Diagnostic performance of perivascular fat attenuation index to predict hemodynamic significance of coronary stenosis: a preliminary coronary computed tomography angiography study.

OBJECTIVE: This study aimed to investigate the association between perivascular fat attenuation index (FAI) and hemodynamic significance of coronary lesions. METHODS: Patients with stable angina who underwent coronary computed tomography (CT) angiography and invasive fractional flow reserve (FFR) measurement within 2 weeks were retrospectively included. Lesion-based perivascular FAI, high-risk plaque features, total plaque volume (TPV), machine learning-based FFRCT, and other parameters were recorded. Lesions with invasive FFR ≤ 0.8 were considered functionally significant. RESULTS: This study included 167 patients with 219 lesions. Diameter stenosis (DS), lesion length, TPV, and perivascular FAI were significantly larger or longer in the group of hemodynamically significant lesions (FFR ≤ 0.8). In addition, smaller FFRCT value was associated with functionally significant lesions (0.720 ± 0.11 vs 0.846 ± 0.10, p < 0.001). No significant difference was found between the hemodynamically significant and insignificant subgroups with respect to CT-derived high-risk plaque features. According to multivariate analysis, DS, TPV, and perivascular FAI were significant predictors of lesion-specific ischemia. When integrating DS, TPV, and perivascular FAI, the area under the curve (AUC) of this combined method was 0.821, which was similar to that of FFRCT (AUC, 0.821 vs 0.850; p = 0.426). The diagnostic accuracy of FFRCT was higher than that of the combined approach, but the difference was statistically insignificant (79.0% vs 74.0%, p = 0.093). CONCLUSIONS: Perivascular FAI was significantly higher for flow-limiting lesions than for non-flow-limiting lesions. The combined use of FAI, TPV, and DS could predict ischemic coronary stenosis with high diagnostic accuracy. KEY POINTS: • Perivascular FAI was significantly higher for flow-limiting lesions than for non-flow-limiting lesions. • Combined use of FAI, plaque volume, and DS provided diagnostic performance comparable to that of machine learning-based FFR CTfor predicting ischemic coronary stenosis. • No significant difference was found between the hemodynamically significant and insignificant subgroups with respect to CT-derived high-risk plaque features.

NAD+-dependent deacetylase SIRT3 regulates mitochondrial protein synthesis by deacetylation of the ribosomal protein MRPL10.

A member of the sirtuin family of NAD(+)-dependent deacetylases, SIRT3, is located in mammalian mitochondria and is important for regulation of mitochondrial metabolism, cell survival, and longevity. In this study, MRPL10 (mitochondrial ribosomal protein L10) was identified as the major acetylated protein in the mitochondrial ribosome. Ribosome-associated SIRT3 was found to be responsible for deacetylation of MRPL10 in an NAD(+)-dependent manner. We mapped the acetylated Lys residues by tandem mass spectrometry and determined the role of these residues in acetylation of MRPL10 by site-directed mutagenesis. Furthermore, we observed that the increased acetylation of MRPL10 led to an increase in translational activity of mitochondrial ribosomes in Sirt3(-/-) mice. In a similar manner, ectopic expression and knockdown of SIRT3 in C2C12 cells resulted in the suppression and enhancement of mitochondrial protein synthesis, respectively. Our findings constitute the first evidence for the regulation of mitochondrial protein synthesis by the reversible acetylation of the mitochondrial ribosome and characterize MRPL10 as a novel substrate of the NAD(+)-dependent deacetylase, SIRT3.

Efficacy of Neuroendoscopic Treatment for Septated Chronic Subdural Hematoma.

