BK Polyomavirus Genomic Integration and Large T Antigen Expression: Evolving Paradigms in Human Oncogenesis.

Human polyomaviruses are ubiquitous, with primary infections that typically occur during childhood and subsequent latency that may last a lifetime. Polyomavirus-mediated disease has been described in immunocompromised patients; its relationship to oncogenesis is poorly understood. We present deep sequencing data from a high-grade BK virus-associated tumor expressing large T antigen. The carcinoma arose in a kidney allograft 6 years after transplantation. We identified a novel genotype 1a BK polyomavirus, called Chapel Hill BK polyomavirus 2 (CH-2), that was integrated into the BRE gene in chromosome 2 of tumor cells. At the chromosomal integration site, viral break points were found, disrupting late BK gene sequences encoding capsid proteins VP1 and VP2/3. Immunohistochemistry and in situ hybridization studies demonstrated that the integrated BK virus was replication incompetent. We propose that the BK virus CH-2 was integrated into the human genome as a concatemer, resulting in alterations of feedback loops and overexpression of large T antigen. Collectively, these findings support the emerging understanding that viral integration is a nearly ubiquitous feature in polyomavirus-associated malignancy and that unregulated large T antigen expression drives a proliferative state that is conducive to oncogenesis. Based on the current observations, we present an updated model of polyomavirus-mediated oncogenesis.

Endothelial cell surface F1-F0 ATP synthase is active in ATP synthesis and is inhibited by angiostatin.

Angiostatin blocks tumor angiogenesis in vivo, almost certainly through its demonstrated ability to block endothelial cell migration and proliferation. Although the mechanism of angiostatin action remains unknown, identification of F(1)-F(O) ATP synthase as the major angiostatin-binding site on the endothelial cell surface suggests that ATP metabolism may play a role in the angiostatin response. Previous studies noting the presence of F(1) ATP synthase subunits on endothelial cells and certain cancer cells did not determine whether this enzyme was functional in ATP synthesis. We now demonstrate that all components of the F(1) ATP synthase catalytic core are present on the endothelial cell surface, where they colocalize into discrete punctate structures. The surface-associated enzyme is active in ATP synthesis as shown by dual-label TLC and bioluminescence assays. Both ATP synthase and ATPase activities of the enzyme are inhibited by angiostatin as well as by antibodies directed against the alpha- and beta-subunits of ATP synthase in cell-based and biochemical assays. Our data suggest that angiostatin inhibits vascularization by suppression of endothelial-surface ATP metabolism, which, in turn, may regulate vascular physiology by established mechanisms. We now have shown that antibodies directed against subunits of ATP synthase exhibit endothelial cell-inhibitory activities comparable to that of angiostatin, indicating that these antibodies function as angiostatin mimetics.

Human La antigen is required for the hepatitis C virus internal ribosome entry site-mediated translation.

The 5'-noncoding region (5'-NCR) of the hepatitis C virus (HCV) RNA genome serves as an internal ribosome entry site (IRES) and mediates translation initiation in a cap-independent manner. Previously, we reported the interaction between La antigen and the HCV IRES, which appeared to occur in the context of initiator AUG. It was further shown that HCV IRES-mediated translation was stimulated in the presence of human La antigen. In this study, we have defined the cis- and trans-acting elements responsible for La-5'-NCR interactions and established the dependence of the HCV IRES efficiency on cellular La antigen. During the La-IRES interaction, initiator AUG but not the neighboring codons was found to be the direct target of La binding. The C terminus effector domain-dependent modulation of La binding to the HCV IRES is demonstrated by deletion and substitution mutagenesis of the protein. An RNA systematic evolution of ligands by exponential enrichment (SELEX), generated against La protein that selectively binds La in HeLa lysates and competes for the protein binding to the 5'-NCR, was used to demonstrate the requirement of La for the HCV IRES function in the context of mono- and dicistronic mRNAs. Sequestration of La antigen by the RNA SELEX in HeLa translation lysates blocked the HCV and poliovirus IRES-mediated translation in vitro. The functional requirement of La protein for the HCV IRES activity was further established in a liver-derived cell line and in an add-back experiment in which the inhibited IRES was rescued by recombinant human La. These results strongly argue for the novel role of La protein during selection of the initiator AUG and its participation during internal initiation of translation of the HCV RNA genome.

