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Abstract Objectives SARS-CoV-2 spike (S) glycoprotein furin cleavage site is a key determinant of SARS-CoV-2 virulence and COVID-19 pathogencity. Located at the S1/S2 junction, it is unique among sarbecoviruses but frequently found among betacoronaviruses. Recent evidence suggests that this site includes two additional functional motifs: a pat7 nuclear localization signal and two flanking O-glycosites. However, a systematic genus and subgenus analysis of spike protein sequences bearing this polyfunctional sequence domain has been missing. Data description Here we report comprehensive sequence data to demonstrate that among spike proteins of genus Betacoronavirus and outside of the SARS-CoV-2 clade a fully analogous S1/S2 domain was found in only one other virus: the artificial MERS infectious clone MERS-MA30, described already in 2017, which was rationally selected from serial passage in genetically humanized mice. As the evolutionarily closest betacoronaviruses outside of the SARS-CoV-2 clade lack all its three functional motifs, these data extend—beyond natural evolution and zoonosis—the current view on SARS-CoV-2 pre-pandemic origins by presenting the analogous S1/S2 MERS-MA30 sequence domain as a precise molecular blueprint for SARS-CoV-2.

Objectives: SARS-CoV-2 spike (S) glycoprotein furin cleavage site is a key determinant of SARS-CoV-2 virulence and COVID-19 pathogencity. Located at the S1/S2 junction, it is unique among sarbecoviruses but frequently found among betacoronaviruses. Recent evidence suggests that this site includes two additional functional motifs: a pat7 nuclear localization signal and two flanking O-glycosites. However, a systematic genus and subgenus analysis of spike protein sequences bearing this polyfunctional sequence domain has been missing. Data description: Here we report comprehensive sequence data to demonstrate that among spike proteins of genus Betacoronavirus and outside of the SARS-CoV-2 clade a fully analogous S1/S2 domain was found in only one other virus: the artificial MERS infectious clone MERS-MA30, described already in 2017, which was rationally selected from serial passage in genetically humanized mice. As the evolutionarily closest betacoronaviruses outside of the SARS-CoV-2 clade lack all its three functional motifs, these data extend—beyond natural evolution and zoonosis—the current view on SARS-CoV-2 pre-pandemic origins by presenting the analogous S1/S2 MERS-MA30 sequence domain as a precise molecular blueprint for SARS-CoV-2. [ABSTRACT FROM AUTHOR]

The appearance of new dominant variants of concern (VOC) of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) threatens the global response to the coronavirus disease 2019 (COVID-19) pandemic. Of these, the alpha variant (also known as B.1.1.7), which appeared initially in the United Kingdom, became the dominant variant in much of Europe and North America in the first half of 2021. The spike (S) glycoprotein of alpha acquired seven mutations and two deletions compared to the ancestral virus, including the P681H mutation adjacent to the polybasic cleavage site, which has been suggested to enhance S cleavage. Here, we show that the alpha spike protein confers a level of resistance to beta interferon (IFN-b) in human lung epithelial cells. This correlates with resistance to an entry restriction mediated by interferon-induced transmembrane protein 2 (IFITM2) and a pronounced infection enhancement by IFITM3. Furthermore, the P681H mutation is essential for resistance to IFN-b and context-dependent resistance to IFITMs in the alpha S. P681H reduces dependence on endosomal cathepsins, consistent with enhanced cell surface entry. However, reversion of H681 does not reduce cleaved spike incorporation into particles, indicating that it exerts its effect on entry and IFN-b downstream of furin cleavage. Overall, we suggest that, in addition to adaptive immune escape, mutations associated with VOC may well also confer a replication and/or transmission advantage through adaptation to resist innate immune mechanisms. IMPORTANCE Accumulating evidence suggests that variants of concern (VOC) of SARS-CoV-2 evolve to evade the human immune response, with much interest focused on mutations in the spike protein that escape from antibodies. However, resistance to the innate immune response is essential for efficient viral replication and transmission. Here, we show that the alpha (B.1.1.7) VOC of SARS-CoV-2 is substantially more resistant to type I interferons than the parental Wuhan-like virus. This correlates with resistance to the antiviral protein IFITM2 and enhancement by its paralogue IFITM3. The key determinant of this is a proline-to-histidine change at position 681 in S adjacent to the furin cleavage site, which in the context of the alpha spike modulates cell entry pathways of SARS-CoV-2. Reversion of the mutation is sufficient to restore interferon and IFITM2 sensitivity, highlighting the dynamic nature of the SARS CoV-2 as it adapts to both innate and adaptive immunity in the huma [ABSTRACT FROM AUTHOR]

Non-alcoholic steatohepatitis (NASH) is a global disease with no effective medication. The fibroblast growth factor 21 (FGF21) can reverse this liver dysfunction, but requires targeted delivery to the liver, which can be achieved via oral administration. Therefore, we fused FGF21 to transferrin (Tf) via a furin cleavage site (F), to promote uptake from the intestine into the portal vein, yielding FGF21-F-Tf, and established its production in both seeds and leaves of commercial Nicotiana tabacum cultivars, compared their expression profile and tested the bioavailability and bioactivity in feeding studies. Since biopharmaceuticals need to be produced in a contained environment, e.g., greenhouses in case of plants, the seed production was increased in this setting from 239 to 380 g m-2 a-1 seed mass with costs of 1.64 € g-1 by side branch induction, whereas leaves yielded 8,193 g m-2 a-1 leave mass at 0.19€ g-1. FGF21-F-Tf expression in transgenic seeds and leaves yielded 6.7 and 5.6 mg kg-1 intact fusion protein, but also 4.5 and 2.3 mg kg-1 additional Tf degradation products. Removing the furin site and introducing the liver-targeting peptide PLUS doubled accumulation of intact FGF21-transferrin fusion protein when transiently expressed in Nicotiana benthamiana from 0.8 to 1.6 mg kg-1, whereas truncation of transferrin (nTf338) and reversing the order of FGF21 and nTf338 increased the accumulation to 2.1 mg kg-1 and decreased the degradation products to 7% for nTf338-FGF21-PLUS. Application of partially purified nTf338-FGF21-PLUS to FGF21-/- mice by oral gavage proved its transfer from the intestine into the blood circulation and acutely affected hepatic mRNA expression. Hence, the medication of NASH via oral delivery of nTf338-FGF21- PLUS containing plants seems possible. [ABSTRACT FROM AUTHOR]

