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Spaceflight entails a variety of environmental and psychological stressors that may have long-term physiological and genomic consequences. Metabolomics, an approach that investigates the terminal metabolic outputs of complex physiological alterations, considers the dynamic state of the human body and allows the identification and quantification of down-stream metabolites linked to up-stream physiological and genomic regulation by stress. Employing a metabolomics-based approach, this study investigated longitudinal metabolic perturbations of male ( n  = 40) and female ( n  = 11) astronauts on 4–6-month missions to the International Space Station (ISS). Proton nuclear magnetic resonance ( 1 H-NMR) spectroscopy followed by univariate, multivariate and machine learning analyses were used on blood serum to examine sex-specific metabolic changes at various time points throughout the astronauts’ missions, and the metabolic effects of long-duration space travel. Space travel resulted in sex-specific changes in energy metabolism, bone mineral and muscle regulation, immunity, as well as macromolecule maintenance and synthesis. Additionally, metabolic signatures suggest differential metabolic responses—especially during the recovery period—with females requiring more time to adjust to return to Earth. These findings provide insight into the perturbations in glucose and amino acid metabolism and macromolecule biosynthesis that result from the stressors of long-duration spaceflight. Metabolomic biomarkers may provide a viable approach to predicting and diagnosing health risks associated with prolonged space travel and other physiological challenges on Earth.

Innate effector mechanisms contribute to the control of viremia and modulate the quality of the adaptive immune response to HIV-1. Altfeld and Gale discuss the concerted actions of PRR signaling, innate immune cells and innate-adaptive crosstalk that direct the outcome of HIV-1 infection. During acute HIV-1 infection, viral pathogen-associated molecular patterns are recognized by pathogen-recognition receptors (PRRs) of infected cells, which triggers a signaling cascade that initiates innate intracellular antiviral defenses aimed at restricting the replication and spread of the virus. This cell-intrinsic response propagates outward via the action of secreted factors such as cytokines and chemokines that activate innate immune cells and attract them to the site of infection and to local lymphatic tissue. Antiviral innate effector cells can subsequently contribute to the control of viremia and modulate the quality of the adaptive immune response to HIV-1. The concerted actions of PRR signaling, specific viral-restriction factors, innate immune cells, innate-adaptive immune crosstalk and viral evasion strategies determine the outcome of HIV-1 infection and immune responses.

Key Points West Nile virus (WNV) continues to pose a significant public health risk throughout most of the world. In the United States, WNV is endemic and the leading cause of mosquito-borne encephalitis. Currently there is no approved vaccine or therapy to prevent or limit WNV infection in humans. Mosquitoes have innate immune programmes, similar to those of mammalian hosts, that function to limit viral replication and spread. In addition, mosquito salivary factors enhance WNV replication, dissemination and virus-induced disease. WNV can cross the blood–brain barrier by one of several routes, including passive transport through the endothelium, infection of the olfactory neurons, transport by infected immune cells, inflammation-induced disruption of blood–brain barrier integrity, and direct axonal retrograde transport from infected peripheral neurons. Both innate and adaptive immune responses are required for controlling WNV replication and protection against a lethal disease outcome. Type I interferons are crucial for eliciting cell-intrinsic immune defences and priming adaptive immune responses during WNV infection. In particular, the RIG-I-like receptor and Toll-like receptor signalling pathways are essential for triggering interferons and immune defences in response to WNV infection. Here, Suthar, Diamond and Gale review recent insights into West Nile virus pathogenesis and the host immune responses that this virus activates. Given the continuing spread of the virus in the Western hemisphere, a better understanding of these host–virus interactions is crucial and should facilitate the development of effective vaccines and therapeutics. West Nile virus (WNV) is an emerging neurotropic flavivirus that is transmitted to humans through the bite of an infected mosquito. WNV has disseminated broadly in the Western hemisphere and now poses a significant public health risk. The continuing spread of WNV, combined with the lack of specific therapeutics or vaccines to combat or prevent infection, imparts a pressing need to identify the viral and host processes that control the outcome of and immunity to WNV infection. Here, we provide an overview of recent research that has revealed the virus–host interface controlling WNV infection and immunity.

In this era of continued emergence of zoonotic virus infections, the rapid development of rodent models represents a critical barrier to public health preparedness, including the testing of antivirus therapy and vaccines. The Middle East respiratory syndrome coronavirus (MERS-CoV) was recently identified as the causative agent of a severe pneumonia. Given the ability of coronavirus to rapidly adapt to new hosts, a major public health concern is that MERS-CoV will further adapt to replication in humans, triggering a pandemic. No small-animal model for this infection is currently available, but studies suggest that virus entry factors can confer virus susceptibility. Here, we show that mice were sensitized to MERS-CoV infection by prior transduction with adenoviral vectors expressing the human host-cell receptor dipeptidyl peptidase 4. Mice developed a pneumonia characterized by extensive inflammatory-cell infiltration with virus clearance occurring 6–8 d after infection. Clinical disease and histopathological changes were more severe in the absence of type-I IFN signaling whereas the T-cell response was required for virus clearance. Using these mice, we demonstrated the efficacy of a therapeutic intervention (poly I:C) and a potential vaccine [Venezuelan equine encephalitis replicon particles expressing MERS-CoV spike protein]. We also found little protective cross-reactivity between MERS-CoV and the severe acute respiratory syndrome-CoV. Our results demonstrate that this system will be useful for MERS-CoV studies and for the rapid development of relevant animal models for emerging respiratory viral infections.

