Ectromelia-encoded virulence factor C15 specifically inhibits antigen presentation to CD4+ T cells post peptide loading.

Smallpox and monkeypox pose severe threats to human health. Other orthopoxviruses are comparably virulent in their natural hosts, including ectromelia, the cause of mousepox. Disease severity is linked to an array of immunomodulatory proteins including the B22 family, which has homologs in all pathogenic orthopoxviruses but not attenuated vaccine strains. We demonstrate that the ectromelia B22 member, C15, is necessary and sufficient for selective inhibition of CD4+ but not CD8+ T cell activation by immunogenic peptide and superantigen. Inhibition is achieved not by down-regulation of surface MHC- II or co-stimulatory protein surface expression but rather by interference with antigen presentation. The appreciable outcome is interference with CD4+ T cell synapse formation as determined by imaging studies and lipid raft disruption. Consequently, CD4+ T cell activating stimulus shifts to uninfected antigen-presenting cells that have received antigen from infected cells. This work provides insight into the immunomodulatory strategies of orthopoxviruses by elucidating a mechanism for specific targeting of CD4+ T cell activation, reflecting the importance of this cell type in control of the virus.

Ectromelia virus induces tubulin cytoskeletal rearrangement in immune cells accompanied by a loss of the microtubule organizing center and increased α-tubulin acetylation.

Ectromelia virus (ECTV) is an orthopoxvirus that productively replicates in dendritic cells (DCs), but its influence on the microtubule (MT) cytoskeleton in DCs is not known. Here, we show that ECTV infection of primary murine granulocyte-macrophage colony stimulating factor-derived bone marrow cells (GM-BM) downregulates numerous genes engaged in MT cytoskeleton organization and dynamics. In infected cells, the MT cytoskeleton undergoes dramatic rearrangement and relaxation, accompanied by disappearance of the microtubule organizing centre (MTOC) and increased acetylation and stabilization of MTs, which are exploited by progeny virions for intracellular transport. This indicates a strong ability of ECTV to subvert the MT cytoskeleton of highly specialized immune cells.

The cGas-Sting Signaling Pathway Is Required for the Innate Immune Response Against Ectromelia Virus.

Activation of the DNA-dependent innate immune pathway plays a pivotal role in the host defense against poxvirus. Cyclic GMP-AMP synthase (cGAS) is a key cytosolic DNA sensor that produces the cyclic dinucleotide cGMP-AMP (cGAMP) upon activation, which triggers stimulator of interferon genes (STING), leading to type I Interferons (IFNs) production and an antiviral response. Ectromelia virus (ECTV) has emerged as a valuable model for investigating the host-Orthopoxvirus relationship. However, the role of cGas-Sting pathway in response to ECTV is not clearly understood. Here, we showed that murine cells (L929 and RAW264.7) mount type I IFN responses to ECTV that are dependent upon cGas, Sting, TANK binding kinase 1 (Tbk1), and interferon regulatory factor 3 (Irf3) signaling. Disruption of cGas or Sting expression in mouse macrophages blocked the type I IFN production and facilitated ECTV replication. Consistently, mice deficient in cGas or Sting exhibited lower type I IFN levels and higher viral loads, and are more susceptible to mousepox. Collectively, our study indicates that the cGas-Sting pathway is critical for sensing of ECTV infection, inducing the type I IFN production, and controlling ECTV replication.

Loss of Actin-Based Motility Impairs Ectromelia Virus Release In Vitro but Is Not Critical to Spread In Vivo.

Ectromelia virus (ECTV) is an orthopoxvirus and the causative agent of mousepox. Like other poxviruses such as variola virus (agent of smallpox), monkeypox virus and vaccinia virus (the live vaccine for smallpox), ECTV promotes actin-nucleation at the surface of infected cells during virus release. Homologs of the viral protein A36 mediate this function through phosphorylation of one or two tyrosine residues that ultimately recruit the cellular Arp2/3 actin-nucleating complex. A36 also functions in the intracellular trafficking of virus mediated by kinesin-1. Here, we describe the generation of a recombinant ECTV that is specifically disrupted in actin-based motility allowing us to examine the role of this transport step in vivo for the first time. We show that actin-based motility has a critical role in promoting the release of virus from infected cells in vitro but plays a minor role in virus spread in vivo. It is likely that loss of microtubule-dependent transport is a major factor for the attenuation observed when A36R is deleted.

