COVID-19関連追加(2021723-2

ウイルスの融合を増強,促進するP681R変異

SARS-CoV-2スパイクP681R変異は,ウイルスの融合を増強・促進する】

Saito A, …, Sato K. SARS-CoV-2 spike P681R mutation enhances and accelerates viral fusion. bioRxiv. June 17, 2021. Preprint.

https://doi.org/10.1101/2021.06.17.448820.

Summary

現在のSARS-CoV-2パンデミックでは,ウイルスゲノムにさまざまな変異が蓄積されており,少なくとも5つのVOCsが人間社会にとって危険なSARS-CoV-2のバリアントと考えられている。新たに出現したVOCであるB.1.617.2系統(delta変異株)は,2021年春にインドで発生したCOVID-19の大流行と密接に関連している.しかし,そのウイルス学的特性はまだ明らかではない.本研究では,B.1.617変異株が高い融合性を持ち、顕著な融合細胞(syncytia)を形成することを示した.Bioinformatic analysesにより,スパイクタンパク質のP681R変異がこの系統で高度に保存されていることが明らかになった.P681R変異は,ウイルス感染性(viral infectivity)を低下させるが,この変異は中和抗体耐性(neutralizing antibody resistance)を付与するさらに我々は,P681R変異は,furinによるスパイク開裂を促進し,細胞と細胞の融合を促進することを明らかにした.これらの結果から,P681R変異は,この最新のVOCのウイルス学的フェノタイプを特徴づけるものであり,ウイルスの病原性と関連している可能性が示唆された.

Highlights

P681R変異B.1.617系統で高度に保存されている.

P681R変異は,SARS-CoV-2S媒介性融合加速・増強する.

P681R変異によるウイルス融合の促進はTMPRSS2によって増強される.

 

 

Figure 1: Molecular phylogenetic and epidemic dynamics of the B.1.617 lineage during the pandemic.

Molecular phylogenetic and epidemic dynamics of the B.1.617 lineage during the pandemic

(A) A phylogenetic tree of the B.1.617 lineage. Bar, 0.0002 substitutions per site. Bootstrap values, **, 100%; *, >70%. The uncollapsed tree is shown in Figure S1. (B-F) Epidemic dynamics of the B.1.617 lineage. (B) The numbers of sequences deposited in GISAID per day for India (orange, left), UK (blue, middle), and the whole world (gray, right). (C-F) The percentages of each lineage deposited per day (C, all B.1.617; D, B.1.617.1; E, B.1.617.2; F, B.1.617.3) from India (orange), the UK (blue) and the whole world (gray) are shown. The date first identified is indicated. The raw data are summarized in Table S1. (G) Proportion of amino acid replacements in the B.1.617 lineage. The top 15 replacements conserved in the S protein of the B.1.617 and its sublineages are summarized. The number in parenthesis indicates the number of sequences included in each panel. The raw data are summarized in Table S2.

 

Figure 2: Virological features of the B.1.617 lineage.

(A) Growth kinetics of two B.1.617 variants and a B.1.1 isolate. Two viral isolates of (GISAID ID: EPI_ISL_2378733) and B.1.617.2 (GISAID ID: EPI_ISL_2378732) and a D614G-bearing B.1.1 isolate (GISAID ID: EPI_ISL_479681) [100 50% tissue culture infectious dose (TCID50)] were inoculated into Vero cells, VeroE6/TMPRSS2 cells and the copy number of viral RNA in the culture supernatant was quantified by real-time RT-PCR. The growth curves of the viruses inoculated are shown. Assays were performed in quadruplicate.

(B) Syncytia formation. The syncytia in infected VeroE6/TMPRSS2 cells were observed at 72 hours postinfection (hpi). (Left) Representative bright-field images of VeroE6/TMPRSS2 cells at 72 hpi are shown. Bars, 100 μm. (Right) The size of floating syncytia in B.1.1-infected (n = 217), B.1.617.1-infected (n = 306) and B.1.617.2-infected (n = 217) cultures are shown. The size of the floating single cells in uninfected culture (n = 177) was also shown as a negative control.

(C) Immunofluorescence staining. VeroE6/TMPRSS2 cells infected with the viruses indicated [multiplicity of infection (MOI) 0.01] were stained with anti-SARS-CoV-2 nucleocapsid (N) (green) and DAPI (blue). (Top) Representative images at 48 hpi are shown. Areas enclosed with squares are enlarged in the bottom panels. DIC, differential interference contrast. Bars, 200 μm for low magnification panels; 50 μm for high magnification panels. (Bottom) The area of N-positive cells in B.1.1-infected (n = 50), B.1.617.1-infected (n = 50) and B.1.617.2-infected (n = 50) cultures are shown.

In A, statistically significant differences (*, P < 0.05) versus the B.1.1 isolate were determined by Student’s t test.

