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Adiponectin-mediated pathways contribute to mammalian homeostasis; however, little is known about adiponectin and adiponectin receptor signaling in arthropods. In this study, we demonstrate that Ixodes scapularis ticks have an adiponectin receptor-like protein (ISARL) but lack adiponectin, suggesting activation by alternative pathways. ISARL expression is significantly upregulated in the tick gut after Borrelia burgdorferi infection, suggesting that ISARL signaling may be co-opted by the Lyme disease agent. Consistent with this, RNA interference (RNAi)-mediated silencing of ISARL significantly reduced the B. burgdorferi burden in the tick. RNA-seq-based transcriptomics and RNAi assays demonstrate that ISARL-mediated phospholipid metabolism by phosphatidylserine synthase I is associated with B. burgdorferi survival. Furthermore, the tick complement C1q-like protein 3 interacts with ISARL, and B. burgdorferi facilitates this process. This study identifies a new tick metabolic pathway that is connected to the life cycle of the Lyme disease spirochete., eLife digest Many countries around the world are seeing an increase in the number of patients diagnosed with Lyme disease, with often serious joint, heart, and neurologic complications. This illness is caused by species of ‘spirochete’ bacteria that live and multiply inside black-legged ticks, and get injected into mammals upon a bite. Ticks are not simply ‘syringes’ however, and a complex relationship is established between spirochetes and their host. This is particularly true since Lyme disease-causing bacteria such as Borrelia burgdorferi rely on ticks to obtain energy and nutrients. Tang, Cao et al. delved into these complex interactions by focusing on the molecular cascades (or pathways) involving adiponectin, a hormone essential for regulating sugar levels and processing fats. Analyses of gene and protein databases highlighted that ticks carry a receptor-like protein for adiponectin but not the hormone itself, suggesting that an alternative pathway is at play. This may involve B. burgdorferi, which gets its fats and sugars from its host. And indeed, experiments showed that ticks produced more of the adiponectin receptor-like protein when they carried B. burgdorferi; conversely, silencing the receptor reduced the number of surviving spirochetes inside the tick. Further exploration showed that the receptor mediates molecular cascades that help to process fat molecules; these are associated with spirochete survival. In addition, the receptor-like protein was activated by C1QL3, a ‘complement 1q domain-contained’ molecule which might be part of the tick energy-making or immune systems. Larger quantities of C1QL3 were found in ticks upon B. burgdorferi infection, suggesting that the spirochete facilitates an interaction that boosts activity of the adiponectin receptor-like protein. Overall, the work by Tang and Cao et al. revealed a new pathway which B. burgdorferi takes advantage of to infect their host and multiply. Targeting this molecular cascade could help to interfere with the life cycle of the spirochete, as well as fight Lyme disease and other insect-borne conditions.

Adiponectin-mediated pathways contribute to mammalian homeostasis; however, little is known about adiponectin and adiponectin receptor signaling in arthropods. In this study, we demonstrate that Ixodes scapularis ticks have an adiponectin receptor-like protein (ISARL) but lack adiponectin – suggesting activation by alternative pathways. ISARL expression is significantly upregulated in the tick gut after Borrelia burgdorferi infection suggesting that ISARL-signaling may be co-opted by the Lyme disease agent. Consistent with this, RNA interference (RNAi)-mediated silencing of ISARL significantly reduced the B. burgdorferi burden in the tick. RNA-seq-based transcriptomics and RNAi assays demonstrate that ISARL-mediated phospholipid metabolism by phosphatidylserine synthase I is associated with B. burgdorferi survival. Furthermore, the tick complement C1q-like protein 3 interacts with ISARL, and B. burgdorferi facilitates this process. This study identifies a new tick metabolic pathway that is connected to the life cycle of the Lyme disease spirochete.Significance StatementAdiponectin binds to adiponectin receptors and participates in glucose and lipid metabolism in mammals. In this study, we found that ticks have an adiponectin receptor-like protein but lack adiponectin. Importantly, we demonstrated that the Lyme disease agent, Borrelia burgdorferi, takes advantage of the adiponectin receptor signaling pathway to establish infection in its arthropod vector, Ixodes scapularis. Our study sheds light on the understanding of Borrelia-tick interactions and provides insights into how a human infectious disease agent may evolve to manipulate host metabolism for its own benefits. Understanding this pathway may lead to new ways to interfere with the Borrelia life cycle, and this mechanism may be applicable to additional microbes that are transmitted by ticks, mosquitoes or other arthropods.

