Murine Py VP1 and VP2 capsid proteins contain integrin ligand motifs. (A) Alignment of the Py A2° VP1 LDV motif with the CS1 fragment of fibronectin (Fn CS1) (GenBank accession number AAD11500) and related Py sequences from aa 128 to 147. (B) Alignment of the Py A2° VP1 DLXXL motif with tenascin (Tn) (GenBank accession number P24821) and related Py sequences from aa 166 to 185. (C) Alignment of the Py A2° VP2 DGE motif with type I collagen (Cl) (GenBank accession number NP_000079) and related VP2 sequences from aa 31 to 50. *, :, and ., identical aa, strongly conserved, and weakly conserved amino acids, respectively, as defined by the CLUSTAL W program. The consensus motifs and the murine Py strains are in boldface. GenBank accession numbers of the analyzed viral proteins are indicated. °, VP1 and VP2 sequences of the Py A2 strain used in this work. Cerco. and Budger., Cercopitheci and Budgerigar fledgling disease viral strains, respectively.

Expression of the α4 integrin subunit enhances Py infectivity in the α4-negative BALB/c 3T3 fibroblasts. (A) Flow cytometric analysis of α4 and β1 subunit surface expression on mock-transfected and α4-BALB/c 3T3 (α4+) cells. Cells were either preincubated (heavy line) or not preincubated with anti-α4 or anti-β1 Abs for 30 min on ice as described in Materials and Methods. After washes, the cells were incubated with PE-conjugated secondary Abs. The histograms display relative cell numbers as a function of relative fluorescence intensities. (B) The α4-expressing BALB/c 3T3 cell line is more susceptible to Py infection than the mock-transfected clone. Mock-transfected and α4-BALB/c 3T3 cells were either pretreated or not pretreated for 1 h at room temperature with 30 μg of anti-α4 or its isotype Ab per ml before infection with Py for 1 h at 37°C. After the virus particles were removed, cells were further incubated for 20 h at 37°C before fixation. Infected cells were stained for LT by indirect immunofluorescence. Results are reported with the value obtained when mock-transfected cells were preincubated with DMEM without competitors (control) considered to be 100% virus infectivity (corresponding to an average of 3% LT positive cells). Data correspond to the means and standard deviations from two independent experiments.

Proposed model for the early interaction of Py with host cells. First, Py interacts with SA-containing cell receptors (step 1). This initial interaction is a prerequisite for subsequent proper recognition of the α4β1 integrin (step 2), either to promote a conformational change of the Py capsid, rendering the LDV motif more accessible to the integrin molecule, or to bring it in closer proximity to the integrin. This second interaction may facilitate the entry of the virus into the cells (step 3). Considering that both α4 and β1 integrin subunits are heavily glycosylated, carrying, in particular, terminal SA residues, integrins may themselves be a component of the SA-containing receptor molecules for Py (steps 1a and 2a) (see text).

Integrin ligands and function-blocking Abs reduce Py infectivity. (A and B) Py infectivity is inhibited by integrin ligands in a dose-dependent manner. Swiss 3T3 cells were pretreated for 1 h at room temperature with various concentrations of integrin specific ligands, i.e., fibronectin (Fn), vitronectin (Vn), collagen (Cl), or tenascin (Tn) (A) or α-chymotryptic fibronectin fragments (Fn40 or Fn120) (B), before infection with virus particles for 1 h at 4°C. The virus inoculum was removed, and cells were further incubated for 20 h at 37°C before fixation. Infected cells were immunostained for LT (see Materials and Methods). Results are presented as the percentage of virus infectivity obtained when cells were preincubated with DMEM without competitors (control). One hundred percent of virus infectivity corresponded to an average of 7 to 10% LT positive cells. Data correspond to the means and standard deviations from two or three independent experiments, each performed in duplicate or triplicate. (C) Inhibition of Py infectivity by integrin-specific Abs. Swiss 3T3 cells were pretreated for 1 h at room temperature with a 30-μg/ml concentration of Abs to specific integrins before infection with Py for 1 h. After virus particles were removed, cells were further incubated for 20 h at 37°C before fixation. Infected cells were stained for LT by indirect immunofluorescence. Results are presented as described for panels A and B. Data correspond to the means and standard deviations from two or three independent experiments, each performed in duplicate or triplicate.

Expression of the α4 integrin subunit enhances cell permissivity to Py at a postattachment step. (A) Addition of α4 function-blocking Abs prior to or after virus adsorption blocks the increased infectivity of Py in α4-BALB/c 3T3 cells. Mock-transfected and α4-BALB/c 3T3 cells were either pretreated or not pretreated for 1 h at 4°C with 30 μg of anti-α4 or its isotype Ab per ml before infection with Py for 1 h at 4°C (left panel) or after infection with Py for 1 h at 4°C (right panel). After the virus particles were removed, cells were further incubated for 20 h at 37°C before fixation and immunostaining for LT. Results are reported with the value obtained when mock-transfected cells were preincubated with DMEM without competitors (control) considered to be 100% virus infectivity (average of 1% LT positive cells). Results of a representative experiment performed in duplicate are reported as described for Fig. 3B. (B) Characterization of the purity and labeling efficiency of biotinylated Py viral particles. Left panel, SDS-PAGE of purified viral particles (500 ng) stained with GelCode Blue staining reagent. Right panel, Western blot analysis of biotinylated viral particles with HRP-conjugated streptavidin. (C) Function-blocking MAb directed to α4 does not affect the binding of biotinylated Py particles to α4-BALB/c 3T3 cells. α4-BALB/c 3T3 cells were pretreated for 30 min on ice with a 30-μg/ml concentration of Ab directed to α4 integrin (right panel) or its isotype (left panel) before treatment with 500 ng of biotinylated viral particles for 1 h at 4°C. Cells incubated with the binding buffer alone are shown as negative control (shaded area). After two washes, cells were incubated for 30 min with PE-conjugated streptavidin. Fixed cells were analyzed by FACS. The histograms display relative cell numbers as a function of relative fluorescence intensities. (D) SA-containing receptors are involved at the attachment level. One microgram of biotinylated viral particles was incubated for 1 h at 4°C with or without NANA and then added to α4-BALB/c 3T3 cells pretreated with 200 mU of neuraminidase (Neu) and left for 1 h at 4°C. Binding of virus on untreated cells is also shown. Cells incubated with the binding buffer alone as a negative control are shown (shaded area). After two washes, cells were incubated for 30 min with PE-conjugated streptavidin. Fixed cells were analyzed by FACS. The histograms display relative cell numbers as a function of relative fluorescence intensities.