Identification of the CD163 protein domains involved in infection of the porcine reproductive and respiratory syndrome virus

Abstract

Scavenger receptor CD163 is a key entry mediator for porcine reproductive and respiratory syndrome virus (PRRSV). To identify the CD163 protein domains involved in PRRSV infection, deletion mutants and chimeric mutants were created. Infection experiments revealed that scavenger receptor cysteine-rich (SRCR) domain 5 (SRCR 5) is essential for PRRSV infection, while the four N-terminal SRCR domains and the cytoplasmic tail are not required. The remaining CD163 protein domains need to be present but can be replaced by corresponding SRCR domains from CD163-L1, resulting in reduced (SRCR 6 and interdomain regions) or unchanged (SRCR 7 to SRCR 9) infection efficiency. In addition, CD163-specific antibodies recognizing SRCR 5 are able to reduce PRRSV infection.

FIG. 1.

CD163 deletion constructs used to identify the essential domains involved in PRRSV infection. (a) Structural domain organization of CD163 (nine extracellular SRCR domains, two PST-rich domains, a transmembrane region, and an intracellular cytoplasmic tail). (b) Domain organization of CD163 deletion mutants. (c) Detection of recombinant CD163 deletion variants in HEK293T cell lysates. Cell lysates were subjected to reducing SDS-PAGE prior to electroblotting and immunodetection with a V5-specific MAb (GenScript). (d) Expression profile of CD163 mutants by using MAb 2A10 (AbD Serotec), PAb AF1607 (R&D Systems), and a V5-specific MAb (GenScript). Immunofluorescence staining was performed first on nonpermeabilized cells to visualize CD163 variants at the cell surface, followed by permeabilization and visualization of surface as well as intracellular recombinant CD163 variants. +, surface and intracellular expression; −, no surface expression, only intracellular expression; NA, not applicable. (e) Twenty-four hours prior to inoculation, HEK293T cells were transfected with sialoadhesin combined with one of the CD163 deletion variants. Transfected cells were inoculated with PRRSV, and 24 h later, cells were fixed with ice-cold methanol and stained. Transfected as well as infected cells were visualized, and the relative percentage of infected cells was calculated, with the original full-length porcine CD163 as a reference. The average numbers of infected cells counted for each CD163 variant were 280 (A), 286 (B), 0 (C), 271 (D), 296 (E), 0 (F), 185 (G), 0 (H), and 0 (I), respectively. (See the text for descriptions of the variants.) Each value represents the mean ± standard deviation for three experiments. Different lowercase letters indicate statistically significant differences between different CD163 mutants (one-way analysis of variance; Tukey B test, α < 0.05).

FIG. 2.

Construction of chimeric constructs via domain swapping between CD163 and CD163-L1 to identify the essential domains involved in PRRSV infection. (a) Structural domain organization of CD163 (white) and CD163-L1 (black). CD163 has been suggested to emerge from CD163-L1 by gene duplication, a process during which three of the first six SRCR domains of CD163-L1 were lost (10, 24). They share six consecutive SRCR domains in the structure k-[b-c-d-e-d]. (b and f) Domain organization of chimeric constructs. Porcine CD163, human CD163, and human CD163-L1 are represented in white, gray, and black, respectively. (c and g) Detection of recombinant chimeric variants in HEK293T cell lysates. Cell lysates were subjected to reducing SDS-PAGE prior to electroblotting and immunodetection with a V5-specific MAb (GenScript). (d and h) Expression profile of CD163 mutants using antibodies MAb 2A10 (AbD Serotec), PAb AF1607 (R&D Systems) and a V5-specific MAb (GenScript). Immunofluorescence staining was performed first on nonpermeabilized cells to visualize CD163 variants at the cell surface, followed by permeabilization and visualization of surface as well as intracellular recombinant CD163 variants. (e and i) Twenty-four hours prior to inoculation, HEK293T cells were transfected with sialoadhesin combined with one of the CD163 chimeric constructs. Transfected cells were inoculated with PRRSV, and 24 h later, cells were fixed with ice-cold methanol and stained. Transfected as well as infected cells were visualized, and the relative percentage of infected cells was calculated, with the full-length porcine CD163 with a V5-His tag as a reference. The average numbers of infected cells counted for each CD163 variant in panel e were 283 (B), 165 (J), 0 (K), 0 (L), 175 (M), 268 (N), 189 (O), 287 (P), 0 (Q), and 107 (R), while the average numbers in panel i were 279 (B), 0 (S), 168 (T), and 243 (U), respectively. (See the text for descriptions of the variants.) Each value represents the mean ± standard deviation for three experiments. Different lowercase letters indicate statistically significant differences between different CD163 mutants (one-way analysis of variance; Tukey B test, α < 0.05).

FIG. 3.

Usage of CD163 variants to deplete a goat polyclonal antibody able to block PRRSV infection in macrophages. (a) The goat PAb AF1607 (R&D Systems) was passaged 16 times on transfected cells expressing different CD163 variants. Depletion of specific antibodies was confirmed via an immunofluorescence staining. Images represent an overlay of several z-sections throughout whole cells. Bar, 50 μm. (b) Depleted antibodies were used to treat macrophages for 1 h at 37°C prior to and during inoculation with PRRSV. Ten hours postinoculation, cells were fixed and infected cells were visualized using MAb P3/27 (32). The relative percentage of infected cells was calculated, with untreated cells as a reference. The average numbers of infected cells counted for each condition were 63 (mock), 30 (no depletion), 27 (depletion on K), 58 (depletion on B), 52 (depletion on S), and 35 (depletion on Q), respectively. Each value represents the mean ± standard deviation for three experiments. Different lowercase letters indicate statistically significant differences between treatments (one-way analysis of variance; Tukey B test, α < 0.05).