A homolog of the variola virus B22 membrane protein contributes to ectromelia virus pathogenicity in the mouse footpad model.

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.

A lack of Fas/FasL signalling leads to disturbances in the antiviral response during ectromelia virus infection.

Ectromelia virus (ECTV) is an orthopoxvirus (OPV) that causes mousepox, the murine equivalent of human smallpox. Fas receptor-Fas ligand (FasL) signaling is involved in apoptosis of immune cells and virus-specific cytotoxicity. The Fas/FasL pathway also plays an important role in controlling the local inflammatory response during ECTV infection. Here, the immune response to the ECTV Moscow strain was examined in Fas (-) (lpr), FasL (-) (gld) and C57BL6 wild-type mice. During ECTV-MOS infection, Fas- and FasL mice showed increased viral titers, decreased total numbers of NK cells, CD4(+) and CD8(+) T cells followed by decreased percentages of IFN-γ expressing NK cells, CD4(+) and CD8(+) T cells in spleens and lymph nodes. At day 7 of ECTV-MOS infection, Fas- and FasL-deficient mice had the highest regulatory T cell (Treg) counts in spleen and lymph nodes in contrast to wild-type mice. Furthermore, at days 7 and 10 of the infection, we observed significantly higher numbers of PD-L1-expressing dendritic cells in Fas (-) and FasL (-) mice in comparison to wild-type mice. Experiments in co-cultures of CD4(+) T cells and bone-marrow-derived dendritic cells showed that the lack of bilateral Fas-FasL signalling led to expansion of Tregs. In conclusion, our results demonstrate that during ECTV infection, Fas/FasL can regulate development of tolerogenic DCs and Tregs, leading to an ineffective immune response.

TLR3 and TLR9 agonists improve postexposure vaccination efficacy of live smallpox vaccines.

Eradication of smallpox and discontinuation of the vaccination campaign resulted in an increase in the percentage of unvaccinated individuals, highlighting the need for postexposure efficient countermeasures in case of accidental or deliberate viral release. Intranasal infection of mice with ectromelia virus (ECTV), a model for human smallpox, is curable by vaccination with a high vaccine dose given up to 3 days postexposure. To further extend this protective window and to reduce morbidity, mice were vaccinated postexposure with Vaccinia-Lister, the conventional smallpox vaccine or Modified Vaccinia Ankara, a highly attenuated vaccine in conjunction with TLR3 or TLR9 agonists. We show that co-administration of the TLR3 agonist poly(I:C) even 5 days postexposure conferred protection, avoiding the need to increase the vaccination dose. Efficacious treatments prevented death, ameliorated disease symptoms, reduced viral load and maintained tissue integrity of target organs. Protection was associated with significant elevation of serum IFNα and anti-vaccinia IgM antibodies, modulation of IFNγ response, and balanced activation of NK and T cells. TLR9 agonists (CpG ODNs) were less protective than the TLR3 agonist poly(I:C). We show that activation of type 1 IFN by poly(I:C) and protection is achievable even without co-vaccination, requiring sufficient amount of the viral antigens of the infective agent or the vaccine. This study demonstrated the therapeutic potential of postexposure immune modulation by TLR activation, allowing to alleviate the disease symptoms and to further extend the protective window of postexposure vaccination.

EVM005: an ectromelia-encoded protein with dual roles in NF-κB inhibition and virulence.

