Studium resistentních kmenů HIV v pacientech léčených inhibitory proteasy. Sekvenování, klonování a charakterisace rekombinantních mutantních proteas. Návrh specifických inhibitorů. Evoluce proteasy a dalších virových genů u HIV positivních pacientů.; Resistant HIV strains in patients treated by protease inhibitors. Sequencing, cloning and analysis of recombinant mutant proteases. Design of a broad specificity inhibitor. Co-evolution of protease and pther viral genes in HIV under selection pressure.

• MeSH
• DNA virů MeSH
• HIV reverzní transkriptasa MeSH
• HIV-1 genetika   MeSH
• virová léková rezistence MeSH

• Konspekt
• Patologie. Klinická medicína
• NLK Obory
• dermatovenerologie
• biologie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu IGA MZ ČR

Z lymfocytů pacientů s AIDS léčených inhibitory proteasy HIV budou sekvenovány a charakterisovány in vitro i ve tkáňové kultuře mutanty proteasy, resistentní vůči původnímu inhibitoru, a otestovány pomocí panelu struktuálně rozdílných inhibitorů.; Resistant mutants of the HIV protease from the blood lymphocytes of AIDS patients treated with protease inhibitors will be amplified, sequenced, characterized in vitro and in tissue culture and tested with a panel of structurally different inhibitors.

• MeSH
• AIDS terapie   MeSH
• antibiotická rezistence MeSH
• molekulární modely MeSH
• molekulární struktura MeSH

• Konspekt
• Virologie
• NLK Obory
• dermatovenerologie
• biologie
• virologie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu IGA MZ ČR

Nicotinamide adenine dinucleotide (NAD) is a critical component of the cellular metabolism and also serves as an alternative 5' cap on various RNAs. However, the function of the NAD RNA cap is still under investigation. We studied NAD capping of RNAs in HIV-1-infected cells because HIV-1 is responsible for the depletion of the NAD/NADH cellular pool and causing intracellular pellagra. By applying the NAD captureSeq protocol to HIV-1-infected and uninfected cells, we revealed that four snRNAs (e.g., U1) and four snoRNAs lost their NAD cap when infected with HIV-1. Here, we provide evidence that the presence of the NAD cap decreases the stability of the U1/HIV-1 pre-mRNA duplex. Additionally, we demonstrate that reducing the quantity of NAD-capped RNA by overexpressing the NAD RNA decapping enzyme DXO results in an increase in HIV-1 infectivity. This suggests that NAD capping is unfavorable for HIV-1 and plays a role in its infectivity.

• MeSH
• HIV infekce * virologie   metabolismus   MeSH
• HIV-1 * MeSH
• lidé MeSH
• malá jadérková RNA * metabolismus   genetika   MeSH
• NAD * metabolismus   MeSH
• RNA čepičky metabolismus   MeSH
• RNA malá jaderná * metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Adhesion G protein-coupled receptors (aGPCRs) play an important role in neurodevelopment, immune defence and cancer; however, their role throughout viral infections is mostly unexplored. We have been searching for specific aGPCRs involved in SARS-CoV-2 infection of mammalian cells. In the present study, we infected human epithelial cell lines derived from lung adenocarcinoma (Calu-3) and colorectal carcinoma (Caco-2) with SARS-CoV-2 in order to analyse changes in the level of mRNA encoding individual aGPCRs at 6 and 12 h post infection. Based on significantly altered mRNA levels, we identified four aGPCR candidates-ADGRB3/BAI3, ADGRD1/GPR133, ADGRG7/GPR128 and ADGRV1/GPR98. Of these receptors, ADGRD1/GPR133 and ADGRG7/GPR128 showed the largest increase in mRNA levels in SARS-CoV-2-infected Calu-3 cells, whereas no increase was observed with heat-inactivated SARS-CoV-2 and virus-cleared conditioned media. Next, using specific siRNA, we downregulated the aGPCR candidates and analysed SARS-CoV-2 entry, replication and infectivity in both cell lines. We observed a significant decrease in the amount of SARS-CoV-2 newly released into the culture media by cells with downregulated ADGRD1/GPR133 and ADGRG7/GPR128. In addition, using a plaque assay, we observed a reduction in SARS-CoV-2 infectivity in Calu-3 cells. In summary, our data suggest that selected aGPCRs might play a role during SARS-CoV-2 infection of mammalian cells.

