Following translation, OPG2-tagged translation products (i.e. inhibited by Ipom-F, whilst several other viral membrane proteins are unaffected (Fig.?2). Similarly, the ER integration of ACE2, an important host receptor for SARS-CoV-2 (Walls et al., 2020), is usually highly sensitive to Ipom-F (Fig.?2). Open in a separate windows Fig. 2. Ipom-F selectively inhibits the ER membrane translocation of SARS-CoV-2 proteins. (A) Schematic of ER import assay using pancreatic microsomes. Following translation, fully translocated or membrane-inserted radiolabelled precursor proteins are recovered and analysed by SDSCPAGE and phosphorimaging. N-glycosylated species were confirmed by treatment with endoglycosidase H (Endo H). (B) Protein precursors of the human angiotensin-converting enzyme 2 (ACE2) and OPG2-tagged versions of the SARS-CoV-2 ORF8 (ORF8COPG2), spike (SCOPG2), envelope (OPG2CE), membrane (MCOPG2) and ORF6 (a doubly OPG2-tagged version, OPG2CORF6COPG2, and two singly OPG2-tagged forms, OPG2CORF6 and ORF6COPG2, with predominant N-glycosylated species in strong) were synthesised in rabbit reticulocyte lysate (RRL) supplemented with ER microsomes without or with Ipom-F (lanes 1 and 3). Phosphorimages of membrane-associated products resolved by SDSCPAGE with representative substrate outlines are shown. N-glycosylation was used to measure the efficiency of membrane translocation or insertion, and N-glycosylated (values shown in the physique) was decided using Dunnett’s multiple comparisons test. ****studies of SARS-CoV-2 protein synthesis at the ER spotlight Ipom-F as a encouraging candidate for the development of a broad-spectrum, host-targeting antiviral agent. Open in a separate windows Fig. 3. SARS-CoV-2 proteins are variably dependent on the Sec61 complex and/or the EMC for ER membrane translocation or insertion. (A) Schematic of ER import assay using control SP cells, or those depleted of a subunit of the Sec61 complex and/or the EMC via siRNA treatment. Following translation, OPG2-tagged translation products (i.e. membrane-associated and non-targeted nascent chains) were immunoprecipitated, resolved by SDSCPAGE and analysed by phosphorimaging. OPG2-tagged variants of the SARS-CoV-2 (B) spike (SCOPG2), (C) ORF8 (ORF8COPG2), (D) envelope (OPG2CE) and (E) ORF6 (OPG2CORF6COPG2) species (labelled as for Fig.?2) were synthesised in rabbit reticulocyte lysate (RRL) supplemented with control SP cells (lanes 1,2) or SP cells with impaired Sec61 and/or EMC function (lanes 3C6). Radiolabelled products were recovered and analysed as in A. Membrane translocation/insertion efficiency was decided using the ratio of RIP2 kinase inhibitor 1 the N-glycosylation of lumenal domains, recognized using Endo H (EH, lane 1), relative to the non-targeting (NT) control (set to 100% translocation/insertion efficiency; dashed lines). Quantitations (means.e.m.; values shown in the physique) determined as for Fig.?2. ****translation system supplemented with canine pancreatic microsomes (Fig.?2A). To facilitate the detection of ER translocation, we altered the viral ORF8, S, envelope (E), membrane (M) and ORF6 proteins by adding an OPG2 tag C an epitope that supports efficient ER lumenal N-glycosylation and enables product recovery via immunoprecipitation, without affecting Ipom-F sensitivity (Fig.?S1A and data not shown). For viral proteins that lack endogenous sites for N-glycosylation, such as the E protein, the ER lumenal OPG2 tag functions as a reporter for ER translocation and enables their recovery by immunoprecipitation. Where viral proteins already contain suitable sites for N-glycosylation (S and M proteins), the cytosolic OPG2 tag is used solely for immunoprecipitation. The identity of the producing N-glycosylated species for each of these OPG2-tagged viral proteins was confirmed by endoglycosidase H (Endo H) treatment of the radiolabelled products associated with the membrane portion prior to SDSCPAGE (Fig.?2B, lanes 1 and 2 in each panel). Using ER lumenal modification of either endogenous N-glycosylation sites (viral S and M proteins) or the appended OPG2 tag (viral E and ORF8 proteins) as a reporter for ER membrane translocation, we found that 1?M Ipom-F strongly inhibited both the translocation of the soluble, secretory-like protein ORF8COPG2 and the integration of the type I transmembrane protein (TMP) SCOPG2, and truncated derivatives thereof (Fig.?2B,C; Fig.?S1C). Furthermore, membrane insertion of the human type I TMP, ACE2, was inhibited to a similar extent (Fig.?2B,C; 70C90% inhibition for these three proteins). These results mirror previous findings showing that precursor proteins bearing N-terminal transmission peptides, and which are therefore obligate clients for the Sec61-translocon, are typically very sensitive to Ipom-F-mediated inhibition.Statistical significance is usually given as n.s., non-significant em P /em 0.1; * em P /em 0.05; ** em P /em 0.01; *** em P /em 0.001; **** em P /em 0.0001. Supplementary Material Supplementary information:Click here to view.