Hüseyin Sirma

Replication of ICP0-Null Mutant Herpes Simplex Virus Type 1 Is Restricted by both PML and Sp100

ABSTRACT Herpes simplex virus type 1 (HSV-1) mutants that fail to express the viral immediate-early protein ICP0 have a pronounced defect in viral gene expression and plaque formation in limited-passage human fibroblasts. ICP0 is a RING finger E3 ubiquitin ligase that induces the degradation of several cellular proteins. PML, the organizer of cellular nuclear substructures known as PML nuclear bodies or ND10, is one of the most notable proteins that is targeted by ICP0. Depletion of PML from human fibroblasts increases ICP0-null mutant HSV-1 gene expression, but not to wild-type levels. In this study, we report that depletion of Sp100, another major ND10 protein, results in a similar increase in ICP0-null mutant gene expression and that simultaneous depletion of both proteins complements the mutant virus to a greater degree. Although chromatin assembly and modification undoubtedly play major roles in the regulation of HSV-1 infection, we found that inhibition of histone deacetylase activity with trichostatin A was unable to complement the defect of ICP0-null mutant HSV-1 in either normal or PML-depleted human fibroblasts. These data lend further weight to the hypothesis that ND10 play an important role in the regulation of HSV-1 gene expression.

Direct Association and Nuclear Import of the Hepatitis B Virus X Protein with the NF-κB Inhibitor IκBα

ABSTRACT The X protein of hepatitis B virus (HBV) is a transcriptional activator which is required for infection and may play an important role in HBV-associated hepatocarcinogenesis. It has been suggested that X acts as a nuclear coactivator or stimulates several signal transduction pathways by acting in the cytoplasm. One of these pathways leads to the nuclear translocation of NF-κB. A recent report indicates that X activates NF-κB by acting on two cytoplasmic inhibitors of this family of transcription factors: IκBα and the precursor/inhibitor p105. We demonstrate here that X directly interacts with IκBα, which is able to transport it to the nucleus by a piggyback mechanism. This transport requires a region of IκBα (the second ankyrin repeat) which has been demonstrated to be involved in its nuclear import following NF-κB activation. Using deletion mutants, we showed that amino acids 249 to 253 of IκBα (located in the C-terminal part of the sixth ankyrin repeat) play a critical role in the interaction with X. This small region overlaps one of the domains of IκBα mediating the interaction with the p50 and p65 subunits of NF-κB and is also close to the nuclear export sequence of IκBα, therefore providing a potential explanation for the nuclear accumulation of IκBα with X. This association can also be observed upon the induction of endogenous IκBα by tumor necrosis factor alpha (TNF-α) treatment of Chang cells expressing X. In accordance with this observation, band shift analysis indicates that X induces a sustained NF-κB activation following TNF-α treatment, probably by preventing the reassociation of newly synthesized nuclear IκBα with DNA-bound NF-κB complexes.

PML nuclear bodies are highly organised DNA-protein structures with a function in heterochromatin remodelling at the G2 phase

We have recently demonstrated that heterochromatin HP1 proteins are aberrantly distributed in lymphocytes of patients with immunodeficiency, centromeric instability and facial dysmorphy (ICF) syndrome. The three HP1 proteins accumulate in one giant body over the 1qh and 16qh juxtacentromeric heterochromatins, which are hypomethylated in ICF. The presence of PML (promyelocytic leukaemia) protein within this body suggests it to be a giant PML nuclear body (PML-NB). The structural integrity of PML-NBs is of major importance for normal cell functioning. Nevertheless, the structural organisation and the functions of these nuclear bodies remain unclear. Here, we take advantage of the large size of the giant body to demonstrate that it contains a core of satellite DNA with proteins being organised in ordered concentric layers forming a sphere around it. We extend these results to normal PML-NBs and propose a model for the general organisation of these structures at the G2 phase. Moreover, based on the presence of satellite DNA and the proteins HP1, BRCA1, ATRX and DAXX within the PML-NBs, we propose that these structures have a specific function: the re-establishment of the condensed heterochromatic state on late-replicated satellite DNA. Our findings that chromatin-remodelling proteins fail to accumulate around satellite DNA in PML-deficient NB4 cells support a central role for PML protein in this cellular function.

