Nucleos(t)ide analogues entecavir (ETV) and tenofovir disoproxil fumarate (TDF) are recommended as first-line monotherapies for chronic hepatitis B (CHB). Multiple HBV genotypes/subgenotypes have been described, but their impact on treatment response remains largely elusive. We investigated the effectiveness of ETV/TDF on HBV/D-subgenotypes, D1/D2/D3/D5, studied the structural/functional differences in subgenotype-specific reverse transcriptase (RT) domains of viral polymerase, and identified novel molecules with robust inhibitory activity on various D-subgenotypes. Transfection of Huh7 cells with full-length D1/D2/D3/D5 and in vitro TDF/ETV susceptibility assays demonstrated that D1/D2 had greater susceptibility to TDF/ETV while D3/D5 exhibited poorer response. Additionally, HBV load was substantially reduced in TDF-treated CHB patients carrying D1/D2 but minimally reduced in D3/D5-infected patients. Comparison of RT sequences of D-subgenotypes led to identification of unique subgenotype-specific residues, and molecular modeling/docking/simulation studies depicted differential bindings of TDF/ETV to the active site of their respective RTs. Replacement of signature residues in D3/D5 HBV clones with corresponding amino acids seen in D1/D2 improved their susceptibility to TDF/ETV. Using high throughput virtual screening, we identified N(9)-[3-fluoro-2-(phosphonomethoxy)propyl] (FPMP) derivatives of purine bases, including N6-substituted (S)-FPMP derivative of 2,6-diaminopurine (DAP) (OB-123-VK), as potential binders of RT of different D-subgenotypes. We synthesized (S)-FPMPG prodrugs (FK-381-FEE/FK-381-SEE/FK-382) and tested their effectiveness along with OB-123-VK. Both OB-123-VK and FK-381-FEE exerted similar antiviral activities against all D-subgenotypes, although FK-381-FEE was more potent. Our study highlighted the natural variation in therapeutic response of D1/D2/D3/D5 and emphasized the need for HBV subgenotype determination before treatment. Novel molecules described here could benefit future design/discovery of pan-D-subgenotypic inhibitors. IMPORTANCE Current treatment of chronic hepatitis B relies heavily on nucleotide/nucleoside analogs in particular, tenofovir disoproxil fumarate (TDF) and entecavir (ETV) to keep HBV replication under control and prevent end-stage liver diseases. However, it was unclear whether the therapeutic effects of TDF/ETV differ among patients infected with different HBV genotypes and subgenotypes. HBV genotype D is the most widespread of all HBV genotypes and multiple D-subgenotypes have been described. We here report that different subgenotypes of HBV genotype-D exhibit variable response toward TDF and ETV and this could be attributed to naturally occurring amino acid changes in the reverse transcriptase domain of the subgenotype-specific polymerase. Further, we identified novel molecules and also synthesized prodrugs that are equally effective on different D-subgenotypes and could facilitate management of HBV/D-infected patients irrespective of D-subgenotype.

• Klíčová slova
• antiviral activity, entecavir, hepatitis B virus D-subgenotypes, high throughput virtual screening, phosphonate prodrug, tenofovir,
• MeSH
• antivirové látky chemie   farmakologie   terapeutické užití   MeSH
• chronická hepatitida B farmakoterapie   virologie   MeSH
• genotyp MeSH
• guanin analogy a deriváty   chemie   farmakologie   terapeutické užití   MeSH
• inhibitory reverzní transkriptasy chemie   farmakologie   terapeutické užití   MeSH
• lidé MeSH
• mutace MeSH
• organofosfonáty chemie   farmakologie   MeSH
• prekurzory léčiv MeSH
• proteinové domény MeSH
• racionální návrh léčiv * MeSH
• reverzní transkriptasa chemie   genetika   MeSH
• tenofovir chemie   farmakologie   terapeutické užití   MeSH
• virová léková rezistence účinky léků   genetika   MeSH
• virová nálož účinky léků   MeSH
• virus hepatitidy B účinky léků   enzymologie   genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antivirové látky MeSH
• entecavir MeSH Prohlížeč
• guanin MeSH
• inhibitory reverzní transkriptasy MeSH
• organofosfonáty MeSH
• prekurzory léčiv MeSH
• reverzní transkriptasa MeSH
• tenofovir MeSH

