Natural Killer Cells: From Innate to Adaptive Features.

Natural killer (NK) cells are innate lymphocytes that provide critical host defense against pathogens and cancer. Originally heralded for their early and rapid effector activity, NK cells have been recognized over the last decade for their ability to undergo adaptive immune processes, including antigen-driven clonal expansion and generation of long-lived memory. This review presents an overview of how NK cells lithely partake in both innate and adaptive responses and how this versatility is manifest in human NK cell-mediated immunity.

An Unbiased Screen for Human Cytomegalovirus Identifies Neuropilin-2 as a Central Viral Receptor.

Characterizing cell surface receptors mediating viral infection is critical for understanding viral tropism and developing antiviral therapies. Nevertheless, due to challenges associated with detecting protein interactions on the cell surface, the host receptors of many human pathogens remain unknown. Here, we build a library consisting of most single transmembrane human receptors and implement a workflow for unbiased and high-sensitivity detection of receptor-ligand interactions. We apply this technology to elucidate the long-sought receptor of human cytomegalovirus (HCMV), the leading viral cause of congenital birth defects. We identify neuropilin-2 (Nrp2) as the receptor for HCMV-pentamer infection in epithelial/endothelial cells and uncover additional HCMV interactors. Using a combination of biochemistry, cell-based assays, and electron microscopy, we characterize the pentamer-Nrp2 interaction and determine the architecture of the pentamer-Nrp2 complex. This work represents an important approach to the study of host-pathogen interactions and provides a framework for understanding HCMV infection, neutralization, and the development of novel anti-HCMV therapies.

Repurposing an endogenous degradation domain for antibody-mediated disposal of cell-surface proteins.

The exquisite specificity of antibodies can be harnessed to effect targeted degradation of membrane proteins. Here, we demonstrate targeted protein removal utilising a protein degradation domain derived from the endogenous human protein Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9). Recombinant antibodies genetically fused to this domain drive the degradation of membrane proteins that undergo constitutive internalisation and recycling, including the transferrin receptor and the human cytomegalovirus latency-associated protein US28. We term this approach PACTAC (PCSK9-Antibody Clearance-Targeting Chimeras).

Novel role of bone morphogenetic protein 9 in innate host responses to HCMV infection.

Herpesviruses modulate immune control to secure lifelong infection. The mechanisms Human Cytomegalovirus (HCMV) employs in this regard can reveal unanticipated aspects of cellular signaling involved in antiviral immunity. Here, we describe a novel relationship between the TGF-ß family cytokine BMP9 and HCMV infection. We identify a cross-talk between BMP9-induced and IFN receptor-mediated signaling, showing that BMP9 boosts the transcriptional response to and antiviral activity of IFNß, thereby enhancing viral restriction. We also show that BMP9 is secreted by human fibroblasts upon HCMV infection. However, HCMV infection impairs BMP9-induced enhancement of the IFNß response, indicating that this signaling role of BMP9 is actively targeted by HCMV. Indeed, transmembrane proteins US18 and US20, which downregulate type I BMP receptors, are necessary and sufficient to cause inhibition of BMP9-mediated boosting of the antiviral response to IFNß. HCMV lacking US18 and US20 is more sensitive to IFNß. Thus, HCMV has a mutually antagonistic relationship with BMP9, which extends the growing body of evidence that BMP signaling is an underappreciated modulator of innate immunity in response to viral infection.

ADAM17 targeting by human cytomegalovirus remodels the cell surface proteome to simultaneously regulate multiple immune pathways.

Human cytomegalovirus (HCMV) is a major human pathogen whose life-long persistence is enabled by its remarkable capacity to systematically subvert host immune defenses. In exploring the finding that HCMV infection up-regulates tumor necrosis factor receptor 2 (TNFR2), a ligand for the pro-inflammatory antiviral cytokine TNFα, we found that the underlying mechanism was due to targeting of the protease, A Disintegrin And Metalloproteinase 17 (ADAM17). ADAM17 is the prototype 'sheddase', a family of proteases that cleaves other membrane-bound proteins to release biologically active ectodomains into the supernatant. HCMV impaired ADAM17 surface expression through the action of two virally-encoded proteins in its UL/b' region, UL148 and UL148D. Proteomic plasma membrane profiling of cells infected with an HCMV double-deletion mutant for UL148 and UL148D with restored ADAM17 expression, combined with ADAM17 functional blockade, showed that HCMV stabilized the surface expression of 114 proteins (P < 0.05) in an ADAM17-dependent fashion. These included reported substrates of ADAM17 with established immunological functions such as TNFR2 and jagged1, but also numerous unreported host and viral targets, such as nectin1, UL8, and UL144. Regulation of TNFα-induced cytokine responses and NK inhibition during HCMV infection were dependent on this impairment of ADAM17. We therefore identify a viral immunoregulatory mechanism in which targeting a single sheddase enables broad regulation of multiple critical surface receptors, revealing a paradigm for viral-encoded immunomodulation.

