Function Of RNA Polymerase Research Articles

Characterization of siRNAs derived from cucumber mosaic virus in infected tobacco plants.

This study characterized the viral small interfering RNAs (vsiRNAs) from Nicotiana tabacum cv. Samsun infected with a cucumber mosaic virus (CMV) 2b-deficient mutant. Most vsiRNAs were 21 -22 nucleotides in length and the 5'-terminal ends were dominated by A and U, respectively. The observed vsiRNAs were heterogeneously distributed throughout the CMV genome; however, most of the vsiRNAs were derived from sense strands, as opposed to antisense strands. These results demonstrate the conserved and specific function of Dicer-like (DCL), Argonaute (AGO) and RNA-dependent RNA polymerase (RDR) proteins in tobacco. Finally, it was revealed that vsiRNAs target abundant host genes, indicating complex roles for CMV vsiRNAs during the development of symptoms.

Investigating RNAP Search Dynamics in Live E. Coli Cells using Single Molecule and Statistical Methods

Transcription is an essential component of gene regulation and one of the most highly regulated processes in the cell. Because bacteria lack a nuclear envelope, genes transcribed by RNA polymerase (RNAP), the key enzyme of transcription, are essentially immediately translated into protein, leaving the kinetics of transcription significantly influencing the resulting protein expression timing and levels. However, while the kinetics of transcription is greatly studied in vitro, the in vivo dynamics of RNAP, including the typical search time and transcription time, are difficult to obtain. Our lab takes advantage of the sensitivity and fast time scale of single-molecule tracking to determine the kinetics of RNAP binding and transcription in live E. coli cells. We used a strain of E. coli in which the β’ subunit (rpoC) of RNAP is tagged with a photoactivatable fluorescent protein, PAmCherry, replacing the endogenous chromosomal copy. We discovered there are three states of RNAP with distinct diffusion coefficients. Using genetic manipulations and drug treatments, we were able to assign each diffusion state to a particular function of RNAP, i.e. RNAP that is bound to the DNA, RNAP that is undergoing rapid association and dissociation from the nucleoid, and freely diffusing RNAP. Further, using a Markov chain Monte Carlo algorithm, we were able to form a hidden Markov model of our data to parse out the dynamics of RNAP switching states, which enabled us to parse out a complete dynamical model of RNAP in living cells. We obtainined a wide variety of important parameters, including the non-specific dwell time, the typical promoter search time, and the typical transcription time for an average gene. These parameters will be useful for future gene regulations studies of all kinds, as well as to help understand general protein-nucleic acid interactions and dynamics within the crowded cytoplasm of a live cell.

Crystal Structure of the Marburg Virus VP35 Oligomerization Domain.

Marburg virus can cause severe disease, with up to 90% human lethality. Its genome is concise, only producing seven proteins. One of the proteins, VP35, is essential for replication of the viral genome and for evasion of host immune responses. VP35 oligomerizes (self-assembles) in order to function, yet the structure by which it assembles has not been visualized. Here we present two crystal structures of this oligomerization domain. In both structures, three copies of VP35 twist about each other to form a coiled coil. This trimeric assembly is in contrast to tetrameric predictions for VP35 of Ebola virus and to known structures of homologous proteins in the measles, mumps, and Nipah viruses. Distinct oligomeric states of the Marburg and Ebola virus VP35 proteins may explain differences between them in polymerase function and immune evasion. These findings may provide a more accurate understanding of the mechanisms governing VP35's functions and inform the design of therapeutics.

Structure and Function of RNA Polymerases and the Transcription Machineries.

In all living organisms, the flow of genetic information is a two-step process: first DNA is transcribed into RNA, which is subsequently used as template for protein synthesis during translation. In bacteria, archaea and eukaryotes, transcription is carried out by multi-subunit RNA polymerases (RNAPs) sharing a conserved architecture of the RNAP core. RNAPs catalyse the highly accurate polymerisation of RNA from NTP building blocks, utilising DNA as template, being assisted by transcription factors during the initiation, elongation and termination phase of transcription. The complexity of this highly dynamic process is reflected in the intricate network of protein-protein and protein-nucleic acid interactions in transcription complexes and the substantial conformational changes of the RNAP as it progresses through the transcription cycle.In this chapter, we will first briefly describe the early work that led to the discovery of multisubunit RNAPs. We will then discuss the three-dimensional organisation of RNAPs from the bacterial, archaeal and eukaryotic domains of life, highlighting the conserved nature, but also the domain-specific features of the transcriptional apparatus. Another section will focus on transcription factors and their role in regulating the RNA polymerase throughout the different phases of the transcription cycle. This includes a discussion of the molecular mechanisms and dynamic events that govern transcription initiation, elongation and termination.

