Posttranslational modifications of HBV core protein.

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. Keywords: hepatitis B virus; posttranslational modifications; HBV core protein; phosphorylation; ubiquitination; arginine methylation.

Characterization of a novel human herpesvirus 8-encoded protein, vIRF-3, that shows homology to viral and cellular interferon regulatory factors.

The genome of the human herpesvirus 8 (HHV-8) contains a cluster of open reading frames (ORFs) encoding proteins with homology to the cellular transcription factors of the interferon regulatory factor (IRF) family. Two of these homologues, vIRF-1 and vIRF-2, were previously identified and functionally analyzed. In this study, we have characterized a novel gene, designated vIRF-3, encoded within the previously predicted ORF K10.5 and our newly identified ORF K10. 6. Northern blotting of RNA extracted from BCBL-1 cells with a vIRF-3-specific probe and reverse transcription-PCR analyses revealed a single transcript of 2.2 kb with a splice present in the coding region. The vIRF-3 mRNA levels in BCBL-1 cells were increased upon 12-O-tetradecanoylphorbol-13-acetate treatment, with kinetics of expression similar to those of the early immediate genes. The vIRF-3 ORF encodes a 73-kDa protein with homology to cellular IRF-4 and HHV-8-encoded vIRF-2 and K11. In transient transfection assays with the IFNACAT reporter, vIRF-3 functioned as a dominant-negative mutant of both IRF-3 and IRF-7 and inhibited virus-mediated transcriptional activity of the IFNA promoter. Similarly, the overexpression of vIRF-3 in mouse L929 cells resulted in inhibition of virus-mediated synthesis of biologically active interferons. These results suggest that by targeting IRF-3 and IRF-7, which play a critical role in the activation of alpha/beta interferon (IFN) genes, HHV-8 has evolved a mechanism by which it directly subverts the functions of IRFs and down-regulates the induction of the IFN genes that are important components of the innate immunity.

Functional analysis of human herpesvirus 8-encoded viral interferon regulatory factor 1 and its association with cellular interferon regulatory factors and p300.

Human herpesvirus 8/Kaposi sarcoma-associated virus (HHV-8/KSHV) contains, in addition to genes required for viral replication, a unique set of nonstructural genes which may be part of viral mimicry and contribute to viral replication and pathogenesis in vivo. Among these, HHV-8 encodes four open reading frames (ORFs) that showed homology to the transcription factors of the interferon regulatory factor (IRF) family. The ORF K9, viral IRF 1 (vIRF-1), has been cloned, and it was shown that, when overexpressed, it down modulates the interferon-mediated transcriptional activation of the interferon-stimulated gene 15 (ISG 15) promoter, and the role of vIRF-1 in viral mimicry was implied. However, the molecular mechanism of this effect has not been clarified. Here, we extend this observation and show that vIRF-1 also downregulates the transcriptional activity of IFNA gene promoter in infected cells by interfering with the transactivating activity of cellular IRFs, including IRF-1 and IRF-3. We further show that ectopic expression of vIRF-1 in NIH 3T3 cells confers resistance to tumor necrosis factor alpha-induced apoptosis. While vIRF-1 is unable to bind DNA with the same specificity as cellular IRFs, we demonstrate by in vitro binding assay that it can associate with the family of cellular IRFs, such as IRF-1 and the interferon consensus sequence binding protein. vIRF-1 interaction domain was localized between amino acids (aa) 152 and 243. While no binding between the full-size IRF-3 and vIRF-1 could be detected by the same assay, we show that vIRF-1 also targets the carboxy-terminal region (aa 1623 to 2414) of the transcriptional coactivator p300 which could also bind IRF-3 and IRF-1. These results demonstrate that vIRF-1 can modulate the transcription of the IFNA genes by direct heterodimerization with members of the IRF family, as well as by competitive binding with cellular transcription factors to the carboxy-terminal region of p300.

Molecular cloning and expression analysis of five novel genes in chromosome 1p36.

