The La protein is an essential RNA-binding protein implicated in different aspects of RNA metabolism. Herein, we report that small interfering (siRNA)-mediated La depletion reduces cell proliferation of different cell lines concomitant with a reduction in cyclin D1 (CCND1) protein. To exclude off-target effects we demonstrate that exogenous La expression in La-depleted cells restores cell proliferation and CCND1 protein levels. In contrast, proliferation of immortalized CCND1 knockout cells is not affected by La depletion, supporting a functional coherence between La, CCND1 and proliferation. Furthermore, we document by reversible in vivo crosslinking and ribonucleoprotein (RNP) immunoprecipitation an association of the La protein with CCND1 messengerRNA and that CCND1 internal ribosome entry site (IRES)-dependent translation is modulated by La protein level within the cell. In addition, we show elevated La protein expression in cervical cancer tissue and its correlation with aberrant CCND1 protein levels in cervical tumor tissue lysates. In conclusion, this study establishes a role of La in cell proliferation and CCND1 expression and demonstrates for the first time an overexpression of the RNA-binding protein La in solid tumors.

Promyelocytic leukemia nuclear bodies (PML-NBs) are implicated in transcriptional regulation. Here we identify a novel transcriptional repressor, PML-associated repressor of transcription (PAROT), which is regulated in its repressor activity through recruitment to PML-NBs. PAROT is a Krüppel-associated box ( KRAB) zinc-finger (ZNF) protein, which comprises an amino terminal KRAB-A and KRAB-B box, a linker domain and 8 tandemly repeated C(2)H(2)-ZNF motifs at its carboxy terminus. Consistent with its domain structure, when tethered to DNA, PAROT represses transcription, and this is partially released by the HDAC inhibitor trichostatin A. PAROT colocalizes with members of the heterochromatin protein 1 (HP1) family and with transcriptional intermediary factor-1beta/KRAB-associated protein 1 (TIF-1beta/KAP1), a transcriptional corepressor for the KRAB-ZNF family. Interestingly, PML isoform IV, in contrast to PML-III, efficiently recruits PAROT and TIF-1beta from heterochromatin to PML-NBs. PML-NB recruitment of PAROT partially releases its transcriptional repressor activity, indicating that PAROT can be regulated through subnuclear compartmentalization. Taken together, our data identify a novel transcriptional repressor and provide evidence for its regulation through association with PML-NBs.

Potent and persistent in vivo anti-HBV activity of chemically modified siRNAs. Morrissey DV, Lockridge JA, Shaw L, Blanchard K, Jensen K, Breen W, Hartsough K, Machemer L, Radka S, Jadhav V, Vaish N, Zinnen S, Vargeese C, Bowman. K, Shaffer CS, Jeffs B, Judge A, MacLachlan I, Polisky B.The efficacy of lipid-encapsulated, chemically modified short interfering RNA (siRNA) targeted to hepatitis B virus (HBV) was examined in an in vivo mouse mode of HBV replication. Stabilized siRNA targeted to the HBV RNA was incorporated into a specialized liposome to form a stable nucleic-acid-lipid particle (SNALP) and administered by intravenous injection into mice carrying replicating HBV. The improved efficacy of siRNA-SNALP compared to unformulated siRNA correlates with a longer half-life in plasma and liver. Three daily intravenous injections of 3 mg/4g/day reduced serum HBV DNA > 1.0 log(10). The reduction in HBV DNA,vas specific, dose-dependent and lasted for up to 7 day; after dosing. Furthermore, reductions were seen in serum RBV DNA for up to 6 weeks With weekly dosing The advances demonstrated here, including persistence of in vivo activity, use of lower doses and reduced dosing RNA. frequency are important steps in making siRNA a clinically viable therapeutic approach.

