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Verticillium wilt and Fusarium wilt, caused by Verticillium dahliae and Fusarium oxysporum, respectively, severely restricted cotton production. It is difficult to control the damage due to large unknown about the interaction between plants and pathogens. Here, we reported the identification of cotton Bsr-k1, which was also designated as GhBsr-k1. The GhBsr-k1 was expressed in the root, stem and leaf. Silencing of GhBsr-k1 improved plant resistance to both V. dahliae and F. oxysporum. In the GhBsr-k1-silenced plants, the expression of phenylpropanoid metabolism related genes were significantly up-regulated comparing with the control, which resulting the increase of lignin deposition. Moreover, the expression level of PALs exhibited highly negative correlation with GhBsr-k1. Collectively, the results suggested GhBsr-k1 acted as a negative regulator during plant resistance against V. dahliae and F. oxysporum. The potential mechanisms beyond GhBsr-k1 in plant defense may related to the regulation of the transcript of lignin deposition genes (GhPAL2, GhPAL5).

Background The virus-induced genome editing (VIGE) system can be used to quickly identify gene functions and generate knock-out libraries as an alternative to the virus-induced gene silencing (VIGS). Although plant virus-mediated VIGE has been shown to have great application prospects, edited genes cannot be transferred to the next generations using this system, as viruses cannot enter into shoot apical meristem (SAM) in plants. Results We developed a novel cotton leaf crumple virus (CLCrV)-mediated VIGE system designed to target BRI1, GL2, PDS genes, and GUS transgene in A. thaliana by transforming Cas9 overexpression (Cas9-OE) A. thaliana. Given the deficiency of the VIGE system, ProYao::Cas9 and Pro35S::Cas9 A. thaliana were transformed by fusing 102 bp FT mRNAs with sgRNAs so as to explore the function of Flowering Locus T (FT) gene in delivering sgRNAs into SAM, thus avoiding tissue culture and stably acquiring heritable mutant offspring. Our results showed that sgRNAs fused with FT mRNA at the 5′ end (FT strategy) effectively enabled gene editing in infected plants and allowed the acquisition of mutations heritable by the next generation, with an efficiency of 4.35–8.79%. In addition, gene-edited offspring by FT-sgRNAs did not contain any components of the CLCrV genome. Conclusions FT strategy can be used to acquire heritable mutant offspring avoiding tissue culture and stable transformation based on the CLCrV-mediated VIGE system in A. thaliana.

SUMMARYThe DNA sensor cyclic GMP-AMP synthase (cGAS) is critical in host antiviral immunity. Vaccinia virus (VACV) is a large cytoplasmic DNA virus that belongs to the poxvirus family. How vaccinia virus antagonizes the cGAS-mediated cytosolic DNA-sensing pathway is largely unknown. In this study, we screened 82 vaccinia viral genes to identify potential viral inhibitors of the cGAS/Stimulator of interferon gene (STING) pathway. We discovered that vaccinia E5 is a virulence factor and a major inhibitor of cGAS that elicits proteasome-dependent cGAS degradation. E5 localizes to the cytoplasm and nuclei of infected cells. Cytosolic E5 triggers K48-linked ubiquitination of cGAS and proteasome-dependent degradation via interacting with cGAS. E5 itself also undergoes ubiquitination and degradation. Deleting the E5R gene from the Modified vaccinia virus Ankara (MVA) genome strongly induces type I IFN production by dendritic cells (DCs) and promotes DC maturation, thereby improving the immunogenicity of the viral vector.