Expression of mutated hepatitis B virus X genes in human hepatocellular carcinomas

To explore the role of hepatitis B virus (HBV) X protein in liver carcinogenesis, independently from its role in viral replication, we have analyzed X gene structure and expression in tumorous and non‐tumorous tissues obtained from 9 hepatitis B surface antigen (HBsAg)‐negative, HBV DNA‐positive patients. HBV replication was undetectable in tumorous tissues. HBV X gene was truncated at its 3′ end in 5 of 9 tumorous tissues and 1 of 8 non‐tumorous livers. Sequence analysis performed on uninterrupted X genes from 3 tumors and 3 surrounding non‐tumorous tissues showed a high rate of mutations, selectively in the tumorous livers. In 1 of the 3 tumors, a frameshift mutation induced a new stop at codon 129. HBV RNAs were tested by reverse transcriptase‐polymerase chain reaction (RT‐PCR) with surface (S), core (C) and X specific primers. X, but not S and C, RNA expression was found in 6 of 8 tumors and in 6 of 7 non‐tumorous tissues. This finding was consistent with immunohistochemical detection of X, but not S and C, antigens in all tumors also expressing X RNA. Our results provide evidence for selective expression of HBV X, but not S and C, RNA and protein in the tumorous and non‐tumorous tissue of HBsAg‐negative, HBV DNA‐positive patients. It also shows that the structure of the X gene is modified (interrupted or highly mutated) in the majority of tumorous livers. Taken together, our findings are consistent with a potential role of mutated X proteins in HBV‐related liver oncogenesis. Int. J. Cancer 80:497–505, 1999.

Proteasome-dependent degradation of Daxx by the viral E1B-55K protein in human adenovirus-infected cells.

ABSTRACT The death-associated protein Daxx found in PML (promyelocytic leukemia protein) nuclear bodies (PML-NBs) is involved in transcriptional regulation and cellular intrinsic antiviral resistence against incoming viruses. We found that knockdown of Daxx in a nontransformed human hepatocyte cell line using RNA interference (RNAi) techniques results in significantly increased adenoviral (Ad) replication, including enhanced viral mRNA synthesis and viral protein expression. This Daxx restriction imposed upon adenovirus growth is counteracted by early protein E1B-55K (early region 1B 55-kDa protein), a multifunctional regulator of cell-cycle-independent Ad5 replication. The viral protein binds to Daxx and induces its degradation through a proteasome-dependent pathway. We show that this process is independent of Ad E4orf6 (early region 4 open reading frame 6), known to promote the proteasomal degradation of cellular p53, Mre11, DNA ligase IV, and integrin α3 in combination with E1B-55K. These results illustrate the importance of the PML-NB-associated factor Daxx in virus growth restriction and suggest that E1B-55K antagonizes innate antiviral activities of Daxx and PML-NBs to stimulate viral replication at a posttranslational level.

PML isoforms I and II participate in PML-dependent restriction of HSV-1 replication

Intrinsic antiviral resistance mediated by constitutively expressed cellular proteins is one arm of defence against virus infection. Promyelocytic leukaemia nuclear bodies (PML-NBs, also known as ND10) contribute to host restriction of herpes simplex virus type 1 (HSV-1) replication via mechanisms that are counteracted by viral regulatory protein ICP0. ND10 assembly is dependent on PML, which comprises several different isoforms, and depletion of all PML isoforms decreases cellular resistance to ICP0-null mutant HSV-1. We report that individual expression of PML isoforms I and II partially reverses the increase in ICP0-null mutant HSV-1 plaque formation that occurs in PML-depleted cells. This activity of PML isoform I is dependent on SUMO modification, its SUMO interaction motif (SIM), and each element of its TRIM domain. Detailed analysis revealed that the punctate foci formed by individual PML isoforms differ subtly from normal ND10 in terms of composition and/or Sp100 modification. Surprisingly, deletion of the SIM motif from PML isoform I resulted in increased colocalisation with other major ND10 components in cells lacking endogenous PML. Our observations suggest that complete functionality of PML is dependent on isoform-specific C-terminal sequences acting in concert.

NS1- and Minute Virus of Mice-Induced Cell Cycle Arrest: Involvement of p53 and p21 cip1

ABSTRACT The nonstructural protein NS1 of the autonomous parvovirus minute virus of mice (MVMp) is cytolytic when expressed in transformed cells. Before causing extensive cell lysis, NS1 induces a multistep cell cycle arrest in G1, S, and G2, well reproducing the arrest in S and G2 observed upon MVMp infection. In this work we investigated the molecular mechanisms of growth inhibition mediated by NS1 and MVMp. We show that NS1-mediated cell cycle arrest correlates with the accumulation of the cyclin-dependent kinase (Cdk) inhibitor p21 cip1 associated with both the cyclin A/Cdk and cyclin E/Cdk2 complexes but in the absence of accumulation of p53, a potent transcriptional activator of p21 cip1 . By comparison, MVMp infection induced the accumulation of both p53 and p21 cip1 . We demonstrate that p53 plays an essential role in the MVMp-induced cell cycle arrest in both S and G2 by using p53 wild-type (+/+) and null (−/−) cells. Furthermore, only the G2 arrest was abrogated in p21 cip1 null (−/−) cells. Together these results show that the MVMp-induced cell cycle arrest in S is p53 dependent but p21 cip1 independent, whereas the arrest in G2 depends on both p53 and its downstream effector p21 cip1 . They also suggest that induction of p21 cip1 by the viral protein NS1 arrests cells in G2 through inhibition of cyclin A-dependent kinase activity.

