Our scientists have published another Cell paper! The effects of SARS-CoV-2 synaptic protein mutation on viral infection and antigen are analyzed in detail.

!--webeditor:page title"--/--- COVID-19 pandemic is a huge threat worldwide.
As of July 3, 2020, 216 countries have reported CASES OF COVID-19, with more than 10 million confirmed cases and about 518,000 deaths, according to the World Health Organization (WHO).
as a pathogen of COVID-19, SARS-CoV-2 causes lower respiratory tract infections that can progress towardsevere acute respiratory syndrome and even multiple organ failure.
SARS-CoV-2 is a single-stranded positive-stranded RNA virus whose genome encodes four structural proteins: stingprotein (S), small protein (E), matrix protein (M), and nuclear shell protein (N).
S protein is a type I fusion protein that forms a tripolymer on the surface of viral particles.
it consists of two sub-bases: S1 is responsible for receptor binding and S2 is responsible for membrane fusion.
SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) as a receptor to enter the target cell.
therefore, the S protein determines the infection of the virus and its transmission in the host.
because this protein is the main antigen that induces a protective immune response, all the vaccines being developed are targeted at it.
clearly, it is essential to closely monitor the antigen changes of S proteins in the virus being transmitted.
Given that it is a highly glycogenized protein, it is also of unquestionable importance to study the effects of site-specific polysaccharides on infection and immune escape.
known RNA virus mutation rate is higher than that of DNA virus.
changes in amino acids in surface proteins can significantly alter viral function and/or interactions with neutralizing antibodies.
, for example, the A226V mutation in the Chikungunya e1 protein promotes the virus's adaptability in the Aedes aegypti mosquito, leading to increased transmission. similarly
, the A82V mutation in the Ebola virus GP protein led to increased viral infection and mortality.
, the four amino acid changes in the highly pathogenic avian influenza virus H5N1 showed increased transmission, while in H7N9 virus, changes in the combination amino acids of A143V/R148K in hemagglutinin reduced the sensitivity of the virus to neutralizing antibodies by more than 10 times.
although SARS-CoV-2 has only recently been found in humans, genetic mutations that encode the S protein are being reported.
as of May 6, 2020, 329 natural variants of S protein have been reported in the public domain.
note, only 13 amino acid sites had mutation rates of more than 0.1%. preliminary studies
suggest that the increase in mortality may be related to the most important D614G mutation.
it is speculated that this mutation may induce conformational changes in the S protein, leading to increased infection.
However, it is not clear whether these reported mutants will affect the infection, transmission, or responsiveness of the virus with neutralizing antibodies.
, mutations that affect viral protein glycogenization are also well documented, and these mutations can also profoundly affect the life cycle of the virus and its interaction with the host.
, for example, N-glycosylation at specific sites of the HIV-1 Env protein is essential for the expression and assembly of Env.
the absence of certain glycogenized sites can reduce the binding of Env proteins to CD4 receptors, thereby disrupting the infectiousness of the resulting viral particles.
, the mutation of glycosylation site mutations can also make the neutralization of viral opponents resistant, while the absence of certain glycogenized sites in H5N1 hemoglobin has been found to affect the cutting, replication, stability and antigen of hemagglutin.
it is worth noting that although the S protein of SARS-CoV-2 has a higher degree of glycogenization and 22 potential N-glycogenization sites, it is still unknown how these glycogenization sites affect viral infection sofa and antibody-mediated neutralization. in a new study
, researchers from the China Food and Drug Research Institute, Beijing Concord College of Medicine and Tsinghua University studied the biological significance of natural variants of amino acid changes in the SARS-CoV-2 S protein, as well as mutants that have amino acid changes at the presumed N-linked glycogenized site. to achieve this
, they constructed 106 S-protein mutants reported in the public domain or those with amino acid changes at the presumed N-linked glycosylation site, and used a high-volume pseudotyped virus system to analyze their infection and responsiveness to neutralizing antibodies.
they report that some natural variants and glycogen mutants have evolved significant infectious and antigen changes.
related findings were published online July 17, 2020 in the journal Cell, with the title "The Impact of the Case of The S. And s.
the authors of the paper are Dr. Youchun Wang and Dr. Weijin Huang of the China Food and Drug Inspection Research Institute.
Figure 1. Description of amino acid changes selected for this study, pictured from Cell, 2020, doi: 10.1016/j.cell.2020.07.012.
using the SARS-CoV-2 strain Wuhan-1 (GenBank: MN908947) as a template, the authors selected three sets of natural variants and glycosylized mutants to build a pseudovirus.
as shown in Figure 1, Group A represents all high-frequency variants of the entire S gene (29 strains) and combination variants with D614G, except in the receptor-binding domain (RBD) region.
Group B includes variants (51 strains) that appear in the RBD.
although eight single mutations in Group A and Group B, namely Q239K, V341I, A435S, K458R, I472V, H519P, A831V and S943T, were found to be present in combination with D614G.
, a pseudo-virus for these eight single-point mutations was also constructed to compare it with the pseudo-virus esconstructed for their two-spot combination mutation with D614G.
Group C consists of 26 mutants with amino acid changes in the presumed glycogenization sites (22 sites), including variants (N74K, N149H, and T719A) and experimental mutants constructed by the authors to analyze the effects on glycogenization.
specifically, 22 single-point mutants were constructed in the lab for all 22 sites (N to Q);
overall, they have built 106 pseudo-viruses, 80 natural variants and 26 glycogenized mutants (Figure 1).
