Steps Of Replication Research Articles

Recovery of Recombinant Marburg Virus by Reverse Genetics.

Reverse genetics systems can be used to study parts or all of the viral life cycle. Recovery systems are reverse genetics systems used for the generation of infectious recombinant viruses that can carry targeted mutations or express reporter genes. The former can help address basic research questions to better understand certain steps of viral replication, while recombinant viruses expressing reporter genes, such as luciferases or fluorescent proteins, can be used for drug screening assays. Accordingly, recombinant viruses can be of great use for a variety of research purposes, including antiviral compound screenings and development of other countermeasures if no virus isolates are available. In this protocol, we describe a T7-driven reverse genetics system to generate recombinant Marburg virus from full-length cDNA.

DciA, the Bacterial Replicative Helicase Loader, promotes LLPS in the presence of ssDNA

The loading of the bacterial replicative helicase DnaB is an essential step for genome replication and depends on the assistance of accessory proteins. Several of these proteins have been identified across the bacterial phyla. DciA is the most common loading protein in bacteria, yet the one whose mechanism is the least understood. We have previously shown that DciA from Vibrio cholerae is composed of a globular domain followed by an unfolded extension and demonstrated its strong affinity for DNA. Here, we characterize the condensates formed by VcDciA upon interaction with a short single-stranded DNA substrate. We demonstrate the fluidity of these condensates using light microscopy and address their network organization through electron microscopy, thereby bridging events to conclude on a liquid-liquid phase separation behavior. Additionally, we observe the recruitment of DnaB in the droplets, concomitant with the release of DciA. We show that the well-known helicase loader DnaC from Escherichia coli is also competent to form these phase-separated condensates in the presence of ssDNA. Our phenomenological data are still preliminary as regards the existence of these condensates in vivo, but open the way for exploring the potential involvement of DciA in the formation of non-membrane compartments within the bacterium to facilitate the assembly of replication players on chromosomal DNA.

AMENAMEVIR: A BREAKTHROUGH IN HERPESVIRUS THERAPY

Amenamevir is a promising antiviral agent with strong activity against herpes simplex virus (HSV) and varicella-zoster virus (VZV). This review article aims to explore its pharmacodynamics, therapeutic potential, and safety profile in detail. Amenamevir functions by inhibiting the viral helicase-primase complex, a crucial step in viral DNA replication, distinguishing it from other antiviral drugs. The article examines the results from clinical trials assessing its efficacy in treating recurrent herpesvirus infections, its pharmacokinetic properties, and its suitability for patients with compromised immune systems. We also discuss the drugs safety and tolerability, identifying common side effects, drug interactions, and contraindications. Furthermore, the review highlights Amenamevirs potential role in managing antiviral resistance and its possible broader applications in the treatment of chronic viral infections. Finally, future directions are outlined, including the exploration of combination therapy and the development of more accessible oral formulations, to optimize the clinical use of Amenamevir.

Lactobacilli-Derived Postmetabolites Are Broad-Spectrum Inhibitors of Herpes Viruses In Vitro.