Summary The TAM receptor, Axl, has been implicated as a candidate entry receptor for Zika virus (ZIKV) infection but has been shown as inessential for virus infection in mice. To probe the role of Axl in murine ZIKV infection, we developed a mouse model lacking the Axl receptor and the interferon alpha/beta receptor (Ifnar−/−Axl−/−), conferring susceptibility to ZIKV. This model validated that Axl is not required for murine ZIKV infection and that mice lacking Axl are resistant to ZIKV pathogenesis. This resistance correlates to lower pro-interleukin-1β production and less apoptosis in microglia of ZIKV-infected mice. This apoptosis occurs through both intrinsic (caspase 9) and extrinsic (caspase 8) manners, and is age dependent, as younger Axl-deficient mice are susceptible to ZIKV pathogenesis. These findings suggest that Axl plays an important role in pathogenesis in the brain during ZIKV infection and indicates a potential role for Axl inhibitors as therapeutics during viral infection., Graphical Abstract, Highlights • IFNAR−/−Axl−/− mice show Axl unnecessary for Zika virus replication in mice • Mice lacking Axl receptor are significantly resistant to Zika virus neuropathogenesis • IFNAR−/−Axl−/− mice have less ZIKV-driven caspase-dependent apoptosis in brain • Axl deficient mice have fewer apoptotic microglia after ZIKV infection, Biological Sciences; Neurotoxicology; Virology

The sperm calcium channel CatSper plays a central role in successful fertilization as a primary Ca2+ gateway into the sperm flagellum. However, CatSper’s complex subunit composition has impeded its reconstitution in vitro and structural elucidation. Here, we applied cryo-electron tomography to visualize the macromolecular organization of the native CatSper channel complex in intact mammalian sperm, as well as identified three additional CatSper-associated proteins. The repeating CatSper units form long zigzag-rows in four nanodomains along the flagella. In both mouse and human sperm, each CatSper repeat consists of a tetrameric pore complex. Murine CatSper contains an additional outwardly directed wing-structure connected to the tetrameric channel. The majority of the extracellular domains form a canopy above each pore-forming channel that interconnects to a zigzag-shaped roof. The intracellular domains link two neighboring channel complexes to a diagonal array. The loss of this intracellular link in Efcab9-/- sperm distorts the longitudinally aligned zigzag pattern and compromises flagellar movement. This work offers unique insights into the mechanisms underlying the assembly and transport of the CatSper complex to generate the nanodomains and provides a long-sought structural basis for understanding CatSper function in the regulation of sperm motility.

Background: Cancer immunotherapy strategies targeting blockade of the CD47-SIRPα immunosuppressive signal have made significant progress in recent years. However, monotherapies have not shown meaningful clinical responses in solid tumors. Therefore, therapeutic combinations are being explored to improve patient outcomes. CD47 is a macrophage checkpoint inhibitor that acts as a “don’t eat me” signal on cancer cells to evade innate immune detection and destruction. Targeted radiation to cancer cells will upregulate calreticulin (CRT), a pro-phagocytic “eat me” signal. We therefore hypothesize that we can enhance the efficacy of anti-CD47 antibodies by combining them with appropriate targeted antibody radioconjugates (ARC). In this experiment we chose to study an anti-HER3 radioconjugate, as HER3 is overexpressed in a variety of cancers including breast, ovarian, lung, gastric and prostate and is associated with poor clinical prognosis. Additionally, upregulation of HER3 is implicated in the acquired resistance against HER1 or HER2 targeted therapies. Here, we demonstrate enhanced therapeutic efficacy of a novel Actinium-225 (225Ac) armed HER3 specific targeting ARC (225Ac-HER3-ARC) and a CD47 blocking antibody (anti-CD47) combination in preclinical solid tumor models. Methods: The anti-HER3 antibody (AT-02) was radiolabeled with 225Ac. 225Ac-HER3-ARC biological activity was evaluated using human recombinant HER3 and receptor positive tumor cell lines. 225Ac-HER3-ARC mediated CRT upregulation and cytotoxicity was evaluated using flow cytometry and MTS assay, respectively. The benefits of the 225Ac-HER3-ARC and anti-CD47 combination to enhance macrophage phagocytosis was evaluated by flow cytometry. We further evaluated the therapeutic benefits of the 225Ac-HER3-ARC and CD47 combination in human HER3+ tumor xenograft mouse model. Results: The 225Ac-HER3-ARC retains similar binding properties to native antibody and demonstrates specific cytotoxicity on tumor cells. CRT was upregulated by 225Ac-HER3-ARC in HER3+ cells. Furthermore, the combination of 225Ac-HER3-ARC and anti-CD47 enhances in vitro macrophage mediated tumor cell phagocytosis compared to each agent alone. Importantly, the in vivo 225Ac-HER3-ARC and CD47 antibody combination shows enhanced antitumor effect with reduced toxicity and improved survival benefit in a human preclinical solid tumor model compared to anti-CD47 agent alone. Conclusions: We demonstrate enhanced efficacy of the 225Ac-HER3-ARC and CD47 blocking antibody combination in vitro and in a preclinical solid tumor animal model. This approach is an encouraging strategy to potentially improve antitumor responses in patients with HER3+ tumors. Consequently, the findings obtained in this study along with the need to develop better therapies for patients with HER3+ tumors support the further preclinical development of HER3-ARC. Citation Format: Denis Beckford-Vera, Jason Li, Caroline Jennings, Megan McCloskey, Amanda Chin, Qing Liang, Jesse Hwang, Monideepa Roy, Mary Chen, Helen Kotanides. Anti-HER3 radioimmunotherapy enhances the anti-tumor effects of CD47 blockade in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 609.

