TransAC4C-a novel interpretable architecture for multi-species identification of N4-acetylcytidine sites in RNA with single-base resolution.

N4-acetylcytidine (ac4C) is a modification found in ribonucleic acid (RNA) related to diseases. Expensive and labor-intensive methods hindered the exploration of ac4C mechanisms and the development of specific anti-ac4C drugs. Therefore, an advanced prediction model for ac4C in RNA is urgently needed. Despite the construction of various prediction models, several limitations exist: (1) insufficient resolution at base level for ac4C sites; (2) lack of information on species other than Homo sapiens; (3) lack of information on RNA other than mRNA; and (4) lack of interpretation for each prediction. In light of these limitations, we have reconstructed the previous benchmark dataset and introduced a new dataset including balanced RNA sequences from multiple species and RNA types, while also providing base-level resolution for ac4C sites. Additionally, we have proposed a novel transformer-based architecture and pipeline for predicting ac4C sites, allowing for highly accurate predictions, visually interpretable results and no restrictions on the length of input RNA sequences. Statistically, our work has improved the accuracy of predicting specific ac4C sites in multiple species from less than 40% to around 85%, achieving a high AUC > 0.9. These results significantly surpass the performance of all existing models.

Molnupiravir increases SARS-CoV-2 genome diversity and complexity: A case-control cohort study.

Molnupiravir, an oral direct-acting antiviral effective in vitro against SARS-CoV-2, has been largely employed during the COVID-19 pandemic, since December 2021. After marketing and widespread usage, a progressive increase in SARS-CoV-2 lineages characterized by a higher transition/transversion ratio, a characteristic signature of molnupiravir action, appeared in the Global Initiative on Sharing All Influenza Data (GISAID) and International Nucleotide Sequence Database Collaboration (INSDC) databases. Here, we assessed the drug effects by SARS-CoV-2 whole-genome sequencing on 38 molnupiravir-treated persistently positive COVID-19 outpatients tested before and after treatment. Seventeen tixagevimab/cilgavimab-treated outpatients served as controls. Mutational analyses confirmed that SARS-CoV-2 exhibits an increased transition/transversion ratio seven days after initiation of molnupiravir. Moreover we observed an increased G->A ratio compared to controls, which was not related to apolipoprotein B mRNAediting enzyme, catalytic polypeptide-like (APOBEC) activity. In addition, we demonstrated for the first time an increased diversity and complexity of the viral quasispecies.

Assessing antiviral treatment efficacy and risk factors for severe COVID-19 in kidney transplant recipients during the Omicron subvariant-dominant period: a retrospective study.

BACKGROUND: Kidney transplant recipients (KTRs) are at risk of severe coronavirus disease 2019 (COVID-19), and even now that Omicron subvariants have become dominant, cases of severe disease are certain to occur. The aims of this retrospective study were to evaluate the efficacy of antiviral treatment for COVID-19 and to identify risk factors for severe disease in KTRs during Omicron subvariant-dominant periods. METHODS: A total of 65 KTRs diagnosed with COVID-19 who received antiviral treatment between July 2022 and September 2023 were analyzed. Mild cases received oral molnupiravir (MP) as outpatient therapy, while moderate or worse cases received intravenous remdesivir (RDV) as inpatient therapy. In principle, mycophenolate mofetil was withdrawn and switched to everolimus. We investigated the efficacy of antiviral treatment and compared the clinical parameters of mild/moderate and severe/critical cases to identify risk factors for severe COVID-19. RESULTS: Among 65 cases, 49 were mild, 6 were moderate, 9 were severe, and 1 was of critical severity. MP was administered to 57 cases; 49 (86%) improved and 8 (14%) progressed. RDV was administered to 16 cases; 14 (87%) improved and 2 (13%) progressed. Seventeen (26%) cases required hospitalization, and none died. Comparisons of the severe/critical group (n = 10) with the mild/moderate group (n = 55) demonstrated that the severe/critical group had a significantly higher median age (64 vs. 53 years, respectively; p = 0.0252), prevalence of diabetes (70% vs. 22%, respectively; p = 0.0047) and overweight/obesity (40% vs. 11%, respectively; p = 0.0393), as well as a significantly longer median time from symptom onset to initial antiviral therapy (3 days vs. 1 day, respectively; p = 0.0026). Multivariate analysis showed that a longer time from symptom onset to initial antiviral treatment was an independent risk factor for severe COVID-19 (p = 0.0196, odds ratio 1.625, 95% confidence interval 1.081-2.441). CONCLUSION: These findings suggest that a longer time from symptom onset to initial antiviral treatment is associated with a higher risk of severe COVID-19 in KTRs. Initiating antiviral treatment as early as possible is crucial for preventing severe outcomes; this represents a valuable insight into COVID-19 management in KTRs.

