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Molnupiravir is an orally available antiviral drug candidate currently in phase III trials for the treatment of patients with COVID-19. Molnupiravir increases the frequency of viral RNA mutations and impairs SARS-CoV-2 replication in animal models and in humans. Here, we establish the molecular mechanisms underlying molnupiravir-induced RNA mutagenesis by the viral RNA-dependent RNA polymerase (RdRp). Biochemical assays show that the RdRp uses the active form of molnupiravir, β- d - N 4 -hydroxycytidine (NHC) triphosphate, as a substrate instead of cytidine triphosphate or uridine triphosphate. When the RdRp uses the resulting RNA as a template, NHC directs incorporation of either G or A, leading to mutated RNA products. Structural analysis of RdRp–RNA complexes that contain mutagenesis products shows that NHC can form stable base pairs with either G or A in the RdRp active center, explaining how the polymerase escapes proofreading and synthesizes mutated RNA. This two-step mutagenesis mechanism probably applies to various viral polymerases and can explain the broad-spectrum antiviral activity of molnupiravir. Quantitative biochemical assays and high-resolution cryo-EM analysis reveal how the COVID-19 antiviral drug candidate molnupiravir causes lethal viral mutagenesis by the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2.

Molnupiravir, an antiviral medication widely used against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), acts by inducing mutations in the virus genome during replication. Most random mutations are likely to be deleterious to the virus and many will be lethal; thus, molnupiravir-induced elevated mutation rates reduce viral load 1 , 2 . However, if some patients treated with molnupiravir do not fully clear the SARS-CoV-2 infections, there could be the potential for onward transmission of molnupiravir-mutated viruses. Here we show that SARS-CoV-2 sequencing databases contain extensive evidence of molnupiravir mutagenesis. Using a systematic approach, we find that a specific class of long phylogenetic branches, distinguished by a high proportion of G-to-A and C-to-T mutations, are found almost exclusively in sequences from 2022, after the introduction of molnupiravir treatment, and in countries and age groups with widespread use of the drug. We identify a mutational spectrum, with preferred nucleotide contexts, from viruses in patients known to have been treated with molnupiravir and show that its signature matches that seen in these long branches, in some cases with onward transmission of molnupiravir-derived lineages. Finally, we analyse treatment records to confirm a direct association between these high G-to-A branches and the use of molnupiravir. A specific class of long phylogenetic branches, distinguished by a high proportion of G-to-A and C-to-T mutations, are almost exclusively found in sequences from 2022, after molnupiravir treatment was introduced, indicating that molnupiravir treatment can give rise to viable mutagenized viruses.

Extrachromosomal DNA (ecDNA) is a major contributor to treatment resistance and poor outcome for patients with cancer 1 , 2 . Here we examine the diversity of ecDNA elements across cancer, revealing the associated tissue, genetic and mutational contexts. By analysing data from 14,778 patients with 39 tumour types from the 100,000 Genomes Project, we demonstrate that 17.1% of tumour samples contain ecDNA. We reveal a pattern highly indicative of tissue-context-based selection for ecDNAs, linking their genomic content to their tissue of origin. We show that not only is ecDNA a mechanism for amplification of driver oncogenes, but it also a mechanism that frequently amplifies immunomodulatory and inflammatory genes, such as those that modulate lymphocyte-mediated immunity and immune effector processes. Moreover, ecDNAs carrying immunomodulatory genes are associated with reduced tumour T cell infiltration. We identify ecDNAs bearing only enhancers, promoters and lncRNA elements, suggesting the combinatorial power of interactions between ecDNAs in trans . We also identify intrinsic and environmental mutational processes linked to ecDNA, including those linked to its formation, such as tobacco exposure, and progression, such as homologous recombination repair deficiency. Clinically, ecDNA detection was associated with tumour stage, more prevalent after targeted therapy and cytotoxic treatments, and associated with metastases and shorter overall survival. These results shed light on why ecDNA is a substantial clinical problem that can cooperatively drive tumour growth signals, alter transcriptional landscapes and suppress the immune system. A study examines the diversity of extrachromosomal DNA elements in cancer, and provides details on the frequency and origin of extrachromosomal DNA and its role in the development of different types of cancer.

