Research on infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) is currently restricted to BSL-3 laboratories. SARS-CoV2 virus-like particles (VLPs) offer a BSL-1, replication-incompetent system that can be used to evaluate virus assembly and virus-cell entry processes in tractable cell culture conditions. Here, we describe a SARS-CoV2 VLP system that utilizes nanoluciferase (Nluc) fragment complementation to track assembly and entry. We utilized the system in two ways. Firstly, we investigated the requirements for VLP assembly. VLPs were produced by concomitant synthesis of three viral membrane proteins, spike (S), envelope (E), and matrix (M), along with the cytoplasmic nucleocapsid (N). We discovered that VLP production and secretion were highly dependent on N proteins. N proteins from related betacoronaviruses variably substituted for the homologous SARS-CoV2 N, and chimeric betacoronavirus N proteins effectively supported VLP production if they contained SARS-CoV2 N carboxy-terminal domains (CTD). This established the CTDs as critical features of virus particle assembly. Secondly, we utilized the system by investigating virus-cell entry. VLPs were produced with Nluc peptide fragments appended to E, M, or N proteins, with each subsequently inoculated into target cells expressing complementary Nluc fragments. Complementation into functional Nluc was used to assess virus-cell entry. We discovered that each of the VLPs were effective at monitoring virus-cell entry, to various extents, in ways that depended on host cell susceptibility factors. Overall, we have developed and utilized a VLP system that has proven useful in identifying SARS-CoV2 assembly and entry features.
【저자키워드】 COVID-19, Release, coronavirus, SARS-CoV2, MERS-CoV, nucleocapsid, D614G, virus-like particles, MHV, virus assembly, HiBiT, LgBiT, NanoBiT technology, 【초록키워드】 coronavirus, susceptibility, peptide, Proteins, virus, Betacoronavirus, Viral, Features, N protein, Factors, envelope, respiratory, homologous, Critical, VLP, assembly, complementary, chimeric, target cells, host cell, target cell, matrix, acute respiratory syndrome, Virus-like particle, acute respiratory syndrome coronavirus, viral membrane, secretion, domain, nLuc, N proteins, offer, VLPs, Complementation, feature, effective, BSL-3, produced, was used, evaluate, inoculated, investigated, supported, can be used, functional, cytoplasmic, dependent on, expressing, CTD, cell culture conditions, 【제목키워드】 coronavirus, coronavirus 2, Particle, respiratory,