TRIM7 inhibits enterovirus replication and promotes emergence of a viral variant with increased pathogenicity.

To control viral infection, vertebrates rely on both inducible interferon responses and less well-characterized cell-intrinsic responses composed of "at the ready" antiviral effector proteins. Here, we show that E3 ubiquitin ligase TRIM7 is a cell-intrinsic antiviral effector that restricts multiple human enteroviruses by targeting viral 2BC, a membrane remodeling protein, for ubiquitination and proteasome-dependent degradation. Selective pressure exerted by TRIM7 results in emergence of a TRIM7-resistant coxsackievirus with a single point mutation in the viral 2C ATPase/helicase. In cultured cells, the mutation helps the virus evade TRIM7 but impairs optimal viral replication, and this correlates with a hyperactive and structurally plastic 2C ATPase. Unexpectedly, the TRIM7-resistant virus has a replication advantage in mice and causes lethal pancreatitis. These findings reveal a unique mechanism for targeting enterovirus replication and provide molecular insight into the benefits and trade-offs of viral evolution imposed by a host restriction factor.

Interferon signaling drives epithelial metabolic reprogramming to promote secondary bacterial infection.

Clinical studies report that viral infections promote acute or chronic bacterial infections at multiple host sites. These viral-bacterial co-infections are widely linked to more severe clinical outcomes. In experimental models in vitro and in vivo, virus-induced interferon responses can augment host susceptibility to secondary bacterial infection. Here, we used a cell-based screen to assess 389 interferon-stimulated genes (ISGs) for their ability to induce chronic Pseudomonas aeruginosa infection. We identified and validated five ISGs that were sufficient to promote bacterial infection. Furthermore, we dissected the mechanism of action of hexokinase 2 (HK2), a gene involved in the induction of aerobic glycolysis, commonly known as the Warburg effect. We report that HK2 upregulation mediates the induction of Warburg effect and secretion of L-lactate, which enhances chronic P. aeruginosa infection. These findings elucidate how the antiviral immune response renders the host susceptible to secondary bacterial infection, revealing potential strategies for viral-bacterial co-infection treatment.

A NIK-SIX signalling axis controls inflammation by targeted silencing of non-canonical NF-κB.

The non-canonical NF-κB signalling cascade is essential for lymphoid organogenesis, B cell maturation, osteoclast differentiation, and inflammation in mammals1,2; dysfunction of this system is associated with human diseases, including immunological disorders and cancer3-6. Although expression of NF-κB-inducing kinase (NIK, also known as MAP3K14) is the rate-limiting step in non-canonical NF-κB pathway activation2,7, the mechanisms by which transcriptional responses are regulated remain largely unknown. Here we show that the sine oculis homeobox (SIX) homologue family transcription factors SIX1 and SIX2 are integral components of the non-canonical NF-κB signalling cascade. The developmentally silenced SIX proteins are reactivated in differentiated macrophages by NIK-mediated suppression of the ubiquitin proteasome pathway. Consequently, SIX1 and SIX2 target a subset of inflammatory gene promoters and directly inhibit the trans-activation function of the transcription factors RELA and RELB in a negative feedback circuit. In support of a physiologically pivotal role for SIX proteins in host immunity, a human SIX1 transgene suppressed inflammation and promoted the recovery of mice from endotoxic shock. In addition, SIX1 and SIX2 protected RAS/P53-driven non-small-cell lung carcinomas from inflammatory cell death induced by SMAC-mimetic chemotherapeutic agents (small-molecule activators of the non-canonical NF-κB pathway). Our findings identify a NIK-SIX signalling axis that fine-tunes inflammatory gene expression programs under both physiological and pathological conditions.

Functional-genomic analysis reveals intraspecies diversification of antiviral receptor transporter proteins in Xenopus laevis.

The Receptor Transporter Protein (RTP) family is present in most, if not all jawed vertebrates. Most of our knowledge of this protein family comes from studies on mammalian RTPs, which are multi-function proteins that regulate cell-surface G-protein coupled receptor levels, influence olfactory system development, regulate immune signaling, and directly inhibit viral infection. However, mammals comprise less than one-tenth of extant vertebrate species, and our knowledge about the expression, function, and evolution of non-mammalian RTPs is limited. Here, we explore the evolutionary history of RTPs in vertebrates. We identify signatures of positive selection in many vertebrate RTP clades and characterize multiple, independent expansions of the RTP family outside of what has been described in mammals. We find a striking expansion of RTPs in the African clawed frog, Xenopus laevis, with 11 RTPs in this species as opposed to 1 to 4 in most other species. RNA sequencing revealed that most X. laevis RTPs are upregulated following immune stimulation. In functional assays, we demonstrate that at least three of these X. laevis RTPs inhibit infection by RNA viruses, suggesting that RTP homologs may serve as antiviral effectors outside of Mammalia.

