Construction and characterization of two SARS‐CoV‐2 minigenome replicon systems

Zhang, Hu; Fischer, Donald R.; Shuda, Masahiro; Moore, Patrick S.; Gao, Shou‐Jiang; Ambrose, Zandrea; Guo, Haitao

doi:10.1002/jmv.27650

Cited by 17 publications

(25 citation statements)

References 67 publications

(173 reference statements)

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“…Notably, a recent study on SARS‐CoV‐2 replicon showed that BAC‐SARS‐CoV‐2 system can generate nonnatural spliced viral RNA species. 29 However, full‐length viral genomic RNA is known to be packaged preferentially. 30 Whether the spliced RNA species might be packaged into ΔS‐S‐Flag or ΔN‐N‐Flag leading to further attenuation of the BAC‐rescued single‐cycle virus requires further analysis.…”

Section: Discussionmentioning

confidence: 99%

Production of single‐cycle infectious SARS‐CoV‐2 through a trans‐complemented replicon

Cheung

Lui

et al. 2022

Journal of Medical Virology

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Single‐cycle infectious virus can elicit close‐to‐natural immune response and memory. One approach to generate single‐cycle severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) is through deletion of structural genes such as spike (S) and nucleocapsid (N). Transcomplementation of the resulting ΔS or ΔN virus through enforced expression of S or N protein in the cells gives rise to a live but unproductive virus. In this study, ΔS and ΔN BAC clones were constructed and their live virions were rescued by transient expression of S and N proteins from the ancestral and the Omicron strains. ΔS and ΔN virions were visualized by transmission electron microscopy. Virion production of ΔS was more efficient than that of ΔN. The coated S protein from ΔS was delivered to infected cells in which the expression of N protein was also robust. In contrast, expression of neither S nor N was detected in ΔN‐infected cells. ΔS underwent viral RNA replication, induced type I interferon (IFN) response, but did not form plaques. Despite RNA replication in cells, ΔS infection did not produce viral progeny in culture supernatant. Interestingly, viral RNA replication was not further enhanced upon overexpression of S protein. Taken together, our work provides a versatile platform for development of single‐cycle vaccines for SARS‐CoV‐2.

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Section: Discussionmentioning

confidence: 99%

Production of single‐cycle infectious SARS‐CoV‐2 through a trans‐complemented replicon

Cheung

Lui

et al. 2022

Journal of Medical Virology

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“…In the study, we designed, constructed, and characterized a dual-promoter-driven and dual-reporter-expressing SARS2 replicon. Our replicon contained the genomic organization from 5′ end to 3′ end: LTR-T7-HHD Rz-5′UTR-NSP1-P2A-fLuc-IRES-NSP2-16-TRS-GFP::Bsr-TRS-N-ORF10-3′UTR-HDV Rz-BGH pA. Over the past two years, several SARS-CoV-2 replicons have been developed [ 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 ]. The general genomic composition of these SARS-CoV-2 replicons includes 5′UTR, ORF1a/1b, a Luc gene or green fluorescence protein (GFP) reporter gene, N, and 3′UTR from 5′ end to 3′ end.…”

Section: Discussionmentioning

confidence: 99%

“…However, they differ in how the replicon RNA is produced. Some replicons have a T7 promoter at the 5′ end, and the replicon RNA has to be synthesized in vitro [ 59 , 60 , 61 , 62 , 63 , 64 ]. Other replicons have the human cytomegalovirus (CMV) immediate-early enhance and promoter at the 5′ end, and the replicon RNA is transcribed from the replicon DNA that is introduced into cells by transfection [ 65 , 66 , 67 , 68 ].…”

Section: Discussionmentioning

confidence: 99%

“…The other possibility is the instability of the engineered replicon so that the replicon RNA loses the ability to self-replicate in a long-term and sustainable manner. Nevertheless, of note is that among all ten published SARS2 replicons thus far, only two SARS2 replicons lead to creation of stable replicon cells [ 59 , 68 ], and the other eight all appear to be transient replicons [ 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 ]. Further understanding of the design and organizational differences between these two groups of SARS2 replicons may lead to identification of viral and host factors necessary for SARS2 replication as well as help construct better SARS2 replicons for anti-SARS2 drug screening and evaluation.…”

Section: Discussionmentioning

confidence: 99%

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A Unique Robust Dual-Promoter-Driven and Dual-Reporter-Expressing SARS-CoV-2 Replicon: Construction and Characterization