• MeSH
• adenokarcinom plic * genetika   virologie   patologie   metabolismus   MeSH
• Caco-2 buňky MeSH
• COVID-19 * genetika   virologie   metabolismus   MeSH
• lidé MeSH
• messenger RNA * genetika   metabolismus   MeSH
• nádorové buněčné linie MeSH
• nádory plic genetika   virologie   patologie   metabolismus   MeSH
• receptory spřažené s G-proteiny * metabolismus   genetika   MeSH
• SARS-CoV-2 * genetika   fyziologie   metabolismus   MeSH
• upregulace * genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cause life-threatening diseases in millions of people worldwide, in particular, in patients with cancer, and there is an urgent need for antiviral agents against this infection. While in vitro activities of artemisinins against SARS-CoV-2 and cancer have recently been demonstrated, no study of artemisinin and/or synthetic peroxide-based hybrid compounds active against both cancer and SARS-CoV-2 has been reported yet. However, the hybrid drug's properties (e. g., activity and/or selectivity) can be improved compared to its parent compounds and effective new agents can be obtained by modification/hybridization of existing drugs or bioactive natural products. In this study, a series of new artesunic acid and synthetic peroxide based new hybrids were synthesized and analyzed in vitro for the first time for their inhibitory activity against SARS-CoV-2 and leukemia cell lines. Several artesunic acid-derived hybrids exerted a similar or stronger potency against K562 leukemia cells (81-83 % inhibition values) than the reference drug doxorubicin (78 % inhibition value) and they were also more efficient than their parent compounds artesunic acid (49.2 % inhibition value) and quinoline derivative (5.5 % inhibition value). Interestingly, the same artesunic acid-quinoline hybrids also show inhibitory activity against SARS-CoV-2 in vitro (EC50 13-19 μm) and no cytotoxic effects on Vero E6 cells (CC50 up to 110 μM). These results provide a valuable basis for design of further artemisinin-derived hybrids to treat both cancer and SARS-CoV-2 infections.

• MeSH
• antivirové látky farmakologie   terapeutické užití   MeSH
• artemisininy * farmakologie   MeSH
• Cercopithecus aethiops MeSH
• chinoliny * terapeutické užití   MeSH
• COVID-19 * MeSH
• farmakoterapie COVID-19 MeSH
• leukemie * farmakoterapie   MeSH
• lidé MeSH
• nádory * farmakoterapie   MeSH
• peroxidy MeSH
• SARS-CoV-2 MeSH
• Vero buňky MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Positive-sense single-stranded RNA (+RNA) viruses have proven to be important pathogens that are able to threaten and deeply damage modern societies, as illustrated by the ongoing COVID-19 pandemic. Therefore, compounds active against most or many +RNA viruses are urgently needed. Here, we present PR673, a helquat-like compound that is able to inhibit the replication of SARS-CoV-2 and tick-borne encephalitis virus in cell culture. Using in vitro polymerase assays, we demonstrate that PR673 inhibits RNA synthesis by viral RNA-dependent RNA polymerases (RdRps). Our results illustrate that the development of broad-spectrum non-nucleoside inhibitors of RdRps is feasible.

