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.
- Klíčová slova
- ATM, ATR, DNA damage response pathway, HBV core protein, serine phosphorylation,
- 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
- Názvy látek
- ATM protein, human MeSH Prohlížeč
- ATM protein MeSH
- checkpoint kinasa 2 MeSH
- CHEK2 protein, human MeSH Prohlížeč
- etoposid MeSH
- hepatitida B - antigeny e MeSH
- hepatitis B virus X protein MeSH Prohlížeč
- peroxid vodíku MeSH
- proteiny virového jádra MeSH
- serin MeSH
- trans-aktivátory MeSH
- virové regulační a přídatné proteiny MeSH
Shortly after entering the cell, HIV-1 copies its genomic RNA into double-stranded DNA in a process known as reverse transcription. This process starts inside a core consisting of an enclosed lattice of capsid proteins that protect the viral RNA from cytosolic sensors and degradation pathways. To accomplish reverse transcription and integrate cDNA into the host cell genome, the capsid shell needs to be disassembled, or uncoated. Premature or delayed uncoating attenuates reverse transcription and blocks HIV-1 infectivity. Small molecules that bind to the capsid lattice of the HIV-1 core and either destabilize or stabilize its structure could thus function as effective HIV-1 inhibitors. To screen for such compounds, we modified our recently developed FAITH assay to allow direct assessment of the stability of in vitro preassembled HIV-1 capsid-nucleocapsid (CANC) tubular particles. This new assay is a high-throughput fluorescence method based on measuring the amount of nucleic acid released from CANC complexes under disassembly conditions. The amount of disassembled CANC particles and released nucleic acid is proportional to the fluorescence signal, from which the relative percentage of CANC stability can be calculated. We consider our assay a potentially powerful tool for in vitro screening for compounds that alter HIV disassembly.
- MeSH
- HIV infekce farmakoterapie MeSH
- HIV-1 účinky léků fyziologie MeSH
- látky proti HIV chemie izolace a purifikace farmakologie MeSH
- lidé MeSH
- nukleokapsida analýza účinky léků MeSH
- proteiny virového jádra chemie genetika metabolismus MeSH
- RNA virová genetika MeSH
- rychlé screeningové testy MeSH
- sekvence aminokyselin MeSH
- sekvence nukleotidů MeSH
- svlékání virového obalu účinky léků genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- látky proti HIV MeSH
- proteiny virového jádra MeSH
- RNA virová MeSH
Nucleotide sequences are presented for 12, 7 and 12 cloned extrachromosomal DNAs by nature harbored in nucleoprotein (NP) complexes forming chicken leukaemic myeloblast (CHLM) post microsomal sediment (POMS) components A, B and C, respectively, and for 11 cloned avian myeloblastosis virus (AMV) DNAs. Analysis of the abundance of sequence motifs significant for eukaryotic chromosomal DNA replication origin (ori) regions (and their initiation zones) has shown that these DNAs are reminiscent of cell DNA fragments enriched in ori sequences (Rao et al., 1990) and/or sequence features of several eukaryotic chromosomal oris containing clusters of modular sequence elements (Dobbs et al., 1994). Accordingly, these DNAs, with an (A + T) content prevalently higher than that of the total cell DNA, revealed the presence of asymmetrically distributed (A + T)-rich stretches, scaffold attachment region (SAR) T consensuses, polypyrimidine nucleotide (poly(Py)) tracts and minimal Saccharomyces cerevisiae autonomously replicating sequence (ARS) consensus, in abundance comparable with that of these sequences of DNA fragments enriched in oris. All these DNAs were found to be enriched also in sequence elements held as primase (Pr) attachment sites. Moreover, DNAs of POMS component B and those of AMV DNA were found to be enriched in the asymmetric pyrimidine (Py) heptanucleotide motif of Waltz et al. (1996) occurring in the initiation zones of ori region. Consequently, these extrachromosomal DNAs, portion of which represents a precursor of AMV DNA, seem to descent from initiation zones of various ori regions of an early replicating chromosomal myeloblast DNA. In addition, a possible explanation of the inclination of these DNAs to form multimers is presented.
- MeSH
- chromozomy chemie genetika MeSH
- cytoplazma chemie MeSH
- deoxyadenosiny chemie MeSH
- DNA nádorová chemie genetika MeSH
- DNA virů chemie genetika MeSH
- kur domácí MeSH
- lidé MeSH
- molekulární sekvence - údaje MeSH
- nádorové buňky kultivované MeSH
- nukleoproteiny chemie genetika MeSH
- proteiny virového jádra chemie genetika MeSH
- ptačí leukóza genetika MeSH
- replikační počátek genetika MeSH
- sekvence nukleotidů MeSH
- sekvenční analýza DNA MeSH
- thymidin chemie MeSH
- virus ptačí myeloblastózy genetika MeSH
- zastoupení bazí MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- deoxyadenosiny MeSH
- DNA nádorová MeSH
- DNA virů MeSH
- nukleoproteiny MeSH
- proteiny virového jádra MeSH
- thymidin MeSH
RNA-synthesizing activities (RNA-SAs) by its nature identical with primase activities (Pr-As) were found to be constantly present in avain myeloblastosis virus (AMV) core isolates. Their endogenous templates are molecules of the virus core-bound host cell DNA (AMV DNA) (Ríman and Beaudreau, 1970) that have been recently recognized as a collection of still active early replicative structures (Ríman et al., 1993b). Like the Pr-As, the RNA-SAs are not inhibited by alpha-amanitin nor by aphidicolin and they show a mutually competitive affinity for ATP and GTP. Their reaction products treated with DNase I are short RNAs similar in length to initiator RNAs (iRNAs), their precursors and degradation products. In AMV core proteins separated in isopycnic CsCl gradients, they are chiefly located in the density region of reverse transcriptase activities (RT-As) but with a distinct peak fraction. Like Pr-As, they are able to use poly(dT) as template and to form, in the presence of [alpha-32P]ATP, products that after DNase I treatment consist of poly(rA) molecules similar in length to iRNA monomers and multimers. Like the Pr-As, they are able to complement E. coli DNA polymerase (pol) I reactions. They occur in the analyzed AMV core proteins as six distinct sedimentation species (PrA-SS). This, together with other relevant properties, indicates the presence of Pr-As associated with molecules of a primase-alpha DNA polymerase enzyme complex, its degradation products and 'free' primase monomers.
- MeSH
- DNA-primasa MeSH
- proteiny virového jádra chemie metabolismus MeSH
- ptáci MeSH
- RNA virová metabolismus MeSH
- RNA-nukleotidyltransferasy chemie metabolismus MeSH
- virus ptačí myeloblastózy enzymologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- DNA-primasa MeSH
- proteiny virového jádra MeSH
- RNA virová MeSH
- RNA-nukleotidyltransferasy MeSH
