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GLUC-tagged Sindbis-like virus XJ-160
Journal of Virological Methods 189 (2013) 235–237
Contents lists available at SciVerse ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Short communication
Development of EGFP/GLUC-tagged Sindbis-like virus XJ-160 Zhu Wuyang, Jiangjiao Li, Huanqin Wang, Ying He, Guodong Liang ∗ State Key Laboratory for Infectious Disease Prevention and Control (SKLID), National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention (IVDC, China CDC), 100 Yingxin Street, Xuan Wu District, Beijing 100052, China
a b s t r a c t Article history: Received 13 November 2012 Received in revised form 15 January 2013 Accepted 28 January 2013 Available online 9 February 2013 Keywords: Sindbis-like virus EGFP GLUC
Based on an infectious clone of Sindbis-like virus XJ-160, recombinant vectors containing a reporter gene (enhanced green fluorescence protein [EGFP] or Gaussia luciferase [GLUC]) were constructed by placing the reporter gene cassette containing the subgenomic promoter behind the 3 terminus of the viral structural protein gene. EGFP/GLUC-tagged Sindbis-like viruses were rescued in BHK-21 cells transfected with transcripts produced from the recombinant vectors. EGFP expression and strong luciferase activity were detected in BHK-21 cells infected with repeated passages of the EGFP/GLUC-tagged viruses, revealing the genetic stability of the chimeric viruses. The EGFP/GLUC-tagged Sindbis viruses reported will contribute to the assessment of viral replication and proliferation, tracking and elucidating Alphavirus-host interactions, and screening for antiviral compounds. © 2013 Elsevier B.V. All rights reserved.
Sindbis virus (SINV) is a prototypical virus of the Alphavirus genus and member of the Togaviridae family, which contains several significant human pathogens and potential bioterrorism or biowarfare agents, including Venezuelan, Eastern, and Western equine encephalitis virus (Vogel et al., 1997; Burgess et al., 2000). Sindbis virus is less pathogenic and rarely causes humanassociate disease; however, it is a good model for investigating the mechanism of Alphavirus replication and proliferation as well as various aspects of the virus-host cell interaction. Alphavirus pathogenicity in host animals and the induction of a cytopathic effect (CPE) in cell cultures are likely to be determined by different structural and nonstructural proteins and many host factor. However, our understanding of the underlying molecular mechanisms involved in the pathogenesis is incomplete. The reporter gene-tagged Sindbis viruses reported in this study will provide a useful tool for deciphering the role of the different viral and host proteins using various imaging techniques, and for the monitoring of CPEs. The Sindbis-like virus XJ-160 (GenBank No. AF103728) was isolated from a pooled sample of Anopheles mosquitoes collected in Xinjiang, China, in 1990 (Liang et al., 2000). The recombinant plasmid pBR-XJ160 is an infectious full-length cDNA clone of XJ-160, from which rescued virus can be obtained by in vitro transcription and transfection. The rescued virus is indistinguishable from XJ-160 in terms of its biological properties, including its plaque morphology, growth kinetics, and suckling mouse neurovirulence (Yang et al., 2005). Using pBR-XJ160 as a basis, recombinant
∗ Corresponding author. E-mail address: [email protected] (G. Liang). 0166-0934/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jviromet.2013.01.015
vectors containing a reporter gene (enhanced green fluorescence protein [EGFP] or Gaussia luciferase [GLUC]) were constructed by placing the reporter gene cassette carrying the subgenomic promoter behind the 3 terminus of the viral structural protein gene (Fig. 1); the recombinant vectors were named pBR-XJ160-EGFP and pBR-XJ160-GLUC, respectively. The correct insertion of the cassette was verified by sequencing of the recombinant clones. The reporter gene-tagged Sindbis viruses were rescued in BHK21 cell transfected with transcripts of the recombinant vectors, as described previously (Zhu et al., 2009). After the transfection of BHK-21 cells with pBR-XJ160-EGFP and BR-XJ160-GLUC transcripts, CPEs similar to those induced by XJ-160 could be seen 24 h post-transfection. According to the names of the reporter genes, the chimeric viruses were designated XJ160-EGFP and XJ160-GLUC, respectively. Sequencing of the complete cDNA of each stock of rescued virus confirmed the absence of any fortuitous mutation (data not shown). To evaluate the effects of the insertions on the propagation characteristics of XJ-160, plaque morphology and growth curves were assessed as described previously (Zhu et al., 2009). As shown in Fig. 2A, XJ160-EGFP and XJ160-GLUC formed clear plaques with similar morphological characteristics to those of the parental virus. Additionally, the growth tendencies of the chimeric viruses were indistinguishable from those of XJ-160, which exhibited rapid propagation from 10 to 40 h post-infection (h.p.i.) and peaked at 40 h.p.i (Fig. 2B). To investigate the functional activity of the chimeric viruses, EGFP assays were performed as described previously (Zhu et al., 2011). EGFP expression was observed by inverted fluorescence microscopy (400×). Continuously enhanced fluorescence was seen in BHK-21 cells infected with XJ160-EGFP at 24–72 h.p.i., while the control was negative (Fig. 3A). A GLUC assay was performed using
236
Z. Wuyang et al. / Journal of Virological Methods 189 (2013) 235–237
Fig. 1. Diagram showing the generation of the reporter gene-tagged Sindbis-like virus XJ-160.
