N-acetyltransferase 10 regulates alphavirus replication via N4-acetylcytidine (ac4C) modification of the lymphocyte antigen six family member E (LY6E) mRNA.

Epitranscriptomic RNA modifications can regulate the stability of mRNA and affect cellular and viral RNA functions. The N4-acetylcytidine (ac4C) modification in the RNA viral genome was recently found to promote viral replication; however, the mechanism by which RNA acetylation in the host mRNA regulates viral replication remains unclear. To help elucidate this mechanism, the roles of N-acetyltransferase 10 (NAT10) and ac4C during the infection and replication processes of the alphavirus, Sindbis virus (SINV), were investigated. Cellular NAT10 was upregulated, and ac4C modifications were promoted after alphavirus infection, while the loss of NAT10 or inhibition of its N-acetyltransferase activity reduced alphavirus replication. The NAT10 enhanced alphavirus replication as it helped to maintain the stability of lymphocyte antigen six family member E mRNA, which is a multifunctional interferon-stimulated gene that promotes alphavirus replication. The ac4C modification was thus found to have a non-conventional role in the virus life cycle through regulating host mRNA stability instead of viral mRNA, and its inhibition could be a potential target in the development of new alphavirus antivirals.IMPORTANCEThe role of N4-acetylcytidine (ac4C) modification in host mRNA and virus replication is not yet fully understood. In this study, the role of ac4C in the regulation of Sindbis virus (SINV), a prototype alphavirus infection, was investigated. SINV infection results in increased levels of N-acetyltransferase 10 (NAT10) and increases the ac4C modification level of cellular RNA. The NAT10 was found to positively regulate SINV infection in an N-acetyltransferase activity-dependent manner. Mechanistically, the NAT10 modifies lymphocyte antigen six family member E (LY6E) mRNA-the ac4C modification site within the 3'-untranslated region (UTR) of LY6E mRNA, which is essential for its translation and stability. The findings of this study demonstrate that NAT10 regulated mRNA stability and translation efficiency not only through the 5'-UTR or coding sequence but also via the 3'-UTR region. The ac4C modification of host mRNA stability instead of viral mRNA impacting the viral life cycle was thus identified, indicating that the inhibition of ac4C could be a potential target when developing alphavirus antivirals.

Impact of natural selection on global patterns of genetic variation, and association with clinical phenotypes, at genes involved in SARS-CoV-2 infection.

We investigated global patterns of genetic variation and signatures of natural selection at host genes relevant to SARS-CoV-2 infection (ACE2, TMPRSS2, DPP4, and LY6E). We analyzed novel data from 2,012 ethnically diverse Africans and 15,997 individuals of European and African ancestry with electronic health records, and integrated with global data from the 1000GP. At ACE2, we identified 41 non-synonymous variants that were rare in most populations, several of which impact protein function. However, three non-synonymous variants were common among Central African hunter-gatherers from Cameroon and are on haplotypes that exhibit signatures of positive selection. We identify strong signatures of selection impacting variation at regulatory regions influencing ACE2 expression in multiple African populations. At TMPRSS2, we identified 13 amino acid changes that are adaptive and specific to the human lineage. Genetic variants that are targets of natural selection are associated with clinical phenotypes common in patients with COVID-19.

Preclinical optimization of Ly6E-targeted ADCs for increased durability and efficacy of anti-tumor response.

Early success with brentuximab vedotin in treating classical Hodgkin lymphoma spurred an influx of at least 20 monomethyl auristatin E (MMAE) antibody-drug conjugates (ADCs) into clinical trials. While three MMAE-ADCs have been approved, most of these conjugates are no longer being investigated in clinical trials. Some auristatin conjugates show limited or no efficacy at tolerated doses, but even for drugs driving initial remissions, tumor regrowth and metastasis often rapidly occur. Here we describe the development of second-generation therapeutic ADCs targeting Lymphocyte antigen 6E (Ly6E) where the tubulin polymerization inhibitor MMAE (Compound 1) is replaced with DNA-damaging agents intended to drive increased durability of response. Comparison of a seco-cyclopropyl benzoindol-4-one (CBI)-dimer (compound 2) to MMAE showed increased potency, activity across more cell lines, and resistance to efflux by P-glycoprotein, a drug transporter commonly upregulated in tumors. Both anti-Ly6E-CBI and -MMAE conjugates drove single-dose efficacy in xenograft and patient-derived xenograft models, but seco-CBI-dimer conjugates showed reduced tumor outgrowth following multiple weeks of treatment, suggesting that they are less susceptible to developing resistance. In parallel, we explored approaches to optimize the targeting antibody. In contrast to immunization with recombinant Ly6E or Ly6E DNA, immunization with virus-like particles generated a high-affinity anti-Ly6E antibody. Conjugates to this antibody improve efficacy versus a previous clinical candidate both in vitro and in vivo with multiple cytotoxics. Conjugation of compound 2 to the second-generation antibody results in a substantially improved ADC with promising preclinical efficacy.

