RESUMO
Roseoloviruses (human herpesvirus 6A [HHV-6A], -6B, and -7) infect >90% of the human population during early childhood and are thought to remain latent or persistent throughout the life of the host. As such, these viruses are among the most pervasive and stealthy of all viruses; they must necessarily excel at escaping immune detection throughout the life of the host, and yet, very little is known about how these viruses so successfully escape host defenses. Here, we characterize the expression, trafficking, and posttranslational modifications of the HHV6B U20 gene product, which is encoded within a block of genes unique to the roseoloviruses. HHV-6B U20 trafficked slowly through the secretory system, receiving several posttranslational modifications to its N-linked glycans, indicative of surface-expressed glycoproteins, and eventually reaching the cell surface before being internalized. Interestingly, U20 is also phosphorylated on at least one Ser, Thr, or Tyr residue. These results provide a framework to understand the role(s) of U20 in evading host defenses. IMPORTANCE The roseolovirus U20 proteins are virus-encoded integral membrane glycoproteins possessing class I major histocompatibility complex (MHC)-like folds. Surprisingly, although U20 proteins from HHV-6A and -6B share 92% identity, recent studies ascribe different functions to HHV6A U20 and HHV6B U20. HHV6A U20 was shown to downregulate NKG2D ligands, while HHV6B U20 was shown to inhibit tumor necrosis factor alpha (TNF-α)-induced apoptosis during nonproductive infection with HHV6B (E. Kofod-Olsen, K. Ross-Hansen, M. H. Schleimann, D. K. Jensen, et al., J Virol 86:11483-11492, 2012, https://doi.org/10.1128/jvi.00847-12; A. E. Chaouat, B. Seliger, O. Mandelboim, D. Schmiedel, Front Immunol 12:714799, 2021, https://doi.org/10.3389/fimmu.2021.714799). Here, we have performed cell biological and biochemical characterization of the trafficking, glycosylation, and posttranslational modifications occurring on HHV6B U20.
Assuntos
Glicoproteínas de Membrana , Infecções por Roseolovirus , Proteínas Virais , Humanos , Herpesvirus Humano 6/genética , Herpesvirus Humano 6/imunologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Infecções por Roseolovirus/imunologia , Infecções por Roseolovirus/virologia , Proteínas Virais/genética , Proteínas Virais/imunologia , Evasão da Resposta ImuneRESUMO
The natural killer cell receptor NKG2D activates NK cells by engaging one of several ligands (NKG2DLs) belonging to either the MIC or ULBP families. Human cytomegalovirus (HCMV) UL16 and UL142 counteract this activation by retaining NKG2DLs and US18 and US20 act via lysomal degradation but the importance of NK cell evasion for infection is unknown. Since NKG2DLs are highly conserved in rhesus macaques, we characterized how NKG2DL interception by rhesus cytomegalovirus (RhCMV) impacts infection in vivo. Interestingly, RhCMV lacks homologs of UL16 and UL142 but instead employs Rh159, the homolog of UL148, to prevent NKG2DL surface expression. Rh159 resides in the endoplasmic reticulum and retains several NKG2DLs whereas UL148 does not interfere with NKG2DL expression. Deletion of Rh159 releases human and rhesus MIC proteins, but not ULBPs, from retention while increasing NK cell stimulation by infected cells. Importantly, RhCMV lacking Rh159 cannot infect CMV-naïve animals unless CD8+ cells, including NK cells, are depleted. However, infection can be rescued by replacing Rh159 with HCMV UL16 suggesting that Rh159 and UL16 perform similar functions in vivo. We therefore conclude that cytomegaloviral interference with NK cell activation is essential to establish but not to maintain chronic infection.
Assuntos
Infecções por Citomegalovirus/imunologia , Citomegalovirus/imunologia , Evasão da Resposta Imune , Células Matadoras Naturais/imunologia , Ativação Linfocitária , Animais , Humanos , Células K562 , Macaca fascicularis , Glicoproteínas de Membrana/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Proteínas Virais/imunologiaRESUMO
NKG2D is an activating receptor expressed on several types of human lymphocytes. NKG2D ligands can be induced upon cell stress and are frequently targeted post-translationally in infected or transformed cells to avoid immune recognition. Virus infection and inflammation alter protein N-glycosylation, and we have previously shown that changes in cellular N-glycosylation are involved in regulation of NKG2D ligand surface expression. The specific mode of regulation through N-glycosylation is, however, unknown. Here we investigated whether direct N-glycosylation of the NKG2D ligand MICA itself is critical for cell surface expression and sought to identify the essential residues. We found that a single N-glycosylation site (Asn(8)) was important for MICA018 surface expression. The frequently expressed MICA allele 008, with an altered transmembrane and intracellular domain, was not affected by mutation of this N-glycosylation site. Mutational analysis revealed that a single amino acid (Thr(24)) in the extracellular domain of MICA018 was essential for the N-glycosylation dependence, whereas the intracellular domain was not involved. The HHV7 immunoevasin, U21, was found to inhibit MICA018 surface expression by affecting N-glycosylation, and the retention was rescued by T24A substitution. Our study reveals N-glycosylation as an allele-specific regulatory mechanism important for regulation of surface expression of MICA018, and we pinpoint the residues essential for this N-glycosylation dependence. In addition, we show that this regulatory mechanism of MICA surface expression is likely targeted during different pathological conditions.