Molnupiravir: Mechanism of action, clinical, and translational science.

Molnupiravir is an oral prodrug of the broadly active, antiviral ribonucleoside analog N-hydroxycytidine (NHC). The primary circulating metabolite NHC is taken up into cells and phosphorylated to NHC-triphosphate (NHC-TP). NHC-TP serves as a competitive substrate for viral RNA-dependent RNA polymerase (RdRp), which results in an accumulation of errors in the viral genome, rendering virus replication incompetent. Molnupiravir has demonstrated activity against SARS-CoV-2 both clinically and preclinically and has a high barrier to development of viral resistance. Little to no molnupiravir is observed in plasma due to rapid hydrolysis to NHC. Maximum concentrations of NHC are reached at 1.5 h following administration in a fasted state. The effective half-life of NHC is 3.3 h, reflecting minimal accumulation in the plasma following twice-daily (Q12H) dosing. The terminal half-life of NHC is 20.6 h. NHC-TP exhibits a flatter profile with a lower peak-to-trough ratio compared with NHC, which supports Q12H dosing. Renal and hepatic pathways are not major routes of elimination, as NHC is primarily cleared by metabolism to uridine and cytidine, which then mix with the endogenous nucleotide pools. In a phase III study of nonhospitalized patients with COVID-19 (MOVe-OUT), 5 days of treatment with 800 mg molnupiravir Q12H significantly reduced the incidence of hospitalization or death compared with placebo. Patients treated with molnupiravir also had a greater reduction in SARS-CoV-2 viral load and improved clinical outcomes, compared with those receiving placebo. The clinical effectiveness of molnupiravir has been further demonstrated in several real-world evidence studies. Molnupiravir is currently authorized or approved in more than 25 countries.

No evidence for ac4C within human mRNA upon data reassessment.

Cytidine acetylation (ac4C) of RNA is a post-transcriptional modification catalyzed by Nat10. Recently, an approach termed RedaC:T was employed to map ac4C in human mRNA, relying on detection of C>T mutations in WT but not in Nat10-KO cells. RedaC:T suggested widespread ac4C presence. Here, we reanalyze RedaC:T data. We find that mismatch signatures are not reproducible, as C>T mismatches are nearly exclusively present in only one of two biological replicates. Furthermore, all mismatch types-not only C>T-are highly enriched in WT samples, inconsistent with an acetylation signature. We demonstrate that the originally observed enrichment in mutations in one of the WT samples is due to its low complexity, resulting in the technical amplification of all classes of mismatch counts. Removal of duplicate reads abolishes the skewed mismatch patterns. These analyses account for the irreproducible mismatch patterns across samples while failing to find evidence for acetylation of RedaC:T sites.

Detection of ac4C in human mRNA is preserved upon data reassessment.

We recently reported the distribution of N4-acetylcytidine (ac4C) in HeLa mRNA at base resolution through chemical reduction and the induction of C:T mismatches in sequencing (RedaC:T-seq). Our results contradicted an earlier report from Schwartz and colleagues utilizing a similar method termed ac4C-seq. Here, we revisit both datasets and reaffirm our findings. Through RedaC:T-seq reanalysis, we establish a low basal error rate at unmodified nucleotides that is not skewed to any specific mismatch type and a prominent increase in C:T substitutions as the dominant mismatch type in both treated wild-type replicates, with a high degree of reproducibility across replicates. In contrast, through ac4C-seq reanalysis, we uncover significant data quality issues including insufficient depth, with one wild-type replicate yielding 2.7 million reads, inconsistencies in reduction efficiencies between replicates, and an overall increase in mismatches involving thymine that could obscure ac4C detection. These analyses bolster the detection of ac4C in HeLa mRNA through RedaC:T-seq.

Secreted novel AID/APOBEC-like deaminase 1 (SNAD1) - a new important player in fish immunology.