The furin cleavage site (FCS), an unusual feature in the SARS-CoV-2 spike protein, has been spotlighted as a factor key to facilitating infection and pathogenesis by increasing spike processing. Similarly, the QTQTN motif directly upstream of the FCS is also an unusual feature for group 2B coronaviruses (CoVs). The QTQTN deletion has consistently been observed in in vitro cultured virus stocks and some clinical isolates. To determine whether the QTQTN motif is critical to SARS-CoV-2 replication and pathogenesis, we generated a mutant deleting the QTQTN motif (?QTQTN). Here, we report that the QTQTN deletion attenuates viral replication in respiratory cells in vitro and attenuates disease in vivo. The deletion results in a shortened, more rigid peptide loop that contains the FCS and is less accessible to host proteases, such as TMPRSS2. Thus, the deletion reduced the efficiency of spike processing and attenuates SARSCoV-2 infection. Importantly, the QTQTN motif also contains residues that are glycosylated, and disruption of its glycosylation also attenuates virus replication in a TMPRSS2-dependent manner. Together, our results reveal that three aspects of the S1/S2 cleavage site--the FCS, loop length, and glycosylation--are required for efficient SARS-CoV-2 replication and pathogenesis. [ABSTRACT FROM AUTHOR]

Background: The striking difference in severity of SARS CoV2 infection among global population is partly attributed to viral factors. With the spike (S) and nucleocapsid (N) are the most immunogenic subunits, genetic diversity and antigenicity of S and N are key players in virulence and in vaccine development. Aim: This paper aims at identifying immunogenic targets for better vaccine development and/or immunotherapy of COVID 19 pandemic. Methods: 18 complete genomes of SARS CoV2 (n = 14), SARS CoV (n = 2) and MERS CoV (n = 2) were examined. Bioinformatics of viral genetics and protein folding allowed functional tuning of NH2 Terminal Domain (NTD) of S protein and development of epitope maps for B and T cell responses. Conclusion: A deletion of amino acid residues Y144 and G107 were discovered in NTD of S protein derived from Indian and French isolates resulting in altered pocket structure exclusively located in NTD and reduced affinity of NTD binding to endogenous nAbs and disrupted NTD mediated cell entry. We therefore, proposed a set of B and T cell epitopes based on Immune Epitope Database, homologous epitopes for nAbs in convalescent plasma post SARS CoV infection and functional domains of S (NTD, Receptor Binding domain and the unique polybasic Furin cleavage site at S1/S2 junction). Nevertheless, laboratory data are required to develop vaccine and immunotherapeutics.

The polybasic furin cleavage site insertion with four amino acid motifs (PRRA) in spike protein's S1/S2 junction site is important in determining viral infectivity, transmission, and host range. However, there is no review so far explaining the effect of the furin cleavage site of the spike protein on SARS‐CoV‐2 replication and pathogenesis in the host and immune responses and vaccination. Therefore, here we specifically focused on genomic evolution and properties of the cleavage site of spike protein in the context of SARS‐CoV‐2 followed by its effect on viral entry, replication, and pathogenesis. We also explored whether the spike protein furin cleavage site affected the host immune responses and SARS‐CoV‐2 vaccination. This review will help to provide novel insights into the effects of polybasic furin cleavage site on the current COVID‐19 pandemic. Highlights: The genomic evolution and properties of the cleavage site of spike protein in the context of SARS‐CoV‐2 are discussed.The effects of furin cleavage site of spike protein on viral entry, replication, and pathogenesis are reviewed.Whether the host immune responses and SARS‐CoV‐2 vaccination are affected by the spike protein furin cleavage site was also discussed. [ABSTRACT FROM AUTHOR]

Among numerous point mutation differences between the SARS-CoV-2 and the bat RaTG13 coronavirus, only the 12-nucleotide furin cleavage site (FCS) exceeds 3 nucleotides. A BLAST search revealed that a 19 nucleotide portion of the SARS-CoV-2 genome encompassing the furin cleavage site is a 100% complementary match to a codon-optimized proprietary sequence that is the reverse complement of the human mutS homolog (MSH3). The reverse complement sequence present in SARS-CoV-2 may occur randomly but other possibilities must be considered. Recombination in an intermediate host is an unlikely explanation. Single stranded RNA viruses such as SARS-CoV-2 utilize negative strand RNA templates in infected cells, which might lead through copy choice recombination with a negative sense SARS-CoV-2 RNA to the integration of the MSH3 negative strand, including the FCS, into the viral genome. In any case, the presence of the 19-nucleotide long RNA sequence including the FCS with 100% identity to the reverse complement of the MSH3 mRNA is highly unusual and requires further investigations.

