Immunomodulators of pathogens frequently affect multiple cellular targets, thus preventing recognition by different immune cells. For instance, the K5 modulator of immune recognition (MIR2) from ...Kaposi sarcoma-associated herpesvirus prevents activation of cytotoxic T cells, natural killer cells, and natural killer T cells by downregulating major histocompatibility complex (MHC) class I molecules, the MHC-like molecule CD1, the cell adhesion molecules ICAM-1 and PECAM, and the co-stimulatory molecule B7.2. K5 belongs to a family of viral- and cellular-membrane-spanning RING ubiquitin ligases. While a limited number of transmembrane proteins have been shown to be targeted for degradation by this family, it is unknown whether additional targets exist. We now describe a quantitative proteomics approach to identify novel targets of this protein family. Using stable isotope labeling by amino acids, we compared the proteome of plasma, Golgi, and endoplasmic reticulum membranes in the presence and absence of K5. Mass spectrometric protein identification revealed four proteins that were consistently underrepresented in the plasma membrane of K5 expression cells: MHC I (as expected), bone marrow stromal antigen 2 (BST-2, CD316), activated leukocyte cell adhesion molecule (ALCAM, CD166) and Syntaxin-4. Downregulation of each of these proteins was independently confirmed by immunoblotting with specific antibodies. We further demonstrate that ALCAM is a bona fide target of both K5 and the myxomavirus homolog M153R. Upon exiting the endoplasmic reticulum, ALCAM is ubiquitinated in the presence of wild-type, but not RING-deficient or acidic motif-deficient, K5, and is targeted for lysosomal degradation via the multivesicular body pathway. Since ALCAM is the ligand for CD6, a member of the immunological synapse of T cells, its removal by viral immune modulators implies a role for CD6 in the recognition of pathogens by T cells. The unbiased global proteome analysis therefore revealed novel immunomodulatory functions of pathogen proteins.
Although the transcription factors IRF-3 and IRF-7 are considered master regulators of type I interferon (IFN) induction and IFN stimulated gene (ISG) expression, Irf3(-/-)×Irf7(-/-) double knockout ...(DKO) myeloid dendritic cells (mDC) produce relatively normal levels of IFN-β after viral infection. We generated Irf3(-/-)×Irf5(-/-)×Irf7(-/-) triple knockout (TKO) mice to test whether IRF-5 was the source of the residual induction of IFN-β and ISGs in mDCs. In pathogenesis studies with two unrelated positive-sense RNA viruses (West Nile virus (WNV) and murine norovirus), TKO mice succumbed at rates greater than DKO mice and equal to or approaching those of mice lacking the type I IFN receptor (Ifnar(-/-)). In ex vivo studies, after WNV infection or exposure to Toll-like receptor agonists, TKO mDCs failed to produce IFN-β or express ISGs. In contrast, this response was sustained in TKO macrophages following WNV infection. To define IRF-regulated gene signatures, we performed microarray analysis on WNV-infected mDC from wild type (WT), DKO, TKO, or Ifnar(-/-) mice, as well as from mice lacking the RIG-I like receptor adaptor protein MAVS. Whereas the gene induction pattern in DKO mDC was similar to WT cells, remarkably, almost no ISG induction was detected in TKO or Mavs(-/-) mDC. The relative equivalence of TKO and Mavs(-/-) responses suggested that MAVS dominantly regulates ISG induction in mDC. Moreover, we showed that MAVS-dependent induction of ISGs can occur through an IRF-5-dependent yet IRF-3 and IRF-7-independent pathway. Our results establish IRF-3, -5, and -7 as the key transcription factors responsible for mediating the type I IFN and ISG response in mDC during WNV infection and suggest a novel signaling link between MAVS and IRF-5.