Objective: Neuroendoscopic treatment is an alternative therapeutic strategy for the treatment of septate chronic subdural hematoma (sCSDH). However, the safety and efficacy of this strategy remain controversial. We compared the clinical outcomes of neuroendoscopic treatment with those of standard (large bone flap) craniotomy for sCSDH reported in our center. Furthermore, the safety and efficacy of the neuroendoscopic treatment procedure for sCSDH were evaluated. Methods: We retrospectively collected the clinical data of 43 patients (37 men and six women) with sCSDH who underwent either neuroendoscopic treatment or standard (large bone flap) craniotomy, such as sex, age, smoking, drinking, medical history, use of antiplatelet drugs, postoperative complications, sCSDH recurrence, length of hospital stay, and postoperative hospital stay. We recorded the surgical procedures and the neurological function recovery prior to surgery and 6 months following the surgical treatment. Results: The enrolled patients were categorized into neuroendoscopic treatment (n = 23) and standard (large bone flap) craniotomy (n = 20) groups. There were no differences in sex, age, smoking, drinking, medical history, antiplatelet drug use, postoperative complications, and sCSDH recurrence between the two groups (p > 0.05). However, the patients in neuroendoscopic treatment group had a shorter length of total hospital stay and postoperative hospital stay as compared with the standard craniotomy group (total hospital stay: 5.26 ± 1.89 vs. 8.15 ± 1.04 days, p < 0.001; postoperative hospital stay: 4.47 ± 1.95 vs. 7.96 ± 0.97 days, p < 0.001). The imaging and Modified Rankin Scale at the 6-month follow-up were satisfactory, and no sCSDH recurrence was reported in the two groups. Conclusions: The findings of this study indicate that neuroendoscopic treatment is safe and effective for sCSDH; it is minimally invasive and could be clinically utilized.

Perivascular fat attenuation index and high-risk plaque features evaluated by coronary CT angiography: relationship with serum inflammatory marker level.

The present study aimed to investigate the association between perivascular fat attenuation index (FAI), high-risk plaque features and serum level of high sensitive C-reactive protein (hs-CRP). Consecutive patients with intermediate pre-test probability of CAD, who were referred for coronary CT angiography (CCTA), were included. High-risk plaque features were assessed by CCTA and included low attenuation plaque (LAP), positive remodeling (PR), napkin-ring sign (NRS) and spotty calcification. Lesion specific perivascular FAI was also measured for all plaques located on major epicardial vessels with diameter ≧ 2 mm. Laboratory test results, including hs-CRP, were recorded. 199 patients with 260 lesions were finally included. NRS and LAP were more commonly present in the group with elevated hs-CRP (35.9% vs. 19.4% and 14.1% vs. 2.6%, both p < 0.05) and more severe stenosis extent was also noted for this group. However, there was no significant difference between the elevated hs-CRP and normal hs-CRP group with respect to other CT parameters, such as lesion length, PR, spotty calcification and focal calcium score. Perivascular FAI failed to show significant difference between the two groups (- 69.8 ± 10.3HU vs. - 70.0 ± 12.0HU, p = 0.953) and there was poor correlation between perivascular FAI and hs-CRP measurements (r = - 0.04, p = 0.510). In conclusion, LAP and NRS are more commonly present in CAD patients with elevated level of hs-CRP. However, perivascular FAI failed to show such correlation with serum level of hs-CRP.

Nuclear suppression of mitochondrial defects in cells without the ND6 subunit.

Previously, we characterized a mouse cell line, 4A, carrying a mitochondrial DNA mutation in the subunit for respiratory complex I, NADH dehydrogenase, in the ND6 gene. This mutation abolished the complex I assembly and disrupted the respiratory function of complex I. We now report here that a galactose-resistant clone, 4AR, was isolated from the cells carrying the ND6 mutation. 4AR still contained the homoplasmic mutation, and apparently there was no ND6 protein synthesis, whereas the assembly of other complex I subunits into complex I was recovered. Furthermore, the respiratory activity and mitochondrial membrane potential were fully recovered. To investigate the genetic origin of this compensation, the mitochondrial DNA (mtDNA) from 4AR was transferred to a new nuclear background. The transmitochondrial lines failed to grow in galactose medium. We further transferred mtDNA with a nonsense mutation at the ND5 gene to the 4AR nuclear background, and a suppression for mitochondrial deficiency was observed. Our results suggest that change(s) in the expression of a certain nucleus-encoded factor(s) can compensate for the absence of the ND6 or ND5 subunit.

38-year-old woman with nasal congestion: A rare case of septum-originated benign fibrous histiocytoma.

