VOPBA of vimentin. Vimentin from bovine lens was separated by SDS-PAGE. The protein was transferred onto a nitrocellulose membrane and incubated with purified PRRSV (lane 1). The protein was incubated with SR-30, a MAb against PRRSV nucleocapsid protein, followed by incubation with the secondary anti-mouse peroxidase-conjugated Ab. The presence of virus binding was detected by the addition of TMB membrane peroxidase substrate (one component). The control reaction was identical, except that no virus was added (lane 2).

Expression profiles of vimentin in different cell lines by Western blot analysis. The total cellular proteins were extracted from each cell line and separated by SDS-PAGE (lane 1, MARC-145; lane 2, BHK-21; lane 3, CRFK; lane 4, MDCK; lane 5, PK-15; lane 6, ST; lane 7, Vero). The proteins were transferred onto a nitrocellulose membrane and incubated with monoclonal anti-vimentin Ab, followed by incubation with peroxidase-labeled anti-mouse IgG (H+L). The protein bands were detected by the addition of TMB membrane peroxidase substrate (one component). The major bands are indicated by arrowheads.

Delivery of simian vimentin into PRRSV-nonsusceptible cell lines. (A) Simian recombinant vimentin or β-galactosidase was delivered into BHK-21 cells (a and b) and CRFK cells (c and d) in the Chariot protein delivery system. Simian vimentin or β-galactosidase recipient cells were infected with PRRSV. The cells were fixed with 80% acetone and stained with FITC-conjugated SDOW-17, a MAb against PRRSV nucleocapsid protein. The cells were examined by fluorescence microscopy and photographed at ×20. (B) A sensitive RT-PCR assay was performed to demonstrate the PRRSV RNA production in simian-vimentin or β-galactosidase recipient cells. M, DNA marker; lane 1, PRRSV-infected MARC-145; lane 2, PRRSV-uninfected MARC-145; lane 3, PRRSV-infected, vimentin recipient BHK-21; lane 4, PRRSV-infected, β-galactosidase recipient BHK-21; lane 5, PRRSV-infected, vimentin recipient CRFK; lane 6, PRRSV-infected, β-galactosidase recipient CRFK.

Separation of 7G10 MAb-bound proteins by 2-D electrophoresis. A 7G10 MAb-bound host cell protein complex was separated by 2-D elecrophoresis. A Readystrip IPG stip was rehydrated in DeStreak rehydration solution containing the protein complex, and isoelectric focusing was performed. The IPG strip was equilibrated, and the proteins were separated by SDS-PAGE. The protein spots were cored and identified by MALDI-TOF MS (PS 101, vimentin; PS 102, no identification; PS 103, cytokeratin 8; PS 104, no identification; PS 105, keratin 18; PS 106, actin; PS 107, hair type II basic keratin).

Alignment of vimentin amino acid sequences. Simian and porcine vimentin amino acid sequences were generated from the cDNA sequences. The amino acid sequences were aligned with human, murine, and bovine vimentin amino acid sequences. The N-terminal region of the porcine vimentin cDNA sequence is absent because it is a partial sequence. The dashes in the porcine sequence represent deletions. Dots represent similarity of amino acid residues.

Localization of vimentin in MARC-145 cells. (A) Subcellular localization of vimentin in MARC-145 cells. The cells were permeabilized with 80% acetone and stained with FITC-conjugated monoclonal anti-vimentin (V9) Ab. The cells were examined by fluorescence microscopy and photographed at ×20. (B) Cell surface expression of vimentin on MARC-145 cells at various time points after infection with PRRSV. The nonpermeabilized cells were stained with polyclonal rabbit anti-vimentin Ab, followed by incubation with FITC-conjugated goat anti-rabbit IgG (H+L) (solid lines). An isotype-matched control is represented by the dotted lines.