Beyond the Surface: Endocytosis of Mosquito-Borne Flaviviruses.

Flaviviruses are a group of positive-sense RNA viruses that are primarily transmitted through arthropod vectors and are capable of causing a broad spectrum of diseases. Many of the flaviviruses that are pathogenic in humans are transmitted specifically through mosquito vectors. Over the past century, many mosquito-borne flavivirus infections have emerged and re-emerged, and are of global importance with hundreds of millions of infections occurring yearly. There is a need for novel, effective, and accessible vaccines and antivirals capable of inhibiting flavivirus infection and ameliorating disease. The development of therapeutics targeting viral entry has long been a goal of antiviral research, but most efforts are hindered by the lack of broad-spectrum potency or toxicities associated with on-target effects, since many host proteins necessary for viral entry are also essential for host cell biology. Mosquito-borne flaviviruses generally enter cells by clathrin-mediated endocytosis (CME), and recent studies suggest that a subset of these viruses can be internalized through a specialized form of CME that has additional dependencies distinct from canonical CME pathways, and antivirals targeting this pathway have been discovered. In this review, we discuss the role and contribution of endocytosis to mosquito-borne flavivirus entry as well as consider past and future efforts to target endocytosis for therapeutic interventions.

Protein LY6E as a candidate for mediating transport of adeno-associated virus across the human blood-brain barrier.

The blood-brain barrier (BBB) is a major obstacle for the treatment of central nervous system (CNS) disorders. Significant progress has been made in developing adeno-associated virus (AAV) variants with increased ability to cross the BBB in mice. However, these variants are not efficacious in non-human primates. Herein, we employed various bioinformatic techniques to identify lymphocyte antigen-6E (LY6E) as a candidate for mediating transport of AAV across the human BBB based on the previously determined mechanism of transport in mice. Our results provide insight into future discovery and optimization of AAV variants for CNS gene delivery in humans.

LY6E Restricts Entry of Human Coronaviruses, Including Currently Pandemic SARS-CoV-2.

C3A is a subclone of the human hepatoblastoma HepG2 cell line with strong contact inhibition of growth. We fortuitously found that C3A was more susceptible to human coronavirus HCoV-OC43 infection than HepG2, which was attributed to the increased efficiency of virus entry into C3A cells. In an effort to search for the host cellular protein(s) mediating the differential susceptibility of the two cell lines to HCoV-OC43 infection, we found that ArfGAP with dual pleckstrin homology (PH) domains 2 (ADAP2), gamma-interferon-inducible lysosome/endosome-localized thiolreductase (GILT), and lymphocyte antigen 6 family member E (LY6E), the three cellular proteins identified to function in interference with virus entry, were expressed at significantly higher levels in HepG2 cells. Functional analyses revealed that ectopic expression of LY6E, but not GILT or ADAP2, in HEK 293 cells inhibited the entry of HCoV-O43. While overexpression of LY6E in C3A and A549 cells efficiently inhibited the infection of HCoV-OC43, knockdown of LY6E expression in HepG2 significantly increased its susceptibility to HCoV-OC43 infection. Moreover, we found that LY6E also efficiently restricted the entry mediated by the envelope spike proteins of other human coronaviruses, including the currently pandemic SARS-CoV-2. Interestingly, overexpression of serine protease TMPRSS2 or amphotericin treatment significantly neutralized the IFN-inducible transmembrane 3 (IFITM3) restriction of human coronavirus (CoV) entry, but did not compromise the effect of LY6E on the entry of human coronaviruses. The work reported herein thus demonstrates that LY6E is a critical antiviral immune effector that controls CoV infection and pathogenesis via a mechanism distinct from other factors that modulate CoV entry.IMPORTANCE Virus entry into host cells is one of the key determinants of host range and cell tropism and is subjected to the control of host innate and adaptive immune responses. In the last decade, several interferon-inducible cellular proteins, including IFITMs, GILT, ADAP2, 25CH, and LY6E, had been identified to modulate the infectious entry of a variety of viruses. Particularly, LY6E was recently identified as a host factor that facilitates the entry of several human-pathogenic viruses, including human immunodeficiency virus, influenza A virus, and yellow fever virus. Identification of LY6E as a potent restriction factor of coronaviruses expands the biological function of LY6E and sheds new light on the immunopathogenesis of human coronavirus infection.