Inhibition of polyglutamine protein aggregation and cell death by novel peptides identified by phage display screening.

Proteins with expanded polyglutamine domains cause eight inherited neurodegenerative diseases, including Huntington's, but the molecular mechanism(s) responsible for neuronal degeneration are not yet established. Expanded polyglutamine domain proteins possess properties that distinguish them from the same proteins with shorter glutamine repeats. Unlike proteins with short polyglutamine domains, proteins with expanded polyglutamine domains display unique protein interactions, form intracellular aggregates, and adopt a novel conformation that can be recognized by monoclonal antibodies. Any of these polyglutamine length-dependent properties could be responsible for the pathogenic effects of expanded polyglutamine proteins. To identify peptides that interfere with pathogenic polyglutamine interactions, we screened a combinatorial peptide library expressed on M13 phage pIII protein to identify peptides that preferentially bind pathologic-length polyglutamine domains. We identified six tryptophan-rich peptides that preferentially bind pathologic-length polyglutamine domain proteins. Polyglutamine-binding peptide 1 (QBP1) potently inhibits polyglutamine protein aggregation in an in vitro assay, while a scrambled sequence has no effect on aggregation. QBP1 and a tandem repeat of QBP1 also inhibit aggregation of polyglutamine-yellow fluorescent fusion protein in transfected COS-7 cells. Expression of QBP1 potently inhibits polyglutamine-induced cell death. Selective inhibition of pathologic interactions of expanded polyglutamine domains with themselves or other proteins may be a useful strategy for preventing disease onset or for slowing progression of the polyglutamine repeat diseases.

Dissection of the LXXLL nuclear receptor-coactivator interaction motif using combinatorial peptide libraries: discovery of peptide antagonists of estrogen receptors alpha and beta.

Recruitment of transcriptional coactivators following ligand activation is a critical step in nuclear receptor-mediated target gene expression. Upon binding an agonist, the receptor undergoes a conformational change which facilitates the formation of a specific coactivator binding pocket within the carboxyl terminus of the receptor. This permits the alpha-helical LXXLL motif within some coactivators to interact with the nuclear receptors. Until recently, the LXXLL motif was thought to function solely as a docking module; however, it now appears that sequences flanking the core motif may play a role in determining receptor selectivity. To address this issue, we used a combinatorial phage display approach to evaluate the role of flanking sequences in influencing these interactions. We sampled more than 10(8) variations of the core LXXLL motif with estradiol-activated estrogen receptor alpha (ERalpha) as a target and found three different classes of peptides. All of these peptides interacted with ERalpha in an agonist-dependent manner and disrupted ERalpha-mediated transcriptional activity when introduced into target cells. Using a series of ERalpha-mutants, we found that these three classes of peptides showed different interaction patterns from each other, suggesting that not all LXXLL motifs are the same and that receptor binding selectivity can be achieved by altering sequences flanking the LXXLL core motif. Most notable in this regard was the discovery of a peptide which, when overexpressed in cells, selectively disrupted ERbeta- but not ERalpha-mediated reporter gene expression. This novel ERbeta-specific antagonist may be useful in identifying and characterizing the ERbeta-regulated process in estradiol-responsive cells. In conclusion, using a combinatorial approach to define cofactor-receptor interactions, we have clearly been able to demonstrate that not all LXXLL motifs are functionally equivalent, a finding which suggests that it may be possible to target receptor-LXXLL interactions to develop receptor-specific antagonists.

Myocarditis in Whipple's disease: an unsuspected cause of symptoms and sudden death.

