RNF185 is required for K27-linked polyubiquitination to downregulate Ebola virus glycoprotein GP1,2 expression by reticulophagy

Abstract Reticulophagy reduces Zaire Ebolavirus (EBOV) glycoprotein GP1,2 expression, and thus increases viral fitness. Reticulophagy degrades endoplasmic reticulum (ER) subdomains containing misfolded proteins to restore the ER proteostasis, but whether degraded cargo proteins are polyubiquitinated is still unknown. Here, we show that the calnexin-calreticulin cycle promoted EBOV-GP1,2 misfolding via PDIA3/ERp57, and that misfolded GP1,2 was degraded via reticulophagy dependent on ATG3, ATG5, and SQSTM1/p62. EBOV-GP1,2 degradation also required its lysine 673 (K673), which served as a substrate for K27-linked ubiquitination. Although E3 ubiquitin ligases RNF26, RNF185, MARCH8, and TRIM25 either degraded or polyubiquitinated EBOV-GP1,2, only RNF185 had both activities. Thus, RNF185 polyubiquitinates misfolded EBOV-GP1,2 in the ER and then SQSTM1/p62 recruits polyubiquitinated GP1,2 into autophagosomes for autophagic degradation. We conclude that ubiquitin is required for reticulophagy, which has played a major role in other autophagic pathways. In addition, EBOV infection should be blocked by enhanced GP1,2 degradation via reticulophagy.