Increased glucocorticoid concentrations in early life cause mitochondrial inefficiency and short telomeres.

Telomeres are DNA structures that protect chromosome ends. However, telomeres shorten during cell replication and at critically low lengths can reduce cell replicative potential, induce cell senescence and decrease fitness. Stress exposure, which elevates glucocorticoid hormone concentrations, can exacerbate telomere attrition. This phenomenon has been attributed to increased oxidative stress generated by glucocorticoids ('oxidative stress hypothesis'). We recently suggested that glucocorticoids could increase telomere attrition during stressful periods by reducing the resources available for telomere maintenance through changes in the metabolic machinery ('metabolic telomere attrition hypothesis'). Here, we tested whether experimental increases in glucocorticoid levels affected telomere length and mitochondrial function in wild great tit (Parus major) nestlings during the energy-demanding early growth period. We monitored resulting corticosterone (Cort) concentrations in plasma and red blood cells, telomere lengths and mitochondrial metabolism (metabolic rate, proton leak, oxidative phosphorylation, maximal mitochondrial capacity and mitochondrial inefficiency). We assessed oxidative damage caused by reactive oxygen species (ROS) metabolites as well as the total non-enzymatic antioxidant protection in plasma. Compared with control nestlings, Cort-nestlings had higher baseline corticosterone, shorter telomeres and higher mitochondrial metabolic rate. Importantly, Cort-nestlings showed increased mitochondrial proton leak, leading to a decreased ATP production efficiency. Treatment groups did not differ in oxidative damage or antioxidants. Hence, glucocorticoid-induced telomere attrition is associated with changes in mitochondrial metabolism, but not with ROS production. These findings support the hypothesis that shortening of telomere length during stressful periods is mediated by glucocorticoids through metabolic rearrangements.

TopBP1 regulates human papillomavirus type 16 E2 interaction with chromatin.

Human papillomavirus type 16 (HPV16) E2 regulates transcription from and replication of the viral genome, in association with viral and cellular factors. HPV16 E2 interacts functionally with TopBP1, a cellular protein essential for the initiation of cellular, and potentially viral, DNA replication. This report demonstrates that the absence of TopBP1 results in the redistribution of HPV16 E2 into an alternative cellular protein complex, resulting in enhanced affinity for chromatin. This redistribution does not significantly alter the ability of HPV16 E2 to either activate or repress transcription. We also show colocalization of both proteins on chromatin at late stages of mitosis, suggesting that TopBP1 could be the mitotic chromatin receptor for HPV16 E2. The possible significance of the results for the regulation of the viral life cycle is discussed.

SF2/ASF binds the human papillomavirus type 16 late RNA control element and is regulated during differentiation of virus-infected epithelial cells.

Pre-mRNA splicing occurs in the spliceosome, which is composed of small ribonucleoprotein particles (snRNPs) and many non-snRNP components. SR proteins, so called because of their C-terminal arginine- and serine-rich domains (RS domains), are essential members of this class. Recruitment of snRNPs to 5' and 3' splice sites is mediated and promoted by SR proteins. SR proteins also bridge splicing factors across exons to help to define these units and have a central role in alternative and enhancer-dependent splicing. Here, we show that the SR protein SF2/ASF is part of a complex that forms upon the 79-nucleotide negative regulatory element (NRE) that is thought to be pivotal in posttranscriptional regulation of late gene expression in human papillomavirus type 16 (HPV-16). However, the NRE does not contain any active splice sites, is located in the viral late 3' untranslated region, and regulates RNA-processing events other than splicing. The level of expression and extent of phosphorylation of SF2/ASF are upregulated with epithelial differentiation, as is subcellular distribution, specifically in HPV-16-infected epithelial cells, and expression levels are controlled, at least in part, by the virus transcription regulator E2.

Intracellular distribution of hepatitis B virus core protein expressed in vitro depends on the sequence of the isolate and the serologic pattern.

Intracellular localization of hepatitis B core antigen (HBcAg) in vivo varies with liver cell damage. Localization of HBcAg was studied using transfection of cloned HBcAg variants. Twenty-six samples were obtained from 14 patients with liver disease; 10 were hepatitis B e antigen positive, and 16 were anti-hepatitis B e (HBe) positive. In hepatitis B e antigen (HBeAg)-positive patients, HBcAg predominantly localized in the nucleus; in anti-HBe-positive patients, it accumulated mainly in the cytoplasm. Of the 13 samples with nuclear localization, 9 were HBeAg positive; 5 of 13 had C-terminus and/or B cell epitope mutations. All but 1 of the 13 samples with predominantly cytoplasmic localization were anti-HBe positive; all 13 had mutations. Reversion of mutant sequences with cytoplasmic expression back to the wild type led to a shifting back to nuclear distribution. Thus, the pattern of HBcAg localization in vitro depends on sequence and the serologic pattern of chronic infection, paralleling the situation in vivo.

The fidelity of HPV16 E1/E2-mediated DNA replication.

