Viral DNA polymerase structures reveal mechanisms of antiviral drug resistance.

DNA polymerases are important drug targets, and many structural studies have captured them in distinct conformations. However, a detailed understanding of the impact of polymerase conformational dynamics on drug resistance is lacking. We determined cryoelectron microscopy (cryo-EM) structures of DNA-bound herpes simplex virus polymerase holoenzyme in multiple conformations and interacting with antivirals in clinical use. These structures reveal how the catalytic subunit Pol and the processivity factor UL42 bind DNA to promote processive DNA synthesis. Unexpectedly, in the absence of an incoming nucleotide, we observed Pol in multiple conformations with the closed state sampled by the fingers domain. Drug-bound structures reveal how antivirals may selectively bind enzymes that more readily adopt the closed conformation. Molecular dynamics simulations and the cryo-EM structure of a drug-resistant mutant indicate that some resistance mutations modulate conformational dynamics rather than directly impacting drug binding, thus clarifying mechanisms that drive drug selectivity.

Assembly and activation of EBV latent membrane protein 1.

Latent membrane protein 1 (LMP1) is the primary oncoprotein of Epstein-Barr virus (EBV) and plays versatile roles in the EBV life cycle and pathogenesis. Despite decades of extensive research, the molecular basis for LMP1 folding, assembly, and activation remains unclear. Here, we report cryo-electron microscopy structures of LMP1 in two unexpected assemblies: a symmetric homodimer and a higher-order filamentous oligomer. LMP1 adopts a non-canonical and unpredicted fold that supports the formation of a stable homodimer through tight and antiparallel intermolecular packing. LMP1 dimers further assemble side-by-side into higher-order filamentous oligomers, thereby allowing the accumulation and specific organization of the flexible cytoplasmic tails for efficient recruitment of downstream factors. Super-resolution microscopy and cellular functional assays demonstrate that mutations at both dimeric and oligomeric interfaces disrupt LMP1 higher-order assembly and block multiple LMP1-mediated signaling pathways. Our research provides a framework for understanding the mechanism of LMP1 and for developing potential therapies targeting EBV-associated diseases.

Exploiting Mucosal Immunity for Antiviral Vaccines.

Mucosal surfaces provide a remarkably effective barrier against potentially dangerous pathogens. Therefore, enhancing mucosal immunity through vaccines-strengthening that first line of defense-holds significant promise for reducing the burden of viral diseases. The large and varied class of viral pathogens, however, continues to present thorny challenges to vaccine development. Two primary difficulties exist: Viruses exhibit a stunning diversity of strategies for evading the host immune response, and even when we understand the nature of effective immune protection against a given virus, eliciting that protection is technically challenging. Only a few mucosal vaccines have surmounted these obstacles thus far. Recent developments, however, could greatly improve vaccine design. In this review, we first sketch out our understanding of mucosal immunity and then compare the herpes simplex virus, human immunodeficiency virus, and influenza virus to illustrate the distinct challenges of developing successful vaccines and to outline potential solutions.

Herpes simplex virus gene therapy for dystrophic epidermolysis bullosa (DEB).

The FDA has recently approved Krystal biotech's beremagene geperpavec (B-VEC, Vyjuvek) to treat the wounds of dystrophic epidermolysis bullosa (DEB) patients. This represents a giant step, not only toward the treatment of this devastating disease, but also for the whole field of non-replicative (nr) recombinant HSV-1 vectors for gene therapy. To view this Bench to Bedside, open or download the PDF.

EBV and MS: The evidence is growing stronger.

Autoimmune cross-reaction of specific antibodies to Epstein-Barr virus (EBV) has been associated with development of multiple sclerosis (MS). In this issue of Cell, Vietzen et al. identify additional immunological regulatory mechanisms that protect against autoimmunity in healthy people but are reduced in MS cases. The results confirm the link between EBV and MS.

Ineffective control of Epstein-Barr-virus-induced autoimmunity increases the risk for multiple sclerosis.

