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Poxviruses represent evolutionary successful infectious agents. As a family, poxviruses can infect a wide variety of species including humans, fish, and insects. While many other viruses are species-specific, an individual poxvirus species is often capable of infecting diverse hosts and cell types. For example, the prototypical poxvirus, vaccinia, is well known to infect numerous human cell types but can also infect cells from divergent hosts like frog neurons. Notably, poxvirus infections result in both detrimental human and animal diseases. The most infamous disease linked to a poxvirus is smallpox caused by variola virus. Poxviruses are large double-stranded DNA viruses, which uniquely replicate in the cytoplasm of cells. The model poxvirus genome encodes ~200 nonoverlapping protein-coding open reading frames (ORFs). Poxvirus gene products impact various biological processes like the production of virus particles, the host range of infectivity, and disease pathogenesis. In addition, poxviruses and their gene products have biomedical application with several species commonly engineered for use as vaccines and oncolytic virotherapy. Nevertheless, we still have an incomplete understanding of the functions associated with many poxvirus genes. In this chapter, we outline evolutionary insights that can complement ongoing studies of poxvirus gene functions and biology, which may serve to elucidate new molecular activities linked to this biomedically relevant class of viruses., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Vaccinia virus (VACV) demonstrates a wide host range, which is determined by its host range genes including the E3L and K3L. The E3L and K3L deletion mutant VACV (VACVΔE3ΔK3) is only able to replicate in cells defective in PKR and RNase L activity. Interestingly, by expressing a K3 ortholog from another poxvirus, the host range of the VACVΔE3ΔK3 can be fine-tuned to specific host species. Accordingly, we developed a novel method for construction of recombinant VACV using the poxvirus K3 protein as a selection marker. This protocol has the advantage of being fast, cheap, and efficient. More importantly, the recombinant VACV constructed using this protocol is highly attenuated due to the absence of the virulent gene E3L., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The antiviral protein kinase R (PKR) is activated by viral double-stranded RNA and phosphorylates translation initiation factor eIF2α, thereby inhibiting translation and virus replication. Most poxviruses contain two PKR inhibitors, called E3 and K3 in vaccinia virus (VACV), which are determinants of viral host range. The prevailing model for E3 function is that it inhibits PKR through the non-specific sequestration of double-stranded (ds) RNA. Our data revealed that Syrian hamster PKR was resistant to E3, which is at odds with the sequestration model. However, Syrian hamster PKR was still sensitive to K3 inhibition. In contrast, Armenian hamster PKR showed opposite sensitivities, being sensitive to E3 and resistant to K3 inhibition. Mutational analyses of hamster PKRs showed that sensitivity to E3 inhibition was largely determined by the region linking the dsRNA-binding domains and the kinase domain of PKR, whereas two amino acid residues in the kinase domain (helix αG) determined sensitivity to K3. The expression of PKRs in congenic cells showed that Syrian hamster PKR containing the two Armenian hamster PKR residues in helix αG was resistant to wild-type VACV infection and that cells expressing either hamster PKR recapitulated the phenotypes observed in species-derived cell lines. The observed resistance of Syrian hamster PKR to E3 explains its host range function and challenges the paradigm that dsRNA-binding PKR inhibitors mainly act by the sequestration of dsRNA.IMPORTANCEThe molecular mechanisms that govern the host range of viruses are incompletely understood. We show that the host range functions of E3 and K3, two host range factors from vaccinia virus, are a result of species-specific interactions with the antiviral protein kinase R (PKR) and that PKR from closely related species displayed dramatic differences in their sensitivities to these viral inhibitors. The current model for E3-mediated PKR inhibition is that E3 non-specifically sequesters double-stranded (ds) RNA to prevent PKR activation. This model does not predict species-specific sensitivity to E3; therefore, our data suggest that the current model is incomplete and that dsRNA sequestration is not the primary mechanism for E3 activity., Competing Interests: The authors declare no conflict of interest.

Ankyrin repeat is a 33-amino acid motif commonly observed in eukaryotes and, to a lesser extent, in prokaryotes and archaea and rarely in viruses. This motif plays a crucial role in regulating various cellular processes like the cell cycle, transcription, cell signaling, and inflammatory responses through interactions between proteins. Poxviruses exhibit a distinctive feature of containing multiple ankyrin repeat proteins within their genomes. All the genera of poxviruses possess these proteins except molluscipox virus, crocodylidpox virus, and red squirrel poxvirus. An intriguing characteristic has generated notable interest in studying the functions of these proteins within poxvirus biology. Within poxviruses, ankyrin repeat proteins exhibit a distinct configuration, featuring ankyrin repeats in the N-terminal region and a cellular F-box homolog in the C-terminal region, which enables interactions with the cellular Skp, Cullin, F-box containing ubiquitin ligase complex. Through the examination of experimental evidences and discussions from current literature, this review elucidates the organization and role of ankyrin repeat proteins in poxviruses. Various research studies have highlighted the significant importance of these proteins in poxviral pathogenesis and, acting as factors that enhance virulence. Consequently, they represent viable targets for developing genetically altered viruses with decreased virulence, thus displaying potential as candidates for vaccines and antiviral therapeutic development contributing to safer and more effective strategies against poxviral infections., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Animal and bacterial cells sense and defend against viral infections using evolutionarily conserved antiviral signaling pathways. Here, we show that viruses overcome host signaling using mechanisms of immune evasion that are directly shared across the eukaryotic and prokaryotic kingdoms of life. Structures of animal poxvirus proteins that inhibit host cGAS-STING signaling demonstrate architectural and catalytic active-site homology shared with bacteriophage Acb1 proteins, which inactivate CBASS anti-phage defense. In bacteria, phage Acb1 proteins are viral enzymes that degrade host cyclic nucleotide immune signals. Structural comparisons of poxvirus protein-2'3'-cGAMP and phage Acb1-3'3'-cGAMP complexes reveal a universal mechanism of host nucleotide immune signal degradation and explain kingdom-specific additions that enable viral adaptation. Chimeric bacteriophages confirm that animal poxvirus proteins are sufficient to evade immune signaling in bacteria. Our findings identify a mechanism of immune evasion conserved between animal and bacterial viruses and define shared rules that explain host-virus interactions across multiple kingdoms of life., Competing Interests: Declaration of interests J.A.D. is a co-founder of Caribou Biosciences, Editas Medicine, Scribe Therapeutics, Intellia Therapeutics, and Mammoth Biosciences. J.A.D. is a scientific advisory board member of Vertex, Caribou Biosciences, Intellia Therapeutics, Scribe Therapeutics, Mammoth Biosciences, Algen Biotechnologies, Felix Biosciences, The Column Group, and Inari Agriculture. J.A.D. is Chief Science Advisor to Sixth Street, a Director at Johnson & Johnson, and Altos and Tempus and has research projects sponsored by Apple Tree Partners and Roche., (Copyright © 2024 Elsevier Inc. All rights reserved.)

BEND family transcription factors directly interact with DNA through BEN domains and have been found across metazoan species. Interestingly, certain insect and mammalian viruses have also hijacked Bend genes into their genome. However, the phylogenetic classification and evolution of these viral BEN domains remain unclear. Building on our previous finding that in silico method accurately determine the 3D model of BEN domains, we used AlphaFold2 to predict the tertiary structures of poxviral BEN domains for comprehensive homologous comparison. We revealed that the majority of poxviral BEN modules exhibit characteristics of type II BEN. Additionally, electrostatic surface potential analysis found various poxviral BEN domains, including the first BEN of OPG067 in Orthopoxvirus, the third BEN of OPG067 in Yatapoxvirus and the third BEN of MC036R in MCV, have positively charged protein surfaces, indicating a structural basis for DNA loading. Notably, MC036R shares structural resemblance with human BEND3, as they both contain four BEN domains and an intrinsically disordered region. In summary, our discoveries provide deeper insights into the functional roles of BEN proteins within poxviruses., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

In the age of COVID, nucleic acid vaccines have garnered much attention, at least in part, because of the simplicity of construction, production, and flexibility to adjust and adapt to an evolving outbreak. Orthopoxviruses remain a threat on multiple fronts, especially as emerging zoonoses. In response, we developed a DNA vaccine, termed 4pox, that protected nonhuman primates against monkeypox virus (MPXV)-induced severe disease. Here, we examined the protective efficacy of the 4pox DNA vaccine delivered by intramuscular (i.m.) electroporation (EP) in rabbits challenged with aerosolized rabbitpox virus (RPXV), a model that recapitulates the respiratory route of exposure and low dose associated with natural smallpox exposure in humans. We found that 4pox-vaccinated rabbits developed immunogen-specific antibodies, including neutralizing antibodies, and did not develop any clinical disease, indicating protection against aerosolized RPXV. In contrast, unvaccinated animals developed significant signs of disease, including lesions, and were euthanized. These findings demonstrate that an unformulated, nonadjuvanted DNA vaccine delivered i.m. can protect against an aerosol exposure. IMPORTANCE The eradication of smallpox and subsequent cessation of vaccination have left a majority of the population susceptible to variola virus or other emerging poxviruses. This is exemplified by human monkeypox, as evidenced by the increase in reported endemic and imported cases over the past decades. Therefore, a malleable vaccine technology that can be mass produced and does not require complex conditions for distribution and storage is sought. Herein, we show that a DNA vaccine, in the absence of a specialized formulation or adjuvant, can protect against a lethal aerosol insult of rabbitpox virus.

