Institute of Molecular Biology, Friedrich-Loeffler-Institutes, Federal Research Centre for Virus Diseases of Animals, D-17498 Insel Riems, Germany1
Author for correspondence: Günther M. Keil. Fax +49 38351 7219. e-mail Guenther.M.Keil{at}rie.bfav.de
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To test for GFP expression, MDBK-Bu100 cultures were infected with approximately 50 p.f.u. of the isolates BHV-1/eTMIIGFP or BHV-1/eGFP and used for live-cell analysis after development of plaques. In contrast to the very intense and homogeneous BHV-1/eGFP-induced fluorescence (Fig. 1b), cells infected with BHV-1/eTMIIGFP exhibited a weaker fluorescence (Fig. 1a
) with a distribution indistinguishable from the allocation of BHV-1/eGsyn (Kühnle et al., 1998
) expressed glycoprotein G of BRSV (Fig. 1c
) or BHV-1 gB (Fig. 1d
) or BHV-1 gD (not shown) after visualization by indirect immunofluorescence. To quantify the differences in the fluorescence intensities, MDBK-Bu100 cells were infected with BHV-1/eGFP and BHV-1/eTMIIGFP at an m.o.i. of 5. Cells were harvested 16 h later and relative fluorescence was determined by flow cytometry using a Becton-Dickinson FACS-Calibur or with a Fluoroscan II (Labsystems). Both methods revealed that the BHV-1/eGFP-induced fluorescence was about 6-fold higher than that induced by BHV-1/eTMIIGFP. Since Northern blot analyses with RNA from cells infected with the recombinants demonstrated comparable amounts of transcripts encompassing the respective GFP ORF (not shown), it was assumed that the reduced fluorescence is due to the expression of GFP as a membrane protein. This assumption was supported by the results of the immunoprecipitations shown in Fig. 2
, which demonstrated that comparable amounts of GFP-related proteins were expressed in BHV-1/eTMIIGFP- and BHV-1/eGFP-infected cells.
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To determine whether the membrane anchor of the BRSV glycoprotein G is indeed sufficient to target the hybrid protein into the viral membrane, [35S]methionine/cysteine labelled proteins from infected cells and purified BHV-1/eGFP and BHV-1/eTMIIGFP virus particles were immunoprecipitated with a GFP-specific antiserum (Clontech) and, to control the infection and the purity of the virus preparations, BHV-1 gD-specific MAb 21/3/3. Enough antibodies were added to immunoprecipitate all gD or TMIIGFP molecules from the respective lysates. From infected cells, MAb 21/3/3 precipitated the 72 kDa mature gD and the 63 kDa gD precursor encoded by BHV-1/eGFP and BHV-1/eTMIIGFP (Fig. 2d, lanes 1 and 2) and only the mature 72 kDa gD from the respective virions (Fig. 2d
, lanes 5 and 6). The anti-GFP serum precipitated the 28 kDa GFP from BHV-1/eGFP-infected cells (Fig. 2d
, lane 4) and proteins with apparent molecular masses of 37, 35 and 28 kDa after infection with BHV-1/eTMIIGFP (Fig. 2d
, lane 3). Proteins with the same mobility were also detected among purified BHV-1/eTMIIGFP virion proteins (Fig. 2d
, lane 7), suggesting that both mature and incompletely processed TMIIGFP are associated with virus particles. However, the presence of polypeptides migrating at a similar position as GFP among the anti-GFP reactive proteins from BHV-1/eTMIIGFP virions indicates that some degradation of the fusion protein occurs during virion purification and immunoprecipitation. It is also possible that the 28 kDa protein found in BHV-1/eTMIIGFP-infected cells and in virions represents an intracellular cleavage product of TMIIGFP. Whether this protein had retained the BRSV G membrane anchor remains to be determined because BRSV G-specific polyclonal antibodies (Kühnle et al., 1998
) did not react with either form of TMIIGFP. Comparison of the relative intensities between gD and TMIIGFP from infected cells and virions indicates that gD and TMIIGFP were incorporated into cellular and viral membranes with comparable efficiencies, suggesting that gD is not preferentially integrated into the viral envelope.
The faint band at 28 kDa detected after precipitation of proteins from BHV-1/eGFP virions by the anti-GFP serum indicated that GFP molecules were associated with BHV-1/eGFP particles. This assumption was supported by infection of MDBK-Bu100 cells in the presence of cycloheximide (100 µg/ml) to prevent de novo protein synthesis with purified virus preparations of BHV-1/eTMIIGFP and BHV-1/eGFP at an m.o.i. of 10 followed by flow cytometry at 3 h post-infection. The maxima of the relative fluorescence intensities corresponded to 1·2x102 for BHV-1/eTMIIGFP-infected cells and 2·2x101 for BHV-1/eGFP-infected cells using the maximal relative fluorescence determined for wild-type BHV-1 as zero (data not shown). Assuming a comparable particle to p.f.u. ratio and taking into account that GFP fluorescence is more efficient than TMIIGFP fluorescence, approximately 30-fold more molecules of the fusion protein than GFP molecules were associated with virus particles. Although not further analysed, it is assumed that GFP constitutes a component of the tegument of BHV-1/eGFP virions and is released into infected cells after fusion of viral and cellular membranes. Whether this association is linked with the synthesis of GFP as a BHV-1-encoded protein or simply due to the over-expression of the protein needs to be clarified. The presence of cryptic packaging signals within GFP cannot be excluded either.
To demonstrate that TMIIGFP is integrated into the viral envelope in the correct orientation and to provide evidence that GFP is not a component of the membrane of BHV-1/eGFP virus particles, BHV-1/eTMIIGFP and BHV-1/eGFP virions were tested for susceptibility to neutralization by the GFP-specific antiserum (Fig. 3, open symbols) and, as a control, by MAb 21/3/3 (Fig. 3
, closed symbols). Without complement, the anti-GFP serum did not neutralize the recombinants at a 1:100 dilution and had no effect on the infectivity of wild-type BHV-1 at dilutions of 1:100 and higher irrespective of the presence of complement (data not shown). With complement, the anti-GFP serum efficiently neutralized BHV-1/eTMIIGFP virions (Fig. 3
, open circles) but not BHV-1/e GFP virions (Fig. 3
, open squares). Thus, TMIIGFP is integrated in the viral envelope as a class II membrane protein, whereas GFP appears to be a constituent of the tegument of BHV-1/eGFP virions. Both BHV-1 recombinants were comparably neutralized by MAb 21/3/3 (Fig. 3
), indicating that the presence of TMIIGFP in the viral membrane does not interfere with binding of MAb 21/3/3 to gD.
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Recombinant BHV-1/eTMIIGFP and plasmid pROMeTMIIGFP provide helpful tools to prove or disprove this hypothesis which is under discussion (Brideau et al., 1998 ).
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References |
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Received 23 July 1999;
accepted 6 December 1999.