The AID/APOBECs are a group of zinc-dependent cytidine deaminases that catalyse the deamination of bases in nucleic acids, resulting in a cytidine to uridine transition. Secreted novel AID/APOBEC-like deaminases (SNADs), characterized by the presence of a signal peptide are unique among all of intracellular classical AID/APOBECs, which are the central part of antibody diversity and antiviral defense. To date, there is no available knowledge on SNADs including protein characterization, biochemical characteristics and catalytic activity. We used various in silico approaches to define the phylogeny of SNADs, their common structural features, and their potential structural variations in fish species. Our analysis provides strong evidence of the universal presence of SNAD1 proteins/transcripts in fish, in which expression commences after hatching and is highest in anatomical organs linked to the immune system. Moreover, we searched published fish data and identified previously, "uncharacterized proteins" and transcripts as SNAD1 sequences. Our review into immunological research suggests SNAD1 role in immune response to infection or immunization, and interactions with the intestinal microbiota. We also noted SNAD1 association with temperature acclimation, environmental pollution and sex-based expression differences, with females showing higher level. To validate in silico predictions we performed expression studies of several SNAD1 gene variants in carp, which revealed distinct patterns of responses under different conditions. Dual sensitivity to environmental and pathogenic stress highlights its importance in the fish and potentially enhancing thermotolerance and immune defense. Revealing the biological roles of SNADs represents an exciting new area of research related to the role of DNA and/or RNA editing in fish biology.

Proof-of-concept studies with a computationally designed Mpro inhibitor as a synergistic combination regimen alternative to Paxlovid.

As the SARS-CoV-2 virus continues to spread and mutate, it remains important to focus not only on preventing spread through vaccination but also on treating infection with direct-acting antivirals (DAA). The approval of Paxlovid, a SARS-CoV-2 main protease (Mpro) DAA, has been significant for treatment of patients. A limitation of this DAA, however, is that the antiviral component, nirmatrelvir, is rapidly metabolized and requires inclusion of a CYP450 3A4 metabolic inhibitor, ritonavir, to boost levels of the active drug. Serious drug-drug interactions can occur with Paxlovid for patients who are also taking other medications metabolized by CYP4503A4, particularly transplant or otherwise immunocompromised patients who are most at risk for SARS-CoV-2 infection and the development of severe symptoms. Developing an alternative antiviral with improved pharmacological properties is critical for treatment of these patients. By using a computational and structure-guided approach, we were able to optimize a 100 to 250 µM screening hit to a potent nanomolar inhibitor and lead compound, Mpro61. In this study, we further evaluate Mpro61 as a lead compound, starting with examination of its mode of binding to SARS-CoV-2 Mpro. In vitro pharmacological profiling established a lack of off-target effects, particularly CYP450 3A4 inhibition, as well as potential for synergy with the currently approved alternate antiviral, molnupiravir. Development and subsequent testing of a capsule formulation for oral dosing of Mpro61 in B6-K18-hACE2 mice demonstrated favorable pharmacological properties, efficacy, and synergy with molnupiravir, and complete recovery from subsequent challenge by SARS-CoV-2, establishing Mpro61 as a promising potential preclinical candidate.

Comparison of effectiveness and safety of molnupiravir versus sotrovimab for COVID-19: A systematic review and meta-analysis.

BACKGROUND AND AIM: This systematic review and meta-analysis aimed to compare the effectiveness and safety of molnupiravir and sotrovimab in the treatment of patients with coronavirus disease 2019 (COVID-19). METHODS: Cochrane Library, Web of Science, PubMed, medRxiv, and Google Scholar were systematically searched to identify relevant evidence up to December 2023. The risk of bias was assessed using the risk of bias in nonrandomized studies of interventions tool. Data were analyzed using Comprehensive Meta-Analysis (CMA). RESULTS: Our search identified and included 13 studies involving 16166 patients. The meta-analysis revealed a significant difference between the molnupiravir and sotrovimab groups in terms of the mortality rate (odds ratio [OR] = 2.07, 95% confidence interval [CI]: 1.16, 3.70). However, no significant difference was observed between the two groups in terms of hospitalization rate (OR = 0.71, 95% CI: 0.47, 1.06), death or hospitalization rate (OR = 1.51, 95% CI: 0.81, 2.83), and intensive care unit admission (OR = 0.59, 95% CI: 0.07, 4.84). In terms of safety, molnupiravir was associated with a higher incidence of adverse events (OR = 1.67, 95% CI: 1.21, 2.30). CONCLUSION: The current findings indicate that sotrovimab may be more effective than molnupiravir in reducing the mortality rate in COVID-19 patients. However, no statistical difference was observed between the two treatments for other effectiveness outcomes. The certainty of evidence for these findings was rated as low or moderate. Further research is required to provide a better comparison of these interventions in treating COVID-19 patients.