Recent emergence of SARS-CoV-1 variants demonstrates the potential of this virus for targeted evolution, despite its overall genomic stability. Here we show the dynamics and the mechanisms behind the rapid adaptation of SARS-CoV-2 to growth in Vero E6 cells. The selective advantage for growth in Vero E6 cells is due to increased cleavage efficiency by cathepsins at the mutated S1/S2 site. S1/S2 site also constitutes a heparan sulfate (HS) binding motif that influenced virus growth in Vero E6 cells, but HS antagonist did not inhibit virus adaptation in these cells. The entry of Vero E6-adapted virus into human cells is defective because the mutated spike variants are poorly processed by furin or TMPRSS2. Minor subpopulation that lack the furin cleavage motif in the spike protein rapidly become dominant upon passaging through Vero E6 cells, but wild type sequences are maintained at low percentage in the virus swarm and mediate a rapid reverse adaptation if the virus is passaged again on TMPRSS21 human cells. Our data show that the spike protein of SARSCoV- 2 can rapidly adapt itself to available proteases and argue for deep sequence surveillance to identify the emergence of novel variants. IMPORTANCE Recently emerging SARS-CoV-2 variants B.1.1.7 (alpha variant), B.1.617.2 (delta variant), and B.1.1.529 (omicron variant) harbor spike mutations and have been linked to increased virus pathogenesis. The emergence of these novel variants highlights coronavirus adaptation and evolution potential, despite the stable consensus genotype of clinical isolates. We show that subdominant variants maintained in the virus population enable the virus to rapidly adapt to selection pressure. Although these adaptations lead to genotype change, the change is not absolute and genomes with original genotype are maintained in the virus swarm. Thus, our results imply that the relative stability of SARS-CoV-2 in numerous independent clinical isolates belies its potential for rapid adaptation to new conditions. [ABSTRACT FROM AUTHOR]

Among numerous point mutation differences between the SARS-CoV-2 and the bat RaTG13 coronavirus, only the 12-nucleotide furin cleavage site (FCS) exceeds 3 nucleotides. A BLAST search revealed that a 19 nucleotide portion of the SARS-CoV- 2 genome encompassing the furin cleavage site is a 100% complementary match to a codon-optimized proprietary sequence that is the reverse complement of the human mutS homolog (MSH3). The reverse complement sequence present in SARS-CoV-2 may occur randomly but other possibilities must be considered. Recombination in an intermediate host is an unlikely explanation. Single stranded RNA viruses such as SARS- CoV-2 utilize negative strand RNA templates in infected cells, which might lead through copy choice recombination with a negative sense SARS-CoV-2 RNA to the integration of the MSH3 negative strand, including the FCS, into the viral genome. In any case, the presence of the 19-nucleotide long RNA sequence including the FCS with 100% identity to the reverse complement of the MSH3 mRNA is highly unusual and requires further investigations. [ABSTRACT FROM AUTHOR]

• Serum p27 antibody was measured following natural RSV infection in healthy adults. • Anti-p27 antibodies were detected in all healthy adults at all study visits. • Antibodies to the p27 region are generated following natural RSV reinfection. • p27 is not an immunodominant epitope in healthy adults. • Serum IgG anti-RSV/B p27 antibody titers were lower than IgG anti-RSV/A p27 titers. The respiratory syncytial virus (RSV) fusion (F) protein undergoes two furin-cleavage events to become fusion competent, resulting in the release of a twenty-seven amino acid peptide (p27). Recent studies indicate that the p27 region of the F protein was an immunodominant antigen in young children. In this study, we evaluated the kinetics of the serum antibody response to the p27 peptide following natural RSV reinfection in adults. Nineteen healthy adults under sixty-five years of age were enrolled during the 2018–2019 RSV season in Houston, TX. Blood was collected at three study visits and RSV infection status was defined by changes in neutralizing antibody resulting in three groups: uninfected (n = 12), acutely infected (n = 4), and recently infected (n = 3). Serum IgG and IgA antibodies against RSV/A and RSV/B p27 peptides were measured by enzyme-linked immunosorbent assays, and serum p27-like antibodies were detected by a p27 competitive antibody assay. Anti-p27 antibodies were detected in all subjects at each study visit. The measured IgG and IgA anti-p27 antibody levels followed the same pattern as other RSV site-specific and neutralizing antibody responses described for this cohort previously: the uninfected group had stable responses for the duration of the study period, the acutely infected group had a significant increase following RSV infection, and the recently infected group had a decrease in anti-p27 antibody during the study period. These results indicate that antibodies to the p27 region of the F protein are generated following natural RSV reinfection and suggest that some of the F protein is potentially in a partially cleaved state on the surface of virions, expanding on the previous assumption that all of p27 is post-translationally released and not present on mature F. Additionally, antibody responses were significantly lower (1.4–1.5-fold) toward RSV/B than to RSV/A p27 at each study visit, despite being an RSV/B dominant outbreak. Understanding the mechanism for the differences in the magnitude of the RSV/A and RSV/B p27 antibody response may enhance our understanding of the intracellular processing of the F protein. [ABSTRACT FROM AUTHOR]

Compared with other SARS-related coronaviruses (SARSr-CoVs), SARS-CoV-2 possesses a unique furin cleavage site (FCS) in its spike. This has stimulated discussion pertaining to the origin of SARS-CoV-2 because the FCS has been observed to be under strong selective pressure in humans and confers the enhanced ability to infect some cell types and induce cell–cell fusion. Furthermore, scientists have demonstrated interest in studying novel cleavage sites by introducing them into SARSr-CoVs. We review what is known about the SARS-CoV-2 FCS in the context of its pathogenesis, origin, and how future wildlife coronavirus sampling may alter the interpretation of existing data. [ABSTRACT FROM AUTHOR]

