Emergence and spread of a SARS-CoV-2 lineage A variant (A.23.1) with altered spike protein in Uganda

AbstractHere, we report SARS-CoV-2 genomic surveillance from March 2020 until January 2021 in Uganda, a landlocked East African country with a population of approximately 40 million people. We report 322 full SARS-CoV-2 genomes from 39,424 reported SARS-CoV-2 infections, thus representing 0.8% of the reported cases. Phylogenetic analyses of these sequences revealed the emergence of lineage A.23.1 from lineage A.23. Lineage A.23.1 represented 88% of the genomes observed in December 2020, then 100% of the genomes observed in January 2021. The A.23.1 lineage was also reported in 26 other countries. Although the precise changes in A.23.1 differ from those reported in the first three SARS-CoV-2 variants of concern (VOCs), the A.23.1 spike-protein-coding region has changes similar to VOCs including a change at position 613, a change in the furin cleavage site that extends the basic amino acid motif and multiple changes in the immunogenic N-terminal domain. In addition, the A.23.1 lineage has changes in non-spike proteins including nsp6, ORF8 and ORF9 that are also altered in other VOCs. The clinical impact of the A.23.1 variant is not yet clear and it has not been designated as a VOC. However, our findings of emergence and spread of this variant indicate that careful monitoring of this variant, together with assessment of the consequences of the spike protein changes for COVID-19 vaccine performance, are advisable.

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A SARS-CoV-2 lineage A variant (A.23.1) with altered spike has emerged and is dominating the current Uganda epidemic

10.1101/2021.02.08.21251393 ◽

2021 ◽

Cited By ~ 7

Author(s):

Daniel Lule Bugembe ◽

My V.T.Phan ◽

Isaac Ssewanyana ◽

Patrick Semanda ◽

Hellen Nansumba ◽

...

Keyword(s):

African Country ◽

Rapid Assessment ◽

Basic Amino Acid ◽

Spike Protein ◽

Coding Region ◽

Careful Monitoring ◽

East African ◽

Furin Cleavage ◽

Protein Coding ◽

Furin Cleavage Site

Introductory paragraphSARS-CoV-2 genomic surveillance in Uganda provides an opportunity to provide a focused description of the virus evolution in a small landlocked East African country. Here we show a recent shift in the local epidemic with a newly emerging lineage A.23 evolving into A.23.1 which is now dominating the Uganda cases and has spread to 26 other countries. Although the precise changes in A.23.1 as it has adapted are different from the changes in the variants of concern (VOC), the evolution shows convergence on a similar set of proteins. The A.23.1 spike protein coding region has accumulated changes that resemble many of the changes seen in VOC including a change at position 613, a change in the furin cleavage site that extends the basic amino acid motif, and multiple changes in the immunogenic N-terminal domain. In addition, the A.23.1lineage encodes changes in non-spike proteins that other VOC show (nsp6, ORF8 and ORF9). The clinical impact of the A.23.1 variant is not yet clear, however it is essential to continue careful monitoring of this variant, as well as rapid assessment of the consequences of the spike protein changes for vaccine efficacy.

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The Functional Consequences of the Novel Ribosomal Pausing Site in SARS-CoV-2 Spike Glycoprotein RNA

International Journal of Molecular Sciences ◽

10.3390/ijms22126490 ◽

2021 ◽

Vol 22 (12) ◽

pp. 6490

Author(s):

Olga A. Postnikova ◽

Sheetal Uppal ◽

Weiliang Huang ◽

Maureen A. Kane ◽

Rafael Villasmil ◽

...

