–1185. Donofrio, G., Cavirani, S., Taddei, S., van Santen, V.L., 2002. Potential of bovine herpesvirus 4 as a gene delivery vector. J. Virol. Methods 101, 49–61. Egyed, L., 1998. Replication of bovine herpesvirus type 4 in human cells in vitro. J. Clin. Microbiol. 36, 2109–2111. El-Deiry, W.S., Kern, S.E., Pietenpol, J.A., Kinzler, K.W., Vogelstein, B., 1992. Definition of a consensus binding site for p53. Nat. Genet. 1, 45–49. El-Deiry, W.S., Tokino, T., Velculescu, V.E., Levy, D.B., Parsons, R., Trent, J.M., Lin, D., Mercer, W.E., Kinzler, K.W., Vogelstein, B., 1993. WAF1, a potential mediator of p53 tumor suppression. Cell 75, 817–825. Gu, Y., Turck, C.W., Morgan, D.O., 1993. Inhibition of CDK2 activity in vivo by an associated 20K regulatory subunit. Nature 366, 707–710. Harper, J.W., Adami, G.R., Wei, N., Keyomarsi, K., Elledge, S.J., 1993. The p21 Cdkinteracting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell 75, 805–816. Harper, J.W., Elledge, S.J., Keyomarsi, K., Dynlacht, B., Tsai, L., Zhang, P., Dobrowolsky, S., Bai, C., Connell-Crowley, L., Swindell, E., Fox, M.P., Wei, N., 1995. Inhibition of cyclin-dependent kinases by p21. Mol. Biol. Cell. 6, 387–400. Knipe, D.M., 1990. The role of viral and cellular nuclear proteins in herpes simplex virus replication. Adv. Virus Res. 37, 85–123.
A. Capocefalo et al. / Journal of Virological Methods 161 (2009) 308–311 Mohanty, S.B., Hammond, R.C., Lillie, M.G., 1971. A new bovine herpesvirus and its effect on experimentally infected calves. Arch. Gesamte Virusforsch. 34, 394–395. Osorio, F.A., Rock, D.L., Reed, D.E., 1985. Studies on the pathogenesis of a bovine cytomegalo-like virus in an experimental host. J. Gen. Virol. 66, 1941–1951. Peterson, R.B., Goyal, S.M., 1988. Propagation and quantitation of animal herpesviruses in eight cell culture systems. Comp. Immunol. Microbiol. Infect. Dis. 11, 93–98. Resnick-Silverman, L., St Clair, S., Maurer, M., Zhao, K., Manfredi, J.J., 1998. Identification of a novel class of genomic DNA-binding sites suggests a mechanism for selectivity in target gene activation by the tumor suppressor protein p53. Genes Dev. 12, 2102–2107. Shadan, F.F., Cowsert, L.M., Villarreal, L.P., 1994. n-Butyrate, a cell cycle blocker, inhibits the replication of polyomaviruses and papillomaviruses but not that of adenoviruses and herpesviruses. J. Virol. 68, 4785–4796. Tarakanova, V.L., Leung-Pineda, V., Hwang, S., Yang, C.W., Matatall, K., Basson, M., Sun, R., Piwnica-Worms, H., Sleckman, B.P., Virgin 4th, H.W., 2007. Gammaherpesvirus kinase actively initiates a DNA damage response by inducing
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phosphorylation of H2AX to foster viral replication. Cell Host Microbe 1, 275–286. Thiry, E., Bublot, M., Dubuisson, J., Pastoret, P.P., 1989. Bovine herpesvirus-4 (BHV-4) infection in cattle. In: Wittmann, G. (Ed.), Herpesvirus Diseases of Cattle, Horses and Pigs. Kluwer, Boston, Mass, pp. 96–115. Vanderplasschen, A., Goltz, M., Lyaku, J., Benarafa, C., Buhk, H.J., Thiry, E., Pastoret, P.P., 1995. The replication in vitro of the gammaherpesvirus bovine herpesvirus 4 is restricted by its DNA synthesis dependence on the S phase of the cell cycle. Virology 10, 328–340. Vasey, D.B., Wolf, C.R., MacArtney, T., Brown, K., Whitelaw, C.B., 2008. p21-LacZ reporter mice reflect p53-dependent toxic insult. Toxicol. Appl. Pharmacol. 227, 440–450. Zimmermann, W., Broll, H., Ehlers, B., Buhk, H.J., Rosenthal, A., Goltz, M., 2001. Genome sequence of bovine herpesvirus 4, a bovine Rhadinovirus, and identification of an origin of DNA replication. J. Virol. 75, 1186–1194. Xiong, Y., Hannon, G.J., Zhang, H., Casso, D., Kobayashi, R., Beach, D., 1993. p21 is a universal inhibitor of cyclin kinases. Nature 366, 701–704.
