Human immunodeficiency virus (HIV) is a retrovirus, belongs to Lentiviridae family. As long as viral genetic material entering into host cytoplasm, double-strand DNAs synthesis occurs which is catalyzed by reverse transcriptase (RT) with viral plus-strand RNA as template. This reverse transcription is a key link of HIV-1 life cycle and an important target for anti-HIV drug development. The process of reverse transcription can be divided into several steps: formation of minus-strand strong-stop DNA; the first translocation; initiation of plus-strand DNA synthesis; and, the second translocation and the completion of both strands. These steps can be detected individually by using polymerase chain reaction (PCR) according to the amplified products on the region of R/U5, U3, U5/PBS and the sequence between LTR and Gag. In this review, we summarize the principle for detecting stages of HIV-1 reverse transcription by using PCR.
DNA Replication ; genetics ; DNA, Viral ; biosynthesis ; genetics ; HIV Reverse Transcriptase ; genetics ; metabolism ; HIV-1 ; genetics ; metabolism ; Polymerase Chain Reaction ; methods ; RNA, Viral ; genetics ; Reverse Transcription

HBV infections leads to severe public health problems around the world, especially in China. Improved understanding of the molecular mechanisms of HBV reverse transcription is fundamental for optimization of treatment and solution to drug-resistance. Recently, the main structural basis involved in the process of HBV reverse transcription and the cis-elements were revealed by means of biochemistry and genetics. The entire process of reverse transcription is completed mainly through the first template switch mediated by the P- epsilon structure; the second template switch mediated by 5E/3E and M structure; and the third template switch mediated by 5' r / 3' r structure. The important structure and the cis-elements involved in this process are the focus of this review, at the same time, an overview of the progress in relevent studies is demonstrated to show the whole picture of the HBV reverse process.
Animals ; Hepatitis B ; virology ; Hepatitis B virus ; enzymology ; genetics ; metabolism ; Humans ; RNA, Viral ; genetics ; RNA-Directed DNA Polymerase ; genetics ; metabolism ; Reverse Transcription ; Viral Proteins ; genetics ; metabolism

OBJECTIVETo explore the possibility of transacting hepatitis D virus (HDV) ribozyme cleaving in vitro the hepatitis B virus (HBV) mRNA fragments.

METHODSAccording to the established pseudoknot-like structure, its' H1 domain was changed to design the transacting HDV ribozyme Rc1 and Rc2, which targeted the 701-713 site and 776-788 site of HBV C domain. After the chemically synthesised cDNA of the ribozyme was cloned into the vector PGEM-4Z, the transacting HDV ribozyme was transcriped using in vitro transcription technology. The in vitro cleavage characteristics of the ribozyme were studied and the kinetic parameters (Kcat and Km) were determined by Eadie Hofstee plotting.

RESULTSBoth the two ribozymes had the ability to cleave the substrate, the cleavage percentage at 37 degrees for 90 minutes were 50% and 51%. According to the Eadie Hofstee plot, the Km of the Rc1 and Rc2 were 0.61 micromol and 0.58 micromol, the Kcat were 0.64 x min(-1) and 0.60 x min(-1),respectively.

CONCLUSIONSThe cleaving ability of trans-acting HDV ribozyme on non-HDV RNA fragment was tested. The results showed a new potential of the antisense antisense regent for HBV gene therapy.

DNA, Antisense ; genetics ; Genome, Viral ; Hepatitis B virus ; genetics ; Hepatitis Delta Virus ; enzymology ; genetics ; Humans ; RNA, Catalytic ; genetics ; metabolism ; RNA, Messenger ; genetics ; RNA, Viral ; genetics ; Transcription, Genetic

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

Animals ; Cloning, Molecular ; DNA Replication ; DNA, Viral ; genetics ; Gene Expression Regulation, Viral ; Hepatitis B virus ; genetics ; metabolism ; physiology ; Humans ; Trans-Activators ; genetics ; Virus Replication

OBJECTIVETo study the relativity between La protein and the stability of HBV mRNA and the expression of HBV protein.