Mutations occurring in viral polymerase gene of hepatitis B virus (HBV) due to the use of nucleos(t)id analogs reduce the activity of the drugs by causing antiviral resistance. In this study, it was aimed to evaluate mutations responsible for drug resistance and drug resistance mutation rates in patients followed up by the diagnosis of chronic hepatitis B (CHB). A total of 318 CHB patients were included in the study. HBV mutations were detected using the INNO-LiPA commercial kit based on the reverse hybridization principle. Drug resistance mutation was detected in 46.86% (149/318) of the patients. The rates of drug resistance were found 36.79% (117/318) for lamivudine resistance, 12.58% (40/318) for entecavir (ETV), and 7.86% (25/318) for adefovir. In 10 patients, the possible tenofovir (TDF) resistance (3.14%) was found. Single-drug and double-drug resistances were detected in 34.59% and in 11.01% of the patients, respectively. Triple drug resistance was detected in only 1.26% of the patients. Unlike various studies in Turkey and in other countries, remarkable resistance to ETV and TDF were found in this study. The high rate of the probable TDF resistance was striking, with 3.14%.

• MeSH
• antivirové látky farmakologie   terapeutické užití   MeSH
• chronická hepatitida B farmakoterapie   mikrobiologie   MeSH
• dítě MeSH
• DNA virů genetika   MeSH
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mnohočetná virová léková rezistence genetika   MeSH
• mutace MeSH
• předškolní dítě MeSH
• retrospektivní studie MeSH
• reverzní transkriptasa genetika   MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• virová léková rezistence genetika   MeSH
• virus hepatitidy B účinky léků   genetika   MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Turecko MeSH
• Názvy látek
• antivirové látky MeSH
• DNA virů MeSH
• reverzní transkriptasa MeSH

Mason-Pfizer monkey virus (M-PMV), like some other betaretroviruses, encodes a G-patch domain (GPD). This glycine-rich domain, which has been predicted to be an RNA binding module, is invariably localized at the 3' end of the pro gene upstream of the pro-pol ribosomal frameshift sequence of genomic RNAs of betaretroviruses. Following two ribosomal frameshift events and the translation of viral mRNA, the GPD is present in both Gag-Pro and Gag-Pro-Pol polyproteins. During the maturation of the Gag-Pro polyprotein, the GPD transiently remains a C-terminal part of the protease (PR), from which it is then detached by PR itself. The destiny of the Gag-Pro-Pol-encoded GPD remains to be determined. The function of the GPD in the retroviral life cycle is unknown. To elucidate the role of the GPD in the M-PMV replication cycle, alanine-scanning mutational analysis of its most highly conserved residues was performed. A series of individual mutations as well as the deletion of the entire GPD had no effect on M-PMV assembly, polyprotein processing, and RNA incorporation. However, a reduction of the reverse transcriptase (RT) activity, resulting in a drop in M-PMV infectivity, was determined for all GPD mutants. Immunoprecipitation experiments suggested that the GPD is a part of RT and participates in its function. These data indicate that the M-PMV GPD functions as a part of reverse transcriptase rather than protease.

• MeSH
• buněčné linie MeSH
• lidé MeSH
• Masonův-Pfizerův opičí virus chemie   enzymologie   genetika   MeSH
• polyproteiny chemie   genetika   metabolismus   MeSH
• reverzní transkriptasa chemie   genetika   metabolismus   MeSH
• terciární struktura proteinů MeSH
• virové proteiny chemie   genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• polyproteiny MeSH
• reverzní transkriptasa MeSH
• virové proteiny MeSH

Unlike all of the other retrons, the bacterial retron reverse transcriptase RrtE is capable of synthesizing small double-stranded DNA (sdsDNA) from template RNA. In this study, we analyzed the biosynthesis of the sdsDNA by RrtE in detail. We found out that the initiation of reverse transcription was dependent on a novel self-priming mechanism utilizing a free 3'OH of RNA that is reverse-transcribed into sdsDNA. The priming of the sdsDNA synthesis was not dependent on any particular nucleotide being used as a donor of 3'OH (unlike all of the other retrons, which prime from 2'OH of a particular guanosine) or any particular nucleotide being introduced into the sdsDNA first. Due to the relaxed demands for the initiation of reverse transcription, RrtE has the potential to generate dsDNA from different RNA transcripts in vivo.