Whipple's disease (WD) is an uncommonly diagnosed infection caused by the recently characterized bacillus, Tropheryma whippelii. The association of WD with pericarditis and endocarditis is widely recognized, although less attention has been paid to the myocardium as a site of disease. Although the disease was uniformly fatal before antibiotic therapy, current treatment usually results in cure. We report two patients whose deaths were directly related to cardiac involvement by WD and whose underlying disease escaped diagnosis for years. The first, a 60-year-old white woman, suffered a cardiovascular collapse, and lymphocytic myocarditis was demonstrated at autopsy. The second, a 48-year-old black man, had a lengthy history of progressive cardiac failure that terminated in arrhythmia. Extensive myocardial fibrosis, with lymphocytic and granulomatous inflammation, was demonstrated at autopsy. The presence of T. whippelii was confirmed by electron microscopic examination in both cases and by polymerase chain reaction in one. Patients with WD might harbor an undiagnosed lymphocytic or granulomatous myocarditis, and this diagnosis should be considered in the evaluation of cardiac failure.

Identification of focal viral infections by confocal microscopy for subsequent ultrastructural analysis.

A correlative microscopy method for the ultrastructural analysis of focal viral tissue infections is presented. Using a confocal scanning laser microscope, foci of infection are identified in tissue sections prior to embedment; a variety of techniques can be employed for viral detection, including staining with standard histochemical reagents and fluorescently labeled antibodies. Areas of infection identified using confocal microscopy are excised from the tissue sections, embedded, and examined by transmission electron microscopy. Applications of this technique in both diagnostic and basic research settings are described.

Sequences within a small yeast RNA required for inhibition of internal initiation of translation: interaction with La and other cellular proteins influences its inhibitory activity.

We recently reported purification, determination of the nucleotide sequence, and cloning of a 60-nucleotide RNA (I-RNA) from the yeast Saccharomyces cerevisiae which preferentially blocked cap-independent, internal ribosome entry site (IRES)-mediated translation programmed by the poliovirus (PV) 5' untranslated region (UTR). The I-RNA appeared to inhibit IRES-mediated translation by virtue of its ability to bind a 52-kDa polypeptide which interacts with the 5' UTR of viral RNA. We demonstrate here that the HeLa 52-kDa I-RNA-binding protein is immunologically identical to human La autoantigen. Moreover, I-RNA-mediated purified La protein. By using I-RNAs with defined deletions, we have identified sequences of I-RNA required for inhibition of internal initiation of translation. Two smaller fragments of I-RNA (16 and 25 nucleotides) inhibited PV UTR-mediated translation from both monocistronic and bicistronic RNAs. When transfected into HeLa cells, these derivatives of I-RNA inhibited translation of PV RNA. A comparison of protein binding by active and inactive I-RNA mutants demonstrates that in addition to the La protein, three other polypeptides with apparent molecular masses of 80, 70, and 37 kDa may influence the translation-inhibitory activity of I-RNA.

Direct interactions between autoantigen La and human immunodeficiency virus leader RNA.

We have characterized the in vivo and in vitro binding of human La protein to the human immunodeficiency virus type 1 (HIV-1) leader RNA, the trans-activation response element (TAR). In immunoprecipitation studies using anti-La serum, La-TAR ribonucleoproteins were recovered from HIV-1-infected lymphocytes. Further characterization of this interaction revealed that La has preference for the TAR stem. However, TAR RNA recognition tolerated changes in the primary sequence of the stem as long as the secondary structure was conserved. This structural aspect of La-TAR recognition was confirmed in competition studies in which certain homopolymers influenced complex formation while other single-stranded and double-stranded RNAs had no effect. Deletion mutants of recombinant La protein were used to demonstrate that the residues responsible for binding to polymerase III precursor transcripts overlapped the binding domain for the TAR leader RNA. This finding of a direct interaction between La and TAR has functional implications for translational regulation of HIV-1 mRNAs as demonstrated in the accompanying report (Y. V. Svitkin, A. Pause, and N. Sonenberg, J. Virol. 68:7001-7007, 1994).

Eukaryotic transcription termination factor La mediates transcript release and facilitates reinitiation by RNA polymerase III.

