The Epstein-Barr Virus G-Protein-Coupled Receptor Contributes to Immune Evasion by Targeting MHC Class I Molecules for Degradation

Epstein-Barr virus (EBV) is a human herpesvirus that persists as a largely subclinical infection in the vast majority of adults worldwide. Recent evidence indicates that an important component of the persistence strategy involves active interference with the MHC class I antigen processing pathway during the lytic replication cycle. We have now identified a novel role for the lytic cycle gene, BILF1, which encodes a glycoprotein with the properties of a constitutive signaling G-protein-coupled receptor (GPCR). BILF1 reduced the levels of MHC class I at the cell surface and inhibited CD8⁺ T cell recognition of endogenous target antigens. The underlying mechanism involves physical association of BILF1 with MHC class I molecules, an increased turnover from the cell surface, and enhanced degradation via lysosomal proteases. The BILF1 protein of the closely related CeHV15 γ₁-herpesvirus of the Rhesus Old World primate (80% amino acid sequence identity) downregulated surface MHC class I similarly to EBV BILF1. Amongst the human herpesviruses, the GPCR encoded by the ORF74 of the KSHV γ₂-herpesvirus is most closely related to EBV BILF1 (15% amino acid sequence identity) but did not affect levels of surface MHC class I. An engineered mutant of BILF1 that was unable to activate G protein signaling pathways retained the ability to downregulate MHC class I, indicating that the immune-modulating and GPCR-signaling properties are two distinct functions of BILF1. These findings extend our understanding of the normal biology of an important human pathogen. The discovery of a third EBV lytic cycle gene that cooperates to interfere with MHC class I antigen processing underscores the importance of the need for EBV to be able to evade CD8⁺ T cell responses during the lytic replication cycle, at a time when such a large number of potential viral targets are expressed.     

Organization of the variable region of the immunoglobulin heavy-chain gene locus of the rat

We have mapped and annotated the variable region of the immunoglobulin heavy (IGH) gene locus of the Brown Norway (BN) rat (assembly V3.4; Rat Genomic Sequence Consortium). In addition to known variable region genes, we found 12 novel previously unidentified functional IGHV genes and 1 novel functional IGHD gene. In total, the variable region of the rat IGH locus is composed of at least 353 unique IGHV genes, 21 IGHD genes, and 5 IGHJ genes, of which 131, 14, and 4 are potentially functional genes, respectively. Of all species studied so far, the rat seems to have the highest number of functional IGHV genes in the genome. Rat IGHV genes can be classified into 13 IGHV families based on nucleotide sequence identity. The variable region of the BN rat spans a total length of approximately 4.9 Mb and is organized in a typical translocon organization. Like the mouse, members of the various IGHV gene families are more or less grouped together on the genome, albeit some members of IGHV gene families are found intermingled with each other. In the rat, the largest IGHV gene families are IGHV1, IGHV2, and IGHV5. The overall conclusion is that the genomic organization of the variable region of the rat IGH locus is strikingly similar to that of the mouse, illustrating the close evolutionary relationship between these two species.     

Acute norepinephrine reuptake inhibition decreases performance in normal and high ambient temperature.

Combined inhibition of dopamine (DA)/norepinephrine (NE) reuptake improves exercise performance and increases core temperature in the heat. A recent study demonstrated that this effect may primarily be related to increased DA activity. NE reuptake inhibition (NERI), however, has received little attention in humans, certainly in the heat, where central fatigue appears to be a main factor influencing performance. Therefore the present study examines the effect of NERI (reboxetine) on exercise capacity, thermoregulation, and hormonal response in normal and high temperature. Nine healthy well-trained male cyclists participated in this study. Subjects ingested either placebo (Pla; 2 x 8 mg) or reboxetine (Rebox; 2 x 8 mg). Subjects exercised in temperate (18 degrees C) or warm (30 degrees C) conditions and cycled for 60 min at 55% W(max) immediately followed by a time trial (TT; Pla18/Rebox18; Pla30/Rebox30) to measure exercise performance. Acute NERI decreased power output and consequently exercise performance in temperate (P = 0.018) and warm (P = 0.007) conditions. Resting heart rate was significantly elevated by NERI (18 degrees C: P = 0.02; 30 degrees C: P = 0.018). In Rebox18, heart rate was significantly higher than in the Pla18, while in the heat no effect of the drug treatment was reported during exercise. In Rebox30, all hormone concentrations increased during exercise, except for growth hormone (GH), which was significantly lower during exercise. In Rebox18, prolactin (PRL) concentrations were significantly elevated; GH was significantly higher at rest, but significantly lower during exercise. In conclusion, manipulation of the NE system decreases performance and modifies hormone concentrations, thereby indicating a central NE effect of the drug. These findings confirm results from previous studies that predominantly increased DA activity is important in improving performance.     

