DAXX modulates human papillomavirus early gene expression and genome replication in U2OS cells.

BACKGROUND: The human papillomavirus (HPV) genomes can replicate, and are maintained as autonomously replicating extrachromosomal plasmids in human U2OS cells. Previous studies have shown that HPV genomes are transcriptionally active in U2OS cells and can express the viral early proteins required for initiation and establishment of HPV replication. In the present work, we have examined the involvement of cellular DAXX protein in HPV replication in U2OS cells. METHODS: We have used indirect immunofluorescence and FISH analysis in order to study HPV replication compartments in U2OS cells. In addition, we have used siRNA knock-down for examining the effect of the DAXX protein on HPV replication and transcription in U2OS cells. RESULTS: We show that a portion of HPV replication foci are partially co-localized with components of ND10, cellular DAXX and PML proteins. In addition, we demonstrate that the knock-down of the cellular DAXX protein modulates the HPV genome replication and transcription in U2OS cells--papillomavirus replication is reduced in the absence of this component of ND10. CONCLUSIONS: The DAXX protein modulates the early gene expression and the transient replication of HPV genomes in U2OS cells.

Identification and analysis of papillomavirus E2 protein binding sites in the human genome.

Papillomavirus E2 protein is required for the replication and maintenance of viral genomes and transcriptional regulation of viral genes. E2 functions through sequence-specific binding to 12-bp DNA motifs-E2 binding sites (E2BS)-in the virus genome. Papillomaviruses are able to establish persistent infection in their host and have developed a long-term relationship with the host cell in order to guarantee the propagation of the virus. In this study, we have analyzed the occurrence and functionality of E2BSs in the human genome. Our computational analysis indicates that most E2BSs in the human genome are found in repetitive DNA regions and have G/C-rich spacer sequences. Using a chromatin immunoprecipitation approach, we show that human papillomavirus type 11 (HPV11) E2 interacts with a subset of cellular E2BSs located in active chromatin regions. Two E2 activities, sequence-specific DNA binding and interaction with cellular Brd4 protein, are important for E2 binding to consensus sites. E2 binding to cellular E2BSs has a moderate or no effect on cellular transcription. We suggest that the preference of HPV E2 proteins for E2BSs with A/T-rich spacers, which are present in the viral genomes and underrepresented in the human genome, ensures E2 binding to specific binding sites in the virus genome and may help to prevent extensive and possibly detrimental changes in cellular transcription in response to the viral protein.

Antibody response against cancer-testis antigens MAGEA4 and MAGEA10 in patients with melanoma.

Melanoma-associated antigen A (MAGEA) represent a class of tumor antigens that are expressed in a variety of malignant tumors, however, their expression in healthy normal tissues is restricted to germ cells of testis, fetal ovary and placenta. The restricted expression and immunogenicity of these antigens make them ideal targets for immunotherapy in human cancer. In the present study the presence of naturally occurring antibodies against two MAGEA subfamily proteins, MAGEA4 and MAGEA10, was analyzed in patients with melanoma at different stages of disease. Results indicated that the anti-MAGEA4/MAGEA10 immune response in melanoma patients was heterogeneous, with only ~8% of patients having a strong response. Comparing the number of strongly responding patients between different stages of disease revealed that the highest number of strong responses was detected among stage II melanoma patients. These findings support the model that the immune system is involved in the control of melanoma in the early stages of disease.

Biochemical and proteomic characterization of retrovirus Gag based microparticles carrying melanoma antigens.

Extracellular vesicles are membraneous particles released by a variety of cells into the extracellular microenvironment. Retroviruses utilize the cellular vesiculation pathway for virus budding/assembly and the retrovirus Gag protein induces the spontaneous formation of microvesicles or virus-like particles (VLPs) when expressed in the mammalian cells. In this study, five different melanoma antigens, MAGEA4, MAGEA10, MART1, TRP1 and MCAM, were incorporated into the VLPs and their localization within the particles was determined. Our data show that the MAGEA4 and MAGEA10 proteins as well as MCAM are expressed on the surface of VLPs. The compartmentalization of exogenously expressed cancer antigens within the VLPs did not depend on the localization of the protein within the cell. Comparison of the protein content of VLPs by LC-MS/MS-based label-free quantitative proteomics showed that VLPs carrying different cancer antigens are very similar to each other, but differ to some extent from VLPs without recombinant antigen. We suggest that retrovirus Gag based virus-like particles carrying recombinant antigens have a potential to be used in cancer immunotherapy.

Human papillomavirus E2 protein with single activation domain initiates HPV18 genome replication, but is not sufficient for long-term maintenance of virus genome.

The papillomavirus life cycle is regulated by a family of proteins encoded by the E2 open reading frame; E2 proteins regulate viral gene expression, DNA replication and genome maintenance. We have previously shown that the bovine papillomavirus (BPV1) full-length E2 protein forms heterodimers with repressor forms of E2, and these E2 heterodimers serve as activators of transcription and replication during the viral life cycle. In the present study, using the single-chain E2 heterodimer as a model, we show that human papillomavirus (HPV) 11 and 18 E2 heterodimers with single activation domain are able to initiate replication of URR-containing plasmid in transient assay. Single-chain E2 heterodimer in the context of HPV18 genome initiates genome replication, but is not sufficient for long-term replication of HPV18 genome. We also show that HPV18 genome has a capacity to encode truncated E2 repressor E8/E2 which acts as a negative regulator of HPV18 genome replication.