Acute carbon monoxide poisoning: MR imaging findings with clinical correlation.

PURPOSE: To assess the magnetic resonance imaging (MRI) findings, including diffusion-weighted imaging (DWI) in patients with acute carbon monoxide (CO) poisoning and correlate MRI findings with carboxyhemoglobin levels. MATERIALS AND METHODS: The MRI examinations and medical records of seven men with a mean age of 43±16.0years (SD) (range: 25-63 years) with acute CO poisoning were reviewed. MRI examinations were analyzed with respect to lesion location, imaging presentation on T1- and T2-weighted images, and diffusion characteristics on DWI and apparent diffusion coefficient (ADC) maps. We also evaluated clinical features and laboratory findings including the presenting symptoms and signs, carboxyhemoglobin level, and treatment. RESULTS: All seven patients presented with mental status change. The level of carboxyhemoglobin ranged between 8.3% and 34.8% (normal<1.5%). All seven patients (7/7, 100%) showed restricted diffusion of the lesions on ADC maps and bilateral involvement of globus pallidus. The mean ratios of ADC values was 0.63±0.15 (SD) (range: 0.46-0.92) on bilateral globi pallidi. Cerebral cortex, cerebral white matter, cerebellum, hippocampus, amygdala, splenium of corpus callosum, midbrain and insula were also involved. CONCLUSION: Bilateral globi pallidi with restricted diffusion may be a characteristic MRI feature in patients with acute CO poisoning. However, the relationship was not certain between the carboxyhemoglobin levels and the variety or severity of MRI findings.

Associations between Cerebral Embolism and Carotid Intraplaque Hemorrhage during Protected Carotid Artery Stenting.

BACKGROUND AND PURPOSE: Carotid artery stent placement in patients with intraplaque hemorrhage remains controversial because of the incidence of cerebral embolism after the procedure. The purpose of this study is to determine if intraplaque hemorrhage is a significant risk factor for cerebral embolism during carotid artery stent placement. MATERIALS AND METHODS: This prospective study assessed 94 consecutive patients with severe carotid stenosis. These patients underwent preprocedural carotid MR imaging and postprocedural DWI after carotid artery stent placement. Intraplaque hemorrhage was defined as the presence of high signal intensity within the carotid plaque that was >200% of the signal from the adjacent muscle on MPRAGE. We then analyzed the incidence of postprocedural ipsilateral ischemic events on DWI and primary outcomes within 30 days of carotid artery stent placement. RESULTS: Forty-three patients (45.7%) had intraplaque hemorrhage on an MPRAGE image. There was no significant difference in the incidence of postprocedural ipsilateral ischemic events and primary outcomes between the intraplaque hemorrhage and non-intraplaque hemorrhage group. However, postprocedural ipsilateral ischemic events were more frequently observed in the symptomatic group (17/41 [41.5%]) than in the asymptomatic group (8/53 [15.1%]; P = .005). CONCLUSIONS: Intraplaque hemorrhage was not a significant risk factor for cerebral embolism during carotid artery stent placement in patients with severe carotid stenosis. Symptomatic patients should receive more careful treatment during carotid artery stent placement because of the higher risk of postprocedural ipsilateral ischemic events.

SUMOylation of nonstructural 5A protein regulates hepatitis C virus replication.

Viruses exploit cellular SUMOylation machinery to favour their own propagation. We show that NS5A is a target protein of small ubiquitin-like modifier (SUMO) and is SUMOylated at lysine residue 348. We demonstrated that SUMOylation increased protein stability of NS5A by inhibiting ubiquitylation, and SUMOylation was also required for protein interaction with NS5B. These data imply that SUMO modification may contribute to HCV replication. Indeed, silencing of UBC9 impaired HCV replication in Jc1-infected cells, and HCV replication level was also significantly reduced in SUMO-defective subgenomic replicon cells. Taken together, these data indicate that HCV replication is regulated by SUMO modification of NS5A protein. We provide evidence for the first time that HCV exploits host cellular SUMO modification system to favour its own replication.

