Search

This report describes measurement of retention of electroactive ferricyanide (Fe(CN)6^3-) entrapped within structurally stable photopolymerized vesicles composed of diacetylenic lipid 1-palmitoyl-2-(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine (PC8,9PC). Vesicle size, shape, and dispersity were assessed by dynamic laser light scattering and rates of permeation of ferricyanide measured by cyclic voltammetry. Gold disk electrodes modified with 6-mercaptohexanol or 2-mercaptoethanamine (cysteamine) respond with quantitative sensitivity to extravesicular ferricyanide over a concentration range of 10 μM to 0.1 M, are insensitive to entrapped ferricyanide even at high applied oxidative potentials, and resist fouling by vesicles, vesicle fragments, or vesicle-rupturing surfactant. Quantitative changes in ferricyanide peak current over time enabled straightforward determination of ferricyanide permeation rate constants as a function of pH and temperature. At 25 °C, ferricyanide permeability increased from 