Background: HER3 overexpression is reported to be associated with poor survival in breast, ovarian, lung, gastric and prostate cancer. In addition, upregulation of HER3 in response to HER1 or HER2 targeted therapies, is implicated in the acquired resistance against these therapies. Therefore, effective targeting of HER3 can potentially overcome resistance and enhance therapeutic efficacy. Although a number of anti-HER3 antibodies have failed clinical testing with the development focus being shifted to other approaches such as antibody drug conjugates and bispecific antibodies, there are currently no approved HER3-targeted therapies. Here we describe a novel approach that can enhance therapeutic efficacy in HER3+ cancer patients by conjugating an anti-HER3 antibody with the alpha-emitting cytotoxic radioisotope Actinium-225 (225Ac) to create an anti-HER3 antibody radiation conjugate (225Ac-HER3-ARC). Alpha emitting radioisotopes like 225Ac can cause double-strand DNA breaks for which there is no known resistance mechanism. Due to the cytotoxic properties of the radioisotope, lower levels of antibody may be needed, resulting in reduced incidence or less severe toxicities. We hypothesize that targeting HER3 in solid tumors with an ARC will result in tumor specific cell killing especially in a setting where HER-targeting agents are not a viable option. We developed a novel 225Ac-HER3-ARC and evaluated its efficacy in HER3+ in vitro and in vivo tumor models. Methods: AT-02, an anti-HER3 antibody, was conjugated with p-SCN-Bn-DOTA and radiolabeled with 225Ac. 225Ac-HER3-ARC specific binding to HER3 was assessed by ELISA using human recombinant HER3 and by flow cytometry on HER3+ cells. The cytotoxic effect of HER3 ARC was evaluated in a panel of HER3 expressing cells. We further evaluated the maximum tolerated dose and therapeutic efficacy of the ARC in nude mice bearing human HER3+ xenograft tumors. Results: In this study we successfully radiolabeled anti-HER3 with 225Ac. 225Ac-HER3-ARC showed similar binding properties to those of the native antibody by ELISA (HER3-ARC: EC50 = 0.0017 µg/ml, HER3 EC50 = 0.0022 µg/ml) and flow cytometry. Treatment with ARC was cytotoxic to HER3+ cells in a dose-dependent manner (EC50 = 54 kBq/ml). 225Ac-HER3-ARC showed potent in vivo efficacy in preclinical solid tumor xenograft models that was correlated with the in vitro cytotoxicity findings. Treatment with 225Ac-HER3-ARC (7.4 - 22.2 kBq, 200 - 600 nCi) led to complete responses and significantly prolonged survival compared to control groups (p < 0.0001). Conclusions: Our findings demonstrate that targeting HER3 with a novel 225Ac-HER3-ARC results in potent tumor cell cytotoxicity and complete anti-tumor response in HER3 tumor xenograft model. This approach provides a promising therapeutic strategy for HER3 positive tumors and warrants further assessment. Citation Format: Denis Beckford-Vera, Jason Li, Megan McCloskey, Caroline Jennings, Amanda Chin, Qing Liang, Jesse Hwang, Monideepa Roy, Mary Chen, Helen Kotanides. Targeting HER3 receptor positive cancers with a novel anti-HER3 antibody radioconjugate (ARC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3306.

