Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Hepatitis B virus (HBV) replicates by reverse transcription of an RNA intermediate, the pregenomic RNA. The first step of HBV genome replication is the encapsidation of the pregenomic RNA encoding the encapsidation signal, termed epsilon, into the core particles, which is preceded by recognition and binding of HBV DNA polymerase to epsilon. The pregenomic RNA contains two identical epsilon elements due to its terminal redundancy: one near the 5' end and another near the 3' end. Despite the fact that both epsilon elements have an identical sequence, only the 5' epsilon, but not the 3' epsilon, is functional for encapsidation. To understand the molecular nature of this position effect, we made a series of lacZ RNA expression plasmids which contain the epsilon element at various positions from the 5' end of the transcripts. Following transfection, the lacZ RNAs in cytoplasmic core particles were measured by RNase protection assay for encapsidation. The results indicated that the lacZ RNAs with epsilon positioned up to 65 nucleotides from the 5' end were encapsidated, whereas the lacZ RNAs with epsilon positioned further downstream were not. Interestingly, the cap-free lacZ RNA transcribed by T7 RNA polymerase was not encapsidated, implying that the 5' cap structure is required for encapsidation of the pregenomic RNA. We hypothesized that HBV DNA polymerase must somehow recognize the cap structure and/or its associated factors, as well as the 5' epsilon, for encapsidation to occur.
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PMID:Evidence that the 5'-end cap structure is essential for encapsidation of hepatitis B virus pregenomic RNA. 1082 55

Our previous studies have demonstrated that the level of the central transcription factor TATA-binding protein (TBP) is increased in cells expressing the hepatitis B virus (HBV) X protein through the activation of the Ras signaling pathway, which serves to enhance both RNA polymerase I and III promoter activities. To understand the mechanism by which TBP is regulated, we have investigated whether enhanced expression is modulated at the transcriptional level. Nuclear run-on assays revealed that the HBV X protein increases the number of active transcription complexes on the TBP gene. In transient-transfection assays with both transformed and primary hepatocytes, the human TBP promoter was shown to be induced by expression of the HBV X protein in a Ras-dependent manner, requiring both Ral guanine nucleotide dissociation stimulator (RalGDS) and Raf signaling. Transient overexpression of TBP did not affect TBP promoter activity. To further delineate the downstream Ras-mediated events contributing to TBP promoter regulation in primary rat hepatocytes, the best-characterized Ras effectors, Raf, phosphoinositide 3-kinase (PI-3 kinase), and RalGDS, were examined. Activation of either Raf or RalGDS, but not that of PI-3 kinase, was sufficient to induce TBP promoter activity. Both Raf- and RalGDS-mediated induction required the activation of mitogen-activated protein kinase kinase (MEK). In addition, another distinct Ras-activated pathway, which does not require MEK activation, appears to induce TBP promoter activity. Analysis of the DNA sequence requirement within the TBP promoter responsible for these regulatory events defined three distinct regions that modulate the abilities of Raf, RalGDS, and the Ras-dependent, MEK-independent pathways to regulate human TBP promoter activity. Together, these results provide new evidence that TBP can be regulated at the transcriptional level and identify three distinct Ras-activated pathways that modulate this central eukaryotic transcription factor.
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PMID:Transcriptional regulation of the TATA-binding protein by Ras cellular signaling. 1086 57

The preS domains of the hepatitis B virus are hydrophilic polypeptides that have been implicated, among other functions, in the binding of the virus to hepatocytes and in the induction of virus-neutralizing antibodies. A method of overproducing the preS domains of two different subtypes, adw and ayw, has been developed by adding a 6x His tag at the carboxy-terminal end of the polypeptides. Codons for the 6x His were added in reverse primers used to amplify the corresponding cDNAs. The polymerase chain reaction products were cloned into the expression vectors pET-3d (subtype ayw) and pT7-7 (subtype adw), under the control of the inducible bacteriophage T7 RNA polymerase promoter. Upon induction with isopropyl-beta-d-thiogalactopyranoside, proteins were overexpressed and purified by affinity chromatography on a Ni-nitrilotriacetic acid agarose column. This method yielded 20-40 mg of highly pure and very stable proteins per liter of cell culture. Circular dichroism and fluorescence spectroscopy of isolated preS-his-ayw and preS-his-adw, as well as their ability to bind polymerized human serum albumin, indicate that the 6x His tag does not modify the native-like conformation and, therefore, they may be considered as useful tools to study the function of these viral polypeptide regions.
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PMID:Cloning, expression, and purification of histidine-tagged preS domains of hepatitis B virus. 1116 5