Keyword(s):

Amino Acid ◽

Insertion Sequence ◽

Experimental Studies ◽

Spike Protein ◽

Spike Glycoprotein ◽

Furin Cleavage ◽

New Host ◽

S Protein ◽

Furin Cleavage Site ◽

Ribosome Pausing

The SARS-CoV-2 Spike glycoprotein (S protein) acquired a unique new 4 amino acid -PRRA- insertion sequence at amino acid residues (aa) 681–684 that forms a new furin cleavage site in S protein as well as several new glycosylation sites. We studied various statistical properties of the -PRRA- insertion at the RNA level (CCUCGGCGGGCA). The nucleotide composition and codon usage of this sequence are different from the rest of the SARS-CoV-2 genome. One of such features is two tandem CGG codons, although the CGG codon is the rarest codon in the SARS-CoV-2 genome. This suggests that the insertion sequence could cause ribosome pausing as the result of these rare codons. Due to population variants, the Nextstrain divergence measure of the CCU codon is extremely large. We cannot exclude that this divergence might affect host immune responses/effectiveness of SARS-CoV-2 vaccines, possibilities awaiting further investigation. Our experimental studies show that the expression level of original RNA sequence “wildtype” spike protein is much lower than for codon-optimized spike protein in all studied cell lines. Interestingly, the original spike sequence produces a higher titer of pseudoviral particles and a higher level of infection. Further mutagenesis experiments suggest that this dual-effect insert, comprised of a combination of overlapping translation pausing and furin sites, has allowed SARS-CoV-2 to infect its new host (human) more readily. This underlines the importance of ribosome pausing to allow efficient regulation of protein expression and also of cotranslational subdomain folding.

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Viral and Host Attributes Underlying the Origins of Zoonotic Coronaviruses in Bats

Comparative Medicine ◽

10.30802/aalas-cm-21-000027 ◽

2021 ◽

Author(s):

Alison E Stout ◽

Qinghua Guo ◽

Jean K Millet ◽

Gary R Whittaker

Keyword(s):

Viral Pathogenesis ◽

Spike Protein ◽

Future Research ◽

Receptor Binding Domain ◽

Viral Reservoirs ◽

Furin Cleavage ◽

Proteolytic Activation ◽

Furin Cleavage Site ◽

Unique Aspect ◽

Genome Level

With a presumed origin in bats, the COVID-19 pandemic has been a major source of morbidity and mortality in the humanpopulation, and the causative agent, SARS-CoV-2, aligns most closely at the genome level with the bat coronavirusesRaBtCoV4991/RaTG13 and RmYN02. The ability of bats to provide reservoirs of numerous viruses in addition to coronavirusesremains an active area of research. Unique aspects of the physiology of the chiropteran immune system may contributeto the ability of bats to serve as viral reservoirs. The coronavirus spike protein plays important roles in viral pathogenesis and the immune response. Although much attention has focused on the spike receptor-binding domain, a unique aspect of SARS-CoV-2 as compared with its closest relatives is the presence of a furin cleavage site in the S1–S2 region of the spike protein. Proteolytic activation is likely an important feature that allows SARS-CoV-2—and other coronaviruses—to overcome the species barriers and thus cause human disease. The diversity of bat species limits the ability to draw broad conclusions about viral pathogenesis, but comparisons across species and with reference to humans and other susceptible mammals may guide future research in this regard.

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Sequence Analysis Indicates that 2019-nCoV Virus Contains a Putative Furin Cleavage Site at the Boundary of S1 and S2 Domains of Spike Protein

10.31219/osf.io/nkcrf ◽

2020 ◽

Author(s):

Z. Galen WO

Keyword(s):

