Staphylococcus aureus Decreases SUMOylation Host Response to Promote Intramacrophage Survival.

Staphylococcus aureus is a commensal bacterium that causes severe infections in soft tissue and the bloodstream. During infection, S. aureus manipulates host cell response to facilitate its own replication and dissemination. Here, we show that S. aureus significantly decreases the level of SUMOylation, an essential post-translational modification, in infected macrophages 24 h post-phagocytosis. The reduced level of SUMOylation correlates with a decrease in the SUMO-conjugating enzyme Ubc9. The over-expression of SUMO proteins in macrophages impaired bacterial intracellular proliferation and the inhibition of SUMOylation with ML-792 increased it. Together, these findings demonstrated for the first time the role of host SUMOylation response toward S. aureus infection.

African Swine Fever Virus Ubiquitin-Conjugating Enzyme Is an Immunomodulator Targeting NF-κB 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 IKKß. 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.

Loss of fragile site-associated tumor suppressor promotes antitumor immunity via macrophage polarization.

Common fragile sites (CFSs) are specific breakage-prone genomic regions and are present frequently in cancer cells. The (E2-independent) E3 ubiquitin-conjugating enzyme FATS (fragile site-associated tumor suppressor) has antitumor activity in cancer cells, but the function of FATS in immune cells is unknown. Here, we report a function of FATS in tumor development via regulation of tumor immunity. Fats-/- mice show reduced subcutaneous B16 melanoma and H7 pancreatic tumor growth compared with WT controls. The reduced tumor growth in Fats-/- mice is macrophage dependent and is associated with a phenotypic shift of macrophages within the tumor from tumor-promoting M2-like to antitumor M1-like macrophages. In addition, FATS deficiency promotes M1 polarization by stimulating and prolonging NF-κB activation by disrupting NF-κB/IκBα negative feedback loops and indirectly enhances both CD4+ T helper type 1 (Th1) and cytotoxic T lymphocyte (CTL) adaptive immune responses to promote tumor regression. Notably, transfer of Fats-/- macrophages protects mice against B16 melanoma. Together, these data suggest that FATS functions as an immune regulator and is a potential target in cancer immunotherapy.

Antibody toolkit reveals N-terminally ubiquitinated substrates of UBE2W.

The ubiquitin conjugating enzyme UBE2W catalyzes non-canonical ubiquitination on the N-termini of proteins, although its substrate repertoire remains unclear. To identify endogenous N-terminally-ubiquitinated substrates, we discover four monoclonal antibodies that selectively recognize tryptic peptides with an N-terminal diglycine remnant, corresponding to sites of N-terminal ubiquitination. Importantly, these antibodies do not recognize isopeptide-linked diglycine (ubiquitin) modifications on lysine. We solve the structure of one such antibody bound to a Gly-Gly-Met peptide to reveal the molecular basis for its selective recognition. We use these antibodies in conjunction with mass spectrometry proteomics to map N-terminal ubiquitination sites on endogenous substrates of UBE2W. These substrates include UCHL1 and UCHL5, where N-terminal ubiquitination distinctly alters deubiquitinase (DUB) activity. This work describes an antibody toolkit for enrichment and global profiling of endogenous N-terminal ubiquitination sites, while revealing functionally relevant substrates of UBE2W.

Molecular Pathways of Interferon-Stimulated Gene 15: Implications in Cancer.

Human interferon-stimulated gene 15 (ISG15) is a 15-kDa ubiquitin-like protein that can be detected as either free ISG15 or covalently associated with its target proteins through a process termed ISGylation. Interestingly, extracellular free ISG15 has been proposed as a cytokinelike protein, whereas ISGylation is a posttranslational modification. ISG15 is a small protein with implications in some biological processes and pathologies that include cancer. This review highlights the findings of both free ISG15 and protein ISGylation involved in several molecular pathways, emerging as central elements in some cancer types.

Ubiquitin-Conjugating Enzyme 2S Enhances Viral Replication by Inhibiting Type I IFN Production through Recruiting USP15 to Deubiquitinate TBK1.

