key: cord-0688465-aivt4gsu
authors: Wang, Chenxi; Zheng, You; Niu, Zubiao; Jiang, Xiaoyi; Sun, Qiang
title: The virological impacts of SARS-CoV-2 D614G mutation
date: 2021-07-21
journal: J Mol Cell Biol
DOI: 10.1093/jmcb/mjab045
sha: fdd7f623f3d85336723cec995e71705ae720a095
doc_id: 688465
cord_uid: aivt4gsu

The coronavirus diseases 2019 (COVID-19) caused by the infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in December 2019 has caused more than 140 million infections worldwide by the end of April 2021. As an enveloped single-stranded positive-sense RNA virus, SARS-CoV-2 underwent constant evolution that produced novel variants carrying mutation conferring fitness advantages. The current prevalent D614G variant, with glycine substituted for aspartic acid at position 614 in the spike glycoprotein (S protein), is one of such variants that became the main circulating strain worldwide in a short period of time. Over the past year, intensive studies from all over the world had defined the epidemiological characteristics of this highly contagious variant and revealed the underlying mechanisms. This review aims at presenting an overall picture of the impacts of D614G mutation on virus transmission, elucidating the underlying mechanisms of D614G in virus pathogenicity, and providing insights into the development of effective therapeutics.

The pneumonia of coronavirus diseases 2019 was caused by the infection of 1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60  3 2020; Korber et al., 2020) . In this review, we summarized the impact of D614G mutation on the epidemiology of SARS-CoV-2 and the mechanism of its enhanced infectivity and rapid transmission.

We also opened up many interesting questions, aiming to provide new insights into future research on SARS-CoV-2 mutations.

Because of the RNA proofreading ability of the replication mechanism, virus evolution was considered to be slow (Robson et al., 2020) . However, the rapid global spread of SARS-CoV-2 has provided a sufficient opportunity for natural selection to make it possible for rare but beneficial mutations to accumulate. It has been found that mutations of SARS-CoV-2 were accumulated throughout the viral genes that encode the non-structural proteins, including ORF1a, ORF1b, ORF3, and ORF8, and the structural proteins consisting of N and S proteins (Franco-Muñoz et al., 2020; Ozono et al., 2021) , among which mutations in S protein are particularly worthy of attention, because the S protein is responsible for viral entry and its mutations can strongly influence host range, tissue tropism, and pathogenesis (Walls et al., 2020; Zhou et al., 2020) . The availability of thousands of SARS-CoV-2 genome sequences provides a valuable resource that can help to better understand the evolution of the virus across time and location. It was observed that a viral variant in the S1 domain of S protein with a single amino at position 614 rapidly became the most prevalent variant all around the world (Isabel et al., 2020; Korber et al., 2020) . Analyses revealed that D614G mutation was rare before March 2020, but increased rapidly and replaced the original virus as the main variant by April 2020 (Korber et al., 2020 ; Figure 1) . Moreover, compared with other variants, the transition frequency of D614G occurred asynchronously in different regions, which spread more easily and displayed higher frequency in regions of Europe and North America, but not in East Asia (Bhattacharyya et al., 2020; Korber et al., 2020) . Up to now, the demographic distribution analysis indicates that there is no significant difference between the D614G variant and other sequenced SARS-CoV-2 variants (Isabel et al., 2020; Korber et al., 2020) .