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for virus infection through the engagement of the human ACE2 protein 1 and is a major antibody target. Here we show that chronic infection with SARS-CoV-2 leads to viral evolution and reduced sensitivity to neutralizing antibodies in an immunosuppressed individual treated with convalescent plasma, by generating whole-genome ultra-deep sequences for 23 time points that span 101 days and using in vitro techniques to characterize the mutations revealed by sequencing. There was little change in the overall structure of the viral population after two courses of remdesivir during the first 57 days. However, after convalescent plasma therapy, we observed large, dynamic shifts in the viral population, with the emergence of a dominant viral strain that contained a substitution (D796H) in the S2 subunit and a deletion (ΔH69/ΔV70) in the S1 N-terminal domain of the spike protein. As passively transferred serum antibodies diminished, viruses with the escape genotype were reduced in frequency, before returning during a final, unsuccessful course of convalescent plasma treatment. In vitro, the spike double mutant bearing both ΔH69/ΔV70 and D796H conferred modestly decreased sensitivity to convalescent plasma, while maintaining infectivity levels that were similar to the wild-type virus.The spike substitution mutant D796H appeared to be the main contributor to the decreased susceptibility to neutralizing antibodies, but this mutation resulted in an infectivity defect. The spike deletion mutant ΔH69/ΔV70 had a twofold higher level of infectivity than wild-type SARS-CoV-2, possibly compensating for the reduced infectivity of the D796H mutation. These data reveal strong selection on SARS-CoV-2 during convalescent plasma therapy, which is associated with the emergence of viral variants that show evidence of reduced susceptibility to neutralizing antibodies in immunosuppressed individuals. Chronic infection with SARS-CoV-2 leads to the emergence of viral variants that show reduced susceptibility to neutralizing antibodies in an immunosuppressed individual treated with convalescent plasma.

Although it is well known that microbial populations can respond adaptively to challenges from antibiotics, empirical difficulties in distinguishing the roles of de novo mutation and natural selection have left several issues unresolved. Here, we explore the mutational properties of Escherichia coli exposed to long-term sublethal levels of the antibiotic norfloxacin, using a mutation accumulation design combined with whole-genome sequencing of replicate lines. The genome-wide mutation rate significantly increases with norfloxacin concentration. This response is associated with enhanced expression of error-prone DNA polymerases and may also involve indirect effects of norfloxacin on DNA mismatch and oxidative-damage repair. Moreover, we find that acquisition of antibiotic resistance can be enhanced solely by accelerated mutagenesis, i.e., without direct involvement of selection. Our results suggest that antibiotics may generally enhance the mutation rates of target cells, thereby accelerating the rate of adaptation not only to the antibiotic itself but to additional challenges faced by invasive pathogens.

A high tumour mutational burden (hypermutation) is observed in some gliomas 1 – 5 ; however, the mechanisms by which hypermutation develops and whether it predicts the response to immunotherapy are poorly understood. Here we comprehensively analyse the molecular determinants of mutational burden and signatures in 10,294 gliomas. We delineate two main pathways to hypermutation: a de novo pathway associated with constitutional defects in DNA polymerase and mismatch repair (MMR) genes, and a more common post-treatment pathway, associated with acquired resistance driven by MMR defects in chemotherapy-sensitive gliomas that recur after treatment with the chemotherapy drug temozolomide. Experimentally, the mutational signature of post-treatment hypermutated gliomas was recapitulated by temozolomide-induced damage in cells with MMR deficiency. MMR-deficient gliomas were characterized by a lack of prominent T cell infiltrates, extensive intratumoral heterogeneity, poor patient survival and a low rate of response to PD-1 blockade. Moreover, although bulk analyses did not detect microsatellite instability in MMR-deficient gliomas, single-cell whole-genome sequencing analysis of post-treatment hypermutated glioma cells identified microsatellite mutations. These results show that chemotherapy can drive the acquisition of hypermutated populations without promoting a response to PD-1 blockade and supports the diagnostic use of mutational burden and signatures in cancer. Temozolomide therapy seems to lead to mismatch repair deficiency and hypermutation in gliomas, but not to an increase in response to immunotherapy.

The sequencing of AML genomes of eight patients before and after relapse reveals two major patterns of clonal evolution, with chemotherapy appearing to have a role in both patterns. Tumour cell evolution in AML Many patients with acute myeloid leukaemia (AML) achieve remission, but it is often short-lived and the returned disease is usually refractory to therapy. Genome sequencing of eight patients with AML before and after relapse reveals two major patterns of tumour cell evolution. The founding clone survives chemotherapy in all patients, and, in one clonal pattern, it acquires new mutations and expands at relapse. In the other, a subclone surviving from the original tumour expands and then acquires new mutations. Comparisons of relapse-specific and primary tumour mutations point to an increase in transversions, implying DNA damage caused by cytotoxic chemotherapy. This work demonstrates that the AML genome in an individual patient presents a moving target, and highlights the importance of striving to eradicate both the founding clone and all of its subclones. Most patients with acute myeloid leukaemia (AML) die from progressive disease after relapse, which is associated with clonal evolution at the cytogenetic level 1 , 2 . To determine the mutational spectrum associated with relapse, we sequenced the primary tumour and relapse genomes from eight AML patients, and validated hundreds of somatic mutations using deep sequencing; this allowed us to define clonality and clonal evolution patterns precisely at relapse. In addition to discovering novel, recurrently mutated genes (for example, WAC , SMC3 , DIS3 , DDX41 and DAXX ) in AML, we also found two major clonal evolution patterns during AML relapse: (1) the founding clone in the primary tumour gained mutations and evolved into the relapse clone, or (2) a subclone of the founding clone survived initial therapy, gained additional mutations and expanded at relapse. In all cases, chemotherapy failed to eradicate the founding clone. The comparison of relapse-specific versus primary tumour mutations in all eight cases revealed an increase in transversions, probably due to DNA damage caused by cytotoxic chemotherapy. These data demonstrate that AML relapse is associated with the addition of new mutations and clonal evolution, which is shaped, in part, by the chemotherapy that the patients receive to establish and maintain remissions.

