A DNA-based non-infectious replicon system to study SARS-CoV-2 RNA synthesis

Feng, Xiaolong; Zhang, Xiaofan; Jiang, Shuangying; Tang, Yuanwei; Chen, Cheng; Krishna, Parthasarathy Abinand; Wang, Xiaoting; Dai, Junbiao; Zhao, Dan; Xia, Tian; Zeng, Jianyang

doi:10.1016/j.csbj.2022.08.044

Cited by 6 publications

(4 citation statements)

References 56 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Prior presence of the SARS-CoV-2 N structural protein has been shown to improve replicon RNA survival and expression (11). To uncover a possible role for SARS-CoV-2 N in stimulating the T7 RNAP-independent expression of marker genes from the replicon BAC DNA, we used previously generated HEK293T cells lentivirally transduced to express the N protein (12), and we also transiently transfected a mammalian expression plasmid encoding the same protein.…”

Section: Resultsmentioning

confidence: 99%

“…At the moment, it is not clear whether the BAC DNA T7 promoter is functional or not in HEK293T cells expressing T7 RNAP, as the absence of detectable replicon RNA might be due to degradation rather than a lack of synthesis. We are currently developing further a DNA-based SARS-CoV-2 replicon system, that will complement systems that have been described recently (11).…”

Section: Discussionmentioning

confidence: 99%

“…A transformative alternative is the direct expression of replicon RNA from a DNA construct within cells, offering several advantages for reverse genetics as applied to RNA viruses: (i) amplifying and purifying DNA is inexpensive compared to RNA; (ii) DNA transfection is less expensive and more efficient, routinely reaching >90% in some cellular models; (iii) stably maintaining a DNA construct is a reachable goal, through its integration into the cellular genome. Prior attempts at such an objective have been made, for instance by using a CMV promoter to govern replicon expression (11). In this study, we explored the possibility to produce SARS-CoV-2 replicon RNA from a T7 promoter-driven BAC construct introduced into human cells by standard transfection protocols.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

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

View full text Add to dashboard Cite

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.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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

View full text Add to dashboard Cite

show abstract

“…Viral escape will more likely occur when the crRNA inhibitor is applied as “mono-therapy”, but a combination of two or multiple crRNAs can result in additive or possibly even synergistic inhibition. A combinatorial attack will also increase the genetic threshold for the acquisition of crRNA resistance as more mutations will be required, which explains the clinical success of a combination drug therapy for HIV infection [ 56 , 57 , 58 , 59 ]. We selected the best crRNAs (5′UTR-1, nsp3-1, slippery, RdRp-2, helicase-3 and s2m) for a combinatorial approach and tested all possible combinations of two crRNAs in HEK293T cells co-transfected with the Luc reporter (100 ng), the Renilla plasmid (1 ng) and 300 ng of the CRISPR-Cas13d/crRNA constructs.…”

Section: Resultsmentioning

confidence: 99%

Efficient CRISPR-Cas13d-Based Antiviral Strategy to Combat SARS-CoV-2

Hussein

Ramos

Vink

et al. 2023

Viruses

View full text Add to dashboard Cite

The current SARS-CoV-2 pandemic forms a major global health burden. Although protective vaccines are available, concerns remain as new virus variants continue to appear. CRISPR-based gene-editing approaches offer an attractive therapeutic strategy as the CRISPR-RNA (crRNA) can be adjusted rapidly to accommodate a new viral genome sequence. This study aimed at using the RNA-targeting CRISPR-Cas13d system to attack highly conserved sequences in the viral RNA genome, thereby preparing for future zoonotic outbreaks of other coronaviruses. We designed 29 crRNAs targeting highly conserved sequences along the complete SARS-CoV-2 genome. Several crRNAs demonstrated efficient silencing of a reporter with the matching viral target sequence and efficient inhibition of a SARS-CoV-2 replicon. The crRNAs that suppress SARS-CoV-2 were also able to suppress SARS-CoV, thus demonstrating the breadth of this antiviral strategy. Strikingly, we observed that only crRNAs directed against the plus-genomic RNA demonstrated antiviral activity in the replicon assay, in contrast to those that bind the minus-genomic RNA, the replication intermediate. These results point to a major difference in the vulnerability and biology of the +RNA versus −RNA strands of the SARS-CoV-2 genome and provide important insights for the design of RNA-targeting antivirals.

show abstract

Reverse genetics systems for SARS-CoV-2: Development and applications

Cai,

Huang

2023

Virologica Sinica

View full text Add to dashboard Cite

A DNA-based non-infectious replicon system to study SARS-CoV-2 RNA synthesis

Cited by 6 publications

References 56 publications

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

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

Efficient CRISPR-Cas13d-Based Antiviral Strategy to Combat SARS-CoV-2

Reverse genetics systems for SARS-CoV-2: Development and applications

Contact Info

Product

Resources

About