Previous studies on SARS-CoV have shown that the continuous accumulation of S protein mutations affected its affinity for ACE2 and virus transmission and infectivity (Sui et al., 2008; Du et al., 2009) . Therefore, it is conceivable that D614G mutation may also affect the transmission and infectivity of SARS-CoV-2. In agreement with this, the COVID-19 patients infected with G614 variant were significantly higher than those infected with D614 variant in viral loads, as demonstrated by the lower cycle threshold (Ct) in quantitative RT-PCR (Korber et al., 2020;  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60   4 Lorenzo-Redondo et al., 2020; Figure 1 ). However, in contrast with the above conclusion, one study by Isabel et al. (2020) found no significant difference in the average Ct values between D614 and G614 variants. Considering a relatively small selection of samples in the latter study, it is more likely that the patients infected with G614 variant have higher viral loads. Moreover, it was found that SARS-CoV-2 virus with D614G mutation had significantly higher titers than D614 in a variety of cell types no matter for vesicular stomatitis virus or lentiviral pseudotype virus (Diez-Fuertes et al., 2020; Korber et al., 2020) . Consistently, higher infection efficiency was observed for G614 mutation in multiple cells that were infected with similar titers of pseudoviruses produced with the S protein or the intact SARS-CoV-2 carrying either D614 or G614 mutation (Hu et al., 2020; Ogawa et al., 2020; Zhang et al., 2020; Daniloski et al., 2021) . Thus, D614G mutation likely enhanced the infection efficiency of SARS-CoV-2 virus. In addition, D614G increases infectivity on target cells bearing ACE2 orthologs from multiple species, including human (Homo sapiens), Chinese rufous horseshoe bat (Rhinolophus sinicus), Malayan pangolin (Manis javanica), cat (Felis catus), and dog (Canis lupis), indicating that the increased infectivity of D614G is not specific for human ACE2 (Yurkovetskiy et al., 2020) . Furthermore, it was observed that D614G does not change the process of S protein synthesis, processing, and incorporation into SARS-CoV-2 particles, suggesting that the increased infectivity caused by D614G mutation is primarily manifested during the period when the virus enters the target cells post assembly (Yurkovetskiy et al., 2020) . In agreement with this, our research showed that the luciferase activity, an infection reporter, was higher in cells infected with S-G614 pseudovirus than those infected with S-D614 pseudovirus; moreover, the luciferase activities were positively correlated with membrane fusion index . These findings suggested that D614G mutation might promote SARS-CoV-2 infection through membrane fusion-mediated host entry. In addition to in vitro experiments, researchers also explored the effects of D614G mutation on SARS-CoV-2 viruses in different animal models such as the golden Syrian hamster and ferret (Plante et al., 2020; Zhou et al., 2021) . Results showed that the replication, infectivity, and fitness activities of SARS-CoV-2 with D614G mutation were increased. The same conclusion was obtained in a primary human airway tissue model too.

Given that D614G mutation can enhance the replication and infectivity of SARS-CoV-2 virus, efforts were carried out to explore the underlying mechanisms. We summarize the current studies on the mechanisms at three levels: cytological, molecular, and structural. 

In the process of SARS-CoV-2 virus infection, the virus first binds to the host cell receptor ACE2, which interacts with S protein to mediate membrane fusion and host entry. It was speculated that D614G mutation in S protein may increase the infectivity of SARS-CoV-2 virus by enhancing membrane fusion. To verify the above view, Ogawa et al. (2020) , by co-culturing 293T cells expressing S protein (D614 or G614) with cells expressing ACE2, observed an enhanced membrane fusion in S-G614-expressing cells. This was independently validated in our research on syncytia formation in cells stably expressing ACE2 (293T-ACE2 and Hela-ACE2). Results showed that S-G614 exhibited a significantly stronger ability to induce syncytium formation, as evidenced by increased frequency and size (syncytia with more nuclei) of syncytia formation (Figure 2 ). In line with this, the viruses pseudotyped with S-G614 demonstrated higher infectivity than did S-D614-pseudotyped viruses . Other researches also support the above notion that the G614 variant is more effective at membrane fusion than wild-type D614, which is likely to be responsible for the higher infectivity of SARS-CoV-2 and the global transmission (Chen et al., 2020a; Zhang et al., 2021a) . Nevertheless, one study notably showed that the difference in fusion activity between D614 and G614 gradually decreased with the increase of the amount of transfected S protein, which indicates that the high expression of S protein may compensate the inferior infectivity of S-D614 protein (Zhang et al., 2021a) .

Similar to SARS-CoV, SARS-CoV-2 binds to ACE2 through the receptor-binding domain (RBD) in the S1 domain of S protein (Walls et al., 2020) . The binding affinity between S protein and ACE2 was considered to be associated with virus transmissibility and infectivity .