Macrophage scavenger receptor 1 (MSR1) mediates the endocytosis of modified low-density lipoproteins and plays an important antiviral role. However, the molecular mechanism underlying MSR1 antiviral actions remains elusive. We report that MSR1 activates autophagy to restrict infection of Chikungunya virus (CHIKV), an arthritogenic alphavirus that causes acute and chronic crippling arthralgia. Msr1 expression was rapidly upregulated after CHIKV infection in mice. Msr1 knockout mice had elevated viral loads and increased susceptibility to CHIKV arthritis along with a normal type I IFN response. Induction of LC3 lipidation by CHIKV, a marker of autophagy, was reduced in Msr1−/− cells. Mechanistically, MSR1 interacted with ATG12 through its cytoplasmic tail and this interaction was enhanced by CHIKV nsP1 protein. MSR1 repressed CHIKV replication through ATG5-ATG12-ATG16L1 and this was dependent on the FIP200-and-WIPI2-binding domain, but not the WD40 domain of ATG16L1. Our results elucidate an antiviral role for MSR1 involving the autophagic function of ATG5-ATG12-ATG16L1., Using Msr1 knockout mice, Long Yang et al. demonstrate that macrophage scavenger receptor 1 (MSR1) activates autophagy to restrict the proliferation of Chikungunya virus, an alphavirus that causes crippling joint stiffness. This study provides insights into how host cellular machinery fights off Chikungunya virus.

Macrophage scavenger receptor 1 (MSR1) mediates the endocytosis of modified low-density lipoproteins and plays an important antiviral role. However, the molecular mechanism underlying MSR1 antiviral actions remains elusive. Herein, we report that MSR1 activates autophagy to restrict infection of Chikungunya virus (CHIKV), an arthritogenic alphavirus that causes acute and chronic crippling arthralgia. Msr1 expression was rapidly upregulated after CHIKV infection in mice. Msr1 knockout mice had elevated viral loads and increased susceptibility to CHIKV arthritis along with a normal type I IFN response. Induction of LC3 lipidation by CHIKV, a marker of autophagy, was reduced in Msr1-/- cells. Mechanistically, MSR1 interacted with ATG12 through its cytoplasmic tail and this interaction was enhanced by CHIKV nsP1 protein. MSR1 repressed CHIKV replication through ATG5-ATG12-ATG16L1 and this was dependent on the FIP200-and-WIPI2-binding domain, but not the WD40 domain of ATG16L1. Our results elucidate an antiviral role for MSR1 involving the autophagic function of ATG5-ATG12-ATG16L1.

BackgroundOne mechanism that tumors use to escape immunosurveillance is the overexpression of CD47, which inhibits the macrophage mediated phagocytosis pathway. Although blockade of the CD47-SIRPα axis is a promising approach to enhance tumor targeted phagocytosis, anti-CD47 monotherapies have not shown meaningful responses in clinical studies of solid tumors. Combination cancer therapies aim to increase the probability of response in settings of resistance by combining drugs with different mechanisms of action. Antibody radioconjugates (ARCs) specifically target and deliver therapeutic radiation directly to cancer cells. We rationalized that the immunogenic and cytotoxic properties of ARCs will upregulate calreticulin (CRT), a pro-phagocytic signal, thereby synergizing with CD47 blocking therapies to enhance phagocytosis and antitumor activity. Here for the first time, we demonstrate the combination benefit of a HER2 specific targeting ARC and a CD47 blocking antibody to enhance therapeutic efficacy in preclinical solid tumor models.MethodsThe anti-HER2 antibody trastuzumab was conjugated with p-SCN-DOTA and radiolabeled with Ac-225 or Lu-177. The biological activity of both radioconjugates was evaluated using human recombinant HER2 and receptor positive tumor cell lines. The cytotoxic effect of radioconjugates and the ability to upregulate CRT was evaluated using XTT assay and flow cytometry, respectively, in a panel of HER2 expressing cells. To evaluate the synergy of anti-HER2 ARC and CD47 antibody combination in vitro, a flow cytometry macrophage phagocytosis assay was developed. We further evaluated the antitumor synergy in vivo between anti-HER2 ARC and CD47 antibody in human HER2 positive tumor xenograft mouse model.ResultsThe anti-HER2 ARCs have similar binding properties to native antibody and demonstrate specific cytotoxicity. Importantly, we observe ARC-mediated CRT upregulation in HER2 expressing cells. Furthermore, the combination of HER2 targeting ARC and CD47 blocking antibody enhances in vitro macrophage mediated tumor cell phagocytosis compared to each agent alone. Remarkably, the in vivo anti-HER2 ARC and CD47 antibody combination shows enhanced therapeutic effect with reduced toxicity and improved survival benefit in a human preclinical solid tumor model.ConclusionsHere for the first time, we demonstrate enhanced therapeutic efficacy between an anti-HER2 ARC and CD47 blocking antibody combination in a preclinical solid tumor model. The finding suggests that ARC mediated upregulation of CRT potentiates the pro-phagocytic signal and synergizes with the anti-CD47 mode of action thereby enhancing antitumor immune response. This combination mechanism provides a very promising strategy to improve therapeutic responses in patients harboring solid tumors and warrants further preclinical evaluation.Ethics ApprovalAll animal experiments were approved by IACUC.