INTRODUCTION: Benign fibrous histiocytoma (BFH) is a mesenchymal tumor composed of fibroblasts and histiocytes, which normally occurs in the bone tissue and/or the skin of the limbs. BFH of the nasal septum is rare worldwide. PATIENT CONCERNS: A 38-year-old female patient attended our clinic with congestion in her right nasal cavity. DIAGNOSES: Endoscopic examination revealed a mass within the right nasal passage. Immunohistochemical staining demonstrated cells labeled with VIM (+), CD68PGM1 (+), CD34 (-), Actin (-), PCK (-), EMA (-), Desmin (-), SMA (-), S-100 (-), P63 (-), ALK-1 (-), beta-catenia (+), and KI67 (+). Clinical and histopathological examinations showed that the mass was a septum-originated BFH. INTERVENTIONS: For the treatment of BFH of the nasal septum, the mass was removed endoscopically using a transnasal approach. OUTCOMES: No recurrence was observed over 30 months of follow-up. CONCLUSION: To prevent the recurrence wide local resection with histological clear margin and long-term follow-up with an endoscope are necessary.

Specific point mutations may not accumulate with aging in the mouse mitochondrial DNA control region.

Increasing evidence suggests that mitochondrial function declines during aging in various tissues and in a wide range of organisms. This correlates with an age-dependent large accumulation of specific point mutations in the mtDNA control region that was reported recently in human fibroblast and skeletal muscle. However, evaluations of aging-related mtDNA mutations in other model animal systems. In this study, we analyzed mtDNA control regions of brain, skeletal muscle, heart, and other tissues from aged mice, in search of specific point mutations. A 948-bp fragment covering the entire mtDNA control region from various tissues of mice at the age of 25-26 months was sequenced. The sequence analysis was accomplished with a newly developed program Mutation Quantifier, which was able to accurately detect mutations with frequencies as low as 3%. Probably due to the relative shorter life-span, unlike what has been reported in human mtDNA, our results indicated there might be no significant accumulation of specific mutations in mouse mtDNA control region during aging.

Restoration of mitochondrial function in cells with complex I deficiency.

The mammalian mitochondrial NADH dehydrogenase (complex I) is the major entry point for the electron transport chain. It is the largest and most complicated respiratory complex consisting of at least 46 subunits, 7 of which are encoded by mitochondrial DNA (mtDNA). Deficiency in complex I function has been associated with various human diseases including neurodegenerative diseases and the aging process. To explore ways to restore mitochondrial function in complex I-deficient cells, various cell models with mutations in genes encoding subunits for complex I have been established. In this paper, we discuss various approaches to recover mitochondrial activity, the complex I activity in particular, in cultured cells.

Prediction of synchronous colorectal cancers by computed tomography in subjects receiving an incomplete colonoscopy: a single-center study.

AIM: To assess the value of computed tomography (CT) for diagnosis of synchronous colorectal cancers (SCRCs) involving incomplete colonoscopy. METHODS: A total of 2123 cases of colorectal cancer (CRC) were reviewed and divided into two groups according to whether a complete or incomplete colonoscopy was performed. CT results and final histological findings were compared to calculate the sensitivity and specificity associated with CT for detection of SCRCs following complete vs incomplete colonoscopy. Factors affecting the CT detection were also analyzed. RESULTS: Three hundred and seventy-four CRC patients underwent incomplete colonoscopy and 1749 received complete colonoscopy. Fifty-six cases of SCRCs were identified by CT, and 36 were missed. In the incomplete colonoscopy group, the sensitivity and specificity of CT were 44.8% and 93.6%, respectively. The positive and negative predictive values were 23.6% and 95.0%, respectively. In contrast, the sensitivity and specificity of CT for the complete colonoscopy group were 68.3% and 97.0%, while the positive and negative predictive values were 22.2% and 98.7%, respectively. In both groups, the mean maximum dimension of the concurrent cancers identified in the CT-negative cases was shorter than in the CT-positive cases (incomplete group: P = 0.02; complete group: P < 0.01) Topographical proximity to synchronous cancers was identified as a risk factor for missed diagnosis (P = 0.03). CONCLUSION: CT has limited sensitivity in detecting SCRCs in patients receiving incomplete colonoscopy. Patients with risk factors and negative CT results should be closely examined and monitored.