Compared with SARS-CoV, SARS-CoV-2 was found to be more infectious, and its affinity for ACE2-binding is 10-to 20-fold higher than that of the former (Wrapp et al., 2020) . Although D614G is located at the C-terminus of the S1 domain outside of the RBD, this single mutation was believed to enhance S-ACE2 interaction by promoting the open conformation of RBD (Yurkovetskiy et al., 2020) , which may conceivably increase S-ACE2 binding. Consistent with this idea, Zhou et al. (2021) reported that S-G614 monomer could bind to ACE2 with higher affinity than did S-D614 monomer (Zhou et al., 2021) . However, more studies using S trimer, a functional form of S protein, showed that the interaction between S-G614 and ACE2 was not enhanced or (Yurkovetskiy et al., 2020; Zhang et al., 2020; Zhang et al., 2021a) , arguing against that the increased infectivity of D614G mutation may be attributed to altered binding affinity between S and ACE2.

In addition to binding to host cell receptors, the activation of S protein by host cell proteases is another key factor in regulating infectivity and pathogenicity. For efficient host entry of SARS-CoV-2 virus, S protein needs to be cleaved by furin at residues 682-685 into two domains including S1 and S2, which is thought to be important for the conformational transformation required for receptor binding or the exposure of another cleavage site (Walls et al., 2020) . The cleavage by TMPRSS2 of S2 domain creating the S2' fragment, probably subsequent to furin cleavage, is related to the activation of membrane fusion (Bestle et al., 2020) . Therefore, the sensitivity of S protein to protease-mediated processing may also be responsible for the enhanced infectivity caused by D614G mutation. It was found that S-G614 was more stable and resistant to proteolytic cleavage in the process of S protein synthesis and virus assembly when the receptor ACE2 was absent, which is consistent with high transmission of the SARS-CoV-2 with D614G mutation Daniloski et al., 2021) . However, during SARS-CoV-2 infection when S protein bound to ACE2, D614G mutation increased the shedding of S1, leading to unsheathe of S2 that promotes membrane fusion and host entry of SARS-CoV-2. Thus, D614G mutation may impose a unique bimodular effect on the stability of S protein, i.e. stabilizing S protein on the virions in the absence of ACE2 and increasing S1 shedding upon ACE2 engagement on the cell surface ( Figure 2 ) as proposed previously . The increased shedding of S1 upon D614G mutation is believed to result in enhanced cleavage of S protein by proteases (Chen et al., 2020a; Jiang et al., 2020; Zhang et al., 2020; Gobeil et al., 2021) . Consistent with the above results, Mohammad et al. (2020) found that D614G mutation increased the binding affinity of furin and S protein by using PRODIGY server, thermodynamic analysis, and molecular dynamics (MD) simulations. Thus, D614G mutation may improve the S1/S2 cleavage efficiency by increasing the interaction between S protein and furin protease, thereby enhancing viral entry into host cells, and ultimately resulting in higher infectivity (Figure 1) . However, it is worth noting that one study showed no difference in S cleavage mode between the two kinds of exocellular SARS-CoV-2 virions in western blotting assay, contrary to the observation reported in the pseudovirus study (Hou et al., 2020) . Therefore, the infection modes of pseudovirus and wild SARS-CoV-2 virus might be different, further pointing to essential validation of results from pseudovirus by the authentic SARS-CoV-2 virus. In addition to the above two cleavage sites, another novel serine protease (elastase-2) cleavage site at residues 615-616 of S-G614 glycoprotein was identified using the method of PROSPER, which is thought to be also important for virus infectivity. Compared with S-D614 pseudovirus, S-G614 pseudovirus more effectively infected 293T-ACE2 cells that had been pre-treated with elastase, indicating that D614G mutation can also facilitate elastase-enhanced viral infectivity (Hu et al., 2020 ; Figure 1 ). However, McAuley et al. (2020) disagreed with that elastase-mediated S1/S2 cleavage promoted S-G614-mediated infectivity, because MD simulations models suggested that, though furin can access the S1/S2 cleavage site, the elastase cleavage site introduced by D614G mutation cannot be accessed because of its interface positioning and glycan blocking, which can be disturbed by the separation of S1 trimer cap and S2.

