Unanticipated Antigens: Translation Initiation at CUG with Leucine

Major histocompatibility class I molecules display tens of thousands of peptides on the cell surface for immune surveillance by T cells. The peptide repertoire represents virtually all cellular translation products, and can thus reveal a foreign presence inside the cell. These peptides are derived from not only conventional but also cryptic translational reading frames, including some without conventional AUG codons. To define the mechanism that generates these cryptic peptides, we used T cells as probes to analyze the peptides generated in transfected cells. We found that when CUG acts as an alternate initiation codon, it can be decoded as leucine rather than the expected methionine residue. The leucine start does not depend on an internal ribosome entry site–like mRNA structure, and its efficiency is enhanced by the Kozak nucleotide context. Furthermore, ribosomes scan 5′ to 3′ specifically for the CUG initiation codon in a eukaryotic translation initiation factor 2–independent manner. Because eukaryotic translation initiation factor 2 is frequently targeted to inhibit protein synthesis, this novel translation mechanism allows stressed cells to display antigenic peptides. This initiation mechanism could also be used at non-AUG initiation codons often found in viral transcripts as well as in a growing list of cellular genes

Genetic influences on schizophrenia and subcortical brain volumes: large-scale proof-of-concept and roadmap for future studies

Schizophrenia is a devastating psychiatric illness with high heritability. Brain structure and function differ, on average, between schizophrenia cases and healthy individuals. As common genetic associations are emerging for both schizophrenia and brain imaging phenotypes, we can now use genome-wide data to investigate genetic overlap. Here we integrated results from common variant studies of schizophrenia (33,636 cases, 43,008 controls) and volumes of several (mainly subcortical) brain structures (11,840 subjects). We did not find evidence of genetic overlap between schizophrenia risk and subcortical volume measures either at the level of common variant genetic architecture or for single genetic markers. The current study provides proof-of-concept (albeit based on a limited set of structural brain measures), and defines a roadmap for future studies investigating the genetic covariance between structural/functional brain phenotypes and risk for psychiatric disorders

Reversible Inactivation and Desiccation Tolerance of Silicified Viruses

Long-distance host-independent virus dispersal is poorly understood, especially for viruses found in isolated ecosystems. Todemonstrate a possible dispersal mechanism, we show that bacteriophage T4, archaeal virus Sulfolobus spindle-shaped virus Kamchatka, and vaccinia virus are reversibly inactivated by mineralization in silica under conditions similar to volcanic hotsprings. In contrast, bacteriophage PRD1 is not silicified. Moreover, silicification provides viruses with remarkable desiccationresistance, which could allow extensive aerial dispersal

Unanticipated antigens: translation initiation at CUG with leucine.

Major histocompatibility class I molecules display tens of thousands of peptides on the cell surface for immune surveillance by T cells. The peptide repertoire represents virtually all cellular translation products, and can thus reveal a foreign presence inside the cell. These peptides are derived from not only conventional but also cryptic translational reading frames, including some without conventional AUG codons. To define the mechanism that generates these cryptic peptides, we used T cells as probes to analyze the peptides generated in transfected cells. We found that when CUG acts as an alternate initiation codon, it can be decoded as leucine rather than the expected methionine residue. The leucine start does not depend on an internal ribosome entry site-like mRNA structure, and its efficiency is enhanced by the Kozak nucleotide context. Furthermore, ribosomes scan 5' to 3' specifically for the CUG initiation codon in a eukaryotic translation initiation factor 2-independent manner. Because eukaryotic translation initiation factor 2 is frequently targeted to inhibit protein synthesis, this novel translation mechanism allows stressed cells to display antigenic peptides. This initiation mechanism could also be used at non-AUG initiation codons often found in viral transcripts as well as in a growing list of cellular genes

A self-help program for memory CD8+ T cells: positive feedback via CD40-CD40L signaling as a critical determinant of secondary expansion.

