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. 1998 May;72(5):4492-7.
doi: 10.1128/JVI.72.5.4492-4497.1998.

Detection of viral proteins after infection of cultured hepatocytes with rabbit hemorrhagic disease virus

M König  1 , H J Thiel, G Meyers
Affiliations

Detection of viral proteins after infection of cultured hepatocytes with rabbit hemorrhagic disease virus

M König et al. J Virol. 1998 May.

Abstract

The calicivirus rabbit hemorrhagic disease virus (RHDV), which replicates predominantly in the livers of infected rabbits, cannot be propagated in tissue culture. To enable the performance of in vitro studies, rabbit hepatocytes were isolated by liver perfusion and gradient centrifugation. After inoculation with purified RHDV, more than 50% of the cells proved to be infected. Protein analyses led to the detection of 13 RHDV-specific polypeptides within the infected cells. These proteins were assigned to defined regions of the viral genome, resulting in a refined model of RHDV genome organization.

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Figures

FIG. 1
FIG. 1
Infection of cultured hepatocytes with RHDV. Overnight cultures were either mock infected (A) or inoculated with gradient-purified virus and subsequently incubated for 24 h (B), 48 h (C, E, and F), or 72 h (D). Immunostaining was performed with monoclonal antibodies directed against RHDV VP60 major capsid protein and anti-mouse immunoglobulin G conjugated to horseradish peroxidase. Cells were counterstained with hematoxylin. (E and F) Higher magnifications of a culture 48 h after infection. Note the noninfected cells (E, bottom region), apparently newly infected cells (E, center), and cells showing a pronounced cytopathic effect (E, left and right margins; F [note condensation and size of nuclei]). Instrumental magnification, ×100 (A to D) or ×1,000 (E and F). The dark staining of the condensed nuclei in panel F resulted from the blue counterstain, which cannot be distinguished from the antibody staining evident on a black and white picture.
FIG. 2
FIG. 2
Immunoprecipitation of proteins from RHDV-infected hepatocytes with a set of antisera raised against bacterial fusion proteins containing defined regions of the polypeptides encoded by the RHDV genome (24). The upper portion of each panel shows the precipitated proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Each lane is labeled with a letter indicating the antiserum used for precipitation. On the right side of each gel, bands discussed in the text are marked with arrows and the designations of the precipitated proteins. The positions of size marker proteins (14C-labeled molecular mass markers; Amersham, Braunschweig, Germany) are indicated on the left (in kilodaltons). A scheme below each gel indicates the reactivities of the different antisera. The ORF1-encoded polyprotein is symbolized by a white bar marked by a scale (in 100-amino-acid increments). Within the polyprotein, the locations of VPg and the RHDV protease (Pro) as well as the positions of amino acid motifs common for RNA virus polymerases (Pol) and helicases (2C-like) are shown. The protein encoded by ORF2 is shown below the ORF1 product. Gray bars labeled with letters indicate the protein segments contained in the different fusion proteins used for generation of the antisera. The exact features of the antisera have been described previously (24). In the lower part of each panel, the putative locations of the precipitated proteins (black bars) with respect to the polyprotein are shown. Please note that the gels of all three panels have been mounted from one protein gel. In panels B and C, the last lane of panel A and the last lane of B, respectively, are included to facilitate comparison of the electrophoretic mobilities of the individual proteins. (A) Proteins precipitated with antisera directed against regions within the amino-terminal one-third of the ORF1-encoded polyprotein. (B) Precipitation products obtained with antisera recognizing different segments of the central region of the polyprotein. Further processing of p29 into p23/2 and X is hypothetical and could occur in different ways, as indicated in the box at the bottom. (C) Proteins precipitated with antisera directed against the carboxy-terminal half of the ORF1-encoded polyprotein and the ORF2 product. Ns, rabbit preimmune serum.
FIG. 3
FIG. 3
Schematic representation of the RHDV-encoded proteins. The upper bars indicate the hypothetical primary translation products of ORF1 and ORF2; the scale represents amino acid numbers. Regions which contain known sequence motifs or already-identified viral proteins are indicated. EG and ET, processing sites determined so far (2, 23, 24). The gray-shaded bars below represent VP10 and the products resulting from processing of the ORF1-encoded polyprotein. The designations of proteins which could not be demonstrated after in vitro translation are written in boldfaced letters. The polypeptides generated by the hypothetical cleavage of p29 are enclosed in a box. The hypothesized further processing of p29 has not been proven; two alternative ways for this processing to occur are indicated. Protein “X” has not been demonstrated.
FIG. 4
FIG. 4
Comparison of the genomic regions encoding part of the nonstructural proteins of RHDV and poliovirus. The genomes are shown as bars, and the locations of individual genes are indicated. The regions shown in gray symbolize those parts of the genomes for which the experiments indicate the existence of a major difference between RHDV and picornaviruses (see also the text and the legend to Fig. 3).
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