Tumor recurrence is a leading cause of cancer mortality. Therapies for recurrent disease may fail, at least in part, because the genomic alterations driving the growth of recurrences are distinct from those in the initial tumor. To explore this hypothesis, we sequenced the exornes of 23 initial low-grade gliomas and recurrent tumors resected from the same patients. In 43% of cases, at least half of the mutations in the initial tumor were undetected at recurrence, including driver mutations in TP53, ATRX, SMARCA4, and BRAF; this suggests that recurrent tumors are often seeded by cells derived from the initial tumor at a very early stage of their evolution. Notably, tumors from 6 of 10 patients treated with the chemotherapeutic drug temozolomide (TMZ) followed an alternative evolutionary path to high-grade glioma. At recurrence, these tumors were hypermutated and harbored driver mutations in the RB (retinoblastoma) and Akt-mTOR (mammalian target of rapamycin) pathways that bore the signature of TMZ-induced mutagenesis.

Maximum-depth sequencing (MDS), a new method of detecting extremely rare variants within a bacterial population, is used to show that mutation rates in Escherichia coli vary across the genome by at least an order of magnitude, and also to uncover mechanisms of antibiotic-induced mutagenesis. Direct measurement of low-level bacterial mutation rates Knowledge of the spontaneous mutation rate of bacteria is important for the study of basic evolutionary processes and of potential value in various clinical settings. De novo mutations in bacteria are a difficult target for high-throughput sequencing, but now Justin Jee et al . describe a new method of detecting extremely rare variants within a bacterial population, termed maximum-depth sequencing (MDS), which can detect extremely rare variants within a bacterial population through error-corrected, high-throughput sequencing. The authors use this method to measure locus-specific mutation rates in Escherichia coli and show that they vary across the genome by at least an order of magnitude. MDS shows that certain types of nucleotide misincorporation occur 10 4 -fold more frequently than the basal mutation rate, but are repaired in vivo and are thus undetectable by conventional methods. Using MDS, the authors also uncover mechanisms of antibiotic-induced mutagenesis. In 1943, Luria and Delbrück used a phage-resistance assay to establish spontaneous mutation as a driving force of microbial diversity 1 . Mutation rates are still studied using such assays, but these can only be used to examine the small minority of mutations conferring survival in a particular condition. Newer approaches, such as long-term evolution followed by whole-genome sequencing 2 , 3 , may be skewed by mutational ‘hot’ or ‘cold’ spots 3 , 4 . Both approaches are affected by numerous caveats 5 , 6 , 7 . Here we devise a method, maximum-depth sequencing (MDS), to detect extremely rare variants in a population of cells through error-corrected, high-throughput sequencing. We directly measure locus-specific mutation rates in Escherichia coli and show that they vary across the genome by at least an order of magnitude. Our data suggest that certain types of nucleotide misincorporation occur 10 4 -fold more frequently than the basal rate of mutations, but are repaired in vivo . Our data also suggest specific mechanisms of antibiotic-induced mutagenesis, including downregulation of mismatch repair via oxidative stress, transcription–replication conflicts, and, in the case of fluoroquinolones, direct damage to DNA.

Chemotherapies may increase mutagenesis of healthy cells and change the selective pressures in tissues, thus influencing their evolution. However, their contributions to the mutation burden and clonal expansions of healthy somatic tissues are not clear. Here, exploiting the mutational footprint of some chemotherapies, we explore their influence on the evolution of hematopoietic cells. Cells of Acute Myeloid Leukemia (AML) secondary to treatment with platinum-based drugs show the mutational footprint of these drugs, indicating that non-malignant blood cells receive chemotherapy mutations. No trace of the 5-fluorouracil (5FU) mutational signature is found in AMLs secondary to exposure to 5FU, suggesting that cells establishing the leukemia could be quiescent during treatment. Using the platinum-based mutational signature as a barcode, we determine that the clonal expansion originating the secondary AMLs begins after the start of the cytotoxic treatment. Its absence in clonal hematopoiesis cases is consistent with the start of the clonal expansion predating the exposure to platinum-based drugs. The mutational effects of chemotherapies on healthy cells are unclear. Here, the authors show that the mutational signature of platinum-based drugs -but not 5-fluorouracil- is detectable in secondary acute myeloid leukemia, implying that the clonal expansion begins after the start of therapy.