Genomic and geographical structure of human cytomegalovirus.

Human cytomegalovirus (CMV) has infected humans since the origin of our species and currently infects most of the world's population. Variability between CMV genomes is the highest of any human herpesvirus, yet large portions of the genome are conserved. Here, we show that the genome encodes 74 regions of relatively high variability each with 2 to 8 alleles. We then identified two patterns in the CMV genome. Conserved parts of the genome and a minority (32) of variable regions show geographic population structure with evidence for African or European clustering, although hybrid strains are present. We find no evidence that geographic segregation has been driven by host immune pressure affecting known antigenic sites. Forty-two variable regions show no geographical structure, with similar allele distributions across different continental populations. These "nongeographical" regions are significantly enriched for genes encoding immunomodulatory functions suggesting a core functional importance. We hypothesize that at least two CMV founder populations account for the geographical differences that are largely seen in the conserved portions of the genome, although the timing of separation and direction of spread between the two are not clear. In contrast, the similar allele frequencies among 42 variable regions of the genome, irrespective of geographical origin, are indicative of a second evolutionary process, namely balancing selection that may preserve properties critical to CMV biological function. Given that genetic differences between CMVs are postulated to alter immunogenicity and potentially function, understanding these two evolutionary processes could contribute important information for the development of globally effective vaccines and the identification of novel drug targets.

RNA m6 A modification enzymes shape innate responses to DNA by regulating interferon ß.

Modification of mRNA by N6-adenosine methylation (m6A) on internal bases influences gene expression in eukaryotes. How the dynamic genome-wide landscape of m6A-modified mRNAs impacts virus infection and host immune responses remains poorly understood. Here, we show that type I interferon (IFN) production triggered by dsDNA or human cytomegalovirus (HCMV) is controlled by the cellular m6A methyltrasferase subunit METTL14 and ALKBH5 demethylase. While METTL14 depletion reduced virus reproduction and stimulated dsDNA- or HCMV-induced IFNB1 mRNA accumulation, ALKBH5 depletion had the opposite effect. Depleting METTL14 increased both nascent IFNB1 mRNA production and stability in response to dsDNA. In contrast, ALKBH5 depletion reduced nascent IFNB1 mRNA production without detectably influencing IFN1B mRNA decay. Genome-wide transcriptome profiling following ALKBH5 depletion identified differentially expressed genes regulating antiviral immune responses, while METTL14 depletion altered pathways impacting metabolic reprogramming, stress responses, and aging. Finally, we determined that IFNB1 mRNA was m6A-modified within both the coding sequence and the 3' untranslated region (UTR). This establishes that the host m6A modification machinery controls IFNß production triggered by HCMV or dsDNA. Moreover, it demonstrates that responses to nonmicrobial dsDNA in uninfected cells, which shape host immunity and contribute to autoimmune disease, are regulated by enzymes controlling m6A epitranscriptomic changes.

Human trophoblast stem cells restrict human cytomegalovirus replication.

Placental infection plays a central role in the pathogenesis of congenital human cytomegalovirus (HCMV) infections and is a cause of fetal growth restriction and pregnancy loss. HCMV can replicate in some trophoblast cell types, but it remains unclear how the virus evades antiviral immunity in the placenta and how infection compromises placental development and function. Human trophoblast stem cells (TSCs) can be differentiated into extravillous trophoblasts (EVTs), syncytiotrophoblasts (STBs), and organoids, and this study assessed the utility of TSCs as a model of HCMV infection in the first-trimester placenta. HCMV was found to non-productively infect TSCs, EVTs, and STBs. Immunofluorescence assays and flow cytometry experiments further revealed that infected TSCs frequently only express immediate early viral gene products. Similarly, RNA sequencing found that viral gene expression in TSCs does not follow the kinetic patterns observed during lytic infection in fibroblasts. Canonical antiviral responses were largely not observed in HCMV-infected TSCs and TSC-derived trophoblasts. Rather, infection dysregulated factors involved in cell identity, differentiation, and Wingless/Integrated signaling. Thus, while HCMV does not replicate in TSCs, infection may perturb trophoblast differentiation in ways that could interfere with placental function. IMPORTANCE: Placental infection plays a central role in human cytomegalovirus (HCMV) pathogenesis during pregnancy, but the species specificity of HCMV and the limited availability and lifespan of primary trophoblasts have been persistent barriers to understanding how infection impacts this vital organ. Human trophoblast stem cells (TSCs) represent a new approach to modeling viral infection early in placental development. This study reveals that TSCs, like other stem cell types, restrict HCMV replication. However, infection perturbs the expression of genes involved in differentiation and cell fate determination, pointing to a mechanism by which HCMV could cause placental injury.

