HIV-1 persists lifelong in memory cells of the immune system as latent provirus that rebounds upon treatment interruption. Therefore, the latent reservoir is the main target for an HIV cure. Here, we studied the direct link between integration site and transcription using LEDGINs and Barcoded HIV-ensembles (B-HIVE). LEDGINs are antivirals that inhibit the interaction between HIV-1 integrase and the chromatin-tethering factor LEDGF/p75. They were used as a tool to retarget integration, while the effect on HIV expression was measured with B-HIVE. B-HIVE tracks insert-specific HIV expression by tagging a unique barcode in the HIV genome. We confirmed that LEDGINs retarget integration out of gene-dense and actively transcribed regions. The distance to H3K36me3, the marker recognized by LEDGF/p75, clearly increased. LEDGIN treatment reduced viral RNA expression and increased the proportion of silent provirus. Finally, silent proviruses obtained after LEDGIN treatment were located further away from epigenetic marks associated with active transcription. Interestingly, proximity to enhancers stimulated transcription irrespective of LEDGIN treatment, while the distance to H3K36me3 only changed after treatment with LEDGINs. The fact that proximity to these markers are associated with RNA expression support the direct link between provirus integration site and viral expression.
- MeSH
- buněčné linie MeSH
- chromatin metabolismus MeSH
- histony metabolismus MeSH
- HIV-1 účinky léků genetika metabolismus MeSH
- inhibitory HIV-integrasy farmakologie MeSH
- integrace viru * účinky léků MeSH
- lidé MeSH
- mezibuněčné signální peptidy a proteiny MeSH
- proviry genetika MeSH
- regulace exprese virových genů * účinky léků MeSH
- RNA virová metabolismus MeSH
- umlčování genů * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- chromatin MeSH
- histony MeSH
- inhibitory HIV-integrasy MeSH
- lens epithelium-derived growth factor MeSH Prohlížeč
- mezibuněčné signální peptidy a proteiny MeSH
- RNA virová MeSH
Arthropod-borne flaviviruses are human pathogens of global medical importance, against which no effective small molecule-based antiviral therapy has currently been reported. Arbidol (umifenovir) is a broad-spectrum antiviral compound approved in Russia and China for prophylaxis and treatment of influenza. This compound shows activities against numerous DNA and RNA viruses. The mode of action is based predominantly on impairment of critical steps in virus-cell interactions. Here we demonstrate that arbidol possesses micromolar-level anti-viral effects (EC50 values ranging from 10.57 ± 0.74 to 19.16 ± 0.29 µM) in Vero cells infected with Zika virus, West Nile virus, and tick-borne encephalitis virus, three medically important representatives of the arthropod-borne flaviviruses. Interestingly, no antiviral effects of arbidol are observed in virus infected porcine stable kidney cells (PS), human neuroblastoma cells (UKF-NB-4), and human hepatoma cells (Huh-7 cells) indicating that the antiviral effect of arbidol is strongly cell-type dependent. Arbidol shows increasing cytotoxicity when tested in various cell lines, in the order: Huh-7 < HBCA < PS < UKF-NB-4 < Vero with CC50 values ranging from 18.69 ± 0.1 to 89.72 ± 0.19 µM. Antiviral activities and acceptable cytotoxicity profiles suggest that arbidol could be a promising candidate for further investigation as a potential therapeutic agent in selective treatment of flaviviral infections.
- Klíčová slova
- antiviral activity, arbidol, cell-type dependent antiviral effect, cytotoxicity, flavivirus, umifenovir,
- MeSH
- antivirové látky farmakologie toxicita MeSH
- buněčné linie MeSH
- Cercopithecus aethiops MeSH
- členovci - vektory virologie MeSH
- Flavivirus účinky léků genetika MeSH
- indoly farmakologie toxicita MeSH
- infekce viry z rodu Flavivirus virologie MeSH
- inhibiční koncentrace 50 MeSH
- lidé MeSH
- proteiny virového obalu genetika MeSH
- regulace exprese virových genů účinky léků MeSH
- Vero buňky MeSH
- viabilita buněk účinky léků MeSH
- virus západního Nilu účinky léků genetika MeSH
- virus zika účinky léků genetika MeSH
- viry klíšťové encefalitidy účinky léků genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- antivirové látky MeSH
- indoly MeSH
- proteiny virového obalu MeSH
- umifenovir MeSH Prohlížeč
The development of the Escherichia coli expression system, which was prepared by transferring the F' episome from strain 71/18 to a highly to a transformable F- strain HB101, is described. These new HB101 (F+) cells, which produced high levels of lac repressor, were capable of taking up lactose and grew under strict selection conditions. A relatively simple two-step purification of part of a protein (M(r) 27,000) encoded by the gag gene of HIV-1 in this expression system is described. The supernatant prepared by removal of cell debris was precipitated by 30% saturation of ammonium sulphate. The protein spectrum was characterized by gel electrophoresis, immunoblotting and ion-exchange titration curves. Optimum separation was achieved using a strong anion exchanger (Mono Q) at pH 8.0. The purified protein did not cross-react with antibodies to E. coli.