Type I interferon (IFN) plays an essential role in the host innate immune responses. Several ubiquitin-conjugating enzyme (E2) family members were reported to regulate type I IFN production and host antiviral immune responses. However, the molecular mechanisms are still not fully understood. Here, we report that UBE2S acts as a negative regulator in the type I IFN signaling pathway. Ectopic expression of UBE2S inhibits host antiviral immune responses and enhances viral replications, whereas deficiency of UBE2S enhances host antiviral immune responses and suppresses viral replications both in vitro and in vivo. Inhibition of type І IFN production by UBE2S is independent on its E2 and E3 enzymic activity. Mechanistically, UBE2S interacts with TBK1 and recruits ubiquitin-specific protease 15 (USP15) to remove Lys63 (K63)-linked polyubiquitin chains of TBK1. Our findings reveal a role of the UBE2S-USP15-TBK1 axis in the regulation of host antiviral innate immune responses.

Interaction between Brucella melitensis 16M and small ubiquitin-related modifier 1 and E2 conjugating enzyme 9 in mouse RAW264.7 macrophages.

Brucella is an intracellular pathogen that invades a host and settles in its immune cells; however, the mechanism of its intracellular survival is unclear. Modification of small ubiquitin-related modifier (SUMO) occurs in many cellular activities. E2 conjugating enzyme 9 (Ubc9) is the only reported ubiquitin-conjugating enzyme that links the SUMO molecule with a target protein. Brucella's intracellular survival mechanism has not been studied with respect to SUMO-related proteins and Ubc9. Therefore, to investigate the relationship between Brucella melitensis 16M and SUMO, we constructed plasmids and cells lines suitable for overexpression and knockdown of SUMO1 and Ubc9 genes. Brucella 16M activated SUMO1/Ubc9 expression in a time-dependent manner, and Brucella 16M intracellular survival was inhibited by SUMO1/Ubc9 overexpression and promoted by SUMO1/Ubc9 depletion. In macrophages, Brucella 16M-dependent apoptosis and immune factors were induced by SUMO1/Ubc9 overexpression and restricted by SUMO1/Ubc9 depletion. We noted no effect on the expressions of SUMO1 and Ubc9 in B. melitensis 16M lipopolysaccharide-prestimulated mouse RAW264.7 macrophages. Additionally, intracellular survival of the 16M△VirB2 mutant was lower than that of Brucella 16M (p < 0.05). VirB2 can affect expression levels of Ubc9, thereby increasing intracellular survival of Brucella in macrophages at the late stage of infection. Collectively, our results demonstrate that B. melitensis 16M may use the VirB IV secretion system of Brucella to interact with SUMO-related proteins during infection of host cells, which interferes with SUMO function and promotes pathogen survival in host cells.

A nanobody that recognizes a 14-residue peptide epitope in the E2 ubiquitin-conjugating enzyme UBC6e modulates its activity.

A substantial fraction of eukaryotic proteins is folded and modified in the endoplasmic reticulum (ER) prior to export and secretion. Proteins that enter the ER but fail to fold correctly must be degraded, mostly in a process termed ER-associated degradation (ERAD). Both protein folding in the ER and ERAD are essential for proper immune function. Several E2 and E3 enzymes localize to the ER and are essential for various aspects of ERAD, but their functions and regulation are incompletely understood. Here we identify and characterize single domain antibody fragments derived from the variable domain of alpaca heavy chain-only antibodies (VHHs or nanobodies) that bind to the ER-localized E2 UBC6e, an enzyme implicated in ERAD. One such VHH, VHH05 recognizes a 14 residue stretch and enhances the rate of E1-catalyzed ubiquitin E2 loading in vitroand interferes with phosphorylation of UBC6e in response to cell stress. Identification of the peptide epitope recognized by VHH05 places it outside the E2 catalytic core, close to the position of activation-induced phosphorylation on Ser184. Our data thus suggests a site involved in allosteric regulation of UBC6e's activity. This VHH should be useful not only to dissect the participation of UBC6e in ERAD and in response to cell stress, but also as a high affinity epitope tag-specific reagent of more general utility.

Ubc9 deficiency selectively impairs the functionality of common lymphoid progenitors (CLPs) during bone marrow hematopoiesis.

Hematopoietic development occurs in the bone marrow, and this process begins with hematopoietic stem cells (HSCs). Ubc9 is a unique E2-conjugating enzyme required for SUMOylation, an evolutionarily conserved post-translational modification system. We herein show that a conditional Ubc9 deletion in the hematopoietic system caused decreased thymus weight and reduced lymphocyte to myeloid cell ratio. Importantly, Ubc9 deletion in the hematopoietic system only selectively impaired the development of common lymphoid progenitors (CLPs) in the bone marrow and perturbed their potential to differentiate into lymphocytes, thereby decreasing the number of T/B cells in the periphery. Ubc9 was found to be required for CLP viability, and therefore, Ubc9 deficiency rendered CLPs to undergo apoptosis and attenuated their proliferation. Thus, Ubc9 plays a critical role in the regulation of CLP function during hematopoietic development in the bone marrow.