The ω Subunit Governs RNA Polymerase Stability and Transcriptional Specificity in Staphylococcus aureus.

Staphylococcus aureus is a major human pathogen that causes infection in a wide variety of sites within the human body. Its ability to adapt to the human host and to produce a successful infection requires precise orchestration of gene expression. While DNA-dependent RNA polymerase (RNAP) is generally well characterized, the roles of several small accessory subunits within the complex have yet to be fully explored. This is particularly true for the omega (ω or RpoZ) subunit, which has been extensively studied in Gram-negative bacteria but largely neglected in Gram-positive counterparts. In Escherichia coli, it has been shown that ppGpp binding, and thus control of the stringent response, is facilitated by ω. Interestingly, key residues that facilitate ppGpp binding by ω are not conserved in S. aureus, and consequently, survival under starvation conditions is unaffected by rpoZ deletion. Further to this, ω-lacking strains of S. aureus display structural changes in the RNAP complex, which result from increased degradation and misfolding of the β' subunit, alterations in δ and σ factor abundance, and a general dissociation of RNAP in the absence of ω. Through RNA sequencing analysis we detected a variety of transcriptional changes in the rpoZ-deficient strain, presumably as a response to the negative effects of ω depletion on the transcription machinery. These transcriptional changes translated to an impaired ability of the rpoZ mutant to resist stress and to fully form a biofilm. Collectively, our data underline, for the first time, the importance of ω for RNAP stability, function, and cellular physiology in S. aureus IMPORTANCE: In order for bacteria to adjust to changing environments, such as within the host, the transcriptional process must be tightly controlled. Transcription is carried out by DNA-dependent RNA polymerase (RNAP). In addition to its major subunits (α2ββ') a fifth, smaller subunit, ω, is present in all forms of life. Although this small subunit is well studied in eukaryotes and Gram-negative bacteria, only limited information is available for Gram-positive and pathogenic species. In this study, we investigated the structural and functional importance of ω, revealing key roles in subunit folding/stability, complex assembly, and maintenance of transcriptional integrity. Collectively, our data underline, for the first time, the importance of ω for RNAP function and cellular harmony in S. aureus.

Annotating Protein Functional Residues by Coupling High-Throughput Fitness Profile and Homologous-Structure Analysis.

ABSTRACTIdentification and annotation of functional residues are fundamental questions in protein sequence analysis. Sequence and structure conservation provides valuable information to tackle these questions. It is, however, limited by the incomplete sampling of sequence space in natural evolution. Moreover, proteins often have multiple functions, with overlapping sequences that present challenges to accurate annotation of the exact functions of individual residues by conservation-based methods. Using the influenza A virus PB1 protein as an example, we developed a method to systematically identify and annotate functional residues. We used saturation mutagenesis and high-throughput sequencing to measure the replication capacity of single nucleotide mutations across the entire PB1 protein. After predicting protein stability upon mutations, we identified functional PB1 residues that are essential for viral replication. To further annotate the functional residues important to the canonical or noncanonical functions of viral RNA-dependent RNA polymerase (vRdRp), we performed a homologous-structure analysis with 16 different vRdRp structures. We achieved high sensitivity in annotating the known canonical polymerase functional residues. Moreover, we identified a cluster of noncanonical functional residues located in the loop region of the PB1 β-ribbon. We further demonstrated that these residues were important for PB1 protein nuclear import through the interaction with Ran-binding protein 5. In summary, we developed a systematic and sensitive method to identify and annotate functional residues that are not restrained by sequence conservation. Importantly, this method is generally applicable to other proteins about which homologous-structure information is available.

In vitro promoter recognition by the catalytic subunit of plant phage-type RNA polymerases.

