Interactions between the human RNA polymerase II subunits

As an initial approach to characterizing the molecular structure of the human RNA polymerase II (hRPB), we systematically investigated the protein-protein contacts that the subunits of this enzyme may establish with each other. To this end, we applied a glutathione S-transferase-pulldown assay to extracts from Sf9 insect cells, which were coinfected with all possible combinations of recombinant baculoviruses expressing hRPB subunits, either as untagged polypeptides or as glutathione S-transferase fusion proteins. This is the first comprehensive study of interactions between eukaryotic RNA polymerase subunits; among the 116 combinations of hRPB subunits tested, 56 showed significant to strong interactions, whereas 60 were negative. Within the intricate network of interactions, subunits hRPB3 and hRPB5 play a central role in polymerase organization. These subunits, which are able to homodimerize and to interact, may constitute the nucleation center for polymerase assembly, by providing a large interface to most of the other subunits.     

The carboxyl-terminal region of the human papillomavirus type 16 E1 protein determines E2 protein specificity during DNA replication

The mechanism of DNA replication is conserved among papillomaviruses. The virus-encoded E1 and E2 proteins collaborate to target the origin and recruit host DNA replication proteins. Expression vectors of E1 and E2 proteins support homologous and heterologous papillomaviral origin replication in transiently transfected cells. Viral proteins from different genotypes can also collaborate, albeit with different efficiencies, indicating a certain degree of specificity in E1-E2 interactions. We report that, in the assays of our study, the human papillomavirus type 11 (HPV-11) E1 protein functioned with the HPV-16 E2 protein, whereas the HPV-16 E1 protein exhibited no detectable activity with the HPV-11 E2 protein. Taking advantage of this distinction, we used chimeric E1 proteins to delineate the E1 protein domains responsible for this specificity. Hybrids containing HPV-16 E1 amino-terminal residues up to residue 365 efficiently replicated either viral origin in the presence of either E2 protein. The reciprocal hybrids containing amino-terminal HPV-11 sequences exhibited a high activity with HPV-16 E2 but no activity with HPV-11 E2. Reciprocal hybrid proteins with the carboxyl-terminal 44 residues from either E1 had an intermediate property, but both collaborated more efficiently with HPV-16 E2 than with HPV-11 E2. In contrast, chimeras with a junction in the putative ATPase domain showed little or no activity with either E2 protein. We conclude that the E1 protein consists of distinct structural and functional domains, with the carboxyl-terminal 284 residues of the HPV-16 E1 protein being the primary determinant for E2 specificity during replication, and that chimeric exchanges in or bordering the ATPase domain inactivate the protein.     

A C-terminal helicase domain of the human papillomavirus E1 protein binds E2 and the DNA polymerase alpha-primase p68 subunit

The human papillomavirus (HPV) E1 and E2 proteins bind cooperatively to the viral origin of replication (ori), forming an E1-E2-ori complex that is essential for initiation of DNA replication. All other replication proteins, including DNA polymerase alpha-primase (polalpha-primase), are derived from the host cell. We have carried out a detailed analysis of the interactions of HPV type 16 (HPV-16) E1 with E2, ori, and the four polalpha-primase subunits. Deletion analysis showed that a C-terminal region of E1 (amino acids [aa] 432 to 583 or 617) is required for E2 binding. HPV-16 E1 was unable to bind the ori in the absence of E2, but the same C-terminal domain of E1 was sufficient to tether E1 to the ori via E2. Of the polalpha-primase subunits, only p68 bound E1, and binding was competitive with E2. The E1 region required (aa 397 to 583) was the same as that required for E2 binding but additionally contained 34 N-terminal residues. In confirmation of these differences, we found that a monoclonal antibody, mapping adjacent to the N-terminal junction of the p68-binding region, blocked E1-p68 but not E1-E2 binding. Sequence alignments and secondary-structure prediction for HPV-16 E1 and other superfamily 3 (SF3) viral helicases closely parallel the mapping data in suggesting that aa 439 to 623 constitute a discrete helicase domain. Assuming a common nucleoside triphosphate-binding fold, we have generated a structural model of this domain based on the X-ray structures of the hepatitis C virus and Bacillus stearothermophilus (SF2) helicases. The modelling closely matches the deletion analysis in suggesting that this region of E1 is indeed a structural domain, and our results suggest that it is multifunctional and critical to several stages of HPV DNA replication.     