The general transcription factor IIF (TFIIF) assembled in the initiation complex, and RAP30 of TFIIF, have been shown to associate with RNA polymerase II (pol II), although it remains unclear which pol II subunit is responsible for the interaction. We examined whether TFIIF interacts with RNA polymerase II subunit 5 (RPB5), the exposed domain of which binds transcriptional regulatory factors such as hepatitis B virus X protein and a novel regulatory protein, RPB5-mediating protein. The results demonstrated that RPB5 directly binds RAP30 in vitro using purified recombinant proteins and in vivo in COS1 cells transiently expressing recombinant RAP30 and RPB5. The RAP30-binding region was mapped to the central region (amino acids (aa) 47-120) of RPB5, which partly overlaps the hepatitis B virus X protein-binding region. Although the middle part (aa 101-170) and the N-terminus (aa 1-100) of RAP30 independently bound RPB5, the latter was not involved in the RPB5 binding when RAP30 was present in TFIIF complex. Scanning of the middle part of RAP30 by clustered alanine substitutions and then point alanine substitutions pinpointed two residues critical for the RPB5 binding in in vitro and in vivo assays. Wild type but not mutants Y124A and Q131A of RAP30 coexpressed with FLAG-RAP74 efficiently recovered endogenous RPB5 to the FLAG-RAP74-bound anti-FLAG M2 resin. The recovered endogenous RPB5 is assembled in pol II as demonstrated immunologically. Interestingly, coexpression of the central region of RPB5 and wild type RAP30 inhibited recovery of endogenous pol II to the FLAG-RAP74-bound M2 resin, strongly suggesting that the RAP30-binding region of RPB5 inhibited the association of TFIIF and pol II. The exposed domain of RPB5 interacts with RAP30 of TFIIF and is important for the association between pol II and TFIIF.
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PMID:Direct interaction between the subunit RAP30 of transcription factor IIF (TFIIF) and RNA polymerase subunit 5, which contributes to the association between TFIIF and RNA polymerase II. 1127 33

Ribonuclease H (RNaseH) recognizes and efficiently cleaves the RNA strand of DNA-RNA hybrids, but has no inherent sequence selectivity. However, the formation of DNA-RNA hybrids does require specific sequence recognition. On the basis of this concept, we wondered whether antisense oligonucleotides complementary to target RNA covalently linked to RNase H could be used to direct specific cleavage events mediated by RNase H. The aim of this research was to couple a DNA oligonucleotide to RNase H to confer specificity of ribonuclease activity toward hepatitis B viral (HBV) mRNA. A modified 13-base oligonucleotide that is specific for the DR1 region of HBV mRNA was conjugated to modified E. coli RNase H using a water soluble cross-linker. A 1200 base fragment of HBV RNA including the DR1 region was synthesized as a substrate using T7 RNA polymerase. Incubation of the RNase H-oligonucleotide conjugate with the RNA transcript resulted in cleavage of the HBV mRNA transcript in a concentration dependent manner. Eighty-five percent of substrate was cleaved under optimal conditions. Controls consisting of RNase H alone, oligonucleotide alone, and incubation of the conjugate with an unrelated mRNA substrate resulted in no cleavage activity. RNase H coupled to an HBV antisense oligonucleotide can specifically cleave target HBV transcripts.
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PMID:A ribonuclease H-oligo DNA conjugate that specifically cleaves hepatitis B viral messenger RNA. 1156 95

Histological features of primary liver cancer among atomic-bomb survivors and their relationship to hepatitis B (HBV) and C viral (HCV) infections are of special interest because of the increased risk of liver cancer in persons exposed to ionizing radiation and the high and increasing liver cancer rates in Japan and elsewhere. We conducted a pathology review of liver cancers occurring from 1958 to 1987 among subjects in the 120,321 member cohort of 1945 Hiroshima and Nagasaki residents. A panel of pathologists classified tumor histological types and defined accompanying cirrhotic changes of the liver. Archival tissue samples were assessed for HBV using pathology stains and PCR. Reverse transcriptase (RT) PCR was used to determine HCV status. We used unconditional logistic regression to compare 302 hepatocellular carcinoma (HCC) cases to 53 cholangiocarcinoma (CC) cases, adjusting for age, year of diagnosis, sex and viral status. Cirrhotic changes occurred significantly more often among HCC than CC cases (76% in HCC and 6% in CC). Compared to CC cases, HCC cases were 10.9 times more likely to be HBV-positive (95% confidence interval: 2.1-83.2) and 4.3 times more likely to be HCV-positive (95% confidence interval: 1.1-20.5). No significant differences were found between HCC and CC cases in radiation exposures. The predominance of HCC in the atomic-bomb survivors follows the background liver cancer pattern in Japan. Our findings suggest that HBV and HCV are involved in the pathogenesis of HCC with or without cirrhosis and are significantly less important in that of CC.
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PMID:Liver cancer in atomic-bomb survivors: histological characteristics and relationships to radiation and hepatitis B and C viruses. 1159 79