!--/ewebeditor:!--webeditor: !--:page title" - To determine the infectiousness of these natural variants and glycogenized mutants, the authors initially infected 26 cell lines with pseudo-viruses that carry the SARS-CoV-2 S protein or VSV G protein.
, the two pseudo-viruses differ in infection between the 26 cell lines (Figure 2).
, however, they eventually selected four cell lines --- 293T-hACE2, Huh-7, Vero and LLC-MK2 --- for experiments.
use 106 pseudo-viruses to infect these cell lines, lyse infected cell lines, dilute the lysate of the infected cell line by 10 times, and analyze the luminous value (RLU) diluted by the corresponding lysate.
whichever type of pseudovirus is considered significant when the RLU value of the reference strain Wuhan-1 is four times different.
Figure 2. Selection of susceptible cell lines, pictured from Cell, 2020, doi: 10.1016/j.cell.2020.07.012.
the authors first tested the infection of the 106 pseudo-viruses in the 293T-hACE2 cell line, and the results showed that 22 pseudo-viruses were classified as lowly infectious (16 natural variants and 6 glycogenized mutants), and the RLU value decreased by 4 to 100 times (Figure 3A), of which 13 counterfeit viruses carried mutations in the RBD region.
natural variants V341I and experimental glycosylized mutants (N331Q plus N343Q) are considered non-infectious and are located in the RBD region.
note, the absence of double glycogenization at N331 and N343 bits led to a sharp decline in viral infection (1200 times), while the monosaclysis loss of single sites led to a mild decrease in viral infection: the infection of N331Q decreased only threefold and the infection of N343Q decreased by 20 times.
in addition, the unnatural biglyglycogenization mutationof of RBD (N331Q and N343Q) results in a significant reduction in infection, suggesting that these two glycogenization sites in the RBD region may be involved in receptor binding or maintaining the region's conformation.
Figure 3. An infectious analysis of the missing mutations of natural variants and glycogen sites, pictured from Cell, 2020, doi: 10.1016/j.cell.2020.07.012.
further tested the infectiousness of the remaining 63 natural variants using three other cell lines (Figure 3B-3D).
notable, single-point variant D614G and combination variants of D614G (D614G-L5F, D614G-V341I, D614G-K458R, D614G The I472V, D614G-D936Y, D614G-S939F, and D614G-S943T) showed higher infectious power in all four cell lines than the reference strain.
and no differences were found between the single-point variant D614G and the combined variant of D614G, suggesting that the increased infection was more likely attributable to the D614G itself.
after identifying natural variants of the infection change (hereinafter referred to as infectious variants), the authors then began to study the antigens of these infectious variants using 13 neutral monoclonal antibodies (mAb).
they noted that some amino acid changes in the RBD region showed changes in sensitivity to neutrality mAb (Figure 4).
specific, the A475V reduces sensitivity to mAb 157, 247, CB6, P2C-1F11, B38 and CA1, while the F490L reduces sensitivity to mAb X593, 261-262, H4 and P2B-2F6.
in addition, the V483A is resistant to mAb X593 and P2B-2F6, and the L452R is resistant to mAb X593 and P2B-2F6.
the end, Y508H reduced sensitivity to mAb H014, N439K to mAb H00S022, A831V to mAb B38, D614G-I472V to mAb X593 and D614G-A435S to mAb H014.
In addition, some amino acid changes in the RBD region were observed, including V367F, Q409E, Q414E, I468F, I468T, Y508H and A522V, and were more susceptible to mAb-mediated neutrality.
Figure 4. Analyzing the antigens of natural variants and experimental glycomylate mutants with a group and monoclonal antibodies, pictured from Cell, 2020, doi: 10.1016/j.cell.2020.07.012.
the authors then determine how infectious glycosylized mutants respond to a series of identical mAbs.
mutant N165Q actually becomes more sensitive to mAb P2B-2F6, while N234Q reduces neutrality and sensitivity to different mAbs including mAb 157, 247, CB6, P2C-1F11, H00S022, B38, AB35 and H014.
these results confirm the importance of these two glycogenized sites for receptor binding.
these authors confirm that these mAbs are valuable in the analysis of amino acid changes.
as shown in Figure 4, five mAbs, namely mAb 157, 247, CB6, P2C-1F11 and B38, are not effectively neutralized with A475V and N234Q.
mAb X593 and P2B-2F6 are not effective lying with L452R, V483A and F490L, while mAb P2B-2F6 is more effective at neutralising n165Q.
, mAb H014 cannot neutralise N234Q, Y508H and D614G-A435S, while mAb H4 and 261-262 cannot neutralise the F490L.
, in the end, mAb H00S022 was unable to neutralise the N439K and N234Q.
finally, the authors measured the sensitivity of strains with changes in amino acids to 10 servings of the recovery period serum in patients with COVID-19.
there was no significant change in the sensitivity of all 10 servings of the recovery serum,
, i.e. no change in EC50 value serotonrate was more than 4 times compared to the reference strain, regardless of the increase or decrease in sensitivity (Figure 5A).
However, the neutrality sensitivity of F490L and H519P to three of these serums was found to have decreased by more than four times, while the neutrality of the six natural variants/glycogenized mutants (N149H, N149Q, N165Q, N354D, N709Q and N1173Q) were found to be sensitive to 1 or 2 of the 10 serums tested.