The SARS-CoV-2 Envelope and Membrane proteins modulate maturation and retention of the Spike protein, allowing optimal formation of VLPs in presence of Nucleoprotein
cell biology molecular biology virology
First Author: Boson et al.
Journal/preprint name: Bioarchives
Paper DOI: https://doi.org/10.1101/2020.08.24.260901
Tags: Virology, Cellular biology, Molecular biology
Boson et al. characterised how the intracellular trafficking and maturation of the SARS-CoV-2 S protein are regulated by the other structural proteins E, M and N. Co-expression of E or M were found to decrease S cleavage, alter the N-glycosylation patterns in the S2 subunit and to retain S protein in the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) or cis-Golgi, known CoV assembly sites. The S protein expressed alone was found widely distributed within the cell and to promote syncytia, which was prevented with SARS-CoV-2 infection or co-expression with E or M. In contrast, N protein does not appear to have any effect on S maturation or localisation. While E protein was found to influence the level of S expression by inducing a non-specific retention of glycoproteins in the ER and to slow down the cell secretory pathway; M influenced S localisation through direct interaction with S C-terminal moiety. Finally, all structural proteins proved essential for VLP formation.
SARS-CoV-2 E and M proteins alter S cleavage, maturation and intracellular trafficking.
SARS-CoV-2 E and M proteins promote S retention at ERGIC or cis-Golgi compartments by different mechanisms and induce furin-mediated processing independently of its mechanisms of intracellular retention.
SARS-CoV-2 E protein induces an ER non-specific retention of glycoproteins and slows down the cell secretory pathway
M-mediated retention at the ERGIC requires the C-terminal motif in the S cytoplasmic tail.
SARS-CoV-2 M, E and N are required for VLP formation.
Impact for COVID-19 research:
Understand the virology and cell biology of SARS-CoV2/COVID19
Study Type: in vitro
Important cell lines/viral models used:
Homo sapiens codon optimized SARS-CoV-2 S (Wuhan-Hu-1, GenBank: QHD43419.1) was cloned into pVAX1 vector. The delta 19 truncation of S form was generated by site directed mutagenesis introducing a stop codon after Cys1254. SARS-CoV-2 E, M and N genes 405 (Wuhan-Hu-1, GenBank: QHD43419.1) were synthesized and cloned into pCDNA3.1(+) vectors; VeroE6, 293T, temperature-dependent folding mutant of VSV-G, Huh-7.5 cells, hepatitis C virus (HCV) p7.
Key Techniques: Transfection, Endo-H assessment for intracellular trafficking, western blot, flow cytometry, Immunofluorescence and confocal microscopy, cell-to-cell fusion assay.
There is no housekeeping protein in the western blots (GAPDH, beta-tubulin and beta-actin) in Figure 1 and 2.
In Figure 1, 3B there is no indication of how many times the experiment was performed.
No other marker for any other cell localisation (as for negative control) is shown in the immunofluorescent studies.