Recombinant Cowpox virus DNA-directed RNA polymerase 132 kDa polypeptide (RPO132), partial

What is Recombinant Cowpox virus DNA-directed RNA polymerase 132 kDa polypeptide (RPO132)?

RPO132 is a critical enzyme component of the Cowpox virus (CPXV) transcriptional machinery, responsible for viral gene expression during infection. This 132 kDa polypeptide functions as part of the viral DNA-dependent RNA polymerase complex that transcribes viral genes into mRNA during the viral replication cycle. The protein is encoded by the RPO132 gene (also known as CPXV157 in some CPXV strains) and represents one of the conserved core genes in orthopoxviruses . The recombinant form refers to the protein produced through genetic engineering techniques where the gene encoding RPO132 is inserted into an expression vector and produced in laboratory settings.

The RPO132 protein shares significant sequence homology with RNA polymerase subunits from other orthopoxviruses, including Variola virus (smallpox), Vaccinia virus, and Monkeypox virus, reflecting the evolutionary relationships among these pathogens . This conservation makes RPO132 an important target for comparative studies of orthopoxvirus biology and potentially for broad-spectrum antiviral development. The functional domains of RPO132 include catalytic regions for RNA synthesis and regulatory domains that interact with other viral and cellular factors.

The partial recombinant form typically refers to a truncated version of the protein that contains key functional domains while potentially excluding regions that might interfere with expression or purification efficiency. These recombinant proteins serve as valuable tools for structural studies, enzymatic assays, and immunological research focused on orthopoxvirus biology and pathogenesis.

How does RPO132 function in cowpox virus replication?

RPO132 serves as a critical subunit of the viral RNA polymerase complex that mediates transcription of the cowpox virus genome. During the viral replication cycle, RPO132 works in conjunction with other viral proteins to form the functional RNA polymerase holoenzyme, which recognizes viral promoters and catalyzes the synthesis of viral mRNAs . This process is essential for viral gene expression and subsequent production of viral proteins necessary for replication.

The functional activity of RPO132 occurs within the cytoplasmic viral factories, specialized compartments where viral DNA replication and transcription take place. These factories form distinctive cytoplasmic A-type inclusion bodies that are characteristic of cowpox virus infection . The RNA polymerase complex containing RPO132 is involved in both early and intermediate/late gene transcription, though its activity is regulated by different mechanisms at various stages of infection.

Unlike host cell transcription that occurs in the nucleus, orthopoxvirus transcription takes place entirely in the cytoplasm, requiring the virus to encode its own transcriptional machinery. This unique aspect of poxvirus biology makes RPO132 and other components of the viral RNA polymerase complex potential targets for selective antiviral strategies. The functional importance of RPO132 is underscored by its high conservation among orthopoxviruses, suggesting strong evolutionary pressure to maintain its structure and activity.

What is the genomic context of the RPO132 gene in cowpox virus?

The RPO132 gene is located in the central conserved region of the cowpox virus genome, which contains genes essential for viral replication and basic functions. Specifically, it is positioned in a region spanning from the DNA polymerase gene (E9L in Vaccinia virus nomenclature) to the A24R gene (DNA-dependent RNA polymerase subunit), a segment that is highly conserved among orthopoxviruses and often used for phylogenetic analysis . This conservation reflects the essential nature of these genes for viral viability.

In phylogenetic analyses, the region containing RPO132 has been extracted from cowpox virus and other orthopoxvirus genomes to construct evolutionary trees, demonstrating its utility in understanding orthopoxvirus evolution and relationships . The genomic architecture surrounding RPO132 is characterized by other genes involved in transcription and DNA replication, forming a functional cluster of essential viral genes.

How is Recombinant Cowpox virus RPO132 expressed and purified?

Recombinant Cowpox virus RPO132 can be expressed in multiple host systems, each offering distinct advantages depending on the research application. The most common expression platforms include prokaryotic (E. coli), yeast, baculovirus-insect cell, and mammalian cell systems. E. coli and yeast systems typically provide the highest protein yields and shorter production timelines, making them suitable for applications where post-translational modifications are not critical . For studies requiring functional analysis or structural investigations that depend on proper protein folding, insect or mammalian expression systems are preferable despite their lower yields and longer production times.