Accuracy of replication in the polymerase chain reaction. Comparison between Thermotoga maritima DNA polymerase and Thermus aquaticus DNA polymerase

A lung suppuration may result in a lung bulla with its own course. We report such a case following a Pseudomonas aeruginosa pneumonia of the upper right lobe, after aspiration of gastric contents, in a 21-year-old tracheotomized patient in chronic post-traumatic coma. Mechanical ventilation (IPPV) was indicated because of respiratory insufficiency. The pneumonia was followed by an abscess and later a lung bulla, increasing in size under the effect of mechanical ventilation with progressive mediastinal compression. Surgery was contraindicated because of poor physical status. An acute episode of cardiac tamponade was controlled with an emergency transthoracic drain insertion into the bulla. The course was favourable after a drainage for 23 days and a persisting small cavity in the lung apex. All weaning attempts being unsuccessful, the patient was discharged under home mechanical ventilation. A CT-scan control 6 months later showed a normal lung parenchyma. The various alternative techniques to surgery for treatment of a lung bulla are discussed.     

Human xeroderma pigmentosum group A protein interacts with human replication protein A and inhibits DNA replication

Human replication protein A (RPA; also known as human single-stranded DNA binding protein, or HSSB) is a multisubunit complex involved in both DNA replication and repair. While the role of RPA in replication has been well studied, its function in repair is less clear, although it is known to be involved in the early stages of the repair process. We found that RPA interacts with xeroderma pigmentosum group A complementing protein (XPAC), a protein that specifically recognizes UV-damaged DNA. We examined the effect of this XPAC-RPA interaction on in vitro simian virus 40 (SV40) DNA replication catalyzed by the monopolymerase system. XPAC inhibited SV40 DNA replication in vitro, and this inhibition was reversed by the addition of RPA but not by the addition of DNA polymerase alpha-primase complex, SV40 large tumor antigen, or topoisomerase I. This inhibition did not result from an interaction between XPAC and single-stranded DNA (ssDNA), or from competition between RPA and XPAC for DNA binding, because XPAC does not show any ssDNA binding activity and, in fact, stimulates RPA's ssDNA binding activity. Furthermore, XPAC inhibited DNA polymerase alpha activity in the presence of RPA but not in RPA's absence. These results suggest that the inhibitory effect of XPAC on DNA replication probably occurs through its interaction with RPA.     

Interactions between subunits of the human epithelial sodium channel

The human epithelial sodium channel (hENaC) mediates Na+ transport across the apical membrane of epithelia, and mutations in hENaC result in hypertensive and salt-wasting diseases. In heterologous expression systems, maximal hENaC function requires co-expression of three homologous proteins, the alpha, beta, and gammahENaC subunits, suggesting that hENaC subunits interact to form a multimeric channel complex. Using a co-immunoprecipitation assay, we found that hENaC subunits associated tightly to form homo- and heteromeric complexes and that the association between subunits occurred early in channel biosynthesis. Deletion analysis of gammahENaC revealed that the N terminus was sufficient but not necessary for co-precipitation of alphahENaC, and that both the N terminus and the second transmembrane segment (M2) were required for gamma subunit function. The biochemical studies were supported by functional studies. Co-expression of gamma subunits lacking M2 with full-length hENaC subunits revealed an inhibitory effect on hENaC channel function that appeared to be mediated by the cytoplasmic N terminus of gamma, and was consistent with the assembly of nonfunctional subunits into the channel complex. We conclude that the N terminus of gammahENaC is involved in channel assembly.     

Distribution of alpha 1A, alpha 1B and alpha 1E voltage-dependent calcium channel subunits in the human hippocampus and parahippocampal gyrus

The distribution of voltage-dependent calcium channel subunits in the central nervous system may provide information about the function of these channels. The present study examined the distribution of three alpha-1 subunits, alpha 1A, alpha 1B and alpha 1E, in the normal human hippocampal formation and parahippocampal gyrus using the techniques of in situ hybridization and immunocytochemistry. All three subunit mRNAs appeared to be similarly localized, with high levels of expression in the dentate granule and CA pyramidal layer. At the protein level, alpha 1A, alpha 1B and alpha 1E subunits were differentially localized. In general, alpha 1A-immunoreactivity was most intense in cell bodies and dendritic processes, including dentate granule cells, CA3 pyramidal cells and entorhinal cortex pre-alpha and pri-alpha cells. The alpha 1B antibody exhibited relatively weak staining of cell bodies but stronger staining of neuropil, especially in certain regions of high synaptic density such as the polymorphic layer of the dentate gyrus and the stratum lucidum and radiatum of the CA regions. The alpha 1E staining pattern shared features in common with both alpha 1A and alpha 1B, with strong immunoreactivity in dentate granule, CA3 pyramidal and entorhinal cortex pri-alpha cells, as well as staining of the CA3 stratum lucidum. These findings suggest regions in which particular subunits may be involved in synaptic communication. For example, comparison of alpha 1B and alpha 1E staining in the CA3 stratum lucidum with calbindin-immuno-reactivity suggested that these two calcium channels subunits may be localized presynaptically in mossy fibre terminals and therefore may be involved in neurotransmitter release from these terminals.     