Hepatitis C recognition Innate immunity is an important defence against infection by viruses, triggered by host recognition of 'PAMPS', or pathogen-associated molecular patterns. Saito et al . have now identified a conserved poly-uridine motif in the 3´ non-transcribed region of the hepatitis C virus genome as the relevant PAMP for detection by the RNA helicase RIG-I, a protein previously shown to have an essential function in double-stranded RNA-induced innate antiviral responses. Innate immune defences are essential for the control of virus infection and are triggered through host recognition of viral macromolecular motifs known as pathogen-associated molecular patterns (PAMPs) 1 . Hepatitis C virus (HCV) is an RNA virus that replicates in the liver, and infects 200 million people worldwide 2 . Infection is regulated by hepatic immune defences triggered by the cellular RIG-I helicase. RIG-I binds PAMP RNA and signals interferon regulatory factor 3 activation to induce the expression of interferon-α/β and antiviral/interferon-stimulated genes (ISGs) that limit infection 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 . Here we identify the polyuridine motif of the HCV genome 3′ non-translated region and its replication intermediate as the PAMP substrate of RIG-I, and show that this and similar homopolyuridine or homopolyriboadenine motifs present in the genomes of RNA viruses are the chief feature of RIG-I recognition and immune triggering in human and murine cells 8 . 5′ terminal triphosphate on the PAMP RNA was necessary but not sufficient for RIG-I binding, which was primarily dependent on homopolymeric ribonucleotide composition, linear structure and length. The HCV PAMP RNA stimulated RIG-I-dependent signalling to induce a hepatic innate immune response in vivo , and triggered interferon and ISG expression to suppress HCV infection in vitro . These results provide a conceptual advance by defining specific homopolymeric RNA motifs within the genome of HCV and other RNA viruses as the PAMP substrate of RIG-I, and demonstrate immunogenic features of the PAMP–RIG-I interaction that could be used as an immune adjuvant for vaccine and immunotherapy approaches.

Evasion of host antiviral mechanisms Many cellular messenger RNAs and viral RNAs are methylated at the 2′- O position of the 5′ guanosine cap. The role of this modification in virus infection has been unclear. Michael Diamond and colleagues now show that this form of methylation enables several unrelated viruses to evade innate host antiviral responses through escape from suppression by interferon-stimulated genes. This suggests an evolutionary explanation for 2′- O methylation of cellular mRNA: it may distinguish self from non-self RNA under conditions of infection. Novel classes of pharmacological agents that specifically inhibit cytoplasmic viral 2′- O methyltransferases may be expected to have broad-spectrum antiviral activity. Many cellular and virus messenger RNAs are methylated at the 2′- O positions of the 5′ guanosine cap. The role of 2′- O methylation in virus infection has been unclear. These authors show that this form of methylation enables several unrelated viruses to evade the antiviral effects of genes stimulated by type I interferon. Cellular messenger RNA (mRNA) of higher eukaryotes and many viral RNAs are methylated at the N-7 and 2′- O positions of the 5′ guanosine cap by specific nuclear and cytoplasmic methyltransferases (MTases), respectively. Whereas N-7 methylation is essential for RNA translation and stability 1 , the function of 2′- O methylation has remained uncertain since its discovery 35 years ago 2 , 3 , 4 . Here we show that a West Nile virus (WNV) mutant (E218A) that lacks 2′- O MTase activity was attenuated in wild-type primary cells and mice but was pathogenic in the absence of type I interferon (IFN) signalling. 2′- O methylation of viral RNA did not affect IFN induction in WNV-infected fibroblasts but instead modulated the antiviral effects of IFN-induced proteins with tetratricopeptide repeats (IFIT), which are interferon-stimulated genes (ISGs) implicated in regulation of protein translation. Poxvirus and coronavirus mutants that lacked 2′- O MTase activity similarly showed enhanced sensitivity to the antiviral actions of IFN and, specifically, IFIT proteins. Our results demonstrate that the 2′- O methylation of the 5′ cap of viral RNA functions to subvert innate host antiviral responses through escape of IFIT-mediated suppression, and suggest an evolutionary explanation for 2′- O methylation of cellular mRNA: to distinguish self from non-self RNA. Differential methylation of cytoplasmic RNA probably serves as an example for pattern recognition and restriction of propagation of foreign viral RNA in host cells.