Abundance of Modifications in Mature miRNAs Revealed by LC-MS/MS Method Coupled with a Two-Step Hybridization Purification Strategy.

Accurate detection of endogenous miRNA modifications, such as N6-methyladenosine (m6A), 7-methylguanosine (m7G), and 5-methylcytidine (m5C), poses significant challenges, resulting in considerable uncertainty regarding their presence in mature miRNAs. In this study, we demonstrate for the first time that liquid chromatography coupled with a tandem mass spectrometry (LC-MS/MS) nucleoside analysis method is a practical tool for quantitatively analyzing human miRNA modifications. The newly designed liquid-solid two-step hybridization (LSTH) strategy enhances specificity for miRNA purification, while LC-MS/MS offers robust capability in recognizing modifications and sufficient sensitivity with detection limits ranging from attomoles to low femtomoles. Therefore, it provides a more reliable approach compared to existing techniques for revealing modifications in endogenous miRNAs. With this approach, we characterized m6A, m7G, and m5C modifications in miR-21-5p, Let-7a/e-5p, and miR-10a-5p isolated from cultured cells and observed unexpectedly low abundance (<1% at each site) of these modifications.

Molnupiravir versus favipiravir in at-risk outpatients with COVID-19: A randomized controlled trial in Thailand.

OBJECTIVES: Evaluate and compare the efficacy and safety of molnupiravir and favipiravir in outpatients with mild to moderate COVID-19 and at risk of severe COVID-19. METHODS: In an open-label, parallel-group, multicenter trial in Thailand, participants with moderate COVID-19 and at least one factor associated with severe COVID-19 were randomly assigned 1:1 to receive oral molnupiravir or oral favipiravir (standard of care). Phone calls for remote symptom assessment were made on Days 6, 15, and 29. Participants with worsening symptoms were instructed to return to the hospital. The primary endpoint was pulmonary involvement by Day 29, as evidenced by ≥2 of the following: dyspnea, oxygen saturation <92% or imaging. RESULTS: Nine hundred seventy-seven participants (487 molnupiravir, 490 favipiravir) were enrolled from 8 July 2022 to 19 January 2023. 98% had received ≥1 dose of COVID-19 vaccine and 83% ≥3 doses. By Day 29, pulmonary involvement occurred in 0% (0/483) in molnupiravir arm versus 1% (5/482) in favipiravir arm (-1.0%; Newcombe 95.2% CI: -2.4% to -0.0%; P = 0.021); all-cause death in 0% (0/483) and <1% (1/482); COVID-19 related hospitalization in <1% (1/483) and 1% (3/482); treatment-related adverse event in 1% (5/483) and 1% (4/486); and serious adverse event in 1% (4/483) and 1% (4/486). CONCLUSIONS: Favipiravir and molnupiravir had a similar efficacy and safety profile. Whether either of the two reduced the risk of complications during the omicron era in this population with a low risk of pulmonary involvement and a high vaccine coverage remains unclear. There were no differences in any of the safety endpoints. THAI CLINICAL TRIALS REGISTRY ID: TCTR20230111009.

A comparative study of electropolymerization and photopolymerization for the determination of molnupiravir and their application in an electrochemical sensor via computationally designed molecularly imprinted polymers.