Assuntos
Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/genética , Alelos , Sequência de Aminoácidos , Substituição de Aminoácidos , Asparagina/química , Sítios de Ligação/genética , Proteínas de Transporte/imunologia , Proteínas de Transporte/metabolismo , Linhagem Celular , Membrana Celular/imunologia , Membrana Celular/metabolismo , Glicosilação , Herpesvirus Humano 7/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Treonina/química , Proteínas Virais/imunologia , Proteínas Virais/metabolismoRESUMO
Introduction: Human Herpesvirus 6B (HHV-6B) impedes host immune responses by downregulating class I MHC molecules (MHC-I), hindering antigen presentation to CD8+ T cells. Downregulation of MHC-I disengages inhibitory receptors on natural killer (NK) cells, resulting in activation and killing of the target cell if NK cell activating receptors such as NKG2D have engaged stress ligands upregulated on the target cells. Previous work has shown that HHV-6B downregulates three MHC-like stress ligands MICB, ULBP1, and ULBP3, which are recognized by NKG2D. The U20 glycoprotein of the related virus HHV-6A has been implicated in the downregulation of ULBP1, but the precise mechanism remains undetermined. Methods: We set out to investigate the role of HHV-6B U20 in modulating NK cell activity. We used HHV-6B U20 expressed as a recombinant protein or transduced into target cells, as well as HHV-6B infection, to investigate binding interactions with NK cell ligands and receptors and to assess effects on NK cell activation. Small-angle X-ray scattering was used to align molecular models derived from machine-learning approaches. Results: We demonstrate that U20 binds directly to ULBP1 with sub-micromolar affinity. Transduction of U20 decreases NKG2D binding to ULBP1 at the cell surface but does not decrease ULBP1 protein levels, either at the cell surface or in toto. HHV-6B infection and soluble U20 have the same effect. Transduction of U20 blocks NK cell activation in response to cell-surface ULBP1. Structural modeling of the U20 - ULBP1 complex indicates some similarities to the m152-RAE1γ complex.
Assuntos
Proteínas Ligadas por GPI , Herpesvirus Humano 6 , Células Matadoras Naturais , Ativação Linfocitária , Subfamília K de Receptores Semelhantes a Lectina de Células NK , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Herpesvirus Humano 6/imunologia , Proteínas Ligadas por GPI/metabolismo , Proteínas Ligadas por GPI/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Ativação Linfocitária/imunologia , Ligação Proteica , Proteínas Virais/imunologia , Proteínas Virais/metabolismo , Glicoproteínas/imunologia , Glicoproteínas/metabolismo , Peptídeos e Proteínas de Sinalização IntracelularRESUMO
Herpesviruses have evolved numerous immune evasion strategies to facilitate establishment of lifelong persistent infections. Many herpesviruses encode gene products devoted to preventing viral antigen presentation as a means of escaping detection by cytotoxic T lymphocytes. The human herpesvirus-7 (HHV-7) U21 gene product, for example, is an immunoevasin that binds to class I major histocompatibility complex molecules and redirects them to the lysosomal compartment. Virus infection can also induce the upregulation of surface ligands that activate NK cells. Accordingly, the herpesviruses have evolved a diverse array of mechanisms to prevent NK cell engagement of NK-activating ligands on virus-infected cells. Here we demonstrate that the HHV-7 U21 gene product interferes with NK recognition. U21 can bind to the NK activating ligand ULBP1 and reroute it to the lysosomal compartment. In addition, U21 downregulates the surface expression of the NK activating ligands MICA and MICB, resulting in a reduction in NK-mediated cytotoxicity. These results suggest that this single viral protein may interfere both with CTL-mediated recognition through the downregulation of class I MHC molecules as well as NK-mediated recognition through downregulation of NK activating ligands.
