Activation of the MKK3-p38-MK2-ZFP36 Axis by Coronavirus Infection Restricts the Upregulation of AU-Rich Element-Containing Transcripts in Proinflammatory Responses.

Coronavirus infections induce the expression of multiple proinflammatory cytokines and chemokines. We have previously shown that in cells infected with gammacoronavirus infectious bronchitis virus (IBV), interleukin 6 (IL-6), and IL-8 were drastically upregulated, and the MAP kinase p38 and the integrated stress response pathways were implicated in this process. In this study, we report that coronavirus infection activates a negative regulatory loop that restricts the upregulation of a number of proinflammatory genes. As revealed by the initial transcriptomic and subsequent validation analyses, the anti-inflammatory adenine-uridine (AU)-rich element (ARE)-binding protein, zinc finger protein 36 (ZFP36), and its related family members were upregulated in cells infected with IBV and three other coronaviruses, alphacoronaviruses porcine epidemic diarrhea virus (PEDV), human coronavirus 229E (HCoV-229E), and betacoronavirus HCoV-OC43, respectively. Characterization of the functional roles of ZFP36 during IBV infection demonstrated that ZFP36 promoted the degradation of transcripts coding for IL-6, IL-8, dual-specificity phosphatase 1 (DUSP1), prostaglandin-endoperoxide synthase 2 (PTGS2) and TNF-α-induced protein 3 (TNFAIP3), through binding to AREs in these transcripts. Consistently, knockdown and inhibition of JNK and p38 kinase activities reduced the expression of ZFP36, as well as the expression of IL-6 and IL-8. On the contrary, overexpression of mitogen-activated protein kinase kinase 3 (MKK3) and MAPKAP kinase-2 (MK2), the upstream and downstream kinases of p38, respectively, increased the expression of ZFP36 and decreased the expression of IL-8. Taken together, this study reveals an important regulatory role of the MKK3-p38-MK2-ZFP36 axis in coronavirus infection-induced proinflammatory response. IMPORTANCE Excessive and uncontrolled induction and release of proinflammatory cytokines and chemokines, the so-called cytokine release syndrome (CRS), would cause life-threatening complications and multiple organ failure in severe coronavirus infections, including severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS) and COVID-19. This study reveals that coronavirus infection also induces the expression of ZFP36, an anti-inflammatory ARE-binding protein, promoting the degradation of ARE-containing transcripts coding for IL-6 and IL-8 as well as a number of other proteins related to inflammatory response. Furthermore, the p38 MAP kinase, its upstream kinase MKK3 and downstream kinase MK2 were shown to play a regulatory role in upregulation of ZFP36 during coronavirus infection cycles. This MKK3-p38-MK2-ZFP36 axis would constitute a potential therapeutic target for severe coronavirus infections.

An Engineered Cytidine Deaminase for Biocatalytic Production of a Key Intermediate of the Covid-19 Antiviral Molnupiravir.

The Covid-19 pandemic highlights the urgent need for cost-effective processes to rapidly manufacture antiviral drugs at scale. Here we report a concise biocatalytic process for Molnupiravir, a nucleoside analogue recently approved as an orally available treatment for SARS-CoV-2. Key to the success of this process was the development of an efficient biocatalyst for the production of N-hydroxy-cytidine through evolutionary adaption of the hydrolytic enzyme cytidine deaminase. This engineered biocatalyst performs >85 000 turnovers in less than 3 h, operates at 180 g/L substrate loading, and benefits from in situ crystallization of the N-hydroxy-cytidine product (85% yield), which can be converted to Molnupiravir by a selective 5'-acylation using Novozym 435.

Tipiracil binds to uridine site and inhibits Nsp15 endoribonuclease NendoU from SARS-CoV-2.

SARS-CoV-2 Nsp15 is a uridine-specific endoribonuclease with C-terminal catalytic domain belonging to the EndoU family that is highly conserved in coronaviruses. As endoribonuclease activity seems to be responsible for the interference with the innate immune response, Nsp15 emerges as an attractive target for therapeutic intervention. Here we report the first structures with bound nucleotides and show how the enzyme specifically recognizes uridine moiety. In addition to a uridine site we present evidence for a second base binding site that can accommodate any base. The structure with a transition state analog, uridine vanadate, confirms interactions key to catalytic mechanisms. In the presence of manganese ions, the enzyme cleaves unpaired RNAs. This acquired knowledge was instrumental in identifying Tipiracil, an FDA approved drug that is used in the treatment of colorectal cancer, as a potential anti-COVID-19 drug. Using crystallography, biochemical, and whole-cell assays, we demonstrate that Tipiracil inhibits SARS-CoV-2 Nsp15 by interacting with the uridine binding pocket in the enzyme's active site. Our findings provide new insights for the development of uracil scaffold-based drugs.

Mutation profile of over 4500 SARS-CoV-2 isolations reveals prevalent cytosine-to-uridine deamination on viral RNAs.

Aim: The inference of coronavirus evolution is largely based on mutations in SARS-CoV-2 genome. Misinterpretation of these mutations would mislead people about the evolution of SARS-CoV-2. Materials & methods: With 4521 lines of SARS-CoV-2, we obtained 3169 unique point mutation sites. We counted the numbers and calculated the minor allele frequency (MAF) of each mutation type. Results: Nearly half of the point mutations are C-T mismatches and 20% are A-G mismatches. The MAF of C-T and A-G mismatches is significantly higher than MAF of other mutation types. Conclusion: The excessive C-T mismatches do not resemble the random mutation profile. They are likely to be caused by the cytosine-to-uridine deamination system in hosts.