Elucidating the genetic architecture of DNA methylation to identify promising molecular mechanisms of disease.

DNA methylation commonly occurs at cytosine-phosphate-guanine sites (CpGs) that can serve as biomarkers for many diseases. We analyzed whole genome sequencing data to identify DNA methylation quantitative trait loci (mQTLs) in 4126 Framingham Heart Study participants. Our mQTL mapping identified 94,362,817 cis-mQTLvariant-CpG pairs (for 210,156 unique autosomal CpGs) at P < 1e-7 and 33,572,145 trans-mQTL variant-CpG pairs (for 213,606 unique autosomal CpGs) at P < 1e-14. Using cis-mQTL variants for 1258 CpGs associated with seven cardiovascular disease (CVD) risk factors, we found 104 unique CpGs that colocalized with at least one CVD trait. For example, cg11554650 (PPP1R18) colocalized with type 2 diabetes, and was driven by a single nucleotide polymorphism (rs2516396). We performed Mendelian randomization (MR) analysis and demonstrated 58 putatively causal relations of CVD risk factor-associated CpGs to one or more risk factors (e.g., cg05337441 [APOB] with LDL; MR P = 1.2e-99, and 17 causal associations with coronary artery disease (e.g. cg08129017 [SREBF1] with coronary artery disease; MR P = 5e-13). We also showed that three CpGs, e.g., cg14893161 (PM20D1), are putatively causally associated with COVID-19 severity. To assist in future analyses of the role of DNA methylation in disease pathogenesis, we have posted a comprehensive summary data set in the National Heart, Lung, and Blood Institute's BioData Catalyst.

Covalent Catalytic Strategies for Enzymes That Modify RNA Molecules on their Tripartite Building Blocks.

The tripartite structures of the four 5'-nucleotide monophosphate (NMP) building blocks in all RNAs enable enzyme-catalyzed chemical modifications to three types of sites: the heterocyclic bases via N- and C-methylations and other alkylations, conversion of the N-glycoside linkages of the uridine moiety to the C-C glycoside link in pseudouridines, and the phosphodiester-mediated processes of 5'-capping, splicing, and 3'-tailing of premRNAs. We examine known cases for enzymatic covalent catalytic strategies that entail transient formation and breakdown of covalent enzyme-RNA adducts in each catalytic cycle. One case involves generation of the required carbon nucleophile during C5 methylation of cytosine residues in RNAs. A second examines the mechanism proposed for pseudouridine synthases and for replacement of a guanine residue in tRNAs by queuosine. The third category involves phosphoric anhydride and phosphodiester chemistry by which viral RNAs encode enzymes for making their own mRNA 5'-caps. This strategy includes the recent finding that the SARS-CoV2 proteins assemble a canonical 5',5'-GTP cap on their 28 900 nucleotide genomic RNA to enable its translation as an mRNA by host translational machinery by way of a covalent RNA-viral enzyme intermediate.

Synthesis of novel pyridine and pyrimidine thioglycoside phosphoramidates for the treatment of COVID-19 and influenza A viruses.

A novel series of pyridine, cytosine, and uracil thioglycoside analogs (4a-i, 9a,b, and 13a,b, respectively) and their corresponding phosphoramidates (6a-I, 10a,b, and 14a,b, respectively) were synthesized and assessed for their antiviral inhibitory activities in a dual-pathogen screening protocol against SARS-CoV-2 and influenza A virus (IAV). MTT cytotoxicity (TC50) and plaque reduction assays were used to explore inhibition and cytotoxicity percentage values for H5N1 influenza virus strain and the half-maximal cytotoxic concentration (CC50) and inhibitory concentration (IC50) for SARS-CoV-2 virus. Most of the tested compounds demonstrated dose-dependent inhibition behavior. Both cytosine thioglycoside phosphoramidates 10a and 10b exhibited the most potent profiles with 83% and 86% inhibition at 0.25 µM concentration against H5N1 and IC50 values of 12.16 µM, 14.9 µM against SARS-CoV-2, respectively. Moreover, compounds 10a and 10b have been shown to have the highest selectivity index (SI) among all the tested compounds against SARS-CoV-2 with 28.2 and 26.9 values, respectively.

Stealth Fluorescence Labeling for Live Microscopy Imaging of mRNA Delivery.

Methods for tracking RNA inside living cells without perturbing their natural interactions and functions are critical within biology and, in particular, to facilitate studies of therapeutic RNA delivery. We present a stealth labeling approach that can efficiently, and with high fidelity, generate RNA transcripts, through enzymatic incorporation of the triphosphate of tCO, a fluorescent tricyclic cytosine analogue. We demonstrate this by incorporation of tCO in up to 100% of the natural cytosine positions of a 1.2 kb mRNA encoding for the histone H2B fused to GFP (H2B:GFP). Spectroscopic characterization of this mRNA shows that the incorporation rate of tCO is similar to cytosine, which allows for efficient labeling and controlled tuning of labeling ratios for different applications. Using live cell confocal microscopy and flow cytometry, we show that the tCO-labeled mRNA is efficiently translated into H2B:GFP inside human cells. Hence, we not only develop the use of fluorescent base analogue labeling of nucleic acids in live-cell microscopy but also, importantly, show that the resulting transcript is translated into the correct protein. Moreover, the spectral properties of our transcripts and their translation product allow for their straightforward, simultaneous visualization in live cells. Finally, we find that chemically transfected tCO-labeled RNA, unlike a state-of-the-art fluorescently labeled RNA, gives rise to expression of a similar amount of protein as its natural counterpart, hence representing a methodology for studying natural, unperturbed processing of mRNA used in RNA therapeutics and in vaccines, like the ones developed against SARS-CoV-2.