Key messageWe identified sequence motifs, which enhance or reduce the ability of the Arabidopsis phage-type RNA polymerases RPOTm (mitochondrial RNAP), RPOTp (plastidial RNAP), and RPOTmp (active in both organelles) to recognize their promoters in vitro with help of a ‘specificity loop’. The importance of this data for the evolution and function of the organellar RNA polymerases is discussed.The single-subunit RNA polymerase (RNAP) of bacteriophage T7 is able to perform all steps of transcription without additional transcription factors. Dicotyledonous plants possess three phage-type RNAPs, RPOTm—the mitochondrial RNAP, RPOTp—the plastidial RNAP, and RPOTmp—an RNAP active in both organelles. RPOTm and RPOTp, like the T7 polymerase, are able to recognize promoters, while RPOTmp displays no significant promoter specificity in vitro. To find out which promoter motifs are crucial for recognition by the polymerases we performed in vitro transcription assays with recombinant Arabidopsis RPOTm and RPOTp enzymes. By comparing different truncated and mutagenized promoter constructs, we observed the same minimal promoter sequence supposed to be needed in vivo for transcription initiation. Moreover, we identified elements of core and flanking sequences, which are of critical importance for promoter recognition and activity in vitro. We further intended to reveal why RPOTmp does not efficiently recognize promoters in vitro and if promoter recognition is based on a structurally defined specificity loop of the plant enzymes as described for the yeast and T7 RNAPs. Interestingly, the exchange of only three amino acids within the putative specificity loop of RPOTmp enabled the enzyme for specific promoter transcription in vitro. Thus, also in plant phage-type RNAPs the specificity loop is engaged in promoter recognition. The results are discussed with respect to their relevance for transcription in organello and to the evolution of RPOT enzymes including the divergence of their functions.Electronic supplementary materialThe online version of this article (doi:10.1007/s11103-016-0518-z) contains supplementary material, which is available to authorized users.

Specific threonine-4 phosphorylation and function of RNA polymerase II CTD during M phase progression.

Dynamic phosphorylation of Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 heptad-repeats in the C-terminal domain (CTD) of the large subunit coordinates progression of RNA polymerase (Pol) II through the transcription cycle. Here, we describe an M phase-specific form of Pol II phosphorylated at Thr4, but not at Tyr1, Ser2, Ser5, and Ser7 residues. Thr4 phosphorylated Pol II binds to centrosomes and midbody and interacts with the Thr4-specific Polo-like kinase 1. Binding of Pol II to centrosomes does not require the CTD but may involve subunits of the non-canonical R2TP-Prefoldin-like complex, which bind to and co-localize with Pol II at centrosomes. CTD Thr4 mutants, but not Ser2 and Ser5 mutants, display severe mitosis and cytokinesis defects characterized by multipolar spindles and polyploid cells. We conclude that proper M phase progression of cells requires binding of Pol II to centrosomes to facilitate regulation of mitosis and cytokinesis in a CTD Thr4-P dependent manner.

LDSS-P: an advanced algorithm to extract functional short motifs associated with coordinated gene expression

Identifying functional elements in promoter sequences is a major goal in computational and experimental genome biology. Here, we describe an algorithm, Local Distribution of Short Sequences for Prokaryotes (LDSS-P), to identify conserved short motifs located at specific positions in the promoters of co-expressed prokaryotic genes. As a test case, we applied this algorithm to a symbiotic nitrogen-fixing bacterium, Sinorhizobium meliloti. The LDSS-P profiles that overlap with the 5′ section of the extracytoplasmic function RNA polymerase sigma factor RpoE2 consensus sequences displayed a sharp peak between -34 and -32 from TSS positions. The corresponding genes overlap significantly with RpoE2 targets identified from previous experiments. We further identified several groups of genes that are co-regulated with characterized marker genes. Our data indicate that in S. meliloti, and possibly in other Rhizobiaceae species, the master cell cycle regulator CtrA may recognize an expanded motif (AACCAT), which is positionally shifted from the previously reported CtrA consensus sequence in Caulobacter crescentus. Bacterial one-hybrid experiments showed that base substitution in the expanded motif either increase or decrease the binding by CtrA. These results show the effectiveness of LDSS-P as a method to delineate functional promoter elements.

A capsidless ssRNA virus hosted by an unrelated dsRNA virus.