Herpes viruses are highly contagious agents affecting all classes of vertebrates, thus causing serious health, social, and economic losses. Within the One Health concept, novel therapeutics are extensively studied for both veterinary and human control and management of the infection, but the optimal strategy has not been invented yet. Lactic acid bacteria are key components of the microbiome that are known to play a protective role against pathogens as one of the proposed mechanisms involves compounds released from their metabolic activity. Previously, we reported the anti-herpes effect of postmetabolites isolated from Lactobacilli, and here, we confirm the inhibitory properties of another nine products against the phylogenetically distant human Herpes simplex virus-1 (HSV-1) and fish Koi Herpes virus (KHV) in cell cultures. Cytotoxicity, cytopathic effect inhibition, virucidal effect, the influence on the adsorption stage of the virus to the cells, as well as the protective effect of the postmetabolites on healthy cells were evaluated. The inhibitory effect was more pronounced against HSV-1 than against KHV at all studied viral cycle stages. Regarding the intracellular replicative steps, samples S7, S8, and S9 (Mix group) isolated from Ligilactobacillus salivarius (vaginal strain) demonstrated the most distinct effect with calculated selective indices (SIs) in the range between 69.4 and 77.8 against HSV-1, and from 62.2 to 68.4 against KHV. Bioactive metabolites from various LAB species significantly inhibit extracellular HSV-1 and, to a lesser extent, KHV virions. The blockage of viral adsorption to the host cells was remarkable, as recorded by a decrease in the viral titer with Δlg ≥ 5 in the Mix group for both herpes viruses. The remaining postmetabolites also significantly inhibited viral adsorption to varying degrees with Δlg ≥ 3. Most metabolites also exerted a protective effect on healthy MDBK and CCB cells to subsequent experimental viral infection. Our results reveal new horizons for the application of LAB and their postbiotic products in the prevention and treatment of herpes diseases.

The primary mechanism for highly potent inhibition of HIV-1 maturation by lenacapavir.

Lenacapavir (LEN) is the first-in-class HIV-1 capsid targeting antiretroviral that exhibits multimodal modality to inhibit both early and late steps of viral replication. Our studies here have elucidated previously undescribed structural and mechanistic bases for a highly potent antiviral activity of LEN during viral egress. These findings will inform clinical applications of LEN as a potent HIV-1 maturation inhibitor and aid the development of second-generation inhibitors targeting assembly of the mature viral capsid.

Causal associations of ambient particulate matter 10 and Alzheimer's disease: result from a two-sample multivariable Mendelian randomization study.

Alzheimer's disease (AD) and ambient particulate matter 10 (PM10) have been associated in epidemiological studies. However, the relationship between PM10 and risk of AD has not been proven to be causal. Thus we used two-sample multivariable Mendelian randomization (MR) to examine this relationship. Genome-wide association studies (GWAS) for PM10 from UK Biobank, AD from EBI GWAS and IEU OpenGWAS were used for discovery and replication, respectively. Pooled meta-analysis of the inverse variance weighted (IVW) method was the main method. Sensitivity analyses included MR-Egger regression, weighted median, weighted mode and leave-one-out methods. The multivariable MR model adjusted for education. The MR estimates of causality association were expressed as odds ratios (OR) and corresponding 95% confidence intervals (CI). There were in total 17 and 19 genetic variants associated with PM10 in the discovery and replication steps, respectively. In the univariate MR, pooled meta-analysis of genetically predicted PM10 was associated with a 99% increased risk of AD (95% CI: 1.25, 3.15, p = 0.004) per 1 standard deviation (SD) increment of PM10 by IVW, and in the multivariable MR with pooled meta-analysis, we found that each SD increase in PM10 was associated with a 127% increase in the risk of AD (95% CI: 1.33, 3.86, p = 0.002) after accounting for education levels. Increased PM10 levels were found to be significantly related to an increased risk of AD. This study provided evidence of genetic prediction of a causal relationship between PM10 and the risk of AD, suggesting that air pollution control may have significant implications for the prevention of AD.

Glass-like Relaxation Dynamics during the Disorder-to-Order Transition of Viral Nucleocapsids.

Nucleocapsid self-assembly is an essential yet elusive step in virus replication. Using time-resolved small-angle X-ray scattering on a model icosahedral ssRNA virus, we reveal a previously unreported kinetic pathway. Initially, RNA-bound capsid subunits rapidly accumulate beyond the stoichiometry of native virions. This is followed by a disorder-to-order transition characterized by glass-like relaxation dynamics and the release of excess subunits. Our molecular dynamics simulations, employing a coarse-grained elastic model, confirm the physical feasibility of self-ordering accompanied by subunit release. The relaxation can be modeled by an exponential integral decay on the mean squared radius of gyration, with relaxation times varying within the second range depending on RNA type and subunit concentration. A nanogel model suggests that the initially disordered nucleoprotein complexes quickly reach an equilibrium size, while their mass fractal dimension continues to evolve. Understanding virus self-assembly is not only crucial for combating viral infections, but also for designing synthetic virus-inspired nanocages for drug delivery applications.