Ample evidence indicates that Alu family interspersed elements retrotranspose via primary transcripts synthesized by RNA polymerase III (pol III) and that this transposition sometimes results in genetic disorders in humans. However, Alu primary transcripts can be processed posttranscriptionally, diverting them away from the transposition pathway. The pol III termination signal of a well-characterized murine B1 (Alu-equivalent) element inhibits RNA 3' processing, thereby stabilizing the putative transposition intermediary. We used an immobilized template-based assay to examine transcription termination by VA1, 7SL, and Alu class III templates and the role of transcript release in the pol III terminator-dependent inhibition of processing of B1-Alu transcripts. We found that the RNA-binding protein La confers this terminator-dependent 3' processing inhibition on transcripts released from the B1-Alu template. Using pure recombinant La protein and affinity-purified transcription complexes, we also demonstrate that La facilitates multiple rounds of transcription reinitiation by pol III. These results illustrate an important role for La in RNA production by demonstrating its ability to clear the termination sites of class III templates, thereby promoting efficient use of transcription complexes by pol III. The role of La as a potential regulatory factor in transcript maturation and how this might apply to Alu interspersed elements is discussed.

Internal translation initiation on poliovirus RNA: further characterization of La function in poliovirus translation in vitro.

Initiation of poliovirus RNA translation by internal entry of ribosomes is believed to require the participation of trans-acting factors. The mechanism of action of these factors is poorly defined. The limiting amount of one of these factors, La protein, in rabbit reticulocyte lysates (RRL) has been postulated to partially explain the inefficient translation of poliovirus RNA in this system. To further characterize La activity in translation and to identify other potential limiting factors, we assayed the ability of La protein as well as purified initiation factors, eIF-2, guanine nucleotide exchange factor (GEF), eIF-4A, eIF-4B, eIF-4F, and eIF-3, to stimulate the synthesis of P1, the capsid precursor protein, in poliovirus type 1 (Mahoney) RNA-programmed RRL. Of the proteins tested, only La, GEF, and to some extent eIF-2 stimulated the synthesis of P1. The enhanced translation of P1 in response to La occurred concomitantly with the inhibition of synthesis of most aberrant polypeptides, resulting from initiation in the middle of the genome. Deletion of the carboxy-terminal half (214 amino acids) of La did not decrease its binding to the poliovirus 5' untranslated region but abrogated the stimulatory and correcting activity in translation. In contrast to La, GEF and eIF-2 stimulated the overall translation and increased the synthesis of aberrant products as well as P1. Neither La, GEF, nor any other factor stimulated translation of encephalomyocarditis virus RNA in RRL. The implications of these findings for the mechanism of internal translation initiation on picornavirus RNAs are discussed.

Exploring molecular diversity with combinatorial shape libraries.

Surface technologies based upon selection of ligands from combinatorial libraries herald a revolution in molecular research and drug discovery. Molecular diversity is generated by random combinations of monomeric building blocks to form polymeric conformers that constitute 'shape libraries'. The media for exploring surfaces of target molecules include synthetic or biological polymers consisting of natural or modified amino acids, nucleotides, carbohydrates and other organic materials. Targets can be any biological surface, including enzymes, antibodies, receptors and other regulatory molecules. The power of combinatorial selection is in finding conceptual leads for designing high-affinity ligands and effector molecules for the analysis and manipulation of biochemical interactions.

La autoantigen enhances and corrects aberrant translation of poliovirus RNA in reticulocyte lysate.

Translation initiation on poliovirus RNA occurs by internal binding of ribosomes to a sequence within the 5' untranslated region. We have previously characterized a HeLa cell protein, p52, that binds to a fragment of the poliovirus 5' untranslated region (K. Meerovitch, J. Pelletier, and N. Sonenberg, Genes Dev. 3:1026-1034, 1989). Here we report the purification of the HeLa p52. Protein microsequencing identified p52 as La autoantigen. The La protein is a human antigen that is recognized by antibodies from patients with autoimmune disorders such as systemic lupus erythematosus and Sjögren's syndrome. We show that the La protein stimulates translation of poliovirus RNA, but not brome mosaic virus, tobacco mosaic virus, and alfalfa mosaic virus 4 RNA, translation in a reticulocyte lysate. In addition, La corrects aberrant translation of poliovirus RNA in a reticulocyte lysate. Subcellular immunolocalization showed that La protein is mainly nuclear, but after poliovirus infection, La is redistributed to the cytoplasm. Our results suggest that La protein is involved in poliovirus internal initiation of translation and might function through a similar mechanism in the translation of cellular mRNAs.

