Expression of the bovine papillomavirus type 1 E5B gene reveals a protein-protein interaction of the E5A and E5B gene products.

The bovine papillomavirus type 1 (BPV-1) genome has been shown to contain a small open-reading frame designated E5B (nucleotides 4013-4167) which is predicted to encode a hydrophobic, 52 amino acid protein. In order to detect and characterize the E5B protein, an 18 nucleotide sequence encoding a 6 amino acid epitope was added to the 3' end of the E5B open-reading frame which was then expressed in COS-1 cells using a SV40 vector. Immunoprecipitation, immunofluorescence, and cell fractionation studies identified the E5B protein as a 4-kDa protein and localized it primarily to membranes of the endoplasmic reticulum and nucleus. Unlike the E5A protein of BPV-1, E5B did not form dimers (despite containing a cysteine residue) or form complexes with growth factor receptors such as the PDGF receptor or erb B-2 receptor. Interestingly, the E5B protein formed physical complexes with the hydrophobic E5A oncoprotein, apparently via transmembrane interactions. Additionally, expression of E5B inhibited the transforming capability of BPV-1 E5A. These observations suggest that the expression of this viral protein may play a significant role in BPV/host cell interactions.

Rolling circle amplification: a new approach to increase sensitivity for immunohistochemistry and flow cytometry.

Immunohistochemistry is a method that can provide complementary diagnostic and prognostic information to morphological observations and soluble assays. Sensitivity, specificity, or requirements for arduous sample preparation or signal amplification procedures often limit the application of this approach to routine clinical specimens. Rolling circle amplification (RCA) generates a localized signal via an isothermal amplification of an oligonucleotide circle. The application of this approach to immunohistochemistry could extend the utility of these methods to include a more complete set of immunological and molecular probes. RCA-mediated signal amplification was successfully applied to the sensitive and specific detection of a variety of cell surface antigens (CD3, CD20, and epithelial membrane antigen) and intracellular molecules (vimentin and prostate-specific antigen) within a variety of routinely fixed specimens, as well as samples prepared for flow cytometry. RCA technology, which has an intrinsically wide dynamic range, is a robust and simple procedure that can provide a universal platform for the localization of a wide variety of molecules as a function of either antigenicity or nucleic acid sequence. The use of RCA in this way could enhance the use of markers of current interest as well as permit the integration of emerging information from genomics and proteomics into cell- and tissue-based analyses.

GreA and GreB proteins revive backtracked RNA polymerase in vivo by promoting transcript trimming.

The GreA and GreB proteins of Escherichia coli show a multitude of effects on transcription elongation in vitro, yet their physiological functions are poorly understood. Here, we investigated whether and how these factors influence lateral oscillations of RNA polymerase (RNAP) in vivo, observed at a protein readblock. When RNAP is stalled within an (ATC/TAG)(n) sequence, it appears to oscillate between an upstream and a downstream position on the template, 3 bp apart, with concomitant trimming of the transcript 3' terminus and its re-synthesis. Using a set of mutant E.coli strains, we show that the presence of GreA or GreB in the cell is essential to induce this trimming. We show further that in contrast to a ternary complex that is stabilized at the downstream position, the oscillating complex relies heavily on the GreA/GreB-induced 'cleavage-and-restart' process to become catalytically competent. Clearly, by promoting transcript shortening and re-alignment of the catalytic register, the Gre factors function in vivo to rescue RNAP from being arrested at template positions where the lateral stability of the ternary complex is impaired.

E5 oncoprotein mutants activate phosphoinositide 3-kinase independently of platelet-derived growth factor receptor activation.