The Assembly of HTLV-1-How Does It Differ from HIV-1?

Retroviral assembly is a highly coordinated step in the replication cycle. The process is initiated when the newly synthesized Gag and Gag-Pol polyproteins are directed to the inner leaflet of the plasma membrane (PM), where they facilitate the budding and release of immature viral particles. Extensive research over the years has provided crucial insights into the molecular determinants of this assembly step. It is established that Gag targeting and binding to the PM is mediated by interactions of the matrix (MA) domain and acidic phospholipids such as phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). This binding event, along with binding to viral RNA, initiates oligomerization of Gag on the PM, a process mediated by the capsid (CA) domain. Much of the previous studies have focused on human immunodeficiency virus type 1 (HIV-1). Although the general steps of retroviral replication are consistent across different retroviruses, comparative studies revealed notable differences in the structure and function of viral components. In this review, we present recent findings on the assembly mechanisms of Human T-cell leukemia virus type 1 and highlight key differences from HIV-1, focusing particularly on the molecular determinants of Gag-PM interactions and CA assembly.

Exploring HIV-1 Maturation: A New Frontier in Antiviral Development.

HIV-1 virion maturation is an essential step in the viral replication cycle to produce infectious virus particles. Gag and Gag-Pol polyproteins are assembled at the plasma membrane of the virus-producer cells and bud from it to the extracellular compartment. The newly released progeny virions are initially immature and noninfectious. However, once the Gag polyprotein is cleaved by the viral protease in progeny virions, the mature capsid proteins assemble to form the fullerene core. This core, harboring two copies of viral genomic RNA, transforms the virion morphology into infectious virus particles. This morphological transformation is referred to as maturation. Virion maturation influences the distribution of the Env glycoprotein on the virion surface and induces conformational changes necessary for the subsequent interaction with the CD4 receptor. Several host factors, including proteins like cyclophilin A, metabolites such as IP6, and lipid rafts containing sphingomyelins, have been demonstrated to have an influence on virion maturation. This review article delves into the processes of virus maturation and Env glycoprotein recruitment, with an emphasis on the role of host cell factors and environmental conditions. Additionally, we discuss microscopic technologies for assessing virion maturation and the development of current antivirals specifically targeting this critical step in viral replication, offering long-acting therapeutic options.

HIV-1 Gag Polyprotein Affinity to the Lipid Membrane Is Independent of Its Surface Charge.

The binding of the HIV-1 Gag polyprotein to the plasma membrane is a critical step in viral replication. The association with membranes depends on the lipid composition, but its mechanisms remain unclear. Here, we report the binding of non-myristoylated Gag to lipid membranes of different lipid compositions to dissect the influence of each component. We tested the contribution of phosphatidylserine, PI(4,5)P2, and cholesterol to membrane charge density and Gag affinity to membranes. Taking into account the influence of the membrane surface potential, we quantitatively characterized the adsorption of the protein onto model lipid membranes. The obtained Gag binding constants appeared to be the same regardless of the membrane charge. Furthermore, Gag adsorbed on uncharged membranes, suggesting a contribution of hydrophobic forces to the protein-lipid interaction. Charge-charge interactions resulted in an increase in protein concentration near the membrane surface. Lipid-specific interactions were observed in the presence of cholesterol, resulting in a two-fold increase in binding constants. The combination of cholesterol with PI(4,5)P2 showed cooperative effects on protein adsorption. Thus, we suggest that the affinity of Gag to lipid membranes results from a combination of electrostatic attraction to acidic lipids, providing different protein concentrations near the membrane surface, and specific hydrophobic interactions.