The morphogenesis of vaccinia virus (VACV, family Poxviridae ), the smallpox vaccine, is a complex process involving multiple distinct cellular membranes and resulting in multiple different forms of infectious virion. Efficient release of enveloped virions, which promote systemic spread of infection within hosts, requires the VACV protein E2 but the molecular basis of E2 function remains unclear and E2 lacks sequence homology to any well-characterised family of proteins. We solved the crystal structure of VACV E2 to 2.3 Å resolution, revealing that it comprises two domains with novel folds: an N-terminal annular (ring) domain and a C-terminal globular (head) domain. The C-terminal head domain displays weak structural homology with cellular (pseudo)kinases but lacks conserved surface residues or kinase features, suggesting that it is not enzymatically active, and possesses a large surface basic patch that might interact with phosphoinositide lipid headgroups. Recent deep learning methods have revolutionised our ability to predict the three-dimensional structures of proteins from primary sequence alone. VACV E2 is an exemplar 'difficult' viral protein target for structure prediction, being comprised of multiple novel domains and lacking sequence homologues outside Poxviridae . AlphaFold2 nonetheless succeeds in predicting the structures of the head and ring domains with high and moderate accuracy, respectively, allowing accurate inference of multiple structural properties. The advent of highly accurate virus structure prediction marks a step-change in structural virology and beckons a new era of structurally-informed molecular virology.

Poxviruses have evolved a variety of innate immunity evasion mechanisms, some of which involve poxvirus-encoded E3 ubiquitin ligases and adaptor proteins. Based on their functional domains and ubiquitin transfer mechanisms, these poxvirus-encoded E3 ubiquitin ligases and adaptor proteins can be divided into five categories: PRANC, ANK/BC, BBK, P28/RING, and MARCH proteins. Although the substrates of many poxvirus E3 ubiquitin ligases remain to be discovered, most of the identified substrates are components of the innate immune system. In this review, we discuss the current research progress on poxvirus-encoded E3 ubiquitin ligases and adaptor proteins to provide mechanistic insights into the interplay between these viruses and their hosts., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Cui, Zhang and Zhang.)

Orf virus (ORFV) is the type species of the Parapoxvirus genus of the Poxviridae family. Genetic and functional studies have revealed ORFV has multiple immunomodulatory genes that manipulate innate immune responses, during the early stage of infection. ORF116 is a novel gene of ORFV with hitherto unknown function. Characterization of an ORF116 deletion mutant showed that it replicated in primary lamb testis cells with reduced levels compared to the wild-type and produced a smaller plaque phenotype. ORF116 was shown to be expressed prior to DNA replication. The potential function of ORF116 was investigated by gene-expression microarray analysis in HeLa cells infected with wild-type ORFV or the ORF116 deletion mutant. The analysis of differential cellular gene expression revealed a number of interferon-stimulated genes (ISGs) differentially expressed at either 4 or 6 h post infection. IFI44 showed the greatest differential expression (4.17-fold) between wild-type and knockout virus. Other ISGs that were upregulated in the knockout included RIG-I, IFIT2, MDA5, OAS1, OASL, DDX60, ISG20 and IFIT1 and in addition the inflammatory cytokine IL-8 . These findings were validated by infecting HeLa cells with an ORF116 revertant recombinant virus and analysis of transcript expression by quantitative real time-PCR (qRT-PCR). These observations suggested a role for the ORFV gene ORF116 in modulating the IFN response and inflammatory cytokines. This study represents the first functional analysis of ORF116 .

Vaccinia virus produces two types of virions known as single-membraned intracellular mature virus (MV) and double-membraned extracellular enveloped virus (EV). EV production peaks earlier when initial MVs are further wrapped and secreted to spread infection within the host. However, late during infection, MVs accumulate intracellularly and become important for host-to-host transmission. The process that regulates this switch remains elusive and is thought to be influenced by host factors. Here, we examined the hypothesis that EV and MV production are regulated by the virus through expression of F13 and the MV-specific protein A26. By switching the promoters and altering the expression kinetics of F13 and A26, we demonstrate that A26 expression downregulates EV production and plaque size, thus limiting viral spread. This process correlates with A26 association with the MV surface protein A27 and exclusion of F13, thus reducing EV titers. Thus, MV maturation is controlled by the abundance of the viral A26 protein, independently of other factors, and is rate limiting for EV production. The A26 gene is conserved within vertebrate poxviruses but is strikingly lost in poxviruses known to be transmitted exclusively by biting arthropods. A26-mediated virus maturation thus has the appearance to be an ancient evolutionary adaptation to enhance transmission of poxviruses that has subsequently been lost from vector-adapted species, for which it may serve as a genetic signature. The existence of virus-regulated mechanisms to produce virions adapted to fulfill different functions represents a novel level of complexity in mammalian viruses with major impacts on evolution, adaptation, and transmission. IMPORTANCE Chordopoxviruses are mammalian viruses that uniquely produce a first type of virion adapted to spread within the host and a second type that enhances transmission between hosts, which can take place by multiple ways, including direct contact, respiratory droplets, oral/fecal routes, or via vectors. Both virion types are important to balance intrahost dissemination and interhost transmission, so virus maturation pathways must be tightly controlled. Here, we provide evidence that the abundance and kinetics of expression of the viral protein A26 regulates this process by preventing formation of the first form and shifting maturation toward the second form. A26 is expressed late after the initial wave of progeny virions is produced, so sufficient viral dissemination is ensured, and A26 provides virions with enhanced environmental stability. Conservation of A26 in all vertebrate poxviruses, but not in those transmitted exclusively via biting arthropods, reveals the importance of A26-controlled virus maturation for transmission routes involving environmental exposure.

Modified vaccinia virus Ankara (MVA) was derived by repeated passaging in chick fibroblasts, during which deletions and mutations rendered the virus unable to replicate in most mammalian cells. Marker rescue experiments demonstrated that the host range defect could be overcome by replacing DNA that had been deleted from near the left end of the genome. One virus isolate, however, recovered the ability to replicate in monkey BS-C-1 cells but not human cells without added DNA, suggesting that it arose from a spontaneous mutation. Here, we showed that variants with enhanced ability to replicate in BS-C-1 cells could be isolated by blind passaging of MVA and that in each there was a point mutation leading to an amino acid substitution in the D10 decapping enzyme. The sufficiency of these single mutations to enhance host range was confirmed by constructing recombinant viruses. The D10 mutations occurred at N- or C-terminal locations distal to the active site, suggesting an indirect effect on decapping or on another previously unknown role of D10. Although increased amounts of viral mRNA and proteins were found in BS-C-1 cells infected with the mutants compared to those with parental MVA, the increases were much less than the 1- to 2-log-higher virus yields. Nevertheless, a contributing role for diminished decapping in overcoming the host range defect was consistent with increased replication and viral protein synthesis in BS-C-1 cells infected with an MVA engineered to have active-site mutations that abrogate decapping activity entirely. Optimal decapping may vary depending on the biological context. IMPORTANCE Modified vaccinia virus Ankara (MVA) is an attenuated virus that is approved as a smallpox vaccine and is in clinical trials as a vector for other pathogens. The safety of MVA is due in large part to its inability to replicate in mammalian cells. Although host range restriction is considered a stable feature of the virus, we describe the occurrence of spontaneous mutations in MVA that increase replication considerably in monkey BS-C-1 cells but only slightly in human cells. The mutants contain single nucleotide changes that lead to amino acid substitutions in one of the two decapping enzymes. Although the spontaneous mutations are distant from the decapping enzyme active site, engineered active-site mutations also increased virus replication in BS-C-1 cells. The effects of these mutations on the immunogenicity of MVA vectors remain to be determined.

