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Pathways of apoptosis and their inhibition by VACV. Apoptosis can be induced by signals from either outside (extrinsic) or inside (intrinsic) the cell. The release of cytochrome c and activation of the apoptotic protease activator factor 1 (APAF1) from the mitochondrion to form the apoptosome is central in the induction of apoptosis. The apoptosome then activates a series of initiator and executioner caspases that lead to apoptosis. External stimuli like the Fas ligand or TNF can activate caspases directly. In non-apoptotic cells, the engagement of Fas receptor (FasR) or TNF receptor (TNFR), or intrinsic signals resulting from the withdrawal of growth factors, lead to activation of the pro-apoptotic proteins Bid and Bad. These then sequester antiapoptotic proteins Bcl-2 and Bcl-xL enabling formation of Bax/Bak-mediated mitochondrial pores. This causes cytochrome c release from the mitochondrion. VACV protein N1 targets Bid and Bad upstream in the signalling cascade, whereas VACV protein F1 neutralizes Bak in the mitochondrial membrane. VACV protein B13 inhibits caspase-1 and the apoptotic processes where it is involved. vGAAP is an integral membrane protein present in the Golgi and causes a reduction in Ca 2+ loading of intracellular stores, so that less Ca 2+ can be released by pro-apoptotic pathways.
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Virus infection of mammalian cells is sensed by pattern recognition receptors (PRRs) and leads to an innate immune response that restricts virus replication and induces adaptive immunity. In response, viruses have evolved many countermeasures that enable them to replicate and be transmitted to new hosts despite the host innate immune response. Poxv...
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... and anti-apoptotic members of the Bcl-2 family of proteins. In response to apoptotic signals, pro- apoptotic proteins such as Bad or Bid bind the effector proteins Bax and Bak, which oligomerize in the outer mitochondrial membrane inducing release of cytochrome c into the cytoplasm and formation of the caspase activation platform or apoptosome (Fig. 6). VACV directly subverts the intrinsic pathway by expressing proteins F1 and N1. Protein F1 adopts a Bcl-2-like fold ( Kvansakul et al., 2008) and binds Bak at the mitochondrion ( Wasilenko et al., 2003Wasilenko et al., , 2005Postigo et al., 2006;Kvansakul et al., 2008). F1 also reduces the inflammatory response by binding NLRP-1, an ...
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... WR-VACV has additional mechanisms to modulate apoptosis in infected cells to evade the host immune response [50]. One key mechanism involves the gene expression of B13R, which encodes protein B13 that closely mimics cowpox virus cytokine response modifier A (crmA), with a high amino acid identity of 92% [51]. In THP-1 cell lines, crmA inhibits the IL-1B-converting enzyme (ICE), catalyzing the conversion of pro-1B to mature IL-1B, a potent cytokine released as part of the innate defense [52]. ...
Vaccinia virus is the most successful vaccine in human history and functions as a protective vaccine against smallpox and monkeypox, highlighting the importance of ongoing research into vaccinia due to its genetic similarity to other emergent poxviruses. Moreover, vaccinia’s ability to accommodate large genetic insertions makes it promising for vaccine development and potential therapeutic applications, such as oncolytic agents. Thus, understanding how superior immunity is generated by vaccinia is crucial for designing other effective and safe vaccine strategies. During vaccinia inoculation by scarification, the skin serves as a primary site for the virus–host interaction, with various cell types playing distinct roles. During this process, hematopoietic cells undergo abortive infections, while non-hematopoietic cells support the full viral life cycle. This differential permissiveness to viral replication influences subsequent innate and adaptive immune responses. Dendritic cells (DCs), key immune sentinels in peripheral tissues such as skin, are pivotal in generating T cell memory during vaccinia immunization. DCs residing in the skin capture viral antigens and migrate to the draining lymph nodes (dLN), where they undergo maturation and present processed antigens to T cells. Notably, CD8+ T cells are particularly significant in viral clearance and the establishment of long-term protective immunity. Here, we will discuss vaccinia virus, its continued relevance to public health, and viral strategies permissive to immune escape. We will also discuss key events and populations leading to long-term protective immunity and remaining key gaps.
