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Members of the Old World Arenaviruses primarily utilize α-dystroglycan (α-DAG1) as a cellular receptor for infection. Mutations within the glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV) reduce or abrogate the binding affinity to α-DAG1 and thus influence viral persistence, kinetics, and cell tropism. The observation that α-DAG1 deficient cells are still highly susceptible to low affinity variants, suggests the use of an alternative receptor(s). In this study, we used a genome-wide CRISPR Cas9 knockout screen in DAG1 deficient 293T cells to identify host factors involved in α-DAG1-independent LCMV infection. By challenging cells with vesicular stomatitis virus (VSV), pseudotyped with the GP of LCMV WE HPI (VSV-GP), we identified the heparan sulfate (HS) biosynthesis pathway as an important host factor for low affinity LCMV infection. These results were confirmed by a genetic approach targeting EXTL3, a key factor in the HS biosynthesis pathway, as well as by enzymatic and chemical methods. Interestingly, a single point mutation within GP1 (S153F or Y155H) of WE HPI is sufficient for the switch from DAG1 to HS binding. Furthermore, we established a simple and reliable virus-binding assay, using directly labelled VSV-GP by intramolecular fusion of VSV-P and mWasabi, demonstrating the importance of HS for virus attachment but not entry in Burkitt lymphoma cells after reconstitution of HS expression. Collectively, our study highlights the essential role of HS for low affinity LCMV infection in contrast to their high affinity counterparts. Residual LCMV infection in double knockouts indicate the use of (a) still unknown entry receptor(s).

Several mammalian arenaviruses (mammarenaviruses) cause hemorrhagic fevers in humans and pose serious public health concerns in their endemic regions. Additionally, mounting evidence indicates that the worldwide-distributed, prototypic mammarenavirus, lymphocytic choriomeningitis virus (LCMV), is a neglected human pathogen of clinical significance. Concerns about human-pathogenic mammarenaviruses are exacerbated by of the lack of licensed vaccines, and current anti-mammarenavirus therapy is limited to off-label use of ribavirin that is only partially effective. Detailed understanding of virus/host-cell interactions may facilitate the development of novel anti-mammarenavirus strategies by targeting components of the host-cell machinery that are required for efficient virus multiplication. Here we document the generation of a recombinant LCMV encoding a nucleoprotein (NP) containing an affinity tag (rLCMV/Strep-NP) and its use to capture the NP-interactome in infected cells. Our proteomic approach combined with genetics and pharmacological validation assays identified ATPase Na+/K+ transporting subunit alpha 1 (ATP1A1) and prohibitin (PHB) as pro-viral factors. Cell-based assays revealed that ATP1A1 and PHB are involved in different steps of the virus life cycle. Accordingly, we observed a synergistic inhibitory effect on LCMV multiplication with a combination of ATP1A1 and PHB inhibitors. We show that ATP1A1 inhibitors suppress multiplication of Lassa virus and Candid#1, a live-attenuated vaccine strain of Junín virus, suggesting that the requirement of ATP1A1 in virus multiplication is conserved among genetically distantly related mammarenaviruses. Our findings suggest that clinically approved inhibitors of ATP1A1, like digoxin, could be repurposed to treat infections by mammarenaviruses pathogenic for humans.

Foxp3+ regulatory T (Treg) cells are essential for the maintenance of immune homeostasis and tolerance. During viral infections, Treg cells can limit the immunopathology resulting from excessive inflammation, yet potentially inhibit effective antiviral T cell responses and promote virus persistence. We report here that the fast-replicating LCMV strain Docile triggers a massive expansion of the Treg population that directly correlates with the size of the virus inoculum and its tendency to establish a chronic, persistent infection. This Treg cell proliferation was greatly enhanced in IL-21R-/- mice and depletion of Treg cells partially rescued defective CD8+ T cell cytokine responses and improved viral clearance in some but not all organs. Notably, IL-21 inhibited Treg cell expansion in a cell intrinsic manner. Moreover, experimental augmentation of Treg cells driven by injection of IL-2/anti-IL-2 immune complexes drastically impaired the functionality of the antiviral T cell response and impeded virus clearance. As a consequence, mice became highly susceptible to chronic infection following exposure to low virus doses. These findings reveal virus-driven Treg cell proliferation as potential evasion strategy that facilitates T cell exhaustion and virus persistence. Furthermore, they suggest that besides its primary function as a direct survival signal for antiviral CD8+ T cells during chronic infections, IL-21 may also indirectly promote CD8+ T cell poly-functionality by restricting the suppressive activity of infection-induced Treg cells.

