Panoply™ Human MALT1 Over-expressing Stable Cell Line

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major swine pathogen that evades host antiviral immunity through multiple mechanisms. PRRSV infection can induce macroautophagy/autophagy, thereby promoting viral replication. MALT1, an important immune regulatory factor, is utilized by PRRSV to optimize the infection process at different stages of the viral life cycle. Here, this study confirms that PRRSV-induced autophagy promotes viral proliferation. Furthermore, the autophagosome fusion process is dynamically regulated during PRRSV infection. Importantly, PRRSV-induced MALT1 promotes the fusion of autophagosomes with lysosomes and the formation of autolysosomes, thereby promoting autophagic flux and viral proliferation. Mechanistically, MALT1 regulates autophagy by mediating the MTOR-ULK1 and -TFEB signaling pathways and affecting lysosomal homeostasis. Inhibition of MALT1 using the inhibitor Mi-2 or RNAi leads to increased lysosomal membrane permeability (LMP), thus blocking autophagosome fusion. Moreover, MALT1 overexpression can alleviate PRRSV-induced LMP by inhibiting the production of reactive oxygen species (ROS). Furthermore, blocking autophagic flux significantly inhibits viral release, suggesting that maintaining intact autophagic flux by MALT1 during PRRSV infection is beneficial for successful viral transmission and proliferation.

To investigate the effects of MALT1 on autophagy flux and viral replication, researchers first compared the expression levels of LC3 and SQSTM1 proteins in Marc-145 cells and stable MALT1-overexpressing Marc-145 cells (Marc-145-MALT1) with or without BafA1 treatment (Figure 1A). Compared to Marc-145 cells, Marc-145-MALT1 cells showed relatively lower levels of SQSTM1 and LC3-II, and the BafA1-induced accumulation of LC3-II and SQSTM1 was also attenuated, suggesting that MALT1 overexpression promotes autophagy flux, possibly due to enhanced activity in the later stages of the autophagic process. Consistent with this, confocal microscopy observations showed that BafA1 induced a significant accumulation of LC3 and SQSTM1 puncta, while these puncta were significantly reduced in MALT1-overexpressing cells (red) (Figure 1B and C). Furthermore, RFP-GFP-LC3 transfection experiments showed that MALT1 overexpression alleviated the autophagosome-lysosome fusion blockage caused by BafA1 (Figure 1D). These results confirm that MALT1 promotes autophagy flux by facilitating autophagosome-lysosome fusion. Importantly, MALT1 overexpression promoted PRRSV replication, as detected by intracellular PRRSV-N protein levels and viral titers in the supernatant, while the autophagy inhibitors 3-MA or CQ blocked this promotion (Figure 1E, F), indicating that MALT1 promotes PRRSV replication in an autophagy-dependent manner. These results confirm that MALT1 enhances autophagy flux, thereby promoting PRRSV replication.

Figure 1. MALT1 overexpression promoted autophagy flux, facilitating PRRSV proliferation. (Gu H, et al., 2024)