It is well established that memory CD8 T cells protect susceptible strains of mice from mousepox, a lethal viral disease caused by ectromelia virus (ECTV), the murine counterpart to human variola virus. While mRNA vaccines induce protective antibody (Ab) responses, it is unknown whether they also induce protective memory CD8 T cells. We now show that immunization with different doses of unmodified or N(1)-methylpseudouridine-modified mRNA (modified mRNA) in lipid nanoparticles (LNP) encoding the ECTV gene EVM158 induced similarly strong CD8 T cell responses to the epitope TSYKFESV, albeit unmodified mRNA-LNP had adverse effects at the inoculation site. A single immunization with 10 μg modified mRNA-LNP protected most susceptible mice from mousepox, and booster vaccination increased the memory CD8 T cell pool, providing full protection. Moreover, modified mRNA-LNP encoding TSYKFESV appended to green fluorescent protein (GFP) protected against wild-type ECTV infection while lymphocytic choriomeningitis virus glycoprotein (GP) modified mRNA-LNP protected against ECTV expressing GP epitopes. Thus, modified mRNA-LNP can be used to create protective CD8 T cell-based vaccines against viral infections., (Copyright © 2021 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Necroptosis mediated by Z-nucleic-acid-binding protein (ZBP)1 (also called DAI or DLM1) contributes to innate host defense against viruses by triggering cell death to eliminate infected cells. During infection, vaccinia virus (VACV) protein E3 prevents death signaling by competing for Z-form RNA through an N-terminal Zα domain. In the absence of this E3 domain, Z-form RNA accumulates during the early phase of VACV infection, triggering ZBP1 to recruit receptor interacting protein kinase (RIPK)3 and execute necroptosis. The C-terminal E3 double-strand RNA-binding domain must be retained to observe accumulation of Z-form RNA and induction of necroptosis. Substitutions of Zα from either ZBP1 or the RNA-editing enzyme double-stranded RNA adenosine deaminase (ADAR)1 yields fully functional E3 capable of suppressing virus-induced necroptosis. Overall, our evidence reveals the importance of Z-form RNA generated during VACV infection as a pathogen-associated molecular pattern (PAMP) unleashing ZBP1/RIPK3/MLKL-dependent necroptosis unless suppressed by viral E3., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Poxviruses are enveloped viruses with a linear, double-stranded DNA genome. Viral DNA synthesis is achieved by a functional DNA polymerase holoenzyme composed of three essential proteins. For vaccinia virus (VACV) these are E9, the catalytic subunit, a family B DNA polymerase, and the heterodimeric processivity factor formed by D4 and A20. The A20 protein links D4 to the catalytic subunit. High-resolution structures have been obtained for the VACV D4 protein in complex with an N-terminal fragment of A20 as well as for E9. In addition, biochemical studies provided evidence that a poxvirus-specific insertion (insert 3) in E9 interacts with the C-terminal residues of A20. Here, we provide solution structures of two different VACV A20 C-terminal constructs containing residues 304-426, fused at their C-terminus to either a BAP (Biotin Acceptor Peptide)-tag or a short peptide containing the helix of E9 insert 3. Together with results from titration studies, these structures shed light on the molecular interface between the catalytic subunit and the processivity factor component A20. The interface comprises hydrophobic residues conserved within the Chordopoxvirinae subfamily. Finally, we constructed a HADDOCK model of the VACV A20 304-426 -E9 complex, which is in excellent accordance with previous experimental data., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The vaccine strain against smallpox, vaccinia virus (VACV), is highly immunogenic yet causes relatively benign disease. These attributes are believed to be caused by gene loss in VACV. Using a targeted small interfering RNA (siRNA) screen, we identified a viral inhibitor found in cowpox virus (CPXV) and other orthopoxviruses that bound to the host SKP1-Cullin1-F-box (SCF) machinery and the essential necroptosis kinase receptor interacting protein kinase 3 (RIPK3). This "viral inducer of RIPK3 degradation" (vIRD) triggered ubiquitination and proteasome-mediated degradation of RIPK3 and inhibited necroptosis. In contrast to orthopoxviruses, the distantly related leporipoxvirus myxoma virus (MYXV), which infects RIPK3-deficient hosts, lacks a functional vIRD. Introduction of vIRD into VACV, which encodes a truncated and defective vIRD, enhanced viral replication in mice. Deletion of vIRD reduced CPXV-induced inflammation, viral replication, and mortality, which were reversed in RIPK3- and MLKL-deficient mice. Hence, vIRD-RIPK3 drives pathogen-host evolution and regulates virus-induced inflammation and pathogenesis., Competing Interests: Declaration of interests A U.S. provisional patent application (no. 62/848,255) has been filed for this study., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Viruses require a host for replication and survival and hence are subjected to host immunological pressures. The complement system, a crucial first response of the host immune system, is effective in targeting viruses and virus-infected cells, and boosting the antiviral innate and acquired immune responses. Thus, the system imposes a strong selection pressure on viruses. Consequently, viruses have evolved multiple countermeasures against host complement. A major mechanism employed by viruses to subvert the complement system is encoding proteins that target complement. Since viruses have limited genome size, most of these proteins are multifunctional in nature. In this review, we provide up to date information on the structure and complement regulatory functions of various viral proteins.

The poxviral B1 and B12 proteins are a homologous kinase-pseudokinase pair, which modulates a shared host pathway governing viral DNA replication and antiviral defense. While the molecular mechanisms involved are incompletely understood, B1 and B12 seem to intersect with signaling processes mediated by their cellular homologs termed the vaccinia-related kinases (VRKs). In this study, we expand upon our previous characterization of the B1-B12 signaling axis to gain insights into B12 function. We begin our studies by demonstrating that modulation of B12 repressive activity is a conserved function of B1 orthologs from divergent poxviruses. Next, we characterize the protein interactome of B12 using multiple cell lines and expression systems and discover that the cellular kinase VRK1 is a highly enriched B12 interactor. Using complementary VRK1 knockdown and overexpression assays, we first demonstrate that VRK1 is required for the rescue of a B1-deleted virus upon mutation of B12. Second, we find that VRK1 overexpression is sufficient to overcome repressive B12 activity during B1-deleted virus replication. Interestingly, we also evince that B12 interferes with the ability of VRK1 to phosphoinactivate the host defense protein BAF. Thus, B12 restricts vaccinia virus DNA accumulation in part by repressing the ability of VRK1 to inactivate BAF. Finally, these data establish that a B12-VRK1-BAF signaling axis forms during vaccinia virus infection and is modulated via kinases B1 and/or VRK2. These studies provide novel insights into the complex mechanisms that poxviruses use to hijack homologous cellular signaling pathways during infection. IMPORTANCE Viruses from diverse families encode both positive and negative regulators of viral replication. While their functions can sometimes be enigmatic, investigation of virus-encoded, negative regulators of viral replication has revealed fascinating aspects of virology. Studies of poxvirus-encoded genes have largely concentrated on positive regulators of their replication; however, examples of fitness gains attributed to poxvirus gene loss suggests that negative regulators of poxvirus replication also impact infection dynamics. This study focuses on the vaccinia B12 pseudokinase, a protein capable of inhibiting vaccinia DNA replication. Here, we elucidate the mechanisms by which B12 inhibits vaccinia DNA replication, demonstrating that B12 activates the antiviral protein BAF by inhibiting the activity of VRK1, a cellular modulator of BAF. Combined with previous data, these studies provide evidence that poxviruses govern their replication by employing both positive and negative regulators of viral replication., (Copyright © 2021 American Society for Microbiology.)

Premature apoptosis of cells is a strategy utilized by multicellular organisms to counter microbial threats. Orf virus (ORFV) is a large double-stranded DNA virus belonging to the poxviridae. ORFV encodes for an apoptosis inhibitory protein ORFV125 homologous to B-cell lymphoma 2 or Bcl-2 family proteins, which has been shown to inhibit host cell encoded pro-apoptotic Bcl-2 proteins. However, the structural basis of apoptosis inhibition by ORFV125 remains to be clarified. We show that ORFV125 is able to bind to a range of peptides spanning the BH3 motif of human pro-apoptotic Bcl-2 proteins including Bax, Bak, Puma and Hrk with modest to weak affinity. We then determined the crystal structures of ORFV125 alone as well as bound to the highest affinity ligand Bax BH3 motif. ORFV125 adopts a globular Bcl-2 fold comprising 7 α-helices, and utilizes the canonical Bcl-2 binding groove to engage pro-apoptotic host cell Bcl-2 proteins. In contrast with a previously predicted structure, ORFV125 adopts a domain-swapped dimeric topology, where the α1 helix from one protomer is swapped into a neighbouring unit. Furthermore, ORFV125 differs from the conserved architecture of the Bcl-2 binding groove and instead of α3 helix forming one of the binding groove walls, ORFV125 utilizes an extended α2 helix that comprises the equivalent region of helix α3. This results in a subtle variation of previously observed dimeric Bcl-2 architectures in other poxvirus and human encoded Bcl-2 proteins. Overall, our results provide a structural and mechanistic basis for orf virus-mediated inhibition of host cell apoptosis., (© 2020 The Author(s).)