ESCRT Protein VPS4A Is Required for the Formation of Replication Centers and Replication of Human Coronavirus 229E (HCoV-229E)

Human coronavirus 229E (HCoV-229E) is an alpha coronavirus that infects humans and bats. In common with all positive-strand RNA viruses, 229E infection causes rearrangements of the host’s intracellular membranes to form replication organelles, a highly conserved and vital step in the viral replication cycle. Here, we investigated the role of the ESCRT protein VPS4A in 229E infection. We found that functional VPS4A was required for the formation of replication organelles and localizing viral RNA to these structures in host cells to facilitate viral genome replication. We validated this effect using small molecule inhibitors to VPS4A, significantly reducing virus replication. We also showed that other ESCRTS, like CHMP4B, were required for the virus replication step, whereas VPS37A was involved in the post-replication stages. The absence of a functional VPS4A prevented the remodeling of membranes to form viral replication centers and, therefore, exposed the viral RNA, triggering an inflammatory immune response as indicated by elevated levels of IL-6. Interestingly, we observed the role of VPS4A to be similar for the OC43 coronavirus, indicating it could be conserved across all four coronavirus genera, including SARS-CoV-2. Understanding more about the replication of coronaviruses is imperative to finding more effective ways to control them.

Antiviral Efficacy of Coridothymus capitatus Essential Oil Against HSV-1 and HSV-2.

Coridothymus capitatus is a perennial herb with aromatic leaves and flowers, distinct from Thymus vulgaris in its chemical composition, resulting in a unique Thymus Essential Oil (TEO). A main component of TEO, carvacrol, is known for its antimicrobial and insecticidal activity. Carvacrol has potent antibacterial, antioxidant, anti-inflammatory, and antifungal properties, generating interest in traditional medicine. However, studies on its antiviral activity are limited. Given the rise in viral infections and limitations of synthetic antiviral drugs, natural antiviral agents are promising due to their efficacy, lower resistance development, and reduced side effects. This study assessed the antiviral efficacy of TEO compared to that of pure carvacrol. We tested various viruses, revealing significant inhibitory effects of TEO on the replication of only Simplexvirus humanalpha1 (HSV-1) and Simplexvirus humanalpha2 (HSV-2), with specific interference during the early stages of the viral replication cycle after the adsorption period. TEO exhibited inhibitory effects at doses below the cytotoxic threshold, with IC50 values of 47 μg/mL for HSV-1 and 40 μg/mL for HSV-2. Maximum virus inhibition was achieved when TEO was added within 90 min post-infection, indicating interference with early viral replication steps. These findings highlight the potential of TEO as a natural antiviral agent and suggest further research into its mechanisms and clinical applications.

Quinolinonyl Derivatives as Dual Inhibitors of the HIV-1 Integrase Catalytic Site and Integrase-RNA interactions.

The HIV-1 integrase (IN) plays a critical role in the viral lifecycle by integrating the viral DNA into the host chromosome. The catalytic function of IN has been exploited as a target, with five drugs acting as active site binders (IN strand transfer inhibitors, INSTIs). However, IN mutations conferring low-level resistance to INSTIs have been reported. Therefore, new IN inhibitors with different mechanisms of action are needed. The allosteric inhibition of IN, exerted by allosteric IN inhibitors (ALLINIs), is gaining interest. ALLINIs inhibit IN by inducing aberrant IN multimerization with different mechanisms. Furthermore, recent discoveries unveiled that IN has an under-studied yet equally vital second function. This involves IN binding to the RNA genome in virions, necessary for proper virion maturation. In this work, we describe a series of quinolinonyl derivatives as inhibitors of both the IN catalytic functions and IN-RNA interactions, which impair both early and late steps of viral replication.

Novel sialidase inhibitors suppress mumps virus replication and infection.