... At present, the most likely explanation for what is causing this effect relates to the observation that it is time dependent and dissipates if VACV exposure is delayed for three weeks (Fig 3). Orthopoxviruses like VACV encode a remarkable number of genes capable of inhibiting elements of both the innate and adaptive immune systems [40] including type I/II IFN and NFKB signaling [41,42] and effector T-cell functions (e.g., [43]). Several apoptotic inhibitors are encoded by these viruses [44]. ...
Ionizing radiation (IR) and oncolytic viruses are both used to treat cancer, and the effectiveness of both agents depends upon stimulating an immune response against the tumor. In this study we tested whether combining image guided ionizing radiation (IG-IR) with an oncolytic vaccinia virus (VACV) could yield a better therapeutic response than either treatment alone. ΔF4LΔJ2R VACV grew well on irradiated human and mouse breast cancer cells, and the virus can be combined with 4 or 8 Gy of IR to kill cells in an additive or weakly synergistic manner. To test efficacy in vivo we used immune competent mice bearing orthotopic TUBO mammary tumors. IG-IR worked well with 10 Gy producing 80% complete responses, but this was halved when the tumors were treated with VACV starting 2 days after IG-IR. VACV monotherapy was ineffective in this model. The antagonism was time dependent as waiting for 21 days after IG-IR eliminated the inhibitory effect but without yielding any further benefits over IR alone. In irradiated tumors, VACV replication was also lower, suggesting that irradiation created an environment that did not support infection as well in vivo as in vitro . A study of how four different treatment regimens affected the immune composition of the tumor microenvironment showed that treating irradiated tumors with VACV altered the immunological profiles in tumors exposed to IR or VACV alone. We detected more PD-1 and PD-L1 expression in tumors exposed to IR+VACV but adding an αPD-1 antibody to the protocol did not change the way VACV interferes with IG-IR therapy. VACV encodes many immunosuppressive gene products that may interfere with the ability of radiotherapy to induce an effective anti-tumor immune response through the release of danger-associated molecular patterns. These data suggest that infecting irradiated tumors with VACV, too soon after exposure, may interfere in the innate and linked adaptive immune responses that are triggered by radiotherapy to achieve a beneficial impact.
... 12,13 VACV is a human pathogen and has evolved to evade immune detection by various means, including through the expression of virus proteins that evade interferon (IFN)-mediated antivirus defense. 14 Numerous live, attenuated strains of VACV have been generated in the campaign to eradicate smallpox, providing an important precedent for the safety and tolerability of VACV-based vaccines. 15 The VACV genome can also be modified further to increase tumor cell-specific replication, such as in AZD4820, where deletion of the vaccinia TK and RR genes limits virus replication to cells that express high levels of these enzymes, such as tumor cells. ...
... VACV encodes several immunomodulatory genes that suppress immune cell activation, such as B8R and B19R, which sequester and neutralize IFN-g and type I IFN, respectively. 5,14 As expected, AZD4820 infection of TSCs resulted in the expression of B8R ( Figure 4D), which has been shown to sequester IFN-g in the supernatant of infected cells 5 and probably accounts for the lack of detectable IFN-g in the above experiments. Similarly, IFN-a2a and IFN-b were not induced in significant amounts upon VACV infection of DTCs ( Figures S4C and S4D), consistent with the expression of B18R. ...
Oncolytic viruses are engineered to selectively kill tumor cells and have demonstrated promising results in early-phase clinical trials. To further modulate the innate and adaptive immune system, we generated AZD4820, a vaccinia virus engineered to express interleukin-12 (IL-12), a potent cytokine involved in the activation of natural killer (NK) and T cells and the reprogramming of the tumor immune microenvironment. Testing in cultured human tumor cell lines demonstrated broad in vitro oncolytic activity and IL-12 transgene expression. A surrogate virus expressing murine IL-12 demonstrated antitumor activity in both MC38 and CT26 mouse syngeneic tumor models that responded poorly to immune checkpoint inhibition. In both models, AZD4820 significantly upregulated interferon-gamma (IFN-γ) relative to control mice treated with oncolytic vaccinia virus (VACV)-luciferase. In the CT26 study, 6 of 10 mice had a complete response after treatment with AZD4820 murine surrogate, whereas control VACV-luciferase–treated mice had 0 of 10 complete responders. AZD4820 treatment combined with anti–PD-L1 blocking antibody augmented tumor-specific T cell immunity relative to monotherapies. These findings suggest that vaccinia virus delivery of IL-12, combined with immune checkpoint blockade, elicits antitumor immunity in tumors that respond poorly to immune checkpoint inhibitors.