Shiftless inhibits flavivirus replication in vitro and is neuroprotective in a mouse model of Zika virus pathogenesis.

Flaviviruses such as Zika virus and West Nile virus have the potential to cause severe neuropathology if they invade the central nervous system. The type I interferon response is well characterized as contributing to control of flavivirus-induced neuropathogenesis. However, the interferon-stimulated gene (ISG) effectors that confer these neuroprotective effects are less well studied. Here, we used an ISG expression screen to identify Shiftless (SHFL, C19orf66) as a potent inhibitor of diverse positive-stranded RNA viruses, including multiple members of the Flaviviridae (Zika, West Nile, dengue, yellow fever, and hepatitis C viruses). In cultured cells, SHFL functions as a viral RNA-binding protein that inhibits viral replication at a step after primary translation of the incoming genome. The murine ortholog, Shfl, is expressed constitutively in multiple tissues, including the central nervous system. In a mouse model of Zika virus infection, Shfl-/- knockout mice exhibit reduced survival, exacerbated neuropathological outcomes, and increased viral replication in the brain and spinal cord. These studies demonstrate that Shfl is an important antiviral effector that contributes to host protection from Zika virus infection and virus-induced neuropathological disease.

Antithetic effect of interferon-α on cell-free and cell-to-cell HIV-1 infection.

In HIV-1-infected individuals, transmitted/founder (TF) virus contributes to establish new infection and expands during the acute phase of infection, while chronic control (CC) virus emerges during the chronic phase of infection. TF viruses are more resistant to interferon-alpha (IFN-α)-mediated antiviral effects than CC virus, however, its virological relevance in infected individuals remains unclear. Here we perform an experimental-mathematical investigation and reveal that IFN-α strongly inhibits cell-to-cell infection by CC virus but only weakly affects that by TF virus. Surprisingly, IFN-α enhances cell-free infection of HIV-1, particularly that of CC virus, in a virus-cell density-dependent manner. We further demonstrate that LY6E, an IFN-stimulated gene, can contribute to the density-dependent enhancement of cell-free HIV-1 infection. Altogether, our findings suggest that the major difference between TF and CC viruses can be explained by their resistance to IFN-α-mediated inhibition of cell-to-cell infection and their sensitivity to IFN-α-mediated enhancement of cell-free infection.

Pan-viral specificity of IFN-induced genes reveals new roles for cGAS in innate immunity.

The type I interferon (IFN) response protects cells from viral infection by inducing hundreds of interferon-stimulated genes (ISGs), some of which encode direct antiviral effectors. Recent screening studies have begun to catalogue ISGs with antiviral activity against several RNA and DNA viruses. However, antiviral ISG specificity across multiple distinct classes of viruses remains largely unexplored. Here we used an ectopic expression assay to screen a library of more than 350 human ISGs for effects on 14 viruses representing 7 families and 11 genera. We show that 47 genes inhibit one or more viruses, and 25 genes enhance virus infectivity. Comparative analysis reveals that the screened ISGs target positive-sense single-stranded RNA viruses more effectively than negative-sense single-stranded RNA viruses. Gene clustering highlights the cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS, also known as MB21D1) as a gene whose expression also broadly inhibits several RNA viruses. In vitro, lentiviral delivery of enzymatically active cGAS triggers a STING-dependent, IRF3-mediated antiviral program that functions independently of canonical IFN/STAT1 signalling. In vivo, genetic ablation of murine cGAS reveals its requirement in the antiviral response to two DNA viruses, and an unappreciated contribution to the innate control of an RNA virus. These studies uncover new paradigms for the preferential specificity of IFN-mediated antiviral pathways spanning several virus families.

LY6E protects mice from pathogenic effects of murine coronavirus and SARS-CoV-2.