Dendritic Cell-Based Therapy Using LY6E Peptide with a Putative Role Against Colorectal Cancer.

INTRODUCTION: Albeit early stage gastrointestinal (GI) carcinomas have a good prognosis if treated with surgery, diagnosis is often confirmed at a late stage and efficacious drugs are lacking. Recent progress in immune-based therapies has focused on dendritic cells (DCs), aiming to elicit tumor-specific responses by inducing immunological memory. Our previous microarray study indicated that a biomarker, termed lymphocyte antigen-6E (LY6E), is commonly overexpressed in two potentially lethal GI cancers: those of colon and stomach. In this study, we examined the antigenic potency of LY6E in stimulating DCs. METHODS: Following isolation, differentiation, and maturation of mononuclear cells, DCs were pulsed with LY6E peptide, a protein related to major histocompatibility complex (MHC) class I/II. Subsequently, DCs were co-cultured with mouse splenocytes to assess antigen-specific T-cell proliferation. Elucidated cytotoxic T-lymphocyte responses were assessed using subcutaneous colorectal murine tumor models. RESULTS: Our in vitro results suggest that DCs loaded with LY6E peptide antigen are capable of stimulating and inducing proliferation of murine T-cells. Furthermore, our in vivo results demonstrate that LY6E peptide has a substantial impact on provoking immune responses against induced colon cancer in mice. DISCUSSION: In conclusion, based on the overexpression of LY6E in colorectal, gastric, and pancreatic cancers, the role of this peptide should be further investigated with a goal of developing new therapies for these challenging diseases.

Relating GPI-Anchored Ly6 Proteins uPAR and CD59 to Viral Infection.

The Ly6 (lymphocyte antigen-6)/uPAR (urokinase-type plasminogen activator receptor) superfamily protein is a group of molecules that share limited sequence homology but conserved three-fingered structures. Despite diverse cellular functions, such as in regulating host immunity, cell adhesion, and migration, the physiological roles of these factors in vivo remain poorly characterized. Notably, increasing research has focused on the interplays between Ly6/uPAR proteins and viral pathogens, the results of which have provided new insight into viral entry and virus-host interactions. While LY6E (lymphocyte antigen 6 family member E), one key member of the Ly6E/uPAR-family proteins, has been extensively studied, other members have not been well characterized. Here, we summarize current knowledge of Ly6/uPAR proteins related to viral infection, with a focus on uPAR and CD59. Our goal is to provide an up-to-date view of the Ly6/uPAR-family proteins and associated virus-host interaction and viral pathogenesis.

Emerging Role of LY6E in Virus-Host Interactions.

As a canonical lymphocyte antigen-6/urokinase-type plasminogen activator receptor Ly6/uPAR family protein, lymphocyte antigen 6 complex, locus E (LY6E), plays important roles in immunological regulation, T cell physiology, and oncogenesis. Emerging evidence indicates that LY6E is also involved in the modulation of viral infection. Consequently, viral infection and associated pathogenesis have been associated with altered LY6E gene expression. The interaction between viruses and the host immune system has offered insights into the biology of LY6E. In this review, we summarize the current knowledge of LY6E in the context of viral infection, particularly viral entry.

Emerging Role of Lymphocyte Antigen-6 Family of Genes in Cancer and Immune Cells.

Stem Cell Antigen-1 (Sca-1/Ly6A) was the first identified member of the Lymphocyte antigen-6 (Ly6) gene family. Sca-1 serves as a marker of cancer stem cells and tissue resident stem cells in mice. The Sca-1 gene is located on mouse chromosome 15. While a direct homolog of Sca-1 in humans is missing, human chromosome 8-the syntenic region to mouse chromosome 15-harbors several genes containing the characteristic domain known as LU domain. The function of the LU domain in human LY6 gene family is not yet defined. The LY6 gene family proteins are present on human chromosome 6, 8, 11, and 19. The most interesting of these genes are located on chromosome 8q24.3, a frequently amplified locus in human cancer. Human LY6 genes represent novel biomarkers for poor cancer prognosis and are required for cancer progression in addition to playing an important role in immune escape. Although the mechanism associated with these phenotype is not yet clear, it is timely to review the current literature in order to address the critical need for future advancements in this field. This review will summarize recent findings which describe the role of human LY6 genes-LY6D, LY6E, LY6H, LY6K, PSCA, LYPD2, SLURP1, GML, GPIHBP1, and LYNX1; and their orthologs in mice at chromosome 15.