Multiple sclerosis (MS) is a demyelinating disease of the CNS. Epstein-Barr virus (EBV) contributes to the MS pathogenesis because high levels of EBV EBNA386-405-specific antibodies cross react with the CNS-derived GlialCAM370-389. However, it is unclear why only some individuals with such high autoreactive antibody titers develop MS. Here, we show that autoreactive cells are eliminated by distinct immune responses, which are determined by genetic variations of the host, as well as of the infecting EBV and human cytomegalovirus (HCMV). We demonstrate that potent cytotoxic NKG2C+ and NKG2D+ natural killer (NK) cells and distinct EBV-specific T cell responses kill autoreactive GlialCAM370-389-specific cells. Furthermore, immune evasion of these autoreactive cells was induced by EBV-variant-specific upregulation of the immunomodulatory HLA-E. These defined virus and host genetic pre-dispositions are associated with an up to 260-fold increased risk of MS. Our findings thus allow the early identification of patients at risk for MS and suggest additional therapeutic options against MS.

The immunology of Epstein-Barr virus-induced disease.

Epstein-Barr virus (EBV) is usually acquired silently early in life and carried thereafter as an asymptomatic infection of the B lymphoid system. However, many circumstances disturb the delicate EBV-host balance and cause the virus to display its pathogenic potential. Thus, primary infection in adolescence can manifest as infectious mononucleosis (IM), as a fatal illness that magnifies the immunopathology of IM in boys with the X-linked lymphoproliferative disease trait, and as a chronic active disease leading to life-threatening hemophagocytosis in rare cases of T or natural killer (NK) cell infection. Patients with primary immunodeficiencies affecting the NK and/or T cell systems, as well as immunosuppressed transplant recipients, handle EBV infections poorly, and many are at increased risk of virus-driven B-lymphoproliferative disease. By contrast, a range of other EBV-positive malignancies of lymphoid or epithelial origin arise in individuals with seemingly intact immune systems through mechanisms that remain to be understood.

Epstein-Barr virus: Biology and clinical disease.

Epstein-Barr virus (EBV) is a ubiquitous, oncogenic virus that is associated with a number of different human malignancies as well as autoimmune disorders. The expression of EBV viral proteins and non-coding RNAs contribute to EBV-mediated disease pathologies. The virus establishes life-long latency in the human host and is adept at evading host innate and adaptive immune responses. In this review, we discuss the life cycle of EBV, the various functions of EBV-encoded proteins and RNAs, the ability of the virus to activate and evade immune responses, as well as the neoplastic and autoimmune diseases that are associated with EBV infection in the human population.

Distinct developmental pathways generate functionally distinct populations of natural killer cells.

Natural killer (NK) cells function by eliminating virus-infected or tumor cells. Here we identified an NK-lineage-biased progenitor population, referred to as early NK progenitors (ENKPs), which developed into NK cells independently of common precursors for innate lymphoid cells (ILCPs). ENKP-derived NK cells (ENKP_NK cells) and ILCP-derived NK cells (ILCP_NK cells) were transcriptionally different. We devised combinations of surface markers that identified highly enriched ENKP_NK and ILCP_NK cell populations in wild-type mice. Furthermore, Ly49H+ NK cells that responded to mouse cytomegalovirus infection primarily developed from ENKPs, whereas ILCP_NK cells were better IFNγ producers after infection with Salmonella and herpes simplex virus. Human CD56dim and CD56bright NK cells were transcriptionally similar to ENKP_NK cells and ILCP_NK cells, respectively. Our findings establish the existence of two pathways of NK cell development that generate functionally distinct NK cell subsets in mice and further suggest these pathways may be conserved in humans.

Immunological lessons from CD28 deficiency in humans.

In this issue of Cell, Casanova and colleagues examine three family members with a mutation that results in deficiency of the T cell co-stimulatory molecule CD28. These patients exhibit clinical symptoms due to human papillomavirus-2 and -4 infections, show increased levels of Epstein-Barr virus and cytomegalovirus in the blood, and respond poorly to vaccines.

A Single Transcription Factor Drives Toxoplasma gondii Differentiation.

Microbes that cause persistent infections (e.g., herpes viruses) do so by switching from fast-growing lytic states to slow-growing latent states. Waldman et al. have identified a single transcription factor that governs the switch between the lytic and latent forms of Toxoplasma gondii, a parasite that causes a persistent brain infection.

Clonal expansion and epigenetic inheritance of long-lasting NK cell memory.

Clonal expansion of cells with somatically diversified receptors and their long-term maintenance as memory cells is a hallmark of adaptive immunity. Here, we studied pathogen-specific adaptation within the innate immune system, tracking natural killer (NK) cell memory to human cytomegalovirus (HCMV) infection. Leveraging single-cell multiomic maps of ex vivo NK cells and somatic mitochondrial DNA mutations as endogenous barcodes, we reveal substantial clonal expansion of adaptive NK cells in HCMV+ individuals. NK cell clonotypes were characterized by a convergent inflammatory memory signature enriched for AP1 motifs superimposed on a private set of clone-specific accessible chromatin regions. NK cell clones were stably maintained in specific epigenetic states over time, revealing that clonal inheritance of chromatin accessibility shapes the epigenetic memory repertoire. Together, we identify clonal expansion and persistence within the human innate immune system, suggesting that these mechanisms have evolved independent of antigen-receptor diversification.