During the process of SARS-CoV-2 virus receptor binding and host entry, S protein undergoes a large-scale structural rearrangement, from a prefusion conformation to a post-fusion conformation, which exposes the hydrophobic fusion peptide (FP) promoting cell membrane fusion and virus entry (Cai et al., 2020; Walls et al., 2020; Wrapp et al., 2020) . The S1 subunit comprises N-terminal domain (NTD) and RBD, which is responsible for recognizing and binding to ACE2 and contributes to stabilization of the prefusion conformation (Walls et al., 2020; Gobeil et al., 2021) . The S2 domain contains FP followed by the second protease cleavage site (S2'), which is responsible for membrane fusion (Hoffmann et al., 2020; Gobeil et al., 2021) . In order to interact with ACE2 receptors, the conformation of RBD domains transforms between two states including "closed" (or down) for the state of receptor inaccessibility and "open" (or up) for the state of receptor access (Cai et al., 2020; Shang et al., 2020) .

The D614 residue of wild-type S protein is located at the surface region, where it interacts with the neighboring subunit. Structural analysis indicated that D614 interacts with the neighboring T859 via a hydrogen bond. However, D614G mutation disrupts the hydrogen bond due to loss of side chains (Gupta et al., 2020; Jiang et al., 2020; Korber et al., 2020; Omotuyi et al., 2020; Yurkovetskiy et al., 2020) , possibly enhancing the mainchain flexibility and reducing S1

shedding from viral-membrane-bound S2 Zhang et al., 2020;  Figure 1 ). In addition, the electrostatic bond formed by D614-K854 in the fusion peptide proximal region (FPPR)

is disrupted by D614G mutation as well, contributing to the fixation of RBD in the prefusion conformation (Gupta et al., 2020) . Simultaneously, the D to G mutation may regulate the 1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58 59 60 8 glycosylation of the nearby N616 site, affecting the dynamics of the spatial proximal FP adjacent to the protomer (Hu et al., 2020; Korber et al., 2020) . Moreover, the hydrogen bond between the skeleton amine of the G residue and the carboxyl group of the adjacent amino acid (A647) was shortened, possibly locally stabilizing S protein (Fernández, 2020; Jiang et al., 2020) . Hence, D614G mutation may change the structural conformation of S protein, leading to altered stability and related functions, such as membrane fusion.

Though the open conformation of RBD is necessary for ACE2 binding, the binding site of RBD is partially blocked in the closed conformation (Cai et al., 2020) . D614 is located on the S1 protomer, close to the RBD, so it is not difficult to infer that the D to G mutation may result in the conformational changes of RBD and thus affect its recognition of ACE2 receptor, and ultimately affect the infection process of SARS-CoV-2. Omotuyi et al. (2020) found that the RBD of G614 mutation was preferentially in an open state, while the wild-type (D614) samples were largely in a closed state (Figure 1) . Moreover, compared with the prefusion conformation preferred by D614, the conformation of S2 domain sampling in G614 is mainly post-fusion, providing evidence that D614G mutation of S protein is more available for receptor binding and has better fitness than the wild-type (Omotuyi et al., 2020) . Cryo-electron microscopy (cryo-EM) structures of the SARS-CoV-2 trimeric S protein ectodomain have revealed that the RBD of each protomer could adopt the closed or open conformation independently, resulting in asymmetric trimers (Walls et al., 2020) .

However, D614G mutation can destroy this asymmetry to a certain extent and contribute to symmetrizations from three aspects, such as the inter-protomer contacts, the correlations between the RBDs, and the specific inter-protomer hydrogen bond. Besides, these symmetrizations of the energetics of D614G mutation lead to the higher population of infection-capable (one-up) conformation, enhancing the exposure of RBD to ACE2 binding and 1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60   9 filtered maps, any populations of two or three RBD in the open conformation could not be identified (Gobeil et al., 2021) . It is worth noting that although the above studies are inconsistent in some aspects, it is certain that the RBD of G614 variant tends to increase the open conformation, which is preferentially ACE2-binding. Further studies have shown that D614G mutation not only affected the interaction between RBD and ACE2, but also increased furin binding due to the flexibility of S protein, which conceivably promotes SARS-CoV-2 infection by increasing exposure of S protein to cleavage by furin (Mohammad et al., 2020; Figure 1 ).

