Vaccinia virus protein A49 activates Wnt signalling by targetting the E3 ligase β-TrCP.

Vaccinia virus (VACV) encodes multiple proteins inhibiting the NF-κB signalling pathway. One of these, A49, targets the E3 ubiquitin ligase β-TrCP, which is responsible for the ubiquitylation and consequential proteosomal degradation of IκBα and the release of the NF-κB heterodimer. β-TrCP is a pleiotropic enzyme ubiquitylating multiple cellular substrates, including the transcriptional activator β-catenin. Here we demonstrate that A49 can activate the Wnt signalling pathway, a critical pathway that is involved in cell cycle and cell differentiation, and is controlled by β-catenin. The data presented show that the expression of A49 ectopically or during VACV infection causes accumulation of β-catenin, and that A49 triggering of Wnt signalling is dependent on binding β-TrCP. This is consistent with A49 blocking the ability of β-TrCP to recognise β-catenin and IκBα, and possibly other cellular targets. Thus, A49 targetting of β-TrCP affects multiple cellular pathways, including the NF-κB and Wnt signalling cascades

Vaccinia virus protein A49 is an unexpected member of the B-cell Lymphoma (Bcl)-2 protein family.

Vaccinia virus (VACV) encodes several proteins that inhibit activation of the proinflammatory transcription factor nuclear factor κB (NF-κB). VACV protein A49 prevents translocation of NF-κB to the nucleus by sequestering cellular β-TrCP, a protein required for the degradation of the inhibitor of κB. A49 does not share overall sequence similarity with any protein of known structure or function. We solved the crystal structure of A49 from VACV Western Reserve to 1.8 Å resolution and showed, surprisingly, that A49 has the same three-dimensional fold as Bcl-2 family proteins despite lacking identifiable sequence similarity. Whereas Bcl-2 family members characteristically modulate cellular apoptosis, A49 lacks a surface groove suitable for binding BH3 peptides and does not bind proapoptotic Bcl-2 family proteins Bax or Bak. The N-terminal 17 residues of A49 do not adopt a single well ordered conformation, consistent with their proposed role in binding β-TrCP. Whereas pairs of A49 molecules interact symmetrically via a large hydrophobic surface in crystallo, A49 does not dimerize in solution or in cells, and we propose that this hydrophobic interaction surface may mediate binding to a yet undefined cellular partner. A49 represents the eleventh VACV Bcl-2 family protein and, despite these proteins sharing very low sequence identity, structure-based phylogenetic analysis shows that all poxvirus Bcl-2 proteins are structurally more similar to each other than they are to any cellular or herpesvirus Bcl-2 proteins. This is consistent with duplication and diversification of a single BCL2 family gene acquired by an ancestral poxvirus.This work was supported by Wellcome Trust Principal Research Fellowship 090315 (to G. L. S.), Sir Henry Dale Fellowship 098406/Z/12/Z, jointly funded by the Wellcome Trust and the Royal Society (to S. C. G.), and United Kingdom Medical Research Council Grant G1000099 (to David Stuart).This is the final published version. It first appeared at http://dx.doi.org/10.1074/jbc.M114.62465

Enhancement of CD8(+) T-cell memory by removal of a vaccinia virus nuclear factor-κB inhibitor.

Factors influencing T-cell responses are important for vaccine development but are incompletely understood. Here, vaccinia virus (VACV) protein N1 is shown to impair the development of both effector and memory CD8(+) T cells and this correlates with its inhibition of nuclear factor-κB (NF-κB) activation. Infection with VACVs that either have the N1L gene deleted (vΔN1) or contain a I6E mutation (vN1.I6E) that abrogates its inhibition of NF-κB resulted in increased central and memory CD8(+) T-cell populations, increased CD8(+) T-cell cytotoxicity and lower virus titres after challenge. Furthermore, CD8(+) memory T-cell function was increased following infection with vN1.I6E, with more interferon-γ production and greater protection against VACV infection following passive transfer to naive mice, compared with CD8(+) T cells from mice infected with wild-type virus (vN1.WT). This demonstrates the importance of NF-κB activation within infected cells for long-term CD8(+) T-cell memory and vaccine efficacy. Further, it provides a rationale for deleting N1 from VACV vectors to enhance CD8(+) T-cell immunogenicity, while simultaneously reducing virulence to improve vaccine safety.This work was supported by grants from the Wellcome Trust and the Medical Research Council. GLS is a Wellcome Trust Principal Research Fellow.This is the final version of the article. It first appeared from Wiley via http://dx.doi.org/10.1111/imm.1242

