Poxvirus Replication Research Articles

A Quantitative Real-Time PCR Assay for Measuring Poxvirus Replication and Cell Binding.

Quantitative real-time PCR (qPCR) is a fast and reliable method to quantify viral genomes as a surrogate to titering on monolayers of cells for measuring virus replication. Whether it be for determining the number of virions released, the total number of genomes produced during infection, or the number of virions bound to a cell, qPCR assays can be adapted to quickly enumerate total viral genomes in a broad range of experiments comparing virus replication under different conditions. In addition, qPCR offers several advantages compared to plaque assays including time, linearity over 9 logs, and scalability from tens-to-hundreds of samples, depending on the qPCR machine. Here we describe a qPCR assay for quantifying vaccinia virus' dsDNA genome that can be used to determine the total number of virions produced. Furthermore, we describe a straightforward protocol for a cell-binding assay that is sensitive enough to use with small concentrations of inoculating virions. This protocol is suitable for measuring the cell-binding ability of mutations that affect virus production and infectivity.

Cardiac glycosides inhibit early and late vaccinia virus protein expression.

Cardiac glycosides (CGs) are natural steroid glycosides, which act as inhibitors of the cellular sodium-potassium ATPase pump. Although traditionally considered toxic to human cells, CGs are widely used as drugs for the treatment of cardiovascular-related medical conditions. More recently, CGs have been explored as potential anti-viral drugs and inhibit replication of a range of RNA and DNA viruses. Previously, a compound screen identified CGs that inhibited vaccinia virus (VACV) infection. However, no further investigation of the inhibitory potential of these compounds was performed, nor was there investigation of the stage(s) of the poxvirus lifecycle they impacted. Here, we investigated the anti-poxvirus activity of a broad panel of CGs. We found that all CGs tested were potent inhibitors of VACV replication. Our virological experiments showed that CGs did not impact virus infectivity, binding, or entry. Rather, experiments using recombinant viruses expressing reporter proteins controlled by VACV promoters and arabinoside release assays demonstrated that CGs inhibited early and late VACV protein expression at different concentrations. Lack of virus assembly in the presence of CGs was confirmed using electron microscopy. Thus, we expand our understanding of compounds with anti-poxvirus activity and highlight a yet unrecognized mechanism by which poxvirus replication can be inhibited.

Hesperetin blocks poxvirus replication with a low tendency to select for drug-resistant viral variants.

In this study, we demonstrated the antiviral efficacy of hesperetin against multiple poxviruses, including buffalopox virus (BPXV), vaccinia virus (VACV), and lumpy skin disease virus (LSDV). The time-of-addition and virus step-specific assays indicated that hesperetin reduces the levels of viral DNA, mRNA, and proteins in the target cells. Further, by immunoprecipitation (IP) of the viral RNA from BPXV-infected Vero cells and a cell-free RNA-IP assay, we demonstrated that hesperetin-induced reduction in BPXV protein synthesis is also consistent with diminished interaction between eukaryotic translation initiation factor eIF4E and the 5' cap of viral mRNA. Molecular docking and MD simulation studies were also consistent with the binding of hesperetin to the cap-binding pocket of eIF4E, adopting a conformation similar to m7GTP binding. Furthermore, in a BPXV egg infection model, hesperetin was shown to suppress the development of pock lesions on the chorioallantoic membrane and associated mortality in the chicken embryos. Most importantly, long-term culture of BPXV in the presence of hesperetin did not induce the generation of drug-resistant viral mutants. In conclusion, we, for the first time, demonstrated the antiviral activity of hesperetin against multiple poxviruses, besides providing some insights into its potential mechanisms of action.

FEAR antiviral response pathway is independent of interferons and countered by poxvirus proteins.

The human facilitates chromatin transcription (FACT) complex is a chromatin remodeller composed of human suppressor of Ty 16 homologue (hSpt16) and structure-specific recognition protein-1 subunits that regulates cellular gene expression. Whether FACT regulates host responses to infection remained unclear. We identify a FACT-mediated, interferon-independent, antiviral pathway that restricts poxvirus replication. Cell culture and bioinformatics approaches suggest that early viral gene expression triggers nuclear accumulation of SUMOylated hSpt16 subunits required for the expression of E26 transformation-specific sequence-1 (ETS-1)-a transcription factor that activates virus restriction programs. However, biochemical studies show that poxvirus-encoded A51R proteins block ETS-1 expression by outcompeting structure-specific recognition protein-1 binding to SUMOylated hSpt16 and by tethering SUMOylated hSpt16 to microtubules. Furthermore, A51R antagonism of FACT enhances poxvirus replication in human cells and virulence in mice. Finally, we show that FACT also restricts rhabdoviruses, flaviviruses and orthomyxoviruses, suggesting broad roles for FACT in antiviral immunity. Our study reveals the FACT-ETS-1 antiviral response (FEAR) pathway to be critical for eukaryotic antiviral immunity and describes a unique mechanism of viral immune evasion.

