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1.  Budding of Enveloped Viruses: Interferon-Induced ISG15—Antivirus Mechanisms Targeting the Release Process 
Advances in Virology  2012;2012:532723.
Pathogenic strains of viruses that infect humans are encapsulated in membranes derived from the host cell in which they infect. After replication, these viruses are released by a budding process that requires cell/viral membrane scission. As such, this represents a natural target for innate immunity mechanisms to interdict enveloped virus spread and recent advances in this field will be the subject of this paper.
PMCID: PMC3362814  PMID: 22666250
2.  Mechanism of Inhibition of Retrovirus Release from Cells by Interferon-Induced Gene ISG15 ▿  
Journal of Virology  2011;85(14):7153-7161.
Budding of retroviruses from cell membranes requires ubiquitination of Gag and recruitment of cellular proteins involved in endosome sorting, including endosome sorting complex required for transport III (ESCRT-III) protein complex and vacuolar protein sorting 4 (VPS4) and its ATPase. In response to infection, a cellular mechanism has evolved that blocks virus replication early and late in the budding process through expression of interferon-stimulated gene 15 (ISG15), a dimer homologue of ubiquitin. Interferon treatment of DF-1 cells blocks avian sarcoma/leukosis virus release, demonstrating that this mechanism is functional under physiological conditions. The late block to release is caused in part by a loss in interaction between VPS4 and its coactivator protein LIP5, which is required to promote the formation of the ESCRT III-VPS4 double-hexamer complex to activate its ATPase. ISG15 is conjugated to two different LIP5-ESCRT-III-binding charged multivesicular body proteins, CHMP2A and CHMP5. Upon ISGylation of each, interaction with LIP5 is no longer detected. Two other ESCRT-III proteins, CHMP4B and CHMP6, are also conjugated to ISG15. ISGylation of CHMP2A, CHMP4B, and CHMP6 weakens their binding directly to VPS4, thereby facilitating the release of this protein from the membrane into the cytosol. The remaining budding complex fails to release particles from the cell membrane. Introducing a mutant of ISG15 into cells that cannot be conjugated to proteins prevents the ISG15-dependent mechanism from blocking virus release. CHMP5 is the primary switch to initiate the antiviral mechanism, because removal of CHMP5 from cells prevents ISGylation of CHMP2A and CHMP6.
PMCID: PMC3126601  PMID: 21543490
3.  Rapid evolution of protein kinase PKR alters sensitivity to viral inhibitors 
Protein kinase PKR is activated during viral infection and phosphorylates the α subunit of eukaryotic translation initiation factor 2 (eIF2), leading to inhibition of translation and viral replication. We report fast evolution of the PKR kinase domain in vertebrates, coupled with positive selection of specific sites. Substitution of positively selected residues in human PKR with residues found in related species altered sensitivity to PKR inhibitors from different poxviruses. Species-specific differences in sensitivity to poxviral pseudosubstrate inhibitors were identified between human and mouse PKR, which were traced to positively-selected residues near the eIF2α-binding site. Our findings indicate how an antiviral protein evolved to evade viral inhibition while maintaining its primary function. Moreover, the identified species-specific differences in the susceptibility to viral inhibitors have important implications for studying human infections in non-human model systems.
PMCID: PMC3142916  PMID: 19043413
4.  The gene-reduction effect of chromosomal losses detected in gastric cancers 
BMC Gastroenterology  2010;10:138.
The level of loss of heterozygosity (LOH) that reduces a gene dose and exerts a cell-adverse effect is known to be a parameter for the genetic staging of gastric cancers. This study investigated if the cell-adverse effect induced with the gene reduction was a rate-limiting factor for the LOH events in two distinct histologic types of gastric cancers, the diffuse- and intestinal-types.
The pathologic specimens obtained from 145 gastric cancer patients were examined for the level of LOH using 40 microsatellite markers on eight cancer-associated chromosomes (3p, 4p, 5q, 8p, 9p, 13q, 17p and 18q).
Most of the cancer-associated chromosomes were found to belong to the gene-poor chromosomes and to contain a few stomach-specific genes that were highly expressed. A baseline-level LOH involving one or no chromosome was frequent in diffuse-type gastric cancers. The chromosome 17 containing a relatively high density of genes was commonly lost in intestinal-type cancers but not in diffuse-type cancers. A high-level LOH involving four or more chromosomes tended to be frequent in the gastric cancers with intestinal and mixed differentiation. Disease relapse was common for gastric cancers with high-level LOH through both the hematogenous (38%) and non-hematogenous (36%) routes, and for the baseline-level LOH cases through the non-hematogenous route (67%).
