Integrase plays a critical role in the recombination of viral DNA into the host genome. Therefore, over the past decade, it has been a hot target of drug design in the fight against type 1 human immunodeficiency virus (HIV-1). Bovine immunodeficiency virus (BIV) integrase has the same function as HIV-1 integrase. We have determined crystal structures of the BIV integrase catalytic core domain (CCD) in two different crystal forms at a resolution of 2.45 Å and 2.2 Å, respectively. In crystal form I, BIV integrase CCD forms a back-to-back dimer, in which the two active sites are on opposite sides. This has also been seen in many of the CCD structures of HIV-1 integrase that were determined previously. However, in crystal form II, BIV integrase CCD forms a novel face-to-face dimer in which the two active sites are close to each other. Strikingly, the distance separating the two active sites is approximately 20 Å, a distance that perfectly matches a 5-base pair interval. Based on these data, we propose a model for the interaction of integrase with its target DNA, which is also supported by many published biochemical data. Our results provide important clues for designing new inhibitors against HIV-1.
Animals ; Catalytic Domain ; genetics ; Cattle ; DNA ; genetics ; DNA, Viral ; HIV-1 ; genetics ; metabolism ; Humans ; Immunodeficiency Virus, Bovine ; enzymology ; genetics ; Integrases ; chemistry ; genetics ; metabolism

OBJECTIVEExpress and purify four single-stranded DNA-binding (SSB) proteins, and evaluate the binding of SSB proteins on HCV RNA.

METHODSThe expression plasmids of four SSB proteins were conducted, termed TTH, SSOB, KOD and BL21, respectively. The BL21 (DE3) was transformed by the expression plasmid of TTH, Transetta (DE3) were transformed by the expression plasmid of SSOB, KOD and BL21, then protein expression was induced with IPTG, the expression products were analysised by SDS-PAGE. To evaluate the binding of SSB on HCV RNA, RNA-SSB protein complexes were applied to a 1.2% TAE agarose gel.

RESULTSSuitable competent cells were transformed with the expression plasmids, induced by IPTG. SSB proteins were purified by affinity chromatography, to visualize their purity all SSB proteins were applied to SDS-PAGE analysis. All four proteins showed single clear bands. We have successfully obtained the SSB protein expression plasmid, expressed and purified SSB protein. TAE agarose gel electrophoresis was used to confirm SSB protein-RNA binding activity. The each of SSB-RNA complex migrated more slowly than the sole RNA, which suggested SSB protein could specifically bind to RNA.

CONCLUSIONSWe have expressed and purified four SSB proteins, and for the first time found that SSB protein can bind HCV RNA. Our results may provide a basis for future studies of the novel functions of SSB proteins on RNA.

DNA, Single-Stranded ; genetics ; metabolism ; DNA-Binding Proteins ; chemistry ; isolation & purification ; metabolism ; Hepacivirus ; Hepatitis C ; metabolism ; virology ; Humans ; Molecular Weight ; Protein Binding ; RNA, Viral ; genetics ; metabolism

OBJECTIVETo screen and clone the genes of protein interacting with the N-terminal protein (TP) of hepatitis B virus DNA polymerase.

METHODSTP was amplified by polymerase chain reaction (PCR) and TP bait plasmid was constructed by using yeast two-hybrid system 3, then transformed into yeast AH 109. The transformed yeast was mated with yeast Y187 containing liver cDNA library plasmid in 2 x YPDA medium. Diploid yeast was plated on synthetic dropout medium (SD/-Trp-Leu-His-Ade) and that containing X-alpha-GAL for selecting two times and screening. Plasmids were extracted from blue colonies, and sequence analysis was performed by bioinformatics.

RESULTSForty-seven clones were obtained, these clones included human P36956 sterol regulatory element binding protein-1, RNA polymerase II subunit hsRPB7 mRNA, asialoglycoprotein receptor 2, transcript variant 3, ceruloplasmin (ferroxidase), transmembrane 4 superfamily member 2 and 19 of the hypothetical proteins and so on.

CONCLUSIONGenes encoding TP interacting proteins in hepatocytes were successfully cloned and the results suggest that TP has a wide variety of biological functions.

Cloning, Molecular ; DNA-Directed DNA Polymerase ; chemistry ; genetics ; metabolism ; Gene Library ; Hepatitis B virus ; enzymology ; genetics ; Humans ; Liver ; metabolism ; Plasmids ; genetics ; Protein Binding ; Receptors, Virus ; genetics ; isolation & purification ; metabolism ; Transformation, Genetic ; Two-Hybrid System Techniques ; Viral Proteins ; chemistry ; genetics ; metabolism

