Human cytomegalovirus (HCMV) can establish both nonproductive (latent) and productive (lytic) infections. Many of the proteins expressed during these phases of infection could be expected to be targets of the immune response; however, much of our understanding of the CD8؉ -T-cell response to HCMV is mainly based on the pp65 antigen. Very little is known about T-cell control over other antigens expressed during the different stages of virus infection; this imbalance in our understanding undermines the importance of these antigens in several aspects of HCMV disease pathogenesis. In the present study, an efficient and rapid strategy based on predictive bioinformatics and ex vivo functional T-cell assays was adopted to profile CD8؉ -T-cell responses to a large panel of HCMV antigens expressed during different phases of replication. These studies revealed that CD8؉ -T-cell responses to HCMV often contained multiple antigen-specific reactivities, which were not just constrained to the previously identified pp65 or IE-1 antigens. Unexpectedly, a number of viral proteins including structural, early/late antigens and HCMV-encoded immunomodulators (pp28, pp50, gH, gB, US2, US3, US6, and UL18) were also identified as potential targets for HCMV-specific CD8؉ -T-cell immunity. Based on this extensive analysis, numerous novel HCMV peptide epitopes and their HLA-restricting determinants recognized by these T cells have been defined. These observations contrast with previous findings that viral interference with the antigen-processing pathway during lytic infection would render immediate-early and early/late proteins less immunogenic. This work strongly suggests that successful HCMV-specific immune control in healthy virus carriers is dependent on a strong T-cell response towards a broad repertoire of antigens.
Abstract. Insulin stimulates glucose transport in adipocytes by translocation of the glucose transporter (GLUT-4) from an intracellular site to the cell surface. We have characterized different synaptobrevin/vesicleassociated membrane protein (VAMP) homologues in adipocytes and studied their intracellular distribution with respect to GLUT-4. VAMP-l, VAMP-2, and cellubrevin cDNAs were isolated from a 3T3-L1 adipocyte expression library. VAMP-2 and cellubrevin were: (a) the most abundant isoforms in adipocytes, (b) detectable in all insulin responsive tissues, (c) translocated to the cell surface in response to insulin, and (d) found in immunoadsorbed GLUT-4 vesicles. To further define their intracellular distribution, 3T3-L1 adipocytes were incubated with a transferrin/HRP conjugate (Tf/HRP) and endosomes ablated following addition of DAB and H202. While this resulted in ablation of >90% of the transferrin receptor (TfR) and cellubrevin found in intracellular membranes, 60% of GLUT-4 and 90% of VAMP-2 was not ablated. Immuno-EM on intracellular vesicles from adipocytes revealed that VAMP-2 was colocalized with GLUT-4, whereas only partial colocalization was observed between GLUT-4 and cellubrevin. These studies show that two different v-SNAREs, cellubrevin and VAMP-2, are partially segregated in different intracellular compartments in adipocytes, implying that they may define separate classes of secretory vesicles in these cells. We conclude that a proportion of GLUT-4 is found in recycling endosomes in nonstimulated adipocytes together with cellubrevin and the transferrin receptor. In addition, GLUT-4 and VAMP-2 are selectively enriched in a postendocytic compartment. Further study is required to elucidate the function of this latter compartment in insulin-responsive cells.
Viruses that establish latent infections have evolved unique mechanisms to avoid host immune recognition. Maintenance proteins of these viruses regulate their synthesis to levels sufficient for maintaining persistent infection but below threshold levels for host immune detection. The mechanisms governing this finely tuned regulation of viral latency are unknown. Here we show that mRNAs encoding gammaherpesviral maintenance proteins contain within their open reading frames clusters of unusual structural elements, G-quadruplexes, which are responsible for the cisacting regulation of viral mRNA translation. By studying the Epstein-Barr virus-encoded nuclear antigen 1 (EBNA1) mRNA, we demonstrate that destabilization of G-quadruplexes using antisense oligonucleotides increases EBNA1 mRNA translation. In contrast, pretreatment with a G-quadruplex-stabilizing small molecule, pyridostatin, decreases EBNA1 synthesis, highlighting the importance of G-quadruplexes within virally encoded transcripts as unique regulatory signals