HIV-2/SIV Vpx antagonises NF-κB activation by targeting p65

BACKGROUND: The NF-κB family of transcription factors and associated signalling pathways are abundant and ubiquitous in human immune responses. Activation of NF-κB transcription factors by viral pathogen-associated molecular patterns, such as viral RNA and DNA, is fundamental to anti-viral innate immune defences and pro-inflammatory cytokine production that steers adaptive immune responses. Diverse non-viral stimuli, such as lipopolysaccharide and cytokines, also activate NF-κB and the same anti-pathogen gene networks. Viruses adapted to human cells often encode multiple proteins targeting the NF-κB pathway to mitigate the anti-viral effects of NF-κB-dependent host immunity. RESULTS: In this study we have demonstrated using a variety of assays, in a number of different cell types including primary cells, that plasmid-encoded or virus-delivered simian immunodeficiency virus (SIV) accessory protein Vpx is a broad antagonist of NF-κB signalling active against diverse innate NF-κB agonists. Using targeted Vpx mutagenesis, we showed that this novel Vpx phenotype is independent of known Vpx cofactor DCAF1 and other cellular binding partners, including SAMHD1, STING and the HUSH complex. We found that Vpx co-immunoprecipitated with canonical NF-κB transcription factor p65, but not NF-κB family members p50 or p100, preventing nuclear translocation of p65. We found that broad antagonism of NF-κB activation by Vpx was conserved across distantly related lentiviruses as well as for Vpr from SIV Mona monkey (SIVmon), which has Vpx-like SAMHD1-degradation activity. CONCLUSIONS: We have discovered a novel mechanism by which lentiviruses antagonise NF-κB activation by targeting p65. These findings extend our knowledge of how lentiviruses manipulate universal regulators of immunity to avoid the anti-viral sequelae of pro-inflammatory gene expression stimulated by both viral and extra-viral agonists. Importantly our findings are also relevant to the gene therapy field where virus-like particle associated Vpx is routinely used to enhance vector transduction through antagonism of SAMHD1, and perhaps also through manipulation of NF-κB

Vaccinia Virus BBK E3 Ligase Adaptor A55 Targets Importin-Dependent NF-κB Activation and Inhibits CD8+ T-Cell Memory.

Viral infection of cells is sensed by pathogen recognition receptors that trigger an antiviral innate immune response, and consequently viruses have evolved countermeasures. Vaccinia virus (VACV) evades the host immune response by expressing scores of immunomodulatory proteins. One family of VACV proteins are the BTB-BACK (broad-complex, tram-trac, and bric-a-brac [BTB] and C-terminal Kelch [BACK]) domain-containing, Kelch-like (BBK) family of predicted cullin-3 E3 ligase adaptors: A55, C2, and F3. Previous studies demonstrated that gene A55R encodes a protein that is nonessential for VACV replication yet affects viral virulence in vivo Here, we report that A55 is an NF-κB inhibitor acting downstream of IκBα degradation, preventing gene transcription and cytokine secretion in response to cytokine stimulation. A55 targets the host importin α1 (KPNA2), acting to reduce p65 binding and its nuclear translocation. Interestingly, while A55 was confirmed to coprecipitate with cullin-3 in a BTB-dependent manner, its NF-κB inhibitory activity mapped to the Kelch domain, which alone is sufficient to coprecipitate with KPNA2 and inhibit NF-κB signaling. Intradermal infection of mice with a virus lacking A55R (vΔA55) increased VACV-specific CD8+ T-cell proliferation, activation, and cytotoxicity in comparison to levels of the wild-type (WT) virus. Furthermore, immunization with vΔA55 induced increased protection to intranasal VACV challenge compared to the level with control viruses. In summary, this report describes the first target of a poxvirus-encoded BBK protein and a novel mechanism for DNA virus immune evasion, resulting in increased CD8+ T-cell memory and a more immunogenic vaccine.IMPORTANCE NF-κB is a critical transcription factor in the innate immune response to infection and in shaping adaptive immunity. The identification of host and virus proteins that modulate the induction of immunological memory is important for improving virus-based vaccine design and efficacy. In viruses, the expression of BTB-BACK Kelch-like (BBK) proteins is restricted to poxviruses and conserved within them, indicating the importance of these proteins for these medically important viruses. Using vaccinia virus (VACV), the smallpox vaccine, we report that the VACV BBK protein A55 dysregulates NF-κB signaling by disrupting the p65-importin interaction, thus preventing NF-κB translocation and blocking NF-κB-dependent gene transcription. Infection with VACV lacking A55 induces increased VACV-specific CD8+ T-cell memory and better protection against VACV challenge. Studying viral immunomodulators therefore expands not only our understanding of viral pathogenesis and immune evasion strategies but also of the immune signaling cascades controlling antiviral immunity and the development of immune memory.Wellcome Trus

