The Synthesis of Truncated Polypeptides for Immune Surveillance and Viral Evasion

Background: Cytotoxic T cells detect intracellular pathogens by surveying peptide loaded MHC class I molecules (pMHC I) on the cell surface. Effective immune surveillance also requires infected cells to present pMHC I promptly before viral progeny can escape. Rapid pMHC I presentation apparently occurs because infected cells can synthesize and present peptides from antigenic precursors called defective ribosomal products (DRiPs). The molecular characteristics of DRiPs are not known. Methodology/Principal Findings: Here, using a novel method for detecting antigenic precursors and proteolytic intermediates, we tracked the synthesis and processing of Epstein-Barr Virus encoded nuclear antigen 1 (EBNA1). We find that ribosomes initiated translation appropriately, but rapidly produced DRiPs representing,120 amino acid truncated EBNA1 polypeptides by premature termination. Moreover, specific sequences in EBNA1 mRNA strongly inhibited the generation of truncated DRiPs and pMHC I presentation. Significance: Our results reveal the first characterization of virus DRiPs as truncated translation products. Furthermore

Synthesis, microwave spectra, x-ray structure and high-level theoretical calculations for formamidinium formate

This material is based upon work supported by the National Science Foundation under Grant No. CHE-1057796 at the University of Arizona.An efficient synthesis of formamidinium formate is described. The experimental x-ray structure shows both internal and external H-bonding to surrounding molecules. However, in the gas phase this compound occurs as a doubly hydrogen bonded dimer. This doubly hydrogen-bonded structure is quite different from the solid state structure. Microwave spectra were measured in the 6-14 GHz range using a pulsed-beam Fourier transform microwave spectrometer. The two nonequivalent N-atoms exhibit distinct quadrupole coupling. The rotational, centrifugal distortion and quadrupole coupling constants determined from the spectra have values: A = 5880.05(2), B = 2148.7710(2), C = 1575.23473(13), 1.5 χaa (N1) =1.715( 3), 0.5(χbb- χcc)(N1) = -1.333(4), 1.5χaa (N2) = 0.381(2), 0.25(χbb- χcc)(N2) = -0.0324(2), and DJ = 0.002145(5) MHz. The experimental inertial defect, Δ = -0.243 amu Å2, is consistent with a planar structure. Accurate and precise rotational constants (A, B and C), obtained from the microwave (MW) measurements, were closely reproduced, within 1-2% of the measured values, with the M11 DFT functional theoretical calculations. Detailed comparison of the measured and calculated A, B and C rotational constants confirm the planar doubly hydrogen bonded structure. the nitrogen quadrupole coupling strengths of the monomer are quite different from either of the two nitrogen sites of the dimer. The poor agreement between measured and calculated quadrupole coupling strengths show that the dimer is not locked in the equilibrium structure, but is likely undergoing large amplitude vibrational motion of the hydrogen atoms moving between the N and O atoms involved in the hydrogen bonding.PostprintPeer reviewe

CTL Escape Mediated by Proteasomal Destruction of an HIV-1 Cryptic Epitope

Cytotoxic CD8+ T cells (CTLs) play a critical role in controlling viral infections. HIV-infected individuals develop CTL responses against epitopes derived from viral proteins, but also against cryptic epitopes encoded by viral alternative reading frames (ARF). We studied here the mechanisms of HIV-1 escape from CTLs targeting one such cryptic epitope, Q9VF, encoded by an HIVgag ARF and presented by HLA-B*07. Using PBMCs of HIV-infected patients, we first cloned and sequenced proviral DNA encoding for Q9VF. We identified several polymorphisms with a minority of proviruses encoding at position 5 an aspartic acid (Q9VF/5D) and a majority encoding an asparagine (Q9VF/5N). We compared the prevalence of each variant in PBMCs of HLA-B*07+ and HLA-B*07- patients. Proviruses encoding Q9VF/5D were significantly less represented in HLA-B*07+ than in HLA-B*07- patients, suggesting that Q9FV/5D encoding viruses might be under selective pressure in HLA-B*07+ individuals. We thus analyzed ex vivo CTL responses directed against Q9VF/5D and Q9VF/5N. Around 16% of HLA-B*07+ patients exhibited CTL responses targeting Q9VF epitopes. The frequency and the magnitude of CTL responses induced with Q9VF/5D or Q9VF/5N peptides were almost equal indicating a possible cross-reactivity of the same CTLs on the two peptides. We then dissected the cellular mechanisms involved in the presentation of Q9VF variants. As expected, cells infected with HIV strains encoding for Q9VF/5D were recognized by Q9VF/5D-specific CTLs. In contrast, Q9VF/5N-encoding strains were neither recognized by Q9VF/5N- nor by Q9VF/5D-specific CTLs. Using in vitro proteasomal digestions and MS/MS analysis, we demonstrate that the 5N variation introduces a strong proteasomal cleavage site within the epitope, leading to a dramatic reduction of Q9VF epitope production. Our results strongly suggest that HIV-1 escapes CTL surveillance by introducing mutations leading to HIV ARF-epitope destruction by proteasomes

