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The fate of developing T cells is determined by the strength of T cell receptor (TCR) signal they receive in the thymus. This process is finely regulated through the tuning of positive and negative regulators in thymocytes. The Family with sequence similarity 49 member B (Fam49b) protein is a newly discovered negative regulator of TCR signaling that has been shown to suppress Rac-1 activity in vitro in cultured T cell lines. However, the contribution of Fam49b to the thymic development of T cells is unknown. To investigate this important issue, we generated a novel mouse line deficient in Fam49b (Fam49b-KO). We observed that Fam49b-KO double positive (DP) thymocytes underwent excessive negative selection, whereas the positive selection stage was unaffected. Fam49b deficiency impaired the survival of single positive thymocytes and peripheral T cells. This altered development process resulted in significant reductions in CD4 and CD8 single-positive thymocytes as well as peripheral T cells. Interestingly, a large proportion of the TCRγδ+ and CD8αα+TCRαβ+ gut intraepithelial T lymphocytes were absent in Fam49b-KO mice. Our results demonstrate that Fam49b dampens thymocytes TCR signaling in order to escape negative selection during development, uncovering the function of Fam49b as a critical regulator of the selection process to ensure normal thymocyte development and peripheral T cells survival.

The endoplasmic reticulum aminopeptidase associated with antigen processing (ERAAP) plays a crucial role in shaping the peptide-major histocompatibility complex (MHC) class I repertoire and maintaining immune surveillance. While murine cytomegalovirus (MCMV) has multiple strategies for manipulating the antigen processing pathway to evade immune responses, the host has also developed ways to counter viral immune evasion. In this study, we find that MCMV modulates ERAAP and induces an interferon γ (IFN-γ)-producing CD8+ T cell effector response that targets uninfected ERAAP-deficient cells. We observe that ERAAP downregulation during infection leads to the presentation of the self-peptide FL9 on non-classical Qa-1b, thereby eliciting Qa-1b-restricted QFL T cells to proliferate in the liver and spleen of infected mice. QFL T cells upregulate effector markers upon MCMV infection and are sufficient to reduce viral load after transfer to immunodeficient mice. Our study highlights the consequences of ERAAP dysfunction during viral infection and provides potential targets for anti-viral therapies.

MHC-E restricted CD8 T cells show promise in vaccine settings, but their development and specificity remain poorly understood. Here we focus on a CD8 T cell population reactive to a self-peptide (FL9) bound to mouse MHC-E (Qa-1b) that is presented in response to loss of the MHC I processing enzyme ERAAP, termed QFL T cells. We find that mature QFL thymocytes are predominantly CD8αβ+CD4-, show signs of agonist selection, and give rise to both CD8αα and CD8αβ intraepithelial lymphocytes (IEL), as well as memory phenotype CD8αβ T cells. QFL T cells require the MHC I subunit β-2 microglobulin (β2m), but do not require Qa1b or classical MHC I for positive selection. However, QFL thymocytes do require Qa1b for agonist selection and full functionality. Our data highlight the relaxed requirements for positive selection of an MHC-E restricted T cell population and suggest a CD8αβ+CD4- pathway for development of CD8αα IELs.

Host receptor usage by Kaposi's sarcoma-associated herpesvirus (KSHV) has been best studied using primary microvascular endothelial and fibroblast cells, although the virus infects a wide variety of cell types in culture and in natural infections. In these two infection models, KSHV adheres to the cell though heparan sulfate (HS) binding and then interacts with a complex of EphA2, xCT, and integrins α3β1, αVβ3, and αVβ5 to catalyze viral entry. We dissected this receptor complex at the genetic level with CRISPR-Cas9 to precisely determine receptor usage in two epithelial cell lines. Surprisingly, we discovered an infection mechanism that requires HS and EphA2 but is independent of αV- and β1-family integrin expression. Furthermore, infection appears to be independent of the EphA2 intracellular domain. We also demonstrated that while two other endogenous Eph receptors were dispensable for KSHV infection, transduced EphA4 and EphA5 significantly enhanced infection of cells lacking EphA2.IMPORTANCE Our data reveal an integrin-independent route of KSHV infection and suggest that multiple Eph receptors besides EphA2 can promote and regulate infection. Since integrins and Eph receptors are large protein families with diverse expression patterns across cells and tissues, we propose that KSHV may engage with several proteins from both families in different combinations to negotiate successful entry into diverse cell types.

