Mart Ustav

Brd4 protein has been proposed to act as a cellular receptor for the bovine papillomavirus type 1 (BPV1) E2 protein in the E2-mediated chromosome attachment and mitotic segregation of viral genomes. Here, we provide data that show the involvement of Brd4 in multiple early functions of the BPV1 life cycle, suggest a Brd4-dependent mechanism for E2-dependent transcription activation, and indicate the role of Brd4 in papillomavirus and polyomavirus replication as well as cell-specific utilization of Brd4-linked features in BPV1 DNA replication. Our data also show the potential therapeutic value of the disruption of the E2-Brd4 interaction for the development of antiviral drugs.

Bovine papillomavirus type 1 (BPV1), Epstein-Barr virus (EBV), and human herpesvirus 8 genomes are stably maintained as episomes in dividing host cells during latent infection. The mitotic segregation/partitioning function of these episomes is dependent on single viral protein with specific DNA-binding activity and its multimeric binding sites in the viral genome. In this study we show that, in the presence of all essential viral trans factors, the segregation/partitioning elements from both BPV1 and EBV can provide the stable maintenance function to the mouse polyomavirus (PyV) core origin plasmids but fail to do so in the case of complete PyV origin. Our study is the first which follows BPV1 E2- and minichromosome maintenance element (MME)-dependent stable maintenance function with heterologous replication origins. In mouse fibroblast cell lines expressing PyV large T antigen (LT) and either BPV1 E2 or EBV EBNA1, the long-term episomal replication of plasmids carrying the PyV minimal origin together with the MME or family of repeats (FR) element can be monitored easily for 1 month under nonselective conditions. Our data demonstrate clearly that the PyV LT-dependent replication function and the segregation/partitioning function of the BPV1 or EBV are compatible in certain, but not all, configurations. The quantitative analysis indicates a loss rate of 6% per cell, doubling in the case of MME-dependent plasmids, and 13% in the case of FR-dependent plasmids in nonselective conditions. Our data clearly indicate that maintenance functions from different viruses are principally interexchangeable and can provide a segregation/partitioning function to different heterologous origins in a variety of cells.

Recent studies have suggested that the tethering of viral genomes to host cell chromosomes could provide one of the ways to achieve their nuclear retention and partitioning during extrachromosomal maintenance in dividing cells. The data we present here provide firm evidence that the partitioning of the bovine papillomavirus type 1 (BPV1) genome is dependent on the chromatin attachment process mediated by viral E2 protein and its multiple binding sites. On the other hand, the attachment of E2 and the E2-mediated tethering of reporter plasmids to host chromosomes are not necessarily sufficient for efficient partitioning, suggesting that additional E2-dependent activities might be involved in the latter process. The activity of E2 protein in chromatin attachment and partitioning is more sensitive to the point mutations in the N-terminal domain than its transactivation and replication initiation functions. Therefore, at least part of the interactions of the E2 N-terminal domain with its targets during the chromatin attachment and partitioning processes are likely to involve specific receptors not involved in transactivation and replication activities of the protein. The mutational analysis also indicates that the binding of E2 to chromatin is not achieved through interaction of linear N-terminal subsequences of the E2 protein with putative receptors. Instead, the composite surface elements of the N-terminal domain build up the receptor-binding surface of E2. In this regard, the interaction of BPV1 E2 with its chromosomal targets clearly differs from the interactions of LANA1 protein from Kaposi's sarcoma-associated human herpesvirus and EBNA1 from Epstein-Barr virus with their specific receptors.

The rationale of using bovine papillomavirus-1 (BPV-1) derived vectors in gene therapy protocols lies in their episomal maintenance at intermediate to high copy number, and stable, high-level expression of the gene products. We constructed the BPV-1 based vector harbouring the human low-density lipoprotein receptor (LDLR) gene cDNA and tested its ability to restore the function of the LDLR in the receptor-deficient cell line CHO-ldlA7. The introduced vector p3.7LDL produced functionally active LDL receptors in the receptor-deficient cell line CHO-ldlA7 during the 32-week period of observation as determined by the internalisation assay with the labelled LDL particles. Bovine papillomavirus type-1 (BPV-1)-derived vectors could be suitable for gene therapy due to their episomal maintenance at intermediate to high copy number and stable, high-level expression of the gene products. The constructed BPV-1 based vector p3.7LDL produced functionally active LDL receptors in the LDLR-deficient cell line CHO-ldlA7 during the 32-week period of observation. In vivo experiments should reveal, whether 1–5% transfection efficiency obtained in the current work is sufficient to bring about detectable and clinically significant lowering of the amount of circulating LDL cholesterol particles.

Distribution of hepatitis C virus (HCV) geno(sub)types among 215 Estonian patients hospitalized with acute or chronic hepatitis and with HCV RNA‐positive sera was investigated. For genotyping, both multiplex PCR with subtype‐specific primers of the core region and RFLP analysis of cDNA of the 5′ NCR region were used. These two methods permitted a correct characterization of genotypes, a more truthful characterization of mixed infections, and combined use of single‐tube performances. They revealed, respectively, 200 and 202 (93.0% and 93.9%) HCV‐positive samples of sera, subtype la ‐ 0.9% and 0.9%, lb ‐ 56.3% and 64.2%, 3a ‐ 13.9% and 22.3%, 2a ‐ 6.5% and 5.6%, type 4 ‐0.5% and 0%, mixed infections ‐ 13.5% and 0%, and unidentified ‐ 1.4% and 0.9%. In the majority of cases (84.7%) both methods gave completely or partially concordant results; in mixed infections, as determined by subtype‐specific PCR, only one subtype was revealed by the RFLP method. In the remaining 15.3% of the cases (Ohno ‐ 7.0%, RFLP ‐ 8.3%) only one of the methods was positive. The epidemiological analysis of the dynamics of the subtypes' relative participation may indicate increasing 3a and decreasing lb subtype infection during recent years.

We describe here the use of two newly mapped bovine papillomavirus type 1 (BPV-1) E2 protein epitopes as tags. We constructed several vector plasmids for overexpression as well as for moderate expr...