• MeSH
• COVID-19 * MeSH
• lidé MeSH
• pandemie MeSH
• RNA-dependentní RNA-polymerasa MeSH
• SARS-CoV-2 MeSH
• viry klíšťové encefalitidy * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Chronic hepatitis caused by infection with the Hepatitis B virus is a life-threatening condition. In fact, 1 million people die annually due to liver cirrhosis or hepatocellular carcinoma. Recently, several studies demonstrated a molecular connection between the host DNA damage response (DDR) pathway and HBV replication and reactivation. Here, we investigated the role of Ataxia-telangiectasia-mutated (ATM) and Ataxia telangiectasia and Rad3-related (ATR) PI3-kinases in phosphorylation of the HBV core protein (HBc). We determined that treatment of HBc-expressing hepatocytes with genotoxic agents, e.g., etoposide or hydrogen peroxide, activated the host ATM-Chk2 pathway, as determined by increased phosphorylation of ATM at Ser1981 and Chk2 at Thr68. The activation of ATM led, in turn, to increased phosphorylation of cytoplasmic HBc at serine-glutamine (SQ) motifs located in its C-terminal domain. Conversely, down-regulation of ATM using ATM-specific siRNAs or inhibitor effectively reduced etoposide-induced HBc phosphorylation. Detailed mutation analysis of S-to-A HBc mutants revealed that S170 (S168 in a 183-aa HBc variant) is the primary site targeted by ATM-regulated phosphorylation. Interestingly, mutation of two major phosphorylation sites involving serines at positions 157 and 164 (S155 and S162 in a 183-aa HBc variant) resulted in decreased etoposide-induced phosphorylation, suggesting that the priming phosphorylation at these serine-proline (SP) sites is vital for efficient phosphorylation of SQ motifs. Notably, the mutation of S172 (S170 in a 183-aa HBc variant) had the opposite effect and resulted in massively up-regulated phosphorylation of HBc, particularly at S170. Etoposide treatment of HBV infected HepG2-NTCP cells led to increased levels of secreted HBe antigen and intracellular HBc protein. Together, our studies identified HBc as a substrate for ATM-mediated phosphorylation and mapped the phosphorylation sites. The increased expression of HBc and HBe antigens in response to genotoxic stress supports the idea that the ATM pathway may provide growth advantage to the replicating virus.

• MeSH
• aminokyselinové motivy MeSH
• ATM protein metabolismus   MeSH
• buňky Hep G2 MeSH
• checkpoint kinasa 2 metabolismus   MeSH
• cytoplazma metabolismus   virologie   MeSH
• etoposid farmakologie   MeSH
• fosforylace MeSH
• hepatitida B - antigeny e metabolismus   MeSH
• hepatocyty virologie   MeSH
• lidé MeSH
• peroxid vodíku farmakologie   MeSH
• poškození DNA * MeSH
• proteiny virového jádra chemie   metabolismus   MeSH
• replikace viru účinky léků   MeSH
• serin metabolismus   MeSH
• trans-aktivátory genetika   metabolismus   MeSH
• virové regulační a přídatné proteiny genetika   metabolismus   MeSH
• virus hepatitidy B účinky léků   fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• AIDS * farmakoterapie   komplikace   MeSH
• antiretrovirové látky aplikace a dávkování   terapeutické užití   MeSH
• HIV infekce * farmakoterapie   komplikace   MeSH
• Kaposiho sarkom farmakoterapie   komplikace   MeSH
• látky proti HIV aplikace a dávkování   terapeutické užití   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mnohočetná virová léková rezistence MeSH
• počet CD4 lymfocytů MeSH
• výsledek terapie MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• kazuistiky MeSH

Infection with hepatitis B virus (HBV) often leads to development of chronic liver disease. In fact, 10% of infected adults and almost 90% of infected infants develop chronic hepatitis B associated with severe liver diseases, including acute liver failure, liver cirrhosis or hepatocellular carcinoma. At present there is no effective cure for chronic hepatitis B. The current treatment of chronically infected patients is long-term, expensive and relies on treatment with nucleos(t)ide analogs in combination with immune therapies, that frequently lead to adverse side effects. Recently, the National Institute of Health proposed strategic plan for Trans-NIH research to cure hepatitis B. The key priority is better understanding of HBV life cycle and its interactions with host cell. Due to the fact that HBV is a small double stranded DNA virus encoding only a limited number of proteins, HBV replication widely relies on host cell pathways and proteins. As demonstrated by numerous reports, HBV core protein (HBc) which is the main component of viral nucleocapsid, plays multiple roles in HBV life cycle and is engaged in many protein interaction networks of the host cell. Several recent studies have shown that HBV proteins can be modified by different types of posttranslational modifications (PTMs) that affect their protein-protein interactions, subcellular localization and function. In this review, we discuss diverse PTMs of HBc and their role in regulation of HBc function in the context of HBV replication and pathogenesis.