A role for S-nitrosylation of the SUMO-conjugating enzyme SCE1 in plant immunity.

SUMOylation, the covalent attachment of the small ubiquitin-like modifier (SUMO) to target proteins, is emerging as a key modulator of eukaryotic immune function. In plants, a SUMO1/2-dependent process has been proposed to control the deployment of host defense responses. The molecular mechanism underpinning this activity remains to be determined, however. Here we show that increasing nitric oxide levels following pathogen recognition promote S-nitrosylation of the Arabidopsis SUMO E2 enzyme, SCE1, at Cys139. The SUMO-conjugating activities of both SCE1 and its human homolog, UBC9, were inhibited following this modification. Accordingly, mutation of Cys139 resulted in increased levels of SUMO1/2 conjugates, disabled immune responses, and enhanced pathogen susceptibility. Our findings imply that S-nitrosylation of SCE1 at Cys139 enables NO bioactivity to drive immune activation by relieving SUMO1/2-mediated suppression. The control of global SUMOylation is thought to occur predominantly at the level of each substrate via complex local machineries. Our findings uncover a parallel and complementary mechanism by suggesting that total SUMO conjugation may also be regulated directly by SNO formation at SCE1 Cys139. This Cys is evolutionary conserved and specifically S-nitrosylated in UBC9, implying that this immune-related regulatory process might be conserved across phylogenetic kingdoms.

Neddylation inhibition upregulates PD-L1 expression and enhances the efficacy of immune checkpoint blockade in glioblastoma.

Pevonedistat (MLN4924), a specific NEDD8-activating enzyme inhibitor, has been considered as a promising treatment for glioblastoma, which is currently in Phase I/II clinical trials. On the other hand, inhibition of neddylation pathway substantially upregulates the expression of T cell negative regulator programmed death-ligand 1 (PD-L1), which might account for the potential resistance via evasion of immune surveillance checkpoints. Whether administration of anti-PD-L1 enhances the efficacy of pevonedistat through a cytotoxic T cell-dependent mechanism in glioblastoma needs to be investigated. Here, we report that depletion of neddylation pathway key enzymes markedly elevates PD-L1 expression in glioblastoma cancer cells. Consistently, neddylation inhibitor pevonedistat significantly enhances PD-L1 expression in both glioblastoma cancer cell lines and animal models. Mechanistically, pevonedistat increases PD-L1 mRNA levels mainly through inhibiting Cullin1-F-box and WD repeat domain-containing 7 E3 ligase activity and accumulating c-MYC proteins, a direct transcriptional activator of PD-L1 gene expression. In addition, inhibition of Cullin3 activity by pevonedistat also blocks PD-L1 protein degradation. Importantly, pevonedistat attenuates T cell killing through PD-L1 induction, and blockade of PD-L1 restores the sensitivity of pevonedistat-treated glioblastoma cancer cells to T cell killing. The combination of pevonedistat and anti-PD-L1 therapy compared to each agent alone significantly increased the therapeutic efficacy in vivo. Our study demonstrates inhibition of neddylation pathway suppresses cancer-associated immunity and provides solid evidence to support the combination of pevonedistat and PD-L1/programmed cell death protein 1 immune checkpoint blockade as a potential therapeutic strategy to treat glioblastoma.

The ATP-Binding Cassette Gene ABCF1 Functions as an E2 Ubiquitin-Conjugating Enzyme Controlling Macrophage Polarization to Dampen Lethal Septic Shock.

