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      The proximal origin of SARS-CoV-2


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          To the Editor — Since the first reports of novel pneumonia (COVID-19) in Wuhan, Hubei province, China 1,2 , there has been considerable discussion on the origin of the causative virus, SARS-CoV-2 3 (also referred to as HCoV-19) 4 . Infections with SARS-CoV-2 are now widespread, and as of 11 March 2020, 121,564 cases have been confirmed in more than 110 countries, with 4,373 deaths 5 . SARS-CoV-2 is the seventh coronavirus known to infect humans; SARS-CoV, MERS-CoV and SARS-CoV-2 can cause severe disease, whereas HKU1, NL63, OC43 and 229E are associated with mild symptoms 6 . Here we review what can be deduced about the origin of SARS-CoV-2 from comparative analysis of genomic data. We offer a perspective on the notable features of the SARS-CoV-2 genome and discuss scenarios by which they could have arisen. Our analyses clearly show that SARS-CoV-2 is not a laboratory construct or a purposefully manipulated virus. Notable features of the SARS-CoV-2 genome Our comparison of alpha- and betacoronaviruses identifies two notable genomic features of SARS-CoV-2: (i) on the basis of structural studies 7–9 and biochemical experiments 1,9,10 , SARS-CoV-2 appears to be optimized for binding to the human receptor ACE2; and (ii) the spike protein of SARS-CoV-2 has a functional polybasic (furin) cleavage site at the S1–S2 boundary through the insertion of 12 nucleotides 8 , which additionally led to the predicted acquisition of three O-linked glycans around the site. 1. Mutations in the receptor-binding domain of SARS-CoV-2 The receptor-binding domain (RBD) in the spike protein is the most variable part of the coronavirus genome 1,2 . Six RBD amino acids have been shown to be critical for binding to ACE2 receptors and for determining the host range of SARS-CoV-like viruses 7 . With coordinates based on SARS-CoV, they are Y442, L472, N479, D480, T487 and Y4911, which correspond to L455, F486, Q493, S494, N501 and Y505 in SARS-CoV-2 7 . Five of these six residues differ between SARS-CoV-2 and SARS-CoV (Fig. 1a). On the basis of structural studies 7–9 and biochemical experiments 1,9,10 , SARS-CoV-2 seems to have an RBD that binds with high affinity to ACE2 from humans, ferrets, cats and other species with high receptor homology 7 . Fig. 1 Features of the spike protein in human SARS-CoV-2 and related coronaviruses. a, Mutations in contact residues of the SARS-CoV-2 spike protein. The spike protein of SARS-CoV-2 (red bar at top) was aligned against the most closely related SARS-CoV-like coronaviruses and SARS-CoV itself. Key residues in the spike protein that make contact to the ACE2 receptor are marked with blue boxes in both SARS-CoV-2 and related viruses, including SARS-CoV (Urbani strain). b, Acquisition of polybasic cleavage site and O-linked glycans. Both the polybasic cleavage site and the three adjacent predicted O-linked glycans are unique to SARS-CoV-2 and were not previously seen in lineage B betacoronaviruses. Sequences shown are from NCBI GenBank, accession codes MN908947, MN996532, AY278741, KY417146 and MK211376. The pangolin coronavirus sequences are a consensus generated from SRR10168377 and SRR10168378 (NCBI BioProject PRJNA573298) 29,30 . While the analyses above suggest that SARS-CoV-2 may bind human ACE2 with high affinity, computational analyses predict that the interaction is not ideal 7 and that the RBD sequence is different from those shown in SARS-CoV to be optimal for receptor binding 7,11 . Thus, the high-affinity binding of the SARS-CoV-2 spike protein to human ACE2 is most likely the result of natural selection on a human or human-like ACE2 that permits another optimal binding solution to arise. This is strong evidence that SARS-CoV-2 is not the product of purposeful manipulation. 2. Polybasic furin cleavage site and O-linked glycans The second notable feature of SARS-CoV-2 is a polybasic cleavage site (RRAR) at the junction of S1 and S2, the two subunits of the spike 8 (Fig. 1b). This allows effective cleavage by furin and other proteases and has a role in determining viral infectivity and host range 12 . In addition, a leading proline is also inserted at this site in SARS-CoV-2; thus, the inserted sequence is PRRA (Fig. 1b). The turn created by the proline is predicted to result in the addition of O-linked glycans to S673, T678 and S686, which flank the cleavage site and are unique to SARS-CoV-2 (Fig. 1b). Polybasic cleavage sites have not been observed in related ‘lineage B’ betacoronaviruses, although