Amino acid residues that are important for Hyal2 function as a receptor for jaagsiekte sheep retrovirus

BACKGROUND: Infection by jaagsiekte sheep retrovirus (JSRV) and by enzootic nasal tumor virus (ENTV) depends on cell-surface expression of the virus entry receptor, hyaluronidase 2 (Hyal2). Human Hyal2 binds the envelope (Env) proteins of these viruses and is functional as a receptor, but Hyal2 from mice does not bind Env nor does it mediate entry of either virus. Here we have explored the amino acid determinants that account for the difference in receptor function. RESULTS: Analysis of human-mouse Hyal2 chimeric proteins showed that amino acid differences responsible for the difference in Hyal2 receptor activity were localized to the central third of Hyal2. Human Hyal2 mutants containing single or double amino acid replacements with the respective mouse amino acids were generated across this region and were assayed for activity. None of the single or double mutation reduced the receptor activity of human Hyal2 by more than 10-fold, whereas mouse Hyal2 activity is reduced 1,000-fold from that of human Hyal2. While the 3-dimensional structures of mammalian Hyal2 proteins are unknown, bee venom hyaluronidase shows significant amino acid similarity to human and mouse Hyal2 and its structure has been determined. Many mutations having the largest negative effects on human Hyal2 function mapped to a small region of the bee venom hyaluronidase close to but not overlapping the active site of the enzyme, suggesting that this site represents the binding site for Env. Analysis of synonymous and non-synonymous nucleotide substitutions in the coding sequences of multiple mammalian Hyal2 proteins shows that the proteins are undergoing strong selection for amino acid conservation. We found no evidence for positive selection of amino acid changes that might reflect evolution of mammalian hosts to resist JSRV or ENTV infection. CONCLUSION: These results show that the greatly reduced receptor activity of mouse Hyal2 in comparison to that of human Hyal2 is determined by multiple amino acid changes acting in concert. In particular, no one amino acid change blocks infection. However, the most important amino acids map to a small patch on a predicted 3-dimensional Hyal2 structure, which may represent the binding site for Env

Broadly reactive human CD8 T cells that recognize an epitope conserved between VZV, HSV and EBV

Human herpesviruses are important causes of potentially severe chronic infections for which T cells are believed to be necessary for control. In order to examine the role of virus-specific CD8 T cells against Varicella Zoster Virus (VZV), we generated a comprehensive panel of potential epitopes predicted in silico and screened for T cell responses in healthy VZV seropositive donors. We identified a dominant HLA-A*0201-restricted epitope in the VZV ribonucleotide reductase subunit 2 and used a tetramer to analyze the phenotype and function of epitope-specific CD8 T cells. Interestingly, CD8 T cells responding to this VZV epitope also recognized homologous epitopes, not only in the other α-herpesviruses, HSV-1 and HSV-2, but also the γ-herpesvirus, EBV. Responses against these epitopes did not depend on previous infection with the originating virus, thus indicating the cross-reactive nature of this T cell population. Between individuals, the cells demonstrated marked phenotypic heterogeneity. This was associated with differences in functional capacity related to increased inhibitory receptor expression (including PD-1) along with decreased expression of co-stimulatory molecules that potentially reflected their stimulation history. Vaccination with the live attenuated Zostavax vaccine did not efficiently stimulate a proliferative response in this epitope-specific population. Thus, we identified a human CD8 T cell epitope that is conserved in four clinically important herpesviruses but that was poorly boosted by the current adult VZV vaccine. We discuss the concept of a “pan-herpesvirus” vaccine that this discovery raises and the hurdles that may need to be overcome in order to achieve this

HLA-C Level Is Regulated by a Polymorphic Oct1 Binding Site in the HLA-C Promoter Region