A comparative analysis of molecularly imprinted polymers based on different synthesis techniques was performed for the recognition of molnupiravir (MOL). The polymerizations were performed with 3-thienyl boronic acid (3-TBA) as a functional monomer by electropolymerization (EP) and with guanine methacrylate (GuaM) as a functional monomer by photopolymerization (PP). Morphological and electrochemical characterizations of the developed sensors were investigated to verify the constructed sensors. Moreover, quantum chemical calculations were used to evaluate changes on the electrode surface at the molecular and electronic levels. The dynamic linear range of both designed sensors under optimized experimental conditions was found to be 7.5 × 10-12-2.5 × 10-10 M and 7.5 × 10-13-2.5 × 10-11 M for EP and PP, respectively. The effect of various interfering agents on MOL peak current was assessed for the selectivity of the study. In the presence of 100 times more interfering agents, the RSD and recovery values were determined. The RSD values of GuaM/MOL@MIP/GCE and poly(Py-co-3-PBA)/MOL@MIP/GCE sensors were found to be 1.99% and 1.72%, respectively. Furthermore, the recovery values of the MIP-based sensors were 98.18-102.69% and 98.05-103.72%, respectively. In addition, the relative selectivity coefficient (k') of the proposed sensor was evaluated, and it exhibited good selectivity for MOL with respect to the NIP sensor. The prepared sensor was successfully applied to determine MOL in commercial serum samples and capsule form. In conclusion, the developed sensors provided excellent reproducibility, repeatability, high sensitivity, and selectivity against the MOL molecule.

Physician characteristics associated with antiviral prescriptions for older adults with COVID-19 in Japan: an observational study.

OBJECTIVES: Although guidelines recommend antiviral therapy for outpatients with COVID-19 who are at high risk of progressing to severe conditions, such as older adults, many patients do not receive appropriate treatment. Little is known, however, about the physician factors associated with the prescription of guideline-recommended antiviral therapy for patients with COVID-19. DESIGN: A cross-sectional study. SETTING: Data including outpatient visits in primary care clinics in Japan from April to August 2023. PARTICIPANTS: We analysed 30 953 outpatients aged ≥65 years treated with COVID-19 (mean (SD) age, 75.0 (7.6) years; 17 652 women (57.0%)) in 1394 primary care clinics. OUTCOME MEASURES: The primary outcome was the prescription of guideline-recommended antivirals (ie, nirmatrelvir-ritonavir or molnupiravir), adjusted for patient characteristics, months of visits and regions. RESULTS: Antiviral prescriptions were concentrated among a small proportion of physicians; for example, the top 10% of physicians that had the largest number of nirmatrelvir-ritonavir prescriptions accounted for 92.4% of all nirmatrelvir-ritonavir prescriptions. After adjusting for potential confounders, physicians with higher patient volumes were more likely to prescribe guideline-recommended antivirals to their patients (adjusted OR (aOR) for high vs low volume, 1.76; 95% CI 1.31 to 2.38; adjusted p<0.001). We found no evidence that the likelihood of guideline-recommended antiviral prescription differed based on physicians' gender (aOR for women vs men, 1.24; 95% CI 0.88 to 1.74; adjusted p=0.48) or age (aOR for 45-59 vs <45 years, 1.16; 95% CI 0.87 to 1.54; adjusted p=0.48; aOR for ≥60 vs <45 years, 0.88; 95% CI 0.66 to 1.16; adjusted p=0.48). These patterns were similar when examining nirmatrelvir-ritonavir and molnupiravir separately. CONCLUSIONS: Our findings suggest that provider-level factors, such as the clinical experience of treating the patients with COVID-19, play an important role in the appropriate prescription of antiviral medications for COVID-19 in the primary care setting.

GANSamples-ac4C: Enhancing ac4C site prediction via generative adversarial networks and transfer learning.

RNA modification, N4-acetylcytidine (ac4C), is enzymatically catalyzed by N-acetyltransferase 10 (NAT10) and plays an essential role across tRNA, rRNA, and mRNA. It influences various cellular functions, including mRNA stability and rRNA biosynthesis. Wet-lab detection of ac4C modification sites is highly resource-intensive and costly. Therefore, various machine learning and deep learning techniques have been employed for computational detection of ac4C modification sites. The known ac4C modification sites are limited for training an accurate and stable prediction model. This study introduces GANSamples-ac4C, a novel framework that synergizes transfer learning and generative adversarial network (GAN) to generate synthetic RNA sequences to train a better ac4C modification site prediction model. Comparative analysis reveals that GANSamples-ac4C outperforms existing state-of-the-art methods in identifying ac4C sites. Moreover, our result underscores the potential of synthetic data in mitigating the issue of data scarcity for biological sequence prediction tasks. Another major advantage of GANSamples-ac4C is its interpretable decision logic. Multi-faceted interpretability analyses detect key regions in the ac4C sequences influencing the discriminating decision between positive and negative samples, a pronounced enrichment of G in this region, and ac4C-associated motifs. These findings may offer novel insights for ac4C research. The GANSamples-ac4C framework and its source code are publicly accessible at http://www.healthinformaticslab.org/supp/.