The E5 oncoprotein of bovine papillomavirus type 1 is a Golgi-resident, 44-amino acid polypeptide that can transform fibroblast cell lines by activating endogenous platelet-derived growth factor receptor beta (PDGF-R). However, the recent discovery of E5 mutants that exhibit strong transforming activity but minimal PDGF-R tyrosine phosphorylation indicates that E5 can potentially use additional signal transduction pathway(s) to transform cells. We now show that two classes of E5 mutants, despite poorly activating the PDGF-R, induce tyrosine phosphorylation and activation of phosphoinositide 3-kinase (PI 3-K) and that this activation is resistant to a selective inhibitor of PDGF-R kinase activity, tyrphostin AG1296. Consistent with this independence from PDGF-R signaling, the E5 mutants fail to induce significant cell proliferation in the absence of PDGF, unlike wild-type E5 or the sis oncoprotein. Despite differences in growth factor requirements, however, both wild-type E5 and mutant E5 cell lines form colonies in agarose. Interestingly, activation of PI 3-K occurs without concomitant activation of the ras-dependent mitogen-activated protein kinase pathway. The known ability of constitutively activated PI 3-K to induce anchorage-independent cell proliferation suggests a mechanism by which the mutant E5 proteins transform cells.

Golgi alkalinization by the papillomavirus E5 oncoprotein.

The E5 oncoprotein of bovine papillomavirus type I is a small, hydrophobic polypeptide localized predominantly in the Golgi complex. E5-mediated transformation is often associated with activation of the PDGF receptor (PDGF-R). However, some E5 mutants fail to induce PDGF-R phosphorylation yet retain transforming activity, suggesting an additional mechanism of action. Since E5 also interacts with the 16-kD pore-forming subunit of the vacuolar H(+)-ATPase (V-ATPase), the oncoprotein could conceivably interfere with the pH homeostasis of the Golgi complex. A pH-sensitive, fluorescent bacterial toxin was used to label this organelle and Golgi pH (pH(G)) was measured by ratio imaging. Whereas pH(G) of untreated cells was acidic (6.5), no acidification was detected in E5-transfected cells (pH approximately 7.0). The Golgi buffering power and the rate of H(+) leakage were found to be comparable in control and transfected cells. Instead, the E5-induced pH differential was attributed to impairment of V-ATPase activity, even though the amount of ATPase present in the Golgi complex was unaltered. Mutations that abolished binding of E5 to the 16-kD subunit or that targeted the oncoprotein to the endoplasmic reticulum abrogated Golgi alkalinization and cellular transformation. Moreover, transformation-competent E5 mutants that were defective for PDGF-R activation alkalinized the Golgi lumen. Neither transformation by sis nor src, two oncoproteins in the PDGF-R signaling pathway, affected pH(G). We conclude that alkalinization of the Golgi complex represents a new biological activity of the E5 oncoprotein that correlates with cellular transformation.

Identification of H, K, and N-ras point mutations in stage IB cervical carcinoma.

OBJECTIVE: The ras oncogenes, Harvey (H), Kirsten (K), and neuroblastoma (N), are a family of genes coding for a membrane-associated protein (p21) which possesses inherent guanine triphosphatase (GTPase) activity. Point mutagenesis at codons 12, 13, and 61 has been implicated in ras activation and subsequent cellular transformation. Given the epidemiologic relationship of HPV infection with cervical carcinoma and the tumorigenic interaction of HPV and mutated ras oncogenes, this study was undertaken to identify if mutated ras oncogenes were present in early invasive cervical carcinomas. METHODS: A combination of polymerase chain reaction (PCR) and dot-blot hybridization was used to determine the frequency and types of ras point mutants occurring in cervical carcinoma. Thirty-three patients with early-stage cervical carcinoma were identified. DNA was extracted from archival tumor samples. ras genes were PCR amplified using flanking primers and hybridized with a series of labeled allele-specific oligonucleotides corresponding to wild-type forms of K12,61, N12,13,61, and H12,61, as well as to all combinations of substitution mutations (7 wild-type, 45 mutants). RESULTS: ras mutations were identified in 24.2% of specimens. The detected mutations in H, K, and N-ras all occurred at codon 61. This was not the result of PCR or hybridization artifact in that mutations were detected in position 12 and 13 in appropriate control samples. CONCLUSIONS: Mutant ras has been shown to convert HPV immortalized keratinocytes to the tumorigenic state. Our results indicate that a significant percentage (24.2%) of these early-stage cervical cancers contain activated ras. Additional studies will be needed to evaluate whether codon 61 represents a characteristic "hot-spot" of ras mutation in a subset of cervical carcinoma.

E5 oncoprotein transmembrane mutants dissociate fibroblast transforming activity from 16-kilodalton protein binding and platelet-derived growth factor receptor binding and phosphorylation.

