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Construction of SARS-CoV-2 Virus-Like Particles by Mammalian Expression System.

ABSTRACT: Virus-like particle (VLP) is a self-assembled nanostructure incorporating key viral structural proteins. VLP resembles molecular and morphological features of authentic viruses but is non-infectious and non-replicating due to lack of genetic materials. Successful applications of VLP has been shown in vaccinological and virological research. As an accessibly safe and relevant substitute of naturally pathogenic viruses, the construction of SARS-CoV-2 VLPs is much in demand in the ongoing fight against 2019 Coronavirus disease (COVID-19) pandemics. In the current study, using mammalian expression system, which is advantageous in maintaining correct protein glycosylation patterns, we efficiently constructed SARS-CoV-2 VLPs. We showed that among four SARS-CoV-2 structural proteins, expression of membrane protein (M) and small envelope protein (E) are essential for efficient formation and release of SARS-CoV-2 VLPs. Moreover, the corona-like structure presented in SARS-CoV-2 VLPs from Vero E6 cells is more stable and unified, as compared to those from HEK-293T cells. Our data demonstrate that SARS-CoV-2 VLPs possess molecular and morphological properties of native virion particles, which endow such VLPs with a promising vaccine candidate and a powerful tool for the research of SARS-CoV-2.

SUBMITTER: Xu R

PROVIDER: S-EPMC7409377 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Corrigendum: Construction of SARS-CoV-2 Virus-Like Particles by Mammalian Expression System.

Project description:[This corrects the article DOI: 10.3389/fbioe.2020.00862.].

| S-EPMC7509488 | biostudies-literature

Construction of SARS-CoV-2 virus-like particles in plant.

Project description:The pandemic of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has caused a public health emergency, and research on the development of various types of vaccines is rapidly progressing at an unprecedented development speed internationally. Some vaccines have already been approved for emergency use and are being supplied to people around the world, but there are still many ongoing efforts to create new vaccines. Virus-like particles (VLPs) enable the construction of promising platforms in the field of vaccine development. Here, we demonstrate that non-infectious SARS-CoV-2 VLPs can be successfully assembled by co-expressing three important viral proteins membrane (M), envelop (E) and nucleocapsid (N) in plants. Plant-derived VLPs were purified by sedimentation through a sucrose cushion. The shape and size of plant-derived VLPs are similar to native SARS-CoV-2 VLPs without spike. Although the assembled VLPs do not have S protein spikes, they could be developed as formulations that can improve the immunogenicity of vaccines including S antigens, and further could be used as platforms that can carry S antigens of concern for various mutations.

| S-EPMC8770512 | biostudies-literature

Minimal system for assembly of SARS-CoV-2 virus like particles.

Project description:SARS-CoV-2 virus is the causative agent of COVID-19. Here we demonstrate that non-infectious SARS-CoV-2 virus like particles (VLPs) can be assembled by co-expressing the viral proteins S, M and E in mammalian cells. The assembled SARS-CoV-2 VLPs possess S protein spikes on particle exterior, making them ideal for vaccine development. The particles range in shape from spherical to elongated with a characteristic size of 129 ± 32 nm. We further show that SARS-CoV-2 VLPs dried in ambient conditions can retain their structural integrity upon repeated scans with Atomic Force Microscopy up to a peak force of 1 nN.

| S-EPMC7736577 | biostudies-literature

Construction of a baculovirus-silkworm multigene expression system and its application on producing virus-like particles.

Project description:A new baculovirus-silkworm multigene expression system named Bombyx mori MultiBac is developed and described here, by which multiple expression cassettes can be introduced into the Bombyx mori nuclear polyhedrosis virus (BmNPV) genome efficiently. The system consists of three donor vectors (pCTdual, pRADM and pUCDMIG) and an invasive diaminopimelate (DAP) auxotrophic recipient E. coli containing BmNPV-Bacmid (BmBacmid) with a homologous recombination region, an attTn7 site and a loxp site. Two genes carried by pCTdual are firstly inserted into BmBacmid by homologous recombination, while the other eight genes in pRADM and pUCDMIG are introduced into BmBacmid through Tn7 transposition and cre-loxp recombination. Then the invasive and DAP auxotrophic E. coli carrying recombinant BmBacmid is directly injected into silkworm for expressing heterologous genes in larvae or pupae. Three structural genes of rotavirus and three fluorescent genes have been simultaneously expressed in silkworm larvae using our new system, resulting in the formation of virus-like particles (VLPs) of rotavirus and the color change of larvae. The VLPs were purified from hemolymph by ultracentrifugation using CsCl gradients, with a yield of 12.7 µg per larva. For the great capacity of foreign genes and the low cost of feeding silkworm, this high efficient BmMultiBac expression system provides a suitable platform to produce VLPs or protein complexes.

| S-EPMC3293821 | biostudies-literature

Tilt series of SARS-CoV-2 spike-bearing virus-like particles (VLPs) interacting with hACE2-bearing extracellular vesicles (tEVs), showing various intermediate states of the SARS-CoV-2 spike protein

Project description: Not available

| EMPIAR-10788 | biostudies-other

Cetylpyridinium chloride promotes disaggregation of SARS-CoV-2 virus-like particles.