Viruses typically encode the capsid that encases their genome, while satellite viruses do not encode a replicase and depend on a helper virus for their replication(1). Here, we report interplay between two RNA viruses, yado-nushi virus 1 (YnV1) and yado-kari virus 1 (YkV1), in a phytopathogenic fungus, Rosellinia necatrix(2). YkV1 has a close phylogenetic affinity to positive-sense, single-stranded (+)ssRNA viruses such as animal caliciviruses(3), while YnV1 has an undivided double-stranded (ds) RNA genome with a resemblance to fungal totiviruses(4). Virion transfection and infectious full-length cDNA transformation has shown that YkV1 depends on YnV1 for viability, although it probably encodes functional RNA-dependent RNA polymerase (RdRp). Immunological and molecular analyses have revealed trans-encapsidation of not only YkV1 RNA but also RdRp by the capsid protein of the other virus (YnV1), and enhancement of YnV1 accumulation by YkV1. This study demonstrates interplay in which the capsidless (+)ssRNA virus (YkV1), hijacks the capsid protein of the dsRNA virus (YnV1), and replicates as if it were a dsRNA virus.

Dynamic methylation pattern of cyp19a1a core promoter during zebrafish ovarian folliculogenesis.

In vertebrates, the aromatase coded by the cyp19a1a gene can catalyze the conversion from androgens to estrogens. Thus, the regulatory mechanisms of cyp19a1a gene expression are a critical research field in reproductive endocrinology. In this study, we use zebrafish as a model to study the dynamic methylation levels of the cyp19a1a gene core promoter during zebrafish ovarian folliculogenesis. The results show that there is an apparent fluctuation of the methylation levels of zebrafish cyp19a1a core promoter. Moreover, the methylation levels are inversely correlated with the expression levels of cyp19a1a transcripts when the ovarian follicles develop from PV into the MV stage. Also, the CpG dinucleotides which are close to the transcriptional starting site may have provided a significant blocking effect on inhibiting the transcriptional function of RNA polymerase II. Taken together, the results from the present study strongly suggest that DNA methylation was one of mechanisms that are involved in the regulation of cyp19a1a gene expression during folliculogenesis. This methylation mechanism modifying transcriptional process accompanied with zebrafish ovarian folliculogenesis might also shed new light on the regulation of cyp19a1a expression during the ovarian developmental stage in other vertebrates.

Altered minor-groove hydrogen bonds in DNA block transcription elongation by T7 RNA polymerase.

DNA transcription depends upon the highly efficient and selective function of RNA polymerases (RNAPs). Modifications in the template DNA can impact the progression of RNA synthesis, and a number of DNA adducts, as well as abasic sites, arrest or stall transcription. Nonetheless, data are needed to understand why certain modifications to the structure of DNA bases stall RNA polymerases while others are efficiently bypassed. In this study, we evaluate the impact that alterations in dNTP/rNTP base-pair geometry have on transcription. T7 RNA polymerase was used to study transcription over modified purines and pyrimidines with altered H-bonding capacities. The results suggest that introducing wobble base-pairs into the DNA:RNA heteroduplex interferes with transcriptional elongation and stalls RNA polymerase. However, transcriptional stalling is not observed if mismatched base-pairs do not H-bond. Together, these studies show that RNAP is able to discriminate mismatches resulting in wobble base-pairs, and suggest that, in cases of modifications with minor steric impact, DNA:RNA heteroduplex geometry could serve as a controlling factor for initiating transcription-coupled DNA repair.

TFIIB is only ∼9 Å away from the 5'-end of a trimeric RNA primer in a functional RNA polymerase II preinitiation complex.

Recent X-ray crystallographic studies of Pol II in complex with the general transcription factor (GTF) IIB have begun to provide insights into the mechanism of transcription initiation. These structures have also shed light on the architecture of the transcription preinitiation complex (PIC). However, structural characterization of a functional PIC is still lacking, and even the topological arrangement of the GTFs in the Pol II complex is a matter of contention. We have extended our activity-based affinity crosslinking studies, initially developed to investigate the interaction of bacterial RNA polymerase with σ, to the eukaryotic transcription machinery. Towards that end, we sought to identify GTFs that are within the Pol II active site in a functioning PIC. We provide biochemical evidence that TFIIB is located within ∼9 Å of the −2 site of promoter DNA, where it is positioned to play a role in de novo transcription initiation.

Identification and characterization of the binding site of the respiratory syncytial virus phosphoprotein to RNA-free nucleoprotein.