other human betacoronaviruses, including HKU1 (lineage A), have those sites and predicted O-linked glycans 13 . Given the level of genetic variation in the spike, it is likely that SARS-CoV-2-like viruses with partial or full polybasic cleavage sites will be discovered in other species. The functional consequence of the polybasic cleavage site in SARS-CoV-2 is unknown, and it will be important to determine its impact on transmissibility and pathogenesis in animal models. Experiments with SARS-CoV have shown that insertion of a furin cleavage site at the S1–S2 junction enhances cell–cell fusion without affecting viral entry 14 . In addition, efficient cleavage of the MERS-CoV spike enables MERS-like coronaviruses from bats to infect human cells 15 . In avian influenza viruses, rapid replication and transmission in highly dense chicken populations selects for the acquisition of polybasic cleavage sites in the hemagglutinin (HA) protein 16 , which serves a function similar to that of the coronavirus spike protein. Acquisition of polybasic cleavage sites in HA, by insertion or recombination, converts low-pathogenicity avian influenza viruses into highly pathogenic forms 16 . The acquisition of polybasic cleavage sites by HA has also been observed after repeated passage in cell culture or through animals 17 . The function of the predicted O-linked glycans is unclear, but they could create a ‘mucin-like domain’ that shields epitopes or key residues on the SARS-CoV-2 spike protein 18 . Several viruses utilize mucin-like domains as glycan shields involved immunoevasion 18 . Although prediction of O-linked glycosylation is robust, experimental studies are needed to determine if these sites are used in SARS-CoV-2. Theories of SARS-CoV-2 origins It is improbable that SARS-CoV-2 emerged through laboratory manipulation of a related SARS-CoV-like coronavirus. As noted above, the RBD of SARS-CoV-2 is optimized for binding to human ACE2 with an efficient solution different from those previously predicted 7,11 . Furthermore, if genetic manipulation had been performed, one of the several reverse-genetic systems available for betacoronaviruses would probably have been used 19 . However, the genetic data irrefutably show that SARS-CoV-2 is not derived from any previously used virus backbone 20 . Instead, we propose two scenarios that can plausibly explain the origin of SARS-CoV-2: (i) natural selection in an animal host before zoonotic transfer; and (ii) natural selection in humans following zoonotic transfer. We also discuss whether selection during passage could have given rise to SARS-CoV-2. 1. Natural selection in an animal host before zoonotic transfer As many early cases of COVID-19 were linked to the Huanan market in Wuhan 1,2 , it is possible that an animal source was present at this location. Given the similarity of SARS-CoV-2 to bat SARS-CoV-like coronaviruses 2 , it is likely that bats serve as reservoir hosts for its progenitor. Although RaTG13, sampled from a Rhinolophus affinis bat 1 , is ~96% identical overall to SARS-CoV-2, its spike diverges in the RBD, which suggests that it may not bind efficiently to human ACE2 7 (Fig. 1a). Malayan pangolins (Manis javanica) illegally imported into Guangdong province contain coronaviruses similar to SARS-CoV-2 21 . Although the RaTG13 bat virus remains the closest to SARS-CoV-2 across the genome 1 , some pangolin coronaviruses exhibit strong similarity to SARS-CoV-2 in the RBD, including all six key RBD residues 21 (Fig. 1). This clearly shows that the SARS-CoV-2 spike protein optimized for binding to human-like ACE2 is the result of natural selection. Neither the bat betacoronaviruses nor the pangolin betacoronaviruses sampled thus far have polybasic cleavage sites. Although no animal coronavirus has been identified that is sufficiently similar to have served as the direct progenitor of SARS-CoV-2, the diversity of coronaviruses in bats and other species is massively undersampled. Mutations, insertions and deletions can occur near the S1–S2 junction of coronaviruses 22 , which shows that the polybasic cleavage site can arise by a natural evolutionary process. For a precursor virus to acquire both the polybasic cleavage site and mutations in the spike protein suitable for binding to human ACE2, an animal host would probably have to have a high population density (to allow natural selection to proceed efficiently) and an ACE2-encoding gene that is similar to the human ortholog. 