Nucleocapsid

For the newly identified virus genome, the potential ORFs were predicted and annotated using the conserved signatures of the cleavage sites recognized by coronavirus proteinases, and were processed in the Lasergene software package (v.7.1, DNAstar). The viral genes were aligned using the L-INS-i algorithm implemented in MAFFT (v.7.407)^{37 (link)}.
Phylogenetic analyses were then performed using the nucleotide sequences of various CoV gene datasets: (1) whole genome, (2) ORF1a, (3) ORF1b, (4) nsp5 (3CLpro), (5) RdRp (nsp12), (6) nsp13 (Hel), (7) nsp14 (ExoN), (8) nsp15 (NendoU), (9) nsp16 (O-MT), (10) spike (S) and (11) nucleocapsid (N). Phylogenetic trees were inferred using the maximum likelihood method implemented in the PhyML program (v.3.0)^{38 (link)}, using the generalized time reversible substitution model and subtree pruning and regrafting branch swapping. Bootstrap support values were calculated from 1,000 pseudo-replicate trees. The best-fitting model of nucleotide substitution was determined using MEGA (v.5)^{39 (link)}. Amino acid identities among sequences were calculated using the MegAlign program implemented in the Lasergene software package (v.7.1, DNAstar).

The day before infection, VeroE6 cells were seeded at 10⁴ cells/well into 96-well plates. Plasma samples and antibodies were serial diluted in BA-1, consisting of medium 199 (Lonza) supplemented with 1% BSA and 1× penicillin/streptomycin. Next, the diluted samples were mixed with a constant amount of SARS-CoV-2 and incubated for 60 min at 37°C. The plasma/antibody/virus mix was then directly applied to VeroE6 cells (MOI of ∼0.1 PFU/cell; n = 3) and incubated for 18–20 h at 37°C. This amount of virus gave 30% to 50% infected cells at the end of the assay. Cells were subsequently fixed by adding an equal volume of 7% formaldehyde to the wells, followed by permeabilization with 0.1% Triton X-100 for 10 min. After extensive washing, cells were incubated for 1 h at RT with blocking solution of 5% goat serum in PBS (Jackson ImmunoResearch; catalog no. 005–000-121). A rabbit polyclonal anti-SARS-CoV-2 nucleocapsid antibody (GeneTex; catalog no. GTX135357) was added to the cells at 1:500 dilution in blocking solution and incubated at 4°C overnight. Alternatively, J2, a mouse monoclonal anti-dsRNA antibody (Scicons; catalog no. 10010500) was added to the cells under similar conditions to detect virus infected cells. Goat anti-rabbit Alexa Fluor 594 (Life Technologies; catalog no. A-11012) and goat anti-mouse Alexa Fluor 488 (Life Technologies; catalog no. A-11001) were used as a secondary antibodies at a dilution of 1:2,000. Nuclei were stained with Hoechst 33342 (Thermo Fisher Scientific; catalog no. 62249) at a 1:1,000 dilution. Images were acquired with a fluorescence microscope and analyzed using ImageXpress Micro XLS (Molecular Devices). Using uninfected cells as a control, we estimated that infection could be reliably quantified if ∼0.7% of cells or more were infected.

The instructor provided to the students a brief introduction
to the most important features of the structure of SARS-CoV-2. The
four major structural proteins are displayed: the envelope (E), membrane
(M), nucleocapsid (N), and spike (S) proteins (Figure 1).^{7 (link)}It is highlighted that spike protein (approximately 180–200
kDa) is the surface glycoprotein anchored to the viral membrane that
plays an essential role when the infection process of SARS-CoV-2 takes
place. This protein is a trimer of three identical protomers (Figure 2). Each protomer
contains three segments: a short intracellular tail (IC), a transmembrane
anchor (TM), and a large ectodomain that extends outward from the
virus which is coated with sugar chains to hide the virus from the
immune system^{8 (link)} and comprises S1 and S2
subunits.
Next, the students are invited to study the ectodomain by analyzing
the requested structural features that they must observe manipulating
PyMOL.
Although hundreds of structures of this spike protein
are already
available in the Protein Data Bank, the one with the code 7DWY(9 (link)) has been selected and must be loaded in a PyMOL session.
They are encouraged to distinguish the four different levels of the
protein structures: primary, secondary, tertiary, and quaternary,
changing the representation of the molecule from lines or wireframe
to cartoon.
They must learn how to select individual residues
or different
chains, how to change their colors, how to generate objects, how to
show and hide different parts of the protein, how to measure distances
and angles for bonds, and how to generate surfaces.
They have
to realize that the spike protein is a complex of three
identical chains. A schematic illustration of the spike protein (Figure 3) is given to the
students, and they must recognize every single domain in the ectodomain,
extracting them as different objects and coloring them in the suggested
color.
The S1 subunit has an N-terminal
domain (NTD) and a receptor-binding
domain (RBD) located in the C-terminal domain, which is implied in
recognition and binding to the host cell receptor. S2 consists of
the fusion peptide (FP), two heptad repeats 1 (HR1 and HR2) which
operate the fusion of viral and host membranes, a transmembrane domain
(TM), and a cytoplasmic tail (CT).
When different species of
coronavirus are compared, the S2 subunit
is highly conserved, but the sequence of the S1 subunit varies greatly.
S1 and S2 are connected to the S1/S2 cleavage site in which specific
proteases act. The cleavage transforms the spike protein into a fusion
competent form that suffers several conformational changes and allows
it to anchor to the host membrane leading to the membrane fusion.^{10 (link)}