OBJECTIVETo investigate distinctive features in drug-resistant mutations (DRMs) and interpretations for reverse transcriptase inhibitors (RTIs) between proviral DNA and paired viral RNA in HIV-1-infected patients.

METHODSForty-three HIV-1-infected individuals receiving first-line antiretroviral therapy were recruited to participate in a multicenter AIDS Cohort Study in Anhui and Henan Provinces in China in 2004. Drug resistance genotyping was performed by bulk sequencing and deep sequencing on the plasma and whole blood of 77 samples, respectively. Drug-resistance interpretation was compared between viral RNA and paired proviral DNA.

RESULTSCompared with bulk sequencing, deep sequencing could detect more DRMs and samples with DRMs in both viral RNA and proviral DNA. The mutations M184I and M230I were more prevalent in proviral DNA than in viral RNA (Fisher's exact test, P<0.05). Considering 'majority resistant variants', 15 samples (19.48%) showed differences in drug resistance interpretation between viral RNA and proviral DNA, and 5 of these samples with different DRMs between proviral DNA and paired viral RNA showed a higher level of drug resistance to the first-line drugs. Considering 'minority resistant variants', 22 samples (28.57%) were associated with a higher level of drug resistance to the tested RTIs for proviral DNA when compared with paired viral RNA.

CONCLUSIONCompared with viral RNA, the distinctive information of DRMs and drug resistance interpretations for proviral DNA could be obtained by deep sequencing, which could provide more detailed and precise information for drug resistance monitoring and the rational design of optimal antiretroviral therapy regimens.

Adult ; Antiviral Agents ; pharmacology ; China ; DNA, Viral ; genetics ; metabolism ; Drug Resistance, Viral ; genetics ; Female ; HIV Infections ; drug therapy ; HIV-1 ; drug effects ; genetics ; metabolism ; High-Throughput Nucleotide Sequencing ; Humans ; Male ; Middle Aged ; Mutation ; Proviruses ; genetics ; metabolism ; RNA, Viral ; genetics ; metabolism ; RNA-Directed DNA Polymerase

Avian Myeloblastosis Virus ; enzymology ; Escherichia coli ; genetics ; metabolism ; Genome, Bacterial ; MicroRNAs ; metabolism ; RNA-Directed DNA Polymerase ; genetics ; metabolism ; Real-Time Polymerase Chain Reaction ; Sequence Analysis, RNA

In order to facilitate the detection of newly emerging or rare viral infectious diseases, a negative-strand RNA virus-severe fever with thrombocytopenia syndrome bunyavirus, and a positive-strand RNA virus-dengue virus, were used to investigate RNA viral genome unspecific amplification by multiple displacement amplification technique from clinical samples. Series of 10-fold diluted purified viral RNA were utilized as analog samples with different pathogen loads, after a series of reactions were sequentially processed, single-strand cDNA, double-strand cDNA, double-strand cDNA treated with ligation without or with supplemental RNA were generated, then a Phi29 DNA polymerase depended isothermal amplification was employed, and finally the target gene copies were detected by real time PCR assays to evaluate the amplification efficiencies of various methods. The results showed that multiple displacement amplification effects of single-strand or double-strand cDNA templates were limited, while the fold increases of double-strand cDNA templates treated with ligation could be up to 6 X 10(3), even 2 X 10(5) when supplemental RNA existed, and better results were obtained when viral RNA loads were lower. A RNA viral genome amplification system using multiple displacement amplification technique was established in this study and effective amplification of RNA viral genome with low load was achieved, which could provide a tool to synthesize adequate viral genome for multiplex pathogens detection.
Bunyaviridae Infections ; diagnosis ; virology ; Cell Line ; DNA Ligases ; metabolism ; DNA, Complementary ; analysis ; genetics ; DNA-Directed DNA Polymerase ; metabolism ; Dengue ; diagnosis ; virology ; Dengue Virus ; genetics ; isolation & purification ; Genome, Viral ; genetics ; Humans ; Phlebovirus ; genetics ; isolation & purification ; RNA, Viral ; analysis ; genetics ; Reference Standards ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Viral Load

