Lethal Mutagenesis Research Articles

27. USING A TRIO-BASED GENETIC DESIGN FOR INVESTIGATING THE AETIOLOGY OF ATTENTION DEFICIT HYPERACTIVITY DISORDER (ADHD)

Three fundamental pillars for the understanding of virus variation and population dynamics are Darwinian principles, quantum mechanical uncertainties at the atomic level, and internal interactions and network connections among components of an ensemble. In this closing chapter, such general pillars are applied to non-viral systems, in particular prokaryotic and eukaryotic cellular collectivities, and non-genetic systems such as prions. Similarities and differences in the behavior of viruses and cells are analyzed with regard to adaptive molecular mechanisms and consequences for disease treatment. In particular, differences between antiviral resistance and antibiotic resistance, and the possibility of a lethal mutagenesis approach for cancer are examined in the light of concepts exposed in previous chapters for viruses. Clonality in cellular and viral evolution is discussed, with the focus in a distinction between inconsequential and biologically meaningful recombination in the infectious cycles of viruses. The chapter ends with some general considerations on network connections in biological and non-biological systems, with the possibility that the understanding of interactions within viral populations may open new windows for antiviral interventions.

Mechanism of action of favipiravir against SARS-CoV-2: Mutagenesis or chain termination?

Mechanism of action of favipiravir against SARS-CoV-2: Mutagenesis or chain termination?

Molnupiravir: coding for catastrophe.

Molnupiravir, a wide-spectrum antiviral that is currently in phase 2/3 clinical trials for the treatment of COVID-19, is proposed to inhibit viral replication by a mechanism known as ‘lethal mutagenesis’. Two recently published studies reveal the biochemical and structural bases of how molnupiravir disrupts the fidelity of SARS-CoV-2 genome replication and prevents viral propagation by fostering error accumulation in a process referred to as ‘error catastrophe’.

Distinct Antiretroviral Mechanisms Elicited by a Viral Mutagen

5-aza-cytidine (5-aza-C) has been shown to be a potent human immunodeficiency virus type 1 (HIV-1) mutagen that induces G-to-C hypermutagenesis by incorporation of the reduced form (i.e., 5-aza-dC, 5-aza-dCTP). Evidence to date suggests that this lethal mutagenesis is the primary antiretroviral mechanism for 5-aza-C. To investigate the breadth of application of 5-aza-C as an antiretroviral mutagen, we have conducted a comparative, parallel analysis of the antiviral mechanism of 5-aza-C between HIV-1 and gammaretroviruses – i.e., murine leukemia virus (MuLV) and feline leukemia virus (FeLV). Intriguingly, in contrast to the hallmark G-to-C hypermutagenesis observed with HIV-1, MuLV and FeLV did not reveal the presence of a significant increase in mutational burden, particularly that of G-to-C transversion mutations. The effect of 5-aza-dCTP on DNA synthesis revealed that while HIV-1 RT was not inhibited by 5-aza-dCTP even at 100 µM, 5-aza-dCTP was incorporated and significantly inhibited MuLV RT, generating pause sites and reducing the fully extended product. 5-aza-dCTP was found to be incorporated into DNA by MuLV RT or HIV-1 RT, but only acted as a non-obligate chain terminator for MuLV RT. This biochemical data provides an independent line of experimental evidence in support of the conclusion that HIV-1 and MuLV have distinct primary mechanisms of antiretroviral action with 5-aza-C. Taken together, our data provides striking evidence that an antiretroviral mutagen can have strong potency via distinct mechanisms of action among closely related viruses, unlinking antiviral activity from antiviral mechanism of action.