The prevalent human pathogen, mumps virus (MuV; orthorubulavirus parotitidis) causes various complications and serious sequelae, such as meningitis, encephalitis, deafness, and impaired fertility. Direct-acting antivirals (DAAs) targeting MuV which can prevent mumps and mumps-associated complications and sequelae are yet to be developed. Paramyxoviridae family members, such as MuV, possess viral surface hemagglutinin-neuraminidase (HN) protein with sialidase activity which facilitates efficient viral replication. Therefore, to develop DAAs targeting MuV we synthesized MuV sialidase inhibitors. It is proposed that the viral HN has a single functional site for N-acetylneuraminic acid (Neu5Ac) binding and sialidase activity. Further, the known MuV sialidase inhibitor is an analog of Neu5Ac-2,3-didehydro-2-deoxy-N-acetylneuraminic acid (DANA)-which lacks potency. DANA derivatives with higher MuV sialidase inhibitory potency are lacking. The MuV-HN-Neu5Ac binding site has a hydrophobic cavity adjacent to the C4 position of Neu5Ac. Exploiting this, here, we synthesized DANA derivatives with increasing hydrophobicity at its C4 position and created 3 novel sialidase inhibitors (Compounds 1, 2, and 3) with higher specificity for MuV-HN than DANA; they inhibited MuV replication step to greater extent than DANA. Furthermore, they also inhibited hemagglutination and the MuV infection step. The insight-that these 3 novel DANA derivatives possess linear hydrocarbon groups at the C4-hydroxyl group of DANA-could help develop highly potent sialidase inhibitors with high specificity for MuV sialidase, which may function as direct-acting MuV-specific antivirals.

How does the polymerase of non-segmented negative strand RNA viruses commit to transcription or genome replication?

The Mononegavirales, or non-segmented negative-sense RNA viruses (nsNSVs), includes significant human pathogens, such as respiratory syncytial virus, parainfluenza virus, measles virus, Ebola virus, and rabies virus. Although these viruses differ widely in their pathogenic properties, they are united by each having a genome consisting of a single strand of negative-sense RNA. Consistent with their shared genome structure, the nsNSVs have evolved similar ways to transcribe their genome into mRNAs and replicate it to produce new genomes. Importantly, both mRNA transcription and genome replication are performed by a single virus-encoded polymerase. A fundamental and intriguing question is: how does the nsNSV polymerase commit to being either an mRNA transcriptase or a replicase? The polymerase must become committed to one process or the other either before it interacts with the genome template or in its initial interactions with the promoter sequence at the 3´ end of the genomic RNA. This review examines the biochemical, molecular biology, and structural biology data regarding the first steps of transcription and RNA replication that have been gathered over several decades for different families of nsNSVs. These findings are discussed in relation to possible models that could explain how an nsNSV polymerase initiates and commits to either transcription or genome replication.

Simulation-Guided Conformational Space Exploration to Assess Reactive Conformations of a Ribozyme.

Self-splicing ribozymes are small ribonucleic acid (RNA) enzymes that catalyze their own cleavage through a transphosphoesterification reaction. While this process is involved in some specific steps of viral RNA replication and splicing, it is also of importance in the context of the (putative) first autocatalytic RNA-based systems that could have preceded the emergence of modern life. The uncatalyzed phosphoester bond formation is thermodynamically very unfavorable, and many experimental studies have focused on understanding the molecular features of catalysis in these ribozymes. However, chemical reaction paths are short-lived and not easily characterized by experimental approaches, so molecular simulation approaches appear as an ideal tool to unveil the molecular details of the reaction. Here, we focus on the model hairpin ribozyme. We show that identifying a relevant initial conformation for reactivity studies, which is frequently overlooked in mixed quantum-classical studies that predominantly concentrate on the chemical reaction itself, can be highly challenging. These challenges stem from limitations in both available experimental structures (which are chemically altered to prevent self-cleavage) and the accuracy of force fields, together with the necessity for comprehensive sampling. We show that molecular dynamics simulations, combined with extensive conformational phase space exploration with Hamiltonian replica-exchange simulations, enable us to characterize the relevant conformational basins of the minimal hairpin ribozyme in the ligated state prior to self-cleavage. We find that what is usually considered a canonical reactive conformation with active site geometries and hydrogen-bond patterns that are optimal for the addition-elimination reaction with general acid/general base catalysis is metastable and only marginally populated. The thermodynamically stable conformation appears to be consistent with the expectations of a mechanism that does not require the direct participation of ribozyme residues in the reaction. While these observations may suffer from forcefield inaccuracies, all investigated forcefields lead to the same conclusions upon proper sampling, contrasting with previous investigations on shorter timescales suggesting that at least one reparametrization of the Amber99 forcefield allowed to stabilize aligned active site conformations. Our study demonstrates that identifying the most pertinent reactant state conformation holds equal importance alongside the accurate determination of the thermodynamics and kinetics of the chemical steps of the reaction.