The posttranscriptional regulatory element (PRE) is considered to enhance hepatitis B virus (HBV) gene expression by facilitating the nuclear export of intronless viral subgenomic RNAs. Its role in the RNA metabolism of the viral pregenomic RNA (pgRNA) is currently unknown. We identified a positively cis-acting splicing regulatory element (SRE-1) and present two lines of evidence for its functionality. Firstly, in a heterologous context SRE-1 functionally substitutes for a retroviral bidirectional exonic splicing enhancer (ESE). As expected, SRE-1 is a splicing enhancer also in its natural viral sequence context, since deletion of SRE-1 reduces splicing of pgRNA in cell culture experiments. Secondly, we show that stimulation of HBV RNA splicing by the splicing factor PSF was repressed by the PRE. Analysis of a variety of PSF mutants indicated that RNA-binding and protein-protein interaction were required to enhance splicing. In addition, we show that the PRE contributed to pgRNA stability, but has little influence on its nuclear export. Herein, we report for the first time that the PRE harbors splicing stimulating and inhibiting regulatory elements controlling processing of the viral pregenome. We discuss a model in which the regulation of pgRNA splicing depends on cellular factors interacting with the PRE.

Homeodomain-interacting protein kinase 2 (HIPK2) is involved in transcriptional regulation, growth suppression, and apoptosis. Previous reports showed that HIPK2 can signal cell death via p53, and independently of p53 by activating the c-Jun NH2-terminal kinase (JNK) pathway or mediating CtBP degradation. Here we demonstrate that human HIPK2 is small ubiquitin-related modifier-1 (SUMO-1)-modified in vitro and in vivo at lysine residue 25, a SUMO consensus modification motif conserved in human and mouse HIPK family proteins. SUMO modification of HIPK2 altered neither its nuclear body localization nor its recruitment to promyelocytic leukemia-nuclear bodies. However, SUMO-1 modification inhibited HIPK2-induced JNK activation and p53-independent antiproliferative function. HIPK2 with a mutated SUMO acceptor lysine residue was refractory to inhibition of HIPK2-mediated JNK activation by SUMO-1. Furthermore, we demonstrate that SUMO protease SuPr-1 interacts with HIPK2, and both proteins predominantly colocalize in promyelocytic leukemia-nuclear bodies. SuPr-1 deconjugates SUMO-1 from HIPK2 in vitro and in vivo, which results in modestly increased HIPK2-induced JNK activity. Thus, our data demonstrate that HIPK2 effector function on JNK is modulated through dynamic SUMO-1 modification.

Heron hepatitis B viruses (HHBVs) in three subspecies of free-living great blue herons (Ardea herodias) from Florida, USA, were identified and characterized. Eight of 13 samples were positive in all assays used, whereas sera from egrets, which are also members of the family Ardeidae, were negative in the same assays. Comparative phylogenetic analysis of viral DNA sequences from the preS/S region of previously reported and novel HHBV strains isolated from captive grey herons (Germany) and free-ranging great blue herons (USA), respectively, revealed a strong conservation (95 % sequence similarity) with two separate clusters, implying a common ancestor of all strains. Our data demonstrate for the first time that different subspecies of herons are infected by HHBV and that these infections exist in non-captive birds. Phylogenetic analysis and the fact that the different heron species are geographically isolated populations suggest that lateral transmission, virus adaptation and environmental factors all play a role in HHBV spreading and evolution.

Serum autoantibodies against components of nuclear dots (anti-NDs), namely PML and Sp100, are specifically detected in 20% to 30% of patients with primary biliary cirrhosis (PBC). Although anti-ND antibodies are nonpathogenic, the mechanisms that lead to this unique reactivity are critical to understanding the loss of immune tolerance in PBC. Importantly, Sp100 and PML are both covalently linked to small ubiquitin-related modifiers (SUMOs). Therefore, we investigated whether SUMO proteins are independent autoantigens in PBC and studied 99 PBC sera samples for reactivity against NDs, PML, and Sp100, as well as against SUMO-2 and SUMO-1 recombinant proteins. Autoantibodies against SUMO-2 and SUMO-1 were found in 42% and 15% of anti-ND-positive PBC sera, respectively. Anti-SUMO reactivity was not observed in anti-ND-negative sera. Anti-SUMO-2 autoantibodies were found in 58% of sera containing autoantibodies against both PML and Sp100 and were detected exclusively in sera containing anti-Sp100 autoantibodies. In conclusion, SUMO proteins constitute a novel and independent class of autoantigens in PBC. Furthermore, we believe our data emphasize the post-translational modification of lysine by either lipoylation in the case of AMA or SUMOylation in the case of specific anti-ND autoantibodies as the pivotal site for autoantibody generation in PBC.

