The origin of SARS-CoV-2 furin cleavage site remains a mystery

Authors: By Dr. Liji Thomas, MD Feb 17 2021

The ongoing pandemic of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has largely defied attempts to contain its spread by non-pharmaceutical interventions (NPIs). With the massive loss of life and economic damage, the only way out, in the absence of specific antiviral therapeutics, has been the development of vaccines to achieve population immunity.

A new study on the Preprints server discusses the origin of the furin cleavage site on the SARS-CoV-2 spike protein, which is responsible for the virus’s relatively high infectivity compared to relatives in the betacoronavirus subgenus.

The furin cleavage site

The SARS-CoV-2 is a betacoronavirus, and is most closely related to the bat SARS-related coronavirus (SARSr-CoV) represented by the genome sequence RaTG13, which shares 96% identity with the former. This has made the bat virus the most probable precursor of the virus in current circulation.

The origin of this strain is linked to the emergence of the novel furin cleavage site in the viral spike glycoprotein. The furin is a serine protease widely expressed in human cells, that cleaves the SARS-CoV-2 spike at the interface of its two subunits. It is encoded by a gene on chromosome 15.

Furin acts on substrates with single or paired basic residues during the processing of proteins within cells. Such a polybasic furin cleavage site is found in various proteins from many viruses, including Betacoronavirus Embecoviruses, and the Merbecovirus. However, within the betacoronaviruses of the sarbecovirus lineage B, this type of site is unique to SARS-CoV-2.

The study used a bioinformatic approach using the genomic data available on the National Center for Biotechnological Information (NCBI) databases, to identify the origin of the furin cleavage site.

Same ancestor

They found three coronaviruses that were very similar to the SARS-CoV-2 at the genomic level. These are Pangolin-CoVs (2017, 2019), Bat-SARS-like (CoVZC45, CoVZXC21) and bat RatG13.

The three genomic fingerprints used to identify these matches include fingerprint 1, in the orf1a RNA polymerase gene, including the nsp2 and nsp3 genes; fingerprint 2, at the beginning of S gene, covering the part encoding the N-terminal domain and the receptor-binding domain (RBD) that mediates attachment to the host cell receptor, the angiotensin-converting enzyme 2 (ACE2).; and fingerprint 3, the orf8 gene.

These fingerprints are distinctive to the three closely related coronaviruses only at the RNA level, but the amino acid sequences in the translated proteins are similar to other sarbecoviruses.

The sharing of these genomic sequences indicates their common ancestry, supported by other short sequence features, with one deletion and three insertions. All three strains show the same deletion-insertion pattern at the same four different locations in the spike gene.

Spike gene recombination in a common ancestor

The analysis of the phylogeny of these three strains showed that the first to diverge was the pangolin coronavirus, with the RatG13 being the closest. However, when only the spike is analyzed, there is a high similarity between the pangolin CoV, RaTG13 and SARS-CoV-2.

This may indicate the occurrence of recombination events between the Pangolin-CoV (2017) and RatG13 ancestors. This was followed by the shift of the pangolin CoV to pangolin hosts.Phylogenetic tree of the closely related SARS-CoV-2 coronaviruses based on complete genomesPhylogenetic tree of the closely related SARS-CoV-2 coronaviruses based on complete genomes.

Unique codons encoding arginines in the furin cleavage site

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The furin cleavage site consists of four amino acids PRRA, which are encoded by 12 inserted nucleotides in the S gene. A characteristic feature of this site is an arginine doublet.

This insertion could have occurred by random insertion mutation, recombination or by laboratory insertion. The researchers say the possibility of random insertion is too low to explain the origin of this motif.

Surprisingly, the CGGCGG codons encoding the two arginines of the doublet in SARS-CoV-2 are not found in any of the furin sites in other viral proteins expressed by a wide range of viruses.

Even within the SARS-CoV-2, where arginine is encoded by six codons, only a minority of arginine residues are encoded by the CGG codon. Again, only two of the 42 arginines in the SARS-CoV-2 spike are encoded by this codon – and these are in the PRRA motif.

For recombination to occur, there must be a donor, from another furin site and probably from another virus. In the absence of a known virus containing this arginine doublet encoded by the CGGCGG codons, the researchers discount the recombination theory as the mechanism underlying the emergence of PRRA in SARS-CoV-2.

For More Information: https://www.news-medical.net/news/20210217/The-origin-of-SARS-CoV-2-furin-cleavage-site-remains-a-mystery.aspx

Pathophysiology of COVID-19:

Mechanisms Underlying Disease Severity and Progression

Authors: Mary Kathryn Bohn,1,2, Alexandra Hall,1 Lusia Sepiashvili,1,2, Benjamin Jung,1,2 Shannon, Steele,1 and Khosrow Adeli1,2,3

The global epidemiology of coronavirus disease 2019 (COVID-19) suggests a wide spectrum of clinical severity, ranging from asymptomatic to fatal. Although the clinical and laboratory characteristics of COVID-19 patients have been well characterized, the pathophysiological mechanisms underlying disease severity and progression remain unclear. This review highlights key mechanisms that have been proposed to contribute to COVID-19 progression from viral entry to multisystem organ failure, as well as the central role of the immune response in successful viral clearance or progression to death.