Poxviruses contain large dsDNA genomes encoding numerous open reading frames that manipulate cellular signalling pathways and interfere with the host immune response. The NF-κB signalling cascade is an important mediator of innate immunity and inflammation, and is tightly regulated by ubiquitination at several key points. A critical step in NF-κB activation is the ubiquitination and degradation of the inhibitor of kappaB (IκBα), by the cellular SCFß-TRCP ubiquitin ligase complex. We show here that upon stimulation with TNFα or IL-1ß, Orthopoxvirus-infected cells displayed an accumulation of phosphorylated IκBα, indicating that NF-κB activation was inhibited during poxvirus infection. Ectromelia virus is the causative agent of lethal mousepox, a natural disease that is fatal in mice. Previously, we identified a family of four ectromelia virus genes (EVM002, EVM005, EVM154 and EVM165) that contain N-terminal ankyrin repeats and C-terminal F-box domains that interact with the cellular SCF ubiquitin ligase complex. Since degradation of IκBα is catalyzed by the SCFß-TRCP ubiquitin ligase, we investigated the role of the ectromelia virus ankyrin/F-box protein, EVM005, in the regulation of NF-κB. Expression of Flag-EVM005 inhibited both TNFα- and IL-1ß-stimulated IκBα degradation and p65 nuclear translocation. Inhibition of the NF-κB pathway by EVM005 was dependent on the F-box domain, and interaction with the SCF complex. Additionally, ectromelia virus devoid of EVM005 was shown to inhibit NF-κB activation, despite lacking the EVM005 open reading frame. Finally, ectromelia virus devoid of EVM005 was attenuated in both A/NCR and C57BL/6 mouse models, indicating that EVM005 is required for virulence and immune regulation in vivo.

Increased formation of autophagosomes in ectromelia virus-infected primary culture of murine bone marrow-derived macrophages.

Induction of autophagy by ectromelia virus (ECTV) in primary cultures of bone marrow-derived macrophages (BMDMs) was investigated. The results showed that ECTV infection of BMDMs resulted in increased formation of autophagosomes, increased level of LC3-II protein present in aggregates and extensive cytoplasmic vacuolization. These data indicate an increased autophagic activity in BMDMs during ECTV infection.

Antibody inhibition of a viral type 1 interferon decoy receptor cures a viral disease by restoring interferon signaling in the liver.

Type 1 interferons (T1-IFNs) play a major role in antiviral defense, but when or how they protect during infections that spread through the lympho-hematogenous route is not known. Orthopoxviruses, including those that produce smallpox and mousepox, spread lympho-hematogenously. They also encode a decoy receptor for T1-IFN, the T1-IFN binding protein (T1-IFNbp), which is essential for virulence. We demonstrate that during mousepox, T1-IFNs protect the liver locally rather than systemically, and that the T1-IFNbp attaches to uninfected cells surrounding infected foci in the liver and the spleen to impair their ability to receive T1-IFN signaling, thus facilitating virus spread. Remarkably, this process can be reversed and mousepox cured late in infection by treating with antibodies that block the biological function of the T1-IFNbp. Thus, our findings provide insights on how T1-IFNs function and are evaded during a viral infection in vivo, and unveil a novel mechanism for antibody-mediated antiviral therapy.

Studying NK cell responses to ectromelia virus infections in mice.

Here we describe methods for the in vivo study of antiviral NK cell responses using the mouse Orthopoxvirus ectromelia virus as a model, the agent of mousepox. The methods include those specific for the preparation and use of ectromelia virus such as the production of virus stocks in tissue culture and in live mice, the purification of virus stocks, the titration of virus stocks and virus loads in organs, and the infection of mice. The chapter also includes methods for the specific study of NK cell responses in infected mice such as the preparation of organs (lymph nodes, spleen, and liver) for analysis, the study of NK cell responses by flow cytometry, the adoptive transfer of NK cells, the measurement of NK cell cytolytic activity ex vivo and in vivo, and the determination of NK cell proliferation by bromodeoxyuridine loading or by dilution of carboxyfluorescein diacetate succinimidyl ester (CFSE).

[Orthopoxvirus genes for kelch-like proteins. III. Construction of mousepox (ectromelia) virus variants with targeted gene deletions].

Mousepox (ectromelia) virus genome contains four genes encoding for kelch-like proteins EVM018, EVM027, EVM150 and EVM167. A complete set of insertion plasmids was constructed to allow the production of recombinant ectromelia viruses with targeted deletions of one to four genes of kelch family both individually (single mutants) and in different combinations (double, triple and quadruple mutants). It was shown that deletion of any of the three genes EVMO18, EVM027 or EVM167 resulted in reduction of 50% lethal dose (LD50) by five and more orders in outbred white mice infected intraperitoneally. Deletion of mousepox kelch-gene EVM150 did not influence the virus virulence. Two or more kelch-genes deletion also resulted in high level of attenuation, which could evidently be due to the lack of three genes EVM167, EVM018 and/or EVM027 identified as virulence factors. The local inflammatory process on the model of intradermal injection of mouse ear pinnae (vasodilatation level, hyperemia, cutaneous edema, arterial thrombosis) was significantly more intensive for wild type virus and virulent mutant deltaEVM150 in comparison with avirulent mutant AEVM167.

