Establishment of a reverse genetics system for SARS-CoV-2 using circular polymerase extension reaction

Torii, Shiho; Ono, Chikako; Suzuki, Rigel; Morioka, Yuhei; Anzai, Itsuki; Fauzyah, Yuzy; Maeda, Yusuke; Kamitani, Wataru; Fukuhara, Takasuke; Matsuura, Yoshiharu

doi:10.1016/j.celrep.2021.109014

Cited by 128 publications

(168 citation statements)

References 25 publications

(27 reference statements)

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“…To investigate the effect of RBM mutations on viral replication, we artificially generated recombinant SARS-CoV-2 viruses that harbor the mutations of interest as well as parental recombinant virus by a reverse genetics system (Torii et al, 2021). The nucleotide similarity of SARS-CoV-2 strain WK-521 (GISAID ID: EPI_ISL_408667) (Matsuyama et al, 2020), the backbone of the artificially generated recombinant SARS-CoV-2, to strain Wuhan-Hu-1 (GenBank: NC_045512.2) (Wu et al, 2020) is 99.91% (27 nucleotide difference); the sequences encoding the S protein between these two strains are identical, indicating that strain WK-521 is a SARS-CoV-2 prototype.…”

Section: Promotion Of Sars-cov-2 Replication In Cell Cultures By the L452 Mutationmentioning

confidence: 99%

“…Recombinant SARS-CoV-2 was generated by circular polymerase extension reaction (CPER) as previously described (Torii et al, 2021). In brief, 9 DNA fragments encoding the partial genome of SARS-CoV-2 (strain WK-521; GISAID ID: EPI_ISL_408667) (Matsuyama et al, 2020) were prepared by PCR using PrimeSTAR GXL DNA polymerase (Takara, cat# R050A).…”

Section: Sars-cov-2 Reverse Geneticsmentioning

confidence: 99%

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SARS-CoV-2 spike L452R variant evades cellular immunity and increases infectivity

Motozono

Toyoda

Zahradník

et al. 2021

Cell Host & Microbe

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471

541

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Many SARS-CoV-2 variants with naturally acquired mutations have emerged. These mutations can affect viral properties such as infectivity and immune resistance. Although the sensitivity of naturally occurring SARS-CoV-2 variants to humoral immunity has been investigated, sensitivity to human leukocyte antigen (HLA)-restricted cellular immunity remains largely unexplored. Here, we demonstrate that two recently emerging mutations in the receptor-binding domain of the SARS-CoV-2 spike protein, L452R (in B.1.427/429 and B.1.617) and Y453F (in B.1.1.298), confer escape from HLA-A24-restricted cellular immunity. These mutations reinforce affinity toward the host entry receptor ACE2. Notably, the L452R mutation increases spike stability, viral infectivity, viral fusogenicity, and thereby promotes viral replication. These data suggest that HLA-restricted cellular immunity potentially affects the evolution of viral phenotypes and that a further threat of the SARS-CoV-2 pandemic is escape from cellular immunity.

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Section: Promotion Of Sars-cov-2 Replication In Cell Cultures By the L452 Mutationmentioning

confidence: 99%

Section: Sars-cov-2 Reverse Geneticsmentioning

confidence: 99%

SARS-CoV-2 spike L452R variant evades cellular immunity and increases infectivity

Motozono

Toyoda

Zahradník

et al. 2021

Cell Host & Microbe

Self Cite

471

541

View full text Add to dashboard Cite

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“…HeLa-ACE2-TMPRSS2 cells (JCRB1835 (Kawase et al, 2012) were maintained in Dulbecco's modified Eagle's medium (low glucose) (Sigma-Aldrich, Cat# D6046-500ML) containing 10% FBS, 1 mg/ml G418 and 1% PS. HEK293-C34 cells, the IFNAR1 KO HEK293 cells expressing human ACE2 and TMPRSS2 by doxycycline treatment (Torii et al, 2021), were maintained in Dulbecco's modified Eagle's medium (high glucose) (Sigma-Aldrich, Cat# R8758-500ML) containing 10% FBS, 10 μg/ml blasticidin (InvivoGen, Cat# ant-bl-1) and 1% PS.…”