Plitidepsin, an inhibitor of the cell elongation factor eEF1a, and molnupiravir an analogue of the ribonucleoside cytidine, two new chemical compounds with intense activity against SARS-CoV-2

RESUMENEl conocimiento del ciclo replicativo del SARS-CoV-2 y sus interacciones con las proteínas celulares, ha abierto una nueva posibilidad terapéutica basada en el bloqueo de las esenciales para el virus. El factor de elongación proteico celular eEF1A podría ser una buena diana. Dentro de sus inhibidores naturales están las didemninas y sus compuestos químicos relacionados como la plitidepsina. En cultivo de células humanas este compuesto es capaz de inhibir al virus con una potencia 27,5 veces la del remdesivir. Debe administrarse por vía endovenosa. De los compuestos análogos a los ribonucleósidos, el molnupiravir (MK-4482/EIDD-2801) (hidroxi-citidina) determina una mutagenesis letal sobre el SARS-CoV-2. En animales, tras su administración oral disminuye unas 25.000 veces la carga viral pulmonar y unas 100.000 veces administrado como profilaxis. Evita la transmisión del virus y elimina su presencia en la orofaringe. Ambos compuestos químicos han iniciado los ensayos clínicos en humanos en Fase I/II.

Effect of Favipiravir on some epidemic infections: A mini review

Favipiravir is a modern antiviral drug that can be used in developing viral pandemics, such as the Ebola virus, H1N1 flu, Lassa fever, and haemorrhagic fever outbreaks in Argentina in 2009. A. Chain conclusion Favipiravir is transformed to favipiravir-RTP and inserted into the RNA strand that is elongating. Then, since favipiravir acts as a chain terminator, chain elongation slows down at incorporation site of favipiravir and does not continue. The proofreading enzyme cannot repair this complex of RNA-favipiravir and it will be discarded as excessive RNA, which causes the extinction of the viral genome. Mutagenesis with lethal consequences Ribavirin is used in the RNA elongating process until completion. The single strand of RNA with several ribavirin incorporation sites incorporates ribavirin or acts as mRNA for the production of viral proteins. Due to incorporation of ribavirin onto viral RNA, a mismatching of the base pairs (transition mutation) will occur. Translation of this mutated RNA will then cause mutations in the sequence of amino acid residues of the protein, result in impaired of protein function. Viral proteins that have lost their function are unable to replicate or generate infectious viral particles, and the virus is no longer infectious. As a result, lethal mutagenesis ends viral infection in a different way than chain termination. Favipiravir’s clinical trial against Ebola virus-infected patients has not yet been formally approved, and further research is required. Favipiravir, on the other hand, poses a risk of embryo toxicity and teratogenicity. As a result, the Japanese Ministry of Health, Welfare and Labor have been approved this medication with recommendations for more clinical use until August 15, 2014, when they approved the use of favipiravir due to the Ebola virus epidemic in West Africa. The aim of this review is to investigate the potential impact of favipiravir on infections espectially RNA-virus infections, according to previous published investigations, and thus to encourage rapid approval of this interesting antivirus in emergencies.

Improving properties of the nucleobase analogs T-705/T-1105 as potential antiviral.

In this minireview we describe our work on the improvement of the nucleobase analogs Favipiravir (T-705) und its non-fluorinated derivative T-1105 as influenza and SARS-CoV-2 active compounds. Both nucleobases were converted into nucleotides and then included in our nucleotide prodrugs technologies cycloSal-monophosphates, DiPPro-nucleoside diphosphates and TriPPPro-nucleoside triphosphates. Particularly the DiPPro-derivatives of T-1105-RDP proved to be very active against influenza viruses. T-1105-derivatives in general were found to be more antivirally active as compared to their T-705 counterpart. This may be due to the low chemical stability of all ribosylated derivatives of T-705. The ribosyltriphosphate derivative of T-1105 was studied for the potential to act as a inhibitor of the SARS-CoV-2 RdRp and was found to be an extremely potent compound causing lethal mutagenesis. The pronucleotide technologies, the chemical synthesis, the biophysical properties and the biological effects of the compounds will be addressed as well.

Favipiravir and COVID-19: A Simplified Summary.

A recent outbreak of coronavirus disease 2019 (COVID-19) caused by the novel coronavirus designated as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) started in Wuhan, China, at the end of 2019 and then spread rapidly all over the world. However, there are no specific antiviral therapies for COVID-19, using the agents which approved or in development for other viral infections is one of the potentially quickest ways to find treatment for this new viral infection. Favipiravir is an effective agent that acts as a nucleotide analog that selectively inhibits the viral RNA dependent RNA polymerase or causes lethal mutagenesis upon incorporation into the virus RNA. In view of recent studies and discussion on favipiravir, in this mini review we aimed to summarize the clinical trials studying the efficacy and safety of favipiravir in patients with COVID-19.