Deconvoluting global cytokine signaling networks in natural killer cells.

Cytokine signaling via signal transducer and activator of transcription (STAT) proteins is crucial for optimal antiviral responses of natural killer (NK) cells. However, the pleiotropic effects of both cytokine and STAT signaling preclude the ability to precisely attribute molecular changes to specific cytokine-STAT modules. Here, we employed a multi-omics approach to deconstruct and rebuild the complex interaction of multiple cytokine signaling pathways in NK cells. Proinflammatory cytokines and homeostatic cytokines formed a cooperative axis to commonly regulate global gene expression and to further repress expression induced by type I interferon signaling. These cytokines mediated distinct modes of epigenetic regulation via STAT proteins, and collective signaling best recapitulated global antiviral responses. The most dynamically responsive genes were conserved across humans and mice, which included a cytokine-STAT-induced cross-regulatory program. Thus, an intricate crosstalk exists between cytokine signaling pathways, which governs NK cell responses.

A latent subset of human hematopoietic stem cells resists regenerative stress to preserve stemness.

Continuous supply of immune cells throughout life relies on the delicate balance in the hematopoietic stem cell (HSC) pool between long-term maintenance and meeting the demands of both normal blood production and unexpected stress conditions. Here we identified distinct subsets of human long-term (LT)-HSCs that responded differently to regeneration-mediated stress: an immune checkpoint ligand CD112lo subset that exhibited a transient engraftment restraint (termed latency) before contributing to hematopoietic reconstitution and a primed CD112hi subset that responded rapidly. This functional heterogeneity and CD112 expression are regulated by INKA1 through direct interaction with PAK4 and SIRT1, inducing epigenetic changes and defining an alternative state of LT-HSC quiescence that serves to preserve self-renewal and regenerative capacity upon regeneration-mediated stress. Collectively, our data uncovered the molecular intricacies underlying HSC heterogeneity and self-renewal regulation and point to latency as an orchestrated physiological response that balances blood cell demands with preserving a stem cell reservoir.

Pathogenesis and host control of gammaherpesviruses: lessons from the mouse.

Gammaherpesviruses are lymphotropic viruses that are associated with the development of lymphoproliferative diseases, lymphomas, as well as other nonlymphoid cancers. Most known gammaherpesviruses establish latency in B lymphocytes. Research on Epstein-Barr virus (EBV) and murine gammaherpesvirus 68 (MHV68/γHV68/MHV4) has revealed a complex relationship between virus latency and the stage of B cell differentiation. Available data support a model in which gammaherpesvirus infection drives B cell proliferation and differentiation. In general, the characterized gammaherpesviruses exhibit a very narrow host tropism, which has severely limited studies on the human gammaherpesviruses EBV and Kaposi's sarcoma-associated herpesvirus. As such, there has been significant interest in developing animal models in which the pathogenesis of gammaherpesviruses can be characterized. MHV68 represents a unique model to define the effects of chronic viral infection on the antiviral immune response.

Human TLRs and IL-1Rs in host defense: natural insights from evolutionary, epidemiological, and clinical genetics.

Toll-like receptors (TLRs) and interleukin-1 receptors (IL-1Rs) have TIR intracellular domains that engage two main signaling pathways, via the TIR-containing adaptors MyD88 (which is not used by TLR3) and TRIF (which is used only by TLR3 and TLR4). Extensive studies in inbred mice in various experimental settings have attributed key roles in immunity to TLR- and IL-1R-mediated responses, but what contribution do human TLRs and IL-1Rs actually make to host defense in the natural setting? Evolutionary genetic studies have shown that human intracellular TLRs have evolved under stronger purifying selection than surface-expressed TLRs, for which the frequency of missense and nonsense alleles is high in the general population. Epidemiological genetic studies have yet to provide convincing evidence of a major contribution of common variants of human TLRs, IL-1Rs, or their adaptors to host defense. Clinical genetic studies have revealed that rare mutations affecting the TLR3-TRIF pathway underlie herpes simplex virus encephalitis, whereas mutations in the TIR-MyD88 pathway underlie pyogenic bacterial diseases in childhood. A careful reconsideration of the contributions of TLRs and IL-1Rs to host defense in natura is required.

