Protein disulfide isomerases (PDIs) negatively regulate ebolavirus structural glycoprotein expression in the endoplasmic reticulum (ER) via the autophagy-lysosomal pathway.

ABSTRACT

Zaire ebolavirus (EBOV) causes a severe hemorrhagic fever in humans and non-human primates with high morbidity and mortality. EBOV infection is dependent on its structural glycoprotein (GP), but high levels of GP expression also trigger cell rounding, detachment, and downregulation of many surface molecules that is thought to contribute to its high pathogenicity. Thus, EBOV has evolved an RNA editing mechanism to reduce its GP expression and increase its fitness. We now report that the GP expression is also suppressed at the protein level in cells by protein disulfide isomerases (PDIs). Although PDIs promote oxidative protein folding by catalyzing correct disulfide formation in the endoplasmic reticulum (ER), PDIA3/ERp57 adversely triggered the GP misfolding by targeting GP cysteine residues and activated the unfolded protein response (UPR). Abnormally folded GP was targeted by ER-associated protein degradation (ERAD) machinery and, unexpectedly, was degraded via the macroautophagy/autophagy-lysosomal pathway, but not the proteasomal pathway. PDIA3 also decreased the GP expression from other ebolavirus species but increased the GP expression from Marburg virus (MARV), which is consistent with the observation that MARV-GP does not cause cell rounding and detachment, and MARV does not regulate its GP expression via RNA editing during infection. Furthermore, five other PDIs also had a similar inhibitory activity to EBOV-GP. Thus, PDIs negatively regulate ebolavirus glycoprotein expression, which balances the viral life cycle by maximizing their infection but minimizing their cellular effect. We suggest that ebolaviruses hijack the host protein folding and ERAD machinery to increase their fitness via reticulophagy during infection.Abbreviations 3-MA 3-methyladenine; 4-PBA 4-phenylbutyrate; ACTB ß-actin; ATF activating transcription factor; ATG autophagy-related; BafA1 bafilomycin A1; BDBV Bundibugyo ebolavirus; CALR calreticulin; CANX calnexin; CHX cycloheximide; CMA chaperone-mediated autophagy; ConA concanamycin A; CRISPR clusters of regularly interspaced short palindromic repeats; Cas9 CRISPR-associated protein 9; dsRNA double-stranded RNA; EBOV Zaire ebolavirus; EDEM ER degradation enhancing alpha-mannosidase like protein; EIF2AK3/PERK eukaryotic translation initiation factor 2 alpha kinase 3; Env envelope glycoprotein; ER endoplasmic reticulum; ERAD ER-associated protein degradation; ERN1/IRE1 endoplasmic reticulum to nucleus signaling 1; GP glycoprotein; HA hemagglutinin; HDAC6 histone deacetylase 6; HMM high-molecular-mass; HIV-1 human immunodeficiency virus type 1; HSPA5/BiP heat shock protein family A (Hsp70) member 5; IAV influenza A virus; IP immunoprecipitation; KIF kifenesine; Lac lactacystin; LAMP lysosomal associated membrane protein; MAN1B1/ERManI mannosidase alpha class 1B member 1; MAP1LC3/LC3 microtubule associated protein 1 light chain 3; MARV Marburg virus; MLD mucin-like domain; NHK/SERPINA1 alpha1-antitrypsin variant null (Hong Kong); NTZ nitazoxanide; PDI protein disulfide isomerase; RAVV Ravn virus; RESTV Reston ebolavirus; SARS-CoV severe acute respiratory syndrome coronavirus; SBOV Sudan ebolavirus; sGP soluble GP; SQSTM1/p62 sequestosome 1; ssGP small soluble GP; TAFV Taï Forest ebolavirus; TIZ tizoxanide; TGN thapsigargin; TLD TXN (thioredoxin)-like domain; Ub ubiquitin; UPR unfolded protein response; VLP virus-like particle; VSV vesicular stomatitis virus; WB Western blotting; WT wild-type; XBP1 X-box binding protein 1.