Sheila V. Graham

GENOMIC ALTERATIONS INVOLVING THE C-MYC PROTO-ONCOGENE LOCUS DURING THE EVOLUTION OF A CASE OF CHRONIC GRANULOCYTIC LEUKAEMIA

In a patient with Ph-positive chronic granulocytic leukaemia who had 3 separate episodes of promyelocytic transformation associated with new cytogenetic and genetic changes DNA from the peripheral-blood leucocytes obtained during the second episode of transformation showed an 8-fold to 16-fold amplification of cellular myc proto-oncogene (c-myc) sequences, and rearrangement of these sequences. There was no amplification or rearrangement of c-myc sequences in DNA from leucocytes collected during the chronic phase, nor were these sequences amplified in DNA from leucocytes collected during the third episode of transformation. These observations show that new clones of cells that proliferate during the evolution of a myeloid leukaemia can carry genetic changes in addition to any recognisable karyotypic changes. Such genetic changes might underlie the progression of the disease.

Mechanisms of stage-regulated gene expression in kinetoplastida.

During their life cycle, trypanosomatid parasites of mammals encounter substantially different environments in their hosts and insect vectors, to which they must adapt by undergoing a series of differentiation processes. At the molecular level, these processes must be the direct result of an elaborate series of changes in stage-regulated expression of a wide range of gene products. How are these changes accomplished? In this review, Sheila Graham discusses some recent advances in understanding the mechanisms of gene expression in trypanosomatids, and examines some clues to some intriguingly complex means of regulating life cycle stage-specific gene expression.

HPV-16 E5 down-regulates expression of surface HLA class I and reduces recognition by CD8 T cells

HPV-16 is the major causes of cervical cancer. Persistence of infection is a necessary event for progression of the infection to cancer. Among other factors, virus persistence is due the viral proteins fighting the immune response. HPV-16 E5 down-regulates MHC/HLA class I, which is much reduced on the cell surface and accumulates in the Golgi apparatus in cells expressing E5. This effect is observed also in W12 cells, which mimic early cervical intraepithelial progression to cervical cancer. The functional effect of MHC I down-regulation on human CD8 T cells is not known, because of the need for HLA-matched, HPV-specific T cells that recognise E5 expressing-cells. Here we employ a heterologous cell/MHC I system which uses mouse cells expressing both E5 and HLA-A2, and A2-restricted CTLs; we show that the E5-induced reduction of HLA-A2 has a functional impact by reducing recognition of E5 expressing cells by HPV specific CD8+ T cells.

VSG gene control and infectivity strategy of metacyclic stage Trypanosoma brucei

As the metacyclic trypanosome stage develops in the tsetse fly salivary glands, it initiates expression of variant surface glycoproteins (VSGs) and does so by each cell activating, at random, one from a small subset of metacyclic VSG (M-VSG) genes. Whereas differential activation of individual VSG genes in the bloodstream occurs as a function of time, to evade waves of antibody, it is believed that the aim in the metacyclic stage is simultaneously to generate population diversity. M-VSG genes are activated in their telomeric loci and belong to monocistronic transcription units, unlike all other known trypanosome protein-coding genes, which appear to be transcribed polycistronically. The promoters of these metacyclic expression sites (M-ESs) have the unique property, in this organism, of being switched on and off in a life-cycle stage specific pattern. We have found that the 1.22 M-ES promoter is regulated according to life cycle stage, differential control being exerted through different elements of the promoter and under the influence of its genomic locus. We have characterized in detail the telomeres containing the 1.22 and 1.61 M-ESs. Upstream of the M-ES is a possibly haploid, non-transcribed region with some degenerate sequences homologous with expression site associated genes (ESAGs) that occur in bloodstream VSG expression sites. Further upstream (respectively, 22 and 13 kb upstream of the 1.22 and 1.61 VSG genes) are alpha-amanitin sensitive transcription units that may be polycistrons and are transcribed in all examined life cycle stages. They contain a number of genes. The differences between metacyclic and bloodstream ESs may have important consequences for life cycle regulation, genetic stability, phenotype complexity and adaptability of the metacyclic stage as it infects different host species.

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.

Interaction between Herpes Simplex Virus Type 1 IE63 Protein and Cellular Protein p32

ABSTRACT The herpes simplex virus type 1 (HSV-1) immediate-early gene IE63 (ICP27), the only HSV-1 regulatory gene with a homologue in every mammalian and avian herpesvirus sequenced so far, is a multifunctional protein which regulates transcriptional and posttranscriptional processes. One of its posttranscriptional effects is the inhibition of splicing of viral and cellular transcripts. We previously identified heterogeneous nuclear ribonucleoprotein (hnRNP) K and casein kinase 2 (CK2) as two protein partners of IE63 (H. Bryant et al., J. Biol. Chem. 274:28991–28998, 1999). Here, using a yeast two-hybrid assay, we identify another partner of IE63, the cellular protein p32. Confirmation of this interaction was provided by coimmunoprecipitation from virus-infected cells and recombinant p32 binding assays. A p32-hnRNP K-CK2 complex, which required IE63 to form, was isolated from HSV-1-infected cells, and coimmunoprecipitating p32 was phosphorylated by CK2. Expression of IE63 altered the cytoplasmic distribution of p32, with some now colocalizing with IE63 in the nuclei of infected and transfected cells. As p32 copurifies with splicing factors and can inhibit splicing, we propose that IE63 together with p32, possibly with other IE63 partner proteins, acts to disrupt or regulate pre-mRNA splicing. As well as contributing to host cell shutoff, this effect could facilitate splicing-independent nuclear export of viral transcripts.