Genetic and functional analysis of mitochondrial DNA-encoded complex I genes.

Mammalian mitochondrial NADH dehydrogenase (complex I) is a multimeric complex consisting of at least 45 subunits, 7 of which are encoded by mitochondrial DNA (mtDNA). The function of these subunits is largely unknown. We have established an efficient method to isolate and characterize cells carrying mutations in various mtDNA-encoded complex I genes. With this method, 15 mouse cell lines with deficiencies in complex I-dependent respiration were obtained, and two near-homoplasmic mutations in mouse ND5 and ND6 genes were isolated. Furthermore, by generating a series of cell lines with the same nuclear background but different content of an mtDNA nonsense mutation, we analyzed the genetic and functional thresholds in mouse mitochondria. We found that in wild-type cells, about 40% of ND5 mRNA is in excess of that required to support a normal rate of ND5 subunit synthesis. However, there is no indication of compensatory upsurge in either transcription or translation with the increase in the proportion of mutant ND5 genes. Interestingly, the highest ND5 protein synthesis rate was just sufficient to support the maximum complex I-dependent respiration rate, suggesting a tight regulation at the translational level. In another line of research, we showed that the mitochondrial NADH-quinone oxidoreductase of Saccharomyces cerevisiae (NDI1), although consisting of a single subunit, can completely restore respiratory NADH dehydrogenase activity in mutant human cells that lack the essential mtDNA-encoded subunit ND4. In particular, in these transfected cells, the yeast enzyme becomes integrated into the human respiratory chain and fully restores the capacity of the cells to grow in galactose medium.

Inhibition of hedgehog and androgen receptor signaling pathways produced synergistic suppression of castration-resistant prostate cancer progression.

UNLABELLED: Metastatic prostate cancer is initially treated with androgen ablation therapy, which causes regression of androgen-dependent tumors. However, these tumors eventually relapse resulting in recurrent castration-resistant prostate cancer (CRPC). Currently, there is no effective therapy for CRPC and the molecular mechanisms that lead to the development of CRPC are not well understood. Here, we evaluated the hypothesis that combined inhibition of Hedgehog (Hh) and androgen receptor (AR) signaling will synergistically attenuate the growth of CRPC in vitro and in vivo. Androgen deprivation induced full-length androgen receptor protein levels in CRPC cells, but decreased its nuclear localization and transcriptional activity. However, androgen deprivation also increased a truncated form of androgen receptor (lacking ligand-binding domain) that possessed transcriptional activity in CRPC cells. Androgen deprivation also promoted the expression of Hh signaling components in CRPC cells, xenograft tumors, and the prostate glands of castrated mice. Importantly, although inhibition of either Hh or androgen receptor signaling alone was only moderately effective in blocking CRPC cell growth, combination of an Hh pathway inhibitor and a noncompetitive androgen receptor inhibitor synergistically suppressed the growth of CRPC cells in vitro and in vivo. Finally, noncompetitive inhibition of androgen receptor, but not competitive inhibition, was effective at limiting the activity of truncated androgen receptor leading to the inhibition of CRPC. IMPLICATIONS: Combined therapy using Hh inhibitors and a non-competitive AR inhibitor may limit CRPC growth.

An assembled complex IV maintains the stability and activity of complex I in mammalian mitochondria.

In the mammalian mitochondrial electron transfer system, the majority of electrons enter at complex I, go through complexes III and IV, and are finally delivered to oxygen. Previously we generated several mouse cell lines with suppressed expression of the nuclearly encoded subunit 4 of complex IV. This led to a loss of assembly of complex IV and its defective function. Interestingly, we found that the level of assembled complex I and its activity were also significantly reduced, whereas levels and activity of complex III were normal or up-regulated. The structural and functional dependence of complex I on complex IV was verified using a human cell line carrying a nonsense mutation in the mitochondrially encoded complex IV subunit 1 gene. Our work documents that, although there is no direct electron transfer between them, an assembled complex IV helps to maintain complex I in mammalian cells.