Knowledge about coronaviruses (CoVs) with furin cleavage sites is extremely limited, although these sites mediate the hydrolysis of glycoproteins in plasma membranes required for MERS-CoV or SARS-CoV-2 to enter cells and infect humans. Thus, we have examined the global epidemiology and evolutionary history of SARS-CoV-2 and 248 other CoVs with 86 diversified furin cleavage sites that have been detected in 24 animal hosts in 28 countries since 1954. Besides MERS-CoV and SARS-CoV-2, two of five other CoVs known to infect humans (HCoV-OC43 and HCoV-HKU1) also have furin cleavage sites. In addition, human enteric coronavirus (HECV-4408) has a furin cleavage site and has been detected in humans (first in Germany in 1988), probably via spillover events from bovine sources. In conclusion, the presence of furin cleavage sites might explain the polytropic nature of SARS-CoV-2- and SARS-CoV-2-like CoVs, which would be helpful for ending the COVID-19 pandemic and preventing outbreaks of novel CoVs.

The causative agent of COVID‐19 SARS‐CoV‐2 has led to over 4 million deaths worldwide. Understanding the origin of this coronavirus is important for the prevention of future outbreaks. The dominant point of view that the virus transferred to humans either directly from bats or through an intermediate mammalian host has been challenged by Segreto and Deigin, who claim that the genome of SARS‐CoV‐2 has certain features suggestive of its artificial creation. Following their response to our commentary, here we continue the discussion of the proposed arguments for this hypothesis. We show that neither the existence of a furin cleavage site in SARS‐CoV‐2, nor the presence of specific sequences within the nucleotide insertion encoding that site are evidence for intelligent design. We also explain why existing genetic data, viral diversity and past human history suggest that a natural origin of the virus is the most likely scenario. Genetic evidence suggesting otherwise is yet to be presented. [ABSTRACT FROM AUTHOR]

Knowledge about coronaviruses (CoVs) with furin cleavage sites is extremely limited, although these sites mediate the hydrolysis of glycoproteins in plasma membranes required for MERS-CoV or SARS-CoV-2 to enter cells and infect humans. Thus, we have examined the global epidemiology and evolutionary history of SARS-CoV-2 and 248 other CoVs with 86 diversified furin cleavage sites that have been detected in 24 animal hosts in 28 countries since 1954. Besides MERS-CoV and SARS-CoV-2, two of five other CoVs known to infect humans (HCoV-OC43 and HCoV-HKU1) also have furin cleavage sites. In addition, human enteric coronavirus (HECV-4408) has a furin cleavage site and has been detected in humans (first in Germany in 1988), probably via spillover events from bovine sources. In conclusion, the presence of furin cleavage sites might explain the polytropic nature of SARS-CoV-2- and SARS-CoV-2-like CoVs, which would be helpful for ending the COVID-19 pandemic and preventing outbreaks of novel CoVs. [ABSTRACT FROM AUTHOR]

ABSTRACT Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) possesses a discriminative polybasic cleavage motif in its spike protein that is recognized by the host furin protease. Proteolytic cleavage activates the spike protein, thereby affecting both the cellular entry pathway and cell tropism of SARS-CoV-2. Here, we investigated the impact of the furin cleavage site on viral growth and pathogenesis using a hamster animal model infected with SARS-CoV-2 variants bearing mutations at the furin cleavage site (S gene mutants). In the airway tissues of hamsters, the S gene mutants exhibited low growth properties. In contrast to parental pathogenic SARS-CoV-2, hamsters infected with the S gene mutants showed no body weight loss and only a mild inflammatory response, thereby indicating the attenuated variant nature of S gene mutants. This transient infection was sufficient for inducing protective neutralizing antibodies that cross-react with different SARS-CoV-2 lineages. Consequently, hamsters inoculated with S gene mutants showed resistance to subsequent infection with both the parental strain and the currently emerging SARS-CoV-2 variants belonging to lineages B.1.1.7 and P.1. Taken together, our findings revealed that the loss of the furin cleavage site causes attenuation in the airway tissues of hamsters and highlighted the potential benefits of S gene mutants as potential immunogens. IMPORTANCE SARS-CoV-2 uses its spike protein to enter target cells. The spike protein is cleaved by a host protease, and this event facilitates viral entry and broadens cell tropism. In this study, we employed SARS-CoV-2 mutants lacking the S protein cleavage site and characterized their growth and pathogenicity using hamsters, a laboratory animal model for SARS-CoV-2 infection. These mutants exerted low pathogenicity but induced sufficient levels of neutralizing antibodies in hamsters, which protected hamsters from rechallenge with pathogenic clinical SARS-CoV-2 strains. These virus mutants may be used as protective immunogens against SARS-CoV-2 infection.

Tyshkovskiy and Panchin have recently published a commentary on our paper in which they outline several "points of disagreement with the Segreto/Deigin hypothesis." As our paper is titled "The genetic structure of SARS‐CoV‐2 does not rule out a laboratory origin," points of disagreement should provide evidence that rules out a laboratory origin. However, Tyshkovskiy and Panchin provide no such evidence and instead attempt to criticize our arguments that highlight aspects of SARS‐CoV‐2 that could be consistent with the lab leak hypothesis. Strikingly, Tyshkovskiy and Panchin's main point of criticism is based on a false premise that we have claimed RaTG13 to be a direct progenitor of SARS‐CoV‐2, and their other points of criticism are either not valid, based on flawed mathematical analysis, or are unrelated to our hypotheses. Thus, the genetic structure of SARS‐CoV‐2 remains consistent with both natural or laboratory origin, which means that both the zoonotic and the lab leak hypothesis need to be investigated equally thoroughly. [ABSTRACT FROM AUTHOR]