The specific metabolome profiling of patients infected by SARS-COV-2 supports the key role of tryptophan-nicotinamide pathway and cytosine metabolism.

The biological mechanisms involved in SARS-CoV-2 infection are only partially understood. Thus we explored the plasma metabolome of patients infected with SARS-CoV-2 to search for diagnostic and/or prognostic biomarkers and to improve the knowledge of metabolic disturbance in this infection. We analyzed the plasma metabolome of 55 patients infected with SARS-CoV-2 and 45 controls by LC-HRMS at the time of viral diagnosis (D0). We first evaluated the ability to predict the diagnosis from the metabotype at D0 in an independent population. Next, we assessed the feasibility of predicting the disease evolution at the 7th and 15th day. Plasma metabolome allowed us to generate a discriminant multivariate model to predict the diagnosis of SARS-CoV-2 in an independent population (accuracy > 74%, sensitivity, specificity > 75%). We identified the role of the cytosine and tryptophan-nicotinamide pathways in this discrimination. However, metabolomic exploration modestly explained the disease evolution. Here, we present the first metabolomic study in SARS-CoV-2 patients which showed a high reliable prediction of early diagnosis. We have highlighted the role of the tryptophan-nicotinamide pathway clearly linked to inflammatory signals and microbiota, and the involvement of cytosine, previously described as a coordinator of cell metabolism in SARS-CoV-2. These findings could open new therapeutic perspectives as indirect targets.

Pharmacokinetics and Efficacy of a Potential Smallpox Therapeutic, Brincidofovir, in a Lethal Monkeypox Virus Animal Model.

Smallpox, caused by Variola virus (VARV), was eradicated in 1980; however, VARV bioterrorist threats still exist, necessitating readily available therapeutics. Current preparedness activities recognize the importance of oral antivirals and recommend therapeutics with different mechanisms of action. Monkeypox virus (MPXV) is closely related to VARV, causing a highly similar clinical human disease, and can be used as a surrogate for smallpox antiviral testing. The prairie dog MPXV model has been characterized and used to study the efficacy of antipoxvirus therapeutics, including recently approved TPOXX (tecovirimat). Brincidofovir (BCV; CMX001) has shown antiviral activity against double-stranded DNA viruses, including poxviruses. To determine the exposure of BCV following oral administration to prairie dogs, a pharmacokinetics (PK) study was performed. Analysis of BCV plasma concentrations indicated variability, conceivably due to the outbred nature of the animals. To determine BCV efficacy in the MPXV prairie dog model, groups of animals were intranasally challenged with 9 × 105 plaque-forming units (PFU; 90% lethal dose [LD90]) of MPXV on inoculation day 0 (ID0). Animals were divided into groups based on the first day of BCV treatment relative to inoculation day (ID-1, ID0, or ID1). A trend in efficacy was noted dependent upon treatment initiation (57% on ID-1, 43% on ID0, and 29% on ID1) but was lower than demonstrated in other animal models. Analysis of the PK data indicated that BCV plasma exposure (maximum concentration [Cmax]) and the time of the last quantifiable concentration (AUClast) were lower than in other animal models administered the same doses, indicating that suboptimal BCV exposure may explain the lower protective effect on survival.IMPORTANCE Preparedness activities against highly transmissible viruses with high mortality rates have been highlighted during the ongoing coronavirus disease 2019 (COVID-19) pandemic. Smallpox, caused by variola virus (VARV) infection, is highly transmissible, with an estimated 30% mortality. Through an intensive vaccination campaign, smallpox was declared eradicated in 1980, and routine smallpox vaccination of individuals ceased. Today's current population has little/no immunity against VARV. If smallpox were to reemerge, the worldwide results would be devastating. Recent FDA approval of one smallpox antiviral (tecovirimat) was a successful step in biothreat preparedness; however, orthopoxviruses can become resistant to treatment, suggesting the need for multiple therapeutics. Our paper details the efficacy of the investigational smallpox drug brincidofovir in a monkeypox virus (MPXV) animal model. Since brincidofovir has not been tested in vivo against smallpox, studies with the related virus MPXV are critical in understanding whether it would be protective in the event of a smallpox outbreak.

A Path toward SARS-CoV-2 Attenuation: Metabolic Pressure on CTP Synthesis Rules the Virus Evolution.