• MeSH
• 5' nepřekládaná oblast MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• bakteriální RNA genetika   metabolismus   MeSH
• DNA bakterií biosyntéza   MeSH
• DNA primery chemie   metabolismus   MeSH
• konformace nukleové kyseliny MeSH
• messenger RNA chemie   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• polymerázová řetězová reakce MeSH
• reverzní transkriptasa genetika   metabolismus   MeSH
• ribonukleasa H metabolismus   fyziologie   MeSH
• Salmonella enzymologie   MeSH
• sekvence nukleotidů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 5' nepřekládaná oblast MeSH
• bakteriální proteiny MeSH
• bakteriální RNA MeSH
• DNA bakterií MeSH
• DNA primery MeSH
• messenger RNA MeSH
• reverzní transkriptasa MeSH
• ribonuclease HI MeSH Prohlížeč
• ribonukleasa H MeSH

In Salmonella enterica serovar Typhimurium, retron reverse transcriptase (rrtT), which is part of St 85 retron, is quite ubiquitous and is located in the thdF -yidY intergenic region. In this study, we showed that rrtT is relatively unstable in multidrug resistant, Salmonella genomic island 1 (SGI 1) positive strains. Out of 365 field strains, 55 were free of retron. In 54 of the rrtT negative strains, the excision must have occurred by the same mechanism in which the rrtT together with five other genes was excised. Altogether 8164 bp was missing in the chromosome of the rrtT negative strains. Since the deletion happened exactly between the right inverted repeat of IS 6100 and inside the yieE gene, we propose that intramolecular transposition of IS 6100 followed by homologous recombination was responsible for the excision. Excision of retron together with the right end of SGI 1 may also result in its stabilisation in the Salmonella typhimurium genome. Experimental deletion of rrtT resulted in an accelerated course of infection in orally infected mice. Since the retron excision occurred exclusively in multidrug resistant S. typhimurium, it cannot be excluded that such strains may increase their virulence in the future.

• MeSH
• amplifikace genu MeSH
• antibakteriální látky farmakologie   MeSH
• delece genu MeSH
• integrasy analýza   MeSH
• integrony * MeSH
• mikrobiální testy citlivosti veterinární   MeSH
• mnohočetná bakteriální léková rezistence genetika   MeSH
• molekulární sekvence - údaje MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• náhodné rozdělení MeSH
• polymerázová řetězová reakce metody   veterinární   MeSH
• reverzní transkriptasa genetika   metabolismus   MeSH
• Salmonella typhimurium * účinky léků   enzymologie   genetika   patogenita   MeSH
• salmonelová infekce u zvířat farmakoterapie   MeSH
• sekvence nukleotidů MeSH
• virulence genetika   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antibakteriální látky MeSH
• integrasy MeSH
• reverzní transkriptasa MeSH

This article gives a historical insight into the establishment of suitable models allowing the postulation that chicken Rous sarcoma virus (RSV) becomes integrated in different cells as a provirus. This is documented by the correspondence between two laboratories involved in these investigations. Special attention is paid to RSV-transformed mammalian cells, their virogenic nature, virus rescue by cell fusion, and finally their use for the oncogene v-src characterization. Two sets of experiments are mentioned, which provided an early indication of a transforming gene present in RSV.

• MeSH
• dějiny 20. století MeSH
• genom virový * MeSH
• krysa rodu Rattus MeSH
• kur domácí MeSH
• onkogenní viry genetika   růst a vývoj   MeSH
• proviry genetika   MeSH
• ptačí sarkom genetika   dějiny   virologie   MeSH
• reverzní transkriptasa genetika   dějiny   metabolismus   MeSH
• transformované buněčné linie MeSH
• virová transformace buněk genetika   MeSH
• viry ptačího sarkomu enzymologie   genetika   růst a vývoj   MeSH
• zvířata MeSH
• Check Tag
• dějiny 20. století MeSH
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• historické články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• reverzní transkriptasa MeSH

Bacterial retron reverse transcriptases are unusual enzymes which utilise the same RNA molecule as a template and also as a primer for initiation of the reverse transcription. Except for their relatively frequent presence in Myxococcus spp., they are considered as quite rare proteins. However, in this study we proved that retron reverse transcriptase is frequently found in certain serovars of Salmonella enterica. Using polymerase chain reaction (PCR), in strains of serovar Typhimurium, the rrtT (retron reverse transcriptase Typhimurium) gene was detected in 158 out of 175 tested field strains. On the other hand, in none of the 18 tested serovar Enteritidis strains the rrtT was detected in their genome. Detailed computer analysis allowed us to predict the sequence of msDNA and to propose that the final msDNA is free of any RNA. Furthermore, we predict that there are at least three different classes of retron reverse transcriptases.