2. Natural selection in humans following zoonotic transfer It is possible that a progenitor of SARS-CoV-2 jumped into humans, acquiring the genomic features described above through adaptation during undetected human-to-human transmission. Once acquired, these adaptations would enable the pandemic to take off and produce a sufficiently large cluster of cases to trigger the surveillance system that detected it 1,2 . All SARS-CoV-2 genomes sequenced so far have the genomic features described above and are thus derived from a common ancestor that had them too. The presence in pangolins of an RBD very similar to that of SARS-CoV-2 means that we can infer this was also probably in the virus that jumped to humans. This leaves the insertion of polybasic cleavage site to occur during human-to-human transmission. Estimates of the timing of the most recent common ancestor of SARS-CoV-2 made with current sequence data point to emergence of the virus in late November 2019 to early December 2019 23 , compatible with the earliest retrospectively confirmed cases 24 . Hence, this scenario presumes a period of unrecognized transmission in humans between the initial zoonotic event and the acquisition of the polybasic cleavage site. Sufficient opportunity could have arisen if there had been many prior zoonotic events that produced short chains of human-to-human transmission over an extended period. This is essentially the situation for MERS-CoV, for which all human cases are the result of repeated jumps of the virus from dromedary camels, producing single infections or short transmission chains that eventually resolve, with no adaptation to sustained transmission 25 . Studies of banked human samples could provide information on whether such cryptic spread has occurred. Retrospective serological studies could also be informative, and a few such studies have been conducted showing low-level exposures to SARS-CoV-like coronaviruses in certain areas of China 26 . Critically, however, these studies could not have distinguished whether exposures were due to prior infections with SARS-CoV, SARS-CoV-2 or other SARS-CoV-like coronaviruses. Further serological studies should be conducted to determine the extent of prior human exposure to SARS-CoV-2. 3. Selection during passage Basic research involving passage of bat SARS-CoV-like coronaviruses in cell culture and/or animal models has been ongoing for many years in biosafety level 2 laboratories across the world 27 , and there are documented instances of laboratory escapes of SARS-CoV 28 . We must therefore examine the possibility of an inadvertent laboratory release of SARS-CoV-2. In theory, it is possible that SARS-CoV-2 acquired RBD mutations (Fig. 1a) during adaptation to passage in cell culture, as has been observed in studies of SARS-CoV 11 . The finding of SARS-CoV-like coronaviruses from pangolins with nearly identical RBDs, however, provides a much stronger and more parsimonious explanation of how SARS-CoV-2 acquired these via recombination or mutation 19 . The acquisition of both the polybasic cleavage site and predicted O-linked glycans also argues against culture-based scenarios. New polybasic cleavage sites have been observed only after prolonged passage of low-pathogenicity avian influenza virus in vitro or in vivo 17 . Furthermore, a hypothetical generation of SARS-CoV-2 by cell culture or animal passage would have required prior isolation of a progenitor virus with very high genetic similarity, which has not been described. Subsequent generation of a polybasic cleavage site would have then required repeated passage in cell culture or animals with ACE2 receptors similar to those of humans, but such work has also not previously been described. Finally, the generation of the predicted O-linked glycans is also unlikely to have occurred due to cell-culture passage, as such features suggest the involvement of an immune system 18 . Conclusions In the midst of the global COVID-19 public-health emergency, it is reasonable to wonder why the origins of the pandemic matter. Detailed understanding of how an animal virus jumped species boundaries to infect humans so productively will help in the prevention of future zoonotic events. For example, if SARS-CoV-2 pre-adapted in another animal species, then there is the risk of future re-emergence events. In contrast, if the adaptive process occurred in humans, then even if repeated zoonotic transfers occur, they are unlikely to take off without the same series of mutations. In addition, identifying the closest viral relatives of SARS-CoV-2 circulating in animals will greatly assist studies of viral function. Indeed, the availability of the RaTG13 bat sequence helped reveal key RBD mutations and the polybasic cleavage site. The