The E5 oncoprotein of bovine papillomavirus type 1 is a 44-amino-acid, hydrophobic polypeptide which localizes predominantly in Golgi membranes and appears to transform cells through the activation of tyrosine kinase growth factor receptors. In fibroblasts, E5 interacts with both the 16-kilodalton vacuolar ATPase subunit and the platelet-derived growth factor receptor (PDGF-R) via its hydrophobic transmembrane domain and induces autophosphorylation of the receptor. To further analyze the correlation between E5 biological activity and its ability to bind these cellular proteins, a series of nine E5 transmembrane mutants was evaluated. In 32D mouse hematopoietic cells, there was an incomplete correlation between the abilities of the E5 mutant proteins to associate the PDGF-R and to transform cells. However, all transforming E5 mutant proteins induced PDGF-R tyrosine phosphorylation. In NIH 3T3 and C127 mouse fibroblasts, both transforming and nontransforming E5 mutant proteins were defective for PDGF-R binding. In addition, while most of the transforming E5 proteins induced PDGF-R phosphorylation, one hypertransforming mutant (serine 17) neither bound nor induced receptor autophosphorylation. These findings support the hypothesis that the transformation of fibroblasts by E5 transmembrane mutants can involve alternative cellular targets or potentially independent activities of the E5 protein. In addition, these results underscore the critical role of the transmembrane domain in mediating E5 biological activities.

E5 oncoprotein retained in the endoplasmic reticulum/cis Golgi still induces PDGF receptor autophosphorylation but does not transform cells.

The E5 oncoprotein encoded by bovine papillomavirus type 1 is a homodimeric, hydrophobic polypeptide which is localized predominantly in Golgi membranes and which transforms several cell types apparently by inducing tyrosine phosphorylation of the platelet-derived growth factor receptor (PDGF-R). While the precise mechanism of receptor activation is unknown, E5 associates with several cellular proteins, including PDGF-R and the 16K V-ATPase protein, and induces the preferential phosphorylation of immature, Endo H-sensitive forms of the receptor. To evaluate whether E5 accumulation in the Golgi was requisite for receptor phosphorylation and cell transformation, we sequestered the E5 protein in the endoplasmic reticulum (ER)/cis Golgi by appending the ER retention KDEL sequence to its C-terminus. In transient assays and in cell lines, E5/KDEL protein and E5/KDEL* protein (a defective variant of KDEL), were stable and formed homodimers normally. E5/KDEL*, similar to wt E5, localized to the Golgi and was transformation-proficient. In contrast, E5/KDEL failed to concentrate in the Golgi and was transformation-incompetent. Despite these critical defects, however, E5/KDEL formed stable complexes with immature PDGF-R and 16K and, even more unexpectedly, induced the phosphorylation of both mature and immature PDGF-R on tyrosine residues to the same level as wt E5. These data demonstrate that E5 can bind and induce PDGF-R phosphorylation in the ER/cis Golgi, but that successful mitogenic signalling (and consequent cell transformation) requires the translocation of E5/receptor complexes to distal Golgi compartments.

Vacuolar H(+)-ATPase mutants transform cells and define a binding site for the papillomavirus E5 oncoprotein.

The 16K subunit of the vacuolar H(+)-ATPase binds specifically to the bovine (BPV) and human (HPV) papillomavirus E5 oncoproteins, and it has been suggested that this interaction may contribute to cell transformation (Goldstein, D. J., and Schlegel, R. (1990) EMBO J. 9, 137-146; Goldstein, D. J., Finbow, M. E., Andresson, T., McLean, P., Smith, K., Bubb, V. J., and Schlegel, R. (1991) Nature 352, 347-349; Conrad, M., Bubb, V. J., and Schlegel, R. (1993) J. Virol. 67, 6170-6178; Goldstein, D. J., Toyama, R., Schlegel, R., and Dhar, R. (1992) Virology 190, 889-893). We generated mutations within the 16K protein to define binding domains for BPV-1 E5 as well as to characterize the role of 16K in cell transformation. 16K consists predominantly of 4 transmembrane (TM) domains. We showed that mutations within the TM4 domain severely inhibited E5 binding. More specifically, conversion of glutamic acid 143 to arginine within TM4 severely reduced 16K/E5 binding, suggesting that charged interactions facilitated efficient binding. This hypothesis was confirmed by demonstrating that binding to the defective 16K arginine mutant could be restored by complementary charge mutations in E5; conversion of E5 glutamine 17 to glutamic acid or aspartic acid enhanced interactions with the 16K arginine mutant. Surprisingly, mutants in TM4 not only bound poorly to wild-type E5 but were converted into an oncoprotein and induced anchorage-independent growth of NIH 3T3 cells. These data define glutamic acid 143 in the 16K TM4 domain and glutamine 17 within E5 as important contributors to E5/16K binding and suggest a role for the 16K protein in the regulation of cell proliferation.