Section: Referencesmentioning

confidence: 99%

“…Recombinant SARS-CoV-2 was generated by circular polymerase extension reaction (CPER) as previously described (Motozono et al, 2021;Torii et al, 2021). In brief, 9 DNA fragments encoding the partial genome of SARS-CoV-2 (strain WK-521, PANGO lineage A; GISAID ID: EPI_ISL_408667) (Matsuyama et al, 2020) were prepared by PCR using PrimeSTAR GXL DNA polymerase (Takara, cat# R050A).…”

Section: Sars-cov-2 Reverse Geneticsmentioning

confidence: 99%

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SARS-CoV-2 spike P681R mutation, a hallmark of the Delta variant, enhances viral fusogenicity and pathogenicity

Saito

Nasser

Uriu

et al. 2021

Preprint

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During the current SARS-CoV-2 pandemic, a variety of mutations have been accumulated in the viral genome, and at least five variants of concerns (VOCs) have been considered as the hazardous SARS-CoV-2 variants to the human society. The newly emerging VOC, the B.1.617.2 lineage (delta variant), closely associates with a huge COVID-19 surge in India in Spring 2021. However, its virological property remains unclear. Here, we show that the B.1.617 variants are highly fusogenic and form prominent syncytia. Bioinformatic analyses reveal that the P681R mutation in the spike protein is highly conserved in this lineage. Although the P681R mutation decreases viral infectivity, this mutation confers the neutralizing antibody resistance. Notably, we demonstrate that the P681R mutation facilitates the furin-mediated spike cleavage and enhances and accelerates cell-cell fusion. Our data suggest that the P681R mutation is a hallmark characterizing the virological phenotype of this newest VOC, which may associate with viral pathogenicity.

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Reverse genetics systems for SARS‐CoV‐2

Wang

Peng

Jin

et al. 2022

Journal of Medical Virology

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The ongoing pandemic of coronavirus disease 2019 (COVID‐19) has caused severe public health crises and heavy economic losses. Limited knowledge about this deadly virus impairs our capacity to set up a toolkit against it. Thus, more studies on severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) biology are urgently needed. Reverse genetics systems, including viral infectious clones and replicons, are powerful platforms for viral research projects, spanning many aspects such as the rescues of wild‐type or mutant viral particles, the investigation of viral replication mechanism, the characterization of viral protein functions, and the studies on viral pathogenesis and antiviral drug development. The operations on viral infectious clones are strictly limited in the Biosafety Level 3 (BSL3) facilities, which are insufficient, especially during the pandemic. In contrast, the operation on the noninfectious replicon can be performed in Biosafety Level 2 (BSL2) facilities, which are widely available. After the outbreak of COVID‐19, many reverse genetics systems for SARS‐CoV‐2, including infectious clones and replicons are developed and given plenty of options for researchers to pick up according to the requirement of their research works. In this review, we summarize the available reverse genetics systems for SARS‐CoV‐2, by highlighting the features of these systems, and provide a quick guide for researchers, especially those without ample experience in operating viral reverse genetics systems.

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Establishment of a reverse genetics system for SARS-CoV-2 using circular polymerase extension reaction

Abstract: Highlights d A quick PCR-based reverse genetics system is established for SARS-CoV-2 d SARS-CoV-2 recombinants harboring reporter genes or mutations can be generated

Cited by 128 publications

References 25 publications

SARS-CoV-2 spike L452R variant evades cellular immunity and increases infectivity

SARS-CoV-2 spike L452R variant evades cellular immunity and increases infectivity

SARS-CoV-2 spike P681R mutation, a hallmark of the Delta variant, enhances viral fusogenicity and pathogenicity

Reverse genetics systems for SARS‐CoV‐2

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