Sepsis is a bi-phasic inflammatory disease that threatens approximately 30 million lives and claims over 14 million annually, yet little is known regarding the molecular switches and pathways that regulate this disease. Here, we have described ABCF1, an ATP-Binding Cassette (ABC) family member protein, which possesses an E2 ubiquitin enzyme activity, through which it controls the Lipopolysaccharide (LPS)- Toll-like Receptor-4 (TLR4) mediated gram-negative insult by targeting key proteins for K63-polyubiquitination. Ubiquitination by ABCF1 shifts the inflammatory profile from an early phase MyD88-dependent to a late phase TRIF-dependent signaling pathway, thereby regulating TLR4 endocytosis and modulating macrophage polarization from M1 to M2 phase. Physiologically, ABCF1 regulates the shift from the inflammatory phase of sepsis to the endotoxin tolerance phase, and modulates cytokine storm and interferon-ß (IFN-ß)-dependent production by the immunotherapeutic mediator, SIRT1. Consequently, ABCF1 controls sepsis induced mortality by repressing hypotension-induced renal circulatory dysfunction.

Genetic rescue of lineage-balanced blood cell production reveals a crucial role for STAT3 antiinflammatory activity in hematopoiesis.

Blood cell formation must be appropriately maintained throughout life to provide robust immune function, hemostasis, and oxygen delivery to tissues, and to prevent disorders that result from over- or underproduction of critical lineages. Persistent inflammation deregulates hematopoiesis by damaging hematopoietic stem and progenitor cells (HSPCs), leading to elevated myeloid cell output and eventual bone marrow failure. Nonetheless, antiinflammatory mechanisms that protect the hematopoietic system are understudied. The transcriptional regulator STAT3 has myriad roles in HSPC-derived populations and nonhematopoietic tissues, including a potent antiinflammatory function in differentiated myeloid cells. STAT3 antiinflammatory activity is facilitated by STAT3-mediated transcriptional repression of Ube2n, which encodes the E2 ubiquitin-conjugating enzyme Ubc13 involved in proinflammatory signaling. Here we demonstrate a crucial role for STAT3 antiinflammatory activity in preservation of HSPCs and lineage-balanced hematopoiesis. Conditional Stat3 removal from the hematopoietic system led to depletion of the bone marrow lineage- Sca-1+ c-Kit+ CD150+ CD48- HSPC subset (LSK CD150+ CD48- cells), myeloid-skewed hematopoiesis, and accrual of DNA damage in HSPCs. These responses were accompanied by intrinsic transcriptional alterations in HSPCs, including deregulation of inflammatory, survival and developmental pathways. Concomitant Ube2n/Ubc13 deletion from Stat3-deficient hematopoietic cells enabled lineage-balanced hematopoiesis, mitigated depletion of bone marrow LSK CD150+ CD48- cells, alleviated HSPC DNA damage, and corrected a majority of aberrant transcriptional responses. These results indicate an intrinsic protective role for STAT3 in the hematopoietic system, and suggest that this is mediated by STAT3-dependent restraint of excessive proinflammatory signaling via Ubc13 modulation.

Inhibition of neddylation pathway represses influenza virus replication and pro-inflammatory responses.

The neddylation pathway belongs post-translational modifications and plays important roles in regulating viral infection and replication. To address the relationship of influenza A virus with the neddylation modification pathway, we demonstrate that IAV infection in A549 cells can activate the neddylation modification pathway to increase virus growth and enhance the expression of pro-inflammatory cytokines to increase pathogenicity. The pre-treatment of Nedd8-activating enzyme subunit 1 (NAE1)-specific inhibitor, MLN4924, interferes with Nedd8 conjugation and NF-κB activity. MLN4924 exhibited pronounced antiviral activity against different subtypes of influenza A virus, including classical H1N1 (PR8), H9N2 subtype, and pandemic H1N1 2009 (pdmH1N1) viruses. Through the inhibition of the CRL/NF-κB pathway, MLN4924 could significantly suppress the expression levels of pro-inflammatory cytokines induced by IAVs. These findings suggest that MLN4924 can be developed as a novel antiviral therapy for influenza infection for anti-viral efficacy and anti-inflammation activity.

Sodium Iodate-Induced Mouse Model of Age-Related Macular Degeneration Displayed Altered Expression Patterns of Sumoylation Enzymes E1, E2 and E3.