Predictors of functional outcome after emergency carotid artery stenting and intra-arterial thrombolysis for treatment of acute stroke associated with obstruction of the proximal internal carotid artery and tandem downstream occlusion.

BACKGROUND AND PURPOSE: Patients who develop severe stroke symptoms due to acute internal carotid artery occlusion eventually in combination with a thromboembolic obstruction of the middle cerebral artery incur a major risk of developing extensive MCA infarction with a poor outcome. The purpose of this study was to evaluate the outcome for patients with tandem occlusions in the MCA and/or distal ICA, retrospectively, who had undergone stent implantation in the proximal segment of the ICA in addition to intra-arterial thrombolysis. MATERIALS AND METHODS: Thirty-five patients with tandem occlusions of the MCA and/or distal ICA and acute occlusion of the proximal ICA underwent stent implantation for the proximal ICA occlusion and IAT for the tandem occlusion. Clinical outcome measures were assessed on admission and at discharge by using the National Institutes of Health Stroke Scale as well as 3 months after treatment by using the modified Rankin Scale. RESULTS: The median NIHSS score on admission was 12 (range, 6-22). All patients had patent flow into the M1 and ICA after carotid artery stent placement and IAT. After the procedure, 19 patients (54.3%) were TICI grade III; 7 (20.0%), TICI grade IIb; and 9 (25.7%), TICI grade IIa. Symptomatic intracerebral hemorrhage occurred in 1 patient (2.9%). The overall mortality rate was 11.4% (4/35). At 3-month follow-up, the median NIHSS score was 4 (range, 1-17). NIHSS score at admission and TICI grade were all found to be independently associated with an unfavorable outcome at 3 months. CONCLUSIONS: Initial stroke severity, degree of successful revascularization, and the side of ischemia were found to independently predict the functional outcome at 3 months after treatment.

Nonstructural 5A protein of hepatitis C virus regulates heat shock protein 72 for its own propagation.

We identified heat shock protein 72 (Hsp72) as a host factor that was differentially expressed in cells expressing nonstructural 5A (NS5A) protein. To investigate how NS5A modulates Hsp72 in hepatitis C virus (HCV) life cycle, we examined the role of Hsp72 in HCV replication and virus production. NS5A specifically interacted with Hsp72. Both Hsp72 and nuclear factor of activated T cells 5 (NFAT5) levels were increased in cells expressing NS5A protein. Treatments of N-acetylcysteine and glutathione markedly reduced protein levels of both NFAT5 and Hsp72. Knockdown of NFAT5 resulted in decrease in Hsp72 level in cells expressing NS5A. Importantly, silencing of Hsp72 expression resulted in decrease in both RNA replication and virus production in HCV-infected cells. These data indicate that NS5A modulates Hsp72 via NFAT5 and reactive oxygen species activation for HCV propagation.

Balloon-expandable stent placement in patients with immediate reocclusion after initial successful thrombolysis of acute middle cerebral arterial obstruction.

We present the results of our approach for treating 12 consecutive cases of acute middle cerebral artery (MCA) stroke by performing balloon-expandable stent (BES) placement after immediate reocclusion due to the underlying stenosis after intra-arterial thrombolysis (IAT). We retrospectively reviewed the clinical outcomes of 12 patients with acute MCA stroke who underwent recanalization by BES placement in an underlying stenosis after IAT. The time to treatment, urokinase dose, duration of the procedure, recanalization rates and symptomatic hemorrhage were analyzed. Clinical outcome measures were assessed on admission and at discharge (the National Institutes of Health stroke scores [NIHSS]) as well as three months after treatment (modified Rankin scales [mRS]). The median NIHSS score on admission was 8.6. Four patients received IV rtPA. The median time from symptom onset to IAT was 236 minutes and the median duration of IAT was 62 minutes. The median dose of urokinase was 140,000 units. Initial recanalization after stent deployment (thrombolysis in cerebral ischemia attack grade of II or III) was achieved in all patients. Two patients died in the hospital due to aspiration pneumonia during medical management. In two patients, in-stent reocclusion occurred within 48 hours after stent deployment. At discharge, the median NIHSS score in ten patients (including the patients with reobstruction) was 2.4. The three-month outcome was excellent (mRS, 0-1) in eight patients. In this study, BES deployment was safe and effective in patients with an immediately reoccluded MCA after successful IAT.