Human papillomaviruses (HPV) are causative agents in a variety of human diseases; for example over 99% of cervical carcinomas contain HPV DNA sequences. Often in cervical carcinoma the HPV genome is integrated into the host genome resulting in unregulated expression of the viral transforming proteins E6 and E7. Therefore viral integration is a step toward HPV-induced carcinogenesis. Integration of the HPV genome could occur following double-strand DNA breaks that could arise during viral DNA replication. We investigated the fidelity of HPV 16 E1- and E2-mediated DNA replication of non-damaged and UVC-damaged templates in a variety of cell lines with different genetic backgrounds; C33a (derived from an HPV-negative cervical carcinoma), XP30RO (deficient in the by-pass polymerase eta (poleta)), XP30eta (expressing a restored wild-type poleta), XP12RO (nucleotide excision repair defective), and MRC5 (derived from a 14-week-old human fetus). The results demonstrate that the fidelity of E1- and E2-mediated DNA replication is reflective of the genetic background in which the assays are carried out. For example, restoring poleta to the XP30 cell line results in a 3-fold drop in the number of mutants obtained following replication of a UVC-damaged template. A relatively high percentage of the mutant-replicated molecules arise as a result of genetic rearrangement. This is the first time such studies have been carried out with an HPV replication system, and the results are discussed in the context of the HPV life cycle and what is known about HPV genomes in human cancers.

UVB irradiation reduces the half-life and transactivation potential of the human papillomavirus 16 E2 protein.

Human papillomaviruses (HPV) are causative agents of human cancers including those of the cervix and also of the head and neck; HPV16 is the most commonly found type in these diseases. The viral E2 protein regulates transcription from the viral genome by interacting with DNA-binding sequences in the HPV transcriptional control region; it also regulates replication by interacting with and recruiting the HPV replication factor E1 to the viral origin. Therefore, E2 is essential for the viral life cycle. The E2 protein interacts with several proteins involved in the cellular response to DNA damage including p53, TopBP1, and PARP. We therefore set out to establish whether DNA-damaging agents can regulate E2 activity. Here we show that UVB irradiation downregulates transcriptional activity of both HPV16 and HPV8 E2, while hydroxyurea and etoposide do not. This downregulation of E2 activity is independent of p53 function as it occurs in p53 wild type and null cell types as well as in the presence of functional HPV16 E6 that degrades p53. Using stable cell lines expressing E2 we show that this downregulation of E2 function by UVB is due to a reduction of the E2 protein half-life. The identification of the pathway(s) through which UVB downregulates E2 transcriptional activity and protein levels will present a novel target for the treatment of HPV-related diseases.

Novel cellular interacting partners of the human papillomavirus 16 transcription/replication factor E2.

Human papillomaviruses (HPVs) are causative agents in a number of human diseases. HPV can be divided into two groups: low risk that cause diseases such as genital warts, and high risk that cause ano-genital cancers. Of the high-risk group, HPV16 is the most commonly found in cervical cancer. All HPV encode an E2 protein and this protein regulates transcription from, and replication of, the viral genome making it essential for the viral life cycle. In order to function E2 must interact with cellular proteins; identification of these cellular partners will provide targets for disruption of the viral life cycle and will also provide insights into the processes of transcription and replication. To identify the cellular interacting partners for HPV16 E2, we carried out a yeast two-hybrid screen with the amino-terminus of E2 that is essential for mediating transcription and replication. Here we describe how this screen was carried out and detail the interacting partners that were identified; these include the proteins TopBP1, RACK1, POMP, p27(BBP), ODC antizyme, and Delta-adaptin. Several of these partners have characteristics that make them ideal candidates for mediating E2 function.

A Functional interaction between the human papillomavirus 16 transcription/replication factor E2 and the DNA damage response protein TopBP1.

The human papillomavirus (HPV) transcription/replication factor E2 is essential for the life cycle of HPVs. E2 protein binds to DNA target sequences in the viral long control regions to regulate transcription of the viral genome. It also enhances viral DNA replication by interacting with the viral replication factor E1 and recruiting it to the origin of replication and may also play a more direct role in replication. The cellular proteins with which E2 interacts to carry out these functions are largely unknown. To identify these proteins a yeast two-hybrid screen was carried out with the transcription/replication domain of HPV16 E2. This screen identified several candidate interacting partners for E2 including TopBP1 (topoisomerase II beta-binding protein 1). TopBP1 has eight BRCA1 carboxyl-terminal domains that are found in proteins regulating the DNA damage response, transcription, and replication. Here we demonstrate that HPV16 E2 and TopBP1 interact in vitro and in vivo and that TopBP1 can enhance the ability of E2 to activate transcription and replication. This is the first time that TopBP1 has been shown to function as a transcriptional coactivator and that E2 interacts with TopBP1. Removal of the amino-terminal domain of TopBP1 abolishes coactivation of transcription and replication. This interaction may have functional consequences upon the viral life cycle.