A trypsin-like serine protease is involved in pseudorabies virus invasion through the basement membrane barrier of porcine nasal respiratory mucosa

Abstract

Several alphaherpesviruses breach the basement membrane during mucosal invasion. In the present study, the role of proteases in this process was examined. The serine protease-specific inhibitor AEBSF inhibited penetration of the basement membrane by the porcine alphaherpesvirus pseudorabies virus (PRV) by 88.1% without affecting lateral spread. Inhibitors of aspartic-, cysteine-, and metalloproteases did not inhibit viral penetration of the basement membrane. Further analysis using the Soybean Type I-S trypsin inhibitor for the serine protease subcategory of trypsin-like serine proteases resulted in a 96.9% reduction in plaque depth underneath the basement membrane. These data reveal a role of a trypsin-like serine protease in PRV penetration of the basement membrane.

Figure 1

Plaque latitude and penetration depth underneath the basement membrane (BM) of PRV(89V87) plaques at 20 h pi in mock-treated (white bars) and protease inhibitor-treated (marked bars) porcine nasal respiratory mucosa explants. Explants were treated with a broad-spectrum protease inhibitor cocktail (complete Mini), inhibiting serine, cysteine and metalloproteases, or with an aspartyl protease inhibitor, pepstatin A, at 1 h pi until sampling. Data are represented as means of 10 plaques of duplicate independent experiments + SD (error bars).

Figure 2

Plaque latitude and penetration depth underneath the basement membrane (BM) of PRV(89V87) plaques at 20 h pi in mock-treated (white bars) and protease inhibitor-treated (marked bars) porcine nasal respiratory mucosa explants. Explants were treated with 100 or 250 μM AEBSF, 1 or 10 μM E-64 or 10 μM phosphoramidon at 1 h pi until sampling. Data are represented as means of 10 plaques of triplicate independent experiments + SD (error bars).

Figure 3

Plaque latitude (L) and penetration depth underneath the basement membrane (D) of PRV(89V87) plaques at 20 h pi in mock-treated (white bars) and protease inhibitor-treated (marked bars) porcine nasal respiratory mucosa explants. Explants were mock-treated or treated with a trypsin-like serine protease inhibitor, Soybean Type I-S trypsin inhibitor, or with an elastase-like serine protease inhibitor, elastatinal, at 1 h pi, 3 h pi, 6 h pi, 9 h pi or 12 h pi until sampling. Data are represented as means of plaques observed in 120 sections (20 μm) of triplicate independent experiments + SD (error bars).

Figure 4

Confocal photomicrographs illustrating PRV(89V87) plaques at 20 h pi in (a) mock-treated and (b) Soybean Type I-S trypsin inhibitor-treated porcine nasal respiratory mucosa explants. Green fluorescence visualizes PRV antigens in virus plaques using FITC-conjugated polyclonal PRV-specific antibodies. Collagen IV (component of basement membrane (BM), black dashed line) is visualized using goat anti-collagen IV antibodies, stained afterwards with TexasRed. Figure (a) shows a representative image of two closely apposed viral plaques both invading through the BM. Figure (b) illustrates the blocked viral invasion underneath the BM in the presence of a trypsin-like protease inhibitor. Bar, 20 μm.