Because spike proteins are critical in mediating viral entry into host cells and are the focus of most vaccine strategies [20, 21], we further investigated the role of spike proteins in repair. of DNA damage and its associated V (D) J recombination. Peak proteins are believed to be synthesized in the rough endoplasmic reticulum (ER) . After post-translational modifications such as glycosylation, spike proteins are trafficked through the cell membrane apparatus together with other viral proteins to form the mature virion .
The spike protein contains two major subunits, S1 and S2, as well as several functional domains or repeats  (Figure 2A). In the native state, spike proteins exist as full-length inactive proteins. During viral infection, host cell proteases, such as furin protease, activate protein S by cleaving it into the S1 and S2 subunits, which is necessary for virus entry into the target cell . Furthermore, we explored different subunits of the spike protein to elucidate the functional characteristics necessary for the inhibition of DNA repair.
Only the full-length peak protein strongly inhibited NHEJ and HR repair (Figure 2B-E and Figure S4A, B). Next, we seek to determine whether the spike protein contributes directly to genomic instability by inhibiting DSB repair. We monitor DSB levels using kite tests. After different DNA damage treatments, such as γ-irradiation, doxorubicin treatment, and H2O2 treatment, there is less repair in the presence of the spike protein (Figure 2F, G). Together, these data demonstrate that the spike protein directly affects DNA repair in the nucleus.
3.3. Spike proteins prevent protein recruitment from DNA damage repair checkpoints
To confirm the existence of peak protein in the nucleus, we performed a subcellular fraction analysis and found that peak proteins are not only enriched in the cell membrane fraction, but are also abundant in the nuclear fraction, with detectable expression. even in the chromatin-bound fraction (Figure 3A). We also observe that the spike has three different shapes, the top band is a highly glycosylated spike, the middle band is a full-length spike, and the bottom band is a split spike subunit. Consistent with the comet assay, we also found upregulation of the DNA damage marker, γ-H2A.X, in peak protein overexpressed cells under DNA damage conditions (Figure 3B).
A recent study suggested that spike proteins induce ER stress and ER-associated protein degradation . To exclude the possibility that the spike protein inhibits DNA repair by promoting degradation of the DNA repair protein, we checked the expression of some essential DNA repair proteins in the NHEJ and HR repair pathways and found that these proteins DNA repairers were stable after spike protein overexpression (Figure 3C). To determine how the spike protein inhibits the NHEJ and HR repair pathways, we analyzed the recruitment of BRCA1 and 53BP1, which are the key checkpoint proteins for HR and NHEJ repair, respectively. We found that the spike protein markedly inhibited the formation of BRCA1 and 53BP1 foci (Figure 3D-G). Together, these data show that the full-length spike protein of SARS-CoV-2 inhibits DNA damage repair by hindering the recruitment of the DNA repair protein.
3.4. Peak protein disrupts V (D) J recombination in vitro
Repair of DNA damage, especially NHEJ repair, is essential for V (D) J recombination, which is at the core of B and T cell immunity . To date, many approved SARS-CoV-2 vaccines have been developed, such as mRNA vaccines and adenovirus-COVID-19 vaccines, based on the full-length spike protein . Although it is debatable whether SARS-CoV-2 directly infects lymphocyte precursors [26,27], some reports have shown that infected cells secrete exosomes that can transport SARS-CoV-2 RNA or proteins to target cells [ 28.29]. Furthermore, we tested whether the spike protein reduced NHEJ-mediated V (D) J recombination. To do this, we designed an in vitro V (D) J recombination reporter system according to a previous study  (Figure S5). Compared to the empty vector, overexpression of the spike protein inhibited RAG-mediated V (D) J recombination in this reporter system in vitro.
Our findings provide evidence that the spike protein hijacks the DNA damage repair machinery and the adaptive immune machinery in vitro. We propose a potential mechanism by which spike proteins can affect adaptive immunity by inhibiting DNA damage repair. Although no published evidence has been published that SARS-CoV-2 can infect bone marrow thymocytes or lymphoid cells, our in vitro reporter V (D) J assay shows that the spike protein strongly impeded V (D) recombination. J. Consistent with our results, clinical observations also show that the risk of severe illness or death with COVID-19 increases with age, especially in older adults who are at highest risk .
This may be because SARS-CoV-2 spike proteins can weaken the DNA repair system in older people, thereby preventing V (D) J recombination and adaptive immunity. In contrast, our data provide valuable details on the involvement of spike protein subunits in DNA damage repair, indicating that full-length spike-based vaccines can inhibit V (D) J recombination in B cells, which is also consistent with a recent study that a full-length spike-based vaccine induced lower antibody titers compared to the RBD-based vaccine . This suggests that the use of antigenic epitopes from the spike as a vaccine against SARS-CoV-2 could be safer and more effective than the full spike.
Collectively, we identify one of the potentially important mechanisms for the suppression of SARS-CoV-2 from the host's adaptive immune machinery. Furthermore, our findings also imply a potential side effect of the full-length spike-based vaccine. This work will improve understanding of the pathogenesis of COVID-19 and provide new strategies to design more efficient and safer vaccines.
The following are available online at https://www.mdpi.com/article/10.3390/v13102056/s1, Figure S1: Expression of nuclear localized SARS-CoV-2 proteins in human cells, Figure S2: Effect of SARS nuclear- CoV-2 proteins in the NHEJ– and HR- DNA repair pathway, Figure S3: Nsp1, Nsp5, Nsp13, Nsp14 but do not inhibit cell proliferation, Figure S4: Effect of SARS-CoV-2 spike mutants in the DNA NHEJ– and HR– repair pathway, Figure S5: V (D) J recombination assay in vitro.
H.J. conceived and designed the study. H.J. and Y.-F.M. supervised the study, conducted experiments, and interpreted the data. Writing: preparation of the original draft, H.J .; Writing: review and editing, H.J. and Y.-F.M.; acquisition of financing, Y.-F.M. All authors have read and accepted the published version of the manuscript.
This work was supported by Umeå University, Medical College Planning Grants for COVID-19 (Research Project Number: 3453 16032 for Y.F.M.); Umeå University Lion's Cancer Research Foundation (grants: LP 17–2153, AMP 19–982, and LP 20–2256 to YFM), and base unit ALF funds for research in academic health units and health units. university health in the northern health region (ALF - Basenheten: 2019, 2020, 2021 to YFM).