- MeSH
- chromatografie iontoměničová MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- ELISA MeSH
- Escherichia coli účinky léků genetika metabolismus MeSH
- genové produkty gag biosyntéza izolace a purifikace MeSH
- HIV-1 genetika MeSH
- isopropylthiogalaktosid farmakologie MeSH
- laktosa farmakologie MeSH
- lidé MeSH
- protilátky bakteriální izolace a purifikace MeSH
- regulace exprese virových genů účinky léků MeSH
- rekombinantní proteiny chemie izolace a purifikace MeSH
- virové geny genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- genové produkty gag MeSH
- isopropylthiogalaktosid MeSH
- laktosa MeSH
- protilátky bakteriální MeSH
- rekombinantní proteiny MeSH
We have studied the inhibitory effect of antisense oligodeoxynucleotides on the expression of hepatitis B virus surface antigens. Human hepatoma cell line PLC/PRF/5 harbors several integrated copies of the HBV genome and produces and secretes hepatitis B virus surface antigen (HBsAg) to the medium. Synthetic antisense oligodeoxynucleotides complementary to various regions of the surface antigen gene were synthesized and their ability to block its expression was tested. Oligodeoxynucleotides (17- and 21-mers) complementary to regions covering ATG codons of both preS2 and S genes significantly inhibited preS2 and S protein production. Less efficient inhibition was achieved when the oligonucleotide complementary to the inside S gene region was assayed.
- MeSH
- antisense oligonukleotidy farmakologie MeSH
- buněčné linie MeSH
- ELISA MeSH
- hepatitida B - antigeny povrchové genetika metabolismus MeSH
- hepatocelulární karcinom mikrobiologie MeSH
- lidé MeSH
- messenger RNA metabolismus MeSH
- molekulární sekvence - údaje MeSH
- regulace exprese virových genů účinky léků MeSH
- sekvence nukleotidů MeSH
- virové geny účinky léků MeSH
- virové strukturální proteiny genetika MeSH
- virus hepatitidy B genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- antisense oligonukleotidy MeSH
- hepatitida B - antigeny povrchové MeSH
- messenger RNA MeSH
- virové strukturální proteiny MeSH
Polyoma (Py) virus-specific RNA, synthesized at reduced level in infected cells in the presence of antiviral compound 5-bromo-2'-deoxyuridine (BrdUrd) was characterized in more detail by Northern blot hybridization analysis. The results obtained with total, cytoplasmic and poly(A)RNA, isolated from mouse embryo cell cultures 42 hrs p.i. indicate that BrdUrd (6.34 micrograms/ml) lowers the level of typical classes of major virus DNA transcripts to a similar extent and that no new, atypical transcription products are formed.
- MeSH
- bromodeoxyuridin farmakologie MeSH
- genetická transkripce účinky léků MeSH
- messenger RNA biosyntéza MeSH
- northern blotting MeSH
- poly A biosyntéza MeSH
- Polyomavirus účinky léků genetika metabolismus MeSH
- regulace exprese virových genů účinky léků MeSH
- replikace viru účinky léků MeSH
- RNA virová biosyntéza MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- bromodeoxyuridin MeSH
- messenger RNA MeSH
- poly A MeSH
- RNA virová MeSH
Synthesis of the major DNA-binding protein (ICP8) was investigated in primary rabbit kidney (RK) and Vero cells infected with the syncytial (syn) strain HSZP or with the non-syn strain KOS of herpes simplex virus type 1 (HSV-1). Results showed the following: 1. In contrast to strain KOS, the rate of viral polypeptide synthesis was accelerated in Vero cells infected with strain HSZP. The ICP8 could be detected in the nuclei of cells by one hour post-infection (hr p. i.) where it became associated with the viral DNA (DNase sensitive form). Later on (7 hr p.i.), the synthesis of viral polypeptides decreased and no further translocation of ICP8 from the cytoplasm into the nucleus was observed. 2. Strain HSZP was approx. three times more resistant to the action of phosphonoacetic acid (PAA) than strain KOS. In order to block the synthesis of HSZP gamma-2 polypeptides, a concentration of 600 micrograms PAA/ml had to be used. Under this condition, the HSZP ICP8 was translocated into the cell nucleus at later interval only (7 hr p.i.), and it was still possible to release this polypeptide from the nucleus by DNase treatment. The failure of the HSZP ICP8 to associate with the nuclear matrix (DNase resistant form) of infected cells in the absence of viral DNA replication may reflect its predominant affinity for the viral DNA which, in turn, may be responsible for the observed accelerated synthesis of the HSZP polypeptides in infected Vero cells. 3. In primary RK cells infected with strain HSZP the ICP8 did not translocate into the cell nucleus. Therefore, no gamma-2 polypeptides were synthesized.
- MeSH
- DNA vazebné proteiny MeSH
- DNA virů metabolismus MeSH
- druhová specificita MeSH
- fibroblasty MeSH
- jaderná matrix metabolismus MeSH
- králíci MeSH
- kultivované buňky MeSH
- kyselina fosfonoctová farmakologie MeSH
- posttranslační úpravy proteinů účinky léků MeSH
- regulace exprese virových genů účinky léků MeSH
- replikace DNA MeSH
- Simplexvirus klasifikace účinky léků metabolismus fyziologie MeSH
- Vero buňky MeSH
- virové proteiny biosyntéza MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
- Názvy látek
- DNA vazebné proteiny MeSH
- DNA virů MeSH
- ICP8 protein, Simplexvirus MeSH Prohlížeč
- kyselina fosfonoctová MeSH
- virové proteiny MeSH