• MeSH
• DNA bakterií chemie   MeSH
• konformace nukleové kyseliny MeSH
• molekulární sekvence - údaje MeSH
• operon MeSH
• polymerázová řetězová reakce MeSH
• reverzní transkriptasa genetika   MeSH
• Salmonella typhimurium klasifikace   enzymologie   genetika   MeSH
• sekvence nukleotidů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA bakterií MeSH
• reverzní transkriptasa MeSH

Retron reverse transcriptases are unusual procaryotic enzymes capable of synthesis of low-molecular-weight DNA by reverse transcription. All of the so-far-described DNA species synthesized by retron reverse transcriptases have been identified as multicopy single-stranded DNA. We have shown that Salmonella enterica serovar Enteritidis is also capable of synthesis of the low-molecular-weight DNA by retron reverse transcriptase. Surprisingly, Salmonella serovar Enteritidis-produced low-molecular-weight DNA was shown to be a double-stranded DNA with single-stranded overhangs (sdsDNA). The sdsDNA was 72 nucleotides (nt) long, of which a 38-nt sequence was formed by double-stranded DNA with 19- and 15-nt single-stranded overhangs, respectively. Three open reading frames (ORFs), encoded by the 4,053-bp plasmid, were essential for the production of sdsDNA. These included an ORF with an unknown function, the retron reverse transcriptase, and an ORF encoding the cold shock protein homologue. This plasmid was also able to confer phage resistance onto the host cell by a mechanism which was independent of sdsDNA synthesis.

• MeSH
• bakteriofágy MeSH
• DNA bakterií biosyntéza   genetika   MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• hybridizace nukleových kyselin MeSH
• inzerční mutageneze MeSH
• jednovláknová DNA biosyntéza   genetika   MeSH
• molekulární sekvence - údaje MeSH
• molekulová hmotnost MeSH
• otevřené čtecí rámce MeSH
• plazmidy chemie   genetika   MeSH
• reverzní transkriptasa genetika   MeSH
• Salmonella enterica enzymologie   genetika   virologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA bakterií MeSH
• jednovláknová DNA MeSH
• reverzní transkriptasa MeSH

The gene for bovine leukemia virus (BLV) reverse transcriptase was cloned in prokaryotic expression vector pUC8-2. After fusion of Escherichia coli lacZ gene to different parts of reverse transcriptase we detected expression of new proteins with molecular weights corresponding to the size of the hybrid genes. A coding region most probably responsible for about a hundred-fold decrease in expression of long fusion proteins has been identified. A few possible causes of this phenomenon were tested.

• MeSH
• beta-galaktosidasa genetika   MeSH
• Escherichia coli MeSH
• exprese genu genetika   MeSH
• genetické vektory genetika   MeSH
• geny pol genetika   MeSH
• plazmidy MeSH
• rekombinantní fúzní proteiny biosyntéza   genetika   MeSH
• reverzní transkriptasa genetika   MeSH
• virus bovinní leukemie genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• beta-galaktosidasa MeSH
• rekombinantní fúzní proteiny MeSH
• reverzní transkriptasa MeSH

The permanently BLV-infected FLK cell line 44/2 and FLK sublines were tested for the stability of their BLV and antigen synthesis by three virus markers, p24, gp51, and activity of reverse transcriptase. The extent of BLV production in the FLK line correlated directly with the surface for cell growth in roller and stationary cultures. The synthesis and secretion of non-virus-associated gp51 is especially stimulated in the roller culture, and is largely independent of the quality of the culture medium. The roller culture allows considerable economy of the medium, at the same time retaining its full biological activity. As much as 2 mg of gp51 per litre of culture supernatant was obtained by this procedure. Appropriate markers for estimation of BLV production are p24 and gp51. For this purpose, the activity of reverse transcriptase on its own was not sufficient. The BLV yield differed by one order of magnitude among the 18 cloned FLK sublines. Three sublines have been identified, which showed a comparatively high virus production, under conditions of stationary cultures. The amount or activity of viral markers could not be correlated with the number of BLV proviruses.

• MeSH
• buněčné linie MeSH
• glykoproteiny biosyntéza   genetika   MeSH
• kultivační média MeSH
• ovce MeSH
• proteiny virového jádra biosyntéza   genetika   MeSH
• proteiny virového obalu biosyntéza   genetika   MeSH
• regulace genové exprese MeSH
• replikace viru MeSH
• Retroviridae genetika   MeSH
• reverzní transkriptasa biosyntéza   genetika   MeSH
• virus bovinní leukemie genetika   růst a vývoj   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• glykoproteiny MeSH
• kultivační média MeSH
• proteiny virového jádra MeSH
• proteiny virového obalu MeSH
• reverzní transkriptasa MeSH