METHODSFour specific siRNAs were obtained by transcription in vitro. After transfection with the siRNAs into HepG2.2.15 cells for 3 days, the inhibitive effects of La protein were analyzed by Western blot; the content changes of HBsAg, HBeAg and HBV-DNA were detected by ECL and RT-PCR.

RESULTSIn comparison to normal cells, La protein was less in the cells. There was less La protein in the cells trans-infected with siRNAs. HBsAg, the HBeAg and HBV-DNA secreted by the cells transfected with siRNA were also less than that in the normal cells.

CONCLUSIONThere is a correlation between La protein and HBV mRNA and the expression of HBV protein.

Autoantigens ; metabolism ; Cell Line, Tumor ; DNA, Viral ; Hepatitis B Surface Antigens ; Hepatitis B virus ; genetics ; metabolism ; Humans ; RNA, Messenger ; genetics ; metabolism ; RNA, Small Interfering ; RNA, Viral ; Ribonucleoproteins ; metabolism

Hepatitis C virus (HCV) core protein is considered to be an attractive candidate for development of protective HCV vaccines. However, this protein may attenuate the induction of systemic immune responses due to its immunomodulatory properties. In this study, we constructed a HCV core gene-containing eukaryotic expression plamid pCI-C, and an in vivo-inducible prokaryotic expression plasmid pZW-C, and transformed the recombinant plasmids into an attenuated Salmonella typhimurium aroA strain SL7207. The resulting bacterial strains SL7207/pCI-C and SL7207/pZW-C were used to orally immunize BALB/c mice, and the immune responses specific to HCV core protein were assessed. Immunization with the recombinant bacteria SL7207/pCI-C led to a persistent drop in percentage of CD3 CD4 T cells, and induced a weak anti-core IgG production. Splenocytes from SL7207/pCI-C immunized mice developed a relatively weak proliferation response and inferior cytotoxic activity compared to those from the mice immunized with bacteria SL7207/pZW-C. Boost immunization with SL7207/ pCI-C yielded limited improvement in immune strength, while the boost with bacteria SL7207/pZW-C significantly enhanced the immune response. These results suggest that de novo synthesis of native HCV core protein may blunt the induction of immune responses. Attenuated S. typhimurium carrying HCV core protein could efficiently activate systemic cellular and humoral responses, and may be a promising strategy for the development of core-based HCV vaccines.
Animals ; Hepacivirus ; genetics ; immunology ; Mice ; Plasmids ; genetics ; Salmonella typhimurium ; genetics ; metabolism ; Vaccines, DNA ; immunology ; Viral Core Proteins ; genetics ; immunology ; Viral Hepatitis Vaccines ; genetics ; immunology

We constructed the plasmid containing the integrated open reading frame (ORF) HPV11 genome, and identified its function, to lay foundation for production of transgenic animal models of HPV11. Recombinant plasmid pQE-Trisystem- EGFP/HPV11 (pE/H) and pQE-Trisystem-EGFP/1.1 copy HPV11 (pE/1.1H) were constructed. Then, the human keratinocyte (KC) was transfected by pE/1.1H, pE/H and closed circular HPV11 and detected. The recombinant plasmid was successfully constructed. The expression of HPV11E6 gene was detected in the experimental group. Fluorescence was observed in the pE/1.1H and pE/H group. The HPV11, pE/H, and pE/1.1 enhanced the KC proliferation, with remarkable differences (P < 0.01) from the control group. Amongst the three experimental groups, pE/1.1H was found to be the weakest. The plasmid containing the integrated ORF of HPV11 (1.1 copy HPV11) genome was successfully constructed. The pE/1.1H had the same phenotype of wild-type HPV11 genome. It provided experimental material and methodological guidance for studying the low-risk HPV, as well as for the production of HPV11 transgenic mice.
Cell Proliferation ; Cells, Cultured ; DNA, Viral ; genetics ; Genome, Viral ; genetics ; Human papillomavirus 11 ; genetics ; Humans ; Keratinocytes ; cytology ; metabolism ; Open Reading Frames ; genetics ; Plasmids ; genetics ; Transfection

OBJECTIVETo investigate the cleavage activities of 10-23 DNA enzymes targeting at HBV C gene mRNA in vitro.