(930K, pdf) Reviewer feedback:Click here to view.(189K, pdf) Acknowledgements We thank Quentin Roebuck for technical assistance, Nevan Krogan (UCSF) for SARS-CoV-2 plasmids, Sven Lang (University or college of Saarland) for Sec61 antisera, and Belinda Hall RIP2 kinase inhibitor 1 and Rachel Simmonds (University or college of Surrey) for useful discussions. insertion of the viral spike (S) protein and membrane translocation of the ORF8 protein are both strongly inhibited by Ipom-F, whilst several other viral membrane proteins are unaffected (Fig.?2). Similarly, the ER integration of ACE2, an important host receptor for SARS-CoV-2 (Walls et al., 2020), is usually highly sensitive to Ipom-F (Fig.?2). Open in a separate windows Fig. 2. Ipom-F selectively inhibits the ER membrane translocation of SARS-CoV-2 proteins. (A) Schematic of ER import assay using pancreatic microsomes. Following translation, fully translocated or membrane-inserted radiolabelled precursor proteins are recovered and analysed by SDSCPAGE and phosphorimaging. N-glycosylated species were confirmed by treatment with endoglycosidase H (Endo H). (B) Protein precursors of the human angiotensin-converting enzyme 2 (ACE2) and OPG2-tagged versions of the SARS-CoV-2 ORF8 (ORF8COPG2), spike (SCOPG2), envelope (OPG2CE), membrane (MCOPG2) and ORF6 (a doubly OPG2-tagged version, OPG2CORF6COPG2, and two singly OPG2-tagged forms, OPG2CORF6 and ORF6COPG2, with predominant N-glycosylated species in strong) were synthesised in rabbit reticulocyte lysate (RRL) supplemented with ER microsomes without or with Ipom-F (lanes 1 and 3). Phosphorimages of membrane-associated products resolved by SDSCPAGE with representative substrate outlines are shown. N-glycosylation was used to measure the efficiency of membrane translocation or insertion, and N-glycosylated (values shown in the physique) was decided using Dunnett’s multiple comparisons test. ****studies of SARS-CoV-2 protein synthesis at the ER spotlight Ipom-F as a encouraging candidate for the development of a broad-spectrum, host-targeting antiviral agent. Open in a separate windows Fig. 3. SARS-CoV-2 proteins are variably dependent on the Sec61 complex and/or the EMC for ER membrane translocation or insertion. (A) Schematic of ER import assay using control SP cells, or those depleted of a subunit of the Sec61 complex and/or the EMC via siRNA treatment. Following translation, OPG2-tagged translation products (i.e. membrane-associated and non-targeted nascent chains) were immunoprecipitated, resolved by SDSCPAGE and analysed by phosphorimaging. OPG2-tagged variants of the SARS-CoV-2 (B) spike (SCOPG2), (C) ORF8 (ORF8COPG2), (D) envelope (OPG2CE) RIP2 kinase inhibitor 1 and (E) ORF6 (OPG2CORF6COPG2) species (labelled as for Fig.?2) were synthesised in rabbit reticulocyte lysate (RRL) supplemented with control SP cells (lanes 1,2) or SP cells with impaired Sec61 and/or EMC function (lanes 3C6). Radiolabelled products were recovered and analysed as in A. Membrane translocation/insertion efficiency was decided using the ratio of the N-glycosylation of lumenal domains, recognized using Endo H (EH, lane 1), relative to the non-targeting (NT) control (set to 100% translocation/insertion efficiency; dashed lines). Quantitations (means.e.m.; values shown in the physique) determined as for Fig.?2. ****translation system supplemented with canine pancreatic microsomes (Fig.?2A). To facilitate the detection of ER translocation, we altered the viral ORF8, S, envelope (E), membrane (M) and ORF6 proteins by adding an OPG2 tag C an epitope that supports efficient ER lumenal N-glycosylation and enables product recovery via immunoprecipitation, without affecting Ipom-F sensitivity (Fig.?S1A and data not shown). For viral proteins that lack endogenous sites for N-glycosylation, such as the E protein, the ER lumenal OPG2 tag RIP2 kinase inhibitor 1 acts as a reporter for ER translocation and enables their recovery by immunoprecipitation. Where NEK3 viral proteins already contain suitable sites for N-glycosylation (S and M proteins), the cytosolic OPG2 tag is used solely for immunoprecipitation. The identity of the resulting N-glycosylated species for each of RIP2 kinase inhibitor 1 these OPG2-tagged viral proteins was confirmed by endoglycosidase H (Endo H) treatment of the radiolabelled products associated with the membrane fraction prior to SDSCPAGE (Fig.?2B, lanes 1 and 2 in each panel). Using ER lumenal modification of either endogenous N-glycosylation sites (viral S and M proteins) or the appended OPG2 tag (viral E and ORF8 proteins) as a reporter for ER membrane translocation, we found that 1?M Ipom-F strongly inhibited both the translocation of the soluble, secretory-like protein ORF8COPG2 and the integration of the type I transmembrane protein (TMP) SCOPG2, and truncated derivatives thereof (Fig.?2B,C; Fig.?S1C). Furthermore, membrane insertion of the human type I TMP, ACE2, was inhibited to a similar extent (Fig.?2B,C; 70C90% inhibition for these three proteins)..