genomic features described here may explain in part the infectiousness and transmissibility of SARS-CoV-2 in humans. Although the evidence shows that SARS-CoV-2 is not a purposefully manipulated virus, it is currently impossible to prove or disprove the other theories of its origin described here. However, since we observed all notable SARS-CoV-2 features, including the optimized RBD and polybasic cleavage site, in related coronaviruses in nature, we do not believe that any type of laboratory-based scenario is plausible. More scientific data could swing the balance of evidence to favor one hypothesis over another. Obtaining related viral sequences from animal sources would be the most definitive way of revealing viral origins. For example, a future observation of an intermediate or fully formed polybasic cleavage site in a SARS-CoV-2-like virus from animals would lend even further support to the natural-selection hypotheses. It would also be helpful to obtain more genetic and functional data about SARS-CoV-2, including animal studies. The identification of a potential intermediate host of SARS-CoV-2, as well as sequencing of the virus from very early cases, would similarly be highly informative. Irrespective of the exact mechanisms by which SARS-CoV-2 originated via natural selection, the ongoing surveillance of pneumonia in humans and other animals is clearly of utmost importance.

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          Serological Evidence of Bat SARS-Related Coronavirus Infection in Humans, China

          Dear Editor, Severe acute respiratory syndrome coronavirus (SARS-CoV) is the causative agent of the 2002–2003 SARS pandemic, which resulted in more than 8000 human infections worldwide and an approximately 10% fatality rate (Ksiazek et al. 2003; Peiris et al. 2004). The virus infects both upper airway and alveolar epithelial cells, resulting in mild to severe lung injury in humans (Peiris et al. 2003). During the SARS outbreak investigation, epidemiological evidence of a zoonotic origin of SARS-CoV was identified (Xu et al. 2004). Isolation of SARS-related coronavirus (SARSr-CoVs) from masked palm civets and the detection of SARS-CoV infection in humans working at wet markets where civets were sold suggested that masked palm civets could serve as a source of human infection (Guan et al. 2003). Subsequent work identified genetically diverse SARSr-CoVs in Chinese horseshoe bats (Rhinolophus sinicus) in a county of Yunnan Province, China and provided strong evidence that bats are the natural reservoir of SARS-CoV (Ge et al. 2013; Li et al. 2005; Yang et al. 2016). Since then, diverse SARS-related coronaviruses (SARSr-CoVs) have been detected and reported in bats in different regions globally (Hu et al. 2015). Importantly, SARSr-CoVs that use the SARS-CoV receptor, angiotensin converting enzyme 2 (ACE2) have been isolated (Ge et al. 2013). These results indicate that some SARSr-CoVs may have high potential to infect human cells, without the necessity for an intermediate host. However, to date, no evidence of direct transmission of SARSr-CoVs from bats to people has been reported. In this study, we performed serological surveillance on people who live in close proximity to caves where bats that carry diverse SARSr-CoVs roost. In October 2015, we collected serum samples from 218 residents in four villages in Jinning County, Yunnan province, China (Fig. 1A), located 1.1–6.0 km from two caves (Yanzi and Shitou). We have been conducting longitudinal molecular surveillance of bats for CoVs in these caves since 2011 and have found that they are inhabited by large numbers of bats including Rhinolophus spp., a major reservoir of SARSr-CoVs. This region was not involved in the 2002–2003 SARS outbreaks and none of the subjects exhibited any evident respiratory illness during sampling. Among those sampled, 139 are female and 79 male, and the median age is 48 (range 12–80). Occupational data were obtained for 208 (95.4%) participants: 85.3% farmers and 8.7% students. Most (81.2%) kept or owned livestock or pets, and the majority (97.2%) had a history of exposure to or contact with livestock or wild animals. Importantly, 20 (9.1%) participants witnessed bats flying close to their houses, and one had handled a bat corpse. As a control, we also collected 240 serum samples from random blood donors in 2015 in Wuhan, Hubei Province more than 1000 km away from Jinning (Fig. 1A) and where inhabitants have a much lower likelihood of contact with bats due to its urban setting. None of the donors had knowledge of prior SARS infection or known contact with