METHODS10-23 DNA enzymes named DrzBC-7, DrzBC-8 and DrzBC-9 specific to HBV C gene ORF A1816UG were designed and synthesized. HBV C gene mRNA was obtained by in vitro transcription method. Cleavage activities were observed in vitro. The influence of MgCl2 concentration on RNA cleaving activity was examined with DrzBC-9. Values of kinetic parameters including Km, Kcat and Kcat/Km were calculated accordingly.

RESULTTargeted substrate mRNA with the size of 300 nt was obtained by transcription in vitro. Under the certain cleavage conditions, DrzBC-7, DrzBC-8 and DrzBC-9 all efficiently cleaved target mRNA at specific sites in vitro. Cleavage products of 109 nt and 191 nt were obtained. No cleavage occurred without MgCl2. The most efficient cleavage was obtained at 150 mmol x L(-1) MgCl2. The efficiency of cleavage did not increase when the MgCl2 concentration was more than 200 mmol x L(-1). The kinetic parameters, Km, Kcat and Kcat/Km for DrzBC-9 were 1.4x10(-9) mol x L(-1), 1.6 min(-1) and 1.1x10(9) mol x L(-1) x min(-1), respectively.

CONCLUSION10-23 DNA enzymes targeting at HBV C gene mRNA possess the specific cleavage activities in vitro.

DNA, Catalytic ; metabolism ; DNA, Single-Stranded ; metabolism ; Hepatitis B virus ; enzymology ; genetics ; Open Reading Frames ; RNA, Messenger ; metabolism ; RNA, Viral ; genetics ; metabolism ; Transcription, Genetic

In this study, we constructed the plasmid of Sendai virus (SeV) BB1 strain minigenome with Gaussia luciferase (Gluc) as reporter and compared the rescue efficiency of SeV minigenome mediated by T7 promoter with that by CMV promoter. Firstly, the sequence was designed and synthesized which contained hammerhead ribozyme, sequence composed of the trailer, untranslated region of L gene, untranslated region of N gene, and the leader from SeV, and mutant hepatitis delta virus ribozyme sequence. Then, the synthesized sequence was inserted into pVAX1 containing CMV and T7 promoters and the general vector for SeV minigenome pVAX-miniSeV was obtained. Furthermore, pVAX-miniSeV-Gluc (+) and pVAX-miniSeV-Gluc(-) were obtained by inserting Gluc gene into pVAX-miniSeV. From the supernatant of BHK-21 cell transfected with pVAX-miniSeV-Gluc(+), high level of Gluc expression was detection indicating the normal transcription function of CMV promoter. pVAX-SeV-miniGluc(-) and plasmids expressing N,P and L protein of SeV were co-transfected into BST T7/5 cell which derived from BHK-21 and expressed T7 RNA polymerase stably. And high expression of Gluc was found, which indicated that SeV minigenome was efficiently rescued. However, we failed to repeat the result on BHK-21 cell, implying that T7 promoter and CMV promoter may have different effects on the rescue efficiency of SeV minigenome. Therefore, we further constructed SeV minigenome vectors pT7-miniSeV-Gluc (-) and pCMV-miniSeV-Gluc(-) with single promoter of T7 or CMV. Then, these vectors were transfected into BSR T7/ 5 cells respectively accompanied with the N, P, and L protein expression vectors. The result demonstrated that high expression of Gluc was found in the group of pT7-miniSeV-Gluc(-), which failed in the group of pCMV-miniSeV-Gluc(-). It indicated that T7 promoter significantly increased the rescue efficiency of SeV minigenome. We successfully constructed a SeV minigenome vector with secreted luciferase gene as report er and proved T7 promoter can enhance the rescue efficiency of SeV minigenome, which provides basis for construction of infectious clone containing SeV full-length genome.
Animals ; Cell Line ; Cricetinae ; Cytomegalovirus ; genetics ; DNA-Directed RNA Polymerases ; genetics ; Genome, Viral ; Humans ; Promoter Regions, Genetic ; Respirovirus Infections ; virology ; Sendai virus ; genetics ; physiology ; Viral Proteins ; genetics ; metabolism