Pseudoenzymes are proteins that are evolutionarily related to active enzymes, but lack relevant catalytic activity. As obligate intracellular pathogens, viruses complete their life cycle fully dependent on the cellular supplies of macromolecule and energy. Traditionally, studies of viral proteins sharing high homology with host counterparts reveal insightful mechanisms by which host signaling pathways are delicately regulated. Recent investigations into the action of cellular pseudoenzymes elucidate diverse molecular means how enzymes are differentially controlled under various physiological conditions, hinting to the potential that pathogens may exploit these regulatory modalities. To date, there have been three types of viral pseudoenzymes reported and our understanding concerning their mechanism of regulation is rudimentary at best. However, it is clear that viral pseudoenzymes are emerging with surprising functions in infection and immunity, and we are only at the beginning to understand this new group of enzyme regulators. In this review, we will summarize current knowledge in viral pseudoenzymes and provide a perspective for future research., (© 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Unlike RNA viruses, most DNA viruses replicate their genomes with high-fidelity polymerases that rarely make base substitution errors. Nevertheless, experimental evolution studies have revealed rapid acquisition of adaptive mutations during serial passage of attenuated vaccinia virus (VACV). One way in which adaptation can occur is by an accordion mechanism in which the gene copy number increases followed by base substitutions and, finally, contraction of the gene copy number. Here, we show rapid acquisition of multiple adaptive mutations mediated by a gene-inactivating frameshift mechanism during passage of an attenuated VACV. Attenuation had been achieved by exchanging the VACV A8R intermediate transcription factor gene with the myxoma virus ortholog. A total of seven mutations in six different genes occurred in three parallel passages of the attenuated virus. The most frequent mutations were single-nucleotide insertions or deletions within runs of five to seven As or Ts, although a deletion of 11 nucleotides also occurred, leading to frameshifts and premature stop codons. During 10 passage rounds, the attenuated VACV was replaced by the mutant viruses. At the end of the experiment, virtually all remaining viruses had one fixed mutation and one or more additional mutations. Although nucleotide substitutions in the transcription apparatus accounted for two low-frequency mutations, frameshifts in genes encoding protein components of the mature virion, namely, A26L, G6R, and A14.5L, achieved 74% to 98% fixation. The adaptive role of the mutations was confirmed by making recombinant VACV with A26L or G6R or both deleted, which increased virus replication levels and decreased particle/PFU ratios. IMPORTANCE Gene inactivation is considered to be an important driver of orthopoxvirus evolution. Whereas cowpox virus contains intact orthologs of genes present in each orthopoxvirus species, numerous genes are inactivated in all other members of the genus. Inactivation of additional genes can occur upon extensive passaging of orthopoxviruses in cell culture leading to attenuation in vivo , a strategy for making vaccines. Whether inactivation of multiple viral genes enhances replication in the host cells or has a neutral effect is unknown in most cases. Using an experimental evolution protocol involving serial passages of an attenuated vaccinia virus, rapid acquisition of inactivating frameshift mutations occurred. After only 10 passage rounds, the starting attenuated vaccinia virus was displaced by viruses with one fixed mutation and one or more additional mutations. The high frequency of multiple inactivating mutations during experimental evolution simulates their acquisition during normal evolution and extensive virus passaging to make vaccine strains., (Copyright © 2020 American Society for Microbiology.)

Programmed death of infected cells is used by multicellular organisms to counter viral infections. Sheeppox virus encodes for SPPV14, a potent inhibitor of Bcl-2-mediated apoptosis. We reveal the structural basis of apoptosis inhibition by determining crystal structures of SPPV14 bound to BH3 motifs of proapoptotic Bax and Hrk. The structures show that SPPV14 engages BH3 peptides using the canonical ligand-binding groove. Unexpectedly, Arg84 from SPPV14 forms an ionic interaction with the conserved Asp in the BH3 motif in a manner that replaces the canonical ionic interaction seen in almost all host Bcl-2:BH3 motif complexes. These results reveal the flexibility of virus-encoded Bcl-2 proteins to mimic key interactions from endogenous host signalling pathways to retain BH3 binding and prosurvival functionality., (© 2020 Federation of European Biochemical Societies.)

Protein kinase R plays a key role in innate antiviral immune responses of vertebrate animals. Most mammalian poxviruses encode two PKR antagonists, E3 (dsRNA binding) and K3 (eIF2α homolog) proteins. In this study, the role of K3 family proteins from poxviruses with distinct host tropisms in determining the virus host range was examined in a vaccinia E3L deletion mutant virus. It was found that K3 orthologs from the species-specific poxviruses (taterapox virus, sheeppox virus, myxoma virus, swinepox virus and yaba monkey tumor virus) restored the virus replication competency in cells derived from their natural hosts or related animal species. Further, it was found that the residues located in the helix insert region of the protein, K45 of vaccinia K3 and Y47 of the sheep poxvirus ortholog 011, are critical for the virus host species specificity. These observations demonstrate that poxvirus K3 proteins are major determinants of the virus host specificity., (Copyright © 2019 Elsevier Inc. All rights reserved.)

On-site translation of mRNAs provides an efficient means of subcellular protein localization. In eukaryotic cells, the transport of cellular mRNAs to membraneless sites usually occurs prior to translation and involves specific sequences known as zipcodes that interact with RNA binding and motor proteins. Poxviruses replicate in specialized cytoplasmic factory regions where DNA synthesis, transcription, translation, and virion assembly occur. Some poxviruses embed infectious virus particles outside of factories in membraneless protein bodies with liquid gel-like properties known as A-type inclusions (ATIs) that are comprised of numerous copies of the viral 150-kDa ATI protein. Here, we demonstrate by fluorescent in situ hybridization that these inclusions are decorated with ATI mRNA. On-site translation is supported by the localization of a translation initiation factor eIF4E and by ribosome-bound nascent chain ribopuromycylation. Nascent peptide-mediated anchoring of ribosome-mRNA translation complexes to the inclusions is suggested by release of the mRNA by puromycin, a peptide chain terminator. Following puromycin washout, relocalization of ATI mRNA at inclusions depends on RNA and protein synthesis but requires neither microtubules nor actin polymerization. Further studies show that the ATI mRNAs remain near the sites of transcription in the factory regions when stop codons are introduced near the N terminus of the ATI or large truncations are made at the N or C termini. Instead of using a zipcode, we propose that ATI mRNA localization is mediated by ribosome-bound nascent ATI polypeptides that interact with ATI protein in inclusions and thereby anchor the complex for multiple rounds of mRNA translation. IMPORTANCE Poxvirus genome replication, transcription, translation, and virion assembly occur at sites within the cytoplasm known as factories. Some poxviruses sequester infectious virions outside of the factories in inclusion bodies comprised of numerous copies of the 150-kDa ATI protein, which can provide stability and protection in the environment. We provide evidence that ATI mRNA is anchored by nascent peptides and translated at the inclusion sites rather than in virus factories. Association of ATI mRNA with inclusion bodies allows multiple rounds of local translation and prevents premature ATI protein aggregation and trapping of virions within the factory., (This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.)

IFITs are interferon-induced proteins that can bind 5'-triphosphate or ribose-unmethylated capped ends of mRNA to inhibit translation. Although some viruses avoid IFITs by synthesizing RNAs with eukaryotic-like caps, no viral proteins were known to antagonize IFITs. We show that the N- and C-terminal portions of C9, a protein required for vaccinia virus to resist the human type I interferon-induced state, bind IFITs and ubiquitin regulatory complexes, respectively. Together, the two C9 domains target IFITs for proteasomal degradation, thereby providing interferon resistance similar to that also achieved by knockout of IFITs. Furthermore, ectopic expression of C9 rescues the interferon sensitivity of a vaccinia virus mutant with an inactivated cap 1-specific ribose-methyltransferase that is otherwise unable to express early proteins. In contrast, the C9-deletion mutant expresses early proteins but is blocked by IFITs at the subsequent genome uncoating/replication step. Thus, poxviruses use mRNA cap methylation and proteosomal degradation to defeat multiple antiviral activities of IFITs., (Published by Elsevier Inc.)

Among the repertoire of immunoregulatory proteins encoded by myxoma virus, M013 is a viral homologue of the viral pyrin domain-only protein (vPOP) family. In myeloid cells, M013 protein has been shown to inhibit both the inflammasome and NF-κB signaling pathways by direct binding to ASC1 and NF-κB1, respectively. In this study, a three-dimensional homology model of the M013 pyrin domain (PYD) was built based on similarities to known PYD structures. A distinctive feature of the deduced surface electrostatic map of the M013 PYD is the presence of a negatively region consisting of numerous aspartate and glutamate residues in close proximity. Single-site mutations of aspartate and glutamate residues reveal their role in interactions with ASC-1. The biological significance of charge complementarity in the M013-ASC-1 interaction was further confirmed by functional assays of caspase-1 activation and subsequent secretion of cytokines. M013 also has a unique 33-residue C-terminal tail that follows the N-terminal PYD, and it is enriched in positively charged residues. Deletion of the tail of M013 significantly inhibited the interactions between M013 and NF-κB1, thus compromising the ability of the viral protein to suppress the secretion of pro-inflammatory cytokines. These results demonstrate that vPOP M013 exploits distinct structural motifs to regulate both the inflammasome and NF-κB pathways., (© 2019 Garg et al.)