BackgroundActimab-A, the anti-CD33 antibody lintuzumab armed with the radioisotope Actinium-225 (Ac-225), has demonstrated single agent antileukemic effects in patients with relapsed or refractory acute myeloid leukemia (AML). Up-regulation of CD47, a macrophage checkpoint that suppresses phagocytosis, is one mechanism by which myeloid malignancies such as AML can evade targeting by the innate immune response. Therapeutic blocking antibodies against this pathway have shown early clinical promise. We hypothesized that Actimab-A will enhance phagocytosis in AML cells by specifically upregulating calreticulin (CRT), a pro-phagocytic signal. Moreover, we hypothesized that combination of the anti-CD33 antibody radioconjugate (ARC) and CD47 blocking antibody could act in synergy to enhance therapeutic outcomes in AML compared to single agent. In this study, we examined, for the first time, the potential mechanistic benefit of combining the anti-CD33 ARC armed with Ac-225 or Lutetium-177 (Lu-177) and a CD47 blocking antibody, using in vitro and in vivo human AML preclinical models.MethodsLintuzumab was conjugated with p-SCN-DOTA and radiolabeled with Ac-225 or Lu-177. The biological activity of both radioconjugates was examined using human recombinant CD33 and receptor positive AML cells. The cytotoxic effect of radioconjugates and the ability to upregulate CRT was evaluated using XTT assay and flow cytometry, respectively, in a panel of CD33 expressing cells. To assess the therapeutic combination of anti-CD33 ARC and CD47 antibody in vitro, a flow cytometry macrophage phagocytosis assay was used. We further evaluated the therapeutic efficacy in vivo of anti-CD33 ARC and CD47 antibody combination in human AML mouse model.ResultsThe anti-CD33 ARCs have similar binding properties to native antibody and demonstrate specific cell cytotoxicity. We show ARC-mediated upregulation of cell surface CRT in a panel of CD33 expressing AML cells. Furthermore, the in vitro combination of CD33 targeting ARC and CD47 blocking antibody enhances macrophage mediated phagocytosis of AML cells compared to each monotherapy. Interestingly, the in vivo anti-CD33 ARC and CD47 antibody combination demonstrates a significant increase in survival and reduces toxicity in a human AML preclinical model.ConclusionsOur findings suggest a novel synergistic mechanism whereby the CD33 ARC targeted radiation induces upregulation of CRT, thereby potentiating a pro-phagocytic innate immune response in combination with anti-CD47 blocking antibody. More importantly, clinical translation of this approach could enhance therapeutic efficacy in AML and warrants further preclinical exploration.Ethics ApprovalAll animal studies were approved by IACUC.

Zika virus (ZIKV) can be transmitted by mosquito bite or sexual contact. Using mice that lack the type I interferon receptor, we examined sexual transmission of ZIKV. Electron microscopy analyses showed association of virions with developing sperm within testes as well as with mature sperm within epididymis. When ZIKV-infected male mice were mated with naive female mice, the weight of fetuses at embryonic day 18.5 was significantly reduced compared with the control group. Additionally, we found ocular deformities in a minority of the fetuses. These results suggest that ZIKV causes fetal abnormalities after female mating with an infected male.

The mosquito-borne chikungunya virus (CHIKV) causes acute pain and joint inflammation, and in recent years the virus has caused large epidemics in previously CHIKV-free geographic areas. To advance the understanding of host factors that antagonize CHIKV, we show that synthetic agonist of liver X receptor (LXR-623) inhibits CHIKV replication by upregulating the cholesterol exporter ABCA1 and that endogenous and pharmacological activation of interferon signaling pathway partners with LXR-623 to generate a superior antiviral state.