Although macrophages are armed with potent antibacterial functions, Mycobacterium tuberculosis (Mtb) replicates inside these innate immune cells. Determinants of macrophage intrinsic bacterial control, and the Mtb strategies to overcome them, are poorly understood. To further study these processes, we used an affinity tag purification mass spectrometry (AP-MS) approach to identify 187 Mtb-human protein-protein interactions (PPIs) involving 34 secreted Mtb proteins. This interaction map revealed two factors involved in Mtb pathogenesis-the secreted Mtb protein, LpqN, and its binding partner, the human ubiquitin ligase CBL. We discovered that an lpqN Mtb mutant is attenuated in macrophages, but growth is restored when CBL is removed. Conversely, Cbl-/- macrophages are resistant to viral infection, indicating that CBL regulates cell-intrinsic polarization between antibacterial and antiviral immunity. Collectively, these findings illustrate the utility of this Mtb-human PPI map for developing a deeper understanding of the intricate interactions between Mtb and its host.

Natural killer (NK) cells recognize and kill cancer cells and infected cells by engaging cell surface ligands that are induced preferentially or exclusively on these cells. These ligands are recognized by activating receptors on NK cells, such as NKG2D. In addition to activation by cell surface ligands, the acquisition of optimal effector activity by NK cells is driven in vivo by cytokines and other signals. This review addresses a developing theme in NK cell biology: that NK-activating ligands on cells, and the provision of cytokines and other signals that drive high effector function in NK cells, are driven by abnormalities that arise from transformation or the infected state. The pathways include genomic damage, which causes self DNA to be exposed in the cytosol of affected cells, where it activates the DNA sensor cGAS. The resulting signaling induces NKG2D ligands and also mobilizes NK cell activation. Other key pathways that regulate NKG2D ligands include PI-3 kinase activation, histone acetylation, and the integrated stress response. This review summarizes the roles of these pathways and their relevance in both viral infections and cancer.

Necroptosis is a form of necrotic cell death that requires the activity of the death domain-containing kinase RIP1 and its family member RIP3. Necroptosis occurs when RIP1 is deubiquitinated to form a complex with RIP3 in cells deficient in the death receptor adapter molecule FADD or caspase-8. Necroptosis may play a role in host defense during viral infection as viruses like vaccinia can induce necroptosis while murine cytomegalovirus encodes a viral inhibitor of necroptosis. To see how general the interplay between viruses and necroptosis is, we surveyed seven different viruses. We found that two of the viruses tested, Sendai virus (SeV) and murine gammaherpesvirus-68 (MHV68), are capable of inducing dramatic necroptosis in the fibrosarcoma L929 cell line. We show that MHV68-induced cell death occurs through the cytosolic STING sensor pathway in a TNF-dependent manner. In contrast, SeV-induced death is mostly independent of TNF. Knockdown of the RNA sensing molecule RIG-I or the RIP1 deubiquitin protein, CYLD, but not STING, rescued cells from SeV-induced necroptosis. Accompanying necroptosis, we also find that wild type but not mutant SeV lacking the viral proteins Y1 and Y2 result in the non-ubiquitinated form of RIP1. Expression of Y1 or Y2 alone can suppress RIP1 ubiquitination but CYLD is dispensable for this process. Instead, we found that Y1 and Y2 can inhibit cIAP1-mediated RIP1 ubiquitination. Interestingly, we also found that SeV infection of B6 RIP3-/- mice results in increased inflammation in the lung and elevated SeV-specific T cells. Collectively, these data identify viruses and pathways that can trigger necroptosis and highlight the dynamic interplay between pathogen-recognition receptors and cell death induction.