The human chromosome 1p36 region displays frequent nonrandom chromosomal deletions and translocations in a number of human malignancies; these are thought to inactivate tumor suppressor genes. To identify these putative tumor suppressors we employed exon trapping, cDNA selection, and zoo blot analysis to clone five new genes located in 1p36. Two of these represent novel genes and were designated C1orf1 and xylan 1,4-beta-xylosidase 1 (XBX1). Two further genes represented new members of known gene families: PTPRZ2 was a tyrosine phosphatase and FRAP2 represented a FKBP12-rapamycin-associated protein. The fifth gene identified, ENO1L1, was significantly homologous to c-myc promoter binding protein, MBP-1, and to enolase 1 (ENO1). It colocalized with alpha enolase (ENO1) on a single P1 clone. ENO1L1 differed from both ENO1 and MBP-1 in the organization of its 5' untranslated sequences. Second, MBP-1 contained two single-base insertions not present in either ENO1 or ENO1L1 sequences, which led to a shift in the MBP-1 reading frame. Expression analysis revealed two brain-specific transcripts of 7.9 and 6.5 kb for PTPRZ2. In contrast, C1orf1, FRAP2, ENO1L1, and XBX1 appeared to be expressed ubiquitously in the tissues tested, with transcript sizes of 4.5, 8.7, 1.75, and 4.5 kb, respectively. Using fluorescence in situ hybridization, we mapped the five novel genes relative to chromosome 1p36 breakpoints present in three established tumor cell lines and one nontumor cell line. The karyotypic abnormalities in these cell lines were exploited as chromosomal landmarks; we could thus show that the telomere to centromere gene order was PTPRZ2-(MBP-1/ENO1/ENO1L1)-(C1orf1/XBX1)-+ ++FRAP2. The localization of these genes to a chromosomal region that is prone to deletions in human cancers makes them potential candidate tumor suppressors.

Analysis of location and integrity of the human PITSLRE (p58(cdc2L1)) genes in neuroblastoma cell genomes.

The family of PITSLRE kinase genes, located in chromosome 1p36, has recently been associated with neuroblastoma tumorigenesis. In order to evaluate the role of these genes as putative tumor suppressor genes, we have analyzed the integrity of the coding region in primary tumors and its location relative to a neuroblastoma consensus deletion. A subset of aggressive neuroblastoma tumors with allelic loss of different parts of chromosome 1p were investigated. Single-strand conformation polymorphism (SSCP), heteroduplex (HD) and sequencing analysis of tumor DNA did not reveal any significant changes in the coding region. In particular, a primary tumor with an interstitial allelic deletion in 1p36 did not reveal concomitant loss of heterozygosity of the PITSLRE gene region when analyzed with a C/T DNA sequence polymorphism in exon 5 of PITSLRE1. FISH analysis on neuroblastoma cell lines with small interstitial deletions and with a balanced translocation in 1p36 revealed that the PITSLRE gene cluster was localized distal to the neuroblastoma consensus deletion. against an involvement of the PITSLRE genes in neuroblastoma tumorigenesis.

A genetic profile of Romany (Gypsy) subethnic group from a single region in Slovakia.

The data presented here are on population structure and genetic markers (ABO, RH, MN, HP) in two series of so-called Slovak Romany subethnic groups from a single region (Gemer) in the Southeastern part of Slovakia. The results demonstrate that favourable conditions have existed for population genetic mechanisms operating in isolated populations, namely genetic drift and inbreeding. In addition, an attempt was made to compare the observed data with those available for other Romany populations and for Slovaks.

Distribution of ApoBII, MCT118 (D1S80), YNZ22 (D17S30), and COL2A1 Amp-FLPs (amplified fragment length polymorphisms) in Caucasoid population of Slovakia.

Amp-FLPs are simple and rapid tools for genetic characterization of both individuals and populations. This paper presents allele frequencies of four Amp-FLPs (ApoBII, MCT118, YNZ22, and COL2A1) based on the analysis of more than 100 unrelated Caucasoid Slovaks. The proportion of heterozygotes observed and expected, and the probability that two individuals taken at random from the population would be identical in a given polymorphism (PI), was determined for each Amp-FLP.