In B and C, statistically significant differences versus the B.1.1-infected culture (*, P < 0.05) and uninfected culture (#, P < 0.05) were determined by the Mann-Whitney U test.

 

 

 

Figure 3: Virological features of the P681R-harboring virus.

(A) Chromatograms of the mutated regions of SARS-CoV-2 viruses artificially generated by reverse genetics. Chromatograms of nucleotide positions 23,399-23,407 (left) and 23,600-23,609 (right) of parental SARS-CoV-2 (strain WK-521, PANGO lineage A; GISAID ID: EPI_ISL_408667) and the D614G (A23403G in nucleotide) and P681R (C23604G in nucleotide) mutation are shown.

(B) Growth kinetics of artificially generated viruses. The D614G and D614G/P681R mutant viruses were generated by reverse genetics. These viruses (100 TCID50) were inoculated into Vero cells, VeroE6/TMPRSS2 cells, and HeLa-ACE2/TMPRSS2 cells and the copy number of viral RNA in the culture supernatant was quantified by real-time RT-PCR. The growth curves of the viruses inoculated are shown. Assays were performed in quadruplicate.

(C and D) Syncytia formation. The floating syncytia in infected VeroE6/TMPRSS2 cells at 72 hpi (C) and the adherent syncytia in infected HeLa-ACE2/TMPRSS2 at 48 hpi (D) are shown. In C, The size of floating syncytia in the D614G mutant-infected (n = 63) and the D614G/P681R mutant-infected (n = 126) cultures are shown. The size of the floating single cells in uninfected culture (n = 60) was also shown as a negative control. Bars, 100 μm.

In B, statistically significant differences (*, P < 0.05) versus the D614G virus were determined by Student’s t test.

In C, statistically significant differences versus the D614G mutant-infected culture (*, P < 0.05) and uninfected culture (#, P < 0.05) were determined by the Mann-Whitney U test.

 

Figure 4: Promotion of cell-cell fusion by the P681R mutation.

(A) Western blotting of pseudoviruses. (Left) Representative blots of SARS-CoV-2 full-length S and cleaved S2 proteins as well as HIV-1 p24 capsid as an internal control. kDa, kilodalton. (Right) The ratio of S2 to the full-length S plus S2 proteins on pseudovirus particles.

(B) Pseudovirus assay. The HIV-1-based reporter virus pseudotyped with the SARS-CoV-2 S D614G or D614G/P681R was inoculated into HOS-ACE2 cells or HOS-ACE2/TMPRSS2 cells at 4 different doses (2.5× 105, 5.0 × 105, 1 × 106 and 2 × 106 HiBiT values). Percentages of infectivity compared to the virus pseudotyped with parental S D614G (2 × 106 HiBiT values) in HOS-ACE2 cells are shown. The numbers on the bars of the HOS-ACE2/TMPRSS2 cell data indicate the fold change versus the HOS-ACE2 cell data. Assays were performed in quadruplicate.

(C) Western blotting of the S-expressing cells. (Left) Representative blots of SARS-CoV-2 full-length S and cleaved S2 proteins as well as ACTB as an internal control. kDa, kilodalton. (Right) The ratio of S2 to the full-length S plus S2 proteins in the S-expressing cells.

(D) Flow cytometry of the S-expressing cells. (Left) Representative histogram of the S protein expression on the cell surface. The number in the histogram indicates the mean fluorescence intensity (MFI). (Right) The MFI of surface S on the S-expressing cells.

(E-G) SARS-CoV-2 S-based fusion assay. Effector cells (S-expressing cells) and target cells (ACE2-expressing cells or ACE2/TMPRSS2-expressing cells) were prepared, and the fusion activity was measured as described in STARMETHODS.

(E) Kinetics of fusion activity (experimental data). Assays were performed in quadruplicate, and fusion activity (arbitrary unit) is shown. (F and G) The kinetics of fusion velocity estimated by a mathematical model based on the kinetics of fusion activity data (see STARMETHODS). (G) Initial velocity of the S-mediated fusion. In B, D, and E, statistically significant differences (*, P < 0.05) versus the D614G S were determined by Students t test.

In F and G, statistically significant differences (*, P < 0.05) versus the D614G S were determined by two-sided Welch’s t test.

 

 

Figure 5: Association of the P681R mutation on the sensitivity to Nabs.

Neutralization assay was performed by using three RBD-targeting monoclonal antibodies (clones 8A5, 4A3 and CB6) (A and C) and 19 vaccinated sera (B). NAbs were used for the pseudovirus assay (A and B) and the S-based fusion assay (C). Pseudoviruses and effector cells (S-expressing cells) were treated with serially diluted NAbs or sera as described in STARMETHODS. The raw data of B is shown in Figure S2. NT50, 50% neutralization titer.

In A, the NT50 values of the D614G S (black) and D614G/P681R S (orange) are indicated.

In B, a statistically significant difference versus the D614G virus was determined by Wilcoxon matched-pairs signed rank test.