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. 2009 Dec 23;84(6):3101–3105. doi: 10.1128/JVI.02093-09

FIG. 2.

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).