Development of A MERS-CoV Replicon Cell Line for Antiviral Screening

Chen, Jing; Hu, Bing; Zhao, Kai; Luo, Yun; Lin, Hao; Shi, Zheng Li

doi:10.1007/s12250-020-00341-z

Cited by 8 publications

(4 citation statements)

References 23 publications

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“… 38 N/A MERS-CoV-NP TCTGTCCTGTCTCCGCCAAT Chen et al. 38 N/A human IFNβ CTTGGATTCCTACAAAGAAGCAGC This paper N/A R-human IFNβ TCCTCCTTCTGGAACTGCTGCA This paper N/A F-human ISG15 CTCTGAGCATCCTGGTGAGGAA This paper N/A R-human ISG15 AAGGTCAGCCAGAACAGGTCGT This paper N/A F-human GAPDH ACAACTTTGGTATCGTGGAAGG This paper N/A R-human GAPDH GCCATCACGCCACAGTTTC This paper N/A CoV-FWD3: GGTTGGGAYTAYCCHAARTGTGA Latinne et al. 40 N/A CoV-RVS3: CCATCATCASWYRAATCATCATA Latinne et al.…”

Section: Methodsmentioning

confidence: 99%

A bat MERS-like coronavirus circulates in pangolins and utilizes human DPP4 and host proteases for cell entry

Chen

Yang

et al. 2023

Cell

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Section: Methodsmentioning

confidence: 99%

A bat MERS-like coronavirus circulates in pangolins and utilizes human DPP4 and host proteases for cell entry

Chen

Yang

et al. 2023

Cell

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“…Nluc expression and subgenomic N RNA production were measured in BHK-21 cells transfected with the plasmids. The MERS-CoV replicon was generated in our previous study by modified Nluc replacement [ 17 ].…”

Section: Methodsmentioning

confidence: 99%

Identification of TMEM53 as a novel SADS-CoV restriction factor that targets viral RNA-dependent RNA polymerase

Yao

Luo

Wang

et al. 2023

Emerging Microbes & Infections

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Zoonotic transmission of coronaviruses (CoVs) poses a serious public health threat. Swine acute diarrhea syndrome coronavirus (SADS-CoV), originating from a bat HKU2-related CoV, causes devastating swine diseases and poses a high risk of spillover to humans. Currently, licensed therapeutics that can prevent potential human outbreaks are unavailable. Identifying the cellular proteins that restrict viral infection is imperative for developing effective interventions and therapeutics. We utilized a large-scale human cDNA screening and identified transmembrane protein 53 (TMEM53) as a novel cell-intrinsic SADS-CoV restriction factor. The inhibitory effect of TMEM53 on SADS-CoV infection was found to be independent of canonical type I interferon responses. Instead, TMEM53 interacts with non-structural protein 12 (NSP12) and disrupts viral RNA-dependent RNA polymerase (RdRp) complex assembly by interrupting NSP8-NSP12 interaction, thus suppressing viral RdRp activity and RNA synthesis. Deleting the transmembrane domain of TMEM53 resulted in the abrogation of TMEM53-NSP12 interaction and TMEM53 antiviral activity. Importantly, TMEM53 exhibited broad antiviral activity against multiple HKU2-related CoVs. Our findings reveal a novel role of TMEM53 in SADS-CoV restriction and pave the way to host-directed therapeutics against HKU2-related CoV infection.

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“…This potentially allows the screening for antiviral compounds that target viral replication in cellulo (8). Prior to SARS-CoV-2, replicon systems had been created for other coronaviruses, such as SARS-CoV (sometimes called SARS-CoV-1) as well as the Middle East respiratory syndrome-related coronavirus (MERS-CoV) (9, 10).…”

Section: Introductionmentioning

confidence: 99%

T7 RNA polymerase-independent expression of reporter genes from a T7 promoter-driven SARS-CoV-2 replicon-encoding DNA in human cells

Friedhoff,

Elfayres,

Mérindol

et al. 2024

Preprint

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Replicons, derived from RNA viruses, are genetic constructs retaining essential viral enzyme genes while lacking key structural protein genes. Upon introduction into cells, the genes carried by the replicon RNA are expressed, and the RNA self-replicates, yet viral particle production does not take place. Typically, RNA replicons are transcribed in vitro and are then electroporated in cells. However, it would be advantageous for the replicon to be generated in cells following DNA transfection instead of RNA. In this study, a bacterial artificial chromosome (BAC) DNA encoding a SARS-CoV-2 replicon under control of a T7 promoter was transfected into HEK293T cells engineered to functionally express the T7 RNA polymerase (T7 RNAP). Upon transfection of the BAC DNA, we observed low, but reproducible expression of reporter proteins GFP and luciferase carried by this replicon. Expression of the reporter proteins required linearization of the BAC DNA prior to transfection. Surprisingly, however, expression occurred independently of T7 RNAP. Gene expression was also insensitive to remdesivir treatment, suggesting that it did not involve self-replication of replicon RNA. Similar results were obtained in highly SARS-CoV-2 infection-permissive Calu-3 cells. Strikingly, prior expression of the SARS-CoV-2 N protein boosted expression from transfected SARS-CoV-2 RNA replicon but not from the replicon BAC DNA. In conclusion, transfection of a large DNA encoding a coronaviral replicon led to reproducible replicon gene expression through an unidentified mechanism. These findings highlight a novel pathway toward replicon gene expression from transfected replicon cDNA, offering valuable insights for the development of methods for DNA-based RNA replicon applications.

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Development of A MERS-CoV Replicon Cell Line for Antiviral Screening

Cited by 8 publications

References 23 publications

A bat MERS-like coronavirus circulates in pangolins and utilizes human DPP4 and host proteases for cell entry

A bat MERS-like coronavirus circulates in pangolins and utilizes human DPP4 and host proteases for cell entry

Identification of TMEM53 as a novel SADS-CoV restriction factor that targets viral RNA-dependent RNA polymerase

T7 RNA polymerase-independent expression of reporter genes from a T7 promoter-driven SARS-CoV-2 replicon-encoding DNA in human cells

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