Contents lists available at ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Short communication
p21Waf1/Cip1 as a molecular sensor for BoHV-4 replication A. Capocefalo a , V. Franceschi a , C.B.A. Whitelaw b , D.B. Vasey b , S.G. Lillico b , S. Cavirani a , G. Donofrio a,∗ a b
Facoltà di Medicina Veterinaria, Dipartimento di Salute Animale, Sezione di Malattie Infettive degli Animali, via del Taglio 10, 43100 Parma, Italy Division of Developmental Biology, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin, Midlothian EH25 9PS, Scotland, UK
a b s t r a c t Article history: Received 9 March 2009 Received in revised form 27 May 2009 Accepted 10 June 2009 Available online 18 June 2009 Keywords: BoHV-4 p21Waf1/Cip1 Cellular damage
BoHV-4 replication cycle is dependent on the S-phase of the cell-cycle at the stage of viral DNA synthesis. Because p21 is a rate-limiting regulator of the G1/S-phase transition and up-regulated by DNA-damaging agents, in this study p21 expression in BoHV-4 infected cells was investigated. The p21 promoter was found to be highly activated in a dose- and time-dependent manner following BoHV-4 infection only in cells which are permissive for BoHV-4 replication. Thus p21 expression reports on BoHV-4 replication and could represent a host cell defensive response to infection-associated cellular damage. © 2009 Elsevier B.V. All rights reserved.
Bovine herpesvirus 4 (BoHV-4) is a herpesvirus and members of the Gammaherpesvirinae subfamily. BoHV-4 was first isolated in Europe from animals with respiratory and ocular diseases by Bartha et al. (1966) and later in the United States by Mohanty et al. (1971). BoHV-4 has been isolated from a variety of samples and cells (Egyed, 1998) from healthy cattle and from cattle with abortion, metritis, pneumonia, diarrhea, respiratory infection, and mammary pustular dermatitis (Bartha et al., 1966; Thiry et al., 1989). Although BoHV4 has been demonstrated in many tissues, accumulated evidence suggests that one site of persistence in both the natural and experimental host is cells of the monocyte/macrophage lineage (Osorio et al., 1985). However, the pathogenic role of BoHV-4 remains unclear; the direct correlation between particular strains of BoHV-4 with variable disease conditions is a delicate question, unsolved even through experimental infection. BoHV-4 causes cytopathic effect (CPE) and replicates in a variety of cell lines and primary cultures of various bovine and other animal species in culture (Peterson and Goyal, 1988). In contrast, several other gammaherpesviruses establish a latent infection in vitro. Viral gene expression is restricted to a specific subset of genes and the cells survive and replicate. The viral genome is maintained as a circular episome (Ceserman et al., 1995) and the origin of replication for the circular viral genome, oriP, distinct from the origin of replication used during lytic viral replication, oriLyt, have been identified (Zimmermann et al., 2001). The outcome of BoHV-4 infection depends on the cell type: (i) permissive infection, characterized by strong cytopathic effect and an abundant viral replication and high level of infectious virus production. (ii) Un-permissive
∗ Corresponding author. Tel.: +39 0521032677; fax: +39 0521032672. E-mail address: [email protected] (G. Donofrio). 0166-0934/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2009.06.012
infection, where BoHV-4 infected cells produced low levels of early and late viral RNAs, but caused no cytopathic effect. (iii) Persistent infection, which could follow a permissive infection, surviving and replicating cells were found to be persistently infected, maintaining the viral genome over many passages and producing low levels of infectious virus (Donofrio et al., 2000; Donofrio and van Santen, 2001). In contrast to other viruses, replication of most herpesviruses is generally assumed to be dependent of the stage of the host cell in the cell-cycle (Knipe, 1990; Shadan et al., 1994). The possibility that BoHV-4 replication could