RaTG13, MP789, and RmYN02 are the strains closest to SARS‐CoV‐2, and their existence came to light only after the start of the pandemic. Their genomes have been used to support a natural origin of SARS‐CoV‐2 but after a close examination all of them exhibit several issues. We specifically address the presence in RmYN02 and closely related RacCSxxx strains of a claimed natural PAA/PVA amino acid insertion at the S1/S2 junction of their spike protein at the same position where the PRRA insertion in SARS‐CoV‐2 has created a polybasic furin cleavage site. We show that RmYN02/RacCSxxx instead of the claimed insertion carry a 6‐nucleotide deletion in the region and that the 12‐nucleotide insertion in SARS‐CoV‐2 remains unique among Sarbecoviruses. Also, our analysis of RaTG13 and RmYN02's metagenomic datasets found unexpected reads which could indicate possible contamination. Because of their importance to inferring SARS‐CoV‐2′s origin, we call for a careful reevaluation of RaTG13, MP789 and RmYN02 sequencing records and assembly methods. [ABSTRACT FROM AUTHOR]

When culturing SARS-CoV-2 in the laboratory it is vital to avoid deletions in the gene for the spike protein that could affect the interpretation of experiments.

Virus propagation methods generally use transformed cell lines to grow viruses from clinical specimens, which may force viruses to rapidly adapt to cell culture conditions, a process facilitated by high viral mutation rates. Upon propagation in VeroE6 cells, SARS-CoV-2 may mutate or delete the multibasic cleavage site (MBCS) in the spike protein. Previously, we showed that the MBCS facilitates serine protease-mediated entry into human airway cells (Mykytyn et al., 2021). Here, we report that propagating SARS-CoV-2 on the human airway cell line Calu-3 – that expresses serine proteases – prevents cell culture adaptations in the MBCS and directly adjacent to the MBCS (S686G). Similar results were obtained using a human airway organoid-based culture system for SARS-CoV-2 propagation. Thus, in-depth knowledge on the biology of a virus can be used to establish methods to prevent cell culture adaptation.

Biochemical and structural analyses suggest that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) efficiently infects humans, and the presence of four residues (PRRA) at the S1/S2 site within the spike (S) protein may lead to unexpected tissue or host tropism. Here, we report that SARS-CoV-2 efficiently utilized angiotensin-converting enzyme 2 (ACE2) of 9 species to infect 293T cells. Similarly, pseudoviruses bearing S protein derived from either bat RaTG13 or pangolin GX, two closely related animal coronaviruses, utilized ACE2 of a diverse range of animal species to gain entry. The removal of PRRA from SARS-CoV-2 S protein displayed distinct effects on pseudoviral entry into different cell types. Unexpectedly, the insertion of PRRA into the RaTG13 S protein selectively abrogated the usage of horseshoe bat and pangolin ACE2 but enhanced the usage of mouse ACE2 by the relevant pseudovirus to enter cells. Together, our findings identified a previously unrecognized effect of the PRRA insert on SARS-CoV-2 and RaTG13 S proteins. [ABSTRACT FROM AUTHOR]

The origin of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is the subject of many hypotheses. One of them, proposed by Segreto and Deigin, assumes artificial chimeric construction of SARS‐CoV‐2 from a backbone of RaTG13‐like CoV and receptor binding domain (RBD) of a pangolin MP789‐like CoV, followed by serial cell or animal passage. Here we show that this hypothesis relies on incorrect or weak assumptions, and does not agree with the results of comparative genomics analysis. The genetic divergence between SARS‐CoV‐2 and both its proposed ancestors is too high to have accumulated in a lab, given the timeframe of several years. Furthermore, comparative analysis of S‐protein gene sequences suggests that the RBD of SARS‐CoV‐2 probably represents an ancestral non‐recombinant variant. These and other arguments significantly weaken the hypothesis of a laboratory origin for SARS‐CoV‐2, while the hypothesis of a natural origin is consistent with all available genetic and experimental data. [ABSTRACT FROM AUTHOR]

In December 2019, the world awoke to a new betacoronavirus strain named severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Betacoronavirus consists of A, B, C and D subgroups. Both SARS-CoV and SARS-CoV-2 belong to betacoronavirus subgroup B. In the present study, we divided betacoronavirus subgroup B into the SARS1 and SARS2 classes by six key insertions and deletions (InDels) in betacoronavirus genomes, and identified a recently detected betacoronavirus strains RmYN02 as a recombinant strain across the SARS1 and SARS2 classes, which has potential to generate a new strain with similar risk as SARS-CoV and SARS-CoV-2. By analyzing genomic features of betacoronavirus, we concluded: (1) the jumping transcription and recombination of CoVs share the same molecular mechanism, which inevitably causes CoV outbreaks; (2) recombination, receptor binding abilities, junction furin cleavage sites (FCSs), first hairpins and ORF8s are main factors contributing to extraordinary transmission, virulence and host adaptability of betacoronavirus; and (3) the strong recombination ability of CoVs integrated other main factors to generate multiple recombinant strains, two of which evolved into SARS-CoV and SARS-CoV-2, resulting in the SARS and COVID-19 pandemics. As the most important genomic features of SARS-CoV and SARS-CoV-2, an enhanced ORF8 and a novel junction FCS, respectively, are indispensable clues for future studies of their origin and evolution. The WIV1 strain without the enhanced ORF8 and the RaTG13 strain without the junction FCS “RRAR” may contribute to, but are not the immediate ancestors of SARS-CoV and SARS-CoV-2, respectively.

