Signaling Lymphocytic Activation Molecule Family Member 1 Inhibits Porcine Reproductive and Respiratory Syndrome Virus Replication

Abstract

The porcine reproductive and respiratory syndrome virus (PRRSV) causes a highly contagious disease in domestic swine. Signaling lymphocytic activation molecule family member 1 (SLAMF1) is a costimulatory factor that is involved in innate immunity, inflammation, and infection. Here, we demonstrate that overexpression of the SLAMF1 gene inhibited PRRSV replication significantly and reduced the levels of key signaling pathways, including MyD88, RIG-I, TLR2, TRIF, and inflammatory factors IL-6, IL-1β, IL-8, TNF-β, TNF-α, and IFN-α in vitro. However, the knockdown of the SLAMF1 gene could enhance replication of the PRRSV and the levels of key signaling pathways and inflammatory factors. Overall, our results identify a new, to our knowledge, antagonist of the PRRSV, as well as a novel antagonistic mechanism evolved by inhibiting innate immunity and inflammation, providing a new reference and direction for PRRSV disease resistance breeding.

Keywords: PRRSV; SLAMF1; inflammatory cytokines; innate immunity; replication.

Figure 1

pEGFP-N1-SLAMF increases SLAM level. (a) The PCR product of the SLAM gene. M: DL2000 Marker; Line1~3: gene fragment of SLAM. (b) Double enzyme digestion product of pEGFP-N1-SLAM. M: DL5000 Marker; 1: The result of pEGFP-N1-SLAM digested with a restriction enzyme. (c) Relative SLAM mRNA level was determined by qRT-PCR; Note: the pEGFP-SLAM group compared with the pEGFP-SLAMF1 group: *** means extremely significant difference (p < 0.001). (d) SLAM protein was determined by western blot.

Figure 2

Overexpression of SLAM inhibits PRRSV replication. (a1) Expression of PRRSV−N protein at different time points after PRRSV infection. (a2) The protein expression of SLAM in infected Marc-145 cells at different time points; (b) Effect of overexpression of SLAM and infection with different MOI of PRRSV on PRRSV proliferation at different time points. (c) qRT-PCR for detecting the differential expression multiple of SLAM gene at different time points and different titers after PRRSV infection at 24 h. (c1) Differential expression multiple of PRRSV-infected 24 h SLAM gene with 0.1–1 MOI. (c2) 0.1 Differential expression multiple of SLAM gene 0–72 h after MOI PRRSV infection. Note: *** means extremely significant difference (p < 0.001).

Figure 3

Knockdown of SLAM enhances PRRSV replication. (a1) Different synthetic si-SLAM interfering plasmids were transfected into Marc-145 cells, and the mRNA level of the SLAM gene was detected by qRT-PCR 24 h later. (a2) Si-SLAM-197 was transfected into Marc-145 cells, and the changes in the SLAM gene in the cells were detected by western blot 24 h later. (b) Effect of SLAM gene on PRRSV proliferation was detected by western blot. (c) Effect of siRNA interference SLAM gene on PRRSV proliferation was detected by qRT-PCR. Note: ** means significant difference (p < 0.01), *** means extremely significant difference (p < 0.001).

Figure 4

SLAM gene overexpression inhibits PRRSV-induced inflammation. (a) Influence of SLAM overexpression on key signaling molecules after PRRSV infection. (b) Effects of SLAM overexpression on inflammatory cytokines after PRRSV infection. Note: *** means an extremely significant difference (p < 0.001).

Figure 5

Knockdown of porcine SLAM promotes PRRSV-induced inflammation. (a) The effect of si-SLAM on key signaling molecules after PRRSV infection. (b) The effect of the si-SLAM gene on inflammatory factors after PRRSV infection. Note: * means significant difference (p < 0.05); ** means significant difference (p < 0.01); *** means an extremely significant difference (p < 0.001).