Amino Acid ◽

Cleavage Site ◽

Spike Protein ◽

Sequence Motif ◽

Furin Cleavage ◽

Host Cell Membrane ◽

Virus Family ◽

Prediction Program ◽

Furin Cleavage Site ◽

Novel Coronavirus

The infectious 2019-nCoV virus, which caused the current novel coronavirus pneumonia epidemic outbreak, possesses a unique 4-Amino Acid insert at the boundary of the two subdomains (S1 and S2) of Spike protein based on multiple protein sequence alignment with the large SARS and SARS-related virus family. Using Bat CoV_RaTG13 Spike protein as reference (sharing 97% aa identity) the 4-amino acid insert can be identified as PRRA (AA position 681-684). The effect of the 4-AA insertion is the presence of a furin signature sequence motif (PRRARSV) at the boundary of S1 and S2 domains of spike protein. This sequence motif consists the required Arg residue for P1 and P4 position of Furin site. In addition, it contains Arg at P3 site as well as Ser at P1’ site of furin motif. This sequence motif matches Aerolysin furin site in FurinDB and was predicted to be moderately strong (score 0.62) by ProP, a protease cleavage site prediction program. This finding suggests that the infectious 2019-nCoV virus, unlike SARS viruses, may be processed via cellular furin recognition and cleavage of the spike protein before host cell membrane fusion and entry. This putative furin site in spike protein of 2019-nCoV virus, if proven to be functional, suggests the potential of looking into agents inhibiting furin as therapeutic mean for the treatment of the novel coronavirus pneumonia.

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First detection and molecular characterization of kobuvirus from a dog in Turkey

Medycyna Weterynaryjna ◽

10.21521/mw.6248 ◽

2019 ◽

Vol 75 (05) ◽

pp. 6248-2019

Author(s):

ZEYNEP AKKUTAY-YOLDAR ◽

TAYLAN KOÇ B. ◽

ÇIĞDEM OĞUZOĞLU T.

Keyword(s):

Molecular Characterization ◽

Rectal Swab ◽

Clinical Symptoms ◽

Phylogenetic Analyses ◽

Amino Acid Sequences ◽

Coding Region ◽

Gastrointestinal Infections ◽

Protein Coding ◽

Pcr Product ◽

Domestic Carnivores

Canine kobuvirus (CaKVs) is a newly emerging virus detected in dogs in several countries. However, kobuvirus infection has not yet been described in domestic carnivores in Turkey. In this study, we tested blood and rectal swab samples to determine the presence of kobuvirus in a dog with clinical symptoms by reverse transcription-polymerase chain reaction (RT-PCR), using 3D (RNA polymerase) region primers of canine kobuviruses. To provide molecular characterization data, the Maximum Likelihood (ML) method was used for the phylogenetic analyses. The PCR product of the partial protein-coding region of the 3D protein gene from the rectal swab was amplified, purified, and sequenced. Phylogenetic analysis of amino acid sequences suggests that our CaKV strain was closely related to US-CaKVs,and placed on a monophyletic clade as a sister branch localized in the CaKV cluster. These results indicate that CaKV exists in dogs in Turkey. With a similarity of 94.2–96.1%, it is like other CaKVs. To our knowledge, this is the first report of CaKV infection of a dog by in Turkey. Further studies are needed to determine its role in dog gastrointestinal infections.

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Variation of mitochondrial minichromosome composition in Hoplopleura lice (Phthiraptera: Hoplopleuridae) from rats

10.21203/rs.3.rs-42350/v5 ◽

2020 ◽

Author(s):

Yi-Tian Fu ◽

Yu Nie ◽

De-Yong Duan ◽

Guo-Hua Liu

Keyword(s):

Phylogenetic Analyses ◽

Amino Acid Sequences ◽

Coding Region ◽

Illumina Platform ◽

Protein Coding ◽

Protein Coding Genes ◽

Blood Sucking ◽

The Family ◽

Sucking Lice ◽

Mt Genome

Abstract Background: The family Hoplopleuridae contains at least 183 species of blood-sucking lice, which widely parasitize both mice and rats. Fragmented mitochondrial (mt) genomes have been reported in two rat lice (Hoplopleura kitti and H. akanezumi) from this family, but some minichromosomes were unidentified in their mt genomes.Methods: We sequenced the mt genome of the rat louse Hoplopleura sp. with an Illumina platform and compared its mt genome organization with H. kitti and H. akanezumi.Results: Fragmented mt genome of the rat louse Hoplopleura sp. contains 37 genes which are on 12 circular mt minichromosomes. Each mt minichromosome is 1.8–2.7 kb long and contains 1–5 genes and one large non-coding region. The gene content and arrangement of mt minichromosomes of Hoplopleura sp. (n = 3) and H. kitti (n = 3) are different from those in H. akanezumi (n = 3). Phylogenetic analyses based on the deduced amino acid sequences of the eight protein-coding genes showed that the Hoplopleura sp. was more closely related to H. akanezumi than to H. kitti, and then they formed a monophyletic group.Conclusions: Comparison among the three rat lice revealed variation in the composition of mt minichromosomes within the genus Hoplopleura. Hoplopleura sp. is the first species from the family Hoplopleuridae for which a complete fragmented mt genome has been sequenced. The new data provide useful genetic markers for studying the population genetics, molecular systematics and phylogenetics of blood-sucking lice.