The Epstein-Barr nuclear antigen-1 (EBNA1) protein of Epstein-Barr virus is important for the replication, segregation, and transcriptional activation of latent Epstein-Barr virus genomes; has been implicated in host cell immortalization; and avoids proteasomal processing and cell-surface presentation. To gain insight into how EBNA1 fulfills these functions, we have profiled cellular protein interactions with EBNA1 using EBNA1 affinity chromatography and tandem affinity purification (TAP) of EBNA1 complexes from human cells (TAPtagging). We discovered several new specific cellular protein interactions with EBNA1, including interactions with HAUSP/USP7, NAP1, template-activating factor-I/SET, CK2, and PRMT5, all of which play important cell regulatory roles. The ubiquitin-specific protease USP7 is a known target of herpes simplex virus, and the USP7-binding region of EBNA1 was mapped to amino acids 395-450. A mutation in EBNA1 that selectively disrupted binding to USP7 was found to cause a 4-fold increase in EBNA1 replication activity but had no effect on EBNA1 turnover and cell-surface presentation. The results suggest that USP7 can regulate the replication function of EBNA1 and that EBNA1 may influence cellular events by sequestering key regulatory proteins.Epstein-Barr virus (EBV) 1 is a ubiquitous human ␥-herpesvirus that persists for the life of the host. As part of its latent infectious cycle, EBV immortalizes the host cell and, in doing so, predisposes the cell to malignant transformation. As a result, EBV is associated with several types of cancer. EBV genomes are maintained in latently infected replicating cells as circular DNA episomes that replicate once per cell cycle and segregate stably during cell division (reviewed in Refs. 1 and 2). Epstein-Barr nuclear antigen-1 (EBNA1) is the only viral protein required to maintain the EBV genomes in proliferating cells, which it does by binding to recognition sites in the FR (family of repeats) and DS (dyad symmetry) elements of the latent origin of DNA replication, oriP (3, 4). EBNA1 binding to the DS element is necessary to initiate DNA replication from this element (5). EBNA1 binding to the FR element is important for the partitioning of the EBV episomes during cell division and also activates the expression of other viral latency genes (6). In addition to its functions at oriP, EBNA1 has been shown to repress its own transcription (7) and to promote the development of B-cell lymphomas in transgenic mice, suggesting a direct role for EBNA1 in cell transformation (8).While fulfilling all of its functions, EBNA1 avoids detection by host cytotoxic T-lymphocytes. This ability to hide from the immune system is biologically important, as it enables the persistence of latently infected cells that express EBNA1 in the absence of other EBV antigens. The failure of EBNA1 to elicit a cytotoxic T-lymphocyte response is due to lack of proteasomal processing, which prevents the presentation of EBNA1 by major histocompatibility complex class I molecules on the cell surfa...
We have previously identified three mammalian Sec1/ Munc-18 homologues in adipocytes (Tellam, J. T., McIntosh, S., and James, D. E. (1995) J. Biol. Chem. 270, 5857-5863). These proteins are thought to modulate the interaction between vesicle membrane and target membrane soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and thus regulate intracellular vesicular transport. This study aimed to further characterize these Munc-18 isoforms and to define their potential role in the trafficking of GLUT-4 in adipocytes, a process reported to involve the vesicle membrane SNARE, VAMP-2. Using an in vitro binding assay with recombinant fusion proteins, we show that Munc-18a and Munc-18b bind to syntaxin-1A, -2, and -3, while Munc-18c binds only to syntaxin-2 and -4. The specific interaction between Munc-18c and syntaxin-4 is of interest because aside from syntaxin-1A, which is not expressed in adipocytes, syntaxin-4 is the only syntaxin that binds to VAMP-2. Using a three-way binding assay, it was shown that Munc-18c inhibits the binding of syntaxin-4 to VAMP-2. The subcellular distribution of syntaxin-4 and Munc-18c was almost identical, both being enriched in the plasma membrane, and both exhibiting an insulin-dependent movement out of an intracellular membrane fraction similar to that observed for GLUT-4. Munc-18b had a similar distribution to Munc-18c and so may also be involved in vesicle transport to the cell surface, whereas Munc-18a was undetectable by immunoblotting in adipocytes. Microinjection of a syntaxin-4 antibody into 3T3-L1 adipocytes blocked the insulin-dependent recruitment of GLUT-4 to the cell surface. These data suggest that syntaxin-4/Munc-18c/VAMP-2 may play a role in the docking/fusion of intracellular GLUT-4-containing vesicles with the cell surface in adipocytes.In adipose tissue and muscle, the glucose transporter isoform 4, GLUT-4, is translocated from an intracellular vesicular pool to the cell surface in response to insulin (1, 2), a process that plays a major role in whole body glucose homeostasis. To understand the molecular mechanisms governing this vesicular transport system, it will be necessary to identify and characterize the individual components of the trafficking machinery. The SNARE hypothesis (reviewed in Ref.3) provides a working model for studies of vesicle targeting and fusion in adipocytes. Vesicle-associated membrane protein VAMP or synaptobrevin (v-SNAREs) 1 present on the transport vesicle and syntaxin (t-SNAREs) on the acceptor membrane form a complex, which also includes the synaptosomal-associated protein-25 (SNAP-25), soluble N-ethylmaleimide-sensitive factor attachment protein (␣-SNAP), and N-ethylmaleimide-sensitive factor (NSF). This complex may facilitate the docking and/or fusion of distinct membrane compartments, the specificity being provided by the pairing of unique v-and t-SNAREs at different loci throughout the cell. Each of the SNAREs belong to large gene families. For example, in mammalian cells seven different syntaxins (4, 5), ...
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