Oral 1-O-octadecyl-2-O-benzyl-sn-glycero-3-cidofovir targets the lung and is effective against a lethal respiratory challenge with ectromelia virus in mice.

Hexadecyloxypropyl-cidofovir (HDP-CDV) has been shown to be orally active against lethal infection with orthopoxviruses including, mousepox, cowpox, vaccinia and rabbitpox. The alkoxyalkyl group provides oral absorption and reduces greatly the amount of drug reaching the kidney, the site of CDV's dose limiting toxicity. However, the amount of HDP-CDV detected in lung, an important site of early poxvirus replication, is low and the reduction of viral titers in surviving animals is reduced moderately compared with the liver where poxvirus titers are virtually undetectable. We synthesized a novel glycerol ester of CDV, 1-O-octadecyl-2-O-benzyl-sn-glycero-3-CDV (ODBG-CDV), and compared its oral pharmacokinetics with that of HDP-CDV. Surprisingly, ODBG-CDV levels in lung are much higher and liver levels are reduced, suggesting that the compound is transported in small intestinal lymph instead the portal vein. ODBG-CDV has excellent in vitro activity in cells infected with ectromelia virus (ECTV). In mice infected with a lethal aerosol or intranasal challenge of ECTV, HDP-CDV and ODBG-CDV are equally effective in preventing death from disease. Other drugs esterified to 1-O-octadecyl-2-O-benzyl-sn-glycerol or 1-O-octadecyl-2-O-benzyl-sn-glycerol-3-phosphate may provide lung targeting for treatment of microbial or neoplastic diseases while reducing first pass removal by the liver during oral absorption.

Involvement of Fas and FasL in Ectromelia virus-induced apoptosis in mouse brain.

In this study we showed that the virulent Moscow strain of Ectromelia virus (ECTV-MOS) infection leads to induction of apoptosis in the BALB/c mouse central nervous system. ECTV-MOS-infected cells and inflammation sites were found in brain parenchyma between 5 and 15 days after footpad infection with ECTV-MOS. Infected cells consisted of microglia and monocytes, astrocytes and oligodendrocytes and these type of cells underwent apoptosis within 5-15 days post infection (d.p.i.). The highest number of apoptotic cells was found at 5 and 10 d.p.i. and represented mainly microglia (61.4% and 38.6% of apoptotic cells, respectively) and astrocytes (21% and 8.9%, respectively). The number of apoptotic oligodendrocytes was 5.4% and 4.5%, respectively. Fluorometric assays demonstrated involvement of caspase-1, -3 and -8 but not caspase-9 in apoptosis in ECTV-MOS-infected mouse brains. Expression of Fas/FasL was significantly increased on ECTV-MOS-infected cells between 5 and 15 d.p.i., whereas Fas was up-regulated also on the surrounding, non-infected cells. Taking together we may conclude that ECTV-MOS infection of microglia and astrocytes leads to local inflammation resulting in Fas/FasL up-regulation and apoptosis, which limits mouse central nervous system infection with ECTV-MOS.

Selective penetration and pharmacological activity of lactosaminated albumin conjugates of adenine arabinoside 5-monophosphate (ara-AMP) in mouse liver.

With the aim of improving the chemotherapeutic index of adenine arabinoside 5-monophosphate (ara-AMP) in the treatment of chronic hepatitis B, this drug was conjugated with lactosaminated serum albumin (L-SA), a neoglycoprotein which only enters into hepatocytes where it is digested in lysosomes. In mice, the L-[3H]SA-ara-AMP conjugates, intravenously injected, selectively penetrated the liver, only small quantities were taken up by cells of spleen, bone marrow, intestine, and brain. After administration of the conjugate to mice with Ectromelia virus hepatitis, ara-AMP was selectively concentrated in liver in a pharmacologically active form. If L-SA-ara-AMP conjugates behave in man as in mouse, their administration to patients with chronic hepatitis B should result in a selective concentration of ara-AMP in liver with a more efficient inhibition of virus replication accompanied by lower toxicity for other tissues.