... This hypothesis is also supported by the observations that resting B lymphocytes need to be activated to become permissive for a productive VV infection [47] and that lymphocyte infection with VV is toxic, even with UV-inactivated virus [48]. Therefore, inhibition of protein synthesis by virion-associated F17 may be beneficial for the vaccinia virus to counter host immunity immediately after entry, well before the early expression of several viral proteins dedicated to this function [49]. In conclusion, our findings provide important insight into a novel functional role of the F17 protein. ...
Vaccinia virus (Orthopoxvirus) F17 protein is a major virion structural phosphoprotein having a molecular weight of 11 kDa. Recently, it was shown that F17 synthesised in infected cells interacts with mTOR subunits to evade cell immunity and stimulate late viral protein synthesis. Several years back, we purified an 11 kDa protein that inhibited protein synthesis in reticulocyte lysate from virions, and that possesses all physico-chemical properties of F17 protein. To investigate this discrepancy, we used defective vaccinia virus particles devoid of the F17 protein (designated iF17− particles) to assess their ability to inhibit protein synthesis. To this aim, we purified iF17− particles from cells infected with a vaccinia virus mutant which expresses F17 only in the presence of IPTG. The SDS-PAGE protein profiles of iF17− particles or derived particles, obtained by solubilisation of the viral membrane, were similar to that of infectious iF17 particles. As expected, the profiles of full iF17− particles and those lacking the viral membrane were missing the 11 kDa F17 band. The iF17− particles did attach to cells and injected their viral DNA into the cytoplasm. Co-infection of the non-permissive BSC40 cells with a modified vaccinia Ankara (MVA) virus, expressing an mCherry protein, and iF17− particles, induced a strong mCherry fluorescence. Altogether, these experiments confirmed that the iF17− particles can inject their content into cells. We measured the rate of protein synthesis as a function of the multiplicity of infection (MOI), in the presence of puromycin as a label. We showed that iF17− particles did not inhibit protein synthesis at high MOI, by contrast to the infectious iF17 mutant. Furthermore, the measured efficiency to inhibit protein synthesis by the iF17 mutant virus generated in the presence of IPTG, was threefold to eightfold lower than that of the wild-type WR virus. The iF17 mutant contained about threefold less F17 protein than wild-type WR. Altogether these results strongly suggest that virion-associated F17 protein is essential to mediate a stoichiometric inhibition of protein synthesis, in contrast to the late synthesised F17. It is possible that this discrepancy is due to different phosphorylation states of the free and virion-associated F17 protein.
... Furthermore, many viral genes have immunomodulatory functions, aimed at subverting the pathogen recognition systems in the cell, as well as counteracting antiviral signaling and effectors (i.e. IFN-stimulated genes -ISGs-) when activated [4][5][6][7] . ...
... IL32 and CCL2). However, we did not detect the overexpression of interferon (IFN) and IFN-stimulated genes (ISGs) in these populations, likely a consequence of virus-mediated repression at the protein level 6 . Previous studies have shown that cytokine production in virus-infected populations is often confined to small subpopulations of abortively infected cells [33][34][35] . ...
... This observation raises the possibility of delayed translation dynamics for specific mRNAs, and the stabilization or destabilization of specific host factors at the protein level. Poxviruses, including VV, encode E3 ubiquitin ligases and adaptor proteins known to target immune factors, including NF-kB, MHC-I, and CD4 6,96,97 . However, most of the targets for these proteins remain unknown. ...
Poxviruses are a large group of DNA viruses with exclusively cytoplasmic life cycles and complex gene expression programs. A number of systems-level studies have analyzed bulk transcriptome and proteome changes upon poxvirus infection, but the cell-to-cell heterogeneity of the transcriptomic response, and the subcellular resolution of proteomic changes have remained unexplored. Here, we measured single-cell transcriptomes of Vaccinia virus-infected populations of HeLa cells and immortalized human fibroblasts, resolving the cell-to-cell heterogeneity of infection dynamics and host responses within those cell populations. We further integrated our transcriptomic data with changes in the levels and subcellular localization of the host and viral proteome throughout the course of Vaccinia virus infection. Our findings from single-cell RNA sequencing indicate conserved transcriptome changes independent of the cellular context, including widespread host shutoff, heightened expression of cellular transcripts implicated in stress responses, the rapid accumulation of viral transcripts, and the robust activation of antiviral pathways in bystander cells. While most host factors were co-regulated at the RNA and protein level, we identified a subset of factors where transcript and protein levels were discordant in infected cells; predominantly factors involved in transcriptional and post-transcriptional mRNA regulation. In addition, we detected the relocalization of several host proteins such as TENT4A, NLRC5, and TRIM5, to different cellular compartments in infected cells. Collectively, our comprehensive data provide spatial and temporal resolution of the cellular and viral transcriptomes and proteomes and offer a robust foundation for in-depth exploration of virus-host interactions in poxvirus-infected cells.