Introduction

Coronavirus disease 2019 (COVID-19) is caused by a novel beta-coronavirus known as Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). As of June 15, 2020, the number of global confirmed cases has surpassed 8 million, with over 400,000 reported mortalities. The unparalleled pathogenicity and global impact of this pandemic has rapidly engaged the scientific community in urgently needed research. Preliminary reports from the Chinese Center for Disease Control and Prevention have estimated that the large majority of confirmed SARS-CoV-2 cases are mild (81%), with ~14% progressing to severe pneumonia and 5% developing acute respiratory distress syndrome (ARDS), sepsis, and/or multisystem organ failure (MOF) (144). Although more data is urgently needed to elucidate the global epidemiology of COVID-19 (80), a wide spectrum of clinical severity is evident, with most patients able to mount a sufficient and appropriate immune response, ultimately leading to viral clearance and case resolution. However, a significant subset of patients present with severe clinical manifestations, requiring life-supporting treatment (51). The pathophysiological mechanisms behind key events in the progression from mild to severe disease remain unclear, warranting further investigation to inform therapeutic decisions. Here, we review the current literature and summarize key proposed mechanisms of COVID-19 pathophysiological progression (FIGURE 1). Key Pathophysiological Mechanisms: Our Current Understanding Viral Invasion The first step in COVID-19 pathogenesis is viral invasion via its target host cell receptors. SARSCoV-2 viral entry has been described in detail elsewhere (138). In brief, SARS-CoV-2 consists of four main structural glycoproteins: spike (S), membrane (M), envelope (E), and nucleocapsid (N). The M, E, and N proteins are critical for viral particle assembly and release, whereas the S protein is responsible for viral binding and entry into host cells (33, 76, 89, 143, 148). Similar to SARS-CoV, several researchers have identified human angiotensin converting enzyme 2 (ACE2) as an entry receptor for SARS-CoV-2 (75, 99, 148, 156). SARSCoV-2 is mostly transmissible through large respiratory droplets, directly infecting cells of the upper and lower respiratory tract, especially nasal ciliated and alveolar epithelial cells (161). In addition to the lungs, ACE2 is also expressed in various other human tissues, such as the small intestine, kidneys, heart, thyroid, testis, and adipose tissue, indicating the virus may directly infect cells of other organ systems when viremia is present (77). Interestingly, although the S proteins of SARS-CoV-2 and SARSCoV share 72% homology in amino acid sequences, SARS-CoV-2 has been reported to have a higher affinity for the ACE2 receptor (18, 21, 143). Following host cell binding, viral and cell membranes fuse, enabling the virus to enter into the cell (89). For many coronaviruses, including SARS-CoV, host cell binding alone is insufficient to facilitate membrane fusion, requiring S-protein priming or cleavage by host cell proteases or transmembrane serine proteases (9, 10, 90, 94, 108). Indeed, Hoffman and colleagues demonstrated that S-protein priming by transmembrane serine protease 2 (TMPRSS2), which may be substituted by cathepsin B/L, is required to facilitate SARS-CoV-2 entry into host cells (58). In addition, unlike other coronaviruses, SARS-CoV-2 has been reported to possess a furin-like cleavage site in the S-protein domain, located between the S1 and S2 subunits (31, 138). Furin-like proteases are ubiquitously expressed, albeit at low levels, indicating that S-protein priming at this cleavage site may contribute to the widened cell tropism and enhanced transmissibility of SARS-CoV-2 (123). However, whether furin-like protease-mediated cleavage is required for SARS-CoV-2 host entry has yet to be determined. Blocking or inhibiting these processing enzymes may serve as a potential antiviral target (130). Interestingly, SARS-CoV-2 has developed a unique S1/S2 cleavage site in its S protein, characterized by a four-amino acid insertion, which seems to be absent in all other coronaviruses (4). This molecular mimicry has been identified as an efficient evolutionary adaptation that some viruses have acquired for exploiting the host cellular machinery. Once the nucleocapsid is deposited into the cytoplasm of the host cell, the RNA genome is replicated and translated into structural and accessory proteins. Vesicles containing the newly formed viral particles are then transported to and fuse with the plasma membrane, releasing them to infect other host cells in the same fashion (33, 89, 105). Although much progress has been made in our understanding of the mechanisms underlying SARS-CoV-2 invasion, additional research is needed to delineate exactly how cleavage of the S proteins by TMPRSS2 confers viral particle entry as well as how S-protein cleavage by membrane proteases contributes to viral penetration.

For More Information: https://journals.physiology.org/doi/pdf/10.1152/physiol.00019.2020

The furin cleavage site in the SARS-CoV-2 spike protein is required for transmission in ferrets

Authors: WilliamsonJack A. HassardEcco StallerBrian HanleyMichael OsbornMauro GiaccaAndrew D. DavidsonDavid A. Matthews & Wendy S. Barclay

SARS-CoV-2 entry requires sequential cleavage of the spike glycoprotein at the S1/S2 and the S2ʹ cleavage sites to mediate membrane fusion. SARS-CoV-2 has a polybasic insertion (PRRAR) at the S1/S2 cleavage site that can be cleaved by furin. Using lentiviral pseudotypes and a cell-culture-adapted SARS-CoV-2 virus with an S1/S2 deletion, we show that the polybasic insertion endows SARS-CoV-2 with a selective advantage in lung cells and primary human airway epithelial cells, but impairs replication in Vero E6, a cell line used for passaging SARS-CoV-2. Using engineered spike variants and live virus competition assays and by measuring growth kinetics, we find that the selective advantage in lung and primary human airway epithelial cells depends on the expression of the cell surface protease TMPRSS2, which enables endosome-independent virus entry by a route that avoids antiviral IFITM proteins. SARS-CoV-2 virus lacking the S1/S2 furin cleavage site was shed to lower titres from infected ferrets and was not transmitted to cohoused sentinel animals, unlike wild-type virus. Analysis of 100,000 SARS-CoV-2 sequences derived from patients and 24 human postmortem tissues showed low frequencies of naturally occurring mutants that harbour deletions at the polybasic site. Taken together, our findings reveal that the furin cleavage site is an important determinant of SARS-CoV-2 transmission.

For More Information: https://www.nature.com/articles/s41564-021-00908-w#Sec