The cell-adverse effect of gene reduction is more tolerated in intestinal-type gastric cancers than in diffuse-type cancers, and the loss of high-dose genes is associated with hematogenous metastasis.
PMCID: PMC2994793  PMID: 21092121
5.  The Interferon-Induced Gene ISG15 Blocks Retrovirus Release from Cells Late in the Budding Process▿  
Journal of Virology  2010;84(9):4725-4736.
The release of retroviruses from cells requires ubiquitination of Gag and recruitment of cellular proteins involved in endosome sorting, including the ESCRT-III proteins and the Vps4 ATPase. In response to infection, cells have evolved an interferon-induced mechanism to block virus replication through expression of the interferon-stimulated gene 15 (ISG15), a dimer homologue of ubiquitin, which interferes with ubiquitin pathways in cells. Previously, it has been reported that ISG15 expression inhibited the E3 ubiquitin ligase, Nedd4, and prevented association of the ESCRT-I protein Tsg101 with human immunodeficiency virus type 1 (HIV-1) Gag. The budding of avian sarcoma leukosis virus and HIV-1 Gag virus-like particles containing L-domain mutations can be rescued by fusion to ESCRT proteins, which cause entry into the budding pathway beyond these early steps. The release of these fusions from cells was susceptible to inhibition by ISG15, indicating that there was a block late in the budding process. We now demonstrate that the Vps4 protein does not associate with the avian sarcoma leukosis virus or the HIV-1 budding complexes when ISG15 is expressed. This is caused by a loss in interaction between Vps4 with its coactivator protein LIP5 needed to promote the formation of the ESCRT-III-Vps4 double-hexamer complex required for membrane scission and virus release. The inability of LIP5 to interact with Vps4 is the probable result of ISG15 conjugation to the ESCRT-III protein, CHMP5, which regulates the availability of LIP5. Thus, there appear to be multiple levels of ISG15-induced inhibition acting at different stages of the virus release process.
PMCID: PMC2863725  PMID: 20164219
6.  DNA Methylation Patterns of Ulcer-Healing Genes Associated with the Normal Gastric Mucosa of Gastric Cancers 
Journal of Korean Medical Science  2010;25(3):405-417.
Recent evidence suggests that gastric mucosal injury induces adaptive changes in DNA methylation. In this study, the methylation status of the key tissue-specific genes in normal gastric mucosa of healthy individuals and cancer patients was evaluated. The methylation-variable sites of 14 genes, including ulcer-healing genes (TFF1, TFF2, CDH1, and PPARG), were chosen from the CpG-island margins or non-island CpGs near the transcription start sites. The healthy individuals as well as the normal gastric mucosa of 23 ulcer, 21 non-invasive cancer, and 53 cancer patients were examined by semiquantitative methylation-specific polymerase chain reaction (PCR) analysis. The ulcer-healing genes were concurrently methylated with other genes depending on the presence or absence of CpG-islands in the normal mucosa of healthy individuals. Both the TFF2 and PPARG genes were frequently undermethylated in ulcer patients. The over- or intermediate-methylated TFF2 and undermethylated PPARG genes was more common in stage-1 cancer patients (71%) than in healthy individuals (10%; odds ratio [OR], 21.9) and non-invasive cancer patients (21%; OR, 8.9). The TFF2-PPARG methylation pattern of cancer patients was stronger in the older-age group (≥55 yr; OR, 43.6). These results suggest that the combined methylation pattern of ulcer-healing genes serves as a sensitive marker for predicting cancer-prone gastric mucosa.
PMCID: PMC2826743  PMID: 20191040
DNA Methylation; Stomach; Ulcer; Non-Invasive Cancer; Neoplasms
7.  DNA Methylation and Expression Patterns of Key Tissue-specific Genes in Adult Stem Cells and Stomach Tissues 
Journal of Korean Medical Science  2009;24(5):918-929.
