Our work showed that porcine ST3GAL2 was a type II transmembrane protein, localized on the Golgi apparatus of 3D4 / 21 cells. The expression of porcine ST3GAL2 was up-regulated in PRRSV infected 3D4 / 21 cells. Porcine ST3GAL2 may participate in the glycosylation modification of the PRRSV envelope protein GP2a and promote the proliferation of PRRSV. This process may be related to the formation and assembly of the viral envelope on the Golgi apparatus of the host cell. ST3GAL2 may increase the expression of anti-inflammatory factors (IL-4, IL-10) and down-regulate the expression of pro-inflammatory factors (IL-1β, IL-2, IL-6, IL-18, IFN-β, TNF-α) to resist inflammation. The relationship between ST3GAL2 and inflammatory factors and the interaction regulation mechanism needs to be further studied.
Glycosylation is a very common modification of proteins and lipids, and most glycosylation reactions occur in the Golgi apparatus[18]. Although the initial transfer of sugar to glycoproteins or glycolipids is carried out on the ER or ER membrane, the subsequent addition of different sugar groups to form a mature glycan occurs in the Golgi apparatus[28]. The Golgi membrane is covered with glycosyltransferases, glycosidases, and nucleotide sugar transporters[35]. The sequence is usually from the cis-Golgi to the trans-Golgi network, so that each enzyme activity can function on specific substrates generated earlier in the pathway[36]. Based on this characteristic, we verified the subcellular localization of porcine ST3GAL2 sialyltransferases on 3D4 / 21 cells and found that porcine ST3GAL2 is also localized on the Golgi body of the cell.
PRRSV has strong recognition and binding activity for the surface receptors of porcine alveolar macrophages, so it can target PAM to achieve massive proliferation and large-scale infection, causing serious damage to the host's lymphatic system and respiratory tract[22]. The body's immune response is triggered, and a series of related genes are regulated to cause differential expression[19]. At the same time, in order to promote its own proliferation, the viruses will also regulate the synthesis of some proteins to assemble into new viruses[48]. Through different methods, we verified that when PRRSV infects 3D4/21 cells, the transcription level of ST3GAL2 is significantly increased, indicating that ST3GAL2 may be related to the immune response triggered by PRRSV.
In the early stage of infection, the virus mainly attaches to the cell surface by binding to the corresponding receptor or attachment factor on the host cell surface[43]. Sialic acid is used by a large number of viruses as the initial connection receptor, especially the sialic acid on the surface of the virus, which can be recognized by the surface receptors of many cells and mediate the virus to invade the cells[46]. Therefore, in the next experiments, we verified the effect of sialyltransferase STGAL2 on PRRSV proliferation. We found that after overexpressing ST3GAL2 in 3D4/21 cells, the proliferation of PRRSV was significantly up-regulated compared to the control group. However, after transfecting ST3GAL2 specific siRNA to interfere with its expression, the viral load of PRRSV decreased significantly. These results indicated that ST3GAL2 expression was positively correlated with PRRSV proliferation.
GP2a, 3, 4, 5 are the structural proteins of PRRSV, they can be modified by glycosylation, and can also measure the process of virus replication[29]. GP2a structural protein is one of the small structural proteins encoded by ORF2a[49]. Previous studies have shown that GP2 can bind to the virus-cell surface receptor CD163 to help the virus invade the host cell[7]. GP2 forms a GP2-GP3-GP4 multimeric complex with two other small structural proteins, GP3, and GP4, which are believed to be critical for viruses to enter susceptible host cells[3]. In this study, we found that unlike GP3-5 glycoproteins, ST3GAL2, and GP2a have a co-localization and interaction relationship. PRRSV, as an enveloped virus, assembles in the cytoplasm and germinates on the Golgi to obtain the surface envelope, including several structural glycoproteins of PRRSV[17]. Therefore, we speculate that ST3GAL2 may play a role in the formation of the PRRSV envelope by modifying GP2a.
In the process of virus infecting cells, macrophages can secrete a variety of cytokines to enhance phagocytosis and mediate inflammation to exert antiviral effects[25]. Interleukins, interferons, etc. are inflammation-related cytokines, some of which play a role in promoting inflammation[21]. Most of these pro-inflammatory factors are the objects of glycosylation, and the corresponding biological functions are obtained through the action of glycosyltransferases[34]. Anti-inflammatory activity of autoreactive IgG antibodies due to sialylation of Fc glycans[42]. The sialylated Fc selectively binds to type II FcR, inducing the production of IL-33 in regulatory macrophage[26]. IL-33 may activate Th2 helper T cells, trigger the release of anti-inflammatory cytokines, and play an anti-inflammatory role[24]. Therefore, the expression of anti-inflammatory factors may also be related to sialylation to some extent. Based on the above materials, we found that porcine ST3GAL2 may be modified by glycosylation to down-regulate part of the glycosylated pro-inflammatory factors and up-regulate the expression of inflammatory factors. The correlation between ST3GAL2 and inflammatory factors needs to be further explored, and the interaction regulation mechanism between them also needs to be verified by detecting intermediate regulatory factors such as Fc receptors or related transcription factors in its pathway.
Overall, this study initially clarified the regulatory role of ST3GAL2 in PRRSV proliferation and cellular immunity, providing a basis for virus prevention and discovery of the mechanism and scope of ST3GAL2. However, there are still many problems to be explored in the subsequent research, such as the regulatory mechanism of increased expression of ST3GAL2 during PRRSV infection, the mechanism of action of the ST3GAL2 gene, and cellular inflammatory factors. It is believed that with the continuous advancement of research, the exploration of β-galactose-α-1,3- sialyltransferases in the field of the inflammatory response and immune regulation will be more in-depth.