LY6E is an antiviral protein that inhibits coronavirus entry. Its expression in immune cells allows mice to control murine coronavirus infection. However, it is not known which immune cell subsets mediate this control or whether LY6E protects mice from SARS-CoV-2. In this study, we used tissue-specific Cre recombinase expression to ablate Ly6e in distinct immune compartments or in all epiblast-derived cells, and bone marrow chimeras to target Ly6e in a subset of radioresistant cells. Mice lacking Ly6e in Lyz2 -expressing cells and radioresistant Vav1 -expressing cells were more susceptible to lethal murine coronavirus infection. Mice lacking Ly6e globally developed clinical disease when challenged with the Gamma (P.1) variant of SARS-CoV-2. By contrast, wildtype mice and mice lacking type I and type III interferon signaling had no clinical symptoms after SARS-CoV-2 infection. Transcriptomic profiling of lungs from SARS-CoV-2-infected wildtype and Ly6e knockout mice revealed a striking reduction of secretory cell-associated genes in infected knockout mice, including Muc5b , an airway mucin-encoding gene that may protect against SARS-CoV-2-inflicted respiratory disease. Collectively, our study reveals distinct cellular compartments in which Ly6e confers cell intrinsic antiviral effects, thereby conferring resistance to disease caused by murine coronavirus and SARS-CoV-2.

LY6E is a pan-coronavirus restriction factor in the respiratory tract.

LY6E is an antiviral restriction factor that inhibits coronavirus spike-mediated fusion, but the cell types in vivo that require LY6E for protection from respiratory coronavirus infection are unknown. Here we used a panel of seven conditional Ly6e knockout mice to define which Ly6e-expressing cells confer control of airway infection by murine coronavirus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Loss of Ly6e in Lyz2-expressing cells, radioresistant Vav1-expressing cells and non-haematopoietic cells increased susceptibility to murine coronavirus. Global conditional loss of Ly6e expression resulted in clinical disease and higher viral burden after SARS-CoV-2 infection, but little evidence of immunopathology. We show that Ly6e expression protected secretory club and ciliated cells from SARS-CoV-2 infection and prevented virus-induced loss of an epithelial cell transcriptomic signature in the lung. Our study demonstrates that lineage confined rather than broad expression of Ly6e sufficiently confers resistance to disease caused by murine and human coronaviruses.

RTP4 Is a Potent IFN-Inducible Anti-flavivirus Effector Engaged in a Host-Virus Arms Race in Bats and Other Mammals.

Among mammals, bats are particularly rich in zoonotic viruses, including flaviviruses. Certain bat species can be productively yet asymptomatically infected with viruses that cause overt disease in other species. However, little is known about the antiviral effector repertoire in bats relative to other mammals. Here, we report the black flying fox receptor transporter protein 4 (RTP4) as a potent interferon (IFN)-inducible inhibitor of human pathogens in the Flaviviridae family, including Zika, West Nile, and hepatitis C viruses. Mechanistically, RTP4 associates with the flavivirus replicase, binds viral RNA, and suppresses viral genome amplification. Comparative approaches revealed that RTP4 undergoes positive selection, that a flavivirus can mutate to escape RTP4-imposed restriction, and that diverse mammalian RTP4 orthologs exhibit striking patterns of specificity against distinct Flaviviridae members. Our findings reveal an antiviral mechanism that has likely adapted over 100 million years of mammalian evolution to accommodate unique host-virus genetic conflicts.

Oxysterols provide innate immunity to bacterial infection by mobilizing cell surface accessible cholesterol.

Cholesterol 25-hydroxylase (CH25H) is an interferon-stimulated gene that converts cholesterol to the oxysterol 25-hydroxycholesterol (25HC). Circulating 25HC modulates essential immunological processes including antiviral immunity, inflammasome activation and antibody class switching; and dysregulation of CH25H may contribute to chronic inflammatory disease and cancer. Although 25HC is a potent regulator of cholesterol storage, uptake, efflux and biosynthesis, how these metabolic activities reprogram the immunological state of target cells remains poorly understood. Here, we used recently designed toxin-based biosensors that discriminate between distinct pools of plasma membrane cholesterol to elucidate how 25HC prevents Listeria monocytogenes from traversing the plasma membrane of infected host cells. The 25HC-mediated activation of acyl-CoA:cholesterol acyltransferase (ACAT) triggered rapid internalization of a biochemically defined fraction of cholesterol, termed 'accessible' cholesterol, from the plasma membrane while having little effect on cholesterol in complexes with sphingomyelin. We show that evolutionarily distinct bacterial species, L. monocytogenes and Shigella flexneri, exploit the accessible pool of cholesterol for infection and that acute mobilization of this pool by oxysterols confers immunity to these pathogens. The significance of this signal-mediated membrane remodelling pathway probably extends beyond host defence systems, as several other biologically active oxysterols also mobilize accessible cholesterol through an ACAT-dependent mechanism.