Bisbenzimide compounds inhibit the replication of prototype and pandemic potential poxviruses.

We previously identified the bisbenzimide Hoechst 33342 (H42) as a potent multi-stage inhibitor of the prototypic poxvirus, the vaccinia virus (VACV), and several parapoxviruses. A recent report showed that novel bisbenzimide compounds similar in structure to H42 could prevent human cytomegalovirus replication. Here, we assessed whether these compounds could also serve as poxvirus inhibitors. Using virological assays, we show that these bisbenzimide compounds inhibit VACV spread, plaque formation, and the production of infectious progeny VACV with relatively low cell toxicity. Further analysis of the VACV lifecycle indicated that the effective bisbenzimide compounds had little impact on VACV early gene expression but inhibited VACV late gene expression and truncated the formation of VACV replication sites. Additionally, we found that bisbenzimide compounds, including H42, can inhibit both monkeypox and a VACV mutant resistant to the widely used anti-poxvirus drug TPOXX (Tecovirimat). Therefore, the tested bisbenzimide compounds were inhibitors of both prototypic and pandemic potential poxviruses and could be developed for use in situations where anti-poxvirus drug resistance may occur. Additionally, these data suggest that bisbenzimide compounds may serve as broad-activity antiviral compounds, targeting diverse DNA viruses such as poxviruses and betaherpesviruses.IMPORTANCEThe 2022 mpox (monkeypox) outbreak served as a stark reminder that due to the cessation of smallpox vaccination over 40 years ago, most of the human population remains susceptible to poxvirus infection. With only two antivirals approved for the treatment of smallpox infection in humans, the need for additional anti-poxvirus compounds is evident. Having shown that the bisbenzimide H33342 is a potent inhibitor of poxvirus gene expression and DNA replication, here we extend these findings to include a set of novel bisbenzimide compounds that show anti-viral activity against mpox and a drug-resistant prototype poxvirus mutant. These results suggest that further development of bisbenzimides for the treatment of pandemic potential poxviruses is warranted.

Genistein is effective in inhibiting Orf virus infection in vitro by targeting viral RNA polymerase subunit RPO30 protein.

Orf virus (ORFV), a typical member of the genus Parapoxvirus, Poxvirus family, causes a contagious pustular dermatitis in sheep, goats, and humans. Poxviruses encode a multisubunit DNA-dependent RNA polymerase (vRNAP) that carries out viral gene expression in the host cytoplasm, which is a viral factor essential to poxvirus replication. Due to its vital role in viral life, vRNAP has emerged as one of the potential drug targets. In the present study, we investigated the antiviral effect of genistein against ORFV infection. We provided evidence that genistein exerted antiviral effect through blocking viral genome DNA transcription/replication and viral protein synthesis and reducing viral progeny, which were dosedependently decreased in genistein-treated cells. Furthermore, we identified that genistein interacted with the vRNAP RPO30 protein by CETSA, molecular modeling and Fluorescence quenching, a novel antiviral target for ORFV. By blocking vRNAP RPO30 protein using antibody against RPO30, we confirmed that the inhibitory effect exerted by genistein against ORFV infection is mediated through the interaction with RPO30. In conclusion, we demonstrate that genistein effectively inhibits ORFV transcription in host cells by targeting vRNAP RPO30, which might be a promising drug candidate against poxvirus infection.