International audienceDifferential HLA-C levels influence several human diseases, but the mechanisms responsible are incompletely characterized. Using a validated prediction algorithm, we imputed HLA-C cell surface levels in 228 individuals from the 1000 Genomes dataset. We tested 68,726 SNPs within the MHC for association with HLA-C level. The HLA-C promoter region variant, rs2395471, 800 bp upstream of the transcription start site, gave the most significant association with HLA-C levels (p = 4.2 × 10−66). This imputed expression quantitative trait locus, termed impeQTL, was also shown to associate with HLA-C expression in a genome-wide association study of 273 donors in which HLA-C mRNA expression levels were determined by quantitative PCR (qPCR) (p = 1.8 × 10−20) and in two cohorts where HLA-C cell surface levels were determined directly by flow cytometry (n = 369 combined, p < 10−15). rs2395471 is located in an Oct1 transcription factor consensus binding site motif where the A allele is predicted to have higher affinity for Oct1 than the G allele. Mobility shift electrophoresis demonstrated that Oct1 binds to both alleles in vitro, but decreased HLA-C promoter activity was observed in a luciferase reporter assay for rs2395471_G relative to rs2395471_A on a fixed promoter background. The rs2395471 variant accounts for up to 36% of the explained variation of HLA-C level. These data strengthen our understanding of HLA-C transcriptional regulation and provide a basis for understanding the potential consequences of manipulating HLA-C levels therapeutically

The RASSF1A tumor suppressor gene is inactivated in prostate tumors and suppresses growth of prostate carcinoma cells

We analyzed expression status of the recently identified tumor suppressor gene RASSF1A in primary prostate carcinomas and in prostate cell lines. We found complete methylation of the RASSF1A promoter in 63% of primary microdissected prostate carcinomas (7 of 11 samples). The remaining 4 samples (37%) were partially methylated, possibly because of contamination with normal cells. No promoter methylation was observed in matching normal prostate tissues. High levels of RASSF1A transcript and no methylation of RASSF1A promoter were found in explanted primary normal prostate epithelial and stromal cells. Complete silencing and methylation of RASSF1A promoter was observed in five widely used prostate carcinoma cell lines, which acquired the ability to grow in culture spontaneously, including LNCaP, P\C-3, ND-1, DU-145, 22Rv1, and one primary prostate carcinoma immortalized by overexpression of the human telomerase catalytic subunit (RC-58T/hTERT). However, no silencing of RASSF1A was found in four other prostate carcinoma cell lines, which were adapted for cell culture after transformation with human papillomaviral DNA. Suppression of cell growth in vitro was demonstrated after the reintroduction of RASSF1A-expressing construct into LNCaP prostate carcinoma cells. Our data implicate the RASSF1A gene in human prostate tumorigenesis

Donor details and serology.

<p>ND – not detected.</p><p>Phenotype 1 – CD27+ CD28+ perforin− granzyme B−.</p><p>Phenotype 2 – CD27+ CD28+ perforin− granzyme B+.</p><p>Phenotype 3 – CD27− CD28− perforin+ granzyme B+.</p

Expression of inhibitory receptors is associated with increased ILI-specific CD8 T cell frequency but impaired functionality.

<p>A2-ILI-tetramer+ CD8 T cells were co-stained with anti-PD-1 and 2B4, and analysed using flow cytometry. (a) Representative donors with each phenotype are shown. Numbers represent frequency of events as a percentage of CD3+CD8+ lymphocytes. (b) Non-linear regression and Spearman's rank correlation were used to show the association between the frequency of IFN-γ producing A2-ILI+ cells and their frequency of PD-1 and 2B4 co-expression. (c) Non-linear regression and Spearman's rank correlation were used to show the association between the frequency of A2-ILI tetramer+ CD8 T cells and their expression of both PD-1 and 2B4. P-values for Spearman's rank correlation are shown.</p

Summary of demographic data of HLA-A*0201+ subjects.

<p>Summary of demographic data of HLA-A*0201+ subjects.</p

ILI-specific CD8 T cells display functional impairment associated with their phenotype.

<p>(a) PBMCs were stimulated with ILIEGIFFV peptide <i>in vitro</i> for 6 hours then co-stained for intracellular IFN-γ and IL-2. Numbers represent frequency as a percentage of CD8+ lymphocytes. The frequency of (b) IFN-γ+ cells as a proportion of A2-ILI tetramer+ cells and (c) IL-2 producing cells as a percentage of IFN-γ+ CD8 T cells are shown. P-values are derived from Student's t-test. (d) PBMCs from HLA-A*0201+ volunteers were stained with CFSE and stimulated with ILIEGIFFV peptide <i>in vitro</i> for 6 days. A2-ILI tetramer+ cells were then analyzed by flow cytometry. Numbers represent frequency as a percentage of CD8+ lymphocytes. Two representative donors are shown.</p