Negative Self-Regulation of TLR9 Signaling by Its N-Terminal Proteolytic Cleavage Product

TLR signaling is essential to innate immunity against microbial invaders and must be tightly controlled. We have previously shown that TLR9 undergoes proteolytic cleavage processing by lysosomal proteases to generate two distinct fragments. The C-terminal cleavage product plays a critical role in activating TLR9 signaling; however, the precise role of the N-terminal fragment, which remains in lysosomes, in the TLR9 response is still unclear. In this article, we report that the N-terminal cleavage product negatively regulates TLR9 signaling. Notably, the N-terminal fragment promotes the aspartic protease-mediated degradation of the C-terminal fragment in endolysosomes. Furthermore, the N-terminal TLR9 fragment physically interacts with the C-terminal product, thereby inhibiting the formation of homodimers of the C-terminal fragment; this suggests that the monomeric C-terminal product is more susceptible to attack by aspartic proteases. Together, these results suggest that the N-terminal TLR9 proteolytic cleavage product is a negative self-regulator that prevents excessive TLR9 signaling activity.Korea (South). Ministry of Education, Science and Technology (MEST) (National Research Foundation of Korea. Grant 2011-0015372)Korea (South). Ministry of Education, Science and Technology (MEST) (National Research Foundation of Korea. Grant 2010-0009203)Korea. Ministry of Health and Welfare. National Research and Development Program for Cancer Contro

The HCMV membrane glycoprotein US10 selectively targets HLA-G for degradation

Human cytomegalovirus (HCMV) encodes an endoplasmic reticulum (ER)-resident transmembrane glycoprotein, US10, expressed early in the replicative cycle of HCMV as part of the same cluster that encodes the known immunoevasins US2, US3, US6, and US11. We show that US10 down-regulates cell surface expression of HLA-G, but not that of classical class I MHC molecules. The unique and short cytoplasmic tail of HLA-G (RKKSSD) is essential in its role as a US10 substrate, and a tri-leucine motif in the cytoplasmic tail of US10 is responsible for down-regulation of HLA-G. Both the kinetics of HLA-G degradation and the mechanisms responsible appear to be distinct from those used by the US2 and US11 pathways, suggesting the existence of a third route of protein dislocation from the ER. We show that US10-mediated degradation of HLA-G interferes with HLA-G–mediated NK cell inhibition. Given the role of HLA-G in protecting the fetus from attack by the maternal immune system and in directing the differentiation of human dendritic cells to promote the evolution of regulatory T cells, HCMV likely targets the HLA-G–dependent axis of immune recognition no less efficiently than it interferes with classical class I MHC–restricted antigen presentation

Human Cytomegalovirus UL18 Utilizes US6 for Evading the NK and T-Cell Responses

Human cytomegalovirus (HCMV) US6 glycoprotein inhibits TAP function, resulting in down-regulation of MHC class I molecules at the cell surface. Cells lacking MHC class I molecules are susceptible to NK cell lysis. HCMV expresses UL18, a MHC class I homolog that functions as a surrogate to prevent host cell lysis. Despite a high level of sequence and structural homology between UL18 and MHC class I molecules, surface expression of MHC class I, but not UL18, is down regulated by US6. Here, we describe a mechanism of action by which HCMV UL18 avoids attack by the self-derived TAP inhibitor US6. UL18 abrogates US6 inhibition of ATP binding by TAP and, thereby, restores TAP-mediated peptide translocation. In addition, UL18 together with US6 interferes with the physical association between MHC class I molecules and TAP that is required for optimal peptide loading. Thus, regardless of the recovery of TAP function, surface expression of MHC class I molecules remains decreased. UL18 represents a unique immune evasion protein that has evolved to evade both the NK and the T cell immune responses

Determinant for Endoplasmic Reticulum Retention in the Luminal Domain of the Human Cytomegalovirus US3 Glycoprotein

US3 of human cytomegalovirus is an endoplasmic reticulum resident transmembrane glycoprotein that binds to major histocompatibility complex class I molecules and prevents their departure. The endoplasmic reticulum retention signal of the US3 protein is contained in the luminal domain of the protein. To define the endoplasmic reticulum retention sequence in more detail, we have generated a series of deletion and point mutants of the US3 protein. By analyzing the rate of intracellular transport and immunolocalization of the mutants, we have identified Ser(58), Glu(63), and Lys(64) as crucial for retention, suggesting that the retention signal of the US3 protein has a complex spatial arrangement and does not comprise a contiguous sequence of amino acids. We also show that a modified US3 protein with a mutation in any of these amino acids maintains its ability to bind class I molecules; however, such mutated proteins are no longer retained in the endoplasmic reticulum and are not able to block the cell surface expression of class I molecules. These findings indicate that the properties that allow the US3 glycoprotein to be localized in the endoplasmic reticulum and bind major histocompatibility complex class I molecules are located in different parts of the molecule and that the ability of US3 to block antigen presentation is due solely to its ability to retain class I molecules in the endoplasmic reticulum

A Short Isoform of Human Cytomegalovirus US3 Functions as a Dominant Negative Inhibitor of the Full-Length Form

Human cytomegalovirus encodes four unique short (US) region proteins, each of which is independently sufficient for causing the down-regulation of major histocompatibility complex (MHC) class I molecules on the cell surface. This down-regulation enables infected cells to evade recognition by cytotoxic T lymphocytes (CTLs) but makes them vulnerable to lysis by natural killer (NK) cells, which lyse those cells that lack MHC class I molecules. The 22-kDa US3 glycoprotein is able to down-regulate the surface expression of MHC class I molecules by dual mechanisms: direct endoplasmic reticulum retention by physical association and/or tapasin inhibition. The alternative splicing of the US3 gene generates two additional products, including 17-kDa and 3.5-kDa truncated isoforms; however, the functional significance of these isoforms during viral infection is unknown. Here, we describe a novel mode of self-regulation of US3 function that uses the endogenously produced truncated isoform. The truncated isoform itself neither binds to MHC class I molecules nor prevents the full-length US3 from interacting with MHC class I molecules. Instead, the truncated isoform associates with tapasin and competes with full-length US3 for binding to tapasin; thus, it suppresses the action of US3 that causes the disruption of the function of tapasin. Our results indicate that the truncated isoform of the US3 locus acts as a dominant negative regulator of full-length US3 activity. These data reflect the manner in which the virus has developed temporal survival strategies during viral infection against immune surveillance involving both CTLs and NK cells

Granulin Is a Soluble Cofactor for Toll-like Receptor 9 Signaling

Toll-like receptor (TLR) signaling plays a critical role in innate and adaptive immune responses and must be tightly controlled. TLR4 uses LPS binding protein, MD-2, and CD14 as accessories to respond to LPS. We therefore investigated the presence of an analagous soluble cofactor that might assist in the recruitment of CpG oligonucleotides (CpG-ODNs) to TLR9. We report the identification of granulin as an essential secreted cofactor that potentiates TLR9-driven responses to CpG-ODNs. Granulin, an unusual cysteine-rich protein, bound to CpG-ODNs and interacted with TLR9. Macrophages from granulin-deficient mice showed not only impaired delivery of CpG-ODNs to endolysosomal compartments, but also decreased interaction of TLR9 with CpG-ODNs. As a consequence, granulin-deficient macrophages showed reduced responses to stimulation with CpG-ODNs, a trait corrected by provision of exogenous granulin. Thus, we propose that granulin contributes to innate immunity as a critical soluble cofactor for TLR9 signaling.National Institutes of Health (U.S.)Novartis (Firm