Epstein-Barr virus gp42 antibodies reveal sites of vulnerability for receptor binding and fusion to B cells.

Epstein-Barr virus (EBV) causes infectious mononucleosis and is associated with B cell lymphomas. EBV glycoprotein 42 (gp42) binds HLA class II and activates membrane fusion with B cells. We isolated gp42-specific monoclonal antibodies (mAbs), A10 and 4C12, which use distinct mechanisms to neutralize virus infection. mAb A10 was more potent than the only known neutralizing gp42 mAb, F-2-1, in neutralizing EBV infection and blocking binding to HLA class II. mAb 4C12 was similar to mAb A10 in inhibiting glycoprotein-mediated B cell fusion but did not block receptor binding, and it was less effective in neutralizing infection. Crystallographic structures of gH/gL/gp42/A10 and gp42/4C12 complexes revealed two distinct sites of vulnerability on gp42 for receptor binding and B cell fusion. Passive transfer of mAb A10 into humanized mice conferred nearly 100% protection from viremia and EBV lymphomas after EBV challenge. These findings identify vulnerable sites on EBV that may facilitate therapeutics and vaccines.

Inborn Errors of RNA Lariat Metabolism in Humans with Brainstem Viral Infection.

Viruses that are typically benign sometimes invade the brainstem in otherwise healthy children. We report bi-allelic DBR1 mutations in unrelated patients from different ethnicities, each of whom had brainstem infection due to herpes simplex virus 1 (HSV1), influenza virus, or norovirus. DBR1 encodes the only known RNA lariat debranching enzyme. We show that DBR1 expression is ubiquitous, but strongest in the spinal cord and brainstem. We also show that all DBR1 mutant alleles are severely hypomorphic, in terms of expression and function. The fibroblasts of DBR1-mutated patients contain higher RNA lariat levels than control cells, this difference becoming even more marked during HSV1 infection. Finally, we show that the patients' fibroblasts are highly susceptible to HSV1. RNA lariat accumulation and viral susceptibility are rescued by wild-type DBR1. Autosomal recessive, partial DBR1 deficiency underlies viral infection of the brainstem in humans through the disruption of tissue-specific and cell-intrinsic immunity to viruses.

Redox homeostasis maintained by GPX4 facilitates STING activation.

Stimulator-of-interferon genes (STING) is vital for sensing cytosolic DNA and initiating innate immune responses against microbial infection and tumors. Redox homeostasis is the balance of oxidative and reducing reactions present in all living systems. Yet, how the intracellular redox state controls STING activation is unclear. Here, we show that cellular redox homeostasis maintained by glutathione peroxidase 4 (GPX4) is required for STING activation. GPX4 deficiency enhanced cellular lipid peroxidation and thus specifically inhibited the cGAS-STING pathway. Concordantly, GPX4 deficiency inhibited herpes simplex virus-1 (HSV-1)-induced innate antiviral immune responses and promoted HSV-1 replication in vivo. Mechanistically, GPX4 inactivation increased production of lipid peroxidation, which led to STING carbonylation at C88 and inhibited its trafficking from the endoplasmic reticulum (ER) to the Golgi complex. Thus, cellular stress-induced lipid peroxidation specifically attenuates the STING DNA-sensing pathway, suggesting that GPX4 facilitates STING activation by maintaining redox homeostasis of lipids.

The activation trajectory of plasmacytoid dendritic cells in vivo during a viral infection.

Plasmacytoid dendritic cells (pDCs) are a major source of type I interferon (IFN-I). What other functions pDCs exert in vivo during viral infections is controversial, and more studies are needed to understand their orchestration. In the present study, we characterize in depth and link pDC activation states in animals infected by mouse cytomegalovirus by combining Ifnb1 reporter mice with flow cytometry, single-cell RNA sequencing, confocal microscopy and a cognate CD4 T cell activation assay. We show that IFN-I production and T cell activation were performed by the same pDC, but these occurred sequentially in time and in different micro-anatomical locations. In addition, we show that pDC commitment to IFN-I production was marked early on by their downregulation of leukemia inhibitory factor receptor and was promoted by cell-intrinsic tumor necrosis factor signaling. We propose a new model for how individual pDCs are endowed to exert different functions in vivo during a viral infection, in a manner tightly orchestrated in time and space.