BackgroundViral-based immunotherapy can overcome resistance to immune checkpoint blockade (ICB) and fill the unmet needs of many patients with cancer. Oncolytic viruses (OVs) are defined as engineered or naturally occurring viruses that selectively replicate in and kill cancer cells. OVs also induce antitumor immunity. The purpose of this study was to compare the antitumor effects of live oncolytic vaccinia viruses versus the inactivated versions and elucidate their underlying immunological mechanisms.MethodsWe engineered a replication-competent, oncolytic vaccinia virus (OV-GM) by inserting a murine GM-CSF gene into the thymidine kinase locus of a mutant vaccinia E3L∆83N, which lacks the Z-DNA-binding domain of vaccinia virulence factor E3. We compared the antitumor effects of intratumoral (IT) delivery of live OV-GM versus heat-inactivated OV-GM (heat-iOV-GM) in a murine B16-F10 melanoma bilateral implantation model. We also generated vvDD, a well-studied oncolytic vaccinia virus, and compared the antitumor effects of live vvDD vs heat-inactivated vvDD (heat-ivvDD) in a murine A20 B-cell lymphoma bilateral tumor implantation model.ResultsHeat-iOV-GM infection of dendritic cells (DCs) and tumor cells in vitro induced type I interferon and proinflammatory cytokines and chemokines, whereas live OV-GM did not. IT live OV-GM was less effective in generating systemic antitumor immunity compared with heat-iOV-GM. Similar to heat-iOV-GM, the antitumor effects of live OV-GM also require Batf3-dependent CD103+ dendritic cells. When combined with systemic delivery of ICB, IT heat-iOV-GM was more effective in eradicating tumors, compared with live OV-GM. IT heat-ivvDD was also more effective in treating murine A20 B-cell lymphoma, compared with live vvDD.ConclusionsTumor lysis induced by the replication of oncolytic vaccinia virus has a limited effect on the generation of systemic antitumor immunity. The activation of Batf3-dependent CD103+ DCs is critical for antitumor effects induced by both live OV-GM and heat-iOV-GM, with the latter being more potent than live OV-GM in inducing innate and adaptive immunity in both locally injected and distant, non-injected tumors. We propose that evaluations of both innate and adaptive immunity, induced by IT oncolytic viral immunotherapy at injected and non-injected tumors, should be included as potential biomarkers for host responses to viral therapy.

Additional file 6. The DNA sequence of targeted editing of AtBRI1 by FT-AtBRI1-sgRNA.

Additional file 3. The DNA sequence of targeted editing of AtBRI1 by AtBRI1-sgRNA.

Additional file 2. The DNA sequence of targeted editing of AtGL2 by AtGL2-sgRNA.

Additional file 4. CLCrV-mediated targeted knockout of AtPDS.

Additional file 5. Tissue-specific Cas9 system for mutation detection of AtBRI1 and AtGL2.

Additional file 1. Primer sequences involved in this study.

The pulmonary immune system consists of a network of tissue-resident cells as well as immune cells that are recruited to the lungs during infection and/or inflammation. How these immune components function during an acute poxvirus infection is not well understood. Intranasal infection of mice with vaccinia virus causes lethal pneumonia and systemic dissemination. Here we report that vaccinia C7 is a crucial virulence factor that blocks activation of the transcription factor IRF3. We provide evidence that type II alveolar epithelial cells (AECIIs) respond to pulmonary infection of vaccinia virus by inducing IFN-β and IFN-stimulated genes via the activation of the MDA5 and STING-mediated nucleic acid-sensing pathways and the type I IFN positive feedback loop. This leads to the recruitment and activation of CCR2+ inflammatory monocytes in the infected lungs and subsequent differentiation into Lyve1− interstitial macrophages (Lyve1− IMs), which efficiently engulf viral particles and block viral replication. Our results provide insights into how innate immune sensing of viral infection by lung AECIIs influences the activation and differentiation of CCR2+ inflammatory monocytes to defend against pulmonary poxvirus infection.