The poxvirus F17 protein counteracts mitochondrially orchestrated antiviral responses

Poxviruses are unusual DNA viruses that replicate in the cytoplasm. To do so, they encode approximately 100 immunomodulatory proteins that counteract cytosolic nucleic acid sensors such as cGAMP synthase (cGAS) along with several other antiviral response pathways. Yet most of these immunomodulators are expressed very early in infection while many are variable host range determinants, and significant gaps remain in our understanding of poxvirus sensing and evasion strategies. Here, we show that after infection is established, subsequent progression of the viral lifecycle is sensed through specific changes to mitochondria that coordinate distinct aspects of the antiviral response. Unlike other viruses that cause extensive mitochondrial damage, poxviruses sustain key mitochondrial functions including membrane potential and respiration while reducing reactive oxygen species that drive inflammation. However, poxvirus replication induces mitochondrial hyperfusion that independently controls the release of mitochondrial DNA (mtDNA) to prime nucleic acid sensors and enables an increase in glycolysis that is necessary to support interferon stimulated gene (ISG) production. To counter this, the poxvirus F17 protein localizes to mitochondria and dysregulates mTOR to simultaneously destabilize cGAS and block increases in glycolysis. Our findings reveal how the poxvirus F17 protein disarms specific mitochondrially orchestrated responses to later stages of poxvirus replication.

The Antiviral Effect of Berdazimer Sodium on Molluscum Contagiosum Virus Using a Novel In Vitro Methodology.

Molluscum contagiosum (MC) is characterized by skin lesions containing the highly contagious molluscum contagiosum poxvirus (MCV). MCV primarily infects children, with one US Food and Drug Administration (FDA)-approved drug-device treatment in use but no approved medications. Assessing antivirals is hindered by the inability of MCV to replicate in vitro. Here, we use vaccinia virus as a surrogate to provide evidence of the anti-poxvirus properties of berdazimer sodium, a new chemical entity, and the active substance in berdazimer gel, 10.3%, a nitric oxide-releasing topical in phase 3 development for the treatment of MC. We show that berdazimer sodium reduced poxvirus replication and, through a novel methodology, demonstrate that cells infected with drug-treated MCV virions have reduced early gene expression. Specifically, this is accomplished by studying the nuclear factor kappa-light-chain-enhancer of activated B cell (NF-kB)-blocking protein MC160 as an example of an early gene. The results provide a plausible unique antiviral mechanism of action supporting increased MCV resolution observed in patients treated with berdazimer gel, 10.3% and describe a novel methodology that overcomes limitations in investigating MCV response in vitro to a potential new MC topical medication.

Exaptation of Inactivated Host Enzymes for Structural Roles in Orthopoxviruses and Novel Folds of Virus Proteins Revealed by Protein Structure Modeling.

Viruses with large, double-stranded DNA genomes captured the majority of their genes from their hosts at different stages of evolution. The origins of many virus genes are readily detected through significant sequence similarity with cellular homologs. In particular, this is the case for virus enzymes, such as DNA and RNA polymerases or nucleotide kinases, that retain their catalytic activity after capture by an ancestral virus. However, a large fraction of virus genes have no readily detectable cellular homologs, meaning that their origins remain enigmatic. We explored the potential origins of such proteins that are encoded in the genomes of orthopoxviruses, a thoroughly studied virus genus that includes major human pathogens. To this end, we used AlphaFold2 to predict the structures of all 214 proteins that are encoded by orthopoxviruses. Among the proteins of unknown provenance, structure prediction yielded clear indications of origin for 14 of them and validated several inferences that were previously made via sequence analysis. A notable emerging trend is the exaptation of enzymes from cellular organisms for nonenzymatic, structural roles in virus reproduction that is accompanied by the disruption of catalytic sites and by an overall drastic divergence that precludes homology detection at the sequence level. Among the 16 orthopoxvirus proteins that were found to be inactivated enzyme derivatives are the poxvirus replication processivity factor A20, which is an inactivated NAD-dependent DNA ligase; the major core protein A3, which is an inactivated deubiquitinase; F11, which is an inactivated prolyl hydroxylase; and more similar cases. For nearly one-third of the orthopoxvirus virion proteins, no significantly similar structures were identified, suggesting exaptation with subsequent major structural rearrangement that yielded unique protein folds. IMPORTANCE Protein structures are more strongly conserved in evolution than are amino acid sequences. Comparative structural analysis is particularly important for inferring the origins of viral proteins that typically evolve at high rates. We used a powerful protein structure modeling method, namely, AlphaFold2, to model the structures of all orthopoxvirus proteins and compared them to all available protein structures. Multiple cases of recruitment of host enzymes for structural roles in viruses, accompanied by the disruption of catalytic sites, were discovered. However, many viral proteins appear to have evolved unique structural folds.

