Development of Fluorescence-Tagged SARS-CoV-2 Virus-like Particles by a Tri-Cistronic Vector Expression System for Investigating the Cellular Entry of SARS-CoV-2

Abstract

Severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) has caused the pandemic that began late December 2019. The co-expression of SARS-CoV-2 structural proteins in cells could assemble into several types of virus-like particles (VLPs) without a viral RNA genome. VLPs containing S proteins with the structural and functional properties of authentic virions are safe materials to exploit for virus-cell entry and vaccine development. In this study, to generate SARS-CoV-2 VLPs (SCoV2-SEM VLPs) composed of three structural proteins including spike (S), envelop (E) protein and membrane (M) protein, a tri-cistronic vector expression system was established in a cell line co-expressing SARS-CoV-2 S, E and M proteins. The SCoV2-SEM VLPs were harvested from the cultured medium, and three structure proteins were confirmed by Western blot assay. A negative-stain TEM assay demonstrated the size of the SCoV2-SEM VLPs with a diameter of about 90 nm. To further characterize the infectious properties of SCoV2-SEM VLPs, the VLPs (atto647N-SCoV2-SEM VLPs) were fluorescence-labeled by conjugation with atto-647N and visualized under confocal microscopy at a single-particle resolution. The results of the infection assay revealed that atto647N-SCoV2-SEM VLPs attached to the surface of the HEK293T cells at the pre-binding phase in a ACE2-dependent manner. At the post-infection phase, atto647N-SCoV2-SEM VLPs either fused with the cellular membrane or internalized into the cytoplasm with mCherry-rab5-positive early endosomes. Moreover, fusion with the cellular membrane and the internalization with early endosomes could be inhibited by the treatment of camostat (a pharmacological inhibitor of TMPRSS2) and chlorpromazine (an endocytosis inhibitor), respectively. These results elucidated that SCoV2-SEM VLPs behave similarly to the authentic live SARS-CoV-2 virus, suggesting that the development of SCoV2-SEM VLPs provide a realistic and safe experimental model for studying the infectious mechanism of SARS-CoV-2.

Pseudotyping Improves the Yield of Functional SARS-CoV-2 Virus-like Particles (VLPs) as Tools for Vaccine and Therapeutic Development

“…Virus-like particles (VLPs) represent an attractive platform to serve a dual purpose in SARS-CoV-2 vaccine development, offering several unique advantages both as a vaccine candidate and as a replacement for live virus in assays. SARS-CoV-2 VLPs are commonly produced by co-expressing spike (S), envelope (E), and membrane (M) proteins in a host expression system [18][19][20][21]. They mimic the structure of the native SARS-CoV-2 virus but lack viral RNA and the ability to replicate.…”

Interchangeability of the Assays Used to Assess the Activity of Anti-SARS-CoV-2 Monoclonal Antibodies

“…A wide variety of pseudotyped virus particles and cell lines have been used to assess the potency of anti-SARS-CoV-2 mAbs with antiviral activity. Commonly used reagents include retroviral (HIV-1 or murine leukemia virus (MLV)-derived) or rhabdoviral (VSV)-based packaging vector systems and immortalized human cells (embryonic kidney 293T or human lung carcinoma epithelial (A549) cells) expressing hACE2 with or without co-expression of TMPRSS2 [ 9 , 17 , 18 ].…”