Winifred Boner

Hepatitis B Virus Core Protein Mutations Are Concentrated in B Cell Epitopes in Progressive Disease and in T Helper Cell Epitopes during Clinical Remission

The distribution and temporal and clinical features of amino acid substitutions of the core protein of hepatitis B (HB) virus were analyzed, using at least 2 sequential samples from 27 patients. Six patients seroconverted from HBe antigen (HBeAg)-positive to anti-HBe-positive (3 went into remission), and 21 were continuously anti-HBe positive with progressive hepatitis. Precore mutations, which terminate HBeAg translation, all appeared by the second sample. Most core mutations occurred between the first and second samples; significantly fewer occurred after the second. In seroconverters who went into remission, mutations occurred in the T helper epitope from aa 50 to 69 (P = .00045); for anti-HBe-positive patients with ongoing disease, mutations occurred in B cell epitopes (P = .0007 for aa 74-83). An ineffective anti-HBc B cell response accounts for ongoing disease and selection of mutations after seroconversion. In those who remit, mutations in the major T helper epitope allow immune escape, thus minimizing immune-mediated hepatitis.

SF2/ASF Binds the Human Papillomavirus Type 16 Late RNA Control Element and Is Regulated during Differentiation of Virus-Infected Epithelial Cells

ABSTRACT 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.

TopBP1 Regulates Human Papillomavirus Type 16 E2 Interaction with Chromatin

ABSTRACT 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.

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.

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.

Repeated exposure to stressful conditions can have beneficial effects on survival.

Repeated exposure to stressful circumstances is generally thought to be associated with increased pathology and reduced longevity. However, growing lines of evidence suggest that the effects of environmental stressors on survival and longevity depend on a multitude of factors and, under some circumstances, might be positive rather than negative. Here, using the zebra finch (Taeniopygia guttata), we show that repeated exposure to stressful conditions (i.e. unpredictable food availability), which induced no changes in body mass, was associated with a decrease in mortality rate and an increase in the age of death. As expected, the treated birds responded to the unpredictable food supply by increasing baseline glucocorticoid stress hormone secretion and there were no signs of habituation of this hormonal response to the treatment across time. Importantly, and consistent with previous literature, the magnitude of hormone increase induced by the treatment was significant, but relatively mild, since the baseline glucocorticoid concentrations in the treated birds were substantially lower than the peak levels that occur during an acute stress response in this species. Taken together, these data demonstrate that protracted exposure to relatively mild stressful circumstances can have beneficial lifespan effects.

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 η (polη)), XP30η (expressing a restored wild-type polη), 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 polη 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.

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.

Assessing the effects of repeated handling on the physiology and condition of semi‐precocial nestlings

Repeated exposure to elevated levels of glucocorticoids during development can have long‐term detrimental effects on survival and fitness, potentially associated with increased telomere attrition. Nestling birds are regularly handled for ecological research, yet few authors have considered the potential for handling‐induced stress to influence hormonally mediated phenotypic development or bias interpretations of subsequent focal measurements. We experimentally manipulated the handling experience of the semi‐precocial nestlings of European Storm Petrel Hydrobates pelagicus to simulate handling in a typical field study and examined cumulative effects on physiology and condition in late postnatal development. Neither baseline corticosterone (the primary glucocorticoid in birds), telomere length nor body condition varied with the number of handling episodes. The absence of a response could be explained if Storm Petrels did not perceive handling to be stressful or if there is dissociation of the hypothalamic–pituitary–adrenal axis from stressful stimuli in early life. Eliciting a response to a stressor may be maladaptive for cavity‐dwelling young that are unable to escape or defend themselves. Furthermore, avoiding elevated overall glucocorticoid exposure may be particularly important in a long‐lived species, in which accelerated early‐life telomere erosion could impact negatively upon longevity. We propose that the level of colony‐wide disturbance induced by investigator handling of young could be important in underlining species‐specific responses. Storm Petrel nestlings appear unresponsive to investigator handling within the limits of handling in a typical field study and handling at this level should not bias physiological and morphological measurements.