The general protocol for expression and purification involves:

• Cloning the RPO132 gene (full-length or partial) into an appropriate expression vector containing necessary regulatory elements and fusion tags (commonly His-tag, GST, or MBP)

• Transformation or transfection of the expression construct into the chosen host system

• Induction of protein expression under optimized conditions (temperature, induction time, media composition)

• Cell harvest and lysis using methods appropriate for the host system

• Initial capture of the recombinant protein using affinity chromatography based on the fusion tag

• Further purification steps potentially including ion exchange chromatography, size exclusion chromatography, or other techniques to achieve high purity

• Quality control assessment, typically involving SDS-PAGE analysis and mass spectrometry to confirm identity

For Cowpox virus RPO132, purification typically achieves greater than 85% purity as determined by SDS-PAGE analysis . The partial recombinant form refers to selected regions of the protein that maintain critical functional domains while potentially improving expression efficiency or solubility compared to the full-length protein.

What quality control methods are essential for verifying RPO132 functionality?

For physical characterization, SDS-PAGE analysis is the primary method to verify protein size and initial purity, with expectations of at least 85% purity for research applications . This should be complemented by Western blotting using antibodies specific to RPO132 or to epitope tags incorporated in the recombinant construct. Mass spectrometry provides confirmation of protein identity through peptide mass fingerprinting or sequence analysis. Circular dichroism spectroscopy can assess secondary structure integrity, while thermal shift assays evaluate protein stability under various buffer conditions.

Functional characterization is essential and typically involves in vitro RNA polymerase assays measuring the synthesis of RNA from template DNA. These assays can utilize radioisotope-labeled nucleotides or fluorescent detection methods to quantify transcriptional activity. Templates containing authentic cowpox virus promoters are ideal for evaluating promoter-specific activity. For multicomponent assays, recombinant RPO132 must be combined with other components of the viral RNA polymerase complex.

Additional functional tests may include:

• DNA binding assays (electrophoretic mobility shift assays or surface plasmon resonance)

• Protein-protein interaction studies with other viral transcription factors

• Assessment of enzymatic parameters (Km, Vmax, processivity)

• Inhibitor sensitivity profiles for comparative pharmacological studies

For structural integrity assessment, limited proteolysis can map domain organization and stability, while dynamic light scattering evaluates sample homogeneity. Functionality in cell-based systems can be tested through complementation assays in cells infected with defective viruses or through reconstituted transcription systems.

How does expression host selection impact RPO132 activity and structure?

The choice of expression host significantly impacts the structural integrity, functional activity, and yield of recombinant RPO132, necessitating careful consideration based on research objectives. Each expression system presents distinct advantages and limitations that directly influence protein quality.

Yeast expression systems (Saccharomyces cerevisiae or Pichia pastoris) represent an intermediate option, providing some eukaryotic processing capabilities while maintaining relatively high yields. These systems can produce soluble RPO132 with some post-translational modifications, though not necessarily identical to those in mammalian cells during natural viral infection.

For applications requiring maximum biological fidelity, baculovirus-insect cell and mammalian expression systems are preferred despite their lower yields and higher complexity . The baculovirus system efficiently produces large proteins like RPO132 (132 kDa) with most post-translational modifications. Mammalian cell expression provides the most authentic environment for viral protein production but typically results in the lowest yields.

The functional impacts of these expression choices can be observed in enzymatic activity assays, where properly folded RPO132 from eukaryotic systems typically demonstrates higher specific activity compared to bacterially-expressed protein. For structural studies using X-ray crystallography or cryo-EM, the homogeneity and stability of the protein preparation may be more critical than maintaining all post-translational modifications.

How does RPO132 contribute to understanding orthopoxvirus evolution and host range?

RPO132 plays a pivotal role in elucidating orthopoxvirus evolution and host range determination through its high conservation and essential function in viral replication. As a core component of the viral transcriptional machinery, RPO132 is subject to strong evolutionary constraints, making its gene sequence valuable for phylogenetic analyses and evolutionary studies of orthopoxviruses.

Phylogenetic analysis utilizing the genomic region from DNA polymerase (E9L) to RPO132 (A24R) has been instrumental in establishing evolutionary relationships among orthopoxviruses . This region has been extracted from cowpox virus, variola virus, and other orthopoxvirus genomes to construct evolutionary trees that reveal distinct clades correlating with historic geographic distribution patterns . These analyses have contributed to understanding the origins and diversification of orthopoxviruses, including the evolution of host-specific viruses like variola virus (which exclusively infects humans) from ancestral viruses with broader host ranges like cowpox virus.