Hepatitis C virus (HCV) is a positive-strand RNA virus of the Flaviviridae family and a major cause of liver disease worldwide. HCV replicates in the cytoplasm, and the synthesis of viral proteins induces extensive rearrangements of host cell membranes producing structures, collectively termed the membranous web (MW). The MW contains the sites of viral replication and assembly, and we have identified distinct membrane fractions derived from HCV-infected cells that contain replication and assembly complexes enriched for viral RNA and infectious virus, respectively. The complex membrane structure of the MW is thought to protect the viral genome limiting its interactions with cytoplasmic pattern recognition receptors (PRRs) and thereby preventing activation of cellular innate immune responses. Here we show that PRRs, including RIG-I and MDA5, and ribosomes are excluded from viral replication and assembly centers within the MW. Furthermore, we present evidence that components of the nuclear transport machinery regulate access of proteins to MW compartments. We show that the restricted assess of RIG-I to the MW can be overcome by the addition of a nuclear localization signal sequence, and that expression of a NLS-RIG-I construct leads to increased immune activation and the inhibition of viral replication.

RIG-I is a cytosolic pathogen recognition receptor that engages viral RNA in infected cells to trigger innate immune defenses through its adaptor protein MAVS. MAVS resides on mitochondria and peroxisomes, but how its signaling is coordinated among these organelles has not been defined. Here we show that a major site of MAVS signaling is the mitochondrial-associated membrane (MAM), a distinct membrane compartment that links the endoplasmic reticulum to mitochondria. During RNA virus infection, RIG-I is recruited to the MAM to bind MAVS. Dynamic MAM tethering to mitochondria and peroxisomes then coordinates MAVS localization to form a signaling synapse between membranes. Importantly, the hepatitis C virus NS3/4A protease, which cleaves MAVS to support persistent infection, targets this synapse for MAVS proteolysis from the MAM, but not from mitochondria, to ablate RIG-I signaling of immune defenses. Thus, the MAM mediates an intracellular immune synapse that directs antiviral innate immunity.

Persistence of hepatitis C virus contributes to chronic infection, which can lead to liver fibrosis and even liver cancer. Different factors, such as host genetics and immunity and viral immune evasion strategies, account for the outcome of the infection and the patient response to antivirals. This Perspective discusses how the interaction of these factors modulates viral immunity and how they might be used to identify the key targets to mount an effective immune response that will clear the virus and improve drug response. Hepatitis C virus (HCV) is a global public health problem involving chronic infection of the liver, which can cause liver disease and is linked with liver cancer. Viral innate immune evasion strategies and human genetic determinants underlie the transition of acute HCV infection to viral persistence and the support of chronic infection. Host genetic factors, such as sequence polymorphisms in IFNL3 , a gene in the host interferon system, can influence both the outcome of the infection and the response to antiviral therapy. Recent insights into how HCV regulates innate immune signaling within the liver reveal a complex interaction of patient genetic background with viral and host factors of innate immune triggering and control that imparts the outcome of HCV infection and immunity.

Although the transcription factors IRF-3 and IRF-7 are considered master regulators of type I interferon (IFN) induction and IFN stimulated gene (ISG) expression, Irf3(-/-)×Irf7(-/-) double knockout (DKO) myeloid dendritic cells (mDC) produce relatively normal levels of IFN-β after viral infection. We generated Irf3(-/-)×Irf5(-/-)×Irf7(-/-) triple knockout (TKO) mice to test whether IRF-5 was the source of the residual induction of IFN-β and ISGs in mDCs. In pathogenesis studies with two unrelated positive-sense RNA viruses (West Nile virus (WNV) and murine norovirus), TKO mice succumbed at rates greater than DKO mice and equal to or approaching those of mice lacking the type I IFN receptor (Ifnar(-/-)). In ex vivo studies, after WNV infection or exposure to Toll-like receptor agonists, TKO mDCs failed to produce IFN-β or express ISGs. In contrast, this response was sustained in TKO macrophages following WNV infection. To define IRF-regulated gene signatures, we performed microarray analysis on WNV-infected mDC from wild type (WT), DKO, TKO, or Ifnar(-/-) mice, as well as from mice lacking the RIG-I like receptor adaptor protein MAVS. Whereas the gene induction pattern in DKO mDC was similar to WT cells, remarkably, almost no ISG induction was detected in TKO or Mavs(-/-) mDC. The relative equivalence of TKO and Mavs(-/-) responses suggested that MAVS dominantly regulates ISG induction in mDC. Moreover, we showed that MAVS-dependent induction of ISGs can occur through an IRF-5-dependent yet IRF-3 and IRF-7-independent pathway. Our results establish IRF-3, -5, and -7 as the key transcription factors responsible for mediating the type I IFN and ISG response in mDC during WNV infection and suggest a novel signaling link between MAVS and IRF-5.