Abstract Background Proprotein convertase furin is responsible for the processing of a wide variety of precursors consisted of signal peptide, propeptide and mature peptide in mammal. Many precursors processed by furin have important physiological functions and can be recombinantly expressed in Pichia pastoris expression system for research, pharmaceutical and vaccine applications. However, it is not clear whether the furin cleavage sites between the propeptide and mature peptide can be properly processed in P. pastoris, bringing uncertainty for proper expression of the coding DNA sequences of furin precursors containing the propeptides and mature peptides. Results In this study, we evaluated the ability of P. pastoris to process furin cleavage sites and how to improve the cleavage efficiencies of furin cleavage sites in P. pastoris. The results showed that P. pastoris can process furin cleavage sites but the cleavage efficiencies are not high. Arg residue at position P1 or P4 in furin cleavage sites significantly affect cleavage efficiency in P. pastoris. Kex2 protease, but not YPS1, in P. pastoris is responsible for processing furin cleavage sites. Heterologous expression of furin or overexpression of Kex2 in P. pastoris effectively increased cleavage efficiencies of furin cleavage sites. Conclusions Our investigation on the processing of furin cleavage sites provides important information for recombinant expression of furin precursors in P. pastoris. Furin or Kex2 overexpressing strains may be good choices for expressing precursors processed by furin in P. pastoris.

In December 2019, the world awoke to a new betacoronavirus strain named severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Betacoronavirus consists of A, B, C and D subgroups. Both SARS-CoV and SARS-CoV-2 belong to betacoronavirus subgroup B. In the present study, we divided betacoronavirus subgroup B into the SARS1 and SARS2 classes by six key insertions and deletions (InDels) in betacoronavirus genomes, and identified a recently detected betacoronavirus strains RmYN02 as a recombinant strain across the SARS1 and SARS2 classes, which has potential to generate a new strain with similar risk as SARS-CoV and SARS-CoV-2. By analyzing genomic features of betacoronavirus, we concluded: (1) the jumping transcription and recombination of CoVs share the same molecular mechanism, which inevitably causes CoV outbreaks; (2) recombination, receptor binding abilities, junction furin cleavage sites (FCSs), first hairpins and ORF8 s are main factors contributing to extraordinary transmission, virulence and host adaptability of betacoronavirus; and (3) the strong recombination ability of CoVs integrated other main factors to generate multiple recombinant strains, two of which evolved into SARS-CoV and SARS-CoV-2, resulting in the SARS and COVID-19 pandemics. As the most important genomic features of SARS-CoV and SARS-CoV-2, an enhanced ORF8 and a novel junction FCS, respectively, are indispensable clues for future studies of their origin and evolution. The WIV1 strain without the enhanced ORF8 and the RaTG13 strain without the junction FCS "RRA R " may contribute to, but are not the immediate ancestors of SARS-CoV and SARS-CoV-2, respectively. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome‐coronavirus (SARS‐CoV)‐2′s origin is still controversial. Genomic analyses show SARS‐CoV‐2 likely to be chimeric, most of its sequence closest to bat CoV RaTG13, whereas its receptor binding domain (RBD) is almost identical to that of a pangolin CoV. Chimeric viruses can arise via natural recombination or human intervention. The furin cleavage site in the spike protein of SARS‐CoV‐2 confers to the virus the ability to cross species and tissue barriers, but was previously unseen in other SARS‐like CoVs. Might genetic manipulations have been performed in order to evaluate pangolins as possible intermediate hosts for bat‐derived CoVs that were originally unable to bind to human receptors? Both cleavage site and specific RBD could result from site‐directed mutagenesis, a procedure that does not leave a trace. Considering the devastating impact of SARS‐CoV‐2 and importance of preventing future pandemics, researchers have a responsibility to carry out a thorough analysis of all possible SARS‐CoV‐2 origins. [ABSTRACT FROM AUTHOR]

The striking difference in severity of SARS CoV2 infection among global population is partly attributed to viral factors. With the spike (S) and nucleocapsid (N) are the most immunogenic subunits, genetic diversity and antigenicity of S and N are key players in virulence and in vaccine development. This paper aims at identifying immunogenic targets for better vaccine development and/or immunotherapy of COVID 19 pandemic. 18 complete genomes of SARS CoV2 (n = 14), SARS CoV (n = 2) and MERS CoV (n = 2) were examined. Bioinformatics of viral genetics and protein folding allowed functional tuning of NH2 Terminal Domain (NTD) of S protein and development of epitope maps for B and T cell responses. A deletion of amino acid residues Y144 and G107 were discovered in NTD of S protein derived from Indian and French isolates resulting in altered pocket structure exclusively located in NTD and reduced affinity of NTD binding to endogenous nAbs and disrupted NTD mediated cell entry. We therefore, proposed a set of B and T cell epitopes based on Immune Epitope Database, homologous epitopes for nAbs in convalescent plasma post SARS CoV infection and functional domains of S (NTD, Receptor Binding domain and the unique polybasic Furin cleavage site at S1/S2 junction). Nevertheless, laboratory data are required to develop vaccine and immunotherapeutics. [ABSTRACT FROM AUTHOR]