The contribution of RPO132 to host range determination is more complex and likely indirect. While RPO132 itself is not a primary host range determinant, its function in viral transcription must adapt to the intracellular environment of different host species. Subtle variations in RPO132 sequence between orthopoxvirus species may reflect adaptations to optimize viral transcription in specific host cellular contexts. Furthermore, the interaction of RPO132 with host cell factors may influence viral replication efficiency in different species.

Comparative genomic studies have revealed that while cowpox virus has the broadest host range among orthopoxviruses and infects a wide variety of mammals , viruses like variola have extremely restricted host ranges. Analysis of the functional differences in core viral proteins like RPO132 across these viruses contributes to understanding the molecular basis of host restriction. The study of selection pressures on different domains of RPO132 through dN/dS ratio analysis can reveal regions under positive selection that may contribute to host adaptation .

What role does RPO132 research play in developing antiviral strategies against orthopoxviruses?

RPO132 research provides crucial insights for antiviral development against orthopoxviruses through multiple strategic avenues. As a highly conserved and essential enzyme for viral replication, RPO132 represents an attractive target for broad-spectrum antiviral agents that could potentially inhibit multiple orthopoxvirus species, including emerging zoonotic threats like cowpox virus.

The viral RNA polymerase complex, of which RPO132 is a key component, is functionally distinct from host cell RNA polymerases, offering a basis for selective inhibition with minimal host toxicity. This selectivity is critical for therapeutic applications and can be leveraged through detailed structural and biochemical characterization of RPO132. Recombinant RPO132 enables high-throughput screening assays for small molecule inhibitors that specifically target viral transcription.

Several approaches to antiviral development centered on RPO132 include:

• Direct enzymatic inhibition through small molecules that bind to catalytic or regulatory domains

• Disruption of essential protein-protein interactions between RPO132 and other components of the viral transcriptional machinery

• Identification of host factors that interact with RPO132, potentially revealing additional targets for intervention

• Development of RNA interference or antisense strategies targeting RPO132 expression

• Structure-based drug design utilizing high-resolution structural data from recombinant RPO132

The growing concern about cowpox as an emerging zoonotic threat and the cessation of smallpox vaccination, which provided cross-protection against other orthopoxviruses, underscores the importance of developing effective antiviral strategies. Human cowpox cases, particularly in immunocompromised and eczematous patients, can be severe and potentially life-threatening , highlighting the medical need for orthopoxvirus-specific antivirals.

Additionally, RPO132 research contributes to vaccine development efforts by enhancing understanding of viral immunogenicity and identifying conserved epitopes that might elicit cross-protective immunity. Recombinant RPO132 can be used to develop assays for evaluating vaccine efficacy by measuring neutralizing antibodies or T-cell responses that target this essential viral component. This research addresses the public health concerns raised by the emergence of cowpox virus as a zoonotic threat .

How does RPO132 compare functionally to RNA polymerases in other virus families?

RPO132 exhibits distinctive functional characteristics when compared to RNA polymerases from other virus families, reflecting the unique replication strategy of orthopoxviruses. These comparative differences provide important insights into viral evolution and potential selective targets for antiviral development.

Unlike many RNA viruses that encode RNA-dependent RNA polymerases (RdRps), RPO132 is a subunit of a DNA-directed RNA polymerase that transcribes DNA templates into mRNA. This fundamental distinction reflects the different genomic strategies: orthopoxviruses possess double-stranded DNA genomes while many other viruses utilize RNA genomes. The structural organization of the orthopoxvirus RNA polymerase complex, including RPO132, more closely resembles the multi-subunit RNA polymerases of eukaryotes than the single-chain RdRps of RNA viruses.

Functionally, RPO132 and the orthopoxvirus transcription system possess several unique features:

• Complete cytoplasmic localization of transcription, unlike herpesviruses which utilize nuclear transcription machinery

• Early gene transcription occurs within viral cores, while intermediate and late transcription occurs in viral factories

• Orthopoxvirus RNA polymerase requires virus-specific transcription factors that have no counterparts in other virus families

• The presence of a virus-encoded capping enzyme system that processes viral transcripts differently than cellular mechanisms

Compared to RNA polymerases of other large DNA viruses like herpesviruses, orthopoxvirus RNA polymerase functions independently of host nuclear transcription machinery. Herpesviruses largely utilize host RNA polymerase II with viral regulatory factors, while orthopoxviruses encode all components of their transcriptional machinery, including RPO132.