A major focus of current research into SARS-CoV-2 genetics is whether any mutations have the potential to significantly alter viral properties, such as transmission, or disease progression.

Evolutionary theory predicts that most new viral mutations are deleterious and short-lived, whereas mutations that persist and grow in observed frequency may be selectively or advantageous to viral fitness (Bhattacharyya et al., 2020) . Multiple researches have shown that D614G mutation in S protein increased viral fitness, potentially viral loads, and infectivity (Korber et al., 2020; Volz et al., 2021) . However, given the rapid expansion and the threat of SARS-CoV-2 virus to the human society, whether D614G mutation causes more severe symptoms for COVID-19 patients caught more attention.

According to the study by Korber Korber et al. (2020) who analyzed 999 individuals with COVID-19 at the Sheffield Teaching Hospitals, the D614G status showed no significant correlation with the disease severity as measured by hospitalization outcomes by regression analysis.

Consistent with this conclusion, Mak et al. (2020) tracked and analyzed 113 cases in Hong Kong, from January 21, 2020 to June 12, 2020, and also found that D614G has nothing to do with the severe illness (P= 1.000). The latest research also showed that D614G mutation did not lead to higher mortality or clinical severity in infected patients, though the G614 variant can infect much younger individuals (Volz et al., 2021) . In summary, current researches so far show that there is no significant correlation between the G614 variant and more severe disease (Figure 1) .

The S protein of SARS-CoV-2 is not only related to receptor binding and cell fusion but also can stimulate potent cellular and humoral immune responses, which is an important target for the development of vaccines and therapeutics antibodies (Du et al., 2009; Poland et al., 2020) . Up to By evaluating the neutralization activity of serum samples from convalescent patients with COVID-19, no significant difference was identified in protecting against viruses pseudotyped with both S-D614 and S-G614 (Hu et al., 2020; Korber et al., 2020) . However, it is unknown what kind of virus initially infected the convalescent patients with COVID-19. Later, to explore the question about whether D614G mutation could reduce the efficacy of the current vaccine based on the original D614 spike sequence, Plante et al. (2020) used a panel of sera collected from hamsters that were previously infected with D614 SARS-CoV-2 to neutralize heterologous G614 virus and homologous D614 virus, respectively. The results showed that all sera exhibited 1.4-to 2.3-fold higher neutralization titers against G614 virus than against D614 virus. After that, they tested the neutralization potency of 11 human RBD mAbs, which further proved that mAbs exhibited 2.1-fold higher potency (mAb18) in protecting against G614 virus or similar neutralization activities (the other 10 mAbs) against both viruses. In order to evaluate the ability of antibodies produced during SARS-CoV-2 infection to react with S-D614 or S-G614, enzyme linked immunosorbent assay (ELISA) was employed to measure serologic reactivity. As a result, D614G mutation did not alter the performance of IgG, IgM, or IgA seroassay, and all sera from donors who tested positive for D614 antibodies also displayed strong reactivity to G614 spike (Klumpp-Thomas et al., 2020) . INO-4800 was a DNA vaccine that had been demonstrated to protect ferrets from SARS-CoV-2 infection.

D614G mutation had little influence on the protection efficiency of INO-4800 against SARS-CoV-2 infection (McAuley et al., 2020) . So far, all existing studies showed that D614G mutation does not attenuate the antigenicity of S protein, and vaccines and therapeutics antibodies designed against the original S protein would be still effective for the G614 variant (Figure 1) . However, it is worth noting that all the experiments that have been conducted were based on the S-protein-pseudotyped viruses. Therefore, these above conclusions warrant further validation with authentic SARS-CoV-2 viruses.