Project description:BackgroundSARS-CoV-2 is continuously disseminating worldwide. The development of strategies to break transmission is mandatory.Aim of the studyTo investigate the potential of cetylpyridinium chloride (CPC) as a viral inhibitor.MethodsSARS-CoV-2 Virus Like-Particles (VLPs) were incubated with CPC, a potent surfactant routinely included in mouthwash preparations.ResultsConcentrations of 0.05% CPC (w/v) commonly used in mouthwash preparations are sufficient to promote the rupture of SARS-CoV-2 VLP membranes.ConclusionIncluding CPC in mouthwashes could be a prophylactic strategy to keep SARS-CoV-2 from spreading.

| S-EPMC8788378 | biostudies-literature

Production of SARS-CoV-2 Virus-Like Particles in Insect Cells.

Project description:Coronavirus disease (COVID-19) causes a serious threat to human health. Virus-like particles (VLPs) constitute a promising platform in SARS-CoV-2 vaccine development. In this study, the E, M, and S genes were cloned into multiple cloning sites of a new triple expression plasmid with one p10 promoter, two pPH promoters, and three multiple cloning sites. The plasmid was transformed into DH10 BacTMEscherichia coli competent cells to obtain recombinant bacmid. Then the recombinant bacmid was transfected in ExpiSf9TM insect cells to generate recombinant baculovirus. After ExpiSf9TM cells infection with the recombinant baculovirus, the E, M, and S proteins were expressed in insect cells. Finally, SARS-CoV-2 VLPs were self-assembled in insect cells after infection. The morphology and the size of SARS-CoV-2 VLPs are similar to the native virions.

| S-EPMC8227081 | biostudies-literature

Plant-based expression and characterization of SARS-CoV-2 virus-like particles presenting a native spike protein.

Project description:We have investigated the use of transient expression to produce virus-like particles (VLPs) of severe acute respiratory syndrome coronavirus 2, the causative agent of COVID-19, in Nicotiana benthamiana. Expression of a native form of the spike (S) protein, either alone or in combination with the envelope (E) and membrane (M) proteins, all of which were directed to the plant membranes via their native sequences, was assessed. The full-length S protein, together with degradation products, could be detected in total protein extracts from infiltrated leaves in both cases. Particles with a characteristic 'crown-shaped' or 'spiky' structure could be purified by density gradient centrifugation. Enzyme-linked immunosorbent assays using anti-S antibodies showed that threefold higher levels of VLPs containing the full-length S protein were obtained by infiltration with S alone, compared to co-infiltration of S with M and E. The S protein within the VLPs could be cleaved by furin in vitro and the particles showed reactivity with serum from recovering COVID-19 patients, but not with human serum taken before the pandemic. These studies show that the native S protein expressed in plants has biological properties similar to those of the parent virus. We show that the approach undertaken is suitable for the production of VLPs from emerging strains and we anticipate that the material will be suitable for functional studies of the S protein, including the assessment of the effects of specific mutations. As the plant-made material is noninfectious, it does not have to be handled under conditions of high containment.

| S-EPMC9115404 | biostudies-literature

Real-Time Conformational Dynamics of SARS-CoV-2 Spikes on Virus Particles.

Project description:The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) mediates viral entry into cells and is critical for vaccine development against coronavirus disease 2019 (COVID-19). Structural studies have revealed distinct conformations of S, but real-time information that connects these structures is lacking. Here we apply single-molecule fluorescence (Förster) resonance energy transfer (smFRET) imaging to observe conformational dynamics of S on virus particles. Virus-associated S dynamically samples at least four distinct conformational states. In response to human receptor angiotensin-converting enzyme 2 (hACE2), S opens sequentially into the hACE2-bound S conformation through at least one on-path intermediate. Conformational preferences observed upon exposure to convalescent plasma or antibodies suggest mechanisms of neutralization involving either competition with hACE2 for binding to the receptor-binding domain (RBD) or allosteric interference with conformational changes required for entry. Our findings inform on mechanisms of S recognition and conformations for immunogen design.

| S-EPMC7664471 | biostudies-literature

Structural stability of SARS-CoV-2 virus like particles degrades with temperature.

Project description:SARS-CoV-2 is a novel coronavirus which has caused the COVID-19 pandemic. Other known coronaviruses show a strong pattern of seasonality, with the infection cases in humans being more prominent in winter. Although several plausible origins of such seasonal variability have been proposed, its mechanism is unclear. SARS-CoV-2 is transmitted via airborne droplets ejected from the upper respiratory tract of the infected individuals. It has been reported that SARS-CoV-2 can remain infectious for hours on surfaces. As such, the stability of viral particles both in liquid droplets as well as dried on surfaces is essential for infectivity. Here we have used atomic force microscopy to examine the structural stability of individual SARS-CoV-2 virus like particles at different temperatures. We demonstrate that even a mild temperature increase, commensurate with what is common for summer warming, leads to dramatic disruption of viral structural stability, especially when the heat is applied in the dry state. This is consistent with other existing non-mechanistic studies of viral infectivity, provides a single particle perspective on viral seasonality, and strengthens the case for a resurgence of COVID-19 in winter.

| S-EPMC7699159 | biostudies-literature

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