Dissociation of the protein primer and DNA polymerase after initiation of adenovirus DNA replication

Initiation of adenovirus DNA replication occurs by a jumping back mechanism in which the precursor terminal priming protein (pTP) forms a pTP.trinucleotide complex (pTP.CAT) catalyzed by the viral DNA polymerase (pol). This covalent complex subsequently jumps back 3 bases to permit the start of chain elongation. Before initiation, pTP and pol form a tight heterodimer. We investigated the fate of this pTP.pol complex during the various steps in replication. Employing in vitro initiation and elongation on both natural viral templates and synthetic oligonucleotides followed by glycerol gradient separation of the reaction products, we established that pTP and pol are separated during elongation. Whereas pTP.C and pTP. CA were still bound to the polymerase, after the formation of pTP. CAT 60% of the pTP.pol complex had dissociated. Dissociation coincides with a change in sensitivity to inhibitors and in Km for dNTPs, suggesting a conformational change in the polymerase, both in the active site and in the pTP interaction domain. In agreement with this, the polymerase becomes a more efficient enzyme after release of the pTP primer. We also investigated whether the synthesis of a pTP initiation intermediate is confined to three nucleotides. Employing synthetic oligonucleotide templates with a sequence repeat of two nucleotides (GAGAGAGA ... instead of the natural GTAGTA ... ) we show that G5 rather than G3 is used to start, leading to a pTP. tetranucleotide (CTCT) intermediate that subsequently jumps back. This indicates flexibility in the use of the start site with a preference for the synthesis of three or four nucleotides during initiation rather than two.     

Association of the human papillomavirus type 11 E1 protein with histone H1

The E1 and E2 proteins are the only virus-encoded factors required for human papillomavirus (HPV) DNA replication. The E1 protein is a DNA helicase responsible for initiation of DNA replication at the viral origin. Its recruitment to the origin is facilitated by binding to E2, for which specific recognition elements are located at the origin. The remaining replication functions for the virus, provided by the host cell's replication machinery, may be mediated by further interactions with E1 and E2. Histone H1 was identified as an HPV type 11 (HPV-11) E1-binding protein by far-Western blotting and by microsequence analyses of a 34-kDa protein purified by E1 affinity chromatography. E1 also bound in vitro to H1 isolated under native conditions in association with intact nucleosomes. In addition, E1 and H1 were coimmunoprecipitated by an E1 antiserum from a nuclear extract prepared from cells expressing recombinant E1. Bound H1 was displaced from HPV-11 DNA by the addition of E1, suggesting that E1 can promote replication initiation and elongation by alteration of viral chromatin structure and disruption of nucleosomes at the replication fork. Furthermore, a region of the HPV-11 genome containing the origin of replication was identified which had weaker affinity for H1 than that of the remaining genome. This result suggests that the presence of a DNA structure at or near the HPV origin facilitates initiation of DNA replication by exclusion of H1. These results are similar to those of studies of simian virus 40 DNA replication, in which a large T antigen-H1 interaction and an H1-resistant region at the origin of DNA replication have also been demonstrated.     

Competition for DNA binding sites between the short and long forms of E2 dimers underlies repression in bovine papillomavirus type 1 DNA replication control

Papillomaviruses establish a long-term latency in vivo by maintaining their genomes as nuclear plasmids in proliferating cells. Bovine papillomavirus type 1 encodes two proteins required for viral DNA replication: the helicase E1 and the positive regulator E2. The homodimeric E2 is known to cooperatively bind to DNA with E1 to form a preinitiation complex at the origin of DNA replication. The virus also codes for two short forms of E2 that can repress viral functions when overexpressed, and at least one copy of the repressor is required for stable plasmid maintenance in transformed cells. Employing a tetracycline-regulated system to control E1 and E2 production from integrated loci, we show that the short form of E2 negatively regulates DNA replication. We also found that the short form could repress replication in a cell-free replication system and that the repression requires the DNA binding domain of the protein. In contrast, heterodimers of the short and long forms were activators and, by footprint analysis, were shown to be as potent as homodimeric E2 in loading E1 to its cognate site. DNA binding studies show that when E1 levels are low and are dependent upon E2 for occupancy of the origin site, the repressor can block E1-DNA interactions. We conclude that DNA replication modulation results from competition between the different forms of E2 for DNA binding. Given that heterodimers are active and that the repressor form of E2 shows little cooperativity with E1 for DNA binding, this protein is a weak repressor.     