Antibody Fab fragments consist of heavy chain (Hc) and light chain (Lc) polypeptides assembled with a disulphide bond. The production of a recombinant Fab fragment requires the simultaneous expression of two genes encoding both an Hc and an Lc in the same host cell. In the present study, we investigated the production of Fab fragments in lepidopteran insect cells using a bicistronic plasmid vector carrying the Hc and Lc genes linked with a 2A self-cleaving peptide sequence from the porcine teschovirus-1. We also examined the arrangement of a GSG spacer sequence and a furin cleavage site sequence with the 2A sequence. Western blot analysis and enzyme-linked immunosorbent assay (ELISA) of culture supernatants showed that Trichoplusia ni BTI-TN-5B1-4 (High Five) cells transfected with a plasmid in which the Hc and Lc genes were joined by the 2A sequence successfully secreted Fab fragments with antigen-binding activity after self-cleavage of the 2A peptide. The GSG linker enhanced 2A cleavage efficiency, and the furin recognition site was useful for removal of 2A residues from the Hc. Transfection with a single plasmid that contained sequences for GSG, the furin cleavage site, GSG, and the 2A peptide between the Hc and Lc genes exhibited a higher productivity than co-transfection with a set of plasmids separately carrying the Hc or Lc gene. These results demonstrate that bicistronic expression with the appropriate combination of a furin recognition site, GSG linkers, and a 2A peptide may be an effective way to efficiently produce recombinant antibody molecules in insect cells. [ABSTRACT FROM AUTHOR]

Here we investigated the virulence properties of a unique cell-adapted SARS-CoV-2 mutant showing a ten-amino acid deletion encompassing the furin cleavage site of the spike protein (Δ 680 SPRAARSVAS 689 ; Δ680-689-B.1) in comparison to its parental strain (wt-B.1) and two Delta variants (AY.122 and AY.21) of concern. After intranasal inoculation, transgenic K18-hACE2 mice were monitored for 14 days for weight change, lethality, and clinical score; oral swabs were daily collected and tested for the presence of N protein subgenomic RNA. At 3 and 7 dpi mice were also sacrificed and organs collected for molecular, histopathological, and immune response profile investigations. The Δ680-689-B.1-infected mice exhibited reduced shedding, lower virulence at the lung level, and milder pulmonary lesions. In the lung, infection with Δ680-689-B.1 was associated with a significant lower expression of some cytokines at 3 dpi (IL-4, IL-27, and IL-28) and 7 dpi (IL-4, IL-27, IL-28, IFN-γ and IL-1α). • The virulence properties of a SARS-CoV-2 mutant showing a deletion encompassing the S FCS (Δ680-689-B.1) were investigated. • Δ680-689-B.1- infected mice showed reduced shedding and milder pulmonary lesions. • Infection with Δ680-689-B.1 was associated with a significant lower expression of some cytokines at 3 dpi and 7 dpi. • IL-6, GRO-α, G-CSF significantly increased in mice infected with Delta VOC isolates. [ABSTRACT FROM AUTHOR]

The HIV-1 envelope glycoprotein (Env) is synthesized in the endoplasmic reticulum as a trimeric gp160 precursor, which requires proteolytic cleavage by a cellular furin protease to mediate virus-cell fusion. Env is conformationally flexible but controls its transition from the unbound “closed” conformation (State 1) to downstream CD4-bound conformations (States 2/3), which are required for fusion. In particular, HIV-1 has evolved several mechanisms that reduce the premature “opening” of Env which exposes highly conserved epitopes recognized by non-neutralizing antibodies (nnAbs) capable of mediating antibody-dependent cellular cytotoxicity (ADCC). Env cleavage decreases its conformational transitions favoring the adoption of the “closed” conformation. Here we altered the gp160 furin cleavage site to impair Env cleavage and to examine its impact on ADCC responses mediated by plasma from HIV-1-infected individuals. We found that infected primary CD4+ T cells expressing uncleaved, but not wildtype, Env are efficiently recognized by nnAbs and become highly susceptible to ADCC responses mediated by plasma from HIV-1-infected individuals. Thus, HIV-1 limits the exposure of uncleaved Env at the surface of HIV-1-infected cells at least in part to escape ADCC responses.

At the end of 2019, a new highly virulent coronavirus known under the name SARS-CoV-2 emerged as a human pathogen. One key feature of SARS-CoV-2 is the presence of an enigmatic insertion in the spike glycoprotein gene representing a novel multibasic S1/S2 protease cleavage site. The proteolytic cleavage of the spike at this site is essential for viral entry into host cells. However, it has been systematically abrogated in structural studies in order to stabilize the spike in the prefusion state. In this study, multi-microsecond molecular dynamics simulations and ab initio modeling were leveraged to gain insights into the structures and dynamics of the loop containing the S1/S2 protease cleavage site. They unveiled distinct conformations, formations of short helices and interactions of the loop with neighboring glycans that could potentially regulate the accessibility of the cleavage site to proteases and its processing. In most conformations, this loop protrudes from the spike, thus representing an attractive SARS-CoV-2 specific therapeutic target.

It is unknown whether pangolins, the most trafficked mammals, play a role in the zoonotic transmission of bat coronaviruses. We report the circulation of a novel MERS-like coronavirus in Malayan pangolins, named Manis javanica HKU4-related coronavirus (MjHKU4r-CoV). Among 86 animals, four tested positive by pan-CoV PCR, and seven tested seropositive (11 and 12.8%). Four nearly identical (99.9%) genome sequences were obtained, and one virus was isolated (MjHKU4r-CoV-1). This virus utilizes human dipeptidyl peptidase-4 (hDPP4) as a receptor and host proteases for cell infection, which is enhanced by a furin cleavage site that is absent in all known bat HKU4r-CoVs. The MjHKU4r-CoV-1 spike shows higher binding affinity for hDPP4, and MjHKU4r-CoV-1 has a wider host range than bat HKU4-CoV. MjHKU4r-CoV-1 is infectious and pathogenic in human airways and intestinal organs and in hDPP4-transgenic mice. Our study highlights the importance of pangolins as reservoir hosts of coronaviruses poised for human disease emergence. [Display omitted] • A bat HKU4r-CoV (Merbecovirus) MjHKU4r-CoV is circulating in Malayan pangolins • MjHKU4r-CoV-1 uses human DPP4 receptor and can be isolated • MjHKU4r-CoV-1 has a furin cleavage site and a wider host range than bat HKU4-CoV • MjHKU4r-CoV-1 is infectious and pathogenic in human organs and hDPP4-Tg mice A bat MERS-like coronavirus similar to HKU4-CoV is identified circulating in Malayan pangolins. This virus uses human DPP4 as a receptor, bears a furin cleavage site that potentially broadens its host range, and is infectious in human organs and transgenic animals. [ABSTRACT FROM AUTHOR]