Cytochrome c oxidase subunit IV is essential for assembly and respiratory function of the enzyme complex.

Cytochrome c oxidase or complex IV, catalyzes the final step in mitochondrial electron transfer chain, and is regarded as one of the major regulation sites for oxidative phosphorylation. This enzyme is controlled by both nuclear and mitochondrial genomes. Among its 13 subunits, three are encoded by mitochondrial DNA and ten by nuclear DNA. In this work, an RNA interference approach was taken which led to the generation of mouse A9 cell derivatives with suppressed expression of nuclear-encoded subunit IV (COX IV) of this complex. The amounts of this subunit are decrease by 86% to 94% of normal level. A detail biosynthetic and functional analysis of several cell lines with suppressed COX IV expression revealed a loss of assembly of cytochrome c oxidase complex and, correspondingly, a reduction in cytochrome c oxidase-dependent respiration and total respiration. Furthermore, dysfunctional cytochrome c oxidase in the cells leads to a compromised mitochondrial membrane potential, a decreased ATP level, and failure to grow in galactose medium. Interestingly, suppression of COX IV expression also sensitizes the cells to apoptosis. These observations provide the evidence of the essential role of the COX IV subunit for a functional cytochrome c oxidase complex and also demonstrate a tight control of cytochrome c oxidase over oxidative phosphorylation. Finally, our results further shed some insights into the pathogenic mechanism of the diseases caused by dysfunctional cytochrome c oxidase complex.

Maternally inherited nonsyndromic hearing loss is associated with the T7511C mutation in the mitochondrial tRNASerUCN gene in a Japanese family.

We report here the characterization of a Japanese family with maternally transmitted nonsyndromic hearing loss. Fourteen of 21 matrilineal relatives in this family exhibited early or late-onset/progressive but noncongenital hearing impairment with a wide range of severity, ranging from severe to normal hearing. The age-of-onset varies from 3 to 30 years. Sequence analysis of the complete mitochondrial genome in one matrilineal relative of this family revealed the presence of T7511C mutation and other variants. However, the levels of heteroplasmy of T7511C mutation did not correlate with the severity and age-of-onset of hearing loss in this family. Furthermore, none of other mtDNA variants are evolutionarily conserved and implicated to have significantly functional consequence. The absence of the ND1 T3308C and tRNA(Ala) T5655C mutations in this Japanese family but the presence of these mtDNA mutations in an African family with a high penetrance seems to account for different penetrance between two pedigrees. Incomplete penetrance in this family indicates the involvement of modulatory factors in the phenotypic expression of hearing impairment associated with the T7511C mutation. Here, two known variants G79A and G109A in the GJB2 gene were identified in the hearing-impaired and normal hearing matrilineal relatives of this Japanese family. However, the lack of correlation in the severity and age-of-onset in hearing impairment with homozygous or heterozygous G79A or G109A or combination of both variants in the GJB2 gene in those subjects with hearing impairment and normal hearing indicates that those variants of GJB2 gene may not be a modifier of the phenotypic effects of the T7511C mutation in those subjects. Thus, the phenotypic variability in this family is due to the involvement of other modifier factor(s).

Productive lytic replication of a recombinant Kaposi's sarcoma-associated herpesvirus in efficient primary infection of primary human endothelial cells.

Kaposi's sarcoma-associated herpesvirus (KSHV) is linked to the development of Kaposi's sarcoma (KS), a vascular spindle cell tumor primarily consisting of proliferating endothelial cells. Although KSHV has been shown to infect primary human endothelial cells and convert them into spindle shapes, KSHV infection is largely latent, and efforts to establish a highly efficient and sustainable infection system have been unsuccessful. A recombinant KSHV, BAC36, that has high primary-infection efficiency in 293 cells has been obtained (F. C. Zhou, Y. J. Zhang, J. H. Deng, X. P. Wang, H. Y. Pan, E. Hettler, and S. J. Gao, J. Virol. 76:6185-6196, 2002). BAC36 contains a green fluorescent protein cassette which can be used to conveniently monitor viral infection. Here, we describe the establishment of a KSHV lytic-replication-permissive infection cell model using BAC36 virions to infect primary human umbilical vein endothelial cell (HUVEC) cultures. BAC36 infection of HUVEC cultures has as high as 90% primary-infection efficiency and consists of two phases: a permissive phase, in which the cultures undergo active viral lytic replication, producing a large number of virions and concomitantly resulting in large-scale cell death, and a latent phase, in which the surviving cells from the permissive phase switch into latent infection, with a small number of cells undergoing spontaneous viral lytic replication, and proliferate into bundles of spindle cells with KS slit-like spaces. An assay for determining the KSHV titer in a virus preparation has also been developed. The cell model should be useful for examining KSHV infection and replication, as well as for understanding the development of KS.