CpG-island margins and non-island-CpG sites round the transcription start sites of CpG-island-positive and -negative genes are methylated to various degrees in a tissue-specific manner. These methylation-variable CpG sites were analyzed to delineate a relationship between the methylation and transcription of the tissue-specific genes. The level of tissue-specific transcription was estimated by counting the number of the total transcripts in the SAGE (serial analysis of gene expression) database. The methylation status of 12 CpG-island margins and 21 non-island CpG sites near the key tissue-specific genes was examined in pluripotent stromal cells obtained from fat and bone marrow samples as well as in lineage-committed cells from marrow bulk, stomach, colon, breast, and thyroid samples. Of the 33 CpG sites examined, 10 non-island-CpG sites, but none of the CpG-island margins were undermethylated concurrent with tissue-specific expression of their nearby genes. The net methylation of the 33 CpG sites and the net amount of non-island-CpG gene transcripts were high in stomach tissues and low in stromal cells. The present findings suggest that the methylation of the non-island-CpG sites is inversely associated with the expression of the nearby genes, and the concert effect of transitional-CpG methylation is linearly associated with the stomach-specific genes lacking CpG-islands.
PMCID: PMC2752778  PMID: 19794993
DNA Methylation; Stem Cells; Stomach; Tissue-Specific Gene; CpG Islands
8.  A Network of Hydrophobic Residues Impeding Helix αC Rotation Maintains Latency of Kinase Gcn2, Which Phosphorylates the α Subunit of Translation Initiation Factor 2▿  
Molecular and Cellular Biology  2008;29(6):1592-1607.
Kinase Gcn2 is activated by amino acid starvation and downregulates translation initiation by phosphorylating the α subunit of translation initiation factor 2 (eIF2α). The Gcn2 kinase domain (KD) is inert and must be activated by tRNA binding to the adjacent regulatory domain. Previous work indicated that Saccharomyces cerevisiae Gcn2 latency results from inflexibility of the hinge connecting the N and C lobes and a partially obstructed ATP-binding site in the KD. Here, we provide strong evidence that a network of hydrophobic interactions centered on Leu-856 also promotes latency by constraining helix αC rotation in the KD in a manner relieved during amino acid starvation by tRNA binding and autophosphorylation of Thr-882 in the activation loop. Thus, we show that mutationally disrupting the hydrophobic network in various ways constitutively activates eIF2α phosphorylation in vivo and bypasses the requirement for a key tRNA binding motif (m2) and Thr-882 in Gcn2. In particular, replacing Leu-856 with any nonhydrophobic residue activates Gcn2, while substitutions with various hydrophobic residues maintain kinase latency. We further provide strong evidence that parallel, back-to-back dimerization of the KD is a step on the Gcn2 activation pathway promoted by tRNA binding and autophosphorylation. Remarkably, mutations that disrupt the L856 hydrophobic network or enhance hinge flexibility eliminate the need for the conserved salt bridge at the parallel dimer interface, implying that KD dimerization facilitates the reorientation of αC and remodeling of the active site for enhanced ATP binding and catalysis. We propose that hinge remodeling, parallel dimerization, and reorientation of αC are mutually reinforcing conformational transitions stimulated by tRNA binding and secured by the ensuing autophosphorylation of T882 for stable kinase activation.
PMCID: PMC2648240  PMID: 19114556
9.  The 5'-end transitional CpGs between the CpG islands and retroelements are hypomethylated in association with loss of heterozygosity in gastric cancers 
BMC Cancer  2006;6:180.
A loss of heterozygosity (LOH) represents a unilateral chromosomal loss that reduces the dose of highly repetitive Alu, L1, and LTR retroelements. The aim of this study was to determine if the LOH events can affect the spread of retroelement methylation in the 5'-end transitional area between the CpG islands and their nearest retroelements.
The 5'-transitional area of all human genes (22,297) was measured according to the nearest retroelements to the transcription start sites. For 50 gastric cancer specimens, the level of LOH events on eight cancer-associated chromosomes was estimated using the microsatellite markers, and the 5'-transitional CpGs of 20 selected genes were examined by methylation analysis using the bisulfite-modified DNA.
The extent of the transitional area was significantly shorter with the nearest Alu elements than with the nearest L1 and LTR elements, as well as in the extragenic regions containing a higher density of retroelements than in the intragenic regions. The CpG islands neighbouring a high density of Alu elements were consistently hypomethylated in both normal and tumor tissues. The 5'-transitional methylated CpG sites bordered by a low density of Alu elements or the L1 and LTR elements were hypomethylated more frequently in the high-level LOH cases than in the low-level LOH cases.
The 5'-transitional methylated CpG sites not completely protected by the Alu elements were hypomethylated in association with LOH events in gastric cancers. This suggests that an irreversible unbalanced decrease in the genomic dose reduces the spread of L1 methylation in the 5'-end regions of genes.
PMCID: PMC1552088  PMID: 16827945

Results 1-9 (9)