The ability of memory CD8+ T cells to rapidly proliferate and acquire cytolytic activity is critical for protective immunity against intracellular pathogens. The signals that control this recall response remain unclear. We show that CD40L production by memory CD8+ T cells themselves is an essential catalyst for secondary expansion when systemic inflammation is limited. Secondary immunization accompanied by high levels of systemic inflammation results in CD8+ T cell secondary expansion independent of CD4+ T cells and CD40-CD40L signaling. Conversely, when the inflammatory response is limited, memory CD8+ T cell secondary expansion requires CD40L-producing cells, and memory CD8+ T cells can provide this signal. These results demonstrate that vaccination regimens differ in their dependence on CD40L-expressing CD8+ T cells for secondary expansion, and propose that CD40L-expression by CD8+ T cells is a fail-safe mechanism that can promote memory CD8+ T cell secondary expansion when inflammation is limited

Suppression of cell-mediated immunity following recognition of phagosome-confined bacteria.

Listeria monocytogenes is a facultative intracellular pathogen capable of inducing a robust cell-mediated immune response to sub-lethal infection. The capacity of L. monocytogenes to escape from the phagosome and enter the host cell cytosol is paramount for the induction of long-lived CD8 T cell-mediated protective immunity. Here, we show that the impaired T cell response to L. monocytogenes confined within a phagosome is not merely a consequence of inefficient antigen presentation, but is the result of direct suppression of the adaptive response. This suppression limited not only the adaptive response to vacuole-confined L. monocytogenes, but negated the response to bacteria within the cytosol. Co-infection with phagosome-confined and cytosolic L. monocytogenes prevented the generation of acquired immunity and limited expansion of antigen-specific T cells relative to the cytosolic L. monocytogenes strain alone. Bacteria confined to a phagosome suppressed the production of pro-inflammatory cytokines and led to the rapid MyD88-dependent production of IL-10. Blockade of the IL-10 receptor or the absence of MyD88 during primary infection restored protective immunity. Our studies demonstrate that the presence of microbes within a phagosome can directly impact the innate and adaptive immune response by antagonizing the signaling pathways necessary for inflammation and the generation of protective CD8 T cells

The CUG Start Is Blocked by a Heat-Stable Hairpin in the 5′ UTR

<p>COS-7 cells were transfected with cDNA encoding K^b and the indicated constructs. (A and C) The cells were titrated and peptide expression was tested with BCZ103 T cells. (B and D) GFP expression in the transfected cells was assayed by fluorescence-activated cell sorting. GFP fluorescence (shaded histograms) is not observed in untransfected cells (or in cells transfected with a vector not encoding GFP [unpublished data]).</p

The Optimal Nucleotide Context for the CUG Initiation Codon

<div><p>(A and B) The indicated degenerate oligonucleotides were cloned into the pcDNA1 vector. “<u>N</u>” represents any one of T, A, C, and G nucleotides. The CTG or CCC initiation codons are boxed and the peptide coding sequence is indicated by [SEL8]. 96 randomly picked plasmids for the CTG-initiated peptide and an equal number for the CCC-initiated peptide were purified. The plasmids were transfected into COS-7 cells along with the K^b MHC class I molecule, and the T cell response was measured. Each bar represents the T cell response to cells transfected with an individual plasmid.</p> <p>(C) Three sets of 18 representative plasmids, each yielding high, intermediate, and low responses (as shown) were selected for nucleotide sequencing.</p> <p>(D) Summary of the nucleotide sequences of plasmids yielding high, intermediate, and low responses. The left, middle, and right panels, respectively, correspond to the plasmids shown in (C). Each panel shows the percent of each nucleotide found at the –6, –3, and +4 degenerate positions indicated by the “<u>N</u>” in A. For example, the upper left square shows that, of the high T cell-stimulating plasmids, the –6 position was T for 67%, A for 6.7%, C for 20%, G for 6.7%, a pyrimidine (T or C) for 87%, and a purine (A or G) for 13%.</p></div

The Leucine Start Is Enhanced in the Presence of Phosphorylated eIF2α

<div><p>HeLa cells transfected with cDNA encoding K^b together with cDNA encoding either the ATG- or CTG- initiated peptides were treated for 4 h with 50 μM NaAs, with brefeldin A (BfA), or left untreated (UT).</p> <p>(A) Transfected cells treated with NaAs or without (UT) were lysed and tested for phosphorylation of eIF2α by Western blot and for tubulin as a loading control.</p> <p>(B) The transfected cells were titrated and tested for their ability to stimulate BCZ103 T cells.</p></div