Emerging evidence for poxvirus-mediated unfolded protein response: Lumpy skin disease virus maintains self-replication by activating PERK and IRE1 signaling.

The monkeypox epidemic has attracted global attention to poxviruses. The cytoplasmic replication of poxviruses requires extensive protein synthesis, challenging the capacity of the endoplasmic reticulum (ER). However, the role of the ER in the life cycle of poxviruses is unclear. In this study, we demonstrate that infection with the lumpy skin disease virus (LSDV), a member of the poxvirus family, causes ER stress in vivo and in vitro, further facilitating the activation of the unfolded protein response (UPR). Although UPR activation aids in the restoration of the cellular environment, its significance in the LSDV life cycle remains unclear. Furthermore, the significance of ER imbalance for viral replication is also unknown. We show that LSDV replication is hampered by an unbalanced ER environment. In addition, we verify that the LSDV replication depends on the activation of PERK-eIF2α and IRE1-XBP1 signaling cascades rather than ATF6, implying that global translation and reduced XBP1 cleavage are deleterious to LSDV replication. Taken together, these findings indicate that LSDV is involved in the repression of global translational signaling, ER chaperone transcription, and ATF6 cleavage from the Golgi into the nucleus, thereby maintaining cell homeostasis; moreover, PERK and IRE1 activation contribute to LSDV replication. Our findings suggest that targeting UPR elements may be applied in response to infection from LSDV or even other poxviruses, such as monkeypox.

Cidofovir for the Treatment of Molluscum Contagiosum Virus.

That cidofovir, an acyclic nucleoside phosphonate (ANP), was inhibitory to the replication of poxviruses was first demonstrated by De Clercq et al.. That its active metabolite, the diphosphate, was found to be inhibitory to the molluscum contagiosum (M. contagiosum) DNA polymerase was demonstrated by Watanabe and Tamaki. Twelve different independent observations have then indicated that cidofovir administered intravenously, topically or intralesionally is efficacious in the treatment of M. contagiosum mostly in immunosuppressed patients.

In ovo inhibition of avian pox virus replication by mangosteen rind and red ginger ethanolic extracts.

Background and Aim:Avian pox is a contagious disease caused by the avian pox virus (APV). Mangostin and γ-mangostin in mangosteen rind (MR) and gingerol in red ginger (RG) exhibit antiviral activity. In this study, we evaluated the effect of MR and RG ethanolic extracts on APV based on pock lesions on the chorioallantoic membrane (CAM) of specific pathogen-free (SPF) embryonated chicken eggs (ECEs).Materials and Methods:Three APVs from chicken isolates (C1, C2, and C3), one APV from a pigeon isolate (P), 1.5% and 3% MR ethanolic extract, 5% and 10% RG ethanolic extract, and a combination of 1.5% MR and 5% RG at 0.1 mL/egg were inoculated in ovo (7th day incubation, chorioallantoic route) in SPF ECEs. A control group inoculated in ovo with APV alone was also established. Each treatment consisted of three replicates. Parameters including embryo survival, CAM lesions, and average number of pock lesions were determined.Results:In ovo inoculation of MR and RG ethanolic extracts was not harmful to the ECEs and did not induce CAM lesions. The average number of pock lesions in the control group (C1, C2, C3, and P) was 35, 14, 10, and 17, respectively, whereas in all treatment groups, the number was 0, except in the 5% RG group of C1, which had a value of 10.Conclusion:In ovo inoculation of 1.5% and 3% MR, 5% and 10% RG, and the combination of 1.5% MR plus 5% RG ethanolic extracts at 0.1 mL/egg inhibit APV by reducing the number of pock lesions on the CAM of the ECE.

Acrylamide inhibits vaccinia virus through vimentin-independent anti-viral granule formation.

The replication and assembly of vaccinia virus (VACV), the prototypic poxvirus, occurs exclusively in the cytoplasm of host cells. While the role of cellular cytoskeletal components in these processes remains poorly understood, vimentin-a type III intermediate filament-has been shown to associate with viral replication sites and to be incorporated into mature VACV virions. Here, we employed chemical and genetic approaches to further investigate the role of vimentin during the VACV lifecycle. The collapse of vimentin filaments, using acrylamide, was found to inhibit VACV infection at the level of genome replication, intermediate- and late-gene expression. However, we found that CRISPR-mediated knockout of vimentin did not impact VACV replication. Combining these tools, we demonstrate that acrylamide treatment results in the formation of anti-viral granules (AVGs) known to mediate translational inhibition of many viruses. We conclude that vimentin is dispensable for poxvirus replication and assembly and that acrylamide, as a potent inducer of AVGs during VACV infection, serves to bolster cell's anti-viral response to poxvirus infection.

