Cholesterol-dependent Nsp5-endosomes co-trafficking to lysosomes facilitates porcine reproductive and respiratory syndrome virus replication by activating autophagy.

Our previous studies showed that intracellular endosomal vesicles participated in PRRS virions trafficking in the early stage of viral infection, and cholesterol retention in endosomal vesicles disturbed viral replication via blocking PRRSV-endosomal vesicles membrane fusion. However, whether endosomal vesicles were associated with PRRSV protein(s) trafficking and the role of cholesterol in this process was still unclarity. In this study, we sought to elucidate the mechanism of cholesterol in endosomal vesicles-mediated viral protein transportation. The results showed that endosomal vesicles participated in trafficking of PRRSV Nsp5 protein. After being synthesized in endoplasmic reticulum (ER) and Golgi apparatus, Nsp5 was trafficked to early endosomes (EEs), but not endocytic recycling compartments (ERCs), then to late endosomes (LEs), and eventually reached lysosomal compartments, whereas disruption of cholesterol flux or LEs function led to the inability of Nsp5 arriving at lysosomes, where Nsp5 activated cellular autophagy to promote PRRSV replication. Molecular docking predictions revealed that cholesterol could form two hydrogen bonds with 74 alanine and 78 asparagine of Nsp5. After mutating the aforementioned binding sites, the replication efficiency of PRRSV decreased. Subsequently, the role of cholesterol in PRRSV replication was explored. Blocking of cholesterol flux significantly inhibited PRRSV replication. Single virus infection cycle analysis showed that cholesterol flux disorder did not affect virus adsorption, but could inhibit virus entry into host cells and block EEs-LEs-lysosomes mediated trafficking of virions, leading to virions retention in endosomal compartments. The present studies suggest that cholesterol and endosomal vesicles synergistically participate in Nsp5 trafficking to promote PRRSV replication, which may provide new insights for the development of novel antiviral drugs targeting cholesterol metabolism pathways or the improvement of commercial vaccines.