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The Polybasic Cleavage Site in SARS-CoV-2 Spike Modulates Viral Sensitivity to Type I Interferon and IFITM2

Journal of Virology ◽

10.1128/jvi.02422-20 ◽

2021 ◽

Vol 95 (9) ◽

Cited By ~ 4

Author(s):

Helena Winstone ◽

Maria Jose Lista ◽

Alisha C. Reid ◽

Clement Bouton ◽

Suzanne Pickering ◽

...

Keyword(s):

Viral Entry ◽

Cleavage Site ◽

Type I Interferons ◽

Spike Protein ◽

Type I ◽

Furin Cleavage ◽

Independent Manner ◽

Type I Ifns ◽

Furin Cleavage Site ◽

Potent Inhibition

ABSTRACT The cellular entry of severe acute respiratory syndrome-associated coronaviruses types 1 and 2 (SARS-CoV-1 and -2) requires sequential protease processing of the viral spike glycoprotein. The presence of a polybasic cleavage site in SARS-CoV-2 spike at the S1/S2 boundary has been suggested to be a factor in the increased transmissibility of SARS-CoV-2 compared to SARS-CoV-1 by facilitating maturation of the spike precursor by furin-like proteases in the producer cells rather than endosomal cathepsins in the target. We investigate the relevance of the polybasic cleavage site in the route of entry of SARS-CoV-2 and the consequences this has for sensitivity to interferons (IFNs) and, more specifically, the IFN-induced transmembrane (IFITM) protein family that inhibit entry of diverse enveloped viruses. We found that SARS-CoV-2 is restricted predominantly by IFITM2, rather than IFITM3, and the degree of this restriction is governed by route of viral entry. Importantly, removal of the cleavage site in the spike protein renders SARS-CoV-2 entry highly pH and cathepsin dependent in late endosomes, where, like SARS-CoV-1 spike, it is more sensitive to IFITM2 restriction. Furthermore, we found that potent inhibition of SARS-CoV-2 replication by type I but not type II IFNs is alleviated by targeted depletion of IFITM2 expression. We propose that the polybasic cleavage site allows SARS-CoV-2 to mediate viral entry in a pH-independent manner, in part to mitigate against IFITM-mediated restriction and promote replication and transmission. This suggests that therapeutic strategies that target furin-mediated cleavage of SARS-CoV-2 spike may reduce viral replication through the activity of type I IFNs. IMPORTANCE The furin cleavage site in the spike protein is a distinguishing feature of SARS-CoV-2 and has been proposed to be a determinant for the higher transmissibility between individuals, compared to SARS-CoV-1. One explanation for this is that it permits more efficient activation of fusion at or near the cell surface rather than requiring processing in the endosome of the target cell. Here, we show that SARS-CoV-2 is inhibited by antiviral membrane protein IFITM2 and that the sensitivity is exacerbated by deletion of the furin cleavage site, which restricts viral entry to low pH compartments. Furthermore, we find that IFITM2 is a significant effector of the antiviral activity of type I interferons against SARS-CoV-2 replication. We suggest that one role of the furin cleavage site is to reduce SARS-CoV-2 sensitivity to innate immune restriction, and thus, it may represent a potential therapeutic target for COVID-19 treatment development.