Despite the high mutation rate of RNA virus, many mutations of SARS-CoV-2 were believed to be eliminated by the genetic proofreading mechanism (Sevajol et al., 2014) . However, the G614 1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60   11 variant replaced the original D614 variant and became a global pandemic in just two months, indicating that D614G mutation provides a selective advantage for SARS-CoV-2 with more fitness (Hou et al., 2020; Plante et al., 2020;  Figure 1) . In multiple assays, D614G mutation was found to replicate more efficiently and increase the infectivity of SARS-CoV-2 virus both in cells and animal models Korber et al., 2020; Plante et al., 2020; Zhou et al., 2021) .

Mechanistically, the high infectivity of the D614G variant depends partly on its ability to increase host entry, which is largely attributed to altered stability and processing of S protein, leading to enhanced membrane fusion and virus replication (Figure 1) . Nevertheless, virus infectivity is not identical to virus transmission, and detailed analysis should be performed on more patients infected with D614 or G614 variants. Although the high infectivity of the G614 variant contributes to its rapid spread, other factors, such as the geographical distribution of host populations, should also be considered. One recent study showed that the high rate spreading of the G614 variant in Europe and North America is related to a single-nucleotide allele deletion that regulates the expression of TMPRSS2 and MX1 host proteins (Bhattacharyya et al., 2020) . It should be noted that there are several mutations always coming out together with D614G mutation in SARS-CoV-2 variants, including a C-to-T mutation in the 5′ UTR region at position 241, a silent C-to-T mutation at position 3037, and a C-to-T mutation at position 14408 that results in an amino-acid change in RNA-dependent RNA polymerase (RdRp P323L) (Korber et al., 2020) . These mutations, and other accompanying mutations as well, may affect the impacts of D614G mutation and should be taken into account when accessing the infectivity and transmissibility of D614G variants. Last but not least, detection methods and epidemic prevention capabilities are also important factors affecting regional transmission.

Although no significant relationship between D614G mutation and disease severity was identified based primarily on the hospitalization outcomes or the cases of patients with severe symptoms (Korber et al., 2020; Mak et al., 2020) , the latest data in western countries indicated that the D614G variant was accompanied by the increased frequency of neurological symptoms, including hyposmia and hypogeusia that were rarely found in the early stage of SARS-CoV-2 pandemic (Alturki et al., 2020; Butowt et al., 2020 ; Figure 1) . Therefore, it is speculated that the enhanced efficiency of virus binding to host cells in vivo caused by D614G mutation may be responsible for the high incidence of neurological symptoms (Butowt et al., 2020) . Besides, the latest research showed that, even after 6 months of symptom onset, ~76% of the new COVID-19 patients have at least one adverse symptom, 63% of which are mainly fatigue or muscle weakness, (Huang et al., 2021) . Overall, these results suggest that, in future research, more attention should be paid to the correlation between certain symptoms, patient prognosis, and D614G mutation or even other mutations such as N501Y (Leung et al., 2021) .

At present, most vaccines and antibodies in clinical trials were designed based on S-D614, which is sharply incompatible with the global prevalence of the G614 variant. Given that S-G614 is quite different from S-D614 in structure and biological function, the efficacy of the existing antibodies and vaccines may be carefully tested with the G614 variant before approved for clinical application. Fortunately, most antibodies and sera arose from/against S-D614 were found being able to react with the G614 spike antigens, indicating that the site of 614 is not a major epitope and does not affect the antigenicity of other epitopes nearby, and D614G mutation less likely affects the efficacy of vaccines and antibodies (Hou et al., 2020; Klumpp-Thomas et al., 2020; McAuley et al., 2020; Plante et al., 2020; Yurkovetskiy et al., 2020; Guthmiller et al., 2021; Ozono et al., 2021) . Nevertheless, with the ongoing vaccination of larger population, further studies on sera from vaccinees are needed to examine their efficacies on mutated variants including not only D614G but also other newly emerging variants such as the UK VUI-202012/01variant, the South Africa 501.V2 variant, and the Brazil P.1 variant. 1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60 http://mc.manuscriptcentral.com/jmcb Manuscripts submitted to Journal of Molecular Cell Biology   1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60 http://mc.manuscriptcentral.com/jmcb Manuscripts submitted to Journal of Molecular Cell Biology   1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60 http://mc.manuscriptcentral.com/jmcb Manuscripts submitted to Journal of Molecular Cell Biology   1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58 

The bimodular effects of D614G mutation on spike protein. Emergence: G6 1 4 v a r i a n t w a s r a r e b e f o r e Ma r c h 2 0 2 0 , b u t r e p l a c e d t h e original virus as the main variant by April 2020.