Stem cells are maintained by transcriptional programs that promote self-renewal and repress differentiation. Here, we found that the transcription factor c-Myb was essential for generating and maintaining stem cells in the CD8 + T cell memory compartment. Following viral infection, CD8 + T cells lacking Myb underwent terminal differentiation and generated fewer stem cell–like central memory cells than did Myb -sufficient T cells. c-Myb acted both as a transcriptional activator of Tcf7 (which encodes the transcription factor Tcf1) to enhance memory development and as a repressor of Zeb2 (which encodes the transcription factor Zeb2) to hinder effector differentiation. Domain-mutagenesis experiments revealed that the transactivation domain of c-Myb was necessary for restraining differentiation, whereas its negative regulatory domain was critical for cell survival. Myb overexpression enhanced CD8 + T cell memory formation, polyfunctionality and recall responses that promoted curative antitumor immunity after adoptive transfer. These findings identify c-Myb as a pivotal regulator of CD8 + T cell stemness and highlight its therapeutic potential. Stemness is crucial for the maintenance of long-term T cell memory. Gattinoni and colleagues demonstrate that the transcription factor c-Myb is essential for the establishment of a stemness program in the CD8 + T cell memory compartment.

Naïve CD8+ T cells can differentiate into effector (Teff), memory (Tmem) or exhausted (Tex) T cells. These developmental pathways are associated with distinct transcriptional and epigenetic changes that endow cells with different functional capacities and therefore therapeutic potential. The molecular circuitry underlying these developmental trajectories and the extent of heterogeneity within Teff, Tmem and Tex populations remain poorly understood. Here, we used the lymphocytic choriomeningitis virus model of acute-resolving and chronic infection to address these gaps by applying longitudinal single-cell RNA-sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) analyses. These analyses uncovered new subsets, including a subpopulation of Tex cells expressing natural killer cell-associated genes that is dependent on the transcription factor Zeb2, as well as multiple distinct TCF-1+ stem/progenitor-like subsets in acute and chronic infection. These data also revealed insights into the reshaping of Tex subsets following programmed death 1 (PD-1) pathway blockade and identified a key role for the cell stress regulator, Btg1, in establishing the Tex population. Finally, these results highlighted how the same biological circuits such as cytotoxicity or stem/progenitor pathways can be used by CD8+ T cell subsets with highly divergent underlying chromatin landscapes generated during different infections.Wherry and colleagues provide a comparative analysis of paired single-cell RNA-sequencing and single-cell assay for transposase-accessible chromatin sequencing profiles of CD8+ T cells in acute and chronic lymphocytic choriomeningitis virus infections, identifying new features about Tex cell subsets and epigenetic differences from acutely infected precursors seen at early time points in infection.

Inhibiting PD-1:PD-L1 signaling has transformed therapeutic immune restoration. CD4 + T cells sustain immunity in chronic infections and cancer, yet little is known about how PD-1 signaling modulates CD4 + helper T (T H ) cell responses or the ability to restore CD4 + T H -mediated immunity by checkpoint blockade. We demonstrate that PD-1:PD-L1 specifically suppressed CD4 + T H 1 cell amplification, prevents CD4 + T H 1 cytokine production and abolishes CD4 + cytotoxic killing capacity during chronic infection in mice. Inhibiting PD-L1 rapidly restored these functions, while simultaneously amplifying and activating T H 1-like T regulatory cells, demonstrating a system-wide CD4–T H 1 recalibration. This effect coincided with decreased T cell antigen receptor signaling, and re-directed type I interferon (IFN) signaling networks towards dominant IFN-γ-mediated responses. Mechanistically, PD-L1 blockade specifically targeted defined populations with pre-established, but actively suppressed proliferative potential, with limited impact on minimally cycling TCF-1 + follicular helper T cells, despite high PD-1 expression. Thus, CD4 + T cells require unique differentiation and functional states to be targets of PD-L1-directed suppression and therapeutic restoration. Snell et al. examine the heterogeneity of CD4 + T cells in chronic viral infection, showing that PD-L1 blockade enhances a cytotoxic gene program in antigen-specific T H 1 cells and can restore antiviral CD4 + T cell killer function.

Chronic infection can lead to T cell exhaustion. Wherry and colleagues demonstrate that a hierarchy of inhibitory receptors coregulate CD8 + T cell exhaustion during chronic viral infection. T cell exhaustion often occurs during chronic infection and prevents optimal viral control. The molecular pathways involved in T cell exhaustion remain poorly understood. Here we show that exhausted CD8 + T cells are subject to complex layers of negative regulation resulting from the coexpression of multiple inhibitory receptors. Exhausted CD8 + T cells expressed up to seven inhibitory receptors. Coexpression of multiple distinct inhibitory receptors was associated with greater T cell exhaustion and more severe infection. Regulation of T cell exhaustion by various inhibitory pathways was nonredundant, as blockade of the T cell inhibitory receptors PD-1 and LAG-3 simultaneously and synergistically improved T cell responses and diminished viral load in vivo . Thus, CD8 + T cell responses during chronic viral infections are regulated by complex patterns of coexpressed inhibitory receptors.