Endovascular management in patients with acute basilar artery obstruction: low-dose intra-arterial urokinase and mechanical clot disruption.

Mechanical clot disruption for the treatment of acute basilar artery occlusion (BAO) is known to provide a benefit. We aimed to determine the safety, recanalization rate and time-to-flow restoration of mechanical clot disruption and low dose urokinase (UK) infusions for the treatment of patients with acute BAO. Between June 2006 and June 2010, 21 patients with acute BAO underwent endovascular treatment that included angioplasty or stent placement. The time to treatment, duration of the procedure, dose of urokinase (UK), recanalization rates and symptomatic hemorrhages were analyzed. Clinical outcome measures were assessed at admission and at the time of discharge using the National Institutes of Health Stroke Scale (NIHSS) score and at three months after treatment using the modified Rankin Score (mRS). On admission, the median NIHSS score was 13.2. Median time from symptom onset to arrival at hospital was 356 minutes, and median time from symptom onset to intraarterial thrombolysis (IAT) was 49 minutes. We used the following interventional treatment regimens: Intra-arterial (IA) UK and a minimal mechanical procedure (n=14), IA UK with angioplasty (n=1), IA UK with angioplasty and stent placement (n=3) and IA UK with HyperForm (n=3). The recanalization (thrombolysis in cerebral ischemia grade II or III) rate was 90.5% (19/21). There was symptomatic hemorrhage in one patient (4.8%). The median NIHSS score at discharge was 6.3. The three-month outcome was favorable (mRS: 0-2) for 14 patients (66.7%) and poor (mRS: 3-6) for seven patients (33.3%). The overall mortality at three months was 14.3% (three patients died). Low-dose IAT with mechanical clot disruption is a safe and effective treatment for treatment for acute BAO.

Diagnostic efficacy of gadoxetic acid-enhanced MRI in the detection of hepatocellular carcinomas: comparison with gadopentetate dimeglumine.

This study compared the efficacy of gadoxetic acid-enhanced MRI and gadopentetate dimeglumine-enhanced MRI in the detection of small hepatocellular carcinoma (HCC). Both MRI techniques were performed on 43 patients with a total of 59 HCCs (size range, 0.5-2.0 cm), with a mean interval between the two MRI studies of 3 days (range, 2-7 days). Two observers reviewed both data sets in consensus. Diagnostic accuracy and sensitivity were evaluated using the alternative-free response receiver operator characteristic (ROC) method. The gadoxetic acid set showed a trend toward increased area under the ROC curve (Az value = 0.958) compared with the gadopentetate dimeglumine set (Az value = 0.927), but the difference was not significant (p = 0.362). The sensitivity of the gadoxetic acid set (n = 51, 86.4%) was significantly higher than that of the gadopentetate dimeglumine set (n = 38, 64.4%) (p = 0.0001). Gadoxetic acid-enhanced MRI is a more sensitive diagnostic tool for detection of HCC than gadopentetate dimeglumine-enhanced MRI.

Comparison of gadoxetic acid-enhanced MRI and superparamagnetic iron oxide-enhanced MRI for the detection of hepatocellular carcinoma.