be dependent on the cell-cycle was investigated by Vanderplasschen et al. (1995), who showed that the replication in vitro of the BoHV-4 is restricted by its DNA synthesis dependence on the S-phase of the cell-cycle and the following assumptions were stated: (i) cell transition through the S-phase quantitatively increased the rate of BoHV-4 DNA replication. (ii) BoHV-4 DNA synthesis could not be detected in cells arrested in G0. (iii) Drug synchronization of cells before infection increased the percentage of cells expressing L proteins. (iv) Infection of cells arrested in G0 led to few BoHV-4 positive cells. Because the CKI p21Waf1/Cip1 (p21) is a rate-limiting regulator of the G1/S-phase transition (Bartek and Lukas, 2000), its transcriptional regulation in permissive and unpermissive BoHV-4 infected cells was investigated in the present communication. Initially, a bovine macrophage cell line (BoMac, from V. van Santen, Auburn University, USA) susceptible to BoHV-4 infection (Donofrio and van Santen, 2001) was transfected with a reporter construct characterized by a 4 kb murine p21 promoter driving the -galactosidase reporter gene, pX3W (Vasey et al., 2008) (Fig. 1A). BoMac cells from a confluent 25-cm2 flask maintained as a monolayer with growth medium containing 90% DMEM, 10% FBS, 2 mM l-glutamine, 100 IU/ml penicillin and 10 g/ml streptomycin and
A. Capocefalo et al. / Journal of Virological Methods 161 (2009) 308–311
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Fig. 1. (A) Diagram showing the pX3W reporter construct, containing the murine p21 promoter in front of the -galactosidase ORF. (B) Representative phase contrast image of BoHV-4 treated cells expressing -galactosidase and the untreated control. (C) The experiment was repeated three times giving always the same result.
incubated at 37 ◦ C in a humidified atmosphere of 95% air–5% CO2 were electroporated with pX3W (5 g) in 500 l DMEM without serum (Equibio apparatus, 186 V, 960 F, 4-mm gap cuvettes). To monitor the efficiency of transfection a parallel electroporation was performed with pEGFP-C1 (Invitrogen, Milan, Italy). Twenty-four hours post-electroporation, cells were infected with 5 multiplicity of infection of BoHV-4EGFPTK, a recombinant BoHV-4 delivering an enhanced green fluorescent protein (EGFP) expression cassette under the control of human cytomegalovirus (CMV) promoter, inserted into the thymidine kinase (TK) locus of BoHV-4 genome (DN599 strain, ATCC VR631) (Donofrio et al., 2002). This recombinant BoHV-4 allowed a direct monitoring of the infection through EGFP expression. Twenty-four hours post-infection, cells were fixed with 0.2% of glutaraldehyde and stained with X-gal for -galactosidase expression (Vasey et al., 2008). BoHV-4EGFPTK infected cells expressed -galactosidase as shown by blue cells (Fig. 1B), in contrast no blue cells appeared in the uninfected control (Fig. 1C). To be able to quantify better the p21 promoter activity in transfected cells, a luciferase reporter construct was developed by sub-cloning the p21 promoter in front of the luciferase open read-
ing frame (ORF) reporter gene contained into the pGL3-Basic vector (Promega, Milan, Italy), to generate p21-Luc reporter construct, facilitated by generating a new EcoRI site in the pGL3-Basic vector multi-cloning site. A 1 kb kanamycin amplicon obtained by PCR using primers (Sense, 5 -aaacccggtaccgaattcccggaattgccagctggg3 ; Antisense, 5 -gggcccctcgaggaaattgtaagcgttaataat-3 ) containing KpnI and EcoRI sites to the 5 -end and XhoI to the 3 end, was subcloned into KpnI/XhoI cut pGL3-Basic vector. Then, the kanamycin amplicon was removed with EcoRI/XhoI restriction digestion and the 4 kb p21 promoter, excised-out from pX3W with EcoRI/XhoI was ligated into EcoRI/XhoI linearized pGL3-Basic vector to get p21-Luc (Fig. 2A). BoMac cells were transfected by electroporation with p21-Luc and infected with BoHV-4EGFPTK as described above. Cells were harvested at different times after infection (6, 12 and 24 h postinfection) and luciferase activity measured. Luciferase reporter assay was performed with a Dual Luciferase Reporter Assay System kit (Promega, Milan, Italy) with minor modifications. Following treatments, cells were washed with PBS, lysed with 100 l of lysis passive buffer by freeze–thawing at −80 ◦ C. 20 l of the cell lysate was added to 50 l of LAR and luciferase activity was determined