In vitro selection of an RNA epitope immunologically cross-reactive with a peptide.

An antiserum raised against a peptide was used to select a unique RNA species from a degenerate pool of RNAs designed to resemble an autoantibody recognition site in U1 RNA. The peptide and the selected RNA epitope could compete for antibody binding, suggesting that both RNA and peptide epitopes occupy the same or overlapping antigen-combining sites. Thus, the RNA epitope functioned as a specific inhibitor of the antibody-antigen interaction. We demonstrate that the RNA epitope can be used to tag unrelated RNA molecules and also to detect the presence of the antibody. We propose that sequence-specific recognition of RNA by antibodies may involve protein-RNA contacts similar to those occurring in other nucleic acid-binding proteins. In addition, these findings are compatible with the suggestion that nucleic acid-binding autoantibodies may arise through immunological cross-reactivity between proteins and nucleic acids.

RNA recognition: towards identifying determinants of specificity.

Members of a family of proteins containing a conserved approximately 80-amino acid RNA recognition motif (RRM) bind specifically to a wide variety of RNA molecules. Structural studies, in combination with sequence alignments, indicate the structural context of both conserved and non-conserved elements in the motif. These analyses suggest that all RRM proteins share a common fold and a similar protein-RNA interface, and that non-conserved residues contribute additional contacts for sequence-specific RNA recognition.

Further evidence for overlapping transcriptional units in an Escherichia coli cell envelope-cell division gene cluster: DNA sequence and transcriptional organization of the ddl ftsQ region.

A 1.2-kilobase-pair BamHI fragment from a cell envelope-cell division gene cluster of Escherichia coli containing ddl and part of ftsQ was cloned and sequenced, and the sequence was interpreted with the aid of genetic complementation and promoter fusion data for the region. Both ddl and ftsQ were transcribed in the same direction (clockwise on the genetic map). ddl was shown to be capable of independent expression from a promoter of its own, and a promoter was identified within the ddl structural gene. The structural gene of ddl consisted of 918 nucleotides, encoding a 306-residue polypeptide of molecular weight 32,840; the synthesis of a protein of this molecular weight was shown to be directed from the 1.2-kilobase-pair BamHI fragment in minicells. Analysis of the DNA sequence further showed that the termination codon of ddl is separated from the initiation codon of ftsQ by one base, which suggests that these two genes may be translationally coupled when transcription is initiated upstream of ddl. This represents a second instance of potential translational coupling within this gene cluster and also indicates that the ddl and ftsQ transcriptional units must overlap (as has been reported earlier for ftsQ and ftsA and for ftsA and ftsZ).

DNA sequence and transcriptional organization of essential cell division genes ftsQ and ftsA of Escherichia coli: evidence for overlapping transcriptional units.

The DNA sequence of a cloned segment of the Escherichia coli chromosome containing ftsQ, ftsA, and part of the ftsZ gene was determined and interpreted for genetic complementation and promoter fusion data for the region. The contiguous genes ftsQ, ftsA, and ftsZ were transcribed in the same direction (clockwise on the genetic map) and each had at least one associated promoter which allowed it to be transcribed independently of neighboring genes. ftsA and ftsZ possessed promoters within the coding sequences of the juxtaposed upstream structural genes, and a promoter element for ftsA was surrounded by a region of twofold symmetry which corresponded closely to a symmetrical element in the region of a putative ftsZ promoter. The structural gene of ftsQ consisted of 838 nucleotides, encoding a 276-residue amino acid polypeptide of molecular weight 31,400; the structural gene of ftsA consisted of 1,260 nucleotides, encoding a 420-residue amino acid polypeptide of molecular weight 45,400. The observation that the termination codon of ftsQ overlaps with a potential initiation codon for ftsA suggested that these two genes may be translationally coupled when transcription is initiated upstream of the ftsQ coding sequence.