Rapid incorporation of Favipiravir by the fast and permissive viral RNA polymerase complex results in SARS-CoV-2 lethal mutagenesis

The ongoing Corona Virus Disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has emphasized the urgent need for antiviral therapeutics. The viral RNA-dependent-RNA-polymerase (RdRp) is a promising target with polymerase inhibitors successfully used for the treatment of several viral diseases. We demonstrate here that Favipiravir predominantly exerts an antiviral effect through lethal mutagenesis. The SARS-CoV RdRp complex is at least 10-fold more active than any other viral RdRp known. It possesses both unusually high nucleotide incorporation rates and high-error rates allowing facile insertion of Favipiravir into viral RNA, provoking C-to-U and G-to-A transitions in the already low cytosine content SARS-CoV-2 genome. The coronavirus RdRp complex represents an Achilles heel for SARS-CoV, supporting nucleoside analogues as promising candidates for the treatment of COVID-19.

Overcoming nonstructural protein 15-nidoviral uridylate-specific endoribonuclease (nsp15/NendoU) activity of SARS-CoV-2

COVID-19 has become the gravest global public health crisis since the Spanish Flu of 1918. Combination antiviral therapy with repurposed broad-spectrum antiviral agents holds a highly promising immediate treatment strategy, especially given uncertainties of vaccine efficacy and developmental timeline. Here, we describe a novel hypothetical approach: combining available broad-spectrum antiviral agents such as nucleoside analogs with potential inhibitors of NendoU, for example nsp15 RNA substrate mimetics. While only hypothesis-generating, this approach may constitute a ‘double-hit’ whereby two CoV-unique protein elements of the replicase–transcriptase complex are inhibited simultaneously; this may be an Achilles' heel and precipitate lethal mutagenesis in a coronavirus. It remains to be seen whether structurally optimized RNA substrate mimetics in combination with clinically approved and repurposed backbone antivirals can synergistically inhibit this endonuclease in vitro, thus fulfilling the ‘double-hit hypothesis’.

Moderate Amounts of Epistasis are Not Evolutionarily Stable in Small Populations.

High mutation rates select for the evolution of mutational robustness where populations inhabit flat fitness peaks with little epistasis, protecting them from lethal mutagenesis. Recent evidence suggests that a different effect protects small populations from extinction via the accumulation of deleterious mutations. In drift robustness, populations tend to occupy peaks with steep flanks and positive epistasis between mutations. However, it is not known what happens when mutation rates are high and population sizes are small at the same time. Using a simple fitness model with variable epistasis, we show that the equilibrium fitness has a minimum as a function of the parameter that tunes epistasis, implying that this critical point is an unstable fixed point for evolutionary trajectories. In agent-based simulations of evolution at finite mutation rate, we demonstrate that when mutations can change epistasis, trajectories with a subcritical value of epistasis evolve to decrease epistasis, while those with supercritical initial points evolve towards higher epistasis. These two fixed points can be identified with mutational and drift robustness, respectively.

A mathematical study for the development of anti-HIV-1 therapies based on the lethal mutagenesis method

This study tried to mathematically unveil the difficulties associated with an anti-HIV therapy based on the lethal mutagenesis method; two difficulties were identified. The first difficulty of this method was that the efficacy of a mutagen must be over a certain threshold to treat HIV-1 infection, which could explain the apparent failure of the KP-1461 trial. Furthermore, we investigated the influence of the latency of HIV-1 on the treatment period and clarified that the latency increases the treatment period in a manner almost independent of the efficacy of a mutagen. This is the second difficulty associated with the lethal mutagenesis method.

Herpesvirus DNA Polymerase Mutants—How Important Is Faithful Genome Replication?

