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. 1999 Jan;73(1):834-8.
doi: 10.1128/JVI.73.1.834-838.1999.

Feline calicivirus capsid protein expression and capsid assembly in cultured feline cells

K Geissler  1 , K Schneider, A Fleuchaus, C R Parrish, G Sutter, U Truyen
Affiliations

Feline calicivirus capsid protein expression and capsid assembly in cultured feline cells

K Geissler et al. J Virol. 1999 Jan.

Abstract

The capsid protein of feline calicivirus (FCV) was expressed by using plasmids containing cytomegalovirus, simian virus 40, or T7 promoters. The strongest expression was achieved with the T7 promoter and coinfection with vaccinia virus expressing the T7 RNA polymerase (MVA/T7pol). The FCV precursor capsid protein was processed to the mature-size protein, and these proteins were assembled in to virus-like particles.

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Figures

FIG. 1
FIG. 1
Summary of the capsid protein constructs that were used in this study. The truncated mature capsid protein (pCAPI) was cloned into pcDNA3.1, the entire precursor capsid protein gene (pCAPIII) was cloned into pCRII.uni, and the mature truncated capsid protein gene was cloned into pSG5 (pCAPIV). All FCV capsid protein gene constructs contain about 90% of the ORF3 gene. The pT7GFP vector is also depicted. Cloning details are given in the text. EMC, encephalomyocarditis virus leader sequence; T7 Stop, T7 polymerase stop motifs.
FIG. 2
FIG. 2
Western blots of cell lysates of CRFK cells transfected with various constructs and stained with FCV-specific Mab K. (A) Comparison of the expression efficiencies of the CMV, SV40, and T7 promoter constructs. Lane 1, pCAPI (CMV promoter) lysate of about 5 × 106 cells; lane 2, pCAPIV (SV40 promoter) lysate of about 5 × 106 cells; lane 3, pCAPI plus 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. (B) Processing of the capsid protein precursor and the mature capsid protein in CRFK cells. Lane 1, precursor capsid protein gene expressed with MVA/T7. Note the processing of the capsid protein precursor to the 62-kDa mature-size protein; lane 2, expression of the mature truncated capsid protein gene expressed with MVA/T7. (C) Processing of the capsid protein precursor in nonpermissive cell lines: murine L929 cells (lanes 1 to 4) and Vero cells (lanes 5 to 8). Lanes 1 and 5, precursor gene expressed with MVA/T7; Lanes 2 and 6, mature truncated capsid protein gene expressed without MVA/T7; lanes 3 and 7, mature truncated capsid protein expressed with MVA/T7, lanes 4 and 8, MVA/T7-infected control cells.
FIG. 3
FIG. 3
Immunofluorescence of CRFK cells transfected with pT7GFP and coinfection with MVA/T7pol (A and B) or transfected with the FCV capsid protein construct pCAPI (C and D), or pCAPIV (E) and coinfection with MVA/T7 pol. All cells were stained with MAb K. Note the syncytium formation due to MVA virus infection.
FIG. 4
FIG. 4
Immunoblots of CsCl density gradient fractions for purification of empty virus capsids after transfection of the mature capsid protein gene (A) or the capsid protein precursor gene (B). The densities of the respective fractions are indicated at the top. (C) Immunoblot of fraction 5 from both gradients along with purified virions separated in an SDS–10% PAGE gel and detected with FCV MAb K to indicate the size homologies of the respective capsid proteins.
FIG. 5
FIG. 5
Negative-staining electron microscopy of empty virus capsids produced after transfection of the precursor capsid protein gene (A), after transfection of the mature capsid protein gene (B), and after transfection of authentic full virus particles (C).
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