O245 Aims: Liver transplant recipients who develop recurrent hepatitis B virus (HBV) infection despite prophylaxis with Anti-HBs hyperimmunoglobuline (HBIG) are often found to have selected a specific immune escape variant which carries a Gly145Arg substitution in the small surface protein (S-protein) of HBV. Previously, we demonstrated that this variant has in vitro a severe secretion defect which may be responsible for a cytotoxic effect of the virus causing fibrosing cholestatic hepatitis by accumulation of viral proteins and DNA (Hepatology 2003). Here, we investigated how the secretion defective Gly145Arg variant can emerge as the dominant viral population in the serum of patients. Methods: To investigate the interaction between wildtype virus (wt) and the Gly145Arg variant several cotransfection experiments were performed using varying amounts of wt and mutant full-length HBV genomes. In addition, viral constructs expressing only mutant or wt small (S-) or large (L-) surface protein were used for the studies. After transfection of viral genomes in human hepatoma cells Northern, Western and Southern blot analysis were performed to study viral production and secretion. Results: A small amount of wt was able to rescue secretion of the defective Gly145Arg mutant. Notably, the virions secreted after cotransfection of wt with the Gly145Arg mutant contained the secretion defective mutant HBV DNA, but were encoated by wt surface proteins. Further experiments revealed that only wt S-protein, but not wt L-protein was important was rescuing secretion of the Gly145Arg variant. Additional cotransfection experiments using constructs expressing only mutant S-protein and wt revealed that selection of the secretion defective mutant viral DNA was further supported by the fact that mutant S-protein was able to suppress secretion of wt virions. Therefore mutant S-protein had a transdominant negative effect on the secretion of Wt. Conclusions: Despite its secretion defect the immune escape variant Gly145Arg can become the dominant viral population in the serum of patients if it emerges in a mixed population with wt.

Here we provide evidence for an interaction-dependent subnuclear trafficking of the human La (hLa) protein, known as transient interaction partner of a variety of RNAs. Among these, precursor transcripts of certain RNAs are located in the nucleoplasm or nucleolus. Here we examined which functional domains of hLa are involved in its nuclear trafficking. By using green fluorescent-hLa fusion proteins, we discovered a nucleolar localization signal and demonstrated its functionality in a heterologous context. In addition, we revealed that the RRM2 motif of hLa is essential both for its RNA binding competence in vitro and in vivo and its exit from the nucleolus. Our data imply that hLa traffics between different subnuclear compartments, which depend decisively on a functional nucleolar localization signal as well as on RNA binding. Directed trafficking of hLa is fully consistent with its function in the maturation of precursor RNAs located in different subnuclear compartments.

Little is known about cellular determinants essential for human hepatitis B virus infection. Using the duck hepatitis B virus as a model, we first established a sensitive binding assay for both virions and subviral particles and subsequently elucidated the characteristics of the early viral entry steps. The infection itinerary was found to initiate with the attachment of viral particles to a low number of binding sites on hepatocytes (about 10(4) per cell). Virus internalization was fully accomplished in less than 3 h but was then followed by a period of unprecedented length, about 14 h, until completion of nuclear import of the viral genome. Steps subsequent to virus entry depended on both intact microtubules and their dynamic turnover but not on actin cytoskeleton. Notably, cytoplasmic trafficking of viral particles and emergence of nuclear covalently closed circular DNA requires microtubules during entry only at and for specific time periods. Taken together, these data disclose for the first time a series of steps and their kinetics that are essential for the entry of hepatitis B viruses into hepatocytes and are different from those of any other virus reported so far.