The genome of cowpoxvirus (CPXV) could be considered prototypical for orthopoxviridae (OXPV) since it contains many open reading frames (ORFs) absent or lost in other OPXV, including vaccinia virus ...(VACV). These additional ORFs are non-essential for growth in vitro but are expected to contribute to the broad host range, virulence and immune evasion characteristics of CPXV. For instance, unlike VACV, CPXV encodes proteins that interfere with T cell stimulation, either directly or by preventing antigen presentation or co-stimulation. When studying the priming of naïve T cells, we discovered that CPXV, but not VACV, encodes a secreted factor that interferes with activation and proliferation of naïve CD8+ and CD4+ T cells, respectively, in response to anti-CD3 antibodies, but not to other stimuli. Deletion mapping revealed that the inhibitory protein is encoded by CPXV14, a small secreted glycoprotein belonging to the poxvirus immune evasion (PIE) family and containing a smallpoxvirus encoded chemokine receptor (SECRET) domain that mediates binding to chemokines. We demonstrate that CPXV14 inhibition of antibody-mediated T cell activation depends on the presence of Fc-gamma receptors (FcγRs) on bystander cells. In vitro, CPXV14 inhibits FcγR-activation by antigen/antibody complexes by binding to FcγRs with high affinity and immobilized CPXV14 can trigger signaling through FcγRs, particularly the inhibitory FcγRIIB. In vivo, CPXV14-deleted virus showed reduced viremia and virulence resulting in reduced weight loss and death compared to wildtype virus whereas both antibody and CD8+ T cell responses were increased in the absence of CPXV14. Furthermore, no impact of CPXV14-deletion on virulence was observed in mice lacking the inhibitory FcγRIIB. Taken together our results suggest that CPXV14 contributes to virulence and immune evasion by binding to host FcγRs.
Most studies of T lymphocytes focus on recognition of classical major histocompatibility complex (MHC) class I or II molecules presenting oligopeptides, yet there are numerous variations and ...exceptions of biological significance based on recognition of a wide variety of nonclassical MHC molecules. These include αβ and γδ T cells that recognize different class Ib molecules (CD1, MR-1, HLA-E, G, F, et al.) that are nearly monomorphic within a given species. Collectively, these T cells can be considered "unconventional," in part because they recognize lipids, metabolites, and modified peptides. Unlike classical MHC-specific cells, unconventional T cells generally exhibit limited T-cell antigen receptor (TCR) repertoires and often produce innate immune cell-like rapid effector responses. Exploiting this system in new generation vaccines for human immunodeficiency virus (HIV), tuberculosis (TB), other infectious agents, and cancer was the focus of a recent workshop, "Immune Surveillance by Non-classical MHC Molecules: Improving Diversity for Antigens," sponsored by the National Institute of Allergy and Infectious Diseases. Here, we summarize salient points presented regarding the basic immunobiology of unconventional T cells, recent advances in methodologies to measure unconventional T-cell activity in diseases, and approaches to harness their considerable clinical potential.
Through major histocompatibility complex class Ia leader sequence-derived (VL9) peptide binding and CD94/NKG2 receptor engagement, human leucocyte antigen E (HLA-E) reports cellular health to NK ...cells. Previous studies demonstrated a strong bias for VL9 binding by HLA-E, a preference subsequently supported by structural analyses. However, Mycobacteria tuberculosis (Mtb) infection and Rhesus cytomegalovirus-vectored SIV vaccinations revealed contexts where HLA-E and the rhesus homologue, Mamu-E, presented diverse pathogen-derived peptides to CD8
T cells, respectively. Here we present crystal structures of HLA-E in complex with HIV and Mtb-derived peptides. We show that despite the presence of preferred primary anchor residues, HLA-E-bound peptides can adopt alternative conformations within the peptide binding groove. Furthermore, combined structural and mutagenesis analyses illustrate a greater tolerance for hydrophobic and polar residues in the primary pockets than previously appreciated. Finally, biochemical studies reveal HLA-E peptide binding and exchange characteristics with potential relevance to its alternative antigen presenting function in vivo.
PDF