T h e v i r a l l o a d i n t h e u p p e r r e s p i r a t o r y t r a c t o f C OV I D -1 9 patients infected with G614 variant is significantly higher than that of patients infected with D614 variant.

Clinical severity: D614G mutation didn' t l ead to higher mortality or clinical severity in infected patients, though the G6 1 4 v a r i a n t c a n i n f e c t m u c h younger individuals.

Neurological symptoms： G614 variant exhibited increased neurological symptoms, including hyposmia and hypogeusia that were rarely found in the early stage of SARS-CoV-2 pandemic.

The site of 614 is not on a major epitope. Thus, D614G mutation w o u l d u n l i k e l y a t t e n u a t e t h e antigenicity of S glycoprotein and affect the antigenicity of other epitopes nearby.

V a c c i n e s a n d t h e r a p e u t i c s antibodies designed against the original S glycoprotein would be still effective for the G614 variant.

T h e D 6 1 4 G mu t a t i o n t e n d s t o reduce spontaneous S1 shedding from viron particles.

T h e D 6 1 4 G mu t a t i o n t e n d s t o i n c r e a s e S 1 s h e d d i n g u p o n engagement of ACE2 on the target c e l l s u r f a c e ( s e e F i g u r e 2 f o r details).

The D614G mutation makes RBD tend to form an open conformation.

There are two opposing opinions on the affinity between RBD and ACE2: one holds that D614G mutation e n h a n c e s t h e b i n d i n g a f f i n i t y between them, while the other holds that the affinity is weakened.

A novel elastase-2 cleavage site (615/616) was created upon the D 6 1 4 G mu t a t i o n , w h i c h m i g h t increase S processing.

The D614G mutation may promote the interaction between S protein and Furin protease, leading to enhanced processing of S protein by Furin and TMPRSS2 proteases. 1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60 

The 2020 Pandemic: Current SARS-CoV-2 Vaccine Development

Mutations in SARS-CoV-2

TMPRSS2 and furin are both essential for proteolytic activation of SARS-CoV-2 in human airway cells

Global Spread of SARS-CoV-2 Subtype with Spike Protein Mutation D614G is Shaped by Human Genomic Variations that Regulate Expression of TMPRSS2 and MX1 Genes

Chemosensory Dysfunction in COVID-19: Integration of Genetic and Epidemiological Data Points to D614G Spike Protein Variant as a Contributing Factor

Distinct conformational states of SARS-CoV-2 spike protein

Genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting Wuhan. Emerging microbes & infections 9

SARS-CoV-2 D614 and G614 spike variants impair neuronal synapses and exhibit differential fusion ability

Emerging coronaviruses: genome structure, replication, and pathogenesis

Severe acute respiratory syndrome

Approaches and Challenges in SARS-CoV-2 Vaccine Development

The Spike D614G mutation increases SARS-CoV-2 infection of multiple human cell types

A Founder Effect Led Early SARS-CoV-2 Transmission in Spain

A systematic review of SARS-CoV-2 vaccine candidates

The spike protein of SARS-CoV-a target for vaccine and therapeutic development

Structural Impact of Mutation D614G in SARS-CoV-2 Spike Protein: Enhanced Infectivity and Therapeutic Opportunity

Substitutions in Spike and Nucleocapsid proteins of SARS-CoV-2 circulating in South America

Evolution of antibody immunity to SARS-CoV-2

D614G Mutation Alters SARS-CoV-2 Spike Conformation and Enhances Protease Cleavage at the S1/S2 Junction