O244 Fibrosing cholestatic hepatitis (FCH) is a severe clinical and histological manifestation of hepatitis B virus (HBV) infection that most often occurs in HBV infected liver transplant (LTx) recipients. The aggressive clinical course of FCH has been attributed to direct cytopathic liver damage and is associated with widespread infection of hepatocytes with intracellular accumulation of HBV proteins and DNA. It has been speculated that viral mutants emerging after LTx are responsible for the increased pathogenicity of the virus. However, detailed structural analyses of HBV genomes from patients with FCH are not yet available and so far only selected genomic regions in few patients have been investigated. Aims: Analysis of the complete genomic sequence of HBV obtained from LTx patients for the identification of mutations associated with FCH. Methods: HBV DNA of 9 patients with FCH was isolated from blood samples taken before and after LTx and amplified by PCR. Full length HBV genomes were sequenced directly and after cloning. All sequence information was assembled using MAC vector software to obtain the complete genomic sequence of the dominant HBV population. These HBV genomes were compared for genomic differences before and after LTx. Further, HBV genomes present in the FCH sample were analysed in between patients and compared with published HBV sequences. Results: Comparison of the viral populations before and after LTx revealed a sequence variability of 5.9 x 10-3 per nucleotide position per year, which is in the magnitude seen in patients with chronic active HBV infection. Nucleotide changes before and after LTx occurred throughout the whole HBV genome with the pre-S-, S- and Core- regions showing higher rates whereas pre-Core, X and pre-S1 had lower than average (0.53%) rates. The HBV genotype was A in 5, C in 1, and D in 3 patients. Post LTx HBV genomic sequences of all 9 FCH patients showed specific mutations that have been associated with an increased pathogenicity in epidemiologic and / or functional analyses. However, a single mutation seen in all 9 FCH cases was not present. HBV regions with a high rate of mutations were S (7/9), Core-promoter(6/9), pre-Core(5/9), Polymerase(5/9), X(4/9), X-promoter(4/9), and pre-S(4/9). The number of mutated HBV regions per patient varied between 2 and 5 (median 4), a specific combination of mutated regions could not be identified. Conclusions: HBV genomes from all patients with FCH after LTx showed mutations in different genomic regions which have been associated with increased pathogenicity of the virus. No single mutation was identified, but it is likely that the combination of mutations in several genomic regions under the circumstance of immunosuppression and allograft environment is responsible for the viral phenotype causing FCH. In the majority of cases these mutations were not new, but could be identified before LTx at the time of decompensated liver cirrhosis. Thus FCH is most likely caused by HBV populations with complex mutations which are present already before LTX and recur after LTx.

Sera of patients with acute (AH) and chronic active hepatitis (CAH) were tested for anti-hepatitis B virus (HBV) x-protein (HBx) by immunoblotting, using recombinant MS2- and βgal-HBx fusion proteins as substrate. Antibodies against HBx were detected in 5 out of 17 patients with AH at an early stage of infection, and in 13 out of 35 patients with CAH. Positive sera from AH patients showed a relatively weak anti-HBx reactivity when compared to sera from CAH patients. In follow up studies we tested serial serum samples from patients positive for anti-HBx. Patients with AH were observed for 3 to 6 weeks and CAH patients for up to 51 months. In general anti-HBx reactivities appeared to be stable although significant differences in apparent antibody levels were noted when sera from individual patients were compared. Our data further support an early expression of HBx-antigen in HBV-infected individuals. There was no correlation between HBe-antigen and anti-HBx in CAH.

The La protein is a multifunctional RNA-binding protein and has also been suggested to be involved in the stabilization of hepatitis B virus (HBV) RNA. Here we demonstrate that antibodies against the human La protein specifically precipitate HBV RNA from HBV ribonucleoprotein-containing mammalian cell extracts, providing evidence for the association between human La and HBV RNA. Moreover, we report that the turnover of HBV RNA depends on structural features and less on the primary sequence of the La-binding site on the viral RNA. In addition we show that the interaction between human La and HBV RNA in vitro is modulated by accessory factor(s) in a phosphorylation-dependent manner. Taken together these data indicate that both structural features, the composition of La/HBV ribonucleoprotein particles as well as interacting cellular factors, are critical determinants in the regulation of the stability of the HBV RNA.

