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. 2023 May 19;26(5):106601.
doi: 10.1016/j.isci.2023.106601. Epub 2023 Apr 8.

The human E3 ligase RNF185 is a regulator of the SARS-CoV-2 envelope protein

Charles Zou  1 , Hojong Yoon  1   2 , Paul M C Park  1   2 , J J Patten  3 , Jesse Pellman  1   2 , Jeannie Carreiro  1   2 , Jonathan M Tsai  1   2 , Yen-Der Li  1   2 , Shourya S Roy Burman  4   5 , Katherine A Donovan  4   5 , Jessica Gasser  1   2 , Adam S Sperling  1   2   6 , Radosław P Nowak  4   5 , Eric S Fischer  4   5 , Robert A Davey  3 , Benjamin L Ebert  1   2   7 , Mikołaj Słabicki  1   2
Affiliations

The human E3 ligase RNF185 is a regulator of the SARS-CoV-2 envelope protein

Charles Zou et al. iScience. .

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) hijacks multiple human proteins during infection and viral replication. To examine whether any viral proteins employ human E3 ubiquitin ligases, we evaluated the stability of SARS-CoV-2 proteins with inhibition of the ubiquitin proteasome pathway. Using genetic screens to dissect the molecular machinery involved in the degradation of candidate viral proteins, we identified human E3 ligase RNF185 as a regulator of protein stability for the SARS-CoV-2 envelope protein. We found that RNF185 and the SARS-CoV-2 envelope co-localize to the endoplasmic reticulum (ER). Finally, we demonstrate that the depletion of RNF185 significantly increases SARS-CoV-2 viral titer in a cellular model. Modulation of this interaction could provide opportunities for novel antiviral therapies.

Keywords: Cell biology; Virology.

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Conflict of interest statement

M.S. has received research funding from Calico Life Sciences LLC. B.L.E. has received research funding from Celgene, Deerfield, Novartis, and Calico Life Sciences LLC and consulting fees from GRAIL. He is a member of the scientific advisory board and shareholder for Neomorph Inc., TenSixteen Bio, Skyhawk Therapeutics, and Exo Therapeutics. E.S.F. is a founder, member of the scientific advisory board (SAB), and equity holder of Civetta Therapeutics, Proximity Therapeutics, and Neomorph Inc (also board of directors), a SAB member and equity holder in Avilar Therapeutics and Photys Therapeutics, equity holder in Lighthorse Therapeutics, and a consultant to Sanofi, Novartis, Deerfield, Odyssey Therapeutics, and EcoR1 capital. The Fischer laboratory receives or has received research funding from Novartis, Deerfield, Ajax, Interline, and Astellas. K.A.D. is a consultant to Kronos Bio and Neomorph Inc. A.S.S. reports consulting fees from Adaptive Technologies and Roche.

Figures

None
Graphical abstract
Figure 1
Figure 1
The stability of SARS-CoV-2 proteins in HEK293T cells (A) Schematic overview of protein stability assay for SARS-CoV-2 proteins. (B) Flow cytometry analysis of SARS-CoV-2 protein stability reporters after 6 h of treatment with 10 μM of MG132, 1 μM MLN7243, 1 μM MLN4924, or DMSO (bars represent mean, n = 3). Black line represents mean eGFP/mCherry expression of non-treated cells; red line represents a 30% increase of expression.
Figure 2
Figure 2
Functional genomic screens identified RNF185 ligases for SARS-CoV-2 envelope stability (A) Schematic of CRISPR sorting screen of the SARS-CoV-2 protein stability. Reporter cell lines were transduced with Bison CRISPR sgRNA library (713 E1, E2, and E3 ubiquitin ligases, deubiquitinases, and control genes containing 2,852 sgRNAs), cultured for 8 days, then sorted into 4 gates based on GFP/mCherry ratio. (Gate A: bottom 0%–5%; Gate B: bottom 5%–10%; Gate C: top 90%–95%; Gate D: top 95%–100%). (B) CRISPR-Cas9 sorting screen for cells with altered stability of SARS-CoV-2 envelope. Guides were collapsed to gene levels (n = 2, four guides per gene, two-sided empirical ran-sum test-statistic, horizontal dashed line indicates p value = 0.0005, vertical dashed line indicates fold change of 2, vertical dot line indicates fold change of 1.6). Normalized read count for sgRNAs targeting RNF185 is shown. (C) Flow cytometry analysis of degradation of SARS-CoV-2 envelope in a panel of three cell lines after 6 h of treatment with 10 μM of MG132, 1 μM MLN7243, 1 μM MLN4924, or DMSO (∗∗ p value <0.01, ns – not significant). (D) Immunoblots of RNF185 levels after infection with sgRNF185 or sgNTC. (E) Flow cytometry analysis of SARS-CoV-2 envelope reporter stability following single-guide knockout of RNF185 across three different cell lines (∗ p value <0.05, ∗∗ p value <0.01).
Figure 3
Figure 3
Degradation of SARS-CoV-2 envelope protein is mediated by RNF185/TMEM259 complex (A) Fluorescent microscopy of HEK293T cells transiently transfected with iRFP720 tagged RNF185 and GFP tagged envelope proteins. (B) Flow cytometry analysis of CRISPR-Cas9 knocked out TMEM259 compared to a non-targeting control.
Figure 4
Figure 4
RNF185-mediated degradation of the envelope protein is seen in SARS-CoV-2 wild-type, clinical variants as well as SARS-CoV but not MERS (A) Flow cytometry analysis of degradation of clinical variants of SARS-CoV-2 envelope protein following infection of CRISPR guides targeting RNF185. (B) Sequence alignment of envelope protein for SARS-CoV-2, SARS-CoV, and MERS. (C) Flow cytometry analysis of degradation of SARS-CoV-2, SARS-CoV, and MERS envelope protein following infection of CRISPR guides targeting RNF185.
Figure 5
Figure 5
SARS-CoV-2 virus titers in A549-ACE2 cell line upon RNF185 depletion Virus titers were measured by detection of genomes in culture supernatants by PCR-based assay (∗ p value <0.05, ns – not significant). Titers are expressed as genome equivalents per mL of culture supernatant.
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