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

This study is to investigate the mechanism and action characteristics of 6-chloro-3-methyl-4-(2-methyoxycarbonylthiophene-3-sulfonyl)-3, 4-dihydroquinoxa-lin-2-(1 H)-one (XU07011) against HIV-1 replication. XU07011 anti-HIV activity was tested by using VSVG/HIV pseudotype viral system and confirmed by HIV-1 live viruses' infectious assay. Time of addition was used to test HIV-1 reverse transcription process. RNA-dependent DNA polymerase activity and RNase H activity were tested by using enzyme linked immunoabsorbent assay and fluorescence method. Wild type and nine NNRTIs-resistant reverse transcriptase enzymatic models and cell-based pharmacological models were used to evaluate XU07011 bio-characteristics. The results showed that XU07011 inhibited HIV-1 replication with IC50 of (0.057 +/- 0.01) micromol x L(-1) which was comparable to nevirapine [IC50: (0.046 +/- 0.01) micromol x L(-1)]. Mechanism study data indicated that XU07011 blocked HIV-1 reverse transcription process through acting on reverse transcriptase RNA-dependent DNA polymerase with IC 50 of (1.1 +/- 0.3) micromol x L(-1). The compound showed no effect on RNase H activity. XU07011 exhibited better activities comparing with nevirapine on K103N mutated NNRTIs-resistant HIV-1 strains. This study could provide a theoretical basis for novel anti-HIV reagents development.
Anti-HIV Agents ; chemistry ; pharmacology ; Drug Resistance, Viral ; HEK293 Cells ; HIV-1 ; physiology ; Humans ; Inhibitory Concentration 50 ; Molecular Structure ; Nevirapine ; pharmacology ; Quinoxalines ; pharmacology ; RNA-Directed DNA Polymerase ; metabolism ; Ribonuclease H ; metabolism ; Thiophenes ; pharmacology ; Virus Replication ; drug effects

Currently, animal somatic cell reprogramming into the induced pluripotent stem cell (iPS) is one of the hottest research target in the field of cell biology. We focused on the analysis of telomerase reverse transcriptase (TERT) gene expression during goat somatic fibroblasts reprogramming, and investigated the relationship between the expression of TERT and the pluripotency of reprogrammed cells. RNA samples of fetal tissues isolated from Guanzhong milk goat fetus, and the induced goat reprogramming cell clones were used to determine the relative expression levels of TERT by the real-time RT-PCR method. Goat embryonic fibroblasts (GEF) collected from the Guanzhong milk goat with normal karyotype were induced by 4 transcription factors to become reprogramming cells. The expression of TERT in reprogramming cells was detected by Real-time RT-PCR. The results showed that the expression of TERT in testis tissue was higher than that in epithelial tissues (P < 0.01). The expression level of TERT was higher in AP staining positive cells than that in AP staining negative cells (P < 0.01). This result indicated that TERT activity played an important role in cell reprogramming.
Animals ; Cellular Reprogramming ; Fibroblasts ; cytology ; Gene Expression Regulation ; Goats ; Induced Pluripotent Stem Cells ; cytology ; metabolism ; RNA-Directed DNA Polymerase ; genetics ; Telomerase ; metabolism

For obtaining new structural compounds with unique resistance profiles or novel mechanisms of action on HIV-1 from natural products, anti-HIV-1 drug screening models were used in vitro. Norcantharidin (NCTD), a derivative from cantharidin, was found to have inhibitory activities on HIV-1(IIIB) p24 antigen in lymphocyte lines MT-4, CEM and H9. It inhibited HIV-1 strain 018a (sensitive to zidovudine) from replicating with EC50 (50% effective concentration) of 14.9 micromol L(-1) and also inhibited HIV-1 strain 018c (resistant to zidovudine) from replicating with EC50 of 20.2 micromol L(-1) in primary lymphocytes peripheral blood mononuclear cells (PBMC). Norcantharidin showed synergistic activity with zidovudine on HIV-1(IIIB) in MT-4 cells, the combination index was less than 0.3. But, it was not active on HIV-1 integrase, reverse transcriptase or protease in vitro. As the structure of norcantharidin is unique and different from that of all clinic drugs approved, it would be possible to obtain new and effective compounds against HIV-1 with low toxicities after modification of norcantharidin.
Anti-HIV Agents ; pharmacology ; Bridged Bicyclo Compounds, Heterocyclic ; pharmacology ; Cell Line ; Drug Resistance, Viral ; Drug Synergism ; HIV Core Protein p24 ; metabolism ; HIV Integrase ; metabolism ; HIV-1 ; metabolism ; Humans ; Leukocytes, Mononuclear ; cytology ; virology ; Peptide Hydrolases ; metabolism ; RNA-Directed DNA Polymerase ; metabolism ; T-Lymphocytes ; cytology ; virology ; Virus Replication ; Zidovudine ; pharmacology