Advanced cancers remain a therapeutic challenge despite recent progress in targeted therapy and immunotherapy. Novel approaches are needed to alter the tumor immunosuppressive microenvironment and to facilitate the recognition of tumor antigens that leads to antitumor immunity. Poxviruses, such as modified vaccinia virus Ankara (MVA), have potential as immunotherapeutic agents. We show that infection of conventional dendritic cells (DCs) with heat- or ultraviolet-inactivated MVA leads to higher levels of interferon induction than MVA alone through the cGAS (cyclic guanosine monophosphate-adenosine monophosphate synthase)-STING cytosolic DNA-sensing pathway. Intratumoral injection of inactivated MVA (iMVA) was effective and generated adaptive antitumor immunity in murine melanoma and colon cancer models. iMVA-induced antitumor therapy was less effective in STING- or Batf3-deficient mice than in wild-type mice, indicating that both cytosolic DNA sensing and Batf3-dependent CD103+/CD8α+ DCs are essential for iMVA immunotherapy. The combination of intratumoral delivery of iMVA and systemic delivery of immune checkpoint blockade generated synergistic antitumor effects in bilateral tumor implantation models as well as in a unilateral large established tumor model. Our results suggest that inactivated vaccinia virus could be used as an immunotherapeutic agent for human cancers.

SUMMARY The drug DMXAA (5,6-dimethylxanthenone-4-acetic acid) showed therapeutic promise against solid tumors in mouse models but subsequently failed in human clinical trials. DMXAA was later discovered to activate mouse, but not human, STING, an adaptor protein in the cyclic dinucleotide cGAMP-mediated signaling pathway, inducing type I interferon expression. To facilitate the development of compounds that target human STING, we combined structural, biophysical, and cellular assays to study mouse and human chimeric proteins and their interaction with DMXAA. We identified a single substitution (G230I) that enables a DMXAA-induced conformational transition of hSTING from an inactive “open” to an active “closed” state. We also identified a substitution within the binding pocket (Q266I) that cooperates with G230I and the previously identified S162A binding-pocket point substitution, rendering hSTING highly sensitive to DMXAA. These findings should facilitate the reciprocal engineering of DMXAA analogs that bind and stimulate wild-type hSTING and their exploitation for vaccine-adjuvant and anti-cancer drug development., Graphical abstract

O1 IL-15 primes an mTOR-regulated gene-expression program to prolong anti-tumor capacity of human natural killer cells #### Andreas Lundqvist1, Vincent van Hoef1, Xiaonan Zhang1, Erik Wennerberg2, Julie Lorent1, Kristina Witt1, Laia Masvidal Sanz1, Shuo Liang1, Shannon Murray3, Ola Larsson1