AIM: To compare the diagnostic accuracy and sensitivity of gadoxetic acid-enhanced MRI and superparamagnetic iron oxide (SPIO)-enhanced magnetic resonance imaging (MRI) for the detection of hepatocellular carcinoma (HCC). MATERIALS AND METHODS: Eighty-nine patients (118 HCCs) who underwent three-dimensional gadoxetic acid-enhanced MRI and SPIO-enhanced MRI with a mean interval of 4.7 days (range 3-7 days), were included in this study. Two observers reviewed the gadoxetic acid set (unenhanced, early dynamic, 10 and 20 min hepatocyte-phase images) and SPIO set [unenhanced and ferucarbotran-enhanced T1-, T2-turbo spin-echo (TSE), and T2* weighted imaging (WI)] in consensus. Diagnostic accuracy and sensitivity were evaluated using the alternative-free response receiver operator characteristic (ROC) method. RESULTS: The area under ROC curve (Az value) and sensitivity of the gadoxetic acid set (Az 0.964; sensitivity 90.7%) were significantly higher than those of the SPIO set (Az 0.830; sensitivity 84.7%; p<0.05). There were 14 and seven lesions that were verified only on the gadoxetic acid set and only on the SPIO set, respectively. Four HCCs were clearly revealed as hypointense only on gadoxetic acid-enhanced hepatocyte phase imaging, but were occult on other sequences, including the SPIO set. CONCLUSION: Gadoxetic acid-enhanced MRI is better than SPIO-enhanced MRI for the detection of HCCs.

Monoclonal antibody recognizing N-terminal epitope of hepatitis C virus nonstructural 5B inhibits viral RNA replication.

The nonstructural 5B (NS5B) protein of hepatitis C virus (HCV) is an RNA-dependent RNA polymerase (RdRp) with a key role in HCV replication. To characterize the functional roles of NS5B in HCV replication, we produced a panel of 10 monoclonal antibodies (mAbs) directed against NS5B protein from mice immunized with functionally active RdRp. The epitopes of eight mAbs are localized in the middle region (amino acid 240-263) of NS5B protein. On the other hand, the epitopes of two mAbs are mapped to amino acids 67-88 at the N-terminus of NS5B protein. To examine the effects of mAbs on HCV-RNA replication, we performed in vitro RdRp assay using either the 3'-untranslated region (UTR) or the full-length of HCV-RNA as a template in the presence of each mAb. mAbs specific for the middle region of NS5B had no effect on RdRp activity. Surprisingly, mAb recognizing the N-terminal region of NS5B inhibited RdRp activity in a dose-dependent manner. We have confirmed the same result using the other subclass of mAb, whose epitope is also localized to the same N-terminal region of NS5B. These data show that NS5B contains a B-cell epitope located between amino acid residues 67 and 88. Binding of this epitope with an antibody interferes with the enzymatic function of NS5B.

Chondroprotective activity of N-acetylglucosamine in rabbits with experimental osteoarthritis.

OBJECTIVE: To examine the therapeutic efficacy of N-acetylglucosamine (GlcNAc) in rabbits with experimental osteoarthritis (OA). METHODS: Experimental OA was induced in rabbits by anterior cruciate ligament transection (ACLT). In the first study, rabbits (six in each group) received intramuscular injections of GlcNAc or normal saline three times a week starting 1 week postoperatively. In the second study, rabbits (eight in each group) were injected intra-articularly with GlcNAc (either once or twice a week) or normal saline. In the third study, rabbits (seven in each group) were injected intra-articularly twice a week with either GlcNAc, hyaluronan, or normal saline. Animals were killed 8 weeks after ACLT for macroscopic and histological assessment of the knee joints. RESULTS: Intramuscular administration of GlcNAc in rabbits with experimental knee OA did not show chondroprotective effects but showed mild anti-inflammatory activity. In contrast, intra-articular administration of GlcNAc twice a week reduced cartilage degradation. Additionally, intra-articular GlcNAc also suppressed synovitis. Once a week intra-articular injections of GlcNAc did not demonstrate therapeutic efficacy. The chondroprotective efficacy of GlcNAc was better than that of viscosupplementation treatment with hyaluronan. CONCLUSION: Intra-articular GlcNAc has chondroprotective and anti-inflammatory activity in experimental OA.

Anti-inflammatory activities of LDP-392, a dual PAF receptor antagonist and 5-lipoxygenase inhibitor.