These distinctive features of RPO132 and orthopoxvirus transcription provide potential advantages for antiviral targeting. The unique aspects of this system could allow for highly selective inhibition without cross-reactivity against host polymerases or polymerases from unrelated viruses, potentially reducing off-target effects in therapeutic applications.

What are the primary challenges in producing functional recombinant RPO132?

Producing functional recombinant RPO132 presents several significant challenges that researchers must address to obtain biologically relevant results. These challenges stem from the protein's size, complexity, and requirements for proper folding and activity.

The substantial molecular weight of RPO132 (132 kDa) creates inherent expression difficulties in all host systems . Large proteins are often prone to premature termination during translation, resulting in truncated products that contaminate purification preparations. Additionally, the complete protein may be expressed at lower levels than smaller proteins due to the metabolic burden on host cells and the increased probability of errors during extended translation.

Expression system selection presents a critical trade-off between yield and functionality. While E. coli systems can produce higher quantities of recombinant protein with shorter production timelines , the prokaryotic cellular environment often fails to support proper folding of complex eukaryotic viral proteins like RPO132. This frequently results in insoluble inclusion bodies requiring denaturation and refolding procedures that may not fully restore native structure and function. Conversely, eukaryotic expression systems (insect or mammalian cells) better support proper folding but typically yield less protein with longer production timelines .

Functional assessment presents another significant challenge because RPO132 normally functions as part of a multi-subunit complex. In isolation, RPO132 may not demonstrate its native activity, necessitating co-expression or reconstitution with other viral proteins to form a functional RNA polymerase complex. This requirement substantially increases the complexity of experimental design and interpretation.

Additional technical challenges include:

• Protein instability and aggregation during purification and storage

• Batch-to-batch variation in activity and purity

• Difficulties in establishing reliable activity assays that reflect in vivo function

• Limited availability of structural information to guide construct design

• Potential toxicity to expression hosts due to interference with cellular transcription

To address these challenges, researchers often employ strategies such as expressing selected domains rather than the full-length protein, optimizing codon usage for the expression host, using fusion tags to enhance solubility, and developing specialized purification protocols that maintain protein stability. Despite achieving greater than 85% purity through these approaches , obtaining consistently active preparations remains a significant challenge.

How do mutations in RPO132 impact viral fitness and potential zoonotic transmission?

Mutations in RPO132 can significantly impact viral fitness and potential zoonotic transmission through effects on transcriptional efficiency, adaptation to host cellular environments, and interactions with the immune system. Understanding these relationships requires sophisticated evolutionary and functional analyses.

Comparative genomic analyses examining the dN/dS ratio (the ratio of non-synonymous to synonymous substitutions) in RPO132 sequences across orthopoxviruses can identify regions under positive selection that may contribute to host adaptation . These adaptively evolving sites potentially play roles in host range determination and zoonotic potential by optimizing viral transcription in different cellular environments.

The implications for zoonotic transmission are complex:

• RPO132 mutations that enhance viral replication efficiency in new host species could facilitate cross-species transmission

• Adaptive changes that optimize interactions with host-specific transcription factors might expand host range

• Mutations affecting epitopes recognized by cross-protective immunity from vaccination or previous orthopoxvirus exposure could impact susceptibility of host populations

Cowpox virus already demonstrates the broadest host range among orthopoxviruses , suggesting its RPO132 and other essential proteins function efficiently across diverse mammalian cellular environments. This broad functionality may contribute to the virus's emergence as a zoonotic concern, particularly as population immunity from smallpox vaccination wanes . A seroprevalence study in Finland showed that individuals over 50 years of age had orthopoxvirus antibodies, with decreasing seroprevalence in younger age groups, reflecting the cessation of smallpox vaccination and potentially increased susceptibility to infection with cowpox virus .

Experimental approaches to investigate these relationships include:

• Site-directed mutagenesis of RPO132 followed by viral fitness assays in different host cell types

• Chimeric virus construction replacing RPO132 between orthopoxvirus species

• Deep mutational scanning to comprehensively assess the fitness effects of all possible mutations

• Structural biology approaches to understand the molecular basis of host-specific adaptations

These studies have significant implications for predicting and potentially preventing future zoonotic orthopoxvirus outbreaks as human immunity to these viruses continues to decline in the post-smallpox vaccination era.