This report broaches the topic of altered fidelity of DNA replication in herpesvirus mutants described over the past decades. Reduced genome replication fidelity of herpesvirus exonuclease mutants allows studying of virus population dynamics in the absence of exonucleolytic proofreading and can inform us on virus evolution in the face of error-prone genome replication. We recently found that mutations previously described to be lethal for herpes simplex type 1 (HSV-1) caused error-prone genome replication in Marek’s disease virus. This has allowed us to study the influence of augmented genetic diversity on viral population dynamics, replicative fitness, and virulence. We conclude that the use of herpesvirus fidelity mutants allows unprecedented insights into virus evolution driven by low-fidelity replication. More than that, their use allows us to observe accelerated evolution, potentially enabling time-saving screens for the rise of drug- or vaccine-resistant mutants. In addition, we can infer that lethal or suicidal phenotypes observed in low-fidelity herpesvirus mutants are likely a consequence of error-prone genome replication, ultimately leading to lethal mutagenesis of small and isolated virus populations in cell culture.

When sinks become sources: Adaptive colonization in asexuals.

The establishment of a population into a new empty habitat outside of its initial niche is a phenomenon akin to evolutionary rescue in the presence of immigration. It underlies a wide range of processes, such as biological invasions by alien organisms, host shifts in pathogens, or the emergence of resistance to pesticides or antibiotics from untreated areas. We derive an analytically tractable framework to describe the evolutionary and demographic dynamics of asexual populations in a source-sink system. We analyze the influence of several factors on the establishment success in the sink, and on the time until establishment. To this aim, we use a classic phenotype-fitness landscape (Fisher's geometrical model in n dimensions) where the source and sink habitats have different phenotypic optima. In case of successful establishment, the mean fitness in the sink follows a typical four-phases trajectory. The waiting time to establishment is independent of the immigration rate and has a "U-shaped" dependence on the mutation rate, until some threshold where lethal mutagenesis impedes establishment and the sink population remains so. We use these results to get some insight into possible effects of several managementstrategies.

Viral quasispecies.

Viral quasispecies refers to a population structure that consists of extremely large numbers of variant genomes, termed mutant spectra, mutant swarms or mutant clouds. Fueled by high mutation rates, mutants arise continually, and they change in relative frequency as viral replication proceeds. The term quasispecies was adopted from a theory of the origin of life in which primitive replicons) consisted of mutant distributions, as found experimentally with present day RNA viruses. The theory provided a new definition of wild type, and a conceptual framework for the interpretation of the adaptive potential of RNA viruses that contrasted with classical studies based on consensus sequences. Standard clonal analyses and deep sequencing methodologies have confirmed the presence of myriads of mutant genomes in viral populations, and their participation in adaptive processes. The quasispecies concept applies to any biological entity, but its impact is more evident when the genome size is limited and the mutation rate is high. This is the case of the RNA viruses, ubiquitous in our biosphere, and that comprise many important pathogens. In virology, quasispecies are defined as complex distributions of closely related variant genomes subjected to genetic variation, competition and selection, and that may act as a unit of selection. Despite being an integral part of their replication, high mutation rates have an upper limit compatible with inheritable information. Crossing such a limit leads to RNA virus extinction, a transition that is the basis of an antiviral design termed lethal mutagenesis.

Synergistic lethal mutagenesis of hepatitis C virus.

Lethal mutagenesis is an antiviral approach that consists in extinguishing a virus by an excess of mutations acquired during replication in the presence of a mutagenic agent, often a nucleotide analogue. One of its advantages is its broad spectrum nature that renders the strategy potentially effective against emergent RNA viral infections. Here we describe synergistic lethal mutagenesis of hepatitis C virus (HCV) by a combination of favipiravir (T-705) and ribavirin. Synergy has been documented over a broad range of analogue concentrations using the Chou-Talalay method as implemented in the CompuSyn graphics, with average dose reduction index (DRI) above 1 (68.02±101.6 for favipiravir, and 5.83±6.07 for ribavirin), and average combination indices (CI) below 1 (0.52±0.28). Furthermore, analogue concentrations that individually did not extinguish high fitness HCV in ten serial infections, when used in combination they extinguished high fitness HCV in one to two passages. Although both analogues display a preference for G→A and C→U transitions, deep sequencing analysis of mutant spectra indicated a different preference of the two analogues for the mutation sites, thus unveiling a new possible synergy mechanism in lethal mutagenesis. Prospects of synergy among mutagenic nucleotides as a strategy to confront emerging viral infections are discussed.