BTB-Kelch proteins are substrate-specific adaptors for cullin-3 (Cul3) RING-box-based E3 ubiquitin ligases, mediating protein ubiquitylation for subsequent proteasomal degradation. Vaccinia virus encodes three BTB-Kelch proteins: A55, C2, and F3. Viruses lacking A55 or C2 have altered cytopathic effects in cultured cells and altered pathology in vivo Previous studies have shown that the ectromelia virus orthologue of A55 interacts with Cul3 in cells. We report that the N-terminal BTB-BACK (BB) domain of A55 binds directly to the Cul3 N-terminal domain (Cul3-NTD), forming a 2:2 complex in solution. We solved the structure of an A55BB/Cul3-NTD complex from anisotropic crystals diffracting to 2.3/3.7 Å resolution in the best/worst direction, revealing that the overall interaction and binding interface closely resemble the structures of cellular BTB/Cul3-NTD complexes, despite low sequence identity between A55 and cellular BTB domains. Surprisingly, despite this structural similarity, the affinity of Cul3-NTD for A55BB was stronger than for cellular BTB proteins. Glutamate substitution of the A55 residue Ile-48, adjacent to the canonical φ X (D/E) Cul3-binding motif, reduced affinity of A55BB for Cul3-NTD by at least 2 orders of magnitude. Moreover, Ile-48 and the φ X (D/E) motif are conserved in A55 orthologues from other poxviruses, but not in the vaccinia virus proteins C2 or F3. The high-affinity interaction between A55BB and Cul3-NTD suggests that, in addition to directing the Cul3-RING E3 ligase complex to degrade cellular/viral target proteins that are normally unaffected, A55 may also sequester Cul3 from cellular adaptor proteins, thereby protecting substrates of these cellular adaptors from ubiquitylation and degradation., (© 2019 Gao et al.)

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 (EC 50  = 8.4-19.7 μM) and blocked in vitro processive DNA synthesis (IC 50  = 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., (Copyright © 2018. Published by Elsevier B.V.)

Ankyrin repeat (ANK) domains are among the most abundant motifs in eukaryotic proteins. ANK proteins are rare amongst viruses, with the exception of poxviruses, which presumably acquired them from the host via horizontal gene transfer. The architecture of poxvirus ANK proteins is, however, different from that of their cellular counterparts, and this precludes a direct acquisition event. Here we combine bioinformatics analysis and quantitative proteomics to discover a new class of viral ANK proteins with a domain organization that relates to cellular ANK proteins. These noncanonical viral ANK proteins, termed ANK/BC, interact with host Cullin-2 via a C-terminal BC box resembling that of cellular Cullin-2 substrate adaptors such as the von Hippel-Lindau protein. Mutagenesis of the BC box-like sequence abrogates binding to Cullin-2, whereas fusion of this motif to an ANK-only protein confers Cullin-2 association. We demonstrated that these viral ANK/BC proteins are potent immunomodulatory proteins suppressing the activation of the proinflammatory transcription factors NF-κB and interferon (IFN)-responsive factor 3 (IRF-3) and the production of cytokines and chemokines, including interferon, and that association with Cullin-2 is required for optimal inhibitory activity. ANK/BC proteins exist in several orthopoxviruses and cluster into 2 closely related orthologue groups in a phylogenetic lineage that is separate from that of canonical ANK/F-box proteins. Given the existence of cellular proteins with similar architecture, viral ANK/BC proteins may be closely related to the original ANK gene acquired by an ancestral orthopoxvirus. These findings uncover a novel viral strategy to antagonize innate immunity and shed light on the origin of the poxviral ANK protein family. IMPORTANCE Viruses encode multiple proteins aimed at modulating cellular homeostasis and antagonizing the host antiviral response. Most of these genes were originally acquired from the host and subsequently adapted to benefit the virus. ANK proteins are common in eukaryotes but are unusual amongst viruses, with the exception of poxviruses, where they represent one of the largest protein families. We report here the existence of a new class of viral ANK proteins, termed ANK/BC, that provide new insights into the origin of poxvirus ANK proteins. ANK/BC proteins target the host E3 ubiquitin ligase Cullin-2 via a C-terminal BC box domain and are potent suppressors of the production of inflammatory cytokines, including interferon. The existence of cellular ANK proteins whose architecture is similar suggests the acquisition of a host ANK/BC gene by an ancestral orthopoxvirus and its subsequent duplication and adaptation to widen the repertoire of immune evasion strategies., (Copyright © 2018 Odon et al.)

Chemokines interact with glycosaminoglycans (GAGs) at the cellular surface and to specific cell-surface receptors to activate signaling pathways. The GAG interaction allows the formation of a chemotactic gradient of chemokine required for cell haptotaxis and chemokine oligomerization. Poxviruses encode secreted chemokine-binding proteins with no sequence similarity to their cellular counterparts to modulate the host immune system. The E163 protein from ectromelia virus, the causative agent of mousepox, binds chemokines through their GAG-binding domain. In addition, E163 interacts with GAGs to be anchored at the cell surface, but its ability to interfere with chemokine-GAG interactions has not been demonstrated. We report the identification of the GAG-binding regions in E163 and the generation of mutant forms deficient of GAG binding. Chemokine binding assays show that some of the E163 GAG-binding sites are also involved in the interaction with chemokines. By using recombinant GAG-binding mutant forms we demonstrate that E163 prevents the interaction of chemokines with cell-surface GAGs, providing mechanisms for the immunomodulatory activity of the viral chemokine-binding protein E163., (© 2018 Heidarieh and Alcamí.)

Host pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs) detect viruses and other pathogens, inducing production of cytokines that cause inflammation and mobilize cells to control infection. Vaccinia virus (VACV) encodes proteins that antagonize these host innate immune responses, and elucidating the mechanisms of action of these viral proteins helped shed light on PRR signaling mechanisms. The VACV virulence factor E3 is one of the most intensely studied VACV proteins and has multiple effects on host cells, many of which cannot be explained by the currently known cellular targets of E3. Here, we report that E3 expression in human monocytes alters TLR2- and TLR8-dependent cytokine induction, and particularly inhibits interleukin (IL)-6. Using MS, we identified DExD/H-box helicase 9 (DHX9) as an E3 target. Although DHX9 has previously been implicated as a PRR for sensing nucleic acid in dendritic cells, we found no role for DHX9 as a nucleic acid-sensing PRR in monocytes. Rather, DHX9 suppression in these cells phenocopied the effects of E3 expression on TLR2- and TLR8-dependent cytokine induction, in that DHX9 was required for all TLR8-dependent cytokines measured, and for TLR2-dependent IL-6. Furthermore, DHX9 also had a cell- and stimulus-independent role in IL-6 promoter induction. DHX9 enhanced NF-κB-dependent IL-6 promoter activation, which was directly antagonized by E3. These results indicate new roles for DHX9 in regulating cytokines in innate immunity and reveal that VACV E3 disrupts innate immune responses by targeting of DHX9., (© 2018 Dempsey et al.)

Poxviruses encode many proteins with the ability to regulate cellular signaling pathways. One such protein is the vaccinia virus innate immunity modulator E3. Multiple functions have been ascribed to E3, including modulating the cellular response to double-stranded RNA, inhibiting the NF-κB and IRF3 pathways, and dampening apoptosis. Apoptosis serves as a powerful defense against damaged and unwanted cells and is an effective defense against viral infection; many viruses therefore encode proteins that prevent or delay apoptosis. Here, we present data indicating that E3 does not directly inhibit the intrinsic apoptotic pathway; instead, it suppresses apoptosis indirectly by stimulating expression of the viral F1 apoptotic inhibitor. Our data demonstrate that E3 promotes F1 expression by blocking activation of the double-stranded RNA-activated protein kinase R (PKR). F1 mRNA is present in cells infected with E3-null virus, but the protein product does not detectably accumulate, suggesting a block at the translational level. We also show that two 3' coterminal transcripts span the F1 open reading frame (ORF), a situation previously described for the vaccinia virus mRNAs encoding the J3 and J4 proteins. One of these is a conventional monocistronic transcript of the F1L gene, while the other arises by read-through transcription from the upstream F2L gene and does not give rise to appreciable levels of F1 protein. IMPORTANCE Previous studies have shown that E3-deficient vaccinia virus triggers apoptosis of infected cells. Our study demonstrates that this proapoptotic phenotype stems, at least in part, from the failure of the mutant virus to produce adequate quantities of the viral F1 protein, which acts at the mitochondria to directly block apoptosis. Our data establish a regulatory link between the vaccinia virus proteins that suppress the innate response to double-stranded RNA and those that block the intrinsic apoptotic pathway., (Copyright © 2018 American Society for Microbiology.)

Receptor tyrosine kinases (RTKs) are essential components of cell communication pathways utilized from the embryonic to adult stages of life. These transmembrane receptors bind polypeptide ligands, such as growth factors, inducing signalling cascades that control cellular processes such as proliferation, survival, differentiation, motility and inflammation. Many viruses have acquired homologs of growth factors encoded by the hosts that they infect. Production of growth factors during infection allows viruses to exploit RTKs for entry and replication in cells, as well as for host and environmental dissemination. This review describes the genetic diversity amongst virus-derived growth factors and the mechanisms by which RTK exploitation enhances virus survival, then highlights how viral ligands can be used to further understanding of RTK signalling and function during embryogenesis, homeostasis and disease scenarios.