Saliva from the mosquito vector of flaviviruses is capable of changing the local immune environment, leading to an increase in flavivirus-susceptible cells at the infected bite site. In addition, an antibody response to specific salivary gland (SG) components changes the pathogenesis of flaviviruses in human populations. To investigate whether antigenic SG proteins are capable of enhancing infection with Zika virus (ZIKV), a reemerging flavivirus primarily transmitted by the Aedes aegypti mosquito, we screened for antigenic SG proteins using a yeast display library and demonstrate that a previously undescribed SG protein we term neutrophil stimulating factor 1 (NeSt1) activates primary mouse neutrophils ex vivo. Passive immunization against NeSt1 decreases pro-interleukin-1β and CXCL2 expression, prevents macrophages from infiltrating the bite site, protects susceptible IFNAR(−/−) IFNGR(–/–) (AG129) mice from early ZIKV replication, and ameliorates virus-induced pathogenesis. These findings indicate that NeSt1 stimulates neutrophils at the mosquito bite site to change the immune microenvironment, allowing a higher level of early viral replication and enhancing ZIKV pathogenesis. IMPORTANCE When a Zika virus-infected mosquito bites a person, mosquito saliva is injected into the skin along with the virus. Molecules in this saliva can make virus infection more severe by changing the immune system to make the skin a better place for the virus to replicate. We identified a molecule that activates immune cells, called neutrophils, to recruit other immune cells, called macrophages, that the virus can infect. We named this molecule neutrophil-stimulating factor 1 (NeSt1). When we used antibodies to block NeSt1 in mice and then allowed Zika virus-infected mosquitoes to feed on these mice, they survived much better than mice that do not have antibodies against NeSt1. These findings give us more information about how mosquito saliva enhances virus infection, and it is possible that a vaccine against NeSt1 might protect people against severe Zika virus infection.

The stimulator-of-interferon-gene (STING) pathway controls both DNA and RNA virus infection. STING is critical for induction of type I interferons (IFN-I) during DNA virus infection, while the molecular mechanism underlying the anti-RNA virus function of STING remains largely elusive. We show that the STING signaling pathway regulates expression of macrophage scavenger receptor 1 (MSR1), which activates a cell-intrinsic antiviral mechanism through autophagy related ATG5-ATG12. Mice deficient in Sting or Msr1 had increased viral replication and susceptibility to Chikungunya virus (CHIKV)-induced arthritis. Repression of CHIKV replication by MSR1 was dependent on ATG5-ATG12, but independent of other autophagy components such as ULK1 and Beclin 1. MSR1 interacted with ATG5–ATG12 following CHIKV infection, and this interaction was independent of canonical autophagy. Induction of MSR1 expression by CHIKV was partially dependent on the STING signaling. Our results elucidate an antiviral role of the STING-MSR1 axis involving the non-canonical autophagy-related function of ATG5-ATG12.

Mosquito-borne Zika virus (ZIKV) can cause congenital microcephaly and Guillain&ndash, Barr&eacute, syndrome, among other symptoms. Specific treatments and vaccines for ZIKV are not currently available. To further understand the host factors that support ZIKV replication, we used mass spectrometry to characterize mammalian proteins that associate with the ZIKV NS1 protein and identified the TRiC/CCT complex as an interacting partner. Furthermore, the suppression of CCT2, one of the critical components of the TRiC/CCT complex, inhibited ZIKV replication in both mammalian cells and mosquitoes. These results highlight an important role for the TRiC/CCT complex in ZIKV infection, suggesting that the TRiC/CCT complex may be a promising therapeutic target.

Chikungunya virus (CHIKV), an alphavirus spread by Aedes spp. mosquitos, causes severe inflammation and joint pain, progressing to a chronic arthralgic state in a subset of patients. Due to recent global epidemics of CHIKV and the potential for related viruses to cause outbreaks, multiple approaches to combat these pathogens are of interest. We report that SR9009, a synthetic agonist of nuclear receptors Rev-erb α/β, inhibits replication of multiple alphaviruses (CHIKV and O’nyong’nyong virus) mainly by suppressing structural protein synthesis, although viral RNA accumulation is relatively unimpeded. Furthermore, SR9009 reduces the inflammatory response in cultured murine macrophages exposed to alphavirus-infected cells.