Lethal Mutagenesis of Rift Valley Fever Virus Induced by Favipiravir.

Rift Valley fever virus (RVFV) is an emerging, mosquito-borne, zoonotic pathogen with recurrent outbreaks taking a considerable toll in human deaths in many African countries, for which no effective treatment is available. In cell culture studies and with laboratory animal models, the nucleoside analogue favipiravir (T-705) has demonstrated great potential for the treatment of several seasonal, chronic, and emerging RNA virus infections in humans, suggesting applicability to control some viral outbreaks. Treatment with favipiravir was shown to reduce the infectivity of Rift Valley fever virus both in cell cultures and in experimental animal models, but the mechanism of this protective effect is not understood. In this work, we show that favipiravir at concentrations well below the toxicity threshold estimated for cells is able to extinguish RVFV from infected cell cultures. Nucleotide sequence analysis has documented RVFV mutagenesis associated with virus extinction, with a significant increase in G to A and C to U transition frequencies and a decrease of specific infectivity, hallmarks of lethal mutagenesis.

The increasing impact of lethal mutagenesis of viruses.

Selection of viral mutants resistant to compounds used in therapy is a major determinant of treatment failure, a problem akin to antibiotic resistance in bacteria. In this scenario, mutagenic base and nucleoside analogs have entered the picture because they increase the mutation rate of viral populations to levels incompatible with their survival. This antiviral strategy is termed lethal mutagenesis. It has found a major impulse with the observation that some antiviral agents, which initially were considered only inhibitors of virus multiplication, may in effect exert part of their antiviral activity through mutagenesis. Here, we review the conceptual basis of lethal mutagenesis, the evidence of virus extinction through mutagenic nucleotide analogs and prospects for application in antiviral designs.

Population persistence under high mutation rate: From evolutionary rescue to lethal mutagenesis.

Populations may genetically adapt to severe stress that would otherwise cause their extirpation. Recent theoretical work, combining stochastic demography with Fisher's geometric model of adaptation, has shown how evolutionary rescue becomes unlikely beyond some critical intensity of stress. Increasing mutation rates may however allow adaptation to more intense stress, raising concerns about the effectiveness of treatments against pathogens. This previous work assumes that populations are rescued by the rise of a single resistance mutation. However, even in asexual organisms, rescue can also stem from the accumulation of multiple mutations in a single genome. Here, we extend previous work to study the rescue process in an asexual population where the mutation rate is sufficiently high so that such events may be common. We predict both the ultimate extinction probability of the population and the distribution of extinction times. We compare the accuracy of different approximations covering a large range of mutation rates. Moderate increase in mutation rates favors evolutionary rescue. However, larger increase leads to extinction by the accumulation of a large mutation load, a process called lethal mutagenesis. We discuss how these results could help design "evolution-proof" antipathogen treatments that even highly mutable strains could not overcome.

T-705 induces lethal mutagenesis in Ebola and Marburg populations in macaques

Nucleoside analogues (NA) disrupt RNA viral RNA-dependent RNA polymerase (RdRP) function and fidelity for multiple viral families. The mechanism of action (MOA) of T-705 has been attributed alternatively or concurrently to chain termination and lethal mutagenesis depending on the viral species during in vitro studies. In this study, we evaluated the effect of T-705 on the viral population in non-human primates (NHPs) after challenge with Ebola virus (EBOV) or Marburg virus (MARV) to identify the predominant in vivo MOA. We used common virological assays in conjunction with deep sequencing to characterize T-705 effects. T-705 exhibited antiviral activity that was associated with a reduction in specific infectivity and an accumulation of low frequency nucleotide variants in plasma samples collected day 7 post infection. Stranded analysis of deep sequencing data to identify chain termination demonstrated no change in the transcriptional gradient in negative stranded viral reads and minimal changes in positive stranded viral reads in T-705 treated animals, questioning as a MOA in vivo. These findings indicate that lethal mutagenesis is a MOA of T-705 that may serve as an indication of therapeutic activity of NAs for evaluation in clinical settings. This study expands our understanding of MOAs of these compounds for the Filovirus family and provides further evidence that lethal mutagenesis could be a preponderant MOA for this class of therapeutic compounds.