Oncolytic viruses can be engineered to become multifunctional cancer immunotherapeutic agents, used either as monotherapy or in combination with immune checkpoint blockade for antitumor effects. Intratumoral (IT) delivery of oncolytic viruses expressing immunomodulatory agents can alter the tumor immunosuppressive microenvironment and facilitate the proliferation and activation of antitumor effector and memory T cells. Although anti-cytotoxic T lymphocyte protein 4 (CTLA-4) antibody has been approved for the treatment of advanced melanoma, its toxicity profile and modest efficacy as a single agent have limited its use in the clinic. Poxviruses are large cytoplasmic DNA viruses and vaccinia virus is a prototypic poxvirus that has been investigated intensively as an oncolytic virus. Its 200-kb genome size allows large insertions of multiple foreign genes. Vaccinia virus has been used in humans extensively in the past during smallpox vaccination. In this study, we engineered a vaccinia (Western Reserve)-based oncolytic virus that expresses anti-muCTLA-4 antibody and human FMS-like tyrosine kinase 3 ligand (Flt3L) for the treatment of murine implantable melanoma via intratumoral injection. We used a mutant vaccinia virus E3LΔ83N as the parental virus. This virus has a deletion of the Z-DNA-binding domain of E3, a key virulence factor, which results in 1000-fold attenuation of the virus. Through homologous recombination at the thymidine kinase (TK) locus of vaccinia E3LΔ83N virus, we successfully generated an attenuated recombinant virus (E3LΔ83N-TK--hFlt3L-anti-muCTLA-4) with a deletion of TK and an insertion of a cassette that allows the expression of both hFlt3L and anti-muCTLA-4 antibody under the vaccinia synthetic early and late promoter. This virus replicates in murine and human tumor cell lines and expresses desired anti-muCTLA-4 antibody and hFlt3L in murine and human melanoma cell lines. IT delivery of this recombinant vaccinia virus is more efficacious compared with E3LΔ83N-TK- (used either alone or in combination of systemic delivery of anti-CTLA-4 antibody), in eradicating or delaying the growth of both injected tumors and non-injected distant tumors, as well as in prolonging the survival of mice in a murine bilateral B16-F10 melanoma tumor implantation model. Furthermore, immunological analyses of tumor-infiltrating lymphocytes, as well as antitumor T cells in spleens, showed highest numbers of activated Granzyme B+ CD4+ and CD8+ T cells in the non-injected distant tumors and highest numbers of antitumor CD8+ T cells in the spleens of mice treated with IT delivery of E3LΔ83N-TK--hFlt3L-anti-muCTLA-4 compared with those treated with E3LΔ83N-TK--hFlt3L or E3LΔ83N-TK-. No toxicities related to IT E3LΔ83N-TK--hFlt3L-anti-muCTLA-4 have been observed. Taken together, our results demonstrate that IT delivery of oncolytic vaccinia virus expressing anti-CTLA-4 and hFlt3L is a safe and effective strategy to enhance antitumor immunity. Citation Format: Weiyi Wang, Peihong Dai, Ning Yang, Stewart Shuman, Wei Yan, Taha Merghoub, Jedd D. Wolchok, Liang Deng. Oncolytic vaccinia virus expressing immune checkpoint blockade antibody as cancer immunotherapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-306.

Vaccinia virus C7 protein is an important host-range factor for the vaccinia virus life cycle in mammalian cells. Type I IFN plays an important role in host defense of viral infection. However, the role of C7 in immune modulation of the IFN pathway is unclear. Here, we find that C7 functions as a dual inhibitor of IFN production and IFN signaling by interfering with the activation of two transcription factors IRF3 and STAT2. Furthermore, vaccinia virus with deletion of C7 (VACVΔC7L) is non-pathogenic in wild-type C57BL/6J mice in an intranasal infection model. We elucidated the immunological mechanisms underlying the attenuation phenotype of VACVΔC7L, which can be summarized as follows: (i) intranasal infection of VACVΔC7L triggers the production of interferons (IFNs), proinflammatory cytokines and chemokines in bronchoalveolar lavage fluid (BAL), whereas WT VACV does not; (ii) intranasal infection of VACVΔC7L results in the recruitment of dendritic cells, inflammatory monocytes, neutrophils, CD8+, and CD4+ in the BAL, whereas WT VACV does not; and (iii) infection of primary type II lung alveolar epithelial cells (AECs) with VACVΔC7L induces IFN, proinflammatory cytokine and chemokine gene expression and protein secretion, whereas WT VACV does not. However, VACVΔC7L dramatically gained virulence in STAT2 or IFNAR1-deficient mice, with increased titers in the lungs and systemic dissemination of the viruses to the blood and distant organs. Taken together, our results provide evidence that vaccinia C7 is a key virulence factor that antagonize both IFN production and signaling and IFNAR1 and STAT2 are critical in restricting vaccinia viral replication in the lung alveolar epithelial cells.