... VACV has been demonstrated by a wound healing assay to induce two forms of cell movement: (i) fast independent migration of infected cells into the wound (induced by early viral proteins) and (ii) slow migration of cells after the development of long-branched projections (induced by late viral proteins) [69]. Orthopoxviruses encode numerous genes involved in actin cytoskeleton rearrangement, interactions of integrins with the ECM, and cell adhesion [70][71][72][73][74][75], which subsequently may affect cell migration. Therefore, future studies are needed to elucidate the effect of adhesive structure rearrangement on the migration of DCs in the context of ECTV infection. ...
Ectromelia virus (ECTV) is a causative agent of mousepox. It provides a suitable model for studying the immunobiology of orthopoxviruses, including their interaction with the host cell cytoskeleton. As professional antigen-presenting cells, dendritic cells (DCs) control the pericellular environment, capture antigens, and present them to T lymphocytes after migration to secondary lymphoid organs. Migration of immature DCs is possible due to the presence of specialized adhesion structures, such as podosomes or focal adhesions (FAs). Since assembly and disassembly of adhesive structures are highly associated with DCs’ immunoregulatory and migratory functions, we evaluated how ECTV infection targets podosomes and FAs’ organization and formation in natural-host bone marrow-derived DCs (BMDC). We found that ECTV induces a rapid dissolution of podosomes at the early stages of infection, accompanied by the development of larger and wider FAs than in uninfected control cells. At later stages of infection, FAs were predominantly observed in long cellular extensions, formed extensively by infected cells. Dissolution of podosomes in ECTV-infected BMDCs was not associated with maturation and increased 2D cell migration in a wound healing assay; however, accelerated transwell migration of ECTV-infected cells towards supernatants derived from LPS-conditioned BMDCs was observed. We suggest that ECTV-induced changes in the spatial organization of adhesive structures in DCs may alter the adhesiveness/migration of DCs during some conditions, e.g., inflammation.
... Like ASFV, VACV is a double-stranded DNA virus with a large genome that replicates in the cell cytoplasm. Many immune-modulatory genes have been identified in VACV [114]. One immune modulator is the A55 protein, a member of the BBK (broad complex, tram-trac, and bric-a-brac [BTB] and C-terminal Kelch) family [45]. ...
Cellular nucleocytoplasmic trafficking is mediated by the importin family of nuclear transport proteins. The well-characterized importin alpha (IMPA) and importin beta (IMPB) nuclear import pathway plays a crucial role in the innate immune response to viral infection by mediating the nuclear import of transcription factors such as IRF3, NFκB, and STAT1. The nuclear transport of these transcription factors ultimately leads to the upregulation of a wide range of antiviral genes, including IFN and IFN-stimulated genes (ISGs). To replicate efficiently in cells, viruses have developed mechanisms to block these signaling pathways. One strategy to evade host innate immune responses involves blocking the nuclear import of host antiviral transcription factors. By binding IMPA proteins, these viral proteins prevent the nuclear transport of key transcription factors and suppress the induction of antiviral gene expression. In this review, we describe examples of proteins encoded by viruses from several different families that utilize such a competitive inhibition strategy to suppress the induction of antiviral gene expression.
... A recent study demonstrated that deleting three key immune-evasion gene products (C10L, N2L, and C6L) from VACV preserved its replication ability in cancer cells [30]. Proteins encoded by VACV, including C10, A46, N2L, and C6, acted as antagonists in the TLR3-IRF3 signaling pathway in various stages [31]. The deletion of these genes facilitated the phosphorylation of IRF3, a pivotal protein in the activation of TLR3, leading to heightened cytotoxic T lymphocyte (CTL) responses in a syngeneic mouse model. ...