Fig. 2. (A) Diagram showing the p21-luc reporter construct, containing the p21 promoter in front of the luciferase ORF. (B) p21 transiently transfected BoMac cells, BoHV-4 treated (black box) or untreated (gray box) and analyzed for luciferase expression at different time points (6, 12 and 24 h). The fluorescence microscope image showing green fluorescent cells, represent the infection level monitored through EGFP expression conferred by the recombinant BoHV-4 delivering an EGFP expression cassette. (C) p21 transiently transfected BoMac cells, treated with different dilutions and compared with the untreated control cells. The data represents the mean relative luciferase units. Each reaction was done in quadruplicate, and each point represents the average ± standard deviation from three experiments. * P < 0.001, as calculated by Student’s t-test or one-way ANOVA. (For interpretation of the references to color in this figure caption, the reader is referred to the web version of the article.)
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A. Capocefalo et al. / Journal of Virological Methods 161 (2009) 308–311
Fig. 3. (A) p21 transiently transfected HC11 cells, BoHV-4 treated (black box) or untreated (gray box) and analyzed for luciferase expression at different time points (6, 12 and 24 h). (B) p21 transiently transfected RD-4 cells, BoHV-4 treated (black box) or untreated (gray box) and analyzed for luciferase expression at different time points (6, 12 and 24 h). The data represents the mean relative luciferase units. Each reaction was done in quadruplicate, and each point represents the average ± standard deviation from three experiments. * P < 0.001, as calculated by Student’s t-test or one-way ANOVA.
with a PerkinElmer Victor3 Multilabel Counter, according to the manufacturer’s specifications. The p21 promoter was highly activated within 24 h postinfection (Fig. 2b) as observed for -galactosidase reporter system (Fig. 1B). However, further time post-infection was not possible to analyse due to the CPE and cell death induced by viral infection and replication. Hence, to confirm the specificity of this data, a dose dependant activation of p21 promoter by BoHV-4 infection was investigated. BoMac cells were transfected as before with p21Luc and infected with different dilution of BoHV-4EGFPTK and 24 h post-infection analyzed for luciferase activity. As expected, p21 promoter was activated in a dose dependent manner (Fig. 2c). Next, a similar experiment was performed on the HC11 mouse cell line (mouse mammary gland cells) permissive for BoHV-4 replication and on the RD-4 human cell line (human rhabdomyosarcoma cells, obtained from D. Derse, National Cancer Institute, Frederick, MD, USA) unpermissive to BoHV-4 replication. p21 promoter was activated only in BoHV-4 permissive HC11 cells which displayed a CPE (Fig. 3A) but not in RD-4 unpermissive cells which remain healthy (Fig. 3B). Therefore, p21 promoter activation correlated with BoHV4 replication. Viruses have evolved elegant approaches to use optimally limited genetic capacity to manipulate the intracellular environment. One common viral strategy is to usurp cellular signalling pathways to create an optimal environment for viral replication. Herpesvirus genomes are linear DNA molecules that circularize upon entry into the cell. The circular form of viral DNA conserved as a template for rolling-circle type of replication with viral genome concatamers are resolved by yet unidentified endonuclease(s). Replication of DNA is associated with increased levels of linear DNA molecules or abnormal DNA structures within the cell nucleus that could be recognized by cellular sensors of DNA damage and trigger DNA damage signalling. The mechanism of DNA damage signalling induction as well its physiological relevance for murine herpesvirus 68 (MHV68), a gamma herpesvirus highly related to BoHV-4, was provided by Tarakanova et al., 2007. p21 is a key mediator of the growth arrest induced by the tumor suppressor protein p53 in response to DNA damage (El-Deiry et al., 1993). p53 can stimulate transcription of the p21 gene by binding to the two p53-response elements located at positions −2281 to −2262 and −1395 to −1376 in the p21 promoter (El-Deiry et al., 1992; Resnick-Silverman et al., 1998). p21 was originally identified as an inhibitor of cyclin-dependent kinases (CDKs), including CDK2, CDK4 and CDC2 kinase complexes (Gu et al., 1993; Harper et al., 1993; Xiong et al., 1993). Moreover, it was shown that p21 can inhibit CDK3 and CDK6 kinases, and
that p21 is most effective towards G1/S cyclin-CDKs (Harper et al., 1995). Taking into account that BoHV-4 replication is dependent on the S-phase of the cell-cycle and p21 is a rate-limiting regulator of the G1/S-phase transition, p21 promoter activation reflects BoHV-4 replication and it could be hypothesized that p21 up-regulation is an attempt of the infected cells to block viral replication. In this brief communication the induction of p21 following BoHV-4 infection and replication in permissive cells has been shown. Although the viral and cellular factors required for p21 promoter transactivation were not investigated, a new door has been opened to a new insight into BoHV-4 and cellular interaction. Acknowledgement We would like to thank the Fondazione Cariparma (Cassa di Risparmio di Parma, Italy) for founding contribution to the project. References Bartha, A., Juhasz, M., Liebermann, H., 1966. Isolation of a bovine herpesvirus from calves with respiratory disease and keratoconjuntivitis. Acta Vet. Acad. Sci. Hung. 16, 357–358. Bartek, J., Lukas, J., 2000. Pathways governing G1/S transition and their response to DNA damage. FEBS Lett. 490, 117–122. Ceserman, E., Moore, P.S., Rao, P.H., Inghirami, G., Knowles, D.M., Chang, Y., 1995. In vitro establishment and characterization of two acquired immunodeficiency syndrome-related lymphoma cell lines (BC-1 and BC-2) containing Kaposi’s sarcoma-associated herpesvirus-like (KSHV) DNA sequences. Blood 86, 2708–2714. Donofrio, G., Cavirani, S., van Santen, V.L., 2000. Establishment of a cell line persistently infected with bovine herpesvirus-4 by use of a recombinant virus. J. Gen. Virol. 81, 1807–1814. Donofrio, G., van Santen, V.L., 2001. A bovine macrophage cell line supports bovine herpesvirus-4 persistent infection. J. Gen. Virol. 82, 1181–1185. Donofrio, G., Cavirani, S., Taddei, S., van Santen, V.L., 2002. Potential of bovine herpesvirus 4 as a gene delivery vector. J. Virol. Methods 101, 49–61. Egyed, L., 1998. Replication of bovine herpesvirus type 4 in human cells in vitro. J. Clin. Microbiol. 36, 2109–2111. El-Deiry, W.S., Kern, S.E., Pietenpol, J.A., Kinzler, K.W., Vogelstein, B., 1992. Definition of a consensus binding site for p53. Nat. Genet. 1, 45–49. El-Deiry, W.S., Tokino, T., Velculescu, V.E., Levy, D.B., Parsons, R., Trent, J.M., Lin, D., Mercer, W.E., Kinzler, K.W., Vogelstein, B., 1993. WAF1, a potential mediator of p53 tumor suppression. Cell 75, 817–825. Gu, Y., Turck, C.W., Morgan, D.O., 1993. Inhibition of CDK2 activity in vivo by an associated 20K regulatory subunit. Nature 366, 707–710. Harper, J.W., Adami, G.R., Wei, N., Keyomarsi, K., Elledge, S.J., 1993. The p21 Cdkinteracting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell 75, 805–816. Harper, J.W., Elledge, S.J., Keyomarsi, K., Dynlacht, B., Tsai, L., Zhang, P., Dobrowolsky, S., Bai, C., Connell-Crowley, L., Swindell, E., Fox, M.P., Wei, N., 1995. Inhibition of cyclin-dependent kinases by p21. Mol. Biol. Cell. 6, 387–400. Knipe, D.M., 1990. The role of viral and cellular nuclear proteins in herpes simplex virus replication. Adv. Virus Res. 37, 85–123.
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