• MeSH
• fosforylace MeSH
• lidé MeSH
• posttranslační úpravy proteinů MeSH
• proteinarginin-N-methyltransferasy MeSH
• proteiny virového jádra genetika   MeSH
• ubikvitinace MeSH
• virus hepatitidy B * genetika   patogenita   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH
• přehledy MeSH

Úvod: Vstup viru lidské imunitní nedostatečnosti typu 1 (HIV-1) do cílových buněk je umožněn CD4 receptorem a jedním ze dvou koreceptorů CXCR4 nebo CCR5. Delece úseku 32 nukleotidů v genu pro CCR5 (CCR5Δ32) v obou alelách poskytuje silnou, avšak ne absolutní odolnost proti infekci HIV-1 a delece v jedné alele zpomaluje postup nemoci směřující k rozvinutí AIDS. Zde jsme analyzovali prevalenci a roli heterozygotního výskytu CCR5Δ32 na postup onemocnění u HIV pozitivních jedinců v České republice. Metody: Celkem 92 HIV-1 séropozitivních osob včetně 80 Čechů z AIDS centra v Nemocnici Na Bulovce v Praze bylo zařazeno do genotypizace CCR5, která byla součástí studie role HIV fitness na průběh onemocnění. Z periferních mononukleárních buněk pacienta byla získána DNA, která byla použita k amplifikaci pomocí PCR v reálném čase použitím specifických sond, které detekovaly divokou variantu CCR5 a variantu s 32 nt delecí. Podmnožina 74 pacientů bez antiretrovirové léčby, kteří byli sledováni déle než jeden rok, byla použita k určení role heterozygotního fenotypu CCR5 na průběh nemoci. Výsledky: Heterozygotní CCR5Δ32 varianta byla nalezena u 23,8 % z 80 českých HIV-1 séropozitivních osob, což je velmi podobné jako publikovaná 21 % a 24 % prevalence u HIV negativní české populace. Homozygotní mutovaná varianta nebyla nalezena. U skupiny osob s heterozygotním CCR5 fenotypem jsme pozorovali slabě zvýšený průměrný počet CD4-pozitivních T-lymfocytů a nižší průměrné hodnoty virémie v plazmě. Závěr: Celkově, naše studie neukázala žádný zřejmý užitek přítomnosti heterozygotní CCR5Δ32 varianty na přenos HIV a pouze malý užitek na průběh nemoci u české HIV-1 pozitivní kohorty.

Background: Entry of human immunodeficiency virus type 1 (HIV-1) in target cells is enabled by CD4 receptor and one of two co-receptors, CXCR4 or CCR5. Deletion of 32 bp in CCR5 gene (CCR5Δ32) in both alleles provides strong but not absolute resi-stance to HIV-1 infection and deletion in one allele slows disease progression to AIDS. Here, we analyzed the prevalence and the role of CCR5Δ32 heterozygosity on the disease progression in HIV positive patients in the Czech Republic. Patients and methods: A total of 92 HIV-1 seropositive subjects that included 80 Czech individuals from the AIDS center in the Hospital Na Bulovce in Prague were enrolled in CCR5 genoty-ping as a part of a study of the role of HIV fitness on disease progression. DNA was extracted from patient’s peripheral blood mononuclear cells and subjected to real-time PCR with specific probes detecting wild-type and 32 bp-deleted CCR5 variants. A subgroup of 74 antiretroviral therapy-naive patients with more than one year of follow-up was used to determine the role of the CCR5Δ32 heterozygous phenotype in disease progression. Results: CCR5Δ32 was found heterozygous in 23.8% of 80 Czech HIV-1 seropositive individuals which is very similar to 21% and 24% prevalence reported in HIV negative Czech population. Homozygous mutant variant was not detected. In CCR5Δ32 he-terozygous group we observed slightly higher mean CD4+ T-cell count and lower mean plasma viremia levels.Conclusions: Overall, our study indicates no obvious benefit of CCR5Δ32 heterozygosity on HIV transmission and only small benefit on disease progression in the Czech HIV-1 cohort.

• MeSH
• genotypizační techniky MeSH
• HIV-1 * genetika   izolace a purifikace   MeSH
• kohortové studie MeSH
• lidé MeSH
• polymorfismus genetický MeSH
• progrese nemoci MeSH
• receptory CCR5 genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika MeSH