PURPOSE: Protein sumoylation is a highly dynamic and reversible post-translational modification, involving covalently conjugation of the small ubiquitin-like modifier (SUMO) to the lysine residue of the target protein. Similar to ubiquitination, sumoylation is catalyzed by E1, E2 and several E3 ligases. However, sumoylation usually does not cause protein degradation but alter the target function through diverse mechanisms. Increasing evidences have shown that sumoylation plays pivotal roles in the pathogenesis of human diseases, including neuron degeneration, cancer and heart disease, etc. We and others have shown that sumoylation is critically implicated in mouse eye development. However, the expression of sumoylation machinery has not been characterized in normal and pathogenic retina. Worldwide, age-related macular degeneration (AMD) is the leading cause of irreversible blindness in aged person. In the present study, we investigated the expression of the major sumoylation enzymes in normal mice and sodium iodateinduced AMD mouse model. METHODS: Four-week-old C57BL/6J mice were used in our experiment. A sterile 1% NaIO3 solution was freshly prepared in PBS from solid NaIO3. Experimental mice were injected with 70 mg/kg NaIO3, and similar volumes of PBS as control. Eyes were enucleated and immersion in FAA fixation overnight and processed for eye cross-sections. After fixation, cross sections eyes were dehydrated, embedded in paraffin, and 6 mm transverse sections were cut using the rotary microtome. Then paraffin sections were stained with hematoxylin and eosin (H&E), and mouse retinal thickness was observed to assess the histopathologic changes. RESULTS: Significantly declined RNA levels of E1, E2 and E3 ligase PIAS1 in NaIO3-injected mouse RPE one day-post treatment. Consistently, the protein level of PIAS1 was also decreased at this time point. At the late stage of treatment (three days post-injection), significantly reduced expression of E1 enzyme SAE1/UBA2 was detected in NaIO3-injected mouse retinas. In the contrary, dramatically increased E3 ligase RanBP2 was found in the injected-retinas. CONCLUSION: Together, our results demonstrated for the first time the dynamic expression of sumoylation pathway enzymes during the progression of retina degeneration induced by oxidative stress. Dynamic expression of E1, E2 and E3 enzymes were found during the time course of RPE and retina degeneration, which revealed the potential regulatory roles of sumoylation in AMD pathogenesis.

Ube2D3 and Ube2N are essential for RIG-I-mediated MAVS aggregation in antiviral innate immunity.

Innate immunity plays a pivotal role in virus infection. RIG-I senses viral RNA and initiates an effective innate immune response for type I interferon production. To transduce RIG-I-mediated antiviral signalling, a mitochondrial protein MAVS forms prion-like aggregates to activate downstream kinases and transcription factors. However, the activation mechanism of RIG-I is incompletely understood. Here we identify two ubiquitin enzymes Ube2D3 and Ube2N through chromatographic purification as activators for RIG-I on virus infection. We show that together with ubiquitin ligase Riplet, Ube2D3 promotes covalent conjugation of polyubiquitin chains to RIG-I, while Ube2N preferentially facilitates production of unanchored polyubiquitin chains. In the presence of these polyubiquitin chains, RIG-I induces MAVS aggregation directly on the mitochondria. Our data thus reveal two essential polyubiquitin-mediated mechanisms underlying the activation of RIG-I and MAVS for triggering innate immune signalling in response to viral infection in cells.

Innate immune signaling in Drosophila is regulated by transforming growth factor ß (TGFß)-activated kinase (Tak1)-triggered ubiquitin editing.

Coordinated regulation of innate immune responses is necessary in all metazoans. In Drosophila the Imd pathway detects Gram-negative bacterial infections through recognition of diaminopimelic acid (DAP)-type peptidoglycan and activation of the NF-κB precursor Relish, which drives robust antimicrobial peptide gene expression. Imd is a receptor-proximal adaptor protein homologous to mammalian RIP1 that is regulated by proteolytic cleavage and Lys-63-polyubiquitination. However, the precise events and molecular mechanisms that control the post-translational modification of Imd remain unclear. Here, we demonstrate that Imd is rapidly Lys-63-polyubiquitinated at lysine residues 137 and 153 by the sequential action of two E2 enzymes, Ubc5 and Ubc13-Uev1a, in conjunction with the E3 ligase Diap2. Lys-63-ubiquitination activates the TGFß-activated kinase (Tak1), which feeds back to phosphorylate Imd, triggering the removal of Lys-63 chains and the addition of Lys-48 polyubiquitin. This ubiquitin-editing process results in the proteasomal degradation of Imd, which we propose functions to restore homeostasis to the Drosophila immune response.

A Subset of Ubiquitin-Conjugating Enzymes Is Essential for Plant Immunity.