Mutation of the bovine papillomavirus E5 oncoprotein at amino acid 17 generates both high- and low-transforming variants.

The E5 transforming protein of bovine papillomavirus type 1 is a 44-amino-acid, hydrophobic protein which localizes predominantly to Golgi membranes. The E5 transmembrane domain contains a highly conserved glutamine residue at position 17 which, from previous limited mutagenic analysis, appeared essential for transforming activity. In order to determine the specific amino acid requirements at this position, we constructed a series of substitution mutants, representing all classes of amino acids, employing a vector which expressed E5 independently of other bovine papillomavirus gene products. All of the expressed E5 mutant proteins were stable, dimerized normally, and localized to the Golgi. Our results obtained with C127 mouse cells demonstrated that acidic amino acids (and serine) increased E5 transforming activity, whereas basic amino acids greatly inhibited E5 activity. Nonpolar amino acid substitutions were also defective. Interestingly, the relative transforming activities of these E5 mutant proteins changed dramatically when assayed with NIH 3T3 cells, suggesting that an auxiliary cellular protein(s) may modulate E5 transformation or that there are additional or different mechanisms of E5 transformation which are utilized in these two cell lines.

The human T-cell leukemia/lymphotropic virus type I p12I protein cooperates with the E5 oncoprotein of bovine papillomavirus in cell transformation and binds the 16-kilodalton subunit of the vacuolar H+ ATPase.

The human T-cell leukemia/lymphotropic virus type I (HTLV-I) induces T-cell leukemia and transforms human T cells in vitro. A recently identified protein with a molecular weight of 12,000 (12K) (p12I), encoded by single- and double-spliced mRNAs transcribed from the 3' end of the HTLV-I genome, has been shown to localize in the perinuclear compartment and in the cellular endomembranes. The p12I protein exhibits significant amino acid sequence similarity to the E5 oncoprotein of bovine papillomavirus type 1 (BPV-1). Both proteins are very hydrophobic, contain a glutamine residue in the middle of a potential transmembrane region(s), and are localized in similar cellular compartments. Because of these observations, we investigated whether the p12I resemblance to E5 correlated with a similarity in their biological behavior. We expressed the p12I protein to evaluate its ability to functionally cooperate with the BPV-1 E5 oncoprotein and to bind to a cellular target of the E5 protein, the 16K component of the vacuolar H+ ATPase. Cotransfection of the mouse C127 cell line with the p12I and E5 cDNAs showed that although p12I alone could not induce focus formation, it strongly potentiated the transforming activity of E5. In addition, the p12I protein bound to the 16K protein as efficiently as the E5 protein. These findings might provide new insight for potential mechanisms of HTLV-I transformation and suggest that p12I and E5 represent an example of convergent evolution between RNA and DNA viruses.

The BPV-1 E5 protein, the 16 kDa membrane pore-forming protein and the PDGF receptor exist in a complex that is dependent on hydrophobic transmembrane interactions.