Mutations in the slow skeletal muscle fiber myosin heavy chain gene (MYH7) cause laing early-onset distal myopathy (MPD1)

We previously linked Laing-type early-onset autosomal dominant distal myopathy (MPD1) to a 22-cM region of chromosome 14. One candidate gene in the region, MYH7, which is mutated in cardiomyopathy and myosin storage myopathy, codes for the myosin heavy chain of type I skeletal muscle fibers and cardiac ventricles. We have identified five novel heterozygous mutations--Arg1500Pro, Lys1617del, Ala1663Pro, Leu1706Pro, and Lys1729del in exons 32, 34, 35, and 36 of MYH7--in six families with early-onset distal myopathy. All five mutations are predicted, by in silico analysis, to locally disrupt the ability of the myosin tail to form the coiled coil, which is its normal structure. These findings demonstrate that heterozygous mutations toward the 3' end of MYH7 cause Laing-type early-onset distal myopathy. MYH7 is the fourth distal-myopathy gene to have been identified.     

Cyclodextrins differentially mobilize free and esterified cholesterol from primary human foam cell macrophages

Human monocyte-derived foam cell macrophages (HMFCs) are resistant to cholesterol efflux mediated by physiological acceptors. The role of the plasma membrane in regulating depletion of free cholesterol (FC) and of cholesteryl ester (CE) was investigated using cyclodextrins (CDs). HMFCs were incubated in media containing CDs (1.0 mg/ml, approximately 0.7 mM) with low [hydroxypropyl-beta-CD (HP-CD)] or high [trimethyl-beta-CD (TM-CD)] affinity for cholesterol in the presence or absence of phospholipid vesicles (PLVs). Low-affinity HP-CD caused minimal cholesterol efflux on its own, but HP-CD+ PLV depleted cell FC and CE to 54.5 +/- 6.7% of control by 24 h. TM-CD depleted FC at least as well as HP-CD+PLV but without depleting CE, even when combined with PLV. This was not explained by acceptor saturation, instability of PLV vesicles, de novo cholesterol synthesis, kinetically distinct cholesterol pools, or inhibition of CE hydrolysis. TM-CD did, however, deplete CE when lower concentrations of TM-CD were combined with PLV and when acetyl-CoA cholesteryl acyltransferase was inhibited. TM-CD caused much greater depletion of plasma membrane cholesterol than HP-CD without depleting plasma membrane sphingomyelin. It is concluded that differential depletion of plasma membrane cholesterol pools regulates cholesterol efflux and CE clearance in human macrophages.     

Regulation of membrane transport by rab GTPases

Membrane flow through the cell is a highly dynamic process in which intracellular compartments communicate via tubulo-vesicular structures shuttling cargo molecules to their destinations. Transport carriers are formed at a donor compartment and navigate through the cytoplasm to the target organelle, on which they subsequently dock and fuse. Many of these events are regulated by the cooperative action of monomeric rab GTPases and their effector proteins. Research in recent years resulted in the identification of many rab effectors, providing first glimpses how the GTPase switch of individual rab proteins is utilized in discrete transport steps.     

Beta-catenin and TCF mediate cell positioning in the intestinal epithelium by controlling the expression of EphB/ephrinB

In the small intestine, the progeny of stem cells migrate in precise patterns. Absorptive, enteroendocrine, and goblet cells migrate toward the villus while Paneth cells occupy the bottom of the crypts. We show here that beta-catenin and TCF inversely control the expression of the EphB2/EphB3 receptors and their ligand ephrin-B1 in colorectal cancer and along the crypt-villus axis. Disruption of EphB2 and EphB3 genes reveals that their gene products restrict cell intermingling and allocate cell populations within the intestinal epithelium. In EphB2/EphB3 null mice, the proliferative and differentiated populations intermingle. In adult EphB3(-/-) mice, Paneth cells do not follow their downward migratory path, but scatter along crypt and villus. We conclude that in the intestinal epithelium beta-catenin and TCF couple proliferation and differentiation to the sorting of cell populations through the EphB/ephrin-B system.     

Differential effects of ammonium and nitrate on three heathland species

Arnica montana and Cirsium dissectum, typical of species-rich heathlands and acidic grasslands, have declined rapidly in The Netherlands in recent years. Field surveys suggest that the decline is caused by soil acidification as a result of enhanced atmospheric N and S deposition. Therefore, the survival, growth and development of these species were studied in a water culture experiment, using nutrient solutions which differed both in mineral nitrogen form and in ammonium concentration. For comparison, the performance of a third, acid tolerant species, Calluna vulgaris, was studied. The results showed that both Arnica and Cirsium performed better using nitrate than when using ammonium as a sole nitrogen source, whereas ammonium toxicity became apparent when ammonium concentrations were raised above 100 µM. Ammonium toxicity was expressed by an increase in mortality of Arnica plants with increasing ammonium concentrations and by a reduction of biomass in Arnica and Cirsium. Furthermore, cation concentrations in both roots and shoots decreased when ammonium was supplied as a nitrogen source. In contrast, Calluna showed optimal development when using ammonium as a sole nitrogen source. In this species, only root biomass was negatively affected by high ammonium concentrations. The ecological implications of these preferences are discussed in relation to soil acidification.