P VKOVSKI¹, V GASCHEN², M STOFFEL², V THIEL<sup>3

1</sup>Federal Institute of Virology and Immunology, Mittelhäusern and Bern, Switzerland; Graduate School for Cellular and Biomedical Sciences, University of Bern, Switzerland, ²Vetsuisse Faculty, Division Veterinary Anatomy, University of Bern, Bern, Switzerland., ³Federal Institute of Virology and Immunology, Mittelhäusern and Bern, Switzerland; Vetsuisse Faculty, Department of Infectious Diseases and Pathobiology, University of Bern, Bern, Switzerland.

Coronaviruses are positive-sense RNA viruses known to extensively remodel intracellular membranes and induce double-membrane vesicles (DMVs) that support viral RNA synthesis. Transmembrane non-structural proteins 3, nsp4 and nsp6 play an essential role in diverting ER-derived membranes and anchoring the viral replication complex to modified membranes. The potent human CoV inhibitor K22 specifically prevents the formation of DMVs and viral RNA synthesis. Remarkably, K22-resistant hCoV-229E viruses containing substitutions in nsp6 are able to form DMVs, although the latter are reduced in number and structurally impaired.
Our results describe the effects of hCoV-229E nsp6 and nsp6 K22-resistant mutant (nsp6*) on membrane modifications using both immunofluorescence microscopy and electron microscopy (EM). Monitoring of nsp6 expression in MRC5 cells results in a punctate perinuclear accumulation comparable to viral replication complexes. In contrast, K22 treatment prevents accumulation and nsp6 distributes homogenously in the cytosol. Interestingly, K22-resistant nsp6* evenly distributes in the cytosol both in presence or absence of K22. At the ultrastructural level, nsp6 induces massive membrane rearrangements resulting in vesicular structures accumulated in the nucleus vicinity. However, nsp6* is unable to induce such structures. Rather, ER compartments appear highly dilated as a consequence of nsp6* expression. Both phenotypes are disrupted or modulated by the addition of K22. We are currently processing our samples for immuno-EM in order to assess the location of nsp6 and nsp6* constructs to modified membranes.
In addition to hCoV-229E, K22 was also shown to efficiently inhibit the replication of SARS-CoV, MERS-CoV and IBV. Nsp6 constructs of these additional viruses, which display high conservation of their amino-acid sequences, were subjected to IF and EM analyses to determine the membrane remodeling properties of homologous nsp6 proteins. Moreover, the assessment of homologous nsp6 sensitivities towards K22 will establish whether K22 supposedly inhibits CoV replication by interfering with functions of nsp6.