Post-translational modification of ribosomal subunit proteins (RPs) is emerging as an important means of regulating gene expression. Recently, regulatory ubiquitination of small RPs RPS10 and RPS20 by the ubiquitin ligase ZNF598 was found to function in ribosome sensing and stalling on internally polyadenylated mRNAs during ribosome quality control (RQC). Here, we reveal that ZNF598 and RPS10 negatively regulate interferon-stimulated gene (ISG) expression in primary cells, depletion of which induced ISG expression and a broad antiviral state. However, cell lines lacking interferon responses revealed that ZNF598 E3 ligase activity and ubiquitination of RPS20, but not RPS10, were specifically required for poxvirus replication and synthesis of poxvirus proteins whose encoding mRNAs contain unusual 5' poly(A) leaders. Our findings reveal distinct functions for ZNF598 and its downstream RPS targets, one that negatively regulates ISG expression and infection by a range of viruses while the other is positively exploited by poxviruses., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Type I interferons (IFNs) induce expression of more than 300 cellular genes that provide protection against viruses and other pathogens. For survival, viruses evolved defenses to prevent the IFN response or counteract the IFN-induced antiviral state. However, because viruses and cells coevolved, the dynamic relationship between virus and host is difficult to discern. In the present study, we demonstrated that vaccinia virus with a large deletion near the left end of the genome had a diminished ability to replicate in cells that had been pretreated with beta interferon (IFN-β), suggesting that one or more of the missing 17 open reading frames (ORFs) encode an antagonist of the IFN-induced antiviral state. By systematically deleting groups of ORFs and then individual ORFs, the C9L gene was shown to be required for IFN resistance. Replication of the C9L deletion mutant (vΔC9) was impaired in human cells that had been pretreated with IFN-β. Expression of viral early genes occurred, but subsequent events, including genome uncoating, genome replication, and postreplicative gene expression, were inhibited. Expression of the C9 protein occurred prior to genome replication, consistent with an early role in counteracting the IFN-induced antiviral state. C9 contains six ankyrin repeat motifs and a near C-terminal F-box. Mass spectrometry and immunoblotting identified host proteins that copurified with a functional epitope-tagged C9. The most abundant proteins were components of the SCF (CUL1, SKP1, F-box) and signalosome/deneddylation complexes, which interact with each other, suggesting a possible role in proteolysis of one or more interferon-induced proteins. IMPORTANCE Poxviruses comprise a family of large DNA viruses that replicate in the cytoplasm of vertebrate and insect hosts and cause human and zoonotic diseases. In most cases the primary infection is moderated by innate immune defenses. Vertebrates, including fish, amphibians, reptiles, birds, and mammals, all produce type I interferon homologs. In humans, interferon stimulates the synthesis of more than 300 proteins thought to have roles in host defense. Conversely, viruses have evolved means to thwart the host defenses. We are attempting to deconstruct the established virus-host relationship in order to better understand the molecular mechanisms involved. In the present study, we identified a vaccinia virus gene that prevents interferon-mediated inhibition of very early stages of viral replication and is conserved in orthopoxviruses. The viral protein was shown to interact with host proteins involved in proteolysis, suggesting that vaccinia virus may subvert the cellular apparatus for its own defense., (Copyright © 2018 American Society for Microbiology.)

Molluscum contagiosum virus (MCV) causes persistent, benign skin neoplasm in children and adults. MCV is refractive to growth in standard tissue culture and there is no relevant animal model of infection. Here we investigated whether another poxvirus (vaccinia virus; VACV) could be used to examine MCV immunoevasion protein properties in vivo. The MCV MC159L or MC160L genes, which encode NF-κB antagonists, were inserted into an attenuated VACV lacking an NF-κB antagonist (vΔA49), creating vMC159 and vMC160. vMC160 slightly increased vΔA49 virulence in the intranasal and intradermal routes of inoculation. vMC159 infection was less virulent than vΔA49 in both inoculation routes. vMC159-infected ear pinnae did not form lesions, but virus replication still occurred. Thus, the lack of lesions was not due to abortive virus replication. This system provides a new approach to examine MCV immunoevasion proteins within the context of a complete and complex immune system.

Vaccinia virus is unusual among DNA viruses in replicating exclusively in the cytoplasm of infected cells. The single-stranded DNA (ssDNA) binding protein (SSB) I3 is among the replication machinery encoded by the 195-kb genome, although direct genetic analysis of I3 has been lacking. Herein, we describe a complementing cell line (CV1-I3) that fully supports the replication of a null virus (vΔI3) lacking the I3 open reading frame (ORF). In noncomplementing CV1-CAT cells, vΔI3 shows a severe defect in the production of infectious virus (≥200-fold reduction). Early protein synthesis and core disassembly occur normally. However, DNA replication is profoundly impaired (≤0.2% of wild-type [WT] levels), and late proteins do not accumulate. When several other noncomplementing cell lines are infected with vΔI3, the yield of infectious virus is also dramatically reduced (168- to 1,776-fold reduction). Surprisingly, the residual levels of DNA accumulation vary from 1 to 12% in the different cell lines (CV1-CAT < A549 < BSC40 < HeLa); however, any nascent DNA that can be detected is subgenomic in size. Although this subgenomic DNA supports late protein expression, it does not support the production of infectious virions. Electron microscopy (EM) analysis of vΔI3-infected BSC40 cells reveals that immature virions are abundant but no mature virions are observed. Aberrant virions characteristic of a block to genome encapsidation are seen instead. Finally, we demonstrate that a CV1 cell line encoding a previously described I3 variant with impaired ssDNA binding activity is unable to complement vΔI3. This report provides definitive evidence that the vaccinia virus I3 protein is the replicative SSB and is essential for productive viral replication. IMPORTANCE Poxviruses are of historical and contemporary importance as infectious agents, vaccines, and oncolytic therapeutics. The cytoplasmic replication of poxviruses is unique among DNA viruses of mammalian cells and necessitates that the double-stranded DNA (dsDNA) genome encode the viral replication machinery. This study focuses on the I3 protein. As a ssDNA binding protein (SSB), I3 has been presumed to play essential roles in genome replication, recombination, and repair, although genetic analysis has been lacking. Herein, we report the characterization of an I3 deletion virus. In the absence of I3 expression, DNA replication is severely compromised and viral yield profoundly decreased. The production of infectious virus can be restored in a cell line expressing WT I3 but not in a cell line expressing an I3 mutant that is defective in ssDNA binding activity. These data show conclusively that I3 is an essential viral protein and functions as the viral replicative SSB., (Copyright © 2018 American Society for Microbiology.)

The serpin family of serine proteinase inhibitors plays key roles in the maintenance of mammalian homeostasis. Virus-encoded serpins disrupt the balance of mammalian proteases to facilitate virus replication in the infected host. DNA viruses, in particular members of the poxvirus family, have acquired multiple copies of the functional serpins which are essential for viral pathogenesis. Virus-encoded serpins have proven to be very effective inhibitors of host proteases and thus are very attractive candidate molecules as immunomodulatory drugs. With this chapter we explain approaches to identifying immune-modulating viral serpins.

Programmed cell death or apoptosis is an important component of host defense systems against viral infection. The B-cell lymphoma 2 (Bcl-2) proteins family is the main arbiter of mitochondrially mediated apoptosis, and viruses have evolved sequence and structural mimics of Bcl-2 to subvert premature host cell apoptosis in response to viral infection. The sequencing of the canarypox virus genome identified a putative pro-survival Bcl-2 protein, CNP058. However, a role in apoptosis inhibition for CNP058 has not been identified to date. Here, we report that CNP058 is able to bind several host cell pro-death Bcl-2 proteins, including Bak and Bax, as well as several BH3 only-proteins including Bim, Bid, Bmf, Noxa, Puma, and Hrk with high to moderate affinities. We then defined the structural basis for CNP058 binding to pro-death Bcl-2 proteins by determining the crystal structure of CNP058 bound to Bim BH3. CNP058 adopts the conserved Bcl-2 like fold observed in cellular pro-survival Bcl-2 proteins, and utilizes the canonical ligand binding groove to bind Bim BH3. We then demonstrate that CNP058 is a potent inhibitor of ultraviolet (UV) induced apoptosis in a cell culture model. Our findings suggest that CNP058 is a potent inhibitor of apoptosis that is able to bind to BH3 domain peptides from a broad range of pro-death Bcl-2 proteins, and may play a key role in countering premature host apoptosis., Competing Interests: The authors declare no conflict of interest.

Although most enveloped viruses acquire their membrane from the host by budding or by a wrapping process, collective data argue that nucleocytoplasmic large DNA viruses (NCLDVs) may be an exception. The prototype member of NCLDVs, vaccinia virus (VACV) may induce rupture of endoplasmic-reticulum-derived membranes to build an open-membrane sphere that closes after DNA uptake. This unconventional membrane assembly pathway is also used by at least 3 other members of the NCLDVs. In this study, we identify the VACV gene product of A11, as required for membrane rupture, hence for VACV membrane assembly and virion formation. By electron tomography, in the absence of A11, the site of assembly formed by the viral scaffold protein D13 is surrounded by endoplasmic reticulum cisternae that are closed. We use scanning transmission electron microscopy-electron tomography to analyse large volumes of cells and demonstrate that in the absence of A11, no open membranes are detected. Given the pivotal role of D13 in initiating VACV membrane assembly, we also analyse viral membranes in the absence of D13 synthesis and show that this protein is not required for rupture. Finally, consistent with a role in rupture, we show that during wild-type infection, A11 localises predominantly to the small ruptured membranes, the precursors of VACV membrane assembly. These data provide strong evidence in favour of the unusual membrane biogenesis of VACV and are an important step towards understanding its molecular mechanism., (© 2017 John Wiley & Sons Ltd.)