Of the three classes of enzymes involved in ubiquitination, ubiquitin-conjugating enzymes (E2) have been often incorrectly considered to play merely an auxiliary role in the process, and few E2 enzymes have been investigated in plants. To reveal the role of E2 in plant innate immunity, we identified and cloned 40 tomato genes encoding ubiquitin E2 proteins. Thioester assays indicated that the majority of the genes encode enzymatically active E2. Phylogenetic analysis classified the 40 tomato E2 enzymes into 13 groups, of which members of group III were found to interact and act specifically with AvrPtoB, a Pseudomonas syringae pv tomato effector that uses its ubiquitin ligase (E3) activity to suppress host immunity. Knocking down the expression of group III E2 genes in Nicotiana benthamiana diminished the AvrPtoB-promoted degradation of the Fen kinase and the AvrPtoB suppression of host immunity-associated programmed cell death. Importantly, silencing group III E2 genes also resulted in reduced pattern-triggered immunity (PTI). By contrast, programmed cell death induced by several effector-triggered immunity elicitors was not affected on group III-silenced plants. Functional characterization suggested redundancy among group III members for their role in the suppression of plant immunity by AvrPtoB and in PTI and identified UBIQUITIN-CONJUGATING11 (UBC11), UBC28, UBC29, UBC39, and UBC40 as playing a more significant role in PTI than other group III members. Our work builds a foundation for the further characterization of E2s in plant immunity and reveals that AvrPtoB has evolved a strategy for suppressing host immunity that is difficult for the plant to thwart.

The Friend of GATA Transcriptional Co-Regulator, U-Shaped, Is a Downstream Antagonist of Dorsal-Driven Prohemocyte Differentiation in Drosophila.

Recent studies suggest that mammalian hematopoietic stem and progenitor cells (HSPCs) respond directly to infection and inflammatory signaling. These signaling pathways also regulate HSPCs during steady-state conditions (absence of infection), and dysregulation may lead to cancer or age-related loss of progenitor repopulation capacity. Toll-like receptors (TLRs) are a major class of pathogen recognition receptors, and are expressed on the surface of immune effector cells and HSPCs. TLR/NF-κB activation promotes HSPCs differentiation; however, the mechanisms by which this signaling pathway alters the intrinsic transcriptional landscape are not well understood. Although Drosophila prohemocytes are the functional equivalent of mammalian HSPCs, a prohemocyte-specific function for Toll signaling has not been reported. Using Drosophila transgenics, we identified prohemocyte-specific roles for Toll pathway members, Dorsal and Cactus. We showed that Dorsal is required to limit the size of the progenitor pool. Additionally, we showed that activation of Toll signaling in prohemocytes drives differentiation in a manner that is analogous to TLR/NF-κB-driven HSPC differentiation. This was accomplished by showing that over-expression of Dorsal, or knockdown of Cactus, promotes differentiation. We also investigated whether Dorsal and Cactus control prohemocyte differentiation by regulating a key intrinsic prohemocyte factor, U-shaped (Ush), which is known to promote multipotency and block differentiation. We showed that Dorsal repressed Ush expression levels to promote differentiation, whereas Cactus maintained Ush levels to block differentiation. Additionally, we showed that another Toll antagonist, Lesswright, also maintained the level of Ush to block differentiation and promote proliferative quiescence. Collectively, these results identify a novel role for Ush as a downstream target of Toll signaling.

Autophagy Genes Enhance Murine Gammaherpesvirus 68 Reactivation from Latency by Preventing Virus-Induced Systemic Inflammation.

Host genes that regulate systemic inflammation upon chronic viral infection are incompletely understood. Murine gammaherpesvirus 68 (MHV68) infection is characterized by latency in macrophages, and reactivation is inhibited by interferon-γ (IFN-γ). Using a lysozyme-M-cre (LysMcre) expression system, we show that deletion of autophagy-related (Atg) genes Fip200, beclin 1, Atg14, Atg16l1, Atg7, Atg3, and Atg5, in the myeloid compartment, inhibited MHV68 reactivation in macrophages. Atg5 deficiency did not alter reactivation from B cells, and effects on reactivation from macrophages were not explained by alterations in productive viral replication or the establishment of latency. Rather, chronic MHV68 infection triggered increased systemic inflammation, increased T cell production of IFN-γ, and an IFN-γ-induced transcriptional signature in macrophages from Atg gene-deficient mice. The Atg5-related reactivation defect was partially reversed by neutralization of IFN-γ. Thus Atg genes in myeloid cells dampen virus-induced systemic inflammation, creating an environment that fosters efficient MHV68 reactivation from latency.