This work aims to explore the effect of glycolysis on the replication of porcine reproductive and respiratory syndrome virus (PRRSV) in porcine alveolar macrophages (PAMs). The changes of glucose metabolism, PRRSV protein levels, PRRSV titers, and the relative expression levels of genes and proteins in PAMs were analyzed by ELISA, qPCR, virus titration, and Western blotting after PRRSV infection. The effect of hypoxia-inducible factor-1α (HIF-1α) on PRRSV replication was subsequently assessed by specific siRNAs targeting to HIF-1α. The results showed that PRRSV infection enhanced glycolysis, elevated the levels of glucose uptake and lactate in the supernatant (P<0.05 and 0.01, respectively), reduced ATP production (P<0.05), and up-regulated the expression of hexokinase 2 (HK2), 6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase 3 (PFKFB3), and pyruvate kinase isozyme type M2 (PKM2) in PAMs (P<0.05 and 0.01, respectively). Glycolysis inhibitors down-regulated the expression of PRRSV proteins and reduced virus titers (P<0.01). The knockdown of HIF-1α by siRNAs inhibited glycolysis and lowered PRRSV titers (P<0.05). In addition, the interferon pathways inhibited by PRRSV infection were reversed by the inhibition of glycolysis. These findings may facilitate further investigation of the role of glycolysis in PRRSV replication.
Porcine respiratory and reproductive syndrome virus/physiology* ; Glycolysis ; Swine ; Animals ; Virus Replication ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics* ; Macrophages, Alveolar/metabolism* ; Porcine Reproductive and Respiratory Syndrome/virology* ; Cells, Cultured ; RNA, Small Interfering/genetics*

In order to study the signal pathway secreting type Ⅰ interferon in porcine alveolar macrophages (PAMs) infected with porcine circovirus type 2 (PCV2), the protein and the mRNA expression levels of cGAS/STING pathways were analyzed by ELISA, Western blotting and quantitative reverse transcriptase PCR in PAMs infected with PCV2. In addition, the roles of cGAS, STING, TBK1 and NF-κB/P65 in the generation of type I interferon (IFN-I) from PAMs were analyzed by using the cGAS and STING specific siRNA, inhibitors BX795 and BAY 11-7082. The results showed that the expression levels of IFN-I increased significantly at 48 h after infection with PCV2 (P<0.05), the mRNA expression levels of cGAS increased significantly at 48 h and 72 h after infection (P<0.01), the mRNA expression levels of STING increased significantly at 72 h after infection (P<0.01), and the mRNA expression levels of TBK1 and IRF3 increased at 48 h after infection (P<0.01). The protein expression levels of STING, TBK1 and IRF3 in PAMs infected with PCV2 were increased, the content of NF-κB/p65 was decreased, and the nuclear entry of NF-κB/p65 and IRF3 was promoted. After knocking down cGAS or STING expression by siRNA, the expression level of IFN-I was significantly decreased after PCV2 infection for 48 h (P<0.01). BX795 and BAY 11-7082 inhibitors were used to inhibit the expression of IRF3 and NF-κB, the concentration of IFN-I in BX795-treated group was significantly reduced than that of the PCV2 group (P<0.01), while no significant difference was observed between the BAY 11-7028 group and the PCV2 group. The results showed that PAMs infected with PCV2 induced IFN-I secretion through the cGAS/STING/TBK1/IRF3 signaling pathway.
Animals ; Cells, Cultured ; Circovirus ; Interferon Type I/genetics* ; Macrophages, Alveolar/virology* ; Membrane Proteins/metabolism* ; Nucleotidyltransferases/metabolism* ; Signal Transduction ; Swine