HIV-2/SIV Vpx antagonises NF-κB activation by targeting p65.

BACKGROUND: The NF-κB family of transcription factors and associated signalling pathways are abundant and ubiquitous in human immune responses. Activation of NF-κB transcription factors by viral pathogen-associated molecular patterns, such as viral RNA and DNA, is fundamental to anti-viral innate immune defences and pro-inflammatory cytokine production that steers adaptive immune responses. Diverse non-viral stimuli, such as lipopolysaccharide and cytokines, also activate NF-κB and the same anti-pathogen gene networks. Viruses adapted to human cells often encode multiple proteins targeting the NF-κB pathway to mitigate the anti-viral effects of NF-κB-dependent host immunity. RESULTS: In this study we have demonstrated using a variety of assays, in a number of different cell types including primary cells, that plasmid-encoded or virus-delivered simian immunodeficiency virus (SIV) accessory protein Vpx is a broad antagonist of NF-κB signalling active against diverse innate NF-κB agonists. Using targeted Vpx mutagenesis, we showed that this novel Vpx phenotype is independent of known Vpx cofactor DCAF1 and other cellular binding partners, including SAMHD1, STING and the HUSH complex. We found that Vpx co-immunoprecipitated with canonical NF-κB transcription factor p65, but not NF-κB family members p50 or p100, preventing nuclear translocation of p65. We found that broad antagonism of NF-κB activation by Vpx was conserved across distantly related lentiviruses as well as for Vpr from SIV Mona monkey (SIVmon), which has Vpx-like SAMHD1-degradation activity. CONCLUSIONS: We have discovered a novel mechanism by which lentiviruses antagonise NF-κB activation by targeting p65. These findings extend our knowledge of how lentiviruses manipulate universal regulators of immunity to avoid the anti-viral sequelae of pro-inflammatory gene expression stimulated by both viral and extra-viral agonists. Importantly our findings are also relevant to the gene therapy field where virus-like particle associated Vpx is routinely used to enhance vector transduction through antagonism of SAMHD1, and perhaps also through manipulation of NF-κB

African Swine Fever Virus Ubiquitin-Conjugating Enzyme Is an Immunomodulator Targeting NF-kappa B Activation

African swine fever virus (ASFV) is an acute and persistent swine virus with a high economic burden that encodes multiple genes to evade host immune response. In this work, we have revealed that early viral protein UBCv1, the only known conjugating enzyme encoded by a virus, modulates innate immune and inflammatory signaling. Transient overexpression of UBCv1 impaired activation of NF-κB and AP-1 transcription factors induced by several agonists of these pathways. In contrast, activation of IRF3 and ISRE signaling upon stimulation with TRIFΔRIP, cGAS/STING or RIG-I-CARD remained unaltered. Experiments aimed at mapping UBCv1 inhibitory activity indicated that this viral protein acts upstream or at the level step of IKKβ. In agreement with this, UBCv1 was able to block p65 nuclear translocation upon cytokine stimulation, a key event in NF-ĸB signaling. Additionally, A549 stably transduced for UBCv1 showed a significant decrease in the levels of NF-ĸB dependent genes. Interestingly, despite the well-defined capacity of UBCv1 to conjugate ubiquitin chains, a mutant disabled for ubiquitylation activity retained similar immunomodulatory activity as the wild-type enzyme, suggesting that the two functions are segregated. Altogether these data suggest that ASFV UBCv1 manipulates the innate immune response targeting the NF-κB and AP-1 pathways and opens new questions about the multifunctionality of this enzyme

Inhibition of apoptosis and NF-κB activation by vaccinia protein N1 occur via distinct binding surfaces and make different contributions to virulence.