Hepatitis B virus (HBV) gene expression is mainly regulated at the transcription initiation level. The viral X protein (pX) is a transcription coactivator/mediator targeting TFIIB for the recruitment of RNA polymerase II. Here we report a novel pX nuclear target designated HBXAP (hepatitis B virus X-associated protein). HBXAP is a novel cellular nuclear protein containing a PHD (plant homology domain) finger, a domain shared by many proteins that play roles in chromatin remodeling, transcription coactivation, and oncogenesis. pX physically interacts with HBXAP in vitro and in vivo via the HBXAP region containing the PHD finger. At the functional level HBXAP increases HBV transcription in a pX-dependent manner suggesting a role for this interaction in the virus life cycle. Interestingly, HBXAP collaborates with pX in coactivating the transcriptional activator NF-kappaB. Coactivation of NF-kappaB was also observed in tumor necrosis factor alpha-treated cells suggesting that pX-HBXAP functional collaboration localized downstream to the NF-kappaB nuclear import. Collectively our data suggest that pX recruits and potentiates a novel putative transcription coactivator to regulate NF-kappaB. The implication of pX-HBXAP interaction in the development of hepatocellular carcinoma is discussed.
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PMID:Hepatitis B virus pX interacts with HBXAP, a PHD finger protein to coactivate transcription. 1178 98

Antigen-presenting dendritic cells (DCs), which play a major role in the triggering of primary anti viral immune reactions, may also contribute, in some viral models, to the pathogenesis of persistent viral infection. In fact, impaired immune response to hepatitis B virus (HBV)-encoded antigens is seen in patients with chronic hepatitis B (CH-B). The aim of this study was to check the function of DCs in these patients and to investigate the underlying mechanism. DCs were enriched from peripheral blood mononuclear cells by culturing with interleukin (IL)-4 and granulocyte-macrophage colony stimulating factor for 7 days. The stimulatory capacity of DCs were checked in allogenic mixed leukocyte (MLR) reaction. The levels of IL-12 in the culture supernatants were measured by an enzyme-linked immunosorbent assay. HBV DNA and HBV RNA were localized in DCs by polymerase chain reaction (PCR) in situ hybridization and reverse-transcriptase (RT)-PCR in situ hybridization. The stimulatory capacity of DCs in allogenic MLR was significantly lower in patients with CH-B (36321+/-12523 cpm, n=18) compared to that of normal controls (65678+/-11174 cpm, n=18) (p<0.0001). Significantly lower levels of IL-12 were detected in cultures containing DCs from patients with CH-B than normal controls (46.7+/-25.6 versus 122.4+/- 37.1 pg/ml, p<0.0001). In situ hybridization revealed the localization of HBV DNA and HBV RNA in DCs from patients with CH-B. These results indicate that chronic infection by HBV is associated with functional defects of DCs. Localization of HBV DNA and HBV RNA indicates that DCs may constitute an extra hepatic reservoir and possibly of replication of HBV.
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PMID:Impaired function of antigen-presenting dendritic cells in patients with chronic hepatitis B: localization of HBV DNA and HBV RNA in blood DC by in situ hybridization. 1252 72

To study the preparation and cleavage of hairpin ribozyme (HpRz) directed against the transcript of HBV core region in vitro, HRz gene designed by computer targeting the transcript of HBV core gene was cloned into the vector p1.5 between 5'-cis-Rz and 3'-cis-Rz. 32P-labeled HpRz transcript proved whether the vector fit for the preparation of hairpin ribozyme. 32P-labeled pKC transcript containing HBV core region as targets-RNA was transcribed by using T7 RNA polymerase and purified by PAGE. Cold HpRz transcript was incubated with 32P-labeled target-RNAs under different conditions and radioautographed after denaturing polyacrylamide gel electrophoresis. The results showed that HpRz had the ability of cleavage at 37 degrees C and 12 mmol/L MgCl2 and the design of ribozyme was correct. It is concluded that HpRz prepared in vitro possesses specific catalytic activity, indicating that it is possible for HpRz to intracellularly inhibit the replication of HBV. It may be developed into a nucleic acid drug in the treatment of hepatitis B in the future.
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PMID:Anti-viral activity of hairpin ribozyme directed against HBV core region in vitro. 1253 81

In a natural setting, hepatitis delta virus (HDV) is only found in patients that are also infected with hepatitis B virus (HBV). In hepatocytes infected with these two viruses, HDV RNA genomes are assembled using the envelope proteins of HBV. Since 1986, we have known that HDV has a small single-stranded RNA genome with a unique circular conformation that is replicated using a host RNA polymerase. These and other features make HDV and its replication unique, at least among agents that infect animals. This mini-review focuses on advances gained over the last 2-3 years, together with an evaluation of HDV questions that are either unsolved or not yet solved satisfactorily.
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PMID:Replication of human hepatitis delta virus: recent developments. 1270 97


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