Non-synonymous mutations of SARS-CoV-2 leads epitope loss and segregates its variants

SARS-CoV-2 Infection Severity Is Linked to Superior Humoral Immunity against the Spike

SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

SARS-CoV-2 D614G variant exhibits efficient replication ex vivo and transmission in vivo

D614G mutation of SARS-CoV-2 spike protein enhances viral infectivity

6-month consequences of COVID-19 in patients discharged from hospital: a cohort study

Evolutionary and structural analyses of SARS-CoV-2 D614G spike protein mutation now documented worldwide

Bimodular effects of D614G mutation on the spike glycoprotein of SARS-CoV-2 enhance protein processing, membrane fusion, and viral infectivity

Effect of D614G Spike Variant on Immunoglobulin G, M, or A Spike Seroassay Performance. The Journal of infectious diseases

Structure-guided multivalent nanobodies block SARS-CoV-2 infection and suppress mutational escape

Tracking Changes in SARS-CoV-2 Spike: Evidence that D614G Increases Infectivity of the COVID-19 Virus

A novel coronavirus associated with severe acute respiratory syndrome

Early transmissibility assessment of the N501Y mutant strains of SARS-CoV-2 in the United Kingdom

A clade of SARS-CoV-2 viruses associated with lower viral loads in patient upper airways

The D614G substitution in the S gene and clinical information for patients with COVID-19 detected in Hong Kong

Experimental and in silico evidence suggests vaccines are unlikely to be affected by D614G mutation in SARS-CoV-2 spike protein

Higher binding affinity of furin for SARS-CoV-2 spike (S) protein D614G mutant could be associated with higher SARS-CoV-2 infectivity

The D614G mutation in the SARS-CoV2 Spike protein increases infectivity in an ACE2 receptor dependent manner

Atomistic simulation reveals structural mechanisms underlying D614G spike glycoprotein-enhanced fitness in SARS-COV-2

SARS-CoV-2 D614G spike mutation increases entry efficiency with enhanced ACE2-binding affinity

Spike mutation D614G alters SARS-CoV-2 fitness

SARS-CoV-2 immunity: review and applications to phase 3 vaccine candidates

Estimation of incubation period distribution of COVID-19 using disease onset forward time: a novel cross-sectional and forward follow-up study

SARS-CoV-2, SARS-CoV, and MERS-COV: a comparative overview

Coronavirus RNA Proofreading: Molecular Basis and Therapeutic Targeting

Prospect of SARS-CoV-2 spike protein: potential role in vaccine 15 and therapeutic development

Insights into RNA synthesis, capping, and proofreading mechanisms of SARS-coronavirus

Cell entry mechanisms of SARS-CoV-2

Complete map of SARS-CoV-2 RBD mutations that escape the monoclonal antibody LY-CoV555 and its cocktail with LY-CoV016

Broadening of neutralization activity to directly block a dominant antibody-driven SARS-coronavirus evolution pathway

Evaluating the Effects of SARS-CoV-2 Spike Mutation D614G on Transmissibility and Pathogenicity

Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein

Receptor Recognition by the Novel Coronavirus from Wuhan: an Analysis Based on Decade-Long Structural Studies of SARS Coronavirus

Transmission and prevention of SARS-CoV-2

Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation

Structural and Functional Analysis of the D614G SARS-CoV-2 Spike Protein Variant

Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia

Structural impact on SARS-CoV-2 spike protein by D614G substitution

SARS-CoV-2 spike-protein D614G mutation increases virion spike density and infectivity

SARS-CoV-2 spike protein dictates syncytium-mediated lymphocyte elimination

SARS-CoV-2 spike D614G change enhances replication and transmission

SARS-CoV-2 Targets by the pscRNA Profiling of ACE2, TMPRSS2 and Furin Proteases

Author contributions: Q.S. conceived and structured the manuscript; C.W. collected and analyzed the data; C.W. and Q.S. made the draft with inputs from Y.Z., Z.N., and X.J.; Q.S. and C.W. drew the figures with inputs from Y.Z.