Fig. 2. Propagation characteristics of the EGFP/GLUC-tagged viruses. (A) Plaque phenotypes of the chimeric viruses. (B) Growth kinetics of the chimeric viruses.
the Gaussia Luciferase Assay System (Promega, Wisconsin, USA) following the manufacturer’s instructions. As shown in Fig. 3B, GLUC was efficiently expressed in BHK-21 cells infected with XJ160-GLUC. These results indicate that reporter gene-tagged viruses expressing EGFP or GLUC were functional. On the passaging of XJ160-EGFP virus, EGFP expression was continuously observed in cells infected with the virus from five consecutive rounds
of infection of the cells, while no specific green fluorescence was observed in the control group (Fig. 4A). Similarly, a continuously high level of luciferase activity was detected in BHK-21 cells infected with five repeated passages of XJ160-GLUC virus, while only background levels of luciferase were detected in the control group (Fig. 4B), demonstrating the genetic stability of both XJ160-EGFP and XJ160-GLUC.
Fig. 3. Functional activity of the chimeric viruses. (A) Expression of EGFP in BHK-21 cells infected with XJ160-EGFP virus. (B) Expression of GLUC in BHK-21 cells infected with XJ160-GLUC virus.
Z. Wuyang et al. / Journal of Virological Methods 189 (2013) 235–237
237
Fig. 4. Genetic stability of the chimeric viruses. (A) Expression of EGFP in BHK-21 cells infected with repeated passages of XJ160-EGFP virus. (B) Expression of GLUC in BHK-21 cells infected with repeated passages of XJ160-GLUC virus.
In recent years, fluorescent proteins, including variants of green fluorescen protein, have been used widely to study protein dynamics, and they have been extensively used as reporters of virus infection and spread. Cristea et al. (2006) used a chimeric Sindbis virus expressing the viral nsP3 protein tagged with GFP to directly observe nsP3 localization and isolated nsP3-interacting proteins at various times after infection. Atasheva et al. (2007) generated a cDNA library of Sindbis virus genomes with GFP randomly inserted into nsP2 and identified a number of sites that can be used for GFP cloning without a strong effect on virus replication. In this study, we report the tagging of the Sindbis-like virus XJ-160 with EGFP or GLUC. Compared with EGFP, Gaussia luciferase is naturally secreted (Wurdinger et al., 2008) and can therefore be used as a reporter for the quantitative assessment of cells in vivo by measuring its concentration in the blood (Niers et al., 2011). Therefore, XJ160-GLUC, which exhibited good genetic stability, provides an unprecedented means for monitoring host cells infected with the chimeric virus in vivo, and will enhance our understanding of their survival, migration, immunogenicity, and potential pathogenicity in living animals. Overall, the EGFP/GLUC-tagged Sindbis viruses reported will not only enable the rapid, reproducible, and quantitative monitoring of viral infection processes in vivo but can also measure gene expression in vivo, allowing for the screening of antiviral compounds and improved optimization of drug and gene therapies. Acknowledgements This work was supported by the grants from the National Natural Science Foundation of China (No. 30970160; 81160353),