Oncolytic viruses (OVs) have emerged as one of the most promising cancer immunotherapy agents that selectively target and kill cancer cells while sparing normal cells. OVs are from diverse families of viruses and can possess either a DNA or an RNA genome. These viruses also have either a natural or engineered tropism for cancer cells. Oncolytic DNA viruses have the additional advantage of a stable genome and multiple-transgene insertion capability without compromising infection or replication. Herpes simplex virus 1 (HSV-1), a member of the oncolytic DNA viruses, has been approved for the treatment of cancers. This success with HSV-1 was achievable by introducing multiple genetic modifications within the virus to enhance cancer selectivity and reduce the toxicity to healthy cells. Here, we review the natural characteristics of and genetically engineered changes in selected DNA viruses that enhance the tumor tropism of these oncolytic viruses.
... Poxviruses have developed sophisticated strategies to evade the immune system and produce a wide range of proteins that can manipulate host innate and adaptive immune responses of [26]. To investigate whether ECTV encodes proteins that interact with GBP2, RAW264.7 cells were infected with ECTV and subjected to immunoprecipitation/mass spectrometry analysis to identify potential protein interactions with GBPs. ...
The recent spread of the monkeypox virus among humans has heightened concerns regarding orthopoxvirus infections. Consequently, conducting a comprehensive study on the immunobiology of the monkeypox virus is imperative for the development of effective therapeutics. Ectromelia virus (ECTV) closely resembles the genetic and disease characteristics of monkeypox virus, making it a valuable research tool for studying orthopoxvirus–host interactions. Guanylate-binding proteins (GBPs), highly expressed interferon-stimulated genes (ISGs), have antagonistic effects against various intracellular pathogenic microorganisms. Our previous research has shown that GBP2 has a mild but statistically significant inhibitory effect on ECTV infection. The presence of a significant number of molecules in the poxvirus genome that encode the host immune response raises questions about whether it also includes proteins that counteract the antiviral activity of GBP2. Using IP/MS and co-IP technology, we discovered that the poly(A) polymerase catalytic subunit (PAPL) protein of ECTV is a viral regulatory molecule that interacts with GBP2. Further studies have shown that PAPL antagonizes the antiviral activity of GBP2 by reducing its protein levels. Knocking out the PAPL gene of ECTV with the CRISPR/Cas9 system significantly diminishes the replication ability of the virus, indicating the indispensable role of PAPL in the replication process of ECTV. In conclusion, our study presents preliminary evidence supporting the significance of PAPL as a virulence factor that can interact with GBP2.
... GCRV infection can affect the normal physiological functions of host cells, cause cell damage, and induce robust immune responses, which have been fully confirmed in the past [29][30][31]. As shown in Figure 5, different subtypes of GCRV generated independent immune responses at different time points, and cluster analysis further showed that all immune-related genes in DEGs, including Con, GV_I, GV_II, and GV_III groups at 6 and 24 h ( Figure 6A). ...
... In previous studies, CIK cells have been identified as susceptible cell lines for three strains of GCRV [26][27][28][29]. We investigated the expression levels of antiviral-related genes at various time points during infection with these three GCRV strains. ...
Grass carp reovirus (GCRV), one of the most serious pathogens threatening grass carp (Ctenopharyngodon idella), can lead to grass carp hemorrhagic disease (GCHD). Currently, GCRV can be divided into three genotypes, but the comparison of their pathogenic mechanisms and the host responses remain unclear. In this study, we utilized the Ctenopharyngodon idella kidney (CIK) model infected with GCRV to conduct comparative studies on the three genotypes. We observed a cytopathic effect (CPE) in the GCRV-I and GCRV-III groups, whereas the GCRV-II group did not show any CPE. Moreover, a consistent trend in the mRNA expression levels of antiviral-related genes across all experimental groups of CIK cells was detected via qPCR and further explored through RNA-seq analysis. Importantly, GO/KEGG enrichment analysis showed that GCRV-I, -II, and -III could all activate the immune response in CIK cells, but GCRV-II induced more intense immune responses. Intriguingly, transcriptomic analysis revealed a widespread down-regulation of metabolism processes such as steroid biosynthesis, butanoate metabolism, and N-Glycan biosynthesis in infected CIK cells. Overall, our results reveal the CIK cells showed unique responses in immunity and metabolism in the three genotypes of GCRV infection. These results provide a theoretical basis for understanding the pathogenesis and prevention and control methods of GCRV.