SUMO modification of E1B-55K oncoprotein regulates isoform-specific binding to the tumour suppressor protein PML

The E1B-55K product from human adenovirus is a substrate of the small ubiquitin-related modifier (SUMO)-conjugation system. SUMOylation of E1B-55K is required to transform primary mammalian cells in cooperation with adenovirus E1A and to repress p53 tumour suppressor functions. The biochemical consequences of SUMO1 conjugation of 55K have so far remained elusive. Here, we report that E1B-55K physically interacts with different isoforms of the tumour suppressor protein promyelocytic leukaemia (PML). We show that E1B-55K binds to PML isoforms IV and V in a SUMO1-dependent and -independent manner. Interaction with PML-IV promotes the localization of 55K to PML-containing subnuclear structures (PML-NBs). In virus-infected cells, this process is negatively regulated by other viral proteins, indicating that binding to PML is controlled through reversible SUMOylation in a timely coordinated manner. These results together with earlier work are consistent with the idea that SUMOylation regulates targeting of E1B-55K to PML-NBs, known to control transcriptional regulation, tumour suppression, DNA repair and apoptosis. Furthermore, they suggest that SUMO1-dependent modulation of p53-dependent growth suppression through E1B-55K PML-IV interaction has a key role in adenovirus-mediated cell transformation.

The Promoter of Human Telomerase Reverse Transcriptase Is Activated During Liver Regeneration and Hepatocyte Proliferation

BACKGROUND & AIMS Telomerase activity has not been detected in healthy human liver biopsy samples, but it is up-regulated in most human liver tumors. It is not clear whether telomerase is activated in response to acute or chronic liver injury. Telomerase activity is closely associated with expression of its catalytic subunit, telomerase reverse transcriptase (TERT). We analyzed the activity of the human TERT (hTERT) promoter during liver regeneration in vivo and hepatocyte proliferation in vitro. METHODS We used hTERTp-lacZ transgenic mice, which contain an 8.0-kilobase pair fragment of the hTERT gene promoter, to study the role of TERT in liver regeneration following partial hepatectomy. As an in vitro model, we used the HepaRG cell line as a new model system for human hepatocyte proliferation and differentiation. RESULTS Activity of the hTERT promoter increased significantly after partial hepatectomy; it was also induced in hepatocytes, based on immunohistologic analysis. Similar to the in vivo results, telomerase activity and hTERT expression were up-regulated in proliferating HepaRG cells and repressed in response to growth arrest and differentiation. Promoter mapping revealed that a proximal 0.3-kilobase pair fragment contains all elements necessary for regulation of hTERT in HepaRG cells. We identified E2F2 and E2F7 as transcription factors that control the differential expression of hTERT in proliferating hepatocytes, in vitro and in vivo. CONCLUSIONS hTERT is induced in hepatocytes during liver regeneration, indicating a functional role for telomerase in human liver.

Strong and Selective Inhibitors of Hepatitis B Virus Replication among Novel N4-Hydroxy- and 5-Methyl-β-l-Deoxycytidine Analogues

ABSTRACT Novel N4-hydroxy- and 5-methyl-modified β-l-deoxycytidine analogues were synthesized and evaluated as anti-hepatitis B virus (HBV) agents. Their in vitro efficiencies were investigated in HepG2.2.15 cells stably transfected with HBV. β-l-2′,3′-Didehydro-2′,3′-dideoxy-N4-hydroxycytidine (β-l-Hyd4C) was most effective in reducing secreted HBV DNA (50% effective concentration [EC50], 0.03 μM), followed by β-l-2′,3′-dideoxy-3′-thia-N4-hydroxycytidine (EC50, 0.51 μM), β-l-2′,3′-dideoxy-N4-hydroxycytidine (EC50, 0.55 μM), and β-l-5-methyl-2′-deoxycytidine (EC50, 0.9 μM). The inhibition of the presumed target, the HBV DNA polymerase, by the triphosphates of some of the β-l-cytidine derivatives was also assessed. In accordance with the cell culture data, β-l-Hyd4C triphosphate was the most active inhibitor, with a 50% inhibitory concentration of 0.21 μM. The cytotoxicities of some of the 4-NHOH-modified β-l-nucleosides were dramatically lower than those of the corresponding cytidine analogues with the unmodified 4-NH2 group. The 50% cytotoxic concentrations for β-l-Hyd4C in HepG2 and HL-60 cells were 2,500 μM and 3,500 μM, respectively. In summary, our results demonstrate that at least β-l-Hyd4C can be recommended as a highly efficient and extremely selective inhibitor of HBV replication for further investigations.