The Lyme disease agent co-opts adiponectin receptor-mediated signaling in its arthropod vector.

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.

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.

Macrophage scavenger receptor 1 controls Chikungunya virus infection through autophagy in mice.

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.

TRiC/CCT Complex, a Binding Partner of NS1 Protein, Supports the Replication of Zika Virus in Both Mammalians and Mosquitoes.

Mosquito-borne Zika virus (ZIKV) can cause congenital microcephaly and Guillain-Barré 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.

Inhibition of Chikungunya Virus Replication in Primary Human Fibroblasts by Liver X Receptor Agonist.

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.

Aedes aegypti NeSt1 Protein Enhances Zika Virus Pathogenesis by Activating Neutrophils.

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-1ß 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.

Loss of the TAM Receptor Axl Ameliorates Severe Zika Virus Pathogenesis and Reduces Apoptosis in Microglia.

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-1ß 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.

A potent prolyl tRNA synthetase inhibitor antagonizes Chikungunya and Dengue viruses.

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.

Rev-erb Agonist Inhibits Chikungunya and O'nyong'nyong Virus Replication.

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/ß, 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.

Fetal Growth Restriction Caused by Sexual Transmission of Zika Virus in Mice.

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.

TAM Receptors Are Not Required for Zika Virus Infection in Mice.

Tyro3, Axl, and Mertk (TAM) receptors are candidate entry receptors for infection with the Zika virus (ZIKV), an emerging flavivirus of global public health concern. To investigate the requirement of TAM receptors for ZIKV infection, we used several routes of viral inoculation and compared viral replication in wild-type versus Axl-/-, Mertk-/-, Axl-/-Mertk-/-, and Axl-/-Tyro3-/- mice in various organs. Pregnant and non-pregnant mice treated with interferon-α-receptor (IFNAR)-blocking (MAR1-5A3) antibody and infected subcutaneously with ZIKV showed no reliance on TAMs for infection. In the absence of IFNAR-blocking antibody, adult female mice challenged intravaginally with ZIKV showed no difference in mucosal viral titers. Similarly, in young mice that were infected with ZIKV intracranially or intraperitoneally, ZIKV replication occurred in the absence of TAM receptors, and no differences in cell tropism were observed. These findings indicate that, in mice, TAM receptors are not required for ZIKV entry and infection.

Zika virus causes testicular atrophy.

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.

Zika virus productively infects primary human placenta-specific macrophages.

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.

Genetics of War and Truce between Mosquitos and Emerging Viruses.

Arboviruses have made unexpected reappearances in recent years. Unlike viruses that undergo direct transmission, arboviruses utilize an arthropod vector (e.g., mosquitos, sandflies, and ticks) to spread throughout human populations. Here, we provide a snapshot of mosquito susceptibility to viral infection using flaviviruses, alphaviruses, and bunyaviruses as examples of emerging pathogens of global health relevance.

Estrogen-related receptor α is required for efficient human cytomegalovirus replication.

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.

Herpes simplex virus 1 infection activates poly(ADP-ribose) polymerase and triggers the degradation of poly(ADP-ribose) glycohydrolase.

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.

Synthesis of amino acid cofactor in cysteine dioxygenase is regulated by substrate and represents a novel post-translational regulation of activity.

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 gamma-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 (Fe(2+)) and O(2). 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 approximately 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.

Discovery and characterization of a second mammalian thiol dioxygenase, cysteamine dioxygenase.

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.

Overexpression of cysteine dioxygenase reduces intracellular cysteine and glutathione pools in HepG2/C3A cells.

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 CdCl(2). These results demonstrate that CDO is capable of altering intracellular cysteine levels as well as glutathione levels.