The use of hepatitis C virus NS3/4A and secreted alkaline phosphatase to quantitate cell–cell membrane fusion mediated by severe acute respiratory syndrome coronavirus S protein and the receptor angiotensin-converting enzyme 2

Abstract

The membrane fusion process mediated by severe acute respiratory syndrome coronavirus (SARS-CoV) S protein and its cellular receptor angiotensin-converting enzyme 2 (ACE2) had been reconstituted using two Chinese hamster ovary (CHO) cell lines that constitutively express these recombinant proteins separately. This system was applied to develop a quantitative measurement of cell-cell fusion using hepatitis C virus (HCV) NS3/4A protease and a secretion-blocked EGFP-4A/4B-SEAP (EGFP: enhanced green fluorescent protein; 4A/4B: a decapeptide substrate of NS3/4A protease; SEAP: secreted alkaline phosphatase) fusion gene. Both genes were transiently expressed in either of the CHO cell lines, followed by fusion treatment. Significant SEAP activity could be detected in the culture medium only after cell-cell fusion occurred. Cell-cell fusion provides an environment in which the protease encounters its substrate 4A/4B, thereby releasing SEAP from the fusion protein. In this study, we developed a simple, sensitive, and quantitative assay to study the membrane fusion process by measuring the extracellular activity of SEAP.

Development of NS3/4A Protease-Based Reporter Assay Suitable for Efficiently Assessing Hepatitis C Virus Infection

“…This may be due to the inclusion of the DEDEDEDE peptide in Iro's study. Chou et al demonstrated that the DEDEDEDE residues prior to the natural 4A/4B cleavage markedly enhanced the cleavage efficiency (6). However, we cannot exclude the possibility that there are different copy numbers of chromosomally integrated fusion reporter genes in selected reporter cell clones.…”

Antimyogenic Effect of SARS-CoV Spike Protein in C2C12 Myoblasts

“…C2C12-SG cell line, cultured in DM did not form myotubes where as cell line C2C12-E2G [5] expressing ectodomain of Hepatitis C virus E2 protein showed normal myogenesis (SD1 Fig. 1).…”

“…C2C12-SG and C2C12-E2G cell lines were established as previously reported [5]. Plasmid pAM (SD1) containing Myod, PLFR, ZAKI-4R and ACE2R was transfected into C2C12-SG or C2C12 cells using lipofectamine (Invitrogen).…”

“…Plasmid pAM (SD1) containing Myod, PLFR, ZAKI-4R and ACE2R was transfected into C2C12-SG or C2C12 cells using lipofectamine (Invitrogen). Stable transfectants C2C12-ACE2R were selected on Hygromycin B medium while C2C12-SG-MyoD, C2C12-SG-PLFR and C2C12-SG-ZAKI-4R were selected on medium containing both G418 and Hygromycin B. C2C12-SG derivative cell lines were established by pooling maximum of 10 colonies and surface expression of S protein was observed using cell-cell fusion assay [5].…”

“…C2C12 myoblasts are extensively used to study differentiation of skeletal myogenesis [4]. We have previously established a method for quantifying the cell-cell fusion mediated by S-ACE2 interaction [5], C2C12 cell line stably expressing S (C2C12-SG) was used to confirm the process. The key stage of myogenesis involves the fusion of myoblasts to form myotubes.…”