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Feline calicivirus capsid protein expression and self-assembly in cultured feline cells
Veterinary Microbiology 69 (1999) 63±66
Feline calicivirus capsid protein expression and self-assembly in cultured feline cells K. Geisslera, C.R. Parrishb, Karla Schneidera, U. Truyena,b,* a
Institute for Medical Microbiology, Infectious and Epidemic Diseases, Ludwig Maximilians, University Munich, VeterinaÈrstr. 13, 80539 Munich, Germany b James A. Baker Institute for Animal Health, New York State College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA
Abstract Feline calicivirus (FCV) capsid protein was expressed in feline cells employing the vaccinia virus MVA/T7 RNA polymerase system. The precursor protein was processed to a mature size protein that assembled to virus like particles (VLPs). # 1999 Elsevier Science B.V. All rights reserved. Keywords: Feline calicivirus; FCV; Capsid assembly; Virus-like particles; LVP; Vaccinia virus; MVA/T7
Feline calicivirus (FCV) is a well-characterized member of the family Caliciviridae and causes upper respiratory tract infections in cats, and is also associated with polyarthritis and chronic stomatitis. The genome is 7.69 kb in size (Munich laboratory strain FCV-KS20, originally isolated in 1995 from a cat with chronic stomatitis (Geissler et al., 1997)) and has three open reading frames. In FCV the mature 62 kDa capsid protein is generated by posttranslational processing of the capsid protein precursor by the viral 3C-protease. To study capsid protein expression and the mechanisms of protein processing in detail, we cloned both the precursor and the mature capsid protein (pCAPI) gene into various expression vectors (Fig. 1). After transfection of CRFK-cells the level of expression as judged by the intensity of specific immunostaining in Western blot analysis, differed markedly between the constructs (Fig. 2). Transfection of CRFK-cells with plasmid constructs controlled by CMV-promotor gave no expression, and expression under the control of the SV40promotor resulted in a weak protein expression. Due to low levels of FCV capsid protein *
Corresponding author. Tel.: +49-89-2180-2535; fax: +49-89-2180-2155 E-mail address: [email protected] (U. Truyen) 0378-1135/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 1 3 5 ( 9 9 ) 0 0 0 8 9 - 9
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Fig. 1. Summary of the capsid protein constructs that were used in this study. The entire precursor capsid protein gene and part of the ORF 3 was cloned into pCRIII.uni (pCAPIII), and the truncated `mature' capsid protein was cloned into pCRIII.uni or pCDNA3.1 (pCAPI or pCAPII, respectively), or into pSG5 (pCAPIV).
Fig. 2. Western blots of cell lysates of CRFK cells transfected with the various constructs and stained with Mab K. Comparison of the expression efficiency of the CMV, SV40 and T7 promoter constructs. Lane 1: pCAPI (CMV promoters) lysate of about 5 106 cells; Lane 2: pCAPIV (SV40 promoter) lysate of about 5 106 cells; Lane 3: pCAPI + MVA-T7 (T7 promoter) lysate of about 5 105 cells; Lane 4: MVA-T7 infected control cells (5 106 cells); Lane 5: CsCl-gradient purified virus FCV-KS20.
synthesis using expression vectors which rely on nuclear RNA synthesis we tested the hybrid vaccinia/T7 RNA-polymerase expression system based on the vector virus MVAT7pol, which relies on cytoplasmatic expression of recombinant genes. In this system both the transfection of the precursor protein (pCAPIII) and of the pCAPI gave a strong expression (not shown).
K. Geissler et al. / Veterinary Microbiology 69 (1999) 63±66
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Fig. 3. Negative staining electron microscopy of empty virus capsids produced after transfection of the precursor capsid protein gene.
Using the highly efficient MVA/T7 expression system we addressed the question whether the various forms of the FCV capsid protein monomers can selfassemble into virus-like particles After transfection of the gene constructs and coinfection with MVA/T7pol cell lysates were prepared and analyzed in Western-blots after CsCl density gradient separation. Both the pCAPIII and the pCAPI genes led to the formation of virus-like particles (VLPs) that banded at a density of about 1.26 g/ml, consistent with other calicivirus VLPs (Jiang et al., 1992; Laurent et al., 1994). Both VLPs were composed of mature size protein monomers (62 kDa). The capsids were uniform in morphology and size 32±34 nm and indistinguishable from authentic calicivirus virions (Fig. 3). The CMV and SV40-promotor controlled gene expression vectors initiated only weak production of the calicivirus protein. The extremely inefficient production of FCV-capsid protein compared to other expression system may be due to splicing, as numerous potential splice donor and acceptor sites were found within the capsid protein gene sequence. In contrast, using the MVA/T7pol system a high expression efficiency was achieved. With regard to the cytoplasmatic life cycle of caliciviruses the MVA/T7expression system is independent of the transport of plamids to nucleus and avoids splicing events. In our experiments expression of both the precursor capsid protein and a truncated (mature) capsid protein gene with an aminoterminal extra methionine resulted in the expression of the mature capsid protein and assembly into virus-like particles. Beside the capsid protein gene sequences both constructs encode also about 90% of the ORF 3 gene. The function of this protein is still unknown although in RHDV it is believed to represent a second structural protein (Wirblich et al., 1996). A role of ORF 3 for the capsid assembly in our FCV system can therefore not be excluded. Studies with other caliciviruses in baculovirus systems, however, showed that ORF 3 protein is dispensable for the capsid assembly (Williams et al., 1997).
