Cloning of a Passage-Free SARS-CoV-2 Genome and Mutagenesis Using Red Recombination

Herrmann, Alexandra; Jungnickl, Doris; Cordsmeier, Arne; Peter, Antonia Sophia; Überla, Klaus; Ensser, Armin

doi:10.3390/ijms221910188

Cited by 41 publications

(63 citation statements)

References 27 publications

(36 reference statements)

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“…Viral titers were determined by plaque assays on Vero-ACE2 cells. SARS-CoV-2 wild-type and ORF8-deleted bacmid constructs were kindly provided by Armin Ensser (Institute of Virology, Erlangen, Germany) ( 49 ).…”

Section: Methodsmentioning

confidence: 99%

Viral Mimicry of Interleukin-17A by SARS-CoV-2 ORF8

Xia

Shin

et al. 2022

mBio

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Patients infected with SARS-CoV-2 variants lacking open reading frame 8 (ORF8) have been associated with milder infection and disease outcome, but the molecular mechanism behind how this viral accessory protein mediates disease pathogenesis is not yet known. In our study, we revealed that secreted ORF8 protein mimics host IL-17 to activate IL-17 receptors A and C (IL-17RA/C) and induces a significantly stronger inflammatory response than host IL-17A, providing molecular insights into the role of ORF8 in COVID-19 pathogenesis and serving as a potential therapeutic target.

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Section: Methodsmentioning

confidence: 99%

Viral Mimicry of Interleukin-17A by SARS-CoV-2 ORF8

Xia

Shin

et al. 2022

mBio

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“…Recombinant varicella zoster virus strain Oka (VZV-GFP) was generated by the use of bacmid technology, as described elsewhere [ 58 ], and virus propagation, including infection experiments for antiviral testing based on GFP fluorometry, was performed in a cell-associated manner on HFFs under standard conditions [ 26 , 59 , 60 ]. The generation and characterization of the recombinant SARS-CoV-2 virus d6-YFP in which viral ORF6 was replaced with EYFP has been described previously [ 41 , 61 ]. All SARS-CoV-2 infection experiments were performed under BSL-3 conditions.…”

Section: Methodsmentioning

confidence: 99%

Development of a PROTAC-Based Targeting Strategy Provides a Mechanistically Unique Mode of Anti-Cytomegalovirus Activity

Hahn

Hamilton

Wangen

et al. 2021

IJMS

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Human cytomegalovirus (HCMV) is a major pathogenic herpesvirus that is prevalent worldwide and it is associated with a variety of clinical symptoms. Current antiviral therapy options do not fully satisfy the medical needs; thus, improved drug classes and drug-targeting strategies are required. In particular, host-directed antivirals, including pharmaceutical kinase inhibitors, might help improve the drug qualities. Here, we focused on utilizing PROteolysis TArgeting Chimeras (PROTACs), i.e., hetero-bifunctional molecules containing two elements, namely a target-binding molecule and a proteolysis-inducing element. Specifically, a PROTAC that was based on a cyclin-dependent kinase (CDK) inhibitor, i.e., CDK9-directed PROTAC THAL-SNS032, was analyzed and proved to possess strong anti-HCMV AD169-GFP activity, with values of EC50 of 0.030 µM and CC50 of 0.175 µM (SI of 5.8). Comparing the effect of THAL-SNS032 with its non-PROTAC counterpart SNS032, data indicated a 3.7-fold stronger anti-HCMV efficacy. This antiviral activity, as illustrated for further clinically relevant strains of human and murine CMVs, coincided with the mid-nanomolar concentration range necessary for a drug-induced degradation of the primary (CDK9) and secondary targets (CDK1, CDK2, CDK7). In addition, further antiviral activities were demonstrated, such as the inhibition of SARS-CoV-2 replication, whereas other investigated human viruses (i.e., varicella zoster virus, adenovirus type 2, and Zika virus) were found insensitive. Combined, the antiviral quality of this approach is seen in its (i) mechanistic uniqueness; (ii) future options of combinatorial drug treatment; (iii) potential broad-spectrum activity; and (iv) applicability in clinically relevant antiviral models. These novel data are discussed in light of the current achievements of anti-HCMV drug development.

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“…To evaluate how the P323L and G671S mutations affected SARS-CoV-2 fitness and transmission, we used the SARS-CoV-2 Bacmid reverse genetic (RG) system to generate three isogenic SARS-CoV-2 variants (Spike D614G) containing the NSP12 WT, P323L, G671S, or P323L/G617S mutations 24 . We then examined their in vitro replication kinetics in Vero, Vero ACE2/TMPRSS2 , Huh-7, and Calu-3 cells at 33°C or 37°C.…”

Section: Resultsmentioning

confidence: 99%

SARS-CoV-2 variants show temperature-dependent enhanced polymerase activity in the upper respiratory tract and high transmissibility

Kim

Casel

et al. 2022

Preprint

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With the convergent global emergence of SARS-CoV-2 variants of concern (VOC), a precise comparison study of viral fitness and transmission characteristics is necessary for the prediction of dominant VOCs and the development of suitable countermeasures. While airway temperature plays important roles in the fitness and transmissibility of respiratory tract viruses, it has not been well studied with SARS-CoV-2. Here we demonstrate that natural temperature differences between the upper (33℃) and lower (37℃) respiratory tract have profound effects on SARS-CoV-2 replication and transmission. Specifically, SARS-COV-2 variants containing the P323L or P323L/G671S mutation in the NSP12 RNA-dependent RNA polymerase (RdRp) exhibited enhanced RdRp enzymatic activity at 33℃ compared to 37℃ and high transmissibility in ferrets. MicroScale Thermophoresis demonstrated that the NSP12 P323L or P323L/G671S mutation stabilized the NSP12-NSP7-NSP8 complex interaction. Furthermore, reverse genetics-derived SARS-CoV-2 variants containing the NSP12 P323L or P323L/G671S mutation displayed enhanced replication at 33℃, and high transmission in ferrets. This suggests that the evolutionarily forced NSP12 P323L and P323L/G671S mutations of recent SARS-CoV-2 VOC strains are associated with increases of the RdRp complex stability and enzymatic activity, promoting the high transmissibility.

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Cloning of a Passage-Free SARS-CoV-2 Genome and Mutagenesis Using Red Recombination

Cited by 41 publications

References 27 publications

Viral Mimicry of Interleukin-17A by SARS-CoV-2 ORF8

Viral Mimicry of Interleukin-17A by SARS-CoV-2 ORF8

Development of a PROTAC-Based Targeting Strategy Provides a Mechanistically Unique Mode of Anti-Cytomegalovirus Activity

SARS-CoV-2 variants show temperature-dependent enhanced polymerase activity in the upper respiratory tract and high transmissibility

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