Natural Killer (NK) cells are essential for control of viral infection and cancer. NK cells express NKG2D, an activating receptor that directly recognizes NKG2D ligands. These are expressed at low level on healthy cells, but are induced by stresses like infection and transformation. The physiological events that drive NKG2D ligand expression during infection are still poorly understood. We observed that the mouse cytomegalovirus encoded protein m18 is necessary and sufficient to drive expression of the RAE-1 family of NKG2D ligands. We demonstrate that RAE-1 is transcriptionally repressed by histone deacetylase inhibitor 3 (HDAC3) in healthy cells, and m18 relieves this repression by directly interacting with Casein Kinase II and preventing it from activating HDAC3. Accordingly, we found that HDAC inhibiting proteins from human herpesviruses induce human NKG2D ligand ULBP-1. Thus our findings indicate that virally mediated HDAC inhibition can act as a signal for the host to activate NK-cell recognition.

Recognition and elimination of tumor cells by the immune system is crucial for limiting tumor growth. Natural killer (NK) cells become activated when the receptor NKG2D is engaged by ligands that are frequently upregulated in primary tumors and on cancer cell lines. However, the molecular mechanisms driving NKG2D ligand expression on tumor cells are not well defined. Using a forward genetic screen in a tumor-derived human cell line, we identified several novel factors supporting expression of the NKG2D ligand ULBP1. Our results show stepwise contributions of independent pathways working at multiple stages of ULBP1 biogenesis. Deeper investigation of selected hits from the screen showed that the transcription factor ATF4 drives ULBP1 gene expression in cancer cell lines, while the RNA-binding protein RBM4 supports ULBP1 expression by suppressing a novel alternatively spliced isoform of ULBP1 mRNA. These findings offer insight into the stress pathways that alert the immune system to danger.

Lytic gammaherpesvirus infection restricts host gene expression by promoting widespread degradation of cytoplasmic mRNA through the activity of the viral endonuclease SOX. Though generally assumed to be selective for cellular transcripts, the extent to which SOX impacts viral mRNA stability has remained unknown. We addressed this issue using the model murine gammaherpesvirus MHV68 and, unexpectedly, found that all stages of viral gene expression are controlled through mRNA degradation. Using both comprehensive RNA expression profiling and half-life studies we reveal that the levels of the majority of viral mRNAs but not noncoding RNAs are tempered by MHV68 SOX (muSOX) activity. The targeting of viral mRNA by muSOX is functionally significant, as it impacts intracellular viral protein abundance and progeny virion composition. In the absence of muSOX-imposed gene expression control the viral particles display increased cell surface binding and entry as well as enhanced immediate early gene expression. These phenotypes culminate in a viral replication defect in multiple cell types as well as in vivo, highlighting the importance of maintaining the appropriate balance of viral RNA during gammaherpesviral infection. This is the first example of a virus that fails to broadly discriminate between cellular and viral transcripts during host shutoff and instead uses the targeting of viral messages to fine-tune overall gene expression.

The endoplasmic reticulum-associated degradation (ERAD) pathway is responsible for disposing misfolded proteins from the endoplasmic reticulum by inducing their ubiquitination and degradation. Ubiquitination is conventionally observed on lysine residues and has been demonstrated on cysteine residues and protein N-termini. Ubiquitination is fundamental to the ERAD process; however, a mutant T-cell receptor α (TCRα) lacking lysine residues is targeted for the degradation by the ERAD pathway. We have shown that ubiquitination of lysine-less TCRα occurs on internal, non-lysine residues and that the same E3 ligase conjugates ubiquitin to TCRα in the presence or absence of lysine residues. Mass-spectrometry indicates that WT-TCRα is ubiquitinated on multiple lysine residues. Recent publications have provided indirect evidence that serine and threonine residues may be modified by ubiquitin. Using a novel peptide-based stable isotope labeling in cell culture (SILAC) approach, we show that specific lysine-less TCRα peptides become modified. In this study, we demonstrate that it is possible to detect both ester and thioester based ubiquitination events, although the exact linkage on lysine-less TCRα remains elusive. These findings demonstrate that SILAC can be used as a tool to identify modified peptides, even those with novel modifications that may not be detected using conventional proteomic work flows or informatics algorithms.