Anti-herpes virus activity of the carnivorous botanical, Sarracenia purpurea

Herpes simplex virus type-1 (HSV-1), one of the most widely spread human viruses in the Herpesviridae family, causes herpes labialis (cold sores) and keratitis (inflammation of the cornea). Conventional treatment for HSV-1 infection includes pharmaceutical drugs, such as acyclovir and docosonal, which are efficacious but maintain the potential for the development of viral drug resistance. Extracts from the carnivorous pitcher plant, Sarracenia purpurea, have previously been shown to inhibit the replication of HSV-1. In this study, we demonstrate that S. purpurea extracts can inhibit the replication of HSV-1 by two distinct mechanisms of action. These extracts directly inhibit extracellular virions or viral attachment to the human host cell as well as inhibiting the expression of viral immediate-early, early and late genes when added at various times post-infection. This botanical has previously been shown to inhibit the replication of poxviruses through the inhibition of early viral gene transcription. These results support a broader anti-viral activity of S. purpurea extracts against both pox and herpes viruses.

Identification of Vaccinia Virus Inhibitors and Cellular Functions Necessary for Efficient Viral Replication by Screening Bioactives and FDA-Approved Drugs

Four decades after the eradication of smallpox, poxviruses continue to threaten the health of humans and other animals. Vaccinia virus (VACV) was used as the vaccine that successfully eradicated smallpox and is a prototypic member of the poxvirus family. Many cellular pathways play critical roles in productive poxvirus replication. These pathways provide opportunities to expand the arsenal of poxvirus antiviral development by targeting the cellular functions required for efficient poxvirus replication. In this study, we developed and optimized a secreted Gaussia luciferase-based, simplified assay procedure suitable for high throughput screening. Using this procedure, we screened a customized compound library that contained over 3200 bioactives and FDA (Food and Drug Administration)-approved chemicals, most having known cellular targets, for their inhibitory effects on VACV replication. We identified over 140 compounds that suppressed VACV replication. Many of these hits target cellular pathways previously reported to be required for efficient VACV replication, validating the effectiveness of our screening. Importantly, we also identified hits that target cellular functions with previously unknown roles in the VACV replication cycle. Among those in the latter category, we verified the antiviral role of several compounds targeting the janus kinase/signal transducer and activator of transcription-3 (JAK/STAT3) signaling pathway by showing that STAT3 inhibitors reduced VACV replication. Our findings identify pathways that are candidates for use in the prevention and treatment of poxvirus infections and additionally provide a foundation to investigate diverse cellular pathways for their roles in poxvirus replications.

Proteomic and mechanistic dissection of the poxvirus-customized ribosome.

Ribosomes are often viewed as protein synthesis machines that lack intrinsic regulatory capacity. However, studies have established that ribosomes can functionally diversify through changes in the composition of, or post-translational modifications to ribosomal subunit proteins (RPs). We recently found that poxviruses phosphorylate unique sites in the RP, receptor for activated C kinase 1 (RACK1) to enhance viral protein synthesis. Here, we developed approaches for large-scale proteomic analysis of ribosomes isolated from cells infected with different viruses. Beyond RACK1, we identified additional phosphorylation events within RPS2 and RPS28 that arise during poxvirus infection, but not other viruses tested. The modified sites lie within unstructured loop domains that position around the mRNA entry and exit channel, respectively, and site-substitution mutants revealed that each modified residue contributed differently to poxvirus replication. Our findings reveal the broader extent to which poxviruses customize host ribosomes and provide new insights into how ribosomes can functionally diversify.