The E5 oncoprotein of bovine papillomavirus type 1 is a 44 amino acid, highly hydrophobic protein that induces the stable transformation of immortalized murine fibroblasts, presumably through its activation of growth factor receptors. Previous studies have shown that the E5 protein complexes with the 16 kDa (16k) pore-forming protein of vacuolar H(+)-ATPases. This integral membrane protein is essential for the acidification and function of subcellular compartments that process growth factor receptors. Using an SV40 expression system in COS cells, we analyzed whether the E5-16k complexes bind additional cellular proteins, including growth factor receptors. These studies demonstrate that E5 binds to both the 16k protein and the PDGF receptor and that this tri-component complex can be isolated with antibodies specific for each protein. Importantly, the 16k protein bound to the PDGF receptor in the absence of E5, suggesting that E5 binds to the PDGF receptor via its interaction with the 16k protein. An E5 mutant lacking the hydrophilic carboxyl-terminal 14 amino acids retained binding to both 16k and the PDGF receptor, indicating that E5 binds to these proteins through its hydrophobic, membrane-associating domain. These studies reveal that hydrophobic, intramembrane interactions govern the association of E5, 16k and the PDGF receptor, suggesting a ligand-independent mechanism for receptor activation and a potential link between receptor signal transduction pathways and membrane pore activity.

Simultaneous gain and loss of functions caused by a single amino acid substitution in the beta subunit of Escherichia coli RNA polymerase: suppression of nusA and rho mutations and conditional lethality.

Transcript elongation and termination in Escherichia coli is modulated, in part, by the nusA gene product, an acidic protein that interacts not only with RNA polymerase itself but also with ancillary factors, namely the host termination protein Rho and phage lambda antitermination protein, N. The E. coli nusA1 mutant fails to support lambda development due to a specific defect in N-mediated antitermination. Certain rifampicin-resistant (rifR) variants of the nusA1 host support lambda growth. We report here the isolation and pleiotropic properties of one such rifR mutant, ts8, resulting from a single amino acid substitution mutation in rpoB, the structural gene for polymerase beta subunit. ts8 is a recessive lethal mutation that blocks cell growth at 42 degrees. Pulse-labeling and analysis of newly synthesized proteins indicate that the mutant cell is proficient in RNA synthesis at high temperature. Apparently, ts8 causes a loss of some specialized function of RNA polymerase without a gross defect in general transcription activities. ts8 is an allele-specific suppressor of nusA1. It does not suppress nusAsal, nusB5 and nusE71 mutations nor does it bypass the requirement for a functional N gene and the nut site for antitermination and lambda growth. A mutation in the N gene, punA1, that restores lambda growth in the nusA1 mutant host but not in the nusAsal host, compensates for the nusAsal allele in the ts8 mutant. This combined effect of two allele-specific suppressors suggests that they enhance some aspect of polymerase-NusA-N interaction and function. ts8 suppresses the rho15 mutation, but not the rho112 mutation, indicating that it might render RNA polymerase susceptible to the action of a defective Rho protein. Marker rescue analysis has localized ts8 to a 910-bp internal segment of rpoB that encodes the Rif domain. By amplification, cloning and sequencing of this segment of the mutant chromosome we have determined that ts8 contains Phe in place of Ser522, caused by a C to T transition. By gene conversion, we have established that the simultaneous gain and loss of three functions of polymerase is caused by this single amino acid substitution. Clearly, a site in the beta subunit critical for the functioning of both termination and antitermination factors is altered by ts8. The alteration, we imagine, might make this site on polymerase receptive to some factors but repulsive to others.

Nucleic acid splicing events occur frequently during macronuclear development in the protozoan Oxytricha nova and involve the elimination of unique DNA.

During its life cycle, the hypotrichous ciliated protozoan Oxytricha nova transforms a copy of its chromosomal micronucleus into a transcriptionally active macronucleus which contains exclusively linear, gene-sized DNA molecules with an average size of about 2.2 kilobase pairs (kbp). The micronuclear precursors of two macronuclear DNA molecules have been examined. Each was found to contain at least five blocks of DNA sequences that are absent in the mature macronuclear DNA molecule. These blocks of sequences, referred to as internal eliminated sequences (IESs), must be removed by a nucleic acid breakage and joining process during development. The data obtained to date indicate that IESs are common and suggest that greater than 60,000 IES removal events occur during macronuclear development. Additional analyses indicate that IESs represent a portion of the unique micronuclear DNA sequences known to be eliminated during development. Comparisons of the sequences of IESs revealed common organizational features and some limited primary sequence homologies that suggest models for their developmental excision.