CD4-Dependent Modulation of HIV-1 Entry by LY6E.

Lymphocyte antigen 6E (LY6E) is a GPI-anchored, interferon-inducible protein that has been shown to modulate viral infection in a cell type-dependent manner. Our recent work showed that LY6E promotes HIV-1 infection in some high-CD4-expressing cells, including human peripheral blood mononuclear cells (PBMCs) and the SupT1 cell line. In this work, we provide evidence that LY6E inhibits HIV-1 entry and spread in low-CD4-expressing Jurkat cells and human monocyte-derived macrophages (MDMs) through downregulation of the viral receptor CD4. We found that knockdown of LY6E in Jurkat cells and MDMs increases HIV-1 infection, yet overexpression of LY6E in Jurkat cells inhibits HIV-1 entry and replication. LY6E was found to be colocalized with CD4 on the plasma membrane of Jurkat cells and MDMs and enhances CD4 internalization. We artificially manipulated the CD4 level in Jurkat and SupT1 cells and found that overexpression of CD4 in Jurkat cells overcomes the inhibitory effect of LY6E; conversely, blocking the function of CD4 in SupT1 with a neutralizing antibody eliminates the enhancement of LY6E on HIV-1 entry. The CD4-dependent inhibitory phenotype of LY6E in low-CD4-expressing human MDMs can be recapitulated for a panel of transmitted founder viruses and laboratory-adapted HIV-1 strains. Given that HIV-1 can target low-CD4-expressing cells during acute infection yet replicates efficiently in high-CD4-expressing T cells at the late stage of disease, our observation that LY6E differentially modulates HIV-1 replication in a CD4-dependent manner has implications for understanding the complex roles of interferon (IFN)-induced proteins in AIDS pathogenesis.IMPORTANCE The role of IFN-induced genes (ISGs) in viral infection remains incompletely understood. While most ISGs are antiviral, some ISGs have been shown to promote viral infection, including HIV-1 infection. We previously showed that IFN-inducible LY6E protein promotes HIV-1 infection in human PMBCs and high-CD4-expressing SupT1 cells. Here we found that LY6E inhibits HIV-1 entry and replication in low-CD4-expressing MDMs and Jurkat cells. Mechanistically, we demonstrated that LY6E downregulates the cell surface receptor CD4, thus impairing the virus binding to target cells. This is in contrast to the situation of high-CD4-expressing cells, where LY6E predominantly promotes viral membrane fusion. The opposing role of IFN-inducible LY6E in modulating HIV-1 infection highlights the complex roles of ISGs in viral infection and viral pathogenesis.

Flavivirus internalization is regulated by a size-dependent endocytic pathway.

Flaviviruses enter host cells through the process of clathrin-mediated endocytosis, and the spectrum of host factors required for this process are incompletely understood. Here we found that lymphocyte antigen 6 locus E (LY6E) promotes the internalization of multiple flaviviruses, including West Nile virus, Zika virus, and dengue virus. Perhaps surprisingly, LY6E is dispensable for the internalization of the endogenous cargo transferrin, which is also dependent on clathrin-mediated endocytosis for uptake. Since viruses are substantially larger than transferrin, we reasoned that LY6E may be required for uptake of larger cargoes and tested this using transferrin-coated beads of similar size as flaviviruses. LY6E was indeed required for the internalization of transferrin-coated beads, suggesting that LY6E is selectively required for large cargo. Cell biological studies found that LY6E forms tubules upon viral infection and bead internalization, and we found that tubule formation was dependent on RNASEK, which is also required for flavivirus internalization, but not transferrin uptake. Indeed, we found that RNASEK is also required for the internalization of transferrin-coated beads, suggesting it functions upstream of LY6E. These LY6E tubules resembled microtubules, and we found that microtubule assembly was required for their formation and flavivirus uptake. Since microtubule end-binding proteins link microtubules to downstream activities, we screened the three end-binding proteins and found that EB3 promotes virus uptake and LY6E tubularization. Taken together, these results highlight a specialized pathway required for the uptake of large clathrin-dependent endocytosis cargoes, including flaviviruses.

Overexpression of Lymphocyte Antigen 6 Complex, Locus E in Gastric Cancer Promotes Cancer Cell Growth and Metastasis.

