Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2023 Mar 10;42(4):112286. doi: 10.1016/j.celrep.2023.112286

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© 2023 The Author(s)

Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.

PMC Copyright notice

ORF8 homo-dimerization is important for ER-phagy inhibition

(A) Co-precipitation analysis of GFP-ORF8 with ORF8-Strep in HEK293T.

(B) Purified MBP-ORF8 was incubated with purified GST or GST-ORF8, and analysis of the self-interaction by GST pull-down.

(C) Analysis of the homo-dimerization of ORF8 by cross-linking with DSS. Cells were transfected with ORF8-FLAG for 24 h and treated with DSS as indicated for 30 min. Cell lysates were analyzed using WB.

(D) Analysis of the self-interaction of ORF8 with p62 overexpression by coIP assay.

(E) Analysis of the self-interaction of ORF8 under starvation treatment by coIP assay.

(F) Analysis of the homo-dimerization of ORF8 by cross-linking with DSS under starvation treatment. Cells stable expressing ORF8-FLAG were starved for 12 h. Cell lysates were analyzed using WB.

(G) Schematic illustration of ORF8 point mutations.

(H and I) Cross-linking with DSS assay (H) and coIP (I) analyzed ORF8 homo-dimerization. Cells were transfected with indicated plasmids for 24 h.

(J) Interaction between Strep-tagged WT ORF8 and mutants with GFP-ORF8-HA observed in a coIP assay.

(K) Analysis of the homodimerization of ORF8 deletion mutants by cross-linking with DSS and analyzed using WB.

(L) Prediction of ORF8 aggregation domain.

(M and N) Analysis of the homodimerization of ORF8 deletion mutants by coIP (M) and DSS assay (N).

(O) Interaction between GFP-ORF8 and mutants with p62-FLAG observed in a coIP assay.

(P and Q) Interaction between GFP-ORF8 and mutants with FAM134B-HA (P) or ATL3-HA (Q) observed in a coIP assay.

(R) Analysis of the colocalization of GFP-ORF8 and deletion mutant with FAM134B-HA or ATL3-HA with anti-HA or anti-FLAG antibodies using confocal microscopy. Scale bar, 10 μm. The number of colocalization of ORF8/p62 bodies with FAM134B or ATL3 in each cell was counted from 25 cells of three independent experiments. Two-tailed unpaired Student’s t test, ^∗∗∗∗p < 0.0001.

(S) U2OS cells expressing mCherry-Sec61B were transfected with indicated plasmids for 24 h and then starved in EBSS for 12 h. Lysates were analyzed using WB.

(T) HEK293T cells were transfected with indicated plasmids for 24 h. Lysates were analyzed using WB.

(U) Proposed model for the role of ORF8/p62 condensates in viral replication through ER-phagy inhibition.