The RNA genome of respiratory syncytial virus (RSV) is constitutively encapsidated by the viral nucleoprotein N, thus forming a helical nucleocapsid. Polymerization of N along the genomic and antigenomic RNAs is concomitant to replication and requires the preservation of an unassembled monomeric nucleoprotein pool. To this end, and by analogy with Paramyxoviridae and Rhabdoviridae, it is expected that the viral phosphoprotein P acts as a chaperone protein, forming a soluble complex with the RNA-free form of N (N(0)-P complex). Here, we have engineered a mutant form of N that is monomeric, is unable to bind RNA, still interacts with P, and could thus mimic the N(0) monomer. We used this N mutant, designated N(mono), as a substitute for N(0) in order to characterize the P regions involved in the N(0)-P complex formation. Using a series of P fragments, we determined by glutathione S-transferase (GST) pulldown assays that the N and C termini of P are able to interact with N(mono). We analyzed the functional role of amino-terminal residues of P by site-directed mutagenesis, using an RSV polymerase activity assay based on a human RSV minireplicon, and found that several residues were critical for viral RNA synthesis. Using GST pulldown and surface plasmon resonance assays, we showed that these critical residues are involved in the interaction between P[1-40] peptide and N(mono) in vitro. Finally, we showed that overexpression of the peptide P[1-29] can inhibit the polymerase activity in the context of the RSV minireplicon, thus demonstrating that targeting the N(0)-P interaction could constitute a potential antiviral strategy. Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract illness in infants. Since no vaccine or efficient antiviral treatment is available against RSV, it is essential to better understand how the viral machinery functions in order to develop new antiviral strategies. RSV phosphoprotein P, the main RNA polymerase cofactor, is believed to function as a chaperon protein, maintaining N as a nonassembled, RNA-free protein (N(0)) competent for RNA encapsidation. In this paper, we provide the first evidence, to our knowledge, that the N terminus of P contains a domain that binds specifically to this RNA-free form of N. We further show that overexpression of a small peptide spanning this region of P can inhibit viral RNA synthesis. These findings extend our understanding of the function of RSV RNA polymerase and point to a new target for the development of drugs against this virus.

Transcription in Archaea: preparation of Methanocaldococcus jannaschii transcription machinery.

Archaeal RNA polymerase and general transcription factors are more closely related to those of eukaryotes than of bacteria. As such the study of transcription of archaea is important both in terms of examination of the evolution of the transcriptional machinery and as a simplified tool for eukaryotic transcription. In particular, the hyperthermophilic Methanocaldococcus jannaschii provides us with a fully recombinant RNA polymerase system allowing for much more detailed in vitro examination of the roles of different components during the transcription cycle than otherwise possible. The individual subunits of M. jannaschii enzyme are easily expressed and purified from heterologous expression systems. Forming functional RNA polymerase involves simply combining the different subunits under denaturing conditions and slowly removing the denaturant.

Function and control of RNA polymerase II C-terminal domain phosphorylation in vertebrate transcription and RNA processing.

The C-terminal domain of the RNA polymerase II largest subunit (the Rpb1 CTD) is composed of tandem heptad repeats of the consensus sequence Y(1)S(2)P(3)T(4)S(5)P(6)S(7). We reported previously that Thr 4 is phosphorylated and functions in histone mRNA 3'-end formation in chicken DT40 cells. Here, we have extended our studies on Thr 4 and to other CTD mutations by using these cells. We found that an Rpb1 derivative containing only the N-terminal half of the CTD, as well as a similar derivative containing all-consensus repeats (26r), conferred full viability, while the C-terminal half, with more-divergent repeats, did not, reflecting a strong and specific defect in snRNA 3'-end formation. Mutation in 26r of all Ser 2 (S2A) or Ser 5 (S5A) residues resulted in lethality, while Ser 7 (S7A) mutants were fully viable. While S2A and S5A cells displayed defects in transcription and RNA processing, S7A cells behaved identically to 26r cells in all respects. Finally, we found that Thr 4 was phosphorylated by cyclin-dependent kinase 9 in cells and dephosphorylated both in vitro and in vivo by the phosphatase Fcp1.

Attractor computation using interconnected Boolean networks: Testing growth rate models in E. Coli

Boolean networks provide a useful tool to address questions on the structure of large biochemical interactions since they do not require kinetic details and, in addition, a wide range of computational tools and algorithms is available to exactly compute and study the dynamical properties of these models. A recently developed method has shown that the attractors, or asymptotic behavior, of an asynchronous Boolean network can be computed at a much lower cost if the network is written as an interconnection of two smaller modules. We have applied this methodology to study the interconnection of two Boolean models to explore bacterial growth and its interactions with the cellular gene expression machinery, with a focus on growth dynamics as a function of ribosomes, RNA polymerase and other “bulk” proteins inside the cell. The discrete framework permits easier testing of different combinations of biochemical interactions, leading to hypotheses elimination and model discrimination, and thus providing useful insights for the construction of a more detailed dynamical growth model.