Leukotrienes (LTs) and platelet-activating factor (PAF) are important mediators of inflammation and allergy. LDP-392, a novel dual PAF receptor antagonist and 5-lipoxygenase (5-LO) inhibitor, has been identified. LDP-392 is 17.9-fold more potent than zileuton (5-LO inhibitor) in the RBL cytosolic 5-LO assay, and equally potent as MK 287 (PAF receptor antagonist) in the human platelet PAF receptor binding assay. The in vivo dual activities of LDP-392 were confirmed by measuring the inhibition of ex vivo LTB(4)production in rats and PAF-induced hemoconcentration in mice. Intravenous administration of LDP-392 demonstrated greater inhibition than zileuton, BN 50739 or MK 287 on arachidonic acid-induced ear edema and protected mice from LPS-induced lethality. Topical administration of LDP-392, in a dose-dependent manner, inhibited TPA-induced ear edema in mice and UVB-induced erythema in guinea-pigs. These data suggest that LDP-392, as a dual PAF receptor antagonist and 5-LO inhibitor, may be of greater clinical effectiveness.

Hepatitis C virus core protein potentiates TNF-alpha-induced NF-kappaB activation through TRAF2-IKKbeta-dependent pathway.

Previous work has implicated that the core protein of hepatitis C virus (HCV) may play a modulatory effect on NF-kappaB activation induced by TNF-alpha. However, it is unclear how HCV core protein modulates TNF-alpha-induced NK-kappaB activation. Here we show that overexpression of HCV core protein potentiates NF-kappaB activation induced by TNF-alpha. Expression of dominant negative form of TRAF2 inhibits the synergistic effects of HCV core protein on NF-kappaB activation, suggesting that HCV core protein potentiates NF-kappaB activation through TRAF2. Moreover, we demonstrate that HCV core protein potentiates TRAF2-mediated NF-kappaB activation via IKKbeta. In addition, HCV core protein associates with TNF-R1-TRADD-TRAF2 signaling complex, resulting in synergistically activation of NF-kappaB induced by TNF-alpha. Thus, these observations indicate that HCV core protein may play an important role in the regulation of the cellular inflammatory and immune responses through NF-kappaB.

Hepatitis C virus core protein potentiates c-Jun N-terminal kinase activation through a signaling complex involving TRADD and TRAF2.

The hepatitis C virus (HCV) core protein is a multifunctional viral nucleocapsid protein. Previously, it has been demonstrated that the HCV core protein interacts with the cytoplasmic domain of tumor necrosis factor receptor 1 (TNFR1). Since the TNFR1 is engaged in stimulation of transcriptional factor NF-kappaB and AP-1 through activation of IkappaB kinase and c-Jun N-terminal kinase (JNK, or stress-activated protein kinase), respectively, we have examined whether the interaction between core protein and TNFR1 can modulate JNK. In this study, we demonstrate that the HCV core protein synergistically activates TNFalpha-induced JNK at a core concentration dependent manner in human embryonic kidney (HEK) 293 cells. HCV core-mediated synergism of JNK activation was also detected in stable cells expressing HCV core protein. Furthermore, we demonstrate that HCV core protein does not compete with TNF receptor-associated death domain (TRADD) for its interaction with the death domain of TNFR1. Our in vivo data show that HCV core and TRADD form a ternary complex with TNFR1. These findings suggest that the HCV core protein modulates TNFR1 signaling and may, thus, play a role in chronic infection of HCV patients.

Characterization and translational regulation of the arginine decarboxylase gene in carnation (Dianthus caryophyllus L.).