Skin keratinocytes provide a first line of defense against invading microorganisms in two ways: (i) by acting as a physical barrier to pathogen entry and (ii) by initiating a vigorous innate immune response upon sensing danger signals. How keratinocytes detect virus infections and generate antiviral immune responses is not well understood. Orthopoxviruses are dermatotropic DNA viruses that cause lethal disease in humans. Virulence in animal models depends on the virus-encoded bifunctional Z-DNA/double-stranded RNA (dsRNA)-binding protein E3. Here, we report that infection of mouse primary keratinocytes with a vaccinia ΔE3L mutant virus triggers the production of beta interferon (IFN-β), interleukin-6 (IL-6), CCL4, and CCL5. None of these immune mediators is produced by keratinocytes infected with wild-type vaccinia virus. The dsRNA-binding domain of E3 suffices to prevent activation of the innate immune response. ΔE3L induction of IFN-β, IL-6, CCL4, and CCL5 secretion requires mitochondrial antiviral signaling protein (MAVS; an adaptor for the cytoplasmic viral RNA sensors RIG-I and MDA5) and the transcription factor IRF3. IRF3 phosphorylation is induced in keratinocytes infected with ΔE3L, an event that depends on MAVS. The response of keratinocytes to ΔE3L is unaffected by genetic ablation of Toll-like receptor 3 (TLR3), TRIF, TLR9, and MyD88.

The bioterror threat of a smallpox outbreak in an unvaccinated population has mobilized efforts to develop new antipoxviral agents. By screening a library of known drugs, we identified 13 compounds that inhibited vaccinia virus replication at noncytotoxic doses. The anticancer drug mitoxantrone is unique among the inhibitors identified in that it has no apparent impact on viral gene expression. Rather, it blocks processing of viral structural proteins and assembly of mature progeny virions. The isolation of mitoxantrone-resistant vaccinia strains underscores that a viral protein is the likely target of the drug. Whole-genome sequencing of mitoxantrone-resistant viruses pinpointed missense mutations in the N-terminal domain of vaccinia DNA ligase. Despite its favorable activity in cell culture, mitoxantrone administered intraperitoneally at the maximum tolerated dose failed to protect mice against a lethal intranasal infection with vaccinia virus.

Vaccinia virus is a prototypic poxvirus, and during the life cycle of the virus, viral DNA and RNA can be detected by the nucleic acid-sensing pathways to induce the expression of type I IFN and IFN-stimulating genes, which leads to the establishment of antiviral state. Poxviruses have evolved multiple strategies to evade host immunity. Vaccinia E3, which consists of two distinct Z-DNA-binding and dsRNA-binding domains, is a critical virulence factor. Virus lacks E3L gene (ΔE3L) is non-pathogenic in wild-type mice in an intranasal infection model. We have previously reported that ΔE3L infection of murine primary keratinocytes (KCs) induces Ifnb, Il6, Ccl5 and Ccl4 gene expression and protein secretion via a MAVS/IRF3-dependent mechanism. Here we show that MDA5, a cytosolic dsRNA sensor, is essential for the induction of innate immunity by ΔE3L in KCs, whereas PKR, another cytosolic dsRNA sensor, is not. ΔE3L gains virulence in MDA5, MAVS, or IRF3-deficient mice, but not in STING-or MyD88-deficient mice, in an intranasal infection model. The MDA5-, MAVS-, or IRF3-deficient mice lost 20% of weight but all recovered in response to ΔE3L infection. By contrast, both the MDA5 and STING-mediated sensing pathways contribute to host defense against WT vaccinia virus infection. Lastly, whereas ΔE3L virus fails to replicate in wild-type murine primary fibroblasts, it gains replication capacity in cGAS or STING-deficient murine primary fibroblasts. Taken together, these results indicate that vaccinia viral nucleic acids are detected via the cytosolic DNA- and/or RNA-sensing pathways in a cell-type dependent manner, and both pathways play important and non-redundant roles in host defense against vaccinia infection.