Liu

Timani

et al. 2022

Viruses

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The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2, SARS2) remains a great global health threat and demands identification of more effective and SARS2-targeted antiviral drugs, even with successful development of anti-SARS2 vaccines. Viral replicons have proven to be a rapid, safe, and readily scalable platform for high-throughput screening, identification, and evaluation of antiviral drugs against positive-stranded RNA viruses. In the study, we report a unique robust HIV long terminal repeat (LTR)/T7 dual-promoter-driven and dual-reporter firefly luciferase (fLuc) and green fluorescent protein (GFP)-expressing SARS2 replicon. The genomic organization of the replicon was designed with quite a few features that were to ensure the replication fidelity of the replicon, to maximize the expression of the full-length replicon, and to offer the monitoring flexibility of the replicon replication. We showed the success of the construction of the replicon and expression of reporter genes fLuc and GFP and SARS structural N from the replicon DNA or the RNA that was in vitro transcribed from the replicon DNA. We also showed detection of the negative-stranded genomic RNA (gRNA) and subgenomic RNA (sgRNA) intermediates, a hallmark of replication of positive-stranded RNA viruses from the replicon. Lastly, we showed that expression of the reporter genes, N gene, gRNA, and sgRNA from the replicon was sensitive to inhibition by Remdesivir. Taken together, our results support use of the replicon for identification of anti-SARS2 drugs and development of new anti-SARS strategies targeted at the step of virus replication.

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“…[4][5][6][7][8][9][10] The SARS-CoV-2 genome consists of several functional genes, of which nucleocapsid (N) and spike (S) proteins are widely used in SARS-CoV-2 molecular and serological detection. [11][12][13] As the structural proteins, S and N, are expected to show high expression levels during infection. The S glycoprotein of SARS-CoV-2, which protrudes from the virus surface, plays an important role in viral infection through the specific molecular interaction of its receptorbinding domain (RBD) with the human angiotensin-converting enzyme 2 receptor (ACE2).…”

mentioning

confidence: 99%

Ultrarapid and ultrasensitive detection of SARS‐CoV‐2 antibodies in COVID‐19 patients via a 3D‐printed nanomaterial‐based biosensing platform

Ali

Zhang

et al. 2022

Journal of Medical Virology

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Rapid detection of antibodies during infection and after vaccination is critical for the control of infectious outbreaks, understanding immune response, and evaluating vaccine efficacy. In this manuscript, we evaluate a simple ultrarapid test for SARS‐CoV‐2 antibodies in COVID‐19 patients, which gives quantitative results (i.e., antibody concentration) in 10–12 s using a previously reported nanomaterial‐based three‐dimensional (3D)‐printed biosensing platform. This platform consists of a micropillar array electrode fabricated via 3D printing of aerosolized gold nanoparticles and coated with nanoflakes of graphene and specific SARS‐CoV‐2 antigens, including spike S1, S1 receptor‐binding domain (RBD) and nucleocapsid (N). The sensor works on the principle of electrochemical transduction, where the change of sensor impedance is realized by the interactions between the viral proteins attached to the sensor electrode surface and the antibodies. The three sensors were used to test samples from 17 COVID‐19 patients and 3 patients without COVID‐19. Unlike other serological tests, the 3D sensors quantitatively detected antibodies at a concentration as low as picomole within 10–12 s in human plasma samples. We found that the studied COVID‐19 patients had higher concentrations of antibodies to spike proteins (RBD and S1) than to the N protein. These results demonstrate the enormous potential of the rapid antibody test platform for understanding patients' immunity, disease epidemiology and vaccine efficacy, and facilitating the control and prevention of infectious epidemics.

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Construction and characterization of two SARS‐CoV‐2 minigenome replicon systems

Cited by 17 publications

References 67 publications

Production of single‐cycle infectious SARS‐CoV‐2 through a trans‐complemented replicon

Production of single‐cycle infectious SARS‐CoV‐2 through a trans‐complemented replicon

A Unique Robust Dual-Promoter-Driven and Dual-Reporter-Expressing SARS-CoV-2 Replicon: Construction and Characterization

Ultrarapid and ultrasensitive detection of SARS‐CoV‐2 antibodies in COVID‐19 patients via a 3D‐printed nanomaterial‐based biosensing platform

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