SARS patients. Fig. 1 SARSr-CoV serosurveillance. (A) Map of Xiyang town, Jinning County, Yunnan Province, China. Shown here is the location of the 4 villages (Tianjing, Dafengkou, Lvxi, Lvxixin) around 2 bat caves (Yanzi Cave and Shitou Cave) chosen for this study. The map of China is also shown in the inset indicating the location of Wuhan, where the negative control sera were collected, in relation to Jinning, Shenzhen and the capital Beijing. Serological reactivity of serum samples with recombinant SARSr-CoV NP protein. B ELISA test. The dotted line represents the cutoff of the test. C Western blot analysis. Numbers on the left are molecular masses in kDa. His-tagged nucleocapsid protein (NP) of the following viruses were expressed and purified in E. coli for this study: SARSr-CoV Rp3; human coronaviruses (HCoVs) HKU1, OC43, 229E, NL63; Middle East Respiratory Syndrome Coronavirus (MERS-CoV); and Ebola virus (EBOV). In addition, the receptor binding domains (RBD) of the spike protein (S) from SARS-CoV, bat SARSr-CoVs Rp3, WIV1, and SHC014 were produced in mammalian cells (Ge et al. 2013; Yang et al. 2016). Polyclonal antibodies against each of the six NPs were prepared in rabbits as previously published (He et al. 2006). Cross-activity was evaluated with ELISA and Western blot (Supplementary Figures S1, S2). No significant cross-activity was detected among NPs and their corresponding antibodies for Rp3, MERS-CoV, NL63, or 229E. Cross-reaction was detected between OC43 and HKU1 as reported previously (Lehmann et al. 2008). The Rp3 NP was chosen to develop a SARSr-CoV specific ELISA for serosurveillance. Micro-titer plates were coated with 100 ng/well of recombinant Rp3 NP and incubated with human sera in duplicates at a dilution of 1:20, followed by detection with HRP labeled goat anti-human IgG antibodies (Proteintech, Wuhan, China) at a dilution of 1:20,000. The 240 random serum samples collected in Wuhan and two SARS positive samples from Zhujiang Hospital, Southern Medical University (kindly provided by Prof. Xiaoyan Che)1 were used to set a cutoff value. We used the mean OD value of the 240 samples plus three standard deviations to set the cutoff value at 0.41. A total of six positive samples were detected by ELISA (Fig. 1B). The specificity of these positive samples was confirmed by Western blot with recombinant Rp3 NP (Fig. 1C) together with NP of NL63, MERS-CoV and EBOV. The degree of reactivity in Western blot correlated well with the ELISA OD readings, providing further confidence in the ELISA screening method. None of the sera reacted with NPs of either MERS-CoV or EBOV. On the other hand, all 10 human sera (9 from Jinning and 1 from Wuhan), regardless of their Rp3 NP reactivity, reacted strongly with the NL63 NP as expected due to high prevalence of NL63 infection in humans worldwide (Abdul-Rasool and Fielding 2010). We conducted a virus neutralization test for the six positive samples targeting two SARSr-CoVs, WIV1 and WIV16 (Ge et al. 2013; Yang et al. 2016). None of them were able to neutralize either virus. These sera also failed to react by Western blot with any of the recombinant RBD proteins from SARS-CoV or the three bat SARSr-CoVs Rp3, WIV1, and SHC014. We also performed viral nucleic acid detection in oral and fecal swabs and blood cells, and none of these were positive. The demography and travel histories of the six positive individuals (four male, two female) are as follows. Two males (JN162, 45 years old, JN129, 51 years old) are from the Dafengkou village; two males (JN117, 49 years old, JN059, 57 years old) from Lvxi village; and two females (JN053, JN041, both 55 years old), from Tianjing village. In the 12 months prior to the sampling date, JN041 was the only individual who travelled outside of Yunnan, to Shenzhen, a city 1400 km away from her home village (Fig. 1A). JN053 and JN059 had travelled to another county 1.4 km away from their village. JN162 had travelled to Kunming, the capital of Yunnan, 63 km away. JN129 and JN117 had never left the village. It is worth noting that all of them had observed bats flying in their villages. Our study provides the first serological evidence of likely human infection by bat SARSr-CoVs or, potentially, related viruses. The lack of prior exposure to SARS patients by the people surveyed, their lack of prior travel to areas heavily affected by SARS during the outbreak, and the rapid decline of detectable antibodies to SARS-CoV in recovered patients within 2–3 years after infection strongly suggests that positive serology obtained in this study is not due to prior infection with SARS-CoV (Wu et