The interferon-induced protein, IFIT2, requires RNA-binding activity and neuronal expression to protect mice from intranasal vesicular stomatitis virus infection.

The interferon (IFN) system protects mammals from diseases caused by virus infections. IFN synthesis is induced by pattern recognition receptor signaling pathways activated by virus infection. IFN is secreted from the infected cells and acts upon neighboring cells by binding cell surface receptors and triggering induction of hundreds of IFN-stimulated genes and proteins, many of which block different steps of virus replication. The IFN-induced tetratricopeptide repeat proteins (IFIT) are a family of RNA-binding proteins. We and others have previously reported that IFIT2 protects mice from many neurotropic RNA viruses; indeed, Ifit2-/- mice are very susceptible to intranasal or subcutaneous infections with vesicular stomatitis virus (VSV). Here, using a newly generated conditional knockout mouse, we report that ablation of Ifit2 expression only in neuronal cells was sufficient to render mice susceptible to neuropathogenesis caused by intranasal, but not subcutaneous, infection of VSV. Another genetically modified mouse line, expressing a mutant IFIT2 that cannot bind RNA, was as susceptible to VSV infection as Ifit2-/- mice. These results demonstrated that IFIT2 RNA-binding activity is essential for protecting mice against neurological diseases caused by intranasal infection of VSV.IMPORTANCEInterferon's (IFN's) antiviral effects are mediated by the proteins encoded by the interferon-stimulated genes. IFN-stimulated genes (IFIT2) is one such protein, which inhibits replication of many RNA viruses in the mouse brain and the resultant neuropathology. Our study sheds light on how IFIT2 works. By ablating Ifit2 expression only in neuronal cells, using a newly generated conditional knockout mouse line, we showed that Ifit2 induction in the neurons of the infected mouse was necessary for antiviral function of interferon. IFIT2 has no known enzyme activity; instead, it functions by binding to cellular or viral proteins or RNAs. We engineered a new mouse line that expressed a mutant IFIT2 that cannot bind RNA. These mice were very susceptible to infection with vesicular stomatitis virus indicating that the RNA-binding property of IFIT2 was essential for its antiviral function in vivo.

Restricting the level of the proteins essential for the regulation of the initiation step of replication extends the chronological lifespan and reproductive potential in budding yeast

Aging is defined as a progressive decline in physiological integrity, leading to impaired biological function, including fertility, and rising vulnerability to death. Disorders of DNA replication often lead to replication stress and are identified as factors influencing the aging rate. In this study, we aimed to reveal how the cells that lost strict control of the formation of crucial for replication initiation a pre-initiation complex impact the cells’ physiology and aging. As strains with the lower pre-IC control (lowPICC) we used, Saccharomyces cerevisiae heterozygous strains having only one functional copy of genes, encoding essential replication proteins such as Cdc6, Dbf4, Sld3, Sld7, Sld2, and Mcm10. The lowPICC strains exhibited a significant reduction in the respective genes’ mRNA levels, causing cell cycle aberrations and doubling time extensions. Additionally, the reduced expression of the lowPICC genes led to an aberrant DNA damage response, affected cellular and mitochondrial DNA content, extended the lifespan of post-mitotic cells, and increased the yeast’s reproductive potential. Importantly, we also demonstrated a strong negative correlation between the content of cellular macromolecules (RNA, proteins, lipids, polysaccharides) and aging. The data presented here will likely contribute to the future development of therapies for treating various human diseases.