Poxviruses are large cytoplasmic DNA viruses that have potentials as vaccine vectors and oncolytic agents. Although almost all of the oncolytic vaccinia viruses that are being investigated in clinical trials are replication competent, it is still unclear how these viruses exert antitumor immunologic effects, and whether viral replication is even necessary to achieve systemic antitumor efficacy. In this study, we compared therapeutic efficacy of the intratumoral delivery of the live, attenuated, replication competent oncolytic vaccinia virus expressing mGM-CSF (VC-TK−-mGM-CSF)vs. its heat-inactivated counterpart (iVC-TK−-mGM-CSF) in two bilateral tumor implantation models- B16-F10 murine melanoma and MC38 colon adenocarcinoma. We found that intratumoral injection of inactivated virus exerted stronger systemic antitumor effects than the live virus. FACS analyses of tumor infiltrating immune cells revealed that intratumoral injection of iVC-TK−-mGM-CSF lead to increased percentages and numbers of activating effector CD8+ and CD4+ T cells and reduced percentages and numbers of CD4+Foxp3+ regulatory T cells in the non-injected tumors compared with intratumoral delivery of the live virus. Infection of bone marrow-derived dendritic cells (DCs) with iVC-TK−-mGM-CSF induced type I IFN and proinflammatory cytokine and chemokine production and DC maturation, whereas the live virus did not. Taken together, our results demonstrate that intratumoral delivery of inactivated vaccina virus is safer and more efficacious than live oncolytic vaccinia virus expressing mGM-CSF in two murine bilateral tumor implantation models. Our findings have important implications for the design of poxviral-based immunotherapeutics for patients with metastatic cancers. Note: This abstract was not presented at the conference. Citation Format: Weiyi Wang, Peihong Dai, Ning Yang, Stewart Shuman, Taha Merghoub, Jedd D. Wolchok, Liang Deng. Intratumoral delivery of inactivated vaccinia virus is more efficacious than live oncolytic vaccinia virus in murine bilateral tumor implantation models [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A007.

Type I interferon (IFN), well known for its antiviral activity, is a critical component of cancer immune surveillance through its direct actions on cancer cells as well as on tumor microenvironment. Advanced melanoma remains a therapeutic challenge despite recent progress in targeted therapy and immunotherapy. Novel approaches are needed to alter the tumor immune suppressive microenvironment and to facilitate the recognition of tumor antigens that leads to antitumor immunity. Poxviruses are cytosolic DNA viruses that have been investigated as oncolytic and immunotherapeutic agents. We recently reported that the highly attenuated modified vaccinia virus Ankara (MVA), a safe vaccine for smallpox, triggers type I IFN production in conventional dendritic cells (cDCs) via the cytosolic DNA sensor cGAS and its adaptor STING, and that it requires transcription factors IRF3 and IRF7. Here we show that infection of cDCs with heat-inactivated MVA leads to higher levels of IFN induction than MVA. This induction is also mediated by the cytosolic DNA-sensing pathway cGAS/STING/IRF3/IRF7. In addition, we found that intratumoral injection of Heat-MVA caused tumor eradication in a murine B16 melanoma model as well as the generation of adaptive anti-tumor immunity. Furthermore, Heat-MVA-induced anti-tumor therapy is less effective in STING, IRF7, or Batf3-deficient mice than in wild-type mice, indicating that both the innate immune-sensing pathway and CD8α+DCs are essential for Heat-MVA-based immunotherapy. Lastly, the combination of intratumoral delivery of Heat-MVA with systemic delivery of anti-CTLA-4, PD-1 or PD-L1 antibodies achieved enhanced efficacy in tumor eradication and survival than Heat-MVA alone. Our results have strong implications for the development of poxvirus-based cancer immunotherapeutics as well novel strategies to overcome resistance to immune checkpoint blockade therapy. Citation Format: Peihong Dai, Weiyi Wang, Cristian Serna-Tamayo, Dimitriy Zamarin, Stewart Shuman, Taha Merghoub, Jedd D. Wolchok, Liang Deng. Heat-inactivated modified vaccinia virus ankara induces type I IFN and antitumor immunity via the cytosolic DNA-sensing pathway. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B031.