Previously, we have established an epsilon library and selected out a series of RNA aptamers with higher affinity to P protein based on the in vitro Systematic Evolution of Ligands by Exponential Enrichment (SELEX) in duck hepatitis B virus (DHBV) system. In order to study the structural elements within the epsilon that is essential for initiating priming of HBV reverse transcriptase (P protein), all selected aptamers were subjected to in vitro priming assay and RNA secondary structure probing. We found that all those aptamers supporting priming had an undamaged bulge, while those lacking of the bulge no more support priming. Our results suggest an undamaged bulge within Depsilon is indispensable for initiating priming of P protein.
Base Sequence ; Hepatitis B Virus, Duck ; chemistry ; enzymology ; genetics ; Molecular Sequence Data ; Nucleic Acid Conformation ; RNA, Viral ; chemistry ; genetics ; RNA-Directed DNA Polymerase ; genetics ; metabolism ; Reverse Transcription ; Sequence Alignment ; Viral Proteins ; genetics ; metabolism

Murine gammaherpesvirus 68 (MHV-68), a member of the gammaherpesvirus family, replicates robustly in permissive cell lines and is able to infect laboratory mice. MHV-68 has emerged as a model for studying the basic aspects of viral replication and host-virus interactions of its human counterparts. Herpesvirus genome replication is mediated through a cis-element in the viral genome called the origin of lytic replication (oriLyt). A family of transcription factors, CCAAT/enhancer binding proteins (C/EBPs), assists in oriLyt-mediated DNA replication during gammaherpesvirus reactivation. In this study, we examined the role of C/EBPs in gammaherpesvirus DNA replication during de novo infection, using MHV-68 as a model. We found that C/EBP α and β bind to the CCAAT boxes in the MHV-68 oriLyt core region both in vitro and in vivo, as demonstrated by electrophoretic mobility shift assay and chromatin immunoprecipitation assay. A dominant negative form of C/EBPs significantly impaired the lytic replication efficiency of MHV-68 on both the plasmid and genome levels in a replication assay, indicating that functional C/EBPs are required for maximal MHV-68 genome DNA replication. Collectively, our data demonstrate that C/EBPs interact with the oriLyt core region and play an important role in MHV-68 lytic DNA replication during de novo infection.
Animals ; Base Sequence ; CCAAT-Enhancer-Binding Proteins ; genetics ; metabolism ; Cell Line ; Chromatin Immunoprecipitation ; Cricetinae ; DNA Replication ; DNA, Viral ; chemistry ; genetics ; metabolism ; Electrophoretic Mobility Shift Assay ; Genome, Viral ; Herpesviridae Infections ; genetics ; metabolism ; virology ; Humans ; Mice ; Molecular Sequence Data ; Plasmids ; Promoter Regions, Genetic ; Protein Isoforms ; genetics ; metabolism ; Replication Origin ; Rhadinovirus ; genetics ; metabolism ; Viral Proteins ; genetics ; metabolism ; Virus Latency ; genetics

Canine adenovirus type 2 (CAV-2) has been proposed as a vector for recombinant vaccine. Alternatively, it may be an attractive tool for gene transfer due to lack of pre-existing immunity in humans. In this study, a transfer vector based on CAV-2, in which the 1381bp fragment of the E3 region was deleted, and a linker containing the Not I, Cla I, Fse I restriction enzyme sites were cloned into the deleted region. The recombinant CAV-2 genome was released from the plasmids enzyme digestion and transfected into MDCK cells by lipofectamine to obtain the recombinant virus. No significant difference in morphology, hemagglutination and replication between the recombinant and the wide type CAV-2 was found. These results indicated that this recombinant virus CAV-2-deltaE3 (NF) may be an efficient vector for gene transfer and the capacity of the vector for inserted foreign gene was up to 3.3kb.
Adenoviruses, Canine ; genetics ; ultrastructure ; Animals ; Binding Sites ; genetics ; Cell Line ; Cloning, Molecular ; DNA Restriction Enzymes ; metabolism ; DNA, Viral ; chemistry ; genetics ; Dogs ; virology ; Genetic Vectors ; genetics ; Humans ; Lipids ; chemistry ; Microscopy, Electron ; Transfection ; methods ; Virus Replication ; genetics

OBJECTIVETo investigate the distribution of hepatitis B virus (HBV) genotypes in Qingdao, and the relationship of HBV genotypes with the serum HBV-DNA levels and HBV YMDD spontaneous mutation of patients, then to discuss the clinical significance.

METHODSHepatitis B virus genotypes and YMDD spontaneous mutation of 144 patients were detected by real time PCR (Taqman probe), then the results were analyzed by statistical method.

RESULTSOf the 144 patients, 130 (90.3%) were genotype C, 12 (8.3%) were genotype B, and 2 (1.4%) were neither genotype B nor genotype C; 33 (22.9%) were detected to have YMDD mutation, and 25 (75.5%) were YVDD positive, 3 (9.1%) were YIDD positive, 5 (15.2%) were YVDD and YIDD positive. There were no significant differences between clinical diagnosis, serum HBV-DNA levels, YMDD spontaneous mutation and HBV genotypes (P > 0.05).

CONCLUSIONGenotype C is the dominant position for HBV genotype in Qingdao. Untreated patients with chronic hepatitis B have YMDD spontaneous mutation. HBV genotypes have no association with YMDD spontaneous mutation and the development of diseases.