Arboviruses represent a group of pathogens that can spread efficiently throughout human populations by hematophagous arthropod vectors. The mosquito-borne (re)emerging Chikungunya and Dengue viruses belong to the alphavirus and flavivirus genus, respectively, with no approved therapeutics or safe vaccines for humans. Transmitted by the same vector Aedes spp., these viruses cause significant morbidity and mortality in endemic areas. Due to the increasing likelihood of co-circulation and co-infection with viruses, we aimed to identify a pharmacologically targetable host factor that can inhibit multiple viruses and show that a potent antagonist of prolyl tRNA synthetase (halofuginone) suppresses both Chikungunya and Dengue viruses. Host tRNA synthetase inhibition may signify an additional approach to combat present and future epidemic pathogens.

Zika virus replicates in mouse testes and causes testicular atrophy, with implication on sexual transmission and male fertility., Zika virus (ZIKV) is an emerging mosquito-borne flavivirus that has recently been found to cause fetal infection and neonatal abnormalities, including microcephaly and neurological dysfunction. ZIKV persists in the semen months after the acute viremic phase in humans. To further understand the consequences of ZIKV persistence in males, we infected Ifnar1−/− mice via subcutaneous injection of a pathogenic but nonlethal ZIKV strain. ZIKV replication persists within the testes even after clearance from the blood, with interstitial, testosterone-producing Leydig cells supporting virus replication. We found high levels of viral RNA and antigen within the epididymal lumen, where sperm is stored, and within surrounding epithelial cells. Unexpectedly, at 21 days post-infection, the testes of the ZIKV-infected mice were significantly smaller compared to those of mock-infected mice, indicating progressive testicular atrophy. ZIKV infection caused a reduction in serum testosterone, suggesting that male fertility can be affected. Our findings have important implications for nonvector-borne vertical transmission, as well as long-term potential reproductive deficiencies, in ZIKV-infected males.

The strong association of Zika virus infection with congenital defects has led to questions of how a flavivirus is capable of crossing the placental barrier to reach the fetal brain. Here, we demonstrate permissive Zika virus infection of primary human placental macrophages, commonly referred to as Hofbauer cells, and placental villous fibroblasts. We also demonstrate Zika virus infection of Hofbauer cells within the context of the tissue ex vivo using term placental villous explants. In addition to amplifying infectious virus within a usually inaccessible area, the putative migratory activities of Hofbauer cells may aid in dissemination of Zika virus to the fetal brain. Understanding the susceptibility of placenta-specific cell types will aid future work around and understanding of Zika virus–associated pregnancy complications.

Herpes simplex virus 1 infection triggers multiple changes in the metabolism of host cells, including a dramatic decrease in the levels of NAD + . In addition to its role as a cofactor in reduction-oxidation reactions, NAD + is required for certain posttranslational modifications. Members of the poly(ADP-ribose) polymerase (PARP) family of enzymes are major consumers of NAD + , which they utilize to form poly(ADP-ribose) (PAR) chains on protein substrates in response to DNA damage. PAR chains can subsequently be removed by the enzyme poly(ADP-ribose) glycohydrolase (PARG). We report here that the HSV-1 infection-induced drop in NAD + levels required viral DNA replication, was associated with an increase in protein poly(ADP-ribosyl)ation (PARylation), and was blocked by pharmacological inhibition of PARP-1/PARP-2 (PARP-1/2). Neither virus yield nor the cellular metabolic reprogramming observed during HSV-1 infection was altered by the rescue or further depletion of NAD + levels. Expression of the viral protein ICP0, which possesses E3 ubiquitin ligase activity, was both necessary and sufficient for the degradation of the 111-kDa PARG isoform. This work demonstrates that HSV-1 infection results in changes to NAD + metabolism by PARP-1/2 and PARG, and as PAR chain accumulation can induce caspase-independent apoptosis, we speculate that the decrease in PARG levels enhances the auto-PARylation-mediated inhibition of PARP, thereby avoiding premature death of the infected cell.

Cysteine levels are carefully regulated in mammals to balance metabolic needs against the potential for cytotoxicity. It has been postulated that one of the major regulators of intracellular cysteine levels in mammals is cysteine dioxygenase (CDO). Hepatic expression of this catabolic enzyme increases dramatically in response to increased cysteine availability and may therefore be part of a homeostatic response to shunt excess toxic cysteine to more benign metabolites such as sulfate or taurine. Direct experimental evidence, however, is lacking to support the hypothesis that CDO is capable of altering steady-state intracellular cysteine levels. In this study, we expressed either the wild-type (WT) or a catalytically inactivated mutant (H86A) isoform of CDO in HepG2/C3A cells (which do not express endogenous CDO protein) and cultured them in different concentrations of extracellular cysteine. WT CDO, but not H86A CDO, was capable of reducing intracellular cysteine levels in cells incubated in physiologically relevant concentrations of cysteine. WT CDO also decreased the glutathione pool and potentiated the toxicity of CdCl2. These results demonstrate that CDO is capable of altering intracellular cysteine levels as well as glutathione levels.