Replication competent HIV-guided CRISPR screen identifies antiviral factors including targets of the accessory protein Nef

Innate antiviral factors are essential for effective defense against viral pathogens. However, the identity of major restriction mechanisms remains elusive. Current approaches to discover antiviral factors usually focus on the initial steps of viral replication and are limited to a single round of infection. Here, we engineered libraries of >1500 replication-competent HIV-1 constructs each expressing a single gRNAs to target >500 cellular genes for virus-driven discovery of antiviral factors. Passaging in CD4+ T cells robustly enriched HIV-1 encoding sgRNAs against GRN, CIITA, EHMT2, CEACAM3, CC2D1B and RHOA by >50-fold. Using an HIV-1 library lacking the accessory nef gene, we identified IFI16 as a Nef target. Functional analyses in cell lines and primary CD4+ T cells support that the HIV-driven CRISPR screen identified restriction factors targeting virus entry, transcription, release and infectivity. Our HIV-guided CRISPR technique enables sensitive discovery of physiologically relevant cellular defense factors throughout the entire viral replication cycle.

4'-Fluorouridine inhibits alphavirus replication and infection in vitro and in vivo.

Chikungunya virus (CHIKV) is an enveloped, positive-sense RNA virus that has re-emerged to cause millions of human infections worldwide. In humans, acute CHIKV infection causes fever and severe muscle and joint pain. Chronic and debilitating arthritis and joint pain can persist for months to years. To date, there are no approved antivirals against CHIKV. Recently, the ribonucleoside analog 4'-fluorouridine (4'-FlU) was reported as a highly potent orally available inhibitor of SARS-CoV-2, respiratory syncytial virus, and influenza virus replication. In this study, we assessed 4'-FlU's potency and breadth of inhibition against a panel of alphaviruses including CHIKV, and found that it broadly suppressed alphavirus production in cell culture. 4'-FlU acted on the viral RNA replication step, and the first 4 hours post-infection were the critical time for its antiviral effect. In vitro replication assays identified nsP4 as the target of inhibition. In vivo, treatment with 4'-FlU reduced disease signs, inflammatory responses, and viral tissue burden in mouse models of CHIKV and Mayaro virus infection. Treatment initiated at 2 hours post-infection was most effective; however, treatment initiated as late as 24-48 hours post-infection produced measurable antiviral effects in the CHIKV mouse model. 4'-FlU showed effective oral delivery in our mouse model and resulted in the accumulation of both 4'-FlU and its bioactive triphosphate form in tissues relevant to arthritogenic alphavirus pathogenesis. Together, our data indicate that 4'-FlU inhibits CHIKV infection in vitro and in vivo and is a promising oral therapeutic candidate against CHIKV infection.IMPORTANCEAlphaviruses including chikungunya virus (CHIKV) are mosquito-borne positive-strand RNA viruses that can cause various diseases in humans. Although compounds that inhibit CHIKV and other alphaviruses have been identified in vitro, there are no licensed antivirals against CHIKV. Here, we investigated a ribonucleoside analog, 4'-fluorouridine (4'-FlU), and demonstrated that it inhibited infectious virus production by several alphaviruses in vitro and reduced virus burden in mouse models of CHIKV and Mayaro virus infection. Our studies also indicated that 4'-FlU treatment reduced CHIKV-induced footpad swelling and reduced the production of pro-inflammatory cytokines. Inhibition in the mouse model correlated with effective oral delivery of 4'-FlU and accumulation of both 4'-FlU and its bioactive form in relevant tissues. In summary, 4'-FlU exhibits potential as a novel anti-alphavirus agent targeting the replication of viral RNA.