China Mega-Project for Infectious Disease (2011ZX10004-001), the Development Grant of State Key Laboratory for Infectious Disease Prevention and Control (2011SKLID205). References Atasheva, S., Gorchakov, R., English, R., Frolov, I., Frolova1, E., 2007. Development of Sindbis viruses encoding nsP2/GFP chimeric proteins and their application for studying nsP2 functioning. J. Virol. 81, 5046–5057. Burgess, T.H., Steele, K.E., Schoneboom, B.A., Grieder, F.B., 2000. Clinicopathologic features of viral agents of potential use by bioterrorists. Clin. Lab. Med. 121, 475–493. Cristea, I.M., Carroll, J.-W.N., Rout, M.P., Rice, C.M., Chait, B.T., MacDonald, M.R., 2006. Tracking and elucidating Alphavirus–host protein interactions. J. Biol. Chem. 281, 30269–30278. Liang, G.D., Li, L., Zhou, G.L., Fu, S.H., Li, Q.P., Li, F.S., He, H.H., Jin, Q., He, Y., Chen, B.Q., Hou, Y.D., 2000. Isolation and complete nucleotide sequence of a Chinese Sindbis-like virus. J. Gen. Virol. 81, 1347–1351. Niers, J.M., Kerami, M., Pike, L., Lewandrowski, G., Tannous, B.A., 2011. Multimodal in vivo imaging and blood monitoring of intrinsic and extrinsic apoptosis. Mol. Ther. 19, 1090–1096. Vogel, P., Fritz, D.L., Kuehl, K., Davis, K.J., Geisbert, T., 1997. The agents of biological warfare. JAMA 278, 438–439. Wurdinger, T., Badr, C., Pike, L., de Kleine, R., Weissleder, R., Breakefield, X.O., Tannous, B.A., 2008. A secreted luciferase for ex vivo monitoring of in vivo processes. Nat. Methods 5, 171–173. Yang, Y.L., Liang, G.D., Fu, S.H., Tang, Q., Wang, L.H., 2005. Construction and infection analysis of the full-length cDNA clone of XJ-160 virus, the first Sindbis virus isolated in China. Virol. Sin. 20, 173–180. Zhu, W.Y., Yang, Y.L., Fu, S.H., Wang, L.H., Zhai, Y.G., Tang, Q., Liang, G.D., 2009. Substitutions of 169Lys and 173Thr in nonstructural protein 1 influence the infectivity and pathogenicity of XJ-160 virus. Arch. Virol. 154, 245–253. Zhu, W.Y., Li, J.J., Tang, L., Wang, H.Q., Li, J., Fu, J.J., Liang, G.D., 2011. Glycoprotein is enough for Sindbis virus-derived DNA vector to express heterogenous genes. Virol. J. 8, 334.
Contents lists available at SciVerse ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Short communication
Development of EGFP/GLUC-tagged Sindbis-like virus XJ-160 Zhu Wuyang, Jiangjiao Li, Huanqin Wang, Ying He, Guodong Liang ∗ State Key Laboratory for Infectious Disease Prevention and Control (SKLID), National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention (IVDC, China CDC), 100 Yingxin Street, Xuan Wu District, Beijing 100052, China
a b s t r a c t Article history: Received 13 November 2012 Received in revised form 15 January 2013 Accepted 28 January 2013 Available online 9 February 2013 Keywords: Sindbis-like virus EGFP GLUC
Based on an infectious clone of Sindbis-like virus XJ-160, recombinant vectors containing a reporter gene (enhanced green fluorescence protein [EGFP] or Gaussia luciferase [GLUC]) were constructed by placing the reporter gene cassette containing the subgenomic promoter behind the 3 terminus of the viral structural protein gene. EGFP/GLUC-tagged Sindbis-like viruses were rescued in BHK-21 cells transfected with transcripts produced from the recombinant vectors. EGFP expression and strong luciferase activity were detected in BHK-21 cells infected with repeated passages of the EGFP/GLUC-tagged viruses, revealing the genetic stability of the chimeric viruses. The EGFP/GLUC-tagged Sindbis viruses reported will contribute to the assessment of viral replication and proliferation, tracking and elucidating Alphavirus-host interactions, and screening for antiviral compounds. © 2013 Elsevier B.V. All rights reserved.