Although viruses have been implicated in central nervous system (CNS) diseases of unknown etiology, including multiple sclerosis and amyotrophic lateral sclerosis, the reproducible identification of viral triggers in such diseases has been largely unsuccessful. Here, we explore the hypothesis that viruses need not replicate in the tissue in which they cause disease; specifically, that a peripheral infection might trigger CNS pathology. To test this idea, we utilized a transgenic mouse model in which we found that immune cells responding to a peripheral infection are recruited to the CNS, where they trigger neurological damage. In this model, mice are infected with both CNS-restricted measles virus (MV) and peripherally restricted lymphocytic choriomeningitis virus (LCMV). While infection with either virus alone resulted in no illness, infection with both viruses caused disease in all mice, with ∼50% dying following seizures. Co-infection resulted in a 12-fold increase in the number of CD8+ T cells in the brain as compared to MV infection alone. Tetramer analysis revealed that a substantial proportion (>35%) of these infiltrating CD8+ lymphocytes were LCMV-specific, despite no detectable LCMV in CNS tissues. Mechanistically, CNS disease was due to edema, induced in a CD8-dependent but perforin-independent manner, and brain herniation, similar to that observed in mice challenged intracerebrally with LCMV. These results indicate that T cell trafficking can be influenced by other ongoing immune challenges, and that CD8+ T cell recruitment to the brain can trigger CNS disease in the apparent absence of cognate antigen. By extrapolation, human CNS diseases of unknown etiology need not be associated with infection with any particular agent; rather, a condition that compromises and activates the blood-brain barrier and adjacent brain parenchyma can render the CNS susceptible to pathogen-independent immune attack.

During the course of many chronic viral infections, the antiviral T cell response becomes attenuated through a process that is regulated in part by the host. While elevated expression of the immunosuppressive cytokine IL-10 is involved in the suppression of viral-specific T cell responses, the relevant cellular sources of IL-10, as well as the pathways responsible for IL-10 induction, remain unclear. In this study, we traced IL-10 production over the course of chronic lymphocytic choriomeningitis virus (LCMV) infection in an IL-10 reporter mouse line. Using this model, we demonstrated that virus-specific T cells with reduced inflammatory function, particularly Th1 cells, display elevated and sustained IL-10 expression during chronic LCMV infection. Furthermore, ablation of IL-10 from the T cell compartment partially restored T cell function and reduced viral loads in LCMV-infected animals. We found that viral persistence is needed for sustained IL-10 production by Th1 cells and that the transcription factor BLIMP-1 is required for IL-10 expression by Th1 cells. Restimulation of Th1 cells from LCMV-infected mice promoted BLIMP-1 and subsequent IL-10 expression, suggesting that constant antigen exposure likely induces the BLIMP-1/IL-10 pathway during chronic viral infection. Together, these data indicate that effector T cells self-limit their responsiveness during persistent viral infection via an IL-10-dependent negative feedback loop.

Viral infections of central nervous system (CNS) often trigger inflammatory responses that give rise to a wide range of pathological outcomes. The CNS is equipped with an elaborate network of innate immune sentinels (e.g. microglia, macrophages, dendritic cells) that routinely serve as first responders to these infections. The mechanisms that underlie the dynamic programming of these cells following CNS viral infection remain undefined. To gain insights into this programming, we utilized a combination of genomic and two-photon imaging approaches to study a pure innate immune response to a noncytopathic virus (lymphocytic choriomeningitis virus) as it established persistence in the brain. This enabled us to evaluate how global gene expression patterns were translated into myeloid cell dynamics following infection. Two-photon imaging studies revealed that innate myeloid cells mounted a vigorous early response to viral infection characterized by enhanced vascular patrolling and a complete morphological transformation. Interestingly, innate immune activity subsided over time and returned to a quasi-normal state as the virus established widespread persistence in the brain. At the genomic level, early myeloid cell dynamics were associated with massive changes in CNS gene expression, most of which declined over time and were linked to type I interferon signaling (IFN-I). Surprisingly, in the absence of IFN-I signaling, almost no differential gene expression was observed in the nervous system despite increased viral loads. In addition, two-photon imaging studies revealed that IFN-I receptor deficient myeloid cells were unresponsive to viral infection and remained in a naïve state. These data demonstrate that IFN-I engages non-redundant programming responsible for nearly all innate immune activity in the brain following a noncytopathic viral infection. This Achilles' heel could explain why so many neurotropic viruses have acquired strategies to suppress IFN-I.