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K. Geissler et al. / Veterinary Microbiology 69 (1999) 63±66
The formation of virus-like particles from pCAPIII implicates the processing of the precursor that is most likely mediated by the viral 3C protease (Sosnovtsev et al., 1998). As no caliciviral protease is encoded by our constructs the processing has to be mediated by a cellular or vaccinia-virus protease. Our future studies will be aimed to further define the mechanisms of calicivirus assembly and the host specificity of FCV replication. Acknowledgements We thank Gerd Sutter for providing the MVA/T7pol virus. This work was supported in part by a Grant from the Fondation Piere Richard Virbac, and a NATO collaborative research Grant. K.G. was the recipient of a fellowship from the German academic exchange service (DAAD). References Geissler, K., Schneider, K., Platzer, G., Truyen, B., Kaaden, O.-R., Truyen, U., 1997. Genetic and antigenic heterogeneity among feline calicivirus isolates from distinct disease manifestations. Virus Res. 48, 193±206. Jiang, X., Wang, M., Graham, D.Y., Estes, M.K., 1992. Expression, self-assembly, and antigenicity of the Norwalk virus capsid protein. J. Virol. 66, 6527±6532. Laurent, S., Vautherot, J.-F., Madelaine, M.-F., Le Gall, G., Rasschaert, D., 1994. Recombinant rabbit hemorrhagic disease virus capsid protein expressed in baculovirus self-assembles into viruslike particles and induces protection. J. Virol. 68, 6794±6798. Sosnovtsev, S.V., Sosnovtseva, S.A., Green, K.Y., 1998. Cleavage of the feline calicivirus capsid precursor is mediated by a virus-encoded proteinase. J. Virol. 72, 3051±3059. Williams, J.C., Liu, B.L., Lambden, P.R., Clarke, I.N., 1997. Expression of SRSV ORFs 2 and 3: assembly of virus-like particles is independent of ORF3 activity. In: Proceedings of the 1st International Symposium on Caliciviruses, pp. 51±58. Wirblich, C., Thiel, H.-J., Meyers, G., 1996. Genetic map of the calicivirus rabbit hemorrhagic disease virus as deduced from in vitro translation studies. J. Virol. 70, 7974±7983.
Feline calicivirus capsid protein expression and self-assembly in cultured feline cells K. Geisslera, C.R. Parrishb, Karla Schneidera, U. Truyena,b,* a
Institute for Medical Microbiology, Infectious and Epidemic Diseases, Ludwig Maximilians, University Munich, VeterinaÈrstr. 13, 80539 Munich, Germany b James A. Baker Institute for Animal Health, New York State College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA
Abstract Feline calicivirus (FCV) capsid protein was expressed in feline cells employing the vaccinia virus MVA/T7 RNA polymerase system. The precursor protein was processed to a mature size protein that assembled to virus like particles (VLPs). # 1999 Elsevier Science B.V. All rights reserved. Keywords: Feline calicivirus; FCV; Capsid assembly; Virus-like particles; LVP; Vaccinia virus; MVA/T7
Feline calicivirus (FCV) is a well-characterized member of the family Caliciviridae and causes upper respiratory tract infections in cats, and is also associated with polyarthritis and chronic stomatitis. The genome is 7.69 kb in size (Munich laboratory strain FCV-KS20, originally isolated in 1995 from a cat with chronic stomatitis (Geissler et al., 1997)) and has three open reading frames. In FCV the mature 62 kDa capsid protein is generated by posttranslational processing of the capsid protein precursor by the viral 3C-protease. To study capsid protein expression and the mechanisms of protein processing in detail, we cloned both the precursor and the mature capsid protein (pCAPI) gene into various expression vectors (Fig. 1). After transfection of CRFK-cells the level of expression as judged by the intensity of specific immunostaining in Western blot analysis, differed markedly between the constructs (Fig. 2). Transfection of CRFK-cells with plasmid constructs controlled by CMV-promotor gave no expression, and expression under the control of the SV40promotor resulted in a weak protein expression. Due to low levels of FCV capsid protein *
Corresponding author. Tel.: +49-89-2180-2535; fax: +49-89-2180-2155 E-mail address: [email protected] (U. Truyen) 0378-1135/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 1 3 5 ( 9 9 ) 0 0 0 8 9 - 9
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K. Geissler et al. / Veterinary Microbiology 69 (1999) 63±66
Fig. 1. Summary of the capsid protein constructs that were used in this study. The entire precursor capsid protein gene and part of the ORF 3 was cloned into pCRIII.uni (pCAPIII), and the truncated `mature' capsid protein was cloned into pCRIII.uni or pCDNA3.1 (pCAPI or pCAPII, respectively), or into pSG5 (pCAPIV).
