Amy W. Hudson

A Human Herpesvirus 7 Glycoprotein, U21, Diverts Major Histocompatibility Complex Class I Molecules to Lysosomes

ABSTRACT All members of the herpesvirus family persist in their host throughout life. In doing so, herpesviruses exploit a surprising number of different strategies to evade the immune system. Human herpesvirus 7 (HHV-7) is a relatively recently discovered member of the herpesvirus family, and little is known about how it escapes immune detection. Here we show that HHV-7 infection results in premature degradation of major histocompatibility complex class I molecules. We identify and characterize a protein from HHV-7, U21, that binds to and diverts properly folded class I molecules to a lysosomal compartment. Thus, U21 is likely to function in the normal course of HHV-7 infection to downregulate surface class I molecules and prevent recognition of infected cells by cytotoxic T lymphocytes.

The Human Herpesvirus-7 (HHV-7) U21 Immunoevasin Subverts NK-Mediated Cytoxicity through Modulation of MICA and MICB

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.

The ER-lumenal domain of the HHV-7 immunoevasin U21 directs class I MHC molecules to lysosomes.

Like all members of the herpesvirus family, human herpesvirus‐7 has evolved mechanisms to evade immune detection. The human herpesvirus‐7 gene product U21 encodes an immunoevasin that binds to class I major histocompatibility complex molecules and diverts them to a lysosomal compartment. Here we show that the cytoplasmic tail of U21, although sufficient to sequester a heterologous membrane protein (CD4 chimera), has no effect on U21s ability to redirect class I major histocompatibility complex molecules to lysosomes. Instead, the ER‐lumenal domain of U21 is sufficient to redirect class I major histocompatibility complex molecules to the lysosomal compartment. These observations demonstrate a novel viral immunoevasive mechanism for U21, and implicate the ER‐lumenal domain of a type I transmembrane protein in lysosomal sorting.

Human Herpesvirus 7 U21 Downregulates Classical and Nonclassical Class I Major Histocompatibility Complex Molecules from the Cell Surface

ABSTRACT Herpesviruses have evolved numerous strategies to evade detection by the immune system. Notably, most of the herpesviruses interfere with viral antigen presentation to cytotoxic T lymphocytes (CTLs) by removing class I major histocompatibility complex (MHC) molecules from the infected cell surface. Clearly, since the herpesviruses have evolved an extensive array of mechanisms to remove class I MHC molecules from the cell surface, this strategy serves them well. However, class I MHC molecules often serve as inhibitory ligands for NK cells, so viral downregulation of all class I MHC molecules should leave the infected cell open to NK cell attack. Some viruses solve this problem by selectively downregulating certain class I MHC products, leaving other class I products at the cell surface to serve as inhibitory NK cell ligands. Here, we show that human herpesvirus 7 (HHV-7) U21 binds to and downregulates all of the human class I MHC gene products, as well as the murine class I molecule H-2Kb. HHV-7-infected cells must therefore possess other means of escaping NK cell detection.

Insight into the Mechanism of Human Herpesvirus 7 U21-mediated Diversion of Class I MHC Molecules to Lysosomes

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.

Adoptively transferred Vγ9Vδ2 T cells show potent antitumor effects in a preclinical B cell lymphomagenesis model

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.

Natural Killer Cell Evasion Is Essential for Infection by Rhesus Cytomegalovirus.

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.

Human Herpesvirus 7 U21 Tetramerizes To Associate with Class I Major Histocompatibility Complex Molecules

ABSTRACT The U21 gene product from human herpesvirus 7 binds to and redirects class I major histocompatibility complex (MHC) molecules to a lysosomal compartment. The molecular mechanism by which U21 reroutes class I MHC molecules to lysosomes is not known. Here, we have reconstituted the interaction between purified soluble U21 and class I MHC molecules, suggesting that U21 does not require additional cellular proteins to interact with class I MHC molecules. Our results demonstrate that U21, itself predicted to contain an MHC class I-like protein fold, interacts tightly with class I MHC molecules as a tetramer, in a 4:2 stoichiometry. These observations have helped to elucidate a refined model describing the mechanism by which U21 escorts class I MHC molecules to the lysosomal compartment. IMPORTANCE In this report, we show that the human herpesvirus 7 (HHV-7) immunoevasin U21, itself a class I MHC-like protein, binds with high affinity to class I MHC molecules as a tetramer and escorts them to lysosomes, where they are degraded. While many class I MHC-like molecules have been described in detail, this unusual viral class I-like protein functions as a tetramer, associating with class I MHC molecules in a 4:2 ratio, illuminating a functional significance of homooligomerization of a class I MHC-like protein.

Adaptor Protein Complexes AP-1 and AP-3 Are Required by the HHV-7 Immunoevasin U21 for Rerouting of Class I MHC Molecules to the Lysosomal Compartment

The human herpesvirus-7 (HHV-7) U21 gene product binds to class I major histocompatibility complex (MHC) molecules and reroutes them to a lysosomal compartment. Trafficking of integral membrane proteins to lysosomes is mediated through cytoplasmic sorting signals that recruit heterotetrameric clathrin adaptor protein (AP) complexes, which in turn mediate protein sorting in post-Golgi vesicular transport. Since U21 can mediate rerouting of class I molecules to lysosomes even when lacking its cytoplasmic tail, we hypothesize the existence of a cellular protein that contains the lysosomal sorting information required to escort class I molecules to the lysosomal compartment. If such a protein exists, we expect that it might recruit clathrin adaptor protein complexes as a means of lysosomal sorting. Here we describe experiments demonstrating that the μ adaptins from AP-1 and AP-3 are involved in U21-mediated trafficking of class I molecules to lysosomes. These experiments support the idea that a cellular protein(s) is necessary for U21-mediated lysosomal sorting of class I molecules. We also examine the impact of transient versus chronic knockdown of these adaptor protein complexes, and show that the few remaining μ subunits in the cells are eventually able to reroute class I molecules to lysosomes.