Plasmacytoid dendritic cells (pDCs) play important roles in antiviral innate immunity by producing type I interferon (IFN). In this study, we assess the immune responses of primary human pDCs to two poxviruses, vaccinia and myxoma virus. Vaccinia, an orthopoxvirus, was used for immunization against smallpox, a contagious human disease with high mortality. Myxoma virus, a Leporipoxvirus, causes lethal disease in rabbits, but is non-pathogenic in humans. We report that myxoma virus infection of human pDCs induces IFN-α and TNF production, whereas vaccinia infection does not. Co-infection of pDCs with myxoma virus plus vaccinia blocks myxoma induction effects. We find that heat-inactivated vaccinia (Heat-VAC; by incubating the virus at 55°C for 1 h) gains the ability to induce IFN-α and TNF in primary human pDCs. Induction of IFN-α in pDCs by myxoma virus or Heat-VAC is blocked by chloroquine, which inhibits endosomal acidification required for TLR7/9 signaling, and by inhibitors of cellular kinases PI3K and Akt. Using purified pDCs from genetic knockout mice, we demonstrate that Heat-VAC-induced type I IFN production in pDCs requires the endosomal RNA sensor TLR7 and its adaptor MyD88, transcription factor IRF7 and the type I IFN feedback loop mediated by IFNAR1. These results indicate that (i) vaccinia virus, but not myxoma virus, expresses inhibitor(s) of the poxvirus sensing pathway(s) in pDCs; and (ii) Heat-VAC infection fails to produce inhibitor(s) but rather produces novel activator(s), likely viral RNA transcripts that are sensed by the TLR7/MyD88 pathway. Using vaccinia gene deletion mutants, we show that the Z-DNA/RNA binding domain at the N-terminus of the vaccinia immunomodulatory E3 protein is an antagonist of the innate immune response of human pDCs to poxvirus infection and TLR agonists. The myxoma virus ortholog of vaccinia E3 (M029) lacks the N-terminal Z-DNA/RNA binding domain, which might contribute to the immunostimulating properties of myxoma virus.

Langerhans cells (LCs) are antigen-presenting cells in the skin that play sentinel roles in host immune defense by secreting proinflammatory molecules and activating T cells. Here we studied the interaction of vaccinia virus with XS52 cells, a murine epidermis-derived dendritic cell line that serves as a surrogate model for LCs. We found that vaccinia virus productively infects XS52 cells, yet this infection displays an atypical response to anti-poxvirus agents. Whereas adenosine N1-oxide blocked virus production and viral protein synthesis during a synchronous infection, cytosine arabinoside had no effect at concentrations sufficient to prevent virus replication in BSC40 monkey kidney cells. Vaccinia virus infection of XS52 cells not only failed to elicit the production of various cytokines, including tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-6, IL-10, IL-12 p40, alpha interferon (IFN-alpha), and IFN-gamma, it actively inhibited the production of proinflammatory cytokines TNF-alpha and IL-6 by XS52 cells in response to exogenous lipopolysaccharide (LPS) or poly(I:C). Infection with a vaccinia virus mutant lacking the E3L gene resulted in TNF-alpha secretion in the absence of applied stimuli. Infection of XS52 cells or BSC40 cells with the DeltaE3L virus, but not wild-type vaccinia virus, triggered proteolytic decay of IkappaBalpha. These results suggest a novel role for the E3L protein as an antagonist of the NF-kappaB signaling pathway. DeltaE3L-infected XS52 cells secreted higher levels of TNF-alpha and IL-6 in response to LPS and poly(I:C) than did cells infected with the wild-type virus. XS52 cells were productively infected by a vaccinia virus mutant lacking the K1L gene. DeltaK1L-infected cells secreted higher levels of TNF-alpha and IL-6 in response to LPS than wild-type virus-infected cells. Vaccinia virus infection of primary LCs harvested from mouse epidermis was nonpermissive, although a viral reporter protein was expressed in the infected LCs. Vaccinia virus infection of primary LCs strongly inhibited their capacity for antigen-specific activation of T cells. Our results highlight suppression of the skin immune response as a feature of orthopoxvirus infection.