What cutting-edge techniques are advancing structural studies of RPO132?

Cutting-edge structural biology techniques are revolutionizing our understanding of RPO132 structure-function relationships, providing unprecedented insights into its molecular mechanisms and interactions. These advanced approaches overcome historical challenges in studying large, complex viral proteins and enable rational design of inhibitors and vaccines.

Cryo-electron microscopy (cryo-EM) has emerged as a transformative technique for studying large protein complexes like the viral RNA polymerase that includes RPO132. Unlike X-ray crystallography, cryo-EM does not require protein crystallization, circumventing a major bottleneck in structural studies of challenging proteins. Single-particle cryo-EM can now achieve near-atomic resolution (2-3 Å), revealing detailed structural features of RPO132 within the context of the complete polymerase complex. This technique is particularly valuable for visualizing different functional states of the enzyme during the transcription cycle.

Integrative structural biology approaches combine multiple experimental techniques with computational modeling to generate comprehensive structural models. For RPO132, this might include:

AlphaFold2 and other AI-based protein structure prediction methods have dramatically improved our ability to model protein structures. For RPO132, these computational approaches can generate high-confidence models of domains or regions lacking experimental structural data, complementing experimentally determined structures. The integration of predicted models with experimental data provides more complete structural information to guide mechanistic studies and inhibitor design.

Advanced techniques for studying protein-protein and protein-nucleic acid interactions relevant to RPO132 function include:

• Surface plasmon resonance (SPR) and bio-layer interferometry (BLI) for quantitative binding kinetics

• Native mass spectrometry to characterize intact complexes and their stoichiometry

• Fluorescence resonance energy transfer (FRET) to monitor dynamic interactions in real-time

• Single-molecule techniques to observe individual molecular events during transcription

Time-resolved structural methods like time-resolved cryo-EM and X-ray free-electron laser (XFEL) crystallography can capture RPO132 in transient conformational states during the transcription cycle. These approaches provide dynamic information beyond static structures, revealing how the protein changes conformation during catalysis.

These advanced structural biology techniques, often applied to recombinant RPO132 expressed in eukaryotic systems to ensure proper folding and post-translational modifications , are transforming our understanding of orthopoxvirus transcription machinery at the molecular level. The resulting structural insights facilitate structure-based drug design targeting RPO132 and inform the development of next-generation vaccines and therapeutics against emerging orthopoxvirus threats.

How does cowpox virus RPO132 compare to analogous proteins in related orthopoxviruses?

Sequence analysis of RPO132 across orthopoxviruses reveals high conservation levels, typically exceeding 95% amino acid identity among members of the genus. This conservation reflects strong evolutionary constraints on this essential protein. The region containing the RPO132 gene, spanning from the DNA polymerase gene to the RNA polymerase subunit gene, is frequently used for phylogenetic analysis precisely because of its consistency and reliability for establishing evolutionary relationships .

Comparative analysis of RPO132 from different orthopoxviruses shows:

While sequence conservation is high in catalytic domains, more variation occurs in regions that may interact with host-specific factors. These variations potentially contribute to differences in host range and pathogenicity between orthopoxvirus species. For example, cowpox virus infects the widest range of host species among orthopoxviruses , suggesting its RPO132 and other viral proteins function efficiently across diverse cellular environments.

Functionally, the RNA polymerase complex containing RPO132 operates similarly across orthopoxviruses, with virus-specific variations in transcriptional efficiency possibly contributing to differences in replication kinetics and host range. The high conservation of this enzyme makes it a potential target for broad-spectrum antivirals that could be effective against multiple orthopoxviruses, including both established and emerging threats .

Structurally, the functional domains of RPO132 are preserved across orthopoxviruses, including catalytic sites for RNA synthesis and regulatory domains that interact with other components of the transcription complex. These structural similarities facilitate comparative studies and the potential application of insights gained from one orthopoxvirus to others within the genus.

What insights does RPO132 provide into orthopoxvirus pathogenicity and virulence mechanisms?

RPO132 contributes to our understanding of orthopoxvirus pathogenicity and virulence mechanisms through its essential role in viral gene expression and its interactions with host cellular systems. While not traditionally classified as a virulence factor, this essential component of the viral transcriptional machinery influences multiple aspects of viral pathogenesis.