Molluscum contagiosum virus (MCV), the only known extant human-adapted poxvirus, causes a long-duration infection characterized by skin lesions that typically display an absence of inflammation despite containing high titers of live virus. Despite this curious presentation, MCV is very poorly characterized in terms of host-pathogen interactions. The absence of inflammation around MCV lesions suggests the presence of potent inhibitors of human antiviral immunity and inflammation. However, only a small number of MCV immunomodulatory genes have been characterized in detail. It is likely that many more remain to be discovered, given the density of such sequences in other poxvirus genomes. NF-κB activation occurs in response to both virus-induced pattern recognition receptor (PRR) signaling and cellular activation by virus-induced proinflammatory cytokines like tumor necrosis factor and interleukin-1. Activated NF-κB drives cytokine and interferon gene expression, leading to inflammation and virus clearance. We report that MC005, which has no orthologs in other poxvirus genomes, is a novel inhibitor of PRR- and cytokine-stimulated NF-κB activation. MC005 inhibited NF-κB proximal to the IκB kinase (IKK) complex, and unbiased affinity purification revealed that MC005 interacts with the IKK subunit NEMO (NF-κB essential modulator). MC005 binding to NEMO prevents the conformational priming of the IKK complex that occurs when NEMO binds to ubiquitin chains during pathway activation. These data reveal a novel mechanism of poxvirus inhibition of human innate immunity, validate current dynamic models of NEMO-dependent IKK complex activation, and further clarify how the human-adapted poxvirus MCV can so effectively evade antiviral immunity and suppress inflammation to persist in human skin lesions. IMPORTANCE Poxviruses adapt to specific hosts over time, evolving and tailoring elegantly precise inhibitors of the rate-limiting steps within the signaling pathways that control innate immunity and inflammation. These inhibitors reveal new features of the antiviral response, clarify existing models of signaling regulation while offering potent new tools for approaching therapeutic intervention in autoimmunity and inflammatory disease. Molluscum contagiosum virus (MCV) is the only known extant poxvirus specifically adapted to human infection and appears adept at evading normal human antiviral responses, yet it remains poorly characterized. We report the identification of MCV protein MC005 as an inhibitor of the pathways leading to the activation of NF-κB, an essential regulator of innate immunity. Further, identification of the mechanism of inhibition of NF-κB by MC005 confirms current models of the complex way in which NF-κB is regulated and greatly expands our understanding of how MCV so effectively evades human immunity., (Copyright © 2017 American Society for Microbiology.)

All viruses strategically alter the antiviral immune response to their benefit. The vaccinia virus (VACV) K1 protein has multiple immunomodulatory effects in tissue culture models of infection, including NF-κB antagonism. However, the effect of K1 during animal infection is poorly understood. We determined that a K1L -less vaccinia virus (vΔK1L) was less pathogenic than wild-type VACV in intranasal and intradermal models of infection. Decreased pathogenicity was correlated with diminished virus replication in intranasally infected mice. However, in intradermally inoculated ears, vΔK1L replicated to levels nearly identical to those of VACV, implying that the decreased immune response to vΔK1L infection, not virus replication, dictated lesion size. Several lines of evidence support this theory. First, vΔK1L induced slightly less edema than vK1L, as revealed by histopathology and noninvasive quantitative ultrasound technology (QUS). Second, infiltrating immune cell populations were decreased in vΔK1L-infected ears. Third, cytokine and chemokine gene expression was decreased in vΔK1L-infected ears. While these results identified the biological basis for smaller lesions, they remained puzzling; because K1 antagonizes NF-κB in vitro , antiviral gene expression was expected to be higher during vΔK1L infection. Despite these diminished innate immune responses, vΔK1L vaccination induced a protective VACV-specific CD8 + T cell response and protected against a lethal VACV challenge. Thus, vΔK1L is the first vaccinia virus construct reported that caused a muted innate immune gene expression profile and decreased immune cell infiltration in an intradermal model of infection yet still elicited protective immunity. IMPORTANCE The vaccinia virus (VACV) K1 protein inhibits NF-κB activation among its other antagonistic functions. A virus lacking K1 (vΔK1L) was predicted to be less pathogenic because it would trigger a more robust antiviral immune response than VACV. Indeed, vΔK1L was less pathogenic in intradermally infected mouse ear pinnae. However, vΔK1L infection unexpectedly elicited dramatically reduced infiltration of innate immune cells into ears. This was likely due to decreased expression of cytokine and chemokine genes in vΔK1L-infected ears. As such, our finding contradicted observations from cell culture systems. Interestingly, vΔK1L conferred protective immunity against lethal VACV challenge. This suggests that the muted immune response triggered during vΔK1L infection remained sufficient to mount an effective protective response. Our results highlight the complexity and unpredictable nature of virus-host interactions, a relationship that must be understood to better comprehend virus pathogenesis or to manipulate viruses for use as vaccines., (Copyright © 2017 American Society for Microbiology.)

Molluscum contagiosum virus (MCV) is a dermatotropic poxvirus that causes benign skin lesions. MCV lesions persist because of virally encoded immune evasion molecules that inhibit antiviral responses. The MCV MC159 protein suppresses NF-κB activation, a powerful antiviral response, via interactions with the NF-κB essential modulator (NEMO) subunit of the IκB kinase (IKK) complex. Binding of MC159 to NEMO does not disrupt the IKK complex, implying that MC159 prevents IKK activation via an as-yet-unidentified strategy. Here, we demonstrated that MC159 inhibited NEMO polyubiquitination, a posttranslational modification required for IKK and downstream NF-κB activation. Because MCV cannot be propagated in cell culture, MC159 was expressed independent of infection or during a surrogate vaccinia virus infection to identify how MC159 prevented polyubiquitination. Cellular inhibitor of apoptosis protein 1 (cIAP1) is a cellular E3 ligase that ubiquitinates NEMO. Mutational analyses revealed that MC159 and cIAP1 each bind to the same NEMO region, suggesting that MC159 may competitively inhibit cIAP1-NEMO interactions. Indeed, MC159 prevented cIAP1-NEMO interactions. MC159 also diminished cIAP1-mediated NEMO polyubiquitination and cIAP1-induced NF-κB activation. These data suggest that MC159 competitively binds to NEMO to prevent cIAP1-induced NEMO polyubiquitination. To our knowledge, this is the first report of a viral protein disrupting NEMO-cIAP1 interactions to strategically suppress IKK activation. All viruses must antagonize antiviral signaling events for survival. We hypothesize that MC159 inhibits NEMO polyubiquitination as a clever strategy to manipulate the host cell environment to the benefit of the virus. IMPORTANCE Molluscum contagiosum virus (MCV) is a human-specific poxvirus that causes persistent skin neoplasms. The persistence of MCV has been attributed to viral downregulation of host cell immune responses such as NF-κB activation. We show here that the MCV MC159 protein interacts with the NEMO subunit of the IKK complex to prevent NEMO interactions with the cIAP1 E3 ubiquitin ligase. This interaction correlates with a dampening of cIAP1 to polyubiquitinate NEMO and to activate NF-κB. This inhibition of cIAP1-NEMO interactions is a new viral strategy to minimize IKK activation and to control NEMO polyubiquitination. This research provides new insights into mechanisms that persistent viruses may use to cause long-term infection of host cells., (Copyright © 2017 American Society for Microbiology.)

Poxviruses use a complex strategy to escape immune control, by expressing immunomodulatory proteins that could limit their use as vaccine vectors. To test the role of poxvirus NF-κB pathway inhibitors A52, B15, and K7 in immunity, we deleted their genes in an NYVAC (New York vaccinia virus) strain that expresses HIV-1 clade C antigens. After infection of mice, ablation of the A52R , B15R , and K7R genes increased dendritic cell, natural killer cell, and neutrophil migration as well as chemokine/cytokine expression. Revertant viruses with these genes confirmed their role in inhibiting the innate immune system. To different extents, enhanced innate immune responses correlated with increased HIV Pol- and Gag-specific polyfunctional CD8 T cell and HIV Env-specific IgG responses induced by single-, double-, and triple-deletion mutants. These poxvirus proteins thus influence innate and adaptive cell-mediated and humoral immunity, and their ablation offers alternatives for design of vaccine vectors that regulate immune responses distinctly. IMPORTANCE Poxvirus vectors are used in clinical trials as candidate vaccines for several pathogens, yet how these vectors influence the immune system is unknown. We developed distinct poxvirus vectors that express heterologous antigens but lack different inhibitors of the central host-cell signaling pathway. Using mice, we studied the capacity of these viruses to induce innate and adaptive immune responses and showed that these vectors can distinctly regulate the magnitude and quality of these responses. These findings provide important insights into the mechanism of poxvirus-induced immune response and alternative strategies for vaccine vector design., (Copyright © 2017 American Society for Microbiology.)