An shRNA-mediated screen of the 48 human nuclear receptor genes identified multiple candidates likely to influence the production of human cytomegalovirus in cultured human fibroblasts, including the estrogen-related receptor α (ERRα), an orphan nuclear receptor. The 50-kDa receptor and a 76-kDa variant were induced posttranscriptionally following infection. Genetic and pharmacological suppression of the receptor reduced viral RNA, protein, and DNA accumulation, as well as the yield of infectious progeny. In addition, RNAs encoding multiple metabolic enzymes, including enzymes sponsoring glycolysis (enolase 1, triosephosphate isomerase 1, and hexokinase 2), were reduced when the function of ERRα was inhibited in infected cells. Consistent with the effect on RNAs, a substantial number of metabolites, which are normally induced by infection, were either not increased or were increased to a reduced extent in the absence of normal ERRα activity. We conclude that ERRα is needed for the efficient production of cytomegalovirus progeny, and we propose that the nuclear receptor contributes importantly to the induction of a metabolic environment that supports optimal cytomegalovirus replication.

Cysteine dioxygenase (CDO) catalyzes the conversion of cysteine to cysteinesulfinic acid and is important in the regulation of intracellular cysteine levels in mammals and in the provision of oxidized cysteine metabolites such as sulfate and taurine. Several crystal structure studies of mammalian CDO have shown that there is a cross-linked cofactor present in the active site of the enzyme. The cofactor consists of a thioether bond between the γ-sulfur of residue cysteine 93 and the aromatic side chain of residue tyrosine 157. The exact requirements for cofactor synthesis and the contribution of the cofactor to the catalytic activity of the enzyme have yet to be fully described. In this study, therefore, we explored the factors necessary for cofactor biogenesis in vitro and in vivo and examined what effect cofactor formation had on activity in vitro. Like other cross-linked cofactor-containing enzymes, formation of the Cys-Tyr cofactor in CDO required a transition metal cofactor (Fe2+) and O2. Unlike other enzymes, however, biogenesis was also strictly dependent upon the presence of substrate. Cofactor formation was also appreciably slower than the rates reported for other enzymes and, indeed, took hundreds of catalytic turnover cycles to occur. In the absence of the Cys-Tyr cofactor, CDO possessed appreciable catalytic activity, suggesting that the cofactor was not essential for catalysis. Nevertheless, at physiologically relevant cysteine concentrations, cofactor formation increased CDO catalytic efficiency by ∼10-fold. Overall, the regulation of Cys-Tyr cofactor formation in CDO by ambient cysteine levels represents an unusual form of substrate-mediated feed-forward activation of enzyme activity with important physiological consequences.

There are only two known thiol dioxygenase activities in mammals, and they are ascribed to the enzymes cysteine dioxygenase (CDO) and cysteamine (2-aminoethanethiol) dioxygenase (ADO). Although many studies have been dedicated to CDO, resulting in the identification of its gene and even characterization of the tertiary structure of the protein, relatively little is known about cysteamine dioxygenase. The failure to identify the gene for this protein has significantly hampered our understanding of the metabolism of cysteamine, a product of the constitutive degradation of coenzyme A, and the synthesis of taurine, the final product of cysteamine oxidation and the second most abundant amino acid in mammalian tissues. In this study we identified a hypothetical murine protein homolog of CDO (hereafter called ADO) that is encoded by the gene Gm237 and belongs to the DUF1637 protein family. When expressed as a recombinant protein, ADO exhibited significant cysteamine dioxygenase activity in vitro. The reaction was highly specific for cysteamine; cysteine was not oxidized by the enzyme, and structurally related compounds were not competitive inhibitors of the reaction. When overexpressed in HepG2/C3A cells, ADO increased the production of hypotaurine from cysteamine. Similarly, when endogenous expression of the human ADO ortholog C10orf22 in HepG2/C3A cells was reduced by RNA-mediated interference, hypotaurine production decreased. Western blots of murine tissues with an antibody developed against ADO showed that the protein is ubiquitously expressed with the highest levels in brain, heart, and skeletal muscle. Overall, these data suggest that ADO is responsible for endogenous cysteamine dioxygenase activity.