al. 2007). The 2.7% seropositivity for the high risk group of residents living in close proximity to bat colonies suggests that spillover is a relatively rare event, however this depends on how long antibodies persist in people, since other individuals may have been exposed and antibodies waned. During questioning, none of the 6 seropositive subjects could recall any clinical symptoms in the past 12 months, suggesting that their bat SARSr-CoV infection either occurred before the time of sampling, or that infections were subclinical or caused only mild symptoms. Our previous work based on cellular and humanized mouse infection studies suggest that these viruses are less virulent than SARS-CoV (Ge et al. 2013; Menachery et al. 2016; Yang et al. 2016). Masked palm civets appeared to play a role as intermediate hosts of SARS-CoV in the 2002–2003 outbreak (Guan et al. 2003). However, considering that these individuals have a high chance of direct exposure to bat secretion in their villages, this study further supports the notion that some bat SARSr-CoVs are able to directly infect humans without intermediate hosts, as suggested by receptor entry and animal infection studies (Menachery et al. 2016). The failure of these NP ELISA positive sera to either neutralize live virus or react with RBD proteins in Western blot could be explained by at least two hypotheses. First, the immune response to the bat SARSr-CoV S protein may be weaker than that to the NP protein or may wane more rapidly, especially in subclinical infections, resulting in antibody levels is too low to be detected by our assays. Secondly, other bat SARSr-CoV variants may be circulating in bats in these villages that have highly divergent S proteins and have not yet been detected in our surveillance studies. Coronaviruses are known to have a high mutation rate during replication and are prone to recombination if different viruses infect the same individual (Knipe et al. 2013). From our previous studies of bat SARSr-CoVs in the two caves near these villages, we have found genetically highly diverse bat SARSr-CoVs and evidence of frequent coinfection of two or more different SARSr-CoVs in the same bat (Ge et al. 2013). Our current study suggests that our surveillance is not exhaustive, as one would have expected, and that further, more extensive surveillance in this region is warranted. It might also be prudent to combine serological surveillance with molecular surveillance of bats in future, despite the technological challenges that this represents. Electronic supplementary material Below is the link to the electronic supplementary material. Supplementary material 1 (PDF 339 kb)
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            Global aspects of viral glycosylation

            Abstract Enveloped viruses encompass some of the most common human pathogens causing infections of different severity, ranging from no or very few symptoms to lethal disease as seen with the viral hemorrhagic fevers. All enveloped viruses possess an envelope membrane derived from the host cell, modified with often heavily glycosylated virally encoded glycoproteins important for infectivity, viral particle formation and immune evasion. While N-linked glycosylation of viral envelope proteins is well characterized with respect to location, structure and site occupancy, information on mucin-type O-glycosylation of these proteins is less comprehensive. Studies on viral glycosylation are often limited to analysis of recombinant proteins that in most cases are produced in cell lines with a glycosylation capacity different from the capacity of the host cells. The glycosylation pattern of the produced recombinant glycoproteins might therefore be different from the pattern on native viral proteins. In this review, we provide a historical perspective on analysis of viral glycosylation, and summarize known roles of glycans in the biology of enveloped human viruses. In addition, we describe how to overcome the analytical limitations by using a global approach based on mass spectrometry to identify viral O-glycosylation in virus-infected cell lysates using the complex enveloped virus herpes simplex virus type 1 as a model. We underscore that glycans often pay important contributions to overall protein structure, function and immune recognition, and that glycans represent a crucial determinant for vaccine design. High throughput analysis of glycosylation on relevant glycoprotein formulations, as well as data compilation and sharing is therefore important to identify consensus glycosylation patterns for translational applications.