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

To produce the reverse transcriptase of moloney murine leukemia virus (MMLV-RT) through gene recombination, MMLV-rt gene was amplified by polymerase chain reaction (PCR) with specifically designed primers bearing restriction enzyme sites. Five mutation sites increasing the solution of the target protein were introduced through Site-directed mutation. After verification by sequencing, the gene was cloned into the expression vector pET15b to construct the recombinant plasmid pET15b-MMLV-rt. Purified MMLV-RT was obtained by affinity chromatography (Ni3+-NTA beads). Molecular weight and purity of MMLV-RT were analyzed with SDS-PAGE. Enzyme activity was characterized with RT-PCR. We successfully constructed the recombinant plasmid pET15b-MMLV-rt and obtained the MMLV-RT fusion protein with 6His on the N-terminus. Recombinant protein was purified through Ni3+-NTA beads based affinity chromatography, the purity of which was 96%. The Activity of the enzyme was high. MMLV-RT of 96% purity was obtained with the prokaryotic expression technique, which serves as the basis for mass production of this enzyme.
Animals ; Mice ; Moloney murine leukemia virus ; enzymology ; genetics ; RNA-Directed DNA Polymerase ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; metabolism ; Recombination, Genetic

BACKGROUND/AIMS: To develop a novel treatment method for hepatitis B virus (HBV) infection, we aimed to make a human monoclonal antibody inhibiting reverse transcriptase (RT) activity of P protein which was important in HBV replication by using phage display technique. Therefore, we analysed the usability of human monoclonal antibody as a protein based gene therapy. METHODS: Reverse transcriptase/polymerase (RT/POL) functional motif of P protein of HBV was cloned in pMAL-c vector and expressed as maltose binding fusion protein form. The RT/POL recombinant protein (pMRT/POL) was purified by amylose resin column. Using human single chain Fv phage antibody library with 1.1x10(10) size, human antibody against pMRT/POL was selected with BIAcore panning. Selected antibody fragments were analyzed for the activity of RT inhibition. Finally, they were analyzed for the affinity with BIAcore and the complementarity determining regions with nucleotide sequencing. RESULTS: pMRT/POL recombinant protein expressed in E. coli showed RT activity, 1microgram of recombinant protein had an activity equivalent to 5 unit of MMLV RT. By BIAcore panning, we could select 3 clones; POL-A5, POL-B8 and POL-B12. Each clone's RT inhibiting activity were 52-82%, affinity against antigen were 8.15x10(-8) M to 1.75x10(-6) M. CONCLUSIONS: Human monoclonal antibodies produced in this study showed low affinity, but efficiently inhibited the activity of RT in vitro. If POL-A5, POL-B8, and POL-B12 can be converted to intracellular antibody form, it can be used for protein-based gene therapy by inhibiting the replication through the neutralization of polymerase protein of HBV.
Antibodies, Monoclonal/biosynthesis/genetics/*pharmacology ; Complementarity Determining Regions/chemistry ; Gene Products, pol/*antagonists & inhibitors/genetics/immunology ; Genetic Vectors ; Hepatitis B virus/enzymology/genetics ; Humans ; Peptide Library ; RNA-Directed DNA Polymerase/genetics/*immunology ; Recombinant Fusion Proteins/biosynthesis/genetics ; Reverse Transcriptase Inhibitors/chemistry/metabolism/*pharmacology