Fig. 2. Western blots of cell lysates of CRFK cells transfected with the various constructs and stained with Mab K. Comparison of the expression efficiency of the CMV, SV40 and T7 promoter constructs. Lane 1: pCAPI (CMV promoters) lysate of about 5 106 cells; Lane 2: pCAPIV (SV40 promoter) lysate of about 5 106 cells; Lane 3: pCAPI + MVA-T7 (T7 promoter) lysate of about 5 105 cells; Lane 4: MVA-T7 infected control cells (5 106 cells); Lane 5: CsCl-gradient purified virus FCV-KS20.
synthesis using expression vectors which rely on nuclear RNA synthesis we tested the hybrid vaccinia/T7 RNA-polymerase expression system based on the vector virus MVAT7pol, which relies on cytoplasmatic expression of recombinant genes. In this system both the transfection of the precursor protein (pCAPIII) and of the pCAPI gave a strong expression (not shown).
K. Geissler et al. / Veterinary Microbiology 69 (1999) 63±66
65
Fig. 3. Negative staining electron microscopy of empty virus capsids produced after transfection of the precursor capsid protein gene.
Using the highly efficient MVA/T7 expression system we addressed the question whether the various forms of the FCV capsid protein monomers can selfassemble into virus-like particles After transfection of the gene constructs and coinfection with MVA/T7pol cell lysates were prepared and analyzed in Western-blots after CsCl density gradient separation. Both the pCAPIII and the pCAPI genes led to the formation of virus-like particles (VLPs) that banded at a density of about 1.26 g/ml, consistent with other calicivirus VLPs (Jiang et al., 1992; Laurent et al., 1994). Both VLPs were composed of mature size protein monomers (62 kDa). The capsids were uniform in morphology and size 32±34 nm and indistinguishable from authentic calicivirus virions (Fig. 3). The CMV and SV40-promotor controlled gene expression vectors initiated only weak production of the calicivirus protein. The extremely inefficient production of FCV-capsid protein compared to other expression system may be due to splicing, as numerous potential splice donor and acceptor sites were found within the capsid protein gene sequence. In contrast, using the MVA/T7pol system a high expression efficiency was achieved. With regard to the cytoplasmatic life cycle of caliciviruses the MVA/T7expression system is independent of the transport of plamids to nucleus and avoids splicing events. In our experiments expression of both the precursor capsid protein and a truncated (mature) capsid protein gene with an aminoterminal extra methionine resulted in the expression of the mature capsid protein and assembly into virus-like particles. Beside the capsid protein gene sequences both constructs encode also about 90% of the ORF 3 gene. The function of this protein is still unknown although in RHDV it is believed to represent a second structural protein (Wirblich et al., 1996). A role of ORF 3 for the capsid assembly in our FCV system can therefore not be excluded. Studies with other caliciviruses in baculovirus systems, however, showed that ORF 3 protein is dispensable for the capsid assembly (Williams et al., 1997).
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K. Geissler et al. / Veterinary Microbiology 69 (1999) 63±66
The formation of virus-like particles from pCAPIII implicates the processing of the precursor that is most likely mediated by the viral 3C protease (Sosnovtsev et al., 1998). As no caliciviral protease is encoded by our constructs the processing has to be mediated by a cellular or vaccinia-virus protease. Our future studies will be aimed to further define the mechanisms of calicivirus assembly and the host specificity of FCV replication. Acknowledgements We thank Gerd Sutter for providing the MVA/T7pol virus. This work was supported in part by a Grant from the Fondation Piere Richard Virbac, and a NATO collaborative research Grant. K.G. was the recipient of a fellowship from the German academic exchange service (DAAD). References Geissler, K., Schneider, K., Platzer, G., Truyen, B., Kaaden, O.-R., Truyen, U., 1997. Genetic and antigenic heterogeneity among feline calicivirus isolates from distinct disease manifestations. Virus Res. 48, 193±206. Jiang, X., Wang, M., Graham, D.Y., Estes, M.K., 1992. Expression, self-assembly, and antigenicity of the Norwalk virus capsid protein. J. Virol. 66, 6527±6532. Laurent, S., Vautherot, J.-F., Madelaine, M.-F., Le Gall, G., Rasschaert, D., 1994. Recombinant rabbit hemorrhagic disease virus capsid protein expressed in baculovirus self-assembles into viruslike particles and induces protection. J. Virol. 68, 6794±6798. Sosnovtsev, S.V., Sosnovtseva, S.A., Green, K.Y., 1998. Cleavage of the feline calicivirus capsid precursor is mediated by a virus-encoded proteinase. J. Virol. 72, 3051±3059. Williams, J.C., Liu, B.L., Lambden, P.R., Clarke, I.N., 1997. Expression of SRSV ORFs 2 and 3: assembly of virus-like particles is independent of ORF3 activity. In: Proceedings of the 1st International Symposium on Caliciviruses, pp. 51±58. Wirblich, C., Thiel, H.-J., Meyers, G., 1996. Genetic map of the calicivirus rabbit hemorrhagic disease virus as deduced from in vitro translation studies. J. Virol. 70, 7974±7983.