Genome-Wide Mapping of the Distribution of CarD, RNAP σA, and RNAP β on the Mycobacterium smegmatis Chromosome using Chromatin Immunoprecipitation Sequencing.

CarD is an essential mycobacterial protein that binds the RNA polymerase (RNAP) and affects the transcriptional profile of Mycobacterium smegmatis and Mycobacterium tuberculosis [6]. We predicted that CarD was directly regulating RNAP function but our prior experiments had not determined at what stage of transcription CarD was functioning and at which genes CarD interacted with the RNAP. To begin to address these open questions, we performed chromatin immunoprecipitation sequencing (ChIP-seq) to survey the distribution of CarD throughout the M. smegmatis chromosome. The distribution of RNAP subunits β and σA were also profiled. We expected that RNAP β would be present throughout transcribed regions and RNAP σA would be predominantly enriched at promoters based on work in Escherichia coli [3], however this had yet to be determined in mycobacteria. The ChIP-seq analyses revealed that CarD was never present on the genome in the absence of RNAP, was primarily associated with promoter regions, and was highly correlated with the distribution of RNAP σA. The colocalization of σA and CarD led us to propose that in vivo, CarD associates with RNAP initiation complexes at most promoters and is therefore a global regulator of transcription initiation. Here we describe in detail the data from the ChIP-seq experiments associated with the study published by Srivastava and colleagues in the Proceedings of the National Academy of Science in 2013 [5] as well as discuss the findings from this dataset in relation to both CarD and mycobacterial transcription as a whole.The ChIP-seq data have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE48164).

Expression, crystallization and preliminary X-ray crystallographic analysis of DNA-directed RNA polymerase subunit L from Thermococcus onnurineus NA1.

RNA polymerase (RNAP) plays a crucial role in gene expression in all organisms. It is a multiprotein complex that produces primary transcript RNA. Generally, the basal transcription apparatus in archaea is simpler than the eukaryotic RNA polymerase II counterpart. To understand the structure and function of archaeal RNAP, the TON-0309 gene encoding DNA-directed RNA polymerase subunit L (ToRNAP_L) from Thermococcus onnurineus NA1 was cloned and the protein was overexpressed in Escherichia coli, purified and crystallized. The purified protein was crystallized using the hanging-drop vapour-diffusion method and the crystal diffracted to 2.10 Å resolution. The crystal belonged to the hexagonal space group P6122, with unit-cell parameters a = b = 42.3, c = 211.2 Å. One molecule was present in the asymmetric unit, with a corresponding VM of 2.5 Å(3) Da(-1) and a solvent content of 50.0%.

Protection against filovirus diseases by a novel broad-spectrum nucleoside analogue BCX4430

Filoviruses are emerging pathogens and causative agents of viral haemorrhagic fever. Case fatality rates of filovirus disease outbreaks are among the highest reported for any human pathogen, exceeding 90% (ref. 1). Licensed therapeutic or vaccine products are not available to treat filovirus diseases. Candidate therapeutics previously shown to be efficacious in non-human primate disease models are based on virus-specific designs and have limited broad-spectrum antiviral potential. Here we show that BCX4430, a novel synthetic adenosine analogue, inhibits infection of distinct filoviruses in human cells. Biochemical, reporter-based and primer-extension assays indicate that BCX4430 inhibits viral RNA polymerase function, acting as a non-obligate RNA chain terminator. Post-exposure intramuscular administration of BCX4430 protects against Ebola virus and Marburg virus disease in rodent models. Most importantly, BCX4430 completely protects cynomolgus macaques from Marburg virus infection when administered as late as 48 hours after infection. In addition, BCX4430 exhibits broad-spectrum antiviral activity against numerous viruses, including bunyaviruses, arenaviruses, paramyxoviruses, coronaviruses and flaviviruses. This is the first report, to our knowledge, of non-human primate protection from filovirus disease by a synthetic drug-like small molecule. We provide additional pharmacological characterizations supporting the potential development of BCX4430 as a countermeasure against human filovirus diseases and other viral diseases representing major public health threats.