Modified vaccinia virus Ankara (MVA) is an attenuated poxvirus that has been engineered as a vaccine against infectious agents and cancers. Our goal is to understand how MVA modulates innate immunity in dendritic cells (DCs), which can provide insights to vaccine design. In this study, using murine bone marrow-derived dendritic cells, we assessed type I interferon (IFN) gene induction and protein secretion in response to MVA infection. We report that MVA infection elicits the production of type I IFN in murine conventional dendritic cells (cDCs), but not in plasmacytoid dendritic cells (pDCs). Transcription factors IRF3 (IFN regulatory factor 3) and IRF7, and the positive feedback loop mediated by IFNAR1 (IFN alpha/beta receptor 1), are required for the induction. MVA induction of type I IFN is fully dependent on STING (stimulator of IFN genes) and the newly discovered cytosolic DNA sensor cGAS (cyclic guanosine monophosphate-adenosine monophosphate synthase). MVA infection of cDCs triggers phosphorylation of TBK1 (Tank-binding kinase 1) and IRF3, which is abolished in the absence of cGAS and STING. Furthermore, intravenous delivery of MVA induces type I IFN in wild-type mice, but not in mice lacking STING or IRF3. Treatment of cDCs with inhibitors of endosomal and lysosomal acidification or the lysosomal enzyme Cathepsin B attenuated MVA-induced type I IFN production, indicating that lysosomal enzymatic processing of virions is important for MVA sensing. Taken together, our results demonstrate a critical role of the cGAS/STING-mediated cytosolic DNA-sensing pathway for type I IFN induction in cDCs by MVA. We present evidence that vaccinia virulence factors E3 and N1 inhibit the activation of IRF3 and the induction of IFNB gene in MVA-infected cDCs., Author Summary Modified vaccinia virus Ankara (MVA) is an attenuated vaccinia strain with large deletions of the parental genome that render it non-replicative in mammalian cells. MVA is a safe and effective vaccine against both smallpox and monkeypox. MVA has been investigated as a vaccine vector for infectious diseases and cancers. Dendritic cells (DCs) play important roles in innate and adaptive immunity. A better understanding of how MVA is detected by innate immune sensors in DCs would guide the development of more effective MVA-based vaccines. We report our findings that MVA infection induces the production of type I interferon (IFN) in conventional dendritic cells via a cytosolic DNA-sensing pathway mediated by the newly discovered DNA sensor cGAS, its adaptor STING, and transcription factors IRF3 and IRF7. By contrast, wild-type vaccinia virus fails to activate this pathway. Furthermore, we show that vaccinia virulence factors E3 and N1 play inhibitory roles in the cytosolic DNA-sensing pathway.

Summary Binding of dsDNA by cyclic GMP-AMP (cGAMP) synthase (cGAS) triggers formation of the metazoan second messenger c[G(2′,5′)pA(3′,5′)p], which binds the signaling protein STING with subsequent activation of the interferon (IFN) pathway. We show that human hSTING H232 adopts a "closed" conformation upon binding c[G(2′,5′)pA(3′,5′)p] and its linkage isomer c[G(2′,5′)pA(2′,5′)p], as does mouse mSting R231 on binding c[G(2′,5′)pA(3′,5′)p], c[G(3′,5′)pA(3′,5′)p] and the antiviral agent DMXAA, leading to similar "closed" conformations. Comparing hSTING to mSting, 2′,5′-linkage-containing cGAMP isomers were more specific triggers of the IFN pathway compared to the all-3′,5′-linkage isomer. Guided by structural information, we identified a unique point mutation (S162A) placed within the cyclic-dinucleotide-binding site of hSTING that rendered it sensitive to the otherwise mouse-specific drug DMXAA, a conclusion validated by binding studies. Our structural and functional analysis highlights the unexpected versatility of STING in the recognition of natural and synthetic ligands within a small-molecule pocket created by the dimerization of STING.