Lytic replication-defective Kaposi's sarcoma-associated herpesvirus: potential role in infection and malignant transformation.

Defective viruses often have pivotal roles in virus-induced diseases. Although Kaposi's sarcoma-associated herpesvirus (KSHV) is etiologically associated with Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL), defective KSHV has not been reported. Using differential genetic screening methods, we show that defective KSHV is present in KS tumors and PEL cell lines. To investigate the role of defective viruses in KSHV-induced pathogenesis, we isolated and characterized a lytic replication-defective KSHV, KV-1, containing an 82-kb genomic deletion of solely lytic genes. Cells harboring KV-1 escaped G(0)/G(1) apoptosis induced by spontaneous lytic replication occurred in cells infected with regular KSHV but maintained efficient latent replication. Consequently, KV-1-infected cells had phenotypes of enhanced cell proliferation and transformation potentials. Importantly, KV-1 was packaged as infectious virions by using regular KSHV as helpers, and KV-1-like variants were detected in cultures of two of five KSHV cell lines and 1 of 18 KS tumors. These results point to a potential role for defective viruses in the regulation of KSHV infection and malignant transformation.

Hot-spot variations of Kaposi's sarcoma-associated herpesvirus latent nuclear antigen and application in genotyping by PCR-RFLP.

Kaposi's sarcoma-associated herpesvirus (KSHV, human herpesvirus-8) is aetiologically associated with Kaposi's sarcoma and several other lymphoproliferative disorders. The latent nuclear antigen (LNA) encoded by KSHV ORF73 has important functions in virus latent infection and shows molecular polymorphism. Sequence variations were identified in the internal repeat domain (IRD) of ORF73. DNA sequencing of ORF73 from one KSHV-infected cell line, PK-1, revealed that there were 558 bp (30.2%) deletions and 66 (3.6%) point mutations located mainly in repeat region 2, the glutamine-rich region of ORF73 IRD, compared with ORF73 of BC-1 KSHV. Similar sequence variations of ORF73 were also identified in two other isolates. None of the sequence variations caused any translational frame-shift in these four KSHV isolates examined, suggesting that LNA has a conservative function in virus latent infection. The frequent sequence variations in repeat region 2 of ORF73 IRD were also identified by PCR-RFLP genotyping in 26 KSHV isolates, suggesting that this region is a 'hot-spot' for genetic variations. Each Kaposi's sarcoma lesion sample contained one virus genotype with a unique RFLP pattern, indicating that in vivo KSHV infection was established with single predominate genotypes, which was further supported by the presence of invariable genotypes in multifocal lesions from individual KS patients. Four KSHV subtypes were classified based on the RFLP patterns that represent the patterns of DNA sequence variations in the ORF73 IRD. PCR-RFLP genotyping is capable of identifying LNA genetic variations and differentiating individual KSHV isolates, and thus may be useful for KSHV molecular epidemiology studies.

High prevalence of human herpesvirus 8 (HHV-8) infection in south Texas children.