Assuntos
Proteínas de Transporte/metabolismo , Citotoxicidade Imunológica , Herpesvirus Humano 7/patogenicidade , Antígenos de Histocompatibilidade Classe I/metabolismo , Células Matadoras Naturais/imunologia , Proteínas Virais/metabolismo , Apresentação de Antígeno , Linhagem Celular , Proteínas Ligadas por GPI/metabolismo , Células HEK293 , Herpesvirus Humano 7/imunologia , Herpesvirus Humano 7/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/virologia , Lisossomos , Infecções por Roseolovirus/imunologia , Proteínas Virais/imunologiaRESUMO
Human roseolovirus U20 and U21 are type I membrane glycoproteins that have been implicated in immune evasion by interfering with recognition of classical and non-classical MHC proteins. U20 and U21 are predicted to be type I glycoproteins with extracytosolic immunoglobulin-like domains, but detailed structural information is lacking. AlphaFold and RoseTTAfold are next generation machine-learning-based prediction engines that recently have revolutionized the field of computational three-dimensional protein structure prediction. Here, we review the structural biology of viral immunoevasins and the current status of computational structure prediction algorithms. We use these computational tools to generate structural models for U20 and U21 proteins, which are predicted to adopt MHC-Ia-like folds with closed MHC platforms and immunoglobulin-like domains. We evaluate these structural models and place them within current understanding of the structural basis for viral immune evasion of T cell and natural killer cell recognition.
Assuntos
Herpesvirus Humano 6 , Herpesvirus Humano 7 , Infecções por Roseolovirus , Herpesvirus Humano 6/metabolismo , Herpesvirus Humano 7/metabolismo , Humanos , Modelos Estruturais , Proteínas Virais/metabolismoRESUMO
[This corrects the article DOI: 10.3389/fimmu.2022.864898.].
RESUMO
The U21 open reading frame from human herpesvirus-7 encodes a membrane protein that associates with and redirects class I MHC molecules to the lysosomal compartment. The mechanism by which U21 accomplishes this trafficking excursion is unknown. Here we have examined the contribution of localization, glycosylation, domain structure, and the absence of substrate class I MHC molecules on the ability of U21 to traffic to lysosomes. Our results suggest the existence of a cellular protein necessary for U21-mediated rerouting of class I MHC molecules.
Assuntos
Proteínas de Transporte/metabolismo , Glioblastoma/metabolismo , Antígeno HLA-A2/metabolismo , Herpesvirus Humano 7/metabolismo , Lisossomos/metabolismo , Proteínas Virais/metabolismo , Western Blotting , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Diferenciação Celular , Citoplasma/metabolismo , Retículo Endoplasmático/metabolismo , Citometria de Fluxo , Imunofluorescência , Glicosilação , Antígeno HLA-A2/genética , Humanos , Imunoprecipitação , Fragmentos de Peptídeos/metabolismo , Transporte Proteico , Interferência de RNA , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/genética , Microglobulina beta-2/antagonistas & inibidores , Microglobulina beta-2/genética , Microglobulina beta-2/metabolismoRESUMO
Since their independent discovery by Frederick Twort in 1915 and Felix d'Herelle in 1917, bacteriophages have captured the attention of scientists for more than a century. They are the most abundant organisms on the planet, often outnumbering their bacterial hosts by tenfold in a given environment, and they constitute a vast reservoir of unexplored genetic information. The increased prevalence of antibiotic resistant pathogens has renewed interest in the use of naturally obtained phages to combat bacterial infections, aka phage therapy. The development of tools to modify phages, genetically or chemically, combined with their structural flexibility, cargo capacity, ease of propagation, and overall safety in humans has opened the door to a myriad of applications. This review article will introduce readers to many of the varied and ingenious ways in which researchers are modifying phages to move them well beyond their innate ability to target and kill bacteria.
RESUMO
A central issue for adoptive cellular immunotherapy is overcoming immunosuppressive signals to achieve tumor clearance. While γδ T cells are known to be potent cytolytic effectors that can kill a variety of cancers, it is not clear whether they are inhibited by suppressive ligands expressed in tumor microenvironments. Here, we have used a powerful preclinical model where EBV infection drives the de novo generation of human B cell lymphomas in vivo, and autologous T lymphocytes are held in check by PD-1/CTLA-4-mediated inhibition. We show that a single dose of adoptively transferred Vδ2+ T cells has potent antitumor effects, even in the absence of checkpoint blockade or activating compounds. Vδ2+ T cell immunotherapy given within the first 5 days of EBV infection almost completely prevented the outgrowth of tumors. Vδ2+ T cell immunotherapy given more than 3 weeks after infection (after neoplastic transformation is evident) resulted in a dramatic reduction in tumor burden. The immunotherapeutic Vδ2+ T cells maintained low cell surface expression of PD-1 in vivo, and their recruitment to tumors was followed by a decrease in B cells expressing PD-L1 and PD-L2 inhibitory ligands. These results suggest that adoptively transferred PD-1lo Vδ2+ T cells circumvent the tumor checkpoint environment in vivo.