The assembly and function of poxvirus proteins

Proteins play essential roles in biological processes, and atomic details of their structure and function provide an insight into their biological roles and how they can be manipulated for therapeutic purposes. One class of proteins that are still poorly characterized at the structural level are those involved in pox virus replication. This work describes the structural and functional studies of several proteins involved in Tanapox virus replication replication. The Tanapox virus is a poxvirus that is related to the medially important vaccinia, variola and Monkeypox viruses. The variola virus is the causative agent of smallpox, which is estimated to have caused 300–500 million deaths in the 19th century alone and is a potential bioterrorism threat. Structural and functional insights into the proteins and biomolecular complexes that regulate the lifecycle of the virus will aid in the development of therapeutic agents against poxviruses and the development of poxviruses as therapeutic tools against cancer. The main goals of the study were to assess the structure and function of Tanapox proteins using biochemical and biophysical methods. Many Tanapox virus protein construct samples were generated and studied. Nuclear magnetic resonance (NMR) spectroscopy was performed on several samples to assess their structural and conformationally dynamic properties. Circular dichroism spectroscopy, size exclusion chromatography and electrophoretic mobility shift assays were used to determine the roles of these proteins molecular function. The results reveal that Tanapox virus proteins are generally insoluble when expressed alone. While biophysical studies of insoluble proteins are difficult, the samples generated are ideal for generating antibodies for in vivo functional studies.Support or Funding InformationThank you to Dr. Karim Essani for providing Tanapox genome.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Methodical Review on Poxvirus Replication, Genes Responsible for the Development of Infection and Host Immune Response Against the Disease

Poxviruses are among the best known and most feared viruses in the world and their emergence was estimated to be around thousands of years ago. Poxviruses use the majority of their genes for intonation of their host antiviral reaction and, assumed as virulence genes. This review aims to cite information about the replication of poxvirus, genes responsible for causing the disease and host defense mechanism against the virus. Glycosaminoglycan’s (GAGs) is supposed to be the receptors of poxvirus during entrance to cells for replication. More than one enzyme is encoded by several poxviruses for production of DNA, which probably increases genome replication in inactive cells. Based on fluorescent dye visualization, the DNA synthesis of Poxvirus is detected within two hours after infection takes place in a separate juxtanuclear location known as factories. The Acquired immune response is a complex interaction of a number of cell types and the consequently lead to the generation of specific antiviral antibody of B lymphocytes and in virus-specific DTH and cytotoxic responses by T lymphocytes. In poxvirus infection, immunodeficiency syndrome reflects both a primary and secondary immunosuppression. Poxviruses infections have a varied effect on host macromolecular Syntheses and act against interferon activities.

Click Chemistry-Based Labeling of Poxvirus Genomes.

Vaccinia virus packages its dsDNA genome inside its core for protection during the extracellular phases of its life cycle. In the cytoplasm of a newly infected cell the viral genome is released from the core so the viral DNA replication machinery can access it and initiate DNA replication. Vaccinia virus replication sites in the cell cytosol can be detected with conventional DNA staining methods; these, however, do not provide enough specificity to be used for quantitative image analysis or further probing of the replication step. Likewise, the ability to generate recombinant vaccinia viruses with fluorescently tagged proteins has provided insight into many stages of the viral life cycle, but many of the early steps involving the viral genome remain to be elucidated. Nucleotide and nucleoside analogs are traditionally used for probing the cell cycle and investigating other changes in cellular DNA, with the more novel nucleoside analogs providing a better way to label with click chemistry. Here we demonstrate how nucleoside analogs and click chemistry can be used for tracking poxvirus replication in the viral factories, and tracking single viral genomes in infected cells.

Mutation and structure guided discovery of an antiviral small molecule that mimics an essential C-Terminal tripeptide of the vaccinia D4 processivity factor

The smallpox virus (variola) remains a bioterrorism threat since a majority of the human population has never been vaccinated. In the event of an outbreak, at least two drugs against different targets of variola are critical to circumvent potential viral mutants that acquire resistance. Vaccinia virus (VACV) is the model virus used in the laboratory for studying smallpox. The VACV processivity factor D4 is an ideal therapeutic target since it is both essential and specific for poxvirus replication. Recently, we identified a tripeptide (Gly-Phe-Ile) motif at the C-terminus of D4 that is conserved among poxviruses and is necessary for maintaining protein function. In the current work, a virtual screening for small molecule mimics of the tripeptide identified a thiophene lead that effectively inhibited VACV, cowpox virus, and rabbitpox virus in cell culture (EC50 = 8.4–19.7 μM) and blocked in vitro processive DNA synthesis (IC50 = 13.4 μM). Compound-binding to D4 was demonstrated through various biophysical methods and a dose-dependent retardation of the proteolysis of D4 proteins. This study highlights an inhibitor design strategy that exploits a susceptible region of the protein and identifies a novel scaffold for a broad-spectrum poxvirus inhibitor.