The primary contribution of RPO132 to pathogenicity stems from its central role in viral gene expression. As a key component of the viral RNA polymerase complex, RPO132 is required for transcription of all viral genes, including those encoding virulence factors that directly modulate host immune responses. Efficient transcription mediated by RPO132 ensures robust expression of viral immunomodulatory proteins that suppress host cell pro-inflammatory cytokines, reduce antiviral activity, and inhibit antigen presentation on cell surfaces, thereby preventing activation of T-cells . These mechanisms collectively contribute to viral evasion of host immunity and successful establishment of infection.

The temporal regulation of gene expression during orthopoxvirus infection depends on promoter recognition and activity of the viral RNA polymerase complex containing RPO132. This staged expression pattern—early, intermediate, and late genes—is critical for coordinated viral replication and assembly. Any alterations to RPO132 that affect promoter recognition or transcriptional efficiency could potentially impact the timing and levels of virulence factor expression, thereby modulating pathogenicity.

Differences in orthopoxvirus pathogenicity correlate with their host range and virulence patterns. For example:

• Cowpox virus has the broadest host range and variable pathogenicity, causing self-limiting disease in immunocompetent humans but potentially severe disease in immunocompromised individuals

• Variola virus (smallpox) has a restricted host range (humans only) but historically caused devastating disease with high mortality

• Vaccinia virus typically causes mild disease while providing cross-protection against other orthopoxviruses

The role of RPO132 in these differing pathogenicity profiles likely involves subtle variations in transcriptional efficiency within specific host cell environments and potential interactions with host factors that differ between species. While the core enzymatic function of RPO132 is conserved, species-specific adaptations may optimize viral gene expression in preferred hosts.

Furthermore, the cytoplasmic location of orthopoxvirus transcription, mediated by RPO132 and other viral factors, represents a distinctive aspect of pathogenesis strategy. By conducting transcription in cytoplasmic viral factories rather than in the nucleus, orthopoxviruses can potentially evade certain nuclear-associated innate immune responses while creating specialized compartments for efficient viral replication.

What are the implications of cowpox virus emergence as a zoonotic threat for RPO132 research?

The emergence of cowpox virus as a zoonotic threat has significant implications for RPO132 research, creating new imperatives for understanding viral transcription mechanisms and developing countermeasures. As human immunity to orthopoxviruses wanes in the post-smallpox vaccination era, research on essential viral proteins like RPO132 gains increased public health relevance.

Cowpox virus, once primarily an occupational hazard for dairy workers, is now recognized as an emerging zoonotic threat with expanding clinical significance . Human cowpox infections typically cause self-limited disease in immunocompetent individuals but can become severe in immunocompromised patients and those with skin conditions like eczema, particularly children . Two notable outbreaks of human cowpox were traced to infected pet rats in Germany and France between 2008-2011, comprising approximately 40 cases . These incidents highlight the changing epidemiology of cowpox and its potential for unexpected transmission routes.

The cessation of routine smallpox vaccination has created population vulnerability to orthopoxvirus infections due to waning cross-protective immunity. A seroprevalence study in Finland demonstrated that individuals over 50 years old had orthopoxvirus antibodies, with decreasing prevalence in younger age groups . This age-structured immunity pattern suggests increasing population susceptibility to cowpox and other orthopoxviruses, elevating the importance of research on viral mechanisms and countermeasures.

For RPO132 research specifically, these developments have several implications:

• Increased priority for developing antiviral compounds targeting viral transcription machinery, including RPO132

• Enhanced relevance of comparative studies examining RPO132 sequence and functional variations between cowpox strains from different geographic regions and host species

• Greater importance of understanding RPO132 interactions with host factors that may influence zoonotic potential

• Renewed interest in characterizing immune responses targeting RPO132 as part of comprehensive protective immunity against orthopoxviruses

The broad host range of cowpox virus—which infects the widest range of host species among orthopoxviruses —suggests its essential proteins like RPO132 function effectively across diverse mammalian cellular environments. This adaptability likely contributes to its zoonotic potential and provides a scientific rationale for studying how RPO132 maintains functionality across different host species.

Developing recombinant RPO132 as a research tool facilitates the screening and development of antiviral compounds that could address the public health concerns raised by emerging cowpox infections . High-throughput screening platforms using recombinant RPO132 can identify inhibitors of viral transcription that might serve as broad-spectrum orthopoxvirus antivirals. Additionally, structure-based drug design targeting conserved features of RPO132 could yield therapeutics effective against multiple orthopoxvirus species.