The papillomavirus minor capsid protein, L2, induces localization of the major capsid protein, L1, and the viral transcription/replication protein, E2, to PML oncogenic domains

Periodontitis and atherosclerosis have complex etiologies, genetic and gender predispositions, and potentially share many risk factors-the most significant of which may be smoking status. These diseases also have many pathogenic mechanisms in common. It is becoming increasingly clear that infections and chronic inflammatory conditions such as periodontitis may influence the atherosclerotic process. The severity and chronicity of periodontal disease provides a rich source of subgingival microbial and host response products and effects over a long time period. The objective of this review is to consider the mechanisms whereby diseases such as periodontitis, which is chronic and Inflammatory In nature and initiated by microbial plaque, can predispose to atherosclerosis. In common with periodontal disease. the pathogenesis of atherosclerosis is not completely understood and both diseases are currently under Intensive investigation. Two main processes in particular are worthy of consideration and may provide the link between these 2 diseases, namely the lipopolysaccharide-related responses and the hyperresponsive monocyte phenomenon. Insufficient experimental evidence exists, however, to further support these hypotheses at present and clearly more research is needed on both of these processes and the interrelationships between both diseases.     

NMR-based discovery of lead inhibitors that block DNA binding of the human papillomavirus E2 protein

The E2 protein is required for the replication of human papillomaviruses (HPVs), which are responsible for anogenital warts and cervical carcinomas. Using an NMR-based screen, we tested compounds for binding to the DNA-binding domain of the HPV-E2 protein. Three classes of compounds were identified which bound to two distinct sites on the protein. Biphenyl and biphenyl ether compounds containing a carboxylic acid bind to a site near the DNA recognition helix and inhibit the binding of E2 to DNA. Benzophenone-containing compounds which lack a carboxylic acid group bind to the beta-barrel formed by the dimer interface and exhibit negligible effects on the binding of E2 to DNA. Structure-activity relationships from the biphenyl and biphenyl ether compounds were combined to produce a compound [5-(3'-(3",5"-dichlorophenoxy)-phenyl)-2,4-pentadienoic acid] with an IC50 value of approximately 10 microM. This compound represents a useful lead for the development of antiviral agents that interfere with HPV replication and further illustrates the usefulness of the SAR by NMR method in the drug discovery process.     

Association of the antibodies against human papillomavirus 16 E4 and E7 proteins with cervical cancer positive for human papillomavirus DNA

Occurrence of the antibodies against human papillomavirus (HPV) 16 proteins E4 and E7 is specifically but independently associated with cervical cancer. To correlate HPV DNA and antibody data, we examined the biopsy specimens and sera, by polymerase chain reaction (PCR) and by ELISA, respectively, from 51 patients with cervical cancer (including 3 recurrent cases) and 22 with cervical intra-epithelial neoplasia. Consensus primers for the L1 region were used for PCR and bacterially expressed, purified fusion protein HPV-16 E4 and non-fusion protein HPV-16 E7 were used for ELISA. HPV-16 DNA and other HPV types were detected in 17 and 25, respectively, out of 51 cases of cervical cancer. Ten out of the 17 HPV-16-DNA-positives were positive either for anti-E4 or for anti-E7: positivities for anti-E4, for anti-E7, and for both were 6/17, 5/17 and 1/17 respectively. Three anti-E7-positives consisted of those for HPV-33, -52 and -58 DNA, suggesting that limited cross-reaction occurred between the HPV types. Among the HPV-16-DNA-positive cases of cancer, lymph-node or distant metastasis was recorded more frequently in the seropositives than in the seronegatives. Our results show that the HPV-16 anti-E4 or anti-E7 occurs in some, but not in all, of the HPV-16-DNA-positive cases, and support the hypothesis that the presence of the HPV-16 antibodies can be used as a marker for possible metastasis.