In the context of the COVID-19 pandemic, we describe here the singular metabolic background that constrains enveloped RNA viruses to evolve toward likely attenuation in the long term, possibly after a step of increased pathogenicity. Cytidine triphosphate (CTP) is at the crossroad of the processes allowing SARS-CoV-2 to multiply, because CTP is in demand for four essential metabolic steps. It is a building block of the virus genome, it is required for synthesis of the cytosine-based liponucleotide precursors of the viral envelope, it is a critical building block of the host transfer RNAs synthesis and it is required for synthesis of dolichol-phosphate, a precursor of viral protein glycosylation. The CCA 3'-end of all the transfer RNAs required to translate the RNA genome and further transcripts into the proteins used to build active virus copies is not coded in the human genome. It must be synthesized de novo from CTP and ATP. Furthermore, intermediary metabolism is built on compulsory steps of synthesis and salvage of cytosine-based metabolites via uridine triphosphate that keep limiting CTP availability. As a consequence, accidental replication errors tend to replace cytosine by uracil in the genome, unless recombination events allow the sequence to return to its ancestral sequences. We document some of the consequences of this situation in the function of viral proteins. This unique metabolic setup allowed us to highlight and provide a raison d'être to viperin, an enzyme of innate antiviral immunity, which synthesizes 3'-deoxy-3',4'-didehydro-CTP as an extremely efficient antiviral nucleotide.

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.

Mutation Patterns of Human SARS-CoV-2 and Bat RaTG13 Coronavirus Genomes Are Strongly Biased Towards C>U Transitions, Indicating Rapid Evolution in Their Hosts.

The pandemic caused by the spread of SARS-CoV-2 has led to considerable interest in its evolutionary origin and genome structure. Here, we analyzed mutation patterns in 34 human SARS-CoV-2 isolates and a closely related RaTG13 isolated from Rhinolophus affinis (a horseshoe bat). We also evaluated the CpG dinucleotide contents in SARS-CoV-2 and other human and animal coronavirus genomes. Out of 1136 single nucleotide variations (~4% divergence) between human SARS-CoV-2 and bat RaTG13, 682 (60%) can be attributed to C>U and U>C substitutions, far exceeding other types of substitutions. An accumulation of C>U mutations was also observed in SARS-CoV2 variants that arose within the human population. Globally, the C>U substitutions increased the frequency of codons for hydrophobic amino acids in SARS-CoV-2 peptides, while U>C substitutions decreased it. In contrast to most other coronaviruses, both SARS-CoV-2 and RaTG13 exhibited CpG depletion in their genomes. The data suggest that C-to-U conversion mediated by C deamination played a significant role in the evolution of the SARS-CoV-2 coronavirus. We hypothesize that the high frequency C>U transitions reflect virus adaptation processes in their hosts, and that SARS-CoV-2 could have been evolving for a relatively long period in humans following the transfer from animals before spreading worldwide.

Rampant C→U Hypermutation in the Genomes of SARS-CoV-2 and Other Coronaviruses: Causes and Consequences for Their Short- and Long-Term Evolutionary Trajectories.

The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has motivated an intensive analysis of its molecular epidemiology following its worldwide spread. To understand the early evolutionary events following its emergence, a data set of 985 complete SARS-CoV-2 sequences was assembled. Variants showed a mean of 5.5 to 9.5 nucleotide differences from each other, consistent with a midrange coronavirus substitution rate of 3 × 10-4 substitutions/site/year. Almost one-half of sequence changes were C→U transitions, with an 8-fold base frequency normalized directional asymmetry between C→U and U→C substitutions. Elevated ratios were observed in other recently emerged coronaviruses (SARS-CoV, Middle East respiratory syndrome [MERS]-CoV), and decreasing ratios were observed in other human coronaviruses (HCoV-NL63, -OC43, -229E, and -HKU1) proportionate to their increasing divergence. C→U transitions underpinned almost one-half of the amino acid differences between SARS-CoV-2 variants and occurred preferentially in both 5' U/A and 3' U/A flanking sequence contexts comparable to favored motifs of human APOBEC3 proteins. Marked base asymmetries observed in nonpandemic human coronaviruses (U ≫ A > G ≫ C) and low G+C contents may represent long-term effects of prolonged C→U hypermutation in their hosts. The evidence that much of sequence change in SARS-CoV-2 and other coronaviruses may be driven by a host APOBEC-like editing process has profound implications for understanding their short- and long-term evolution. Repeated cycles of mutation and reversion in favored mutational hot spots and the widespread occurrence of amino acid changes with no adaptive value for the virus represent a quite different paradigm of virus sequence change from neutral and Darwinian evolutionary frameworks and are not incorporated by standard models used in molecular epidemiology investigations.IMPORTANCE The wealth of accurately curated sequence data for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), its long genome, and its low substitution rate provides a relatively blank canvas with which to investigate effects of mutational and editing processes imposed by the host cell. The finding that a large proportion of sequence change in SARS-CoV-2 in the initial months of the pandemic comprised C→U mutations in a host APOBEC-like context provides evidence for a potent host-driven antiviral editing mechanism against coronaviruses more often associated with antiretroviral defense. In evolutionary terms, the contribution of biased, convergent, and context-dependent mutations to sequence change in SARS-CoV-2 is substantial, and these processes are not incorporated by standard models used in molecular epidemiology investigations.