The 3' region of Human Papillomavirus type 16 early mRNAs decrease expression

BACKGROUND: High risk human papillomavirus (HR-HPV) infects mucosal surfaces and HR-HPV infection is required for development of cervical cancer. Accordingly, enforced expression of the early HR-HPV proteins can induce immortalisation of human cells. In most cervical cancers and cervical cancer cell lines the HR-HPV double stranded DNA genome has been integrated into the host cell genome. METHODS: We have used a retroviral GUS reporter system to generate pools of stably transfected HaCaT and SiHa cells. The HPV-16 early sequences that are deleted upon integration of the HPV-16 genome was inserted into the 3' UTR of the reporter mRNA. Pools containing thousands of independent integrations were tested for the steady state levels of the reporter mRNA by Real Time PCR and reporter protein by a GUS enzymatic activity assays. In addition, we tested the cellular distribution and half lives of the reporter mRNAs. The integrity of the reporter mRNAs were tested by northern blotting. RESULTS: We show that the 3' region of the HPV-16 early mRNAs (HPV-16 nucleotide (nt.) 2582–4214) act in cis to decrease both mRNA and protein levels. This region seems to affect transcription from the exogenous minimal CMV promoter or processing of the reporter mRNA. The observed repression was most pronounced at the protein level, suggesting that this sequence may also affect translation. For the HPV types: 2, 6, 11, 13, 18, 30, 31, and 35 we have investigated the regulatory effect of the regions corresponding to the HPV-16 nt. 3358–4214. For all types, except HPV-18, the region was found to repress expression by posttranscriptional mechanisms. CONCLUSION: We find that the 3' region of HPV-16 early mRNAs interfere with gene expression. It is therefore possible that the deletion of the 3' part of early HPV-16 mRNAs occurring during cervical oncogenesis could contribute to transformation of cells through deregulation of the viral oncogene synthesis. Moreover, we find that the corresponding region from several other HPV types also repress expression, suggesting that the repression by this region may be a general feature of the HPV life cycle

Glycosylation status of the C. albicans cell wall affects the efficiency of neutrophil phagocytosis and killing but not cytokine signaling

The cell wall of the opportunistic human fungal pathogen, Candida albicans is a complex, layered network of rigid structural polysaccharides composed of β-glucans and chitin that is covered with a fibrillar matrix of highly glycosylated mannoproteins. Poly-morphonuclear cells (PMNs, neutrophils) are the most prevalent circulating phagocytic leukocyte in peripheral blood and they are pivotal in the clearance of invading fungal cells from tissues. The importance of cell-wall mannans for the recognition and uptake of C. albicans by human PMNs was therefore investigated. N- and O-glycosylation-deficient mutants were attenuated in binding and phagocytosis by PMNs and this was associated with reduced killing of C. albicans yeast cells. No differences were found in the production of the respiratory burst enzyme myeloperoxidase (MPO) and the neutrophil chemokine IL-8 in PMNs exposed to control and glycosylation-deficient C. albicans strains. Thus, the significant decrease in killing of glycan-deficient C. albicans strains by PMNs is a consequence of a marked reduction in phagocytosis rather than changes in the release of inflammatory mediators by PMNs

The activity of TRAF RING homo- and heterodimers is regulated by zinc finger 1

Ubiquitin chains linked through lysine63 (K63) play a critical role in inflammatory signalling. Following ligand engagement of immune receptors, the RING E3 ligase TRAF6 builds K63-linked chains together with the heterodimeric E2 enzyme Ubc13-Uev1A. Dimerisation of the TRAF6 RING domain is essential for the assembly of K63-linked ubiquitin chains. Here, we show that TRAF6 RING dimers form a catalytic complex where one RING interacts with a Ubc13~Ubiquitin conjugate, while the zinc finger 1 (ZF1) domain and linker-helix of the opposing monomer contact ubiquitin. The RING dimer interface is conserved across TRAFs and we also show that TRAF5–TRAF6 heterodimers form. Importantly, TRAF5 can provide ZF1, enabling ubiquitin transfer from a TRAF6-bound Ubc13 conjugate. Our study explains the dependence of activity on TRAF RING dimers, and suggests that both homo- and heterodimers mediated by TRAF RING domains have the capacity to synthesise ubiquitin chains

Programmed cell death and its role in inflammation

Cell death plays an important role in the regulation of inflammation and may be the result of inflammation. The maintenance of tissue homeostasis necessitates both the recognition and removal of invading microbial pathogens as well as the clearance of dying cells. In the past few decades, emerging knowledge on cell death and inflammation has enriched our molecular understanding of the signaling pathways that mediate various programs of cell death and multiple types of inflammatory responses. This review provides an overview of the major types of cell death related to inflammation. Modification of cell death pathways is likely to be a logical therapeutic target for inflammatory diseases

Regulation of splicing-associated SR proteins by HPV-16

Abstract HPV-16 (human papillomavirus type 16) is a small dsDNA (double-stranded DNA) virus which infects mucosal epithelial tissue of the cervix. Epithelial tissue is composed of a basal layer of cells, capable of division, and a number of suprabasal layers, wherein the cells become more differentiated the closer to the surface of the epithelium they become. Expression of viral proteins is dependent upon epithelial differentiation status, and, within the HPV-16 genome, several elements have been found which control expression both transcriptionally and post-transcriptionally. Expression of the highly immunogenic capsid proteins, L1 and L2, is restricted to only the most differentiated cells, where immune surveillance is limited. However, L1 and L2 transcripts can be detected in less differentiated cells, suggesting post-transcriptional mechanisms exist to prevent their expression in these cells. Indeed, a number of cis-acting RNA elements have been observed within the HPV-16 late region which may be involved in control of capsid gene expression. Mechanisms controlling HPV-16 capsid gene expression and the cellular RNA-processing factors involved will be the focus of this article. We have demonstrated that, during differentiation of uninfected epithelial cells and tissue, cellular levels of RNAprocessing proteins are down-regulated as cellular function begins to shut down. General down-regulation of these proteins is observed during differentiation of HaCaT cells, a spontaneously immortalized epithelial cell line, which is virus-uninfected [1]. However, late gene expression from HPV-16 (human papillomavirus type 16) requires extensive RNA processing to ensure efficient production of late viral proteins (S.G. Milligan and S.V. Graham, unpublished work). Therefore the virus must regulate at least a subset of the factors controlling RNA processing to ensure completion of its life cycle. One particular family of splicing-related proteins, known as SR proteins (serine-and arginine-rich proteins), are regulated during HPV-16 infection. These are essential for both constitutive and alternative splicing, and bring together protein complexes which form upon 5 and 3 splice sites Abbreviations used: CIN I, cervical intraepithelial neoplasia, grade I; ESE, exonic splicing enhancer; ESS, exonic splicing silencer; hnRNP, heterogeneous nuclear ribonucleoprotein; HPV, human papillomavirus; LRE, late regulatory element; ORF, open reading frame; pAE, early polyadenylation signal; SF2/ASF, splicing factor 2/alternative splicing factor; SR protein, serineand arginine-rich protein. 1 To whom correspondence should be addressed (email [email protected]). splice sites, allowing for regulation of splice patterns through the use of competing ESEs and ESSs and varying levels of these antagonistic factors The key SR protein, SF2/ASF (splicing factor 2/alternative splicing factor), and hnRNP A1 are up-regulated in HPV-16-infected differentiated epithelial cells W12 is a cell line derived from an HPV-16-infected CIN I (cervical intraepithelial neoplasia, grade I; benign cervical lesions that are the precursors to carcinomas), in which the virus genome is episomal and maintains the natural pattern of HPV infectio