Fig 2: TREM2 regulates the expression of ADAM17 and CD163.(A) PAMs were transfected with siNC or siTREM2-1 for 24 h, and cells were collected. The protein levels of CD163 and ADAM17 are shown, as detected by western blot. GAPDH is shown as an internal control. (B) PAMs were transfected with siNC or siTREM2-1 for 12 h and infected with PRRSV (MOI = 1) for an additional 24 h. The CD163, ADAM17 and PRRSV N protein expression is shown, as measured by western blot. GAPDH is shown as an internal control. (C and D) PAMs were transfected with siNC or siTREM2-1 for 24 h, and then mock-infected or infected with PRRSV (MOI = 1) for 24 h. Expression of ADAM17 (C) and CD163 (D) is shown, as measured using qRT-PCR. (E and F) Immunofluorescence analysis is shown to detect the expression of ADAM17 (E) and CD163 (F) after down-regulating TREM2 expression (Bar, 200 µm). (G) Representative histograms for flow cytometry analysis of cell surface CD163 immunostaining for PAMs with the indicated conditions. (H) Corresponding positive cell ratio of cell surface CD163 based on analysis conditions in G. (I) PAMs with TREM2 knockdown were infected with PRRSV (MOI = 1) for the indicated periods. Cells were collected and protein levels of CD163, ADAM17, TREM2, and PRRSV N are shown using western blot analysis. GAPDH is shown as an internal control. (J—L) PAMs with TREM2 knockdown were infected with PRRSV (MOI = 1) in the presence or absence of R406 (5 µM) (J), Wortmannin (1 µM) (K) or BAY11-7082 (10 µM) (L) and western blots are shown to detect protein levels of ADAM17 and CD163. GAPDH is shown as an internal control. Data are representative of the results of three independent experiments (mean ± SE). Significant differences compared to the control group are denoted by * (P < .05), ** (P < .01) and *** (P < .001).

Fig 3: Model showing how PRRSV utilizes viral apoptotic mimicry and TIM-induced macropinocytosis as an alternative pathway to infect host cells. In addition to CME, TIM-1/4 recognizes PRRSV as apoptotic mimicry and induces macropinocytosis via the Rac1/Cdc42-Pak1 signaling pathway. PRRSV enters SNX5-macropinosomes via macropinocytosis as an alternative pathway, where CD163 functions as an indispensable receptor.

Fig 4: Schematic model of TREM2 regulation of PRRSV infection.TREM2 downregulation promotes the recruitment of Syk and the activation of downstream PI3K/Akt and ERK1/2 pathways. Simultaneously, TLR4-NF-?B signaling is triggered early in the viral infection, leading to an intensive and sustained proinflammatory response. Elevated proinflammatory cytokines and type I interferons reduce the expression of CD163 and increase the cleavage of CD163 mediated by ADAM17, which contributes to suppression of PRRSV infection. On the contrary, the cytoplasmic tail of TREM2 interacted with PRRSV Nsp2 to promote infection. Meanwhile, exogenous sTREM2 blocks PRRSV binding to cell receptors via competitively interacting with viral envelope proteins.

Fig 5: Identification of porcine reproductive and respiratory syndrome virus receptors in porcine pulmonary microvascular endothelial cells using immunofluorescence staining. Cells were seeded into 24-well plates and fixed after 24 hr of incubation. Indirect immunofluorescence was performed using anti-CD151 (A), anti-CD163 (B), and anti-CD169 antibodies (C). Bar=100 µm.