Vaccinia virus (VACV) protein N1 is an intracellular virulence factor and belongs to a family of VACV B-cell lymphoma (Bcl)-2-like proteins whose members inhibit apoptosis or activation of pro-inflammatory transcription factors, such as interferon (IFN) regulatory factor-3 (IRF-3) and nuclear factor-κB (NF-κB). Unusually, N1 inhibits both apoptosis and NF-κB activation. To understand how N1 exerts these different functions, we have mutated residues in the Bcl-2-like surface groove and at the interface used to form N1 homodimers. Mutagenesis of the surface groove abolished only the N1 anti-apoptotic activity and protein crystallography showed these mutants differed from wild-type N1 only at the site of mutation. Conversely, mutagenesis of the dimer interface converted N1 to a monomer and affected only inhibition of NF-κB activation. Collectively, these data show that N1 inhibits pro-inflammatory and pro-apoptotic signalling using independent surfaces of the protein. To determine the relative contribution of each activity to virus virulence, mutant N1 alleles were introduced into a VACV strain lacking N1 and the virulence of these viruses was analysed after intradermal and intranasal inoculation in mice. In both models, VACV containing a mutant N1 unable to inhibit apoptosis had similar virulence to wild-type virus, whereas VACV containing a mutant N1 impaired for NF-κB inhibition induced an attenuated infection similar to that of the N1-deleted virus. This indicates that anti-apoptotic activity of N1 does not drive virulence in these in vivo models, and highlights the importance of pro-inflammatory signalling in the immune response against viral infections

Distinct monkeypox virus lineages co-circulating in humans before 2022

The 2022 global mpox outbreak raises questions about how this zoonotic disease established effective human-to-human transmission and its potential for further adaptation. The 2022 outbreak virus is related to an ongoing outbreak in Nigeria originally reported in 2017, but the evolutionary path linking the two remains unclear due to a lack of genomic data between 2018, when virus exportations from Nigeria were first recorded, and 2022, when the global mpox outbreak began. Here, 18 viral genomes obtained from patients across southern Nigeria in 2019-2020 reveal multiple lineages of monkeypox virus (MPXV) co-circulated in humans for several years before 2022, with progressive accumulation of mutations consistent with APOBEC3 activity over time. We identify Nigerian A.2 lineage isolates, confirming the lineage that has been multiply exported to North America independently of the 2022 outbreak originated in Nigeria, and that it has persisted by human-to-human transmission in Nigeria for more than 2 years before its latest exportation. Finally, we identify a lineage-defining APOBEC3-style mutation in all A.2 isolates that disrupts gene A46R, encoding a viral innate immune modulator. Collectively, our data demonstrate MPXV capacity for sustained diversification within humans, including mutations that may be consistent with established mechanisms of poxvirus adaptation.</p

Un siglo de luz : Historia empresarial de Iberdrola

El libro se divide en épocas y, dentro de cada una de ellas, se analizan los avatares de las tres empresas fundadoras de Iberdrola. La parte I tiene carácter de introducción y sus dos capítulos responden al deseo de integrar la historia de Iberdrola, tanto en el sector eléctrico espafíol como en el de otros países de Europa. La parte II abarca hasta el afío 1944, fecha de la fusión entre Saltos del Duero e Hidroeléctrica Ibérica. La parte III comprende los afíos 1944-1973, con la crisis energética como límite diferenciador. La parte IV comprende desde el comienzo de la crisis hasta la fusión de Iberduero e Hidrola, en 1991. La última parte del libro, en la que se hace la historia reciente de la empresa, ha sido escrita por sus principales protagonistas, lo cual afíade un interés muy especial a los capítulos catorce, quince y dieciséis, con los que termina la obra