The endoplasmic reticulum-associated degradation (ERAD) pathway is responsible for disposing misfolded proteins from the endoplasmic reticulum by inducing their ubiquitination and degradation. Ubiquitination is conventionally observed on lysine residues and has been demonstrated on cysteine residues and protein N-termini. Ubiquitination is fundamental to the ERAD process; however, a mutant T-cell receptor α (TCRα) lacking lysine residues is targeted for the degradation by the ERAD pathway. We have shown that ubiquitination of lysine-less TCRα occurs on internal, non-lysine residues and that the same E3 ligase conjugates ubiquitin to TCRα in the presence or absence of lysine residues. Mass-spectrometry indicates that WT-TCRα is ubiquitinated on multiple lysine residues. Recent publications have provided indirect evidence that serine and threonine residues may be modified by ubiquitin. Using a novel peptide-based stable isotope labeling in cell culture (SILAC) approach, we show that specific lysine-less TCRα peptides become modified. In this study, we demonstrate that it is possible to detect both ester and thioester based ubiquitination events, although the exact linkage on lysine-less TCRα remains elusive. These findings demonstrate that SILAC can be used as a tool to identify modified peptides, even those with novel modifications that may not be detected using conventional proteomic work flows or informatics algorithms.

Natural killer (NK) cells are lymphocytes that play a major role in the elimination of virally-infected cells and tumor cells. NK cells recognize and target abnormal cells through activation of stimulatory receptors such as NKG2D. NKG2D ligands are self-proteins, which are absent or expressed at low levels on healthy cells but are induced upon cellular stress, transformation, or viral infection. The exact molecular mechanisms driving expression of these ligands remain poorly understood. Here we show that murine cytomegalovirus (MCMV) infection activates the phosphatidylinositol-3-kinase (PI3K) pathway and that this activation is required for the induction of the RAE-1 family of mouse NKG2D ligands. Among the multiple PI3K catalytic subunits, inhibition of the p110α catalytic subunit blocks this induction. Similarly, inhibition of p110α PI3K reduces cell surface expression of RAE-1 on transformed cells. Many viruses manipulate the PI3K pathway, and tumors frequently mutate the p110α oncogene. Thus, our findings suggest that dysregulation of the PI3K pathway is an important signal to induce expression of RAE-1, and this may represent a commonality among various types of cellular stresses that result in the induction of NKG2D ligands.

During a lytic gammaherpesvirus infection, host gene expression is severely restricted by the global degradation and altered 3' end processing of mRNA. This host shutoff phenotype is orchestrated by the viral SOX protein, yet its functional significance to the viral lifecycle has not been elucidated, in part due to the multifunctional nature of SOX. Using an unbiased mutagenesis screen of the murine gammaherpesvirus 68 (MHV68) SOX homolog, we isolated a single amino acid point mutant that is selectively defective in host shutoff activity. Incorporation of this mutation into MHV68 yielded a virus with significantly reduced capacity for mRNA turnover. Unexpectedly, the MHV68 mutant showed little defect during the acute replication phase in the mouse lung. Instead, the virus exhibited attenuation at later stages of in vivo infections suggestive of defects in both trafficking and latency establishment. Specifically, mice intranasally infected with the host shutoff mutant accumulated to lower levels at 10 days post infection in the lymph nodes, failed to develop splenomegaly, and exhibited reduced viral DNA levels and a lower frequency of latently infected splenocytes. Decreased latency establishment was also observed upon infection via the intraperitoneal route. These results highlight for the first time the importance of global mRNA degradation during a gammaherpesvirus infection and link an exclusively lytic phenomenon with downstream latency establishment.

MHC-E restricted CD8 T cells show promise in vaccine settings, but their development and specificity remain poorly understood. Here we focus on a CD8 T cell population reactive to a self-peptide (FL9) bound to mouse MHC-E (Qa-1b ) that is presented in response to loss of the MHC I processing enzyme ERAAP, termed QFL T cells. We find that mature QFL thymocytes are predominantly CD8αβ+CD4-, show signs of agonist selection, and give rise to both CD8αα and CD8αβ intraepithelial lymphocytes (IEL), as well as memory phenotype CD8αβ T cells. QFL T cells require the MHC I subunit β-2 microglobulin (β2m), but do not require Qa1b or classical MHC I for positive selection. However, QFL thymocytes do require Qa1b for agonist selection and full functionality. Our data highlight the relaxed requirements for positive selection of an MHC-E restricted T cell population and suggest a CD8αβ+CD4- pathway for development of CD8αα IELs. [ABSTRACT FROM AUTHOR]