Abstract: It is often desirable to co-express a reporter protein with a potential therapeutic protein, to verify correct targeting of an expression strategy. Vectors containing a viral self-processing 2A sequence have been reported to drive equimolar expression of two or more transgenes from a single promoter. Here, we report on the co-expression of a secreted antibody fragment and an intracellular reporter protein, enhanced yellow fluorescent protein from lentiviral shuttle plasmids by inserting a furin-2A (F2A) sequence between the two cDNAs, in two different orientations, in the expression cassette. We show that the order of these two transgenes relative to the F2A sequence affects expression levels. Reduced expression of each transgene positioned downstream of F2A, compared with upstream of F2A, was observed (p<0.05). Moreover, protein expression from double-cDNA plasmids was significantly lower than from their corresponding single transgene counterparts (p<0.05). [Copyright &y& Elsevier]

Abstract: Monoclonal antibodies and derivative formats such as Fab′ fragments are used in a broad range of therapeutic, diagnostic and research applications. New systems and methodologies that can improve the production of these proteins are consequently of much interest. Here we present a novel approach for the rapid production of processed Fab′ fragments in a CHO cell line that has been engineered to express the mouse cationic amino acid transporter receptor 1 (mCAT-1). This facilitated the introduction of the target antibody gene through retroviral transfection, rapidly producing stable expression. Using this system, we designed a single retroviral vector construct for the expression of a target Fab′ fragment as a single polypeptide with a furin cleavage site and a FMDV 2A self-cleaving peptide introduced to bridge the light and truncated heavy chain regions. The introduction of these cleavage motifs ensured equimolar expression and processing of the heavy and light domains as exemplified by the production of an active chimeric Fab′ fragment against the Fas receptor, routinely expressed in 1–2mg/L yield in spinner-flask cell cultures. These results demonstrate that this method could have application in the facile production of bioactive Fab′ fragments. [Copyright &y& Elsevier]

FurinDB (freely available online at http://www.nuolan.net/substrates.html) is a database of furin substrates. This database includes experimentally verified furin cleavage sites, substrates, species, experimental methods, original publications of experiments and associated drugs targeting furin substrates. The current database release contains 126 furin cleavage sites from three species: mammals, bacteria and viruses. A main feature of this database is that all furin cleavage sites are recorded as a 20-residue motif, including one core region (eight amino acids, P6-P2') and two flanking solvent accessible regions (eight amino acids, P7-P14, and four amino acids, P3'-P6'), that represent our current understanding of the molecular biology of furin cleavage. This database is important for understanding the molecular evolution and relationships between sequence motifs, 3D structures, cellular functions and physical properties required by furin for cleavage, and for elucidating the molecular mechanisms and the progression of furin cleavage associated human diseases, including pathogenic infections, neurological disorders, tumorigenesis, tumor invasion, angiogenesis, and metastasis. FurinDB database will be a solid addition to the publicly available infrastructure for scientists in the field of molecular biology. [ABSTRACT FROM AUTHOR]

Compared with other SARS-related coronaviruses (SARSr-CoVs), SARS-CoV-2 possesses a unique furin cleavage site (FCS) in its spike. This has stimulated discussion pertaining to the origin of SARS-CoV-2 because the FCS has been observed to be under strong selective pressure in humans and confers the enhanced ability to infect some cell types and induce cell-cell fusion. Furthermore, scientists have demonstrated interest in studying novel cleavage sites by introducing them into SARSr-CoVs. We review what is known about the SARS-CoV-2 FCS in the context of its pathogenesis, origin, and how future wildlife coronavirus sampling may alter the interpretation of existing data., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

SARS-CoV-2 (Severe Acute Respiratory Syndrome-Coronavirus 2) has accumulated multiple mutations during its global circulation. Recently, three SARS-CoV-2 lineages, B.1.1.7 (501Y.V1), B.1.351 (501Y.V2) and B.1.1.28.1 (P.1), have emerged in the United Kingdom, South Africa and Brazil, respectively. Here, we have presented global viewpoint on implications of emerging SARS-CoV-2 variants based on structural–function impact of crucial mutations occurring in its spike (S), ORF8 and nucleocapsid (N) proteins. While the N501Y mutation was observed in all three lineages, the 501Y.V1 and P.1 accumulated a different set of mutations in the S protein. The missense mutational effects were predicted through a COVID-19 dedicated resource followed by atomistic molecular dynamics simulations. Current findings indicate that some mutations in the S protein might lead to higher affinity with host receptors and resistance against antibodies, but not all are due to different antibody binding (epitope) regions. Mutations may, however, result in diagnostic tests failures and possible interference with binding of newly identified anti-viral candidates against SARS-CoV-2, likely necessitating roll out of recurring "flu-like shots" annually for tackling COVID-19. The functional relevance of these mutations has been described in terms of modulation of host tropism, antibody resistance, diagnostic sensitivity and therapeutic candidates. Besides global economic losses, post-vaccine reinfections with emerging variants can have significant clinical, therapeutic and public health impacts. [ABSTRACT FROM AUTHOR]