ABSTRACT Viruses can spread by different mechanisms: via intracellular particles through cell junctions to neighboring cells or via secreted virions to adjacent or remote cells. The observation of clusters of hepadnavirus-infected cells both in vivo and in primary hepatocytes neither proves the first mechanism nor excludes the second. In order to test which mechanism, if not both, is used by hepatitis B viruses in order to spread, we used primary duck hepatocytes and duck hepatitis B virus (DHBV) as an infection model. If extracellular progeny virus alone determines spreading, neutralizing antisera or drugs blocking virus binding to hepatocytes should abolish secondary infection. In order to test this, we used DHBV envelope-specific neutralizing antisera, as well as suramin, a known inhibitor of infection. Both reagents strongly reduced hepatocellular attachment of viral particles and almost completely abolished primary infection, whereas an ongoing intracellular infection was not affected as long as no progeny virus was released. In contrast, incubation of infected primary hepatocytes with these reagents during release of progeny virus completely prevented secondary infection. Moreover, the combination of electron and immunofluorescence microscopy analyses revealed the residence of viral particles in cytoplasmic vesicles preferentially located near the basolateral membrane of infected hepatocytes. Taken together, these data strongly suggest that hepatitis B viruses mainly spread by secreted, extracellular progeny and point to polarized egress of viral particles into intercellular compartments, which restricts their diffusion and favors transmission of virus to adjacent cells.

Hepadnaviruses including human hepatitis B virus (HBV) and duck hepatitis B virus (DHBV) express X proteins, HBx and DHBx, respectively. Both HBx and DHBx are transcriptional activators and modulate cellular signaling in in vitro assays. To test whether the DHBx protein plays a role in virus infection, we compared the in vivo infectivity and growth characteristics of a DHBV3 strain with a stop codon in the X-like ORF (DHBV3-X-K.O.) to those of the wild-type DHBV3 strain. Here we report that the two strains showed no significant difference in (i) their ability to induce infection that resulted in stable viraemia measured by serum surface antigen (DHBsAg) and DHBV DNA, and detection of viral proteins and replicative DNA intermediates in the liver; (ii) the rate of spread of infection in liver and extrahepatic sites after low-dose virus inoculation; and (iii) the ability to produce transient or persistent infection under balanced age/dose conditions designed to detect small differences between the strains. Thus, none of the infection parameters assayed were detectably affected by the X-ORF knockout mutation, raising the question whether DHBx expression plays a physiological role during in vivo infection with wild-type DHBV.

ABSTRACT All hepadnaviruses known so far have a very limited host range, restricted to their natural hosts and a few closely related species. This is thought to be due mainly to sequence divergence in the large envelope protein and species-specific differences in host components essential for virus propagation. Here we report an infection of cranes with a novel hepadnavirus, designated CHBV, that has an unexpectedly broad host range and is only distantly evolutionarily related to avihepadnaviruses of related hosts. Direct DNA sequencing of amplified CHBV DNA as well a sequencing of cloned viral genomes revealed that CHBV is most closely related to, although distinct from, Ross' goose hepatitis B virus (RGHBV) and slightly less closely related to duck hepatitis B virus (DHBV). Phylogenetically, cranes are very distant from geese and ducks and are most closely related to herons and storks. Naturally occurring hepadnaviruses in the last two species are highly divergent in sequence from RGHBV and DHBV and do not infect ducks or do so only marginally. In contrast, CHBV from crane sera and recombinant CHBV produced from LMH cells infected primary duck hepatocytes almost as efficiently as DHBV did. This is the first report of a rather broad host range of an avihepadnavirus. Our data imply either usage of similar or identical entry pathways and receptors by DHBV and CHBV, unusual host and virus adaptation mechanisms, or divergent evolution of the host genomes and cellular components required for virus propagation.