RNA splicing factors regulated by HPV16 during cervical tumour progression

The most prevalent human papillomaviruses (HPVs) causing cervical disease are the ‘high‐risk’ HPV types 16 and 18. All papillomaviruses express a transcription factor, E2, that can regulate viral and cellular gene expression. Recently, we demonstrated high‐risk HPV E2‐mediated transcriptional transactivation of SF2/ASF. This essential oncoprotein is a key member of a family of proteins, the SR proteins, that regulate constitutive and alternative splicing. Tight control of RNA splicing is necessary for the production of wild‐type proteins. So, aberrant expression of SR proteins is involved in the aetiology of a range of human diseases, including cancer. Here we demonstrate epithelial differentiation‐specific control of SF2/ASF in HPV16‐infected keratinocytes in organotypic raft culture and in low‐grade cervical lesions (CIN1). Further, we demonstrate HPV16 infection/differentiation‐induced up‐regulation of a specific subset of SR proteins and present evidence that HPV16 E2 controls expression of SRp20, SC35 and SRp75. Using a series of cell lines that model cervical tumour progression, we show that SF2/ASF, SRp20 and SC35 are specifically up‐regulated in a model of cervical tumour progression. These SR proteins are also over‐expressed in high‐grade cervical lesions, indicating that they may all have oncogenic functions. SR proteins could be useful biomarkers for HPV‐associated disease. Copyright

The relationship between connexins, gap junctions, tissue architecture and tumour invasion, as studied in a novel in vitro model of HPV-16-associated cervical cancer progression.

Disruption of gap junctional intercellular communication (GJIC) and/or connexins (gap junction proteins) is frequently reported in malignant cell lines and tumours. Certain human papillomaviruses (HPV) associated with the development of cancers, especially of the cervix, have previously been reported to downregulate GJIC in vitro. There is also evidence for reduced gap junctions in cervical dysplasia. However, many squamous hyperproliferative conditions, including HPV-induced warts, often show extensive upregulation of certain connexins. The association between HPV and GJIC, and the mechanism and consequence of deregulated GJIC in cervical tumour progression, remains unclear. Therefore, using a variety of nonmalignant and malignant cell lines and an organotypic raft-culture system, we investigated the relationship between HPV, gap junctions and tumour progression. Established cervical tumour cell lines carrying HPV were unable to communicate via gap junctions (when assayed by dye-transfer techniques). This correlated with lack of connexin protein expression, while transfection with connexins 26 or 43 led to functional gap junction membrane plaques. On the other hand, immortal but nonmalignant cell lines that contained episomal or integrated HPV-16, but required feeder-layer and growth-factor support, were consistently well coupled, and expressed multiple connexins at membrane junctions. In vitro selection of feeder-layer and growth-factor-independent variants eventually lead to loss of GJIC, which correlated with loss of membrane and increased cytoplasmic connexin 43 localization. However, this was preceded by loss of differentiation and stromal invasion, as assayed on the organotypic raft-culture model. Using this model, a comparison between noncoupled, well-coupled and connexin-transfected cell lines revealed no firm correlation between GJIC and dysplasia, but GJIC appeared to favour increased stratification. These findings demonstrate that loss of GJIC is frequent, but appears to occur more as a consequence of, rather than being the cause of, epithelial dysplasia, and may be influenced by, but is not directly attributable to, HPV.

Transcriptional regulation of metacyclic variant surface glycoprotein gene expression during the life cycle of Trypanosoma brucei.

In antigenic variation in African trypanosomes, switching of the variant surface glycoprotein (VSG) allows evasion of the mammalian host immune response. Trypanosomes first express the VSG in the tsetse fly vector, at the metacyclic stage, in preparation for transfer into the mammal. In this life cycle stage, a small, specific subset (1 to 2%) of VSGs are activated, and we have shown previously that the system of activation and expression of metacyclic VSG (M-VSG) genes is very different from that used for bloodstream VSG genes (S.V. Graham, K.R. Matthews, P.G. Shiels, and J.D. Barry, Parasitology 101:361-367, 1990). Now we show that unlike other trypanosome genes including bloodstream VSG genes, M-VSG genes are expressed from promoters subject to exclusively transcriptional regulation in a life cycle stage-dependent manner. We have located an M-VSG gene promoter, and we demonstrate that it is specifically up-regulated at the metacyclic stage. This is the first demonstration of gene expression being regulated entirely at the level of transcription among the Kinetoplastida; all other protein-coding genes examined in these organisms are, at least partly, under posttranscriptional control. The distinctive mode of expression of M-VSG genes may be due to a stochastic mechanism for metacyclic VSG activation.

Poly(A)-Binding Protein 1 Partially Relocalizes to the Nucleus during Herpes Simplex Virus Type 1 Infection in an ICP27-Independent Manner and Does Not Inhibit Virus Replication

ABSTRACT Infection of cells by herpes simplex virus type 1 (HSV-1) triggers host cell shutoff whereby mRNAs are degraded and cellular protein synthesis is diminished. However, virus protein translation continues because the translational apparatus in HSV-infected cells is maintained in an active state. Surprisingly, poly(A)-binding protein 1 (PABP1), a predominantly cytoplasmic protein that is required for efficient translation initiation, is partially relocated to the nucleus during HSV-1 infection. This relocalization occurred in a time-dependent manner with respect to virus infection. Since HSV-1 infection causes cell stress, we examined other cell stress inducers and found that oxidative stress similarly relocated PABP1. An examination of stress-induced kinases revealed similarities in HSV-1 infection and oxidative stress activation of JNK and p38 mitogen-activated protein (MAP) kinases. Importantly, PABP relocalization in infection was found to be independent of the viral protein ICP27. The depletion of PABP1 by small interfering RNA (siRNA) knockdown had no significant effect on viral replication or the expression of selected virus late proteins, suggesting that reduced levels of cytoplasmic PABP1 are tolerated during infection.