Human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus, is etiologically associated with Kaposi's sarcoma and other rare malignancies. HHV-8 infection is common in certain areas of Africa and Italy, but occurs in only 0-15% of populations in North America and Europe. The epidemiology and prevalence of HHV-8 infection among children in the United States has not been determined, but is assumed to be low based on limited studies. The objective of this study was to determine the seroprevalence and possible risk factors of HHV-8 infection in children living in south Texas. Questionnaire data were collected and HHV-8 serologic tests were performed from a consecutive, non-probability sample of 123 healthy children (ages 4-13 years) attending general pediatric clinics in south Texas. Serum was tested for HHV-8 antibodies by latent immunofluorescence assay and ORF65 enzyme-linked immunosorbent assay confirmed by immunoblot. HHV-8 prevalence and 95 percent confidence intervals were calculated using standard epidemiologic methods. Logistic regression was used to assess independent risk factors associated with HHV-8 seropositivity. The overall prevalence of HHV-8 infection was 26%. No statistically significant associations were exhibited between HHV-8 prevalence and the variables under study. The prevalence of HHV-8 infection among children in south Texas, particularly among those under the age of 12 years, indicates that non-sexual transmission of this virus is likely to occur among this population. Future investigations of larger study samples will be necessary to develop an understanding of specific routes and risk factors of HHV-8 transmission among children in south Texas.

Identification of a novel cellular transcriptional repressor interacting with the latent nuclear antigen of Kaposi's sarcoma-associated herpesvirus.

The latent nuclear antigen (LNA) of Kaposi's sarcoma-associated herpesvirus (KSHV) has an essential role in viral latent infection. LNA maintains the stability of KSHV episomes and modulates the expression of cellular genes. A novel cellular protein KLIP1 was identified to interact with LNA through yeast two-hybrid screening, and confirmed by a glutathione S-transferase pull down assay. Domain mapping showed that KLIP1 interacted with the N-terminal domain of LNA. Northern blot hybridization with a KLIP1 probe identified a major transcript of 1.8 kb and a minor transcript of 2.8 kb. cDNA library screening and 5'-RACE revealed that the major transcript encoded an open-reading-frame of 1,257 bp and had a 5'-untranslated region of 73 nucleotides. The major KLIP1 transcript was ubiquitously present in different cell types examined. A KLIP1 synthetic peptide antibody detected a doublet of 58-kDa and 63-kDa proteins in a Western blot assay. KLIP1 had two putative nuclear localization signals and showed punctate nuclear localization when expressed as a GFP-fusion protein. KLIP1 interacted with LNA in vivo, as demonstrated by coimmunoprecipitation using KSHV-infected cells and colocalization when they were expressed as GFP- and DsRed-fusion proteins, respectively. Consistent with its interaction with LNA, nuclear localization, and possession of two leucine zipper motifs, KLIP1 behaved like a transcriptional factor and repressed herpes simplex virus thymidine kinase (TK) promoter activity in a mammalian one-hybrid assay. In addition, cotransfection with LNA alleviated the transcriptional repression effect of KLIP1 on TK promoter activity. These results suggest that KLIP1 is a new member of cellular transcriptional repressors, and that LNA is involved in deregulating cellular transcription process.

Transcriptional regulation of Kaposi's sarcoma-associated herpesvirus-encoded oncogene viral interferon regulatory factor by a novel transcriptional silencer, Tis.

Viral interferon regulatory factor (vIRF) encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) has been shown to transform NIH3T3 and Rat-1 cells, inhibit interferon signal transduction, and regulate the expression of KSHV genes. We had previously characterized the vIRF core promoter and defined a 12-O-tetradecanoylphorbol-13-acetate (TPA)-responsive region in the upstream regulatory sequence of vIRF gene. Here, we have further identified a novel transcriptional silencer, named Tis in this region. Tis represses the promoter activities of vIRF and heterologous herpes simplex virus thymidine kinase genes in both position- and orientation-independent manners. Deletion analysis has identified a cis-element of 23 nucleotides that is essential for the negative regulation. Two Tis-binding protein complexes, named vR1 and vR2, were observed by electrophoretic mobility shift assays using nuclear extracts from both KSHV-negative and -positive cell lines. A sequence fragment GAGTTAATAGGTAGAG in the cis-element was shown to be required for the DNA-protein interactions as well as the repression of vIRF promoter activity. Point-mutation analysis identified TTAAT and GTTAATAG as the core sequence motifs for the binding of vR1 and vR2, respectively. These results define the function of a novel transcriptional silencer in the regulation of vIRF gene expression.