A Sensitive Yellow Fever Virus Entry Reporter Identifies Valosin-Containing Protein (VCP/p97) as an Essential Host Factor for Flavivirus Uncoating.

While the basic mechanisms of flavivirus entry and fusion are understood, little is known about the postfusion events that precede RNA replication, such as nucleocapsid disassembly. We describe here a sensitive, conditionally replication-defective yellow fever virus (YFV) entry reporter, YFVΔSK/Nluc, to quantitively monitor the translation of incoming, virus particle-delivered genomes. We validated that YFVΔSK/Nluc gene expression can be neutralized by YFV-specific antisera and requires known flavivirus entry pathways and cellular factors, including clathrin- and dynamin-mediated endocytosis, endosomal acidification, YFV E glycoprotein-mediated fusion, and cellular LY6E and RPLP1 expression. The initial round of YFV translation was shown to require cellular ubiquitylation, consistent with recent findings that dengue virus capsid protein must be ubiquitylated in order for nucleocapsid uncoating to occur. Importantly, translation of incoming YFV genomes also required valosin-containing protein (VCP)/p97, a cellular ATPase that unfolds and extracts ubiquitylated client proteins from large complexes. RNA transfection and washout experiments showed that VCP/p97 functions at a postfusion, pretranslation step in YFV entry. Finally, VCP/p97 activity was required by other flaviviruses in mammalian cells and by YFV in mosquito cells. Together, these data support a critical role for VCP/p97 in the disassembly of incoming flavivirus nucleocapsids during a postfusion step in virus entry.IMPORTANCE Flaviviruses are an important group of RNA viruses that cause significant human disease. The mechanisms by which flavivirus nucleocapsids are disassembled during virus entry remain unclear. Here, we used a yellow fever virus entry reporter, which expresses a sensitive reporter enzyme but does not replicate, to show that nucleocapsid disassembly requires the cellular protein-disaggregating enzyme valosin-containing protein, also known as p97.

LY6E impairs coronavirus fusion and confers immune control of viral disease.

Zoonotic coronaviruses (CoVs) are significant threats to global health, as exemplified by the recent emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 1 . Host immune responses to CoV are complex and regulated in part through antiviral interferons. However, the interferon-stimulated gene products that inhibit CoV are not well characterized 2 . Here, we show that interferon-inducible lymphocyte antigen 6 complex, locus E (LY6E) potently restricts cellular infection by multiple CoVs, including SARS-CoV, SARS-CoV-2, and Middle East respiratory syndrome coronavirus (MERS-CoV). Mechanistic studies revealed that LY6E inhibits CoV entry into cells by interfering with spike protein-mediated membrane fusion. Importantly, mice lacking Ly6e in hematopoietic cells were highly susceptible to murine CoV infection. Exacerbated viral pathogenesis in Ly6e knockout mice was accompanied by loss of hepatic and splenic immune cells and reduction in global antiviral gene pathways. Accordingly, we found that Ly6e directly protects primary B cells and dendritic cells from murine CoV infection. Our results demonstrate that LY6E is a critical antiviral immune effector that controls CoV infection and pathogenesis. These findings advance our understanding of immune-mediated control of CoV in vitro and in vivo , knowledge that could help inform strategies to combat infection by emerging CoV.

LY6E impairs coronavirus fusion and confers immune control of viral disease.

Zoonotic coronaviruses (CoVs) are substantial threats to global health, as exemplified by the emergence of two severe acute respiratory syndrome CoVs (SARS-CoV and SARS-CoV-2) and Middle East respiratory syndrome CoV (MERS-CoV) within two decades1-3. Host immune responses to CoVs are complex and regulated in part through antiviral interferons. However, interferon-stimulated gene products that inhibit CoVs are not well characterized4. Here, we show that lymphocyte antigen 6 complex, locus E (LY6E) potently restricts infection by multiple CoVs, including SARS-CoV, SARS-CoV-2 and MERS-CoV. Mechanistic studies revealed that LY6E inhibits CoV entry into cells by interfering with spike protein-mediated membrane fusion. Importantly, mice lacking Ly6e in immune cells were highly susceptible to a murine CoV-mouse hepatitis virus. Exacerbated viral pathogenesis in Ly6e knockout mice was accompanied by loss of hepatic immune cells, higher splenic viral burden and reduction in global antiviral gene pathways. Accordingly, we found that constitutive Ly6e directly protects primary B cells from murine CoV infection. Our results show that LY6E is a critical antiviral immune effector that controls CoV infection and pathogenesis. These findings advance our understanding of immune-mediated control of CoV in vitro and in vivo-knowledge that could help inform strategies to combat infection by emerging CoVs.