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Gene of the month: the 2019-nCoV/SARS-CoV-2 novel coronavirus spike protein

Journal of Clinical Pathology ◽

10.1136/jclinpath-2020-206658 ◽

2020 ◽

Vol 73 (7) ◽

pp. 366-369 ◽

Cited By ~ 23

Author(s):

Tahir S Pillay

Keyword(s):

Receptor Binding ◽

Transmembrane Domain ◽

Vaccine Development ◽

Protein S ◽

Respiratory Illness ◽

Spike Protein ◽

Furin Cleavage ◽

Furin Cleavage Site ◽

Cellular Entry ◽

Novel Coronavirus

The year 2020 has seen a major and sustained outbreak of a novel betacoronavirus (severe acute respiratory syndrome (SARS)-coronavirus (CoV)-2) infection that causes fever, severe respiratory illness and pneumonia, a disease called COVID-19. At the time of writing, the death toll was greater than 120 000 worldwide with more than 2 million documented infections. The genome of the CoV encodes a number of structural proteins that facilitate cellular entry and assembly of virions, of which the spike protein S appears to be critical for cellular entry. The spike protein guides the virus to attach to the host cell. The spike protein contains a receptor-binding domain (RBD), a fusion domain and a transmembrane domain. The RBD of spike protein S binds to Angiotensin Converting Enzyme 2 (ACE2) to initiate cellular entry. The spike protein of SARS-CoV-2 shows more than 90% amino acid similarity to the pangolin and bat CoVs and these also use ACE2 as a receptor. Binding of the spike protein to ACE2 exposes the cleavage sites to cellular proteases. Cleavage of the spike protein by transmembrane protease serine 2 and other cellular proteases initiates fusion and endocytosis. The spike protein contains an addition furin cleavage site that may allow it to be ‘preactivated’ and highly infectious after replication. The fundamental role of the spike protein in infectivity suggests that it is an important target for vaccine development, blocking therapy with antibodies and diagnostic antigen-based tests. This review briefly outlines the structure and function of the 2019 novel CoV/SARS-CoV-2 spike protein S.

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Metalloproteinase-dependent and TMPRSS2-independnt cell surface entry pathway of SARS-CoV-2 requires the furin-cleavage site and the S2 domain of spike protein

10.1101/2021.12.14.472513 ◽

2021 ◽

Author(s):

Mizuki Yamamoto ◽

Jin Gohda ◽

Ayako Kobayashi ◽

Keiko Tomita ◽

Youko Hirayama ◽

...

Keyword(s):

Cell Surface ◽

Spike Protein ◽

Dependent Manner ◽

Furin Cleavage ◽

Entry Pathway ◽

Furin Cleavage Site ◽

Metalloproteinase Inhibitors ◽

A Cell ◽

A Disintegrin And Metalloproteinase ◽

Human Coronaviruses

The ongoing global vaccination program to prevent SARS-CoV-2 infection, the causative agent of COVID-19, has had significant success. However, recently virus variants have emerged that can evade the immunity in a host achieved through vaccination. Consequently, new therapeutic agents that can efficiently prevent infection from these new variants, and hence COVID-19 spread are urgently required. To achieve this, extensive characterization of virus-host cell interactions to identify effective therapeutic targets is warranted. Here, we report a cell surface entry pathway of SARS-CoV-2 that exists in a cell type-dependent manner is TMPRSS2-independent but sensitive to various broad-spectrum metalloproteinase inhibitors such as marimastat and prinomastat. Experiments with selective metalloproteinase inhibitors and gene-specific siRNAs revealed that a disintegrin and metalloproteinase 10 (ADAM10) is partially involved in the metalloproteinase pathway. Consistent with our finding that the pathway is unique to SARS-CoV-2 among highly pathogenic human coronaviruses, both the furin cleavage motif in the S1/S2 boundary and the S2 domain of SARS-CoV-2 spike protein are essential for metalloproteinase-dependent entry. In contrast, the two elements of SARS-CoV-2 independently contributed to TMPRSS2-dependent S2 priming. The metalloproteinase pathway is involved in SARS-CoV-2-induced syncytia formation and cytopathicity, leading us to theorize that it is also involved in the rapid spread of SARS-CoV-2 and the pathogenesis of COVID-19. Thus, targeting the metalloproteinase pathway in addition to the TMPRSS2 and endosome pathways could be an effective strategy by which to cure COVID-19 in the future.

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