Adult ; Aged ; Aged, 80 and over ; China ; DNA-Directed DNA Polymerase ; chemistry ; genetics ; metabolism ; Female ; Genotype ; Hepatitis B virus ; classification ; enzymology ; genetics ; isolation & purification ; Humans ; Male ; Middle Aged ; Mutation ; Viral Proteins ; chemistry ; genetics ; metabolism ; Young Adult

The purpose of this study is to elucidate the molecular mechanism of one-way serological reaction between XJ-160 virus and SINV by recombinant viruses which exchanged the glycoprotein genes individually or simultaneously. Three recombinant viruses were obtained based on the whole-length infectious cDNA clone of XJ-160 virus. The infectivity and pathogenesis to BHK-21 cells and animals were studied and the gene which controlled this one-way serological reaction phenomenon was searched by MCPENT. The results showed that the E2 glycoprotein was the main factor which influenced the growth rate, plaque morphology and pathogenicity of BHK-21 cells and suckling mice. The results of MCPENT showed that the E2 glycoprotein of SINV played a major role in this one-way serological reaction phenomenon. Our study identified the SINE2 gene was the determined gene for one way serological reaction between XJ-160 virus and SINV, and this research laid the foundation for further analysis of the genomic structure and function of SINV.
Alphavirus ; genetics ; immunology ; physiology ; Amino Acid Sequence ; Animals ; Cell Line ; DNA, Recombinant ; genetics ; Female ; Genetic Engineering ; Glycoproteins ; chemistry ; metabolism ; Mice ; Mice, Inbred BALB C ; Molecular Sequence Data ; Neutralization Tests ; Sindbis Virus ; immunology ; Viral Load ; Viral Proteins ; chemistry ; metabolism

OBJECTIVETo establish a sensitive and specific technique for detecting HBV DNA in serum using HBV DNA probe labeled directly by alkaline phosphatase (AlkPhos Direc probe).

METHODSThe probe that purified HBV DNA sequence was labeled directly by alkaline phosphatase and chemiluminescent substrate CDP-star for AP was used in the hybridization assay. HBV DNA was detected by autoradiography on the film. The test compared the chemiluminescen dot blot hybridization assay for 80 samples with digoxigenin-labeled HBV DNA probe detective method. The correlation of 70 samples test results between fluorescent quantitative HBV DNA PCR method and dot blot hybridization assay by AlkPhos Direc probe was analysed.

RESULTSThe sensitivity of the probe labeled directly by alkaline phosphatase was 10pg at least. The coincidence was 100% compared with digoxigenin-labeled HBV DNA probe detection. A correlation coefficient of HBV DNA quantitative results between fluorescent quantitative HBV DNA PCR (QPCR) method and dot blot hybridization assay by AlkPhos Direc probe was 0.98 (P<0.01).

CONCLUSIONSThe method detecting HBV DNA in serum by HBV DNA AlkPhos Direc probe is sensitive and specific. The results between two methods with AlkPhos Direc and digoxigenin-labeled HBV DNA probe are coincident completely. The correlation of HBV DNA quantitative results between fluorescent QPCR method and dot blot hybridization assay by AlkPhos Direc probe is satisfactory.

Alkaline Phosphatase ; chemistry ; metabolism ; Animals ; DNA Probes ; chemistry ; genetics ; DNA, Viral ; blood ; genetics ; Hepatitis B ; blood ; diagnosis ; virology ; Hepatitis B virus ; genetics ; Humans ; Molecular Diagnostic Techniques ; methods ; standards ; Polymerase Chain Reaction ; methods ; Sensitivity and Specificity

External Guide Sequences (EGSs) represents a novel nucleic acid based gene interference approach to modulate gene expression. They are oligonucleotides that consist of a sequence complementary to a target mRNA and recruit intracellular RNase P for specific degradation of the target RNA. DNA-based EGS1386 with a size of 12 nt was chemically synthesized to target the mRNA coding for the UL49 gene of human cytomegalovirus (HCMV). The DNA-based EGS1386 molecule efficiently directed human RNase P to cleave the target mRNA sequence in vitro. A reduction of more than 50% in the levels of UL49 expression was observed in human cells treated with the DNA-based EGS1386 targeted UL49 assayed by fluorescent quantization PCR and Western blotting. This results showed that the DNA-EGS1386 can effectively guide the RNase P cut the target mRNA. Therefore, DNA-EGS can develop into a new gene silencing technology and potential of the anti-viral reagents.
Base Sequence ; Cytomegalovirus ; drug effects ; genetics ; metabolism ; Cytomegalovirus Infections ; enzymology ; virology ; DNA, Viral ; genetics ; Directed Molecular Evolution ; methods ; Gene Expression Regulation, Viral ; Humans ; Nucleic Acid Conformation ; Oligodeoxyribonucleotides ; genetics ; pharmacology ; RNA, Guide ; chemistry ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Ribonuclease P ; genetics ; metabolism ; Viral Structural Proteins ; genetics ; metabolism