Arginine decarboxylase (ADC; EC 4.1.1.9) is a key enzyme in polyamine biosynthesis in plants. We characterized a carnation genomic clone, gDcADC8, in which the deduced polypeptide of ADC was 725 amino acids with a molecular mass of 77.7 kDa. The unusually long 5'-UTR that contained a short upstream open reading frame (uORF) of seven amino acids (MQKSLHI) was predicted to form an extensive secondary structure (free energy of approximately -117 kcal mol-1) using the Zuker m-fold algorithm. The result that an ADC antibody detected two bands of 45 and 33 kDa in a petal extract suggested the full length of the 78 kDa polypeptide precursor converted into two polypeptides in the processing reaction. To investigate the role of the transcript leader in translation, in vitro transcription/translation reactions with various constructs of deletion and mutation were performed using wheat germ extract. The ADC transcript leader affected positively downstream translation in both wheatgerm extract and primary transformant overexpressing ADC gene. It was demonstrated that heptapeptide (8.6 kDa) encoded by the ADC uORF was synthesized in vitro. Both uORF peptide, and the synthetic heptapeptide MQKSLHI of the uORF, repressed the translation of downstream ORF. Mutation of the uORF ATG codon alleviated the inhibitory effect. ORF translation was not affected by either a frame-shift mutation in uORF or a random peptide. To our knowledge, this is the first report to provide evidence that a uORF may inhibit the translation of a downstream ORF, not only in cis but also in trans, and that the leader sequence of the ADC gene is important for efficient translation.

Hepatitis C virus RNA polymerase and NS5A complex with a SNARE-like protein.

Hepatitis C virus (HCV) NS5A is a phosphoprotein that possesses a cryptic trans-activation activity. To investigate its potential role in viral replication, we searched for the cellular proteins interacting with NS5A protein by yeast two-hybrid screening of a human hepatocyte cDNA library. We identified a newly discovered soluble N-ethylmaleimide-sensitive factor attachment protein receptor-like protein termed human vesicle-associated membrane protein-associated protein of 33 kDa (hVAP-33). In vitro binding assay and in vivo coimmunoprecipitation studies confirmed the interaction between hVAP-33 and NS5A. Interestingly, hVAP-33 was also shown to interact with NS5B, the viral RNA-dependent RNA polymerase. NS5A and NS5B bind to different domains of hVAP-33: NS5A binds to the C-terminus, whereas NS5B binds to the N-terminus of hVAP-33. Immunofluorescent staining showed a significant colocalization of hVAP-33 with both NS5A and NS5B proteins. hVAP-33 contains a coiled-coil domain followed by a membrane-spanning domain at its C-terminus. Cell fractionation analysis revealed that hVAP-33 is predominantly associated with the ER, the Golgi complex, and the prelysosomal membrane, consistent with its potential role in intracellular membrane trafficking. These interactions provide a mechanism for membrane association of the HCV RNA replication complex and further suggest that NS5A is a part of the viral RNA replication complex.

Cell cycle arrest mediated by hepatitis delta antigen.

Hepatitis delta antigen (HDAg) is the only viral-encoded protein of the hepatitis delta virus (HDV). This protein has been extensively characterized with respect to its biochemical and functional properties. However, the molecular mechanism responsible for persistent HDV infection is not yet clear. Previously, we reported that overexpression of HDAg protects insect cells from baculovirus-induced cytolysis [Hwang, S.B. Park, K.-J. and Kim, Y.S. (1998) Biochem. Biophys. Res. Commun. 244, 652-658]. Here we report that HDAg mediates cell cycle arrest when overexpressed in recombinant baculovirus-infected insect cells. Flow cytometry analysis has shown that HDAg expression in Spodoptera frugiperda cells causes an accumulation of substantial amounts of polyploid DNA in the absence of cell division. This phenomenon may be partly responsible for the persistent infection of chronic HDV patients.

Characterization of mRNA for hepatitis delta antigen: exclusion of the full-length antigenomic RNA as an mRNA.