A broadly neutralizing human monoclonal antibody generated from transgenic mice immunized with HCMV particles limits virus infection and proliferation.

Human cytomegalovirus (HCMV) is a ß-herpesvirus that poses severe disease risk for immunocompromised patients who experience primary infection or reactivation. Development and optimization of safe and effective anti-HCMV therapeutics is of urgent necessity for the prevention and treatment of HCMV-associated diseases in diverse populations. The use of neutralizing monoclonal antibodies (mAbs) to limit HCMV infection poses a promising therapeutic strategy, as anti-HCMV mAbs largely inhibit infection by targeting virion glycoprotein complexes. In contrast, the small-molecule compounds currently approved for patients (e.g., ganciclovir, letermovir, and maribavir) target later stages of the HCMV life cycle. Here, we present a broadly neutralizing human mAb, designated 1C10, elicited from a VelocImmune mouse immunized with infectious HCMV particles. Clone 1C10 neutralizes infection after virion binding to cells by targeting gH/gL envelope complexes and potently reduces infection of diverse HCMV strains in fibroblast, trophoblast, and epithelial cells. Antibody competition assays found that 1C10 recognizes a region of gH associated with broad neutralization and binds to soluble pentamer in the low nanomolar range. Importantly, 1C10 treatment significantly reduced virus proliferation in both fibroblast and epithelial cells. Further, the combination treatment of mAb 1C10 with ganciclovir reduced HCMV infection and proliferation in a synergistic manner. This work characterizes a neutralizing human mAb for potential use as a HCMV treatment, as well as a possible therapeutic strategy utilizing combination-based treatments targeting disparate steps of the viral life cycle. Collectively, the findings support an antibody-based therapy to effectively treat patients at risk for HCMV-associated diseases. IMPORTANCE: Human cytomegalovirus is a herpesvirus that infects a large proportion of the population and can cause significant disease in diverse patient populations whose immune systems are suppressed or compromised. The development and optimization of safe anti-HCMV therapeutics, especially those that have viral targets and inhibition mechanisms different from current HCMV treatments, are of urgent necessity to better public health. Human monoclonal antibodies (mAbs) that prevent HCMV entry of cells were identified by immunizing transgenic mice and screened for broad and effective neutralization capability. Here, we describe one such mAb, which was found to target gH/gL envelope complexes and effectively limit HCMV infection and dissemination. Further, administration of the antibody in combination with the antiviral drug ganciclovir inhibited HCMV in a synergistic manner, highlighting this approach and the use of anti-HCMV mAbs more broadly, as a potential therapeutic strategy for the treatment of diverse patient populations.

Human cytomegalovirus glycoprotein variants governing viral tropism and syncytium formation in epithelial cells and macrophages.

Human cytomegalovirus (HCMV) displays a broad cell tropism, and the infection of biologically relevant cells such as epithelial, endothelial, and hematopoietic cells supports viral transmission, systemic spread, and pathogenesis in the human host. HCMV strains differ in their ability to infect and replicate in these cell types, but the genetic basis of these differences has remained incompletely understood. In this study, we investigated HCMV strain VR1814, which is highly infectious for epithelial cells and macrophages and induces cell-cell fusion in both cell types. A VR1814-derived bacterial artificial chromosome (BAC) clone, FIX-BAC, was generated many years ago but has fallen out of favor because of its modest infectivity. By sequence comparison and genetic engineering of FIX, we demonstrate that the high infectivity of VR1814 and its ability to induce syncytium formation in epithelial cells and macrophages depends on VR1814-specific variants of the envelope glycoproteins gB, UL128, and UL130. We also show that UL130-neutralizing antibodies inhibit syncytium formation, and a FIX-specific mutation in UL130 is responsible for its low infectivity by reducing the amount of the pentameric glycoprotein complex in viral particles. Moreover, we found that a VR1814-specific mutation in US28 further increases viral infectivity in macrophages, possibly by promoting lytic rather than latent infection of these cells. Our findings show that variants of gB and the pentameric complex are major determinants of infectivity and syncytium formation in epithelial cells and macrophages. Furthermore, the VR1814-adjusted FIX strains can serve as valuable tools to study HCMV infection of myeloid cells.IMPORTANCEHuman cytomegalovirus (HCMV) is a major cause of morbidity and mortality in transplant patients and the leading cause of congenital infections. HCMV infects various cell types, including epithelial cells and macrophages, and some strains induce the fusion of neighboring cells, leading to the formation of large multinucleated cells called syncytia. This process may limit the exposure of the virus to host immune factors and affect pathogenicity. However, the reason why some HCMV strains exhibit a broader cell tropism and why some induce cell fusion more than others is not well understood. We compared two closely related HCMV strains and provided evidence that small differences in viral envelope glycoproteins can massively increase or decrease the virus infectivity and its ability to induce syncytium formation. The results of the study suggest that natural strain variations may influence HCMV infection and pathogenesis in humans.