Viruses are used as expression vectors for protein synthesis, immunology research, vaccines, and therapeutics. Advantages of poxvirus vectors include the accommodation of large amounts of heterologous DNA, the presence of a cytoplasmic site of transcription, and high expression levels. On the other hand, competition of approximately 200 viral genes with the target gene for expression and immune recognition may be disadvantageous. We describe a vaccinia virus (VACV) vector that uses an early promoter to express the bacteriophage T7 RNA polymerase; has the A23R intermediate transcription factor gene deleted, thereby restricting virus replication to complementing cells; and has a heterologous gene regulated by a T7 promoter. In noncomplementing cells, viral early gene expression and DNA replication occurred normally but synthesis of intermediate and late proteins was prevented. Nevertheless, the progeny viral DNA provided templates for abundant expression of heterologous genes regulated by a T7 promoter. Selective expression of the Escherichia coli lac repressor gene from an intermediate promoter reduced transcription of the heterologous gene specifically in complementing cells, where large amounts might adversely impact VACV replication. Expression of heterologous proteins mediated by the A23R deletion vector equaled that of a replicating VACV, was higher than that of a nonreplicating modified vaccinia virus Ankara (MVA) vector used for candidate vaccines in vitro and in vivo , and was similarly immunogenic in mice. Unlike the MVA vector, the A23R deletion vector still expresses numerous early genes that can restrict immunogenicity as demonstrated here by the failure of the prototype vector to induce interferon alpha. By deleting immunomodulatory genes, we anticipate further improvements in the system. IMPORTANCE Vaccines provide an efficient and effective way of preventing infectious diseases. Nevertheless, new and better vaccines are needed. Vaccinia virus, which was used successfully as a live vaccine to eradicate smallpox, has been further attenuated and adapted as a recombinant vector for immunization against other pathogens. However, since the initial description of this vector system, only incremental improvements largely related to safety have been implemented. Here we described novel modifications of the platform that increased expression of the heterologous target gene and decreased expression of endogenous vaccinia virus genes while providing safety by preventing replication of the candidate vaccine except in complementing cells used for vector propagation., (Copyright © 2017 Wyatt et al.)

Programmed cell death or apoptosis of infected host cells is an important defense mechanism in response to viral infections. This process is regulated by proapoptotic and prosurvival members of the B-cell lymphoma 2 (Bcl-2) protein family. To counter premature death of a virus-infected cell, poxviruses use a range of different molecular strategies including the mimicry of prosurvival Bcl-2 proteins. One such viral prosurvival protein is the fowlpox virus protein FPV039, which is a potent apoptosis inhibitor, but the precise molecular mechanism by which FPV039 inhibits apoptosis is unknown. To understand how fowlpox virus inhibits apoptosis, we examined FPV039 using isothermal titration calorimetry, small-angle X-ray scattering, and X-ray crystallography. Here, we report that the fowlpox virus prosurvival protein FPV039 promiscuously binds to cellular proapoptotic Bcl-2 and engages all major proapoptotic Bcl-2 proteins. Unlike other identified viral Bcl-2 proteins to date, FPV039 engaged with cellular proapoptotic Bcl-2 with affinities comparable with those of Bcl-2's endogenous cellular counterparts. Structural studies revealed that FPV039 adopts the conserved Bcl-2 fold observed in cellular prosurvival Bcl-2 proteins and closely mimics the structure of the prosurvival Bcl-2 family protein Mcl-1. Our findings suggest that FPV039 is a pan-Bcl-2 protein inhibitor that can engage all host BH3-only proteins, as well as Bcl-2-associated X, apoptosis regulator (Bax) and Bcl-2 antagonist/killer (Bak) proteins to inhibit premature apoptosis of an infected host cell. This work therefore provides a mechanistic platform to better understand FPV039-mediated apoptosis inhibition., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Vaccinia virus is the prototypic poxvirus. The 192 kilobase double-stranded DNA viral genome encodes most if not all of the viral replication machinery. The vaccinia virus DNA polymerase is encoded by the E9L gene. Sequence analysis indicates that E9 is a member of the B family of replicative polymerases. The enzyme has both polymerase and 3'-5' exonuclease activities, both of which are essential to support viral replication. Genetic analysis of E9 has identified residues and motifs whose alteration can confer temperature-sensitivity, drug resistance (phosphonoacetic acid, aphidicolin, cytosine arabinsode, cidofovir) or altered fidelity. The polymerase is involved both in DNA replication and in recombination. Although inherently distributive, E9 gains processivity by interacting in a 1:1 stoichiometry with a heterodimer of the A20 and D4 proteins. A20 binds to both E9 and D4 and serves as a bridge within the holoenzyme. The A20/D4 heterodimer has been purified and can confer processivity on purified E9. The interaction of A20 with D4 is mediated by the N'-terminus of A20. The D4 protein is an enzymatically active uracil DNA glycosylase. The DNA-scanning activity of D4 is proposed to keep the holoenzyme tethered to the DNA template but allow polymerase translocation. The crystal structure of D4, alone and in complex with A20 1-50 and/or DNA has been solved. Screens for low molecular weight compounds that interrupt the A20 1-50 /D4 interface have yielded hits that disrupt processive DNA synthesis in vitro and/or inhibit plaque formation. The observation that an active DNA repair enzyme is an integral part of the holoenzyme suggests that DNA replication and repair may be coupled., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Myxoma virus (MYXV) is Leporipoxvirus that possesses a specific rabbit-restricted host tropism but exhibits a much broader  cellular host range in cultured cells. MYXV is able to efficiently  block all aspects of the type I interferon (IFN)-induced  antiviral  state  in rabbit cells, partially in  human  cells  and  very  poorly  in  mouse  cells.  The mechanism(s) of this species-specific inhibition of  type I IFN-induced antiviral state is not well understood. Here we demonstrate that MYXV encoded  protein  M029, a truncated relative of the vaccinia virus (VACV) E3 double-stranded RNA (dsRNA)  binding  protein  that  inhibits  protein  kinase  R (PKR),  can  also  antagonize the type I IFN-induced  antiviral state in a highly species-specific manner. In cells pre-treated with type I IFN prior to  infection,  MYXV  exploits  M029  to  overcome  the  induced  antiviral  state completely in rabbit cells,  partially  in  human  cells,  but  not at all in mouse cells. However, in cells pre-infected with MYXV,  IFN-induced  signaling  is fully  inhibited  even  in the  absence  of M029 in cells from all three species,  suggesting  that  other  MYXV  protein(s)  apart  from  M029  block  IFN  signaling  in  a  speciesindependent  manner.  We  also  show  that  the  antiviral  state  induced in rabbit, human or mouse cells  by  type  I IFN  can  inhibit M029-knockout MYXV even when PKR is genetically knocked-out, suggesting  that  M029  targets  other  host  proteins  for  this  antiviral state inhibition. Thus, the MYXV  dsRNA  binding  protein  M029  not  only  antagonizes  PKR  from  multiple  species  but  also blocks the  type I IFN antiviral state independently of PKR in a highly species-specific fashion.

Most poxviruses encode a homolog of a ~200,000-kDa membrane protein originally identified in variola virus. We investigated the importance of the ectromelia virus (ECTV) homolog C15 in a natural infection model. In cultured mouse cells, the replication of a mutant virus with stop codons near the N-terminus (ECTV-C15Stop) was indistinguishable from a control virus (ECTV-C15Rev). However, for a range of doses injected into the footpads of BALB/c mice there was less mortality with the mutant. Similar virus loads were present at the site of infection with mutant or control virus whereas there was less ECTV-C15Stop in popliteal and inguinal lymph nodes, spleen and liver indicating decreased virus spread and replication. The latter results were supported by immunohistochemical analyses. Decreased spread was evidently due to immune modulatory activity of C15, rather than to an intrinsic viral function, as the survival of infected mice depended on CD4+ and CD8+ T cells., (Published by Elsevier Inc.)

Mousepox is caused by the orthopoxvirus ectromelia virus (ECTV), and is thought to be transmitted via skin abrasions. We studied the ECTV virulence factor N1 following subcutaneous infection of mousepox-susceptible BALB/c mice. In this model, ECTV lacking N1L gene was attenuated more than 1000-fold compared with wild-type virus and replication was profoundly reduced as early as four days after infection. However, in contrast to data from an intranasal model, N1 protein was not required for virus dissemination. Further, neither T cell nor cytokine responses were enhanced in the absence of N1. Together with the early timing of reduced virus titres, this suggests that in a cutaneous model, N1 exerts its function at the level of infected cells or in the inhibition of the very earliest effectors of innate immunity., (Copyright © 2016 Elsevier B.V. All rights reserved.)