Time-dependent increase in ribosome processivity

We created a novel tripartite reporter RNA to separately and simultaneously examine ribosome translation rates at the 5′- and 3′-ends of a large open reading frame (ORF) in vitro in HeLa cell lysates. The construct contained Renilla luciferase (RLuc), β-galactosidase and firefly luciferase (FLuc) ORFs linked in frame and separated by a viral peptide sequence that causes cotranslational scission of emerging peptide chains. The length of the ORF contributed to low ribosome processivity, a low number of initiating ribosomes completing translation of the entire ORF. We observed a time-dependent increase in FLuc production rate that was dependent on a poly(A) tail and poly(A)-binding protein, but was independent of eIF4F function. Stimulation of FLuc production occurred earlier on shorter RNA templates. Cleavage of eIF4G at times after ribosome loading on templates occurred did not cause immediate cessation of 5′-RLuc translation; rather, a delay was observed that shortened when shorter templates were translated. Electron microscopic analysis of polysome structures in translation lysates revealed a time-dependent increase in ribosome packing and contact that correlated with increased processivity on the FLuc ORF. The results suggest that ORF transit combined with PABP function contribute to interactions between ribosomes that increase or sustain processivity on long ORFs

Non-conventional sources of peptides presented by MHC class I

Effectiveness of immune surveillance of intracellular viruses and bacteria depends upon a functioning antigen presentation pathway that allows infected cells to reveal the presence of an intracellular pathogen. The antigen presentation pathway uses virtually all endogenous polypeptides as a source to produce antigenic peptides that are eventually chaperoned to the cell surface by MHC class I molecules. Intriguingly, MHC I molecules present peptides encoded not only in the primary open reading frames but also those encoded in alternate reading frames. Here, we review recent studies on the generation of cryptic pMHC I. We focus on the immunological significance of cryptic pMHC I, and the novel translational mechanisms that allow production of these antigenic peptides from unconventional sources

Epstein Barr Virus-Encoded EBNA1 Interference with MHC Class I Antigen Presentation Reveals a Close Correlation between mRNA Translation Initiation and Antigen Presentation

Viruses are known to employ different strategies to manipulate the major histocompatibility (MHC) class I antigen presentation pathway to avoid recognition of the infected host cell by the immune system. However, viral control of antigen presentation via the processes that supply and select antigenic peptide precursors is yet relatively unknown. The Epstein-Barr virus (EBV)-encoded EBNA1 is expressed in all EBV-infected cells, but the immune system fails to detect and destroy EBV-carrying host cells. This immune evasion has been attributed to the capacity of a Gly-Ala repeat (GAr) within EBNA1 to inhibit MHC class I restricted antigen presentation. Here we demonstrate that suppression of mRNA translation initiation by the GAr in cis is sufficient and necessary to prevent presentation of antigenic peptides from mRNAs to which it is fused. Furthermore, we demonstrate a direct correlation between the rate of translation initiation and MHC class I antigen presentation from a certain mRNA. These results support the idea that mRNAs, and not the encoded full length proteins, are used for MHC class I restricted immune surveillance. This offers an additional view on the role of virus-mediated control of mRNA translation initiation and of the mechanisms that control MHC class I restricted antigen presentation in general

Cancer Genome Sequencing and Its Implications for Personalized Cancer Vaccines

New DNA sequencing platforms have revolutionized human genome sequencing. The dramatic advances in genome sequencing technologies predict that the $1,000 genome will become a reality within the next few years. Applied to cancer, the availability of cancer genome sequences permits real-time decision-making with the potential to affect diagnosis, prognosis, and treatment, and has opened the door towards personalized medicine. A promising strategy is the identification of mutated tumor antigens, and the design of personalized cancer vaccines. Supporting this notion are preliminary analyses of the epitope landscape in breast cancer suggesting that individual tumors express significant numbers of novel antigens to the immune system that can be specifically targeted through cancer vaccines

Identification of Keratinocyte Growth Factor as a Target of microRNA-155 in Lung Fibroblasts: Implication in Epithelial-Mesenchymal Interactions

International audienceBACKGROUND: Epithelial-mesenchymal interactions are critical in regulating many aspects of vertebrate embryo development, and for the maintenance of homeostatic equilibrium in adult tissues. The interactions between epithelium and mesenchyme are believed to be mediated by paracrine signals such as cytokines and extracellular matrix components secreted from fibroblasts that affect adjacent epithelia. In this study, we sought to identify the repertoire of microRNAs (miRNAs) in normal lung human fibroblasts and their potential regulation by the cytokines TNF-alpha, IL-1beta and TGF-beta. METHODOLOGY/PRINCIPAL FINDINGS: MiR-155 was significantly induced by inflammatory cytokines TNF-alpha and IL-1beta while it was down-regulated by TGF-beta. Ectopic expression of miR-155 in human fibroblasts induced modulation of a large set of genes related to "cell to cell signalling", "cell morphology" and "cellular movement". This was consistent with an induction of caspase-3 activity and with an increase in cell migration in fibroblasts tranfected with miR-155. Using different miRNA bioinformatic target prediction tools, we found a specific enrichment for miR-155 predicted targets among the population of down-regulated transcripts. Among fibroblast-selective targets, one interesting hit was keratinocyte growth factor (KGF, FGF-7), a member of the fibroblast growth factor (FGF) family, which owns two potential binding sites for miR-155 in its 3'-UTR. Luciferase assays experimentally validated that miR-155 can efficiently target KGF 3'-UTR. Site-directed mutagenesis revealed that only one out of the 2 potential sites was truly functional. Functional in vitro assays experimentally validated that miR-155 can efficiently target KGF 3'-UTR. Furthermore, in vivo experiments using a mouse model of lung fibrosis showed that miR-155 expression level was correlated with the degree of lung fibrosis. CONCLUSIONS/SIGNIFICANCE: Our results strongly suggest a physiological function of miR-155 in lung fibroblasts. Altogether, this study implicates this miRNA in the regulation by mesenchymal cells of surrounding lung epithelium, making it a potential key player during tissue injury