Hepatitis delta virus (HDV) encodes a single protein, the hepatitis delta antigen (HDAg), which is thought to be translated from a 0. 8-kb RNA of antigenomic sense. This subgenomic RNA species is present in very small amounts in HDV-infected liver tissues and in cultured cells infected or transfected with HDV, and in some cases it cannot be detected at all. In contrast, HDAg protein is present in large amounts in all natural and experimental models of HDV infection. This study addresses whether other HDV RNA species, such as the antigenomic-sense, genome-size HDV RNA can also serve as the mRNA for HDAg synthesis. Taking advantage of the ability of herpes simplex virus (HSV) to degrade only polyadenylated mRNAs, we examined the effect of HSV coinfection on HDAg synthesis. It was shown that HSV infection did degrade the subgenomic 0.8-kb HDV mRNA but not HDV genome-length RNA. Under such conditions, HDAg synthesis was completely inhibited. Furthermore, the genome-length HDV RNA was found not to be associated with polysomes. Finally, in vitro translation studies demonstrated that HDAg could not be translated directly from the genome-length antigenomic-sense HDV RNA. These results suggest that only the subgenomic RNA species of HDV possesses properties characteristic of the mRNA for HDAg and that the genome-length RNA cannot be used for translating HDAg. In addition, we found that HDV RNA replication did not depend on de novo HDAg synthesis.

TR4 orphan receptor crosstalks to chicken ovalbumin upstream protein-transcription factor and thyroid hormone receptor to induce the transcriptional activity of the human immunodeficiency virus type 1 long-terminal repeat.

Here we investigate the roles of human testicular orphan receptors, TR2 and TR4, on the gene regulation of the long-terminal repeat of the human immunodeficiency virus type 1 (HIV-LTR). In gel-retardation assays, a palindromic element at the 5'-end of HIV-LTR,5'-AGGGGTCAGATATCCACTGACCTTT-3',showed high affinity to TR2 and TR4 with an equilibrium dissociation constant (Kd) of 1.11 +/- 0.48 (n = 3) and 0.52 +/- 0.12 nM (n = 3), respectively. Interestingly, each half-site of the palindromic element is sufficient to compete with the binding of the labeled palindromic element to TR2 or TR4 with an equilibrium inhibition constant (ki) around 10 nM. However, the transiently expressed TR2 or TR4 in Chinese hamster ovary (CHO) cells or Japanese quail muscle myoblasts (QM7) cells showed no activity in regulating the transcriptional activity of the chloramphenicol acetyltransferase (CAT) reporter gene inserted downstream of the HIV-LTR promoter. Although both TR2 and TR4 showed no effect on CAT activity by itself, our data showed only the TR4 could crosstalk to the chicken ovalbumin upstream protein-transcription factor (COUP-TF1) and thyroid hormone receptor (TR alpha 1), and potentiated the transcriptional activity of HIV-LTR on the CAT reporter gene regulated by COUP-TF1 and TR alpha 1. These results indicate that TR4, but not TR2, may couple to other nuclear receptors in the upregulation of the HIV replication.

Hepatitis C virus core protein interacts with heterogeneous nuclear ribonucleoprotein K.

Hepatitis C virus (HCV) core protein, a component of viral nucleocapsid, has been shown to modulate cellular and viral promoter activities. To identify potential cellular targets for HCV core protein, a human liver cDNA library was screened for core-interacting proteins using the yeast two-hybrid system. Among the proteins identified was heterogeneous nuclear ribonucleoprotein K (hnRNP K), which has been demonstrated to be a transcriptional regulator. The interaction of HCV core protein with hnRNP K was confirmed by glutathione S-transferase fusion protein binding assay, protein-protein blotting assay, and coimmunoprecipitation in vitro and in vivo. Additionally, these two proteins were shown to be partially colocalized in the nucleus. The hnRNP K-binding site in HCV core protein was mapped to the region from amino acid residues 25-91, a hydrophilic area near the N terminus. The HCV core protein-binding domain was located within amino acid residues 250 to 392, which contain the three proline-rich domains, of hnRNP K. Furthermore, HCV core protein relieved the suppression effect of hnRNP K on the activity of the human thymidine kinase gene promoter. The specific binding of HCV core protein to hnRNP K suggests that multiple functions of hnRNP K may be disrupted by the core protein during HCV infection and thus explains, in part, the pathogenesis of HCV.