The host-targeted antiviral drug Zapnometinib exhibits a high barrier to the development of SARS-CoV-2 resistance.

Host targeting antiviral drugs (HTA) are directed against cellular mechanisms which can be exploited by viruses. These mechanisms are essential for viral replication, because missing functions cannot be compensated by the virus. However, this assumption needs experimental proof. Here we compared the HTA Zapnometinib (ZMN), with direct acting antivirals (DAA) (Remdesivir (RDV), Molnupiravir (MPV), Nirmatrelvir (NTV), Ritonavir (RTV), Paxlovid PAX)), in terms of their potency to induce reduced drug susceptibilities in SARS-CoV-2. During serial passage of δ-B1.617.2 adaptation to all DAAs occurred, while the inhibitory capacity of ZMN was not altered. Known single nucleotide polymorphisms (SNPs) responsible for partial resistances were found for RDV, NTV and PAX. Additionally, the high mutagenic potential of MPV was confirmed and decreased drug efficacies were found for the first time. Reduced DAA efficacy did not alter the inhibitory potential of ZMN. These results show that ZMN confers a high barrier towards the development of viral resistance and has the potential to act against partially DAA-insensitive viruses.

ß-D-N4-hydroxycytidine (NHC, EIDD-1931) inhibits chikungunya virus replication in mosquito cells and ex vivo Aedes aegypti guts, but not when ingested during blood-feeding.

Chikungunya virus (CHIKV) is a mosquito-borne virus transmitted by Aedes mosquitoes. While there are no antiviral therapies currently available to treat CHIKV infections, several licensed oral drugs have shown significant anti-CHIKV activity in cells and in mouse models. However, the efficacy in mosquitoes has not yet been assessed. Such cross-species antiviral activity could be favorable, since virus inhibition in the mosquito vector might prevent further transmission to vertebrate hosts. Here, we explored the antiviral effect of ß-d-N4-hydroxycytidine (NHC, EIDD-1931), the active metabolite of molnupiravir, on CHIKV replication in Aedes aegypti mosquitoes. Antiviral assays in mosquito cells and in ex vivo cultured mosquito guts showed that NHC had significant antiviral activity against CHIKV. Exposure to a clinically relevant concentration of NHC did not affect Ae. aegypti lifespan when delivered via a bloodmeal, but it slightly reduced the number of eggs developed in the ovaries. When mosquitoes were exposed to a blood meal containing both CHIKV and NHC, the compound did not significantly reduce virus infection and dissemination in the mosquitoes. This was confirmed by modelling and could be explained by pharmacokinetic analysis, which revealed that by 6 h post-blood-feeding, 90% of NHC had been cleared from the mosquito bodies. Our data show that NHC inhibited CHIKV replication in mosquito cells and gut tissue, but not in vivo when mosquitoes were provided with a CHIKV-infectious bloodmeal spiked with NHC. The pipeline presented in this study offers a suitable approach to identify anti-arboviral drugs that may impede replication in mosquitoes.

ac4C: a fragile modification with stabilizing functions in RNA metabolism.

In recent years, concerted efforts to map and understand epitranscriptomic modifications in mRNA have unveiled new complexities in the regulation of gene expression. These studies cumulatively point to diverse functions in mRNA metabolism, spanning pre-mRNA processing, mRNA degradation, and translation. However, this emerging landscape is not without its intricacies and sources of discrepancies. Disparities in detection methodologies, divergent interpretations of functional outcomes, and the complex nature of biological systems across different cell types pose significant challenges. With a focus of N4-acetylcytidine (ac4C), this review endeavors to unravel conflicting narratives by examining the technological, biological, and methodological factors that have contributed to discrepancies and thwarted research progress. Our goal is to mitigate detection inconsistencies and establish a unified model to elucidate the contribution of ac4C to mRNA metabolism and cellular equilibrium.

Effect of molnupiravir on SARS-CoV-2 evolution in immunocompromised patients: a retrospective observational study.