LY6E mediates an evolutionarily conserved enhancement of virus infection by targeting a late entry step.

Interferons (IFNs) contribute to cell-intrinsic antiviral immunity by inducing hundreds of interferon-stimulated genes (ISGs). In a screen to identify antiviral ISGs, we unexpectedly found that LY6E, a member of the LY6/uPAR family, enhanced viral infection. Here, we show that viral enhancement by ectopically expressed LY6E extends to several cellular backgrounds and affects multiple RNA viruses. LY6E does not impair IFN antiviral activity or signaling, but rather promotes viral entry. Using influenza A virus as a model, we narrow the enhancing effect of LY6E to uncoating after endosomal escape. Diverse mammalian orthologs of LY6E also enhance viral infectivity, indicating evolutionary conservation of function. By structure-function analyses, we identify a single amino acid in a predicted loop region that is essential for viral enhancement. Our study suggests that LY6E belongs to a class of IFN-inducible host factors that enhance viral infectivity without suppressing IFN antiviral activity.

A CRISPR screen identifies IFI6 as an ER-resident interferon effector that blocks flavivirus replication.

The endoplasmic reticulum (ER) is an architecturally diverse organelle that serves as a membrane source for the replication of multiple viruses. Flaviviruses, including yellow fever virus, West Nile virus, dengue virus and Zika virus, induce unique single-membrane ER invaginations that house the viral replication machinery1. Whether this virus-induced ER remodelling is vulnerable to antiviral pathways is unknown. Here, we show that flavivirus replication at the ER is targeted by the interferon (IFN) response. Through genome-scale CRISPR screening, we uncovered an antiviral mechanism mediated by a functional gene pairing between IFI6 (encoding IFN-α-inducible protein 6), an IFN-stimulated gene cloned over 30 years ago2, and HSPA5, which encodes the ER-resident heat shock protein 70 chaperone BiP. We reveal that IFI6 is an ER-localized integral membrane effector that is stabilized through interactions with BiP. Mechanistically, IFI6 prophylactically protects uninfected cells by preventing the formation of virus-induced ER membrane invaginations. Notably, IFI6 has little effect on other mammalian RNA viruses, including the related Flaviviridae family member hepatitis C virus, which replicates in double-membrane vesicles that protrude outwards from the ER. These findings support a model in which the IFN response is armed with a membrane-targeted effector that discriminately blocks the establishment of virus-specific ER microenvironments that are required for replication.

NLRX1 Helps HIV Avoid a STING Operation.

Immune evasion by HIV-1 during acute infection is critical for the establishment of latency. Barouch et al. (2016) and Guo et al. (2016) demonstrate in independent studies that Nod-like receptor X1 (NLRX1) may facilitate early systemic dissemination of HIV-1 by inhibiting the virus-triggered innate immune response.

Tracing conidial fate and measuring host cell antifungal activity using a reporter of microbial viability in the lung.

Fluorescence can be harnessed to monitor microbial fate and to investigate functional outcomes of individual microbial cell-host cell encounters at portals of entry in native tissue environments. We illustrate this concept by introducing fluorescent Aspergillus reporter (FLARE) conidia that simultaneously report phagocytic uptake and fungal viability during cellular interactions with the murine respiratory innate immune system. Our studies using FLARE conidia reveal stepwise and cell-type-specific requirements for CARD9 and Syk, transducers of C-type lectin receptor and integrin signals, in neutrophil recruitment, conidial uptake, and conidial killing in the lung. By achieving single-event resolution in defined leukocyte populations, the FLARE method enables host cell profiling on the basis of pathogen uptake and killing and may be extended to other pathogens in diverse model host organisms to query molecular, cellular, and pharmacologic mechanisms that shape host-microbe interactions.