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              Proteolytic activation of the spike protein at a novel RRRR/S motif is implicated in furin-dependent entry, syncytium formation, and infectivity of coronavirus infectious bronchitis virus in cultured cells.

              The spike (S) protein of the coronavirus (CoV) infectious bronchitis virus (IBV) is cleaved into S1 and S2 subunits at the furin consensus motif RRFRR(537)/S in virus-infected cells. In this study, we observe that the S2 subunit of the IBV Beaudette strain is additionally cleaved at the second furin site (RRRR(690)/S) in cells expressing S constructs and in virus-infected cells. Detailed time course experiments showed that a peptide furin inhibitor, decanoyl-Arg-Val-Lys-Arg-chloromethylketone, blocked both viral entry and syncytium formation. Site-directed mutagenesis studies revealed that the S1/S2 cleavage by furin was not necessary for, but could promote, syncytium formation by and infectivity of IBV in Vero cells. In contrast, the second site is involved in the furin dependence of viral entry and syncytium formation. Mutations of the second site from furin-cleavable RRRR/S to non-furin-cleavable PRRRS and AAARS, respectively, abrogated the furin dependence of IBV entry. Instead, a yet-to-be-identified serine protease(s) was involved, as revealed by protease inhibitor studies. Furthermore, sequence analysis of CoV S proteins by multiple alignments showed conservation of an XXXR/S motif, cleavable by either furin or other trypsin-like proteases, at a position equivalent to the second IBV furin site. Taken together, these results suggest that proteolysis at a novel XXXR/S motif in the S2 subunit might be a common mechanism for the entry of CoV into cells.

                Author and article information

                Nat Med
                Nat. Med
                Nature Medicine
                Nature Publishing Group US (New York )
                17 March 2020
                : 1-3
                [1 ]ISNI 0000000122199231, GRID grid.214007.0, Department of Immunology and Microbiology, , The Scripps Research Institute, ; La Jolla, CA USA
                [2 ]Scripps Research Translational Institute, La Jolla, CA USA
                [3 ]ISNI 0000 0004 1936 7988, GRID grid.4305.2, Institute of Evolutionary Biology, , University of Edinburgh, ; Edinburgh, UK
                [4 ]ISNI 0000000419368729, GRID grid.21729.3f, Center for Infection and Immunity, , Mailman School of Public Health of Columbia University, ; New York, NY USA
                [5 ]ISNI 0000 0004 1936 834X, GRID grid.1013.3, Marie Bashir Institute for Infectious Diseases and Biosecurity, School of Life and Environmental Sciences and School of Medical Sciences, , The University of Sydney, ; Sydney, Australia
                [6 ]ISNI 0000 0001 2217 8588, GRID grid.265219.b, Tulane University, School of Medicine, Department of Microbiology and Immunology, ; New Orleans, LA USA
                [7 ]ISNI 0000 0004 5901 1919, GRID grid.505518.c, Zalgen Labs, ; Germantown, MD USA
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                This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.


                molecular evolution,computational biology and bioinformatics
                molecular evolution, computational biology and bioinformatics


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