INTRODUCTION: Continued SARS-CoV-2 infection among immunocompromised individuals is likely to play a role in generating genomic diversity and the emergence of novel variants. Antiviral treatments such as molnupiravir are used to mitigate severe COVID-19 outcomes, but the extended effects of these drugs on viral evolution in patients with chronic infections remain uncertain. This study investigates how molnupiravir affects SARS-CoV-2 evolution in immunocompromised patients with prolonged infections. METHODS: The study included five immunocompromised patients treated with molnupiravir and four patients not treated with molnupiravir (two immunocompromised and two non-immunocompromised). We selected patients who had been infected by similar SARS-CoV-2 variants and with high-quality genomes across timepoints to allow comparison between groups. Throat and nasopharyngeal samples were collected in patients up to 44 days post treatment and were sequenced using tiled amplicon sequencing followed by variant calling. The UShER pipeline and University of California Santa Cruz genome viewer provided insights into the global context of variants. Treated and untreated patients were compared, and mutation profiles were visualised to understand the impact of molnupiravir on viral evolution. FINDINGS: Patients treated with molnupiravir showed a large increase in low-to-mid-frequency variants in as little as 10 days after treatment, whereas no such change was observed in untreated patients. Some of these variants became fixed in the viral population, including non-synonymous mutations in the spike protein. The variants were distributed across the genome and included unique mutations not commonly found in global omicron genomes. Notably, G-to-A and C-to-T mutations dominated the mutational profile of treated patients, persisting up to 44 days post treatment. INTERPRETATION: Molnupiravir treatment in immunocompromised patients led to the accumulation of a distinctive pattern of mutations beyond the recommended 5 days of treatment. Treated patients maintained persistent PCR positivity for the duration of monitoring, indicating clear potential for transmission and subsequent emergence of novel variants. FUNDING: Australian Research Council.

New conjugates based on N4-hydroxycytidine with more potent antiviral efficacy in vitro than EIDD-2801 against SARS-CoV-2 and other human coronaviruses.

The spread of COVID-19 continues due to genetic variation in SARS-CoV-2. Highly mutated variants of SARS-CoV-2 have an increased transmissibility and immune evasion. Due to the emergence of various new variants of the virus, there is an urgent need to develop broadly effective specific drugs for therapeutic strategies for the prevention and treatment of COVID-19. Molnupiravir (EIDD-2801, MK-4482), is an orally bioavailable ribonucleoside analogue of ß-D-N4-hydroxycytidine (NHC), has demonstrated efficacy against SARS-CoV-2 and was recently approved for COVID-19 treatment. To improve antiviral potency of NHC, we developed a panel of NHC conjugates with lipophilic vectors and ester derivatives with amino- and carboxylic-acids. Most of the synthesized compounds had comparable or higher (2-20 times) antiviral activity than EIDD-2801, against different lineages of SARS-CoV-2, MERS-CoV, seasonal coronaviruses OC43 and 229E, as well as bovine coronavirus. For further studies, we assessed the most promising compound in terms of activity, simplicity and cost of synthesis - NHC conjugate with phenylpropionic acid (SN_9). SN_9 has shown high efficacy in prophylactic, therapeutic and transmission models of COVID-19 infection in hamsters. Importantly, SN_9 profoundly inhibited virus replication in the lower respiratory tract of hamsters and transgenic mice infected with the Omicron sublineages XBB.1.9.1, XBB.1.16 and EG.5.1.1. These data indicate that SN_9 represents a promising antiviral drug candidate for COVID-19 treatment, and NHC modification strategies deserve further investigation as an approach to develop prodrugs against various coronaviruses.

N4-acetylcytidine (ac4C) promotes mRNA localization to stress granules.

Stress granules are an integral part of the stress response that are formed from non-translating mRNAs aggregated with proteins. While much is known about stress granules, the factors that drive their mRNA localization are incompletely described. Modification of mRNA can alter the properties of the nucleobases and affect processes such as translation, splicing and localization of individual transcripts. Here, we show that the RNA modification N4-acetylcytidine (ac4C) on mRNA associates with transcripts enriched in stress granules and that stress granule localized transcripts with ac4C are specifically translationally regulated. We also show that ac4C on mRNA can mediate localization of the protein NOP58 to stress granules. Our results suggest that acetylation of mRNA regulates localization of both stress-sensitive transcripts and RNA-binding proteins to stress granules and adds to our understanding of the molecular mechanisms responsible for stress granule formation.