BACKGROUND/AIMS: Lymphocyte antigen 6 complex, locus E (LY6E) is a member of the lymphostromal cell membrane Ly6 superfamily protein. The present study investigated the clinical significance and potential biological function of LY6E in gastric cancer (GC). METHODS: LY6E mRNA and protein expressions in human GC tissues and GC cells were tested. Relationship between LY6E expression and the GC patients' clinicopathologic characteristics was analyzed. LY6E was silenced by siRNA in the cultured GC cells. RESULTS: The RNA expression microarray profiling assay results demonstrated that LY6E mRNA was significantly increased in multiple human GC tumor tissues. Immunohistochemistry (IHC) staining analysis revealed that 59 of 75 (78.7%) GC specimens were LY6E positive. LY6E over-expression in human GC was correlated with the histology grade, AJCC stage, N classification, lymphatic invasion, and tumor location. Notably, functional LY6E expression was also detected in AGS and other established GC cell lines. LY6E knockdown by targeted-siRNA inhibited AGS cell survival and proliferation. Meanwhile, the LY6E siRNA induced G1-S cell cycle arrest and apoptosis in AGC cells. Additionally, AGC cell migration was also inhibited by LY6E knockdown. Expressions of tumor-suppressing proteins, including PTEN (phosphatase and tensin homolog) and E-Cadherin, were increased in LY6E-silenced AGS cells. CONCLUSION: LY6E over-expression in GC is potentially required for cancer cell survival, proliferation and migration.

Interferon-inducible LY6E Protein Promotes HIV-1 Infection.

LY6E is a glycosylphosphatidylinositol-anchored, IFN-inducible protein that regulates T lymphocytes proliferation, differentiation, and development. Single-nucleotide polymorphism rs2572886 in the LY6 family protein locus has been shown to associate with accelerated progression to AIDS. In this study, we show that LY6E promotes HIV, type 1 (HIV-1) infection by enhancing viral entry and gene expression. Knockdown of LY6E in human peripheral blood mononuclear, SupT1, and THP-1 cells diminishes HIV-1 replication. Virion-cell and cell-cell fusion experiments revealed that LY6E promotes membrane fusion of the viral entry step. Interestingly, we find that LTR-driven HIV-1 gene expression is also enhanced by LY6E, suggesting additional roles of LY6E in HIV-1 replication. HIV-1 infection induces LY6E expression in human peripheral blood mononuclear cells, concomitant with increased production of type I IFN and some classical IFN-stimulated genes. Altogether, our results demonstrate that IFN-inducible LY6E promotes HIV-1 entry and replication and highlight a positive regulatory role of IFN-induced proteins in HIV-1 infection. Our work emphasizes the complexity of IFN-mediated signaling in HIV-host interaction and AIDS pathogenesis.

LY6E: a conductor of malignant tumor growth through modulation of the PTEN/PI3K/Akt/HIF-1 axis.

Lymphocyte antigen 6 complex, locus E (LY6E) has been implicated in the malignant progression of various types of cancers; however, the underlying mechanism remains unclear. Here, we identified LY6E as an activator of HIF-1 and revealed their mechanistic and functional links in malignant tumor growth. The aberrant overexpression of LY6E increased HIF-1α gene expression principally at the transcription level. This, in turn, led to the expression of the pro-angiogenic factors, VEGFA and PDGFB, through decreases in the expression levels of PTEN mRNA and subsequent activation of the PI3K/Akt pathway. The LY6E-HIF-1 axis functioned to increase tumor blood vessel density and promoted tumor growth in immunodeficient mice. LY6E expression levels were significantly higher in human breast cancers than in normal breast tissues, and were strongly associated with the poor prognoses of various cancer patients. Our results characterized LY6E as a novel conductor of tumor growth through its modulation of the PTEN/PI3K/Akt/HIF-1 axis and demonstrated the validity of targeting this pathway for cancer therapy.

Ly6e expression is restricted to syncytiotrophoblast cells of the mouse placenta.

In the present study, we characterized the expression of lymphocyte antigen 6, locus E (Ly6e) in mouse placental trophoblast. We identified Ly6e mRNA expression in trophoblast stem (TS) cells by a gene expression screen. In vivo, Ly6e was first detectable by mRNA in situ hybridization in the chorion beginning at E8.5 with spatial expression similar to Syncytin a (Syna). At later stages of gestation, Ly6e was restricted to syncytiotrophoblast in the labyrinth. Northern blot confirmed that Ly6e was expressed in both undifferentiated and differentiated TS cell cultures but that its expression increased with differentiation. FACS analysis confirmed these results and allowed us to isolate LY6E⁺ cells, which we found to express Syna at a much higher level than did LY6E⁻ cells. Our findings suggest that LY6E is expressed in differentiated syncytiotrophoblast and may also be useful as an early marker, expressed in progenitors of this cell-type.