Sindbis virus (SINV) is a prototypical virus of the Alphavirus genus and member of the Togaviridae family, which contains several significant human pathogens and potential bioterrorism or biowarfare agents, including Venezuelan, Eastern, and Western equine encephalitis virus (Vogel et al., 1997; Burgess et al., 2000). Sindbis virus is less pathogenic and rarely causes humanassociate disease; however, it is a good model for investigating the mechanism of Alphavirus replication and proliferation as well as various aspects of the virus-host cell interaction. Alphavirus pathogenicity in host animals and the induction of a cytopathic effect (CPE) in cell cultures are likely to be determined by different structural and nonstructural proteins and many host factor. However, our understanding of the underlying molecular mechanisms involved in the pathogenesis is incomplete. The reporter gene-tagged Sindbis viruses reported in this study will provide a useful tool for deciphering the role of the different viral and host proteins using various imaging techniques, and for the monitoring of CPEs. The Sindbis-like virus XJ-160 (GenBank No. AF103728) was isolated from a pooled sample of Anopheles mosquitoes collected in Xinjiang, China, in 1990 (Liang et al., 2000). The recombinant plasmid pBR-XJ160 is an infectious full-length cDNA clone of XJ-160, from which rescued virus can be obtained by in vitro transcription and transfection. The rescued virus is indistinguishable from XJ-160 in terms of its biological properties, including its plaque morphology, growth kinetics, and suckling mouse neurovirulence (Yang et al., 2005). Using pBR-XJ160 as a basis, recombinant
∗ Corresponding author. E-mail address: [email protected] (G. Liang). 0166-0934/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jviromet.2013.01.015
vectors containing a reporter gene (enhanced green fluorescence protein [EGFP] or Gaussia luciferase [GLUC]) were constructed by placing the reporter gene cassette carrying the subgenomic promoter behind the 3 terminus of the viral structural protein gene (Fig. 1); the recombinant vectors were named pBR-XJ160-EGFP and pBR-XJ160-GLUC, respectively. The correct insertion of the cassette was verified by sequencing of the recombinant clones. The reporter gene-tagged Sindbis viruses were rescued in BHK21 cell transfected with transcripts of the recombinant vectors, as described previously (Zhu et al., 2009). After the transfection of BHK-21 cells with pBR-XJ160-EGFP and BR-XJ160-GLUC transcripts, CPEs similar to those induced by XJ-160 could be seen 24 h post-transfection. According to the names of the reporter genes, the chimeric viruses were designated XJ160-EGFP and XJ160-GLUC, respectively. Sequencing of the complete cDNA of each stock of rescued virus confirmed the absence of any fortuitous mutation (data not shown). To evaluate the effects of the insertions on the propagation characteristics of XJ-160, plaque morphology and growth curves were assessed as described previously (Zhu et al., 2009). As shown in Fig. 2A, XJ160-EGFP and XJ160-GLUC formed clear plaques with similar morphological characteristics to those of the parental virus. Additionally, the growth tendencies of the chimeric viruses were indistinguishable from those of XJ-160, which exhibited rapid propagation from 10 to 40 h post-infection (h.p.i.) and peaked at 40 h.p.i (Fig. 2B). To investigate the functional activity of the chimeric viruses, EGFP assays were performed as described previously (Zhu et al., 2011). EGFP expression was observed by inverted fluorescence microscopy (400×). Continuously enhanced fluorescence was seen in BHK-21 cells infected with XJ160-EGFP at 24–72 h.p.i., while the control was negative (Fig. 3A). A GLUC assay was performed using
236
Z. Wuyang et al. / Journal of Virological Methods 189 (2013) 235–237
Fig. 1. Diagram showing the generation of the reporter gene-tagged Sindbis-like virus XJ-160.
Fig. 2. Propagation characteristics of the EGFP/GLUC-tagged viruses. (A) Plaque phenotypes of the chimeric viruses. (B) Growth kinetics of the chimeric viruses.
the Gaussia Luciferase Assay System (Promega, Wisconsin, USA) following the manufacturer’s instructions. As shown in Fig. 3B, GLUC was efficiently expressed in BHK-21 cells infected with XJ160-GLUC. These results indicate that reporter gene-tagged viruses expressing EGFP or GLUC were functional. On the passaging of XJ160-EGFP virus, EGFP expression was continuously observed in cells infected with the virus from five consecutive rounds
of infection of the cells, while no specific green fluorescence was observed in the control group (Fig. 4A). Similarly, a continuously high level of luciferase activity was detected in BHK-21 cells infected with five repeated passages of XJ160-GLUC virus, while only background levels of luciferase were detected in the control group (Fig. 4B), demonstrating the genetic stability of both XJ160-EGFP and XJ160-GLUC.
Fig. 3. Functional activity of the chimeric viruses. (A) Expression of EGFP in BHK-21 cells infected with XJ160-EGFP virus. (B) Expression of GLUC in BHK-21 cells infected with XJ160-GLUC virus.
Z. Wuyang et al. / Journal of Virological Methods 189 (2013) 235–237
237
Fig. 4. Genetic stability of the chimeric viruses. (A) Expression of EGFP in BHK-21 cells infected with repeated passages of XJ160-EGFP virus. (B) Expression of GLUC in BHK-21 cells infected with repeated passages of XJ160-GLUC virus.
In recent years, fluorescent proteins, including variants of green fluorescen protein, have been used widely to study protein dynamics, and they have been extensively used as reporters of virus infection and spread. Cristea et al. (2006) used a chimeric Sindbis virus expressing the viral nsP3 protein tagged with GFP to directly observe nsP3 localization and isolated nsP3-interacting proteins at various times after infection. Atasheva et al. (2007) generated a cDNA library of Sindbis virus genomes with GFP randomly inserted into nsP2 and identified a number of sites that can be used for GFP cloning without a strong effect on virus replication. In this study, we report the tagging of the Sindbis-like virus XJ-160 with EGFP or GLUC. Compared with EGFP, Gaussia luciferase is naturally secreted (Wurdinger et al., 2008) and can therefore be used as a reporter for the quantitative assessment of cells in vivo by measuring its concentration in the blood (Niers et al., 2011). Therefore, XJ160-GLUC, which exhibited good genetic stability, provides an unprecedented means for monitoring host cells infected with the chimeric virus in vivo, and will enhance our understanding of their survival, migration, immunogenicity, and potential pathogenicity in living animals. Overall, the EGFP/GLUC-tagged Sindbis viruses reported will not only enable the rapid, reproducible, and quantitative monitoring of viral infection processes in vivo but can also measure gene expression in vivo, allowing for the screening of antiviral compounds and improved optimization of drug and gene therapies. Acknowledgements This work was supported by the grants from the National Natural Science Foundation of China (No. 30970160; 81160353),
China Mega-Project for Infectious Disease (2011ZX10004-001), the Development Grant of State Key Laboratory for Infectious Disease Prevention and Control (2011SKLID205). References Atasheva, S., Gorchakov, R., English, R., Frolov, I., Frolova1, E., 2007. Development of Sindbis viruses encoding nsP2/GFP chimeric proteins and their application for studying nsP2 functioning. J. Virol. 81, 5046–5057. Burgess, T.H., Steele, K.E., Schoneboom, B.A., Grieder, F.B., 2000. Clinicopathologic features of viral agents of potential use by bioterrorists. Clin. Lab. Med. 121, 475–493. Cristea, I.M., Carroll, J.-W.N., Rout, M.P., Rice, C.M., Chait, B.T., MacDonald, M.R., 2006. Tracking and elucidating Alphavirus–host protein interactions. J. Biol. Chem. 281, 30269–30278. Liang, G.D., Li, L., Zhou, G.L., Fu, S.H., Li, Q.P., Li, F.S., He, H.H., Jin, Q., He, Y., Chen, B.Q., Hou, Y.D., 2000. Isolation and complete nucleotide sequence of a Chinese Sindbis-like virus. J. Gen. Virol. 81, 1347–1351. Niers, J.M., Kerami, M., Pike, L., Lewandrowski, G., Tannous, B.A., 2011. Multimodal in vivo imaging and blood monitoring of intrinsic and extrinsic apoptosis. Mol. Ther. 19, 1090–1096. Vogel, P., Fritz, D.L., Kuehl, K., Davis, K.J., Geisbert, T., 1997. The agents of biological warfare. JAMA 278, 438–439. Wurdinger, T., Badr, C., Pike, L., de Kleine, R., Weissleder, R., Breakefield, X.O., Tannous, B.A., 2008. A secreted luciferase for ex vivo monitoring of in vivo processes. Nat. Methods 5, 171–173. Yang, Y.L., Liang, G.D., Fu, S.H., Tang, Q., Wang, L.H., 2005. Construction and infection analysis of the full-length cDNA clone of XJ-160 virus, the first Sindbis virus isolated in China. Virol. Sin. 20, 173–180. Zhu, W.Y., Yang, Y.L., Fu, S.H., Wang, L.H., Zhai, Y.G., Tang, Q., Liang, G.D., 2009. Substitutions of 169Lys and 173Thr in nonstructural protein 1 influence the infectivity and pathogenicity of XJ-160 virus. Arch. Virol. 154, 245–253. Zhu, W.Y., Li, J.J., Tang, L., Wang, H.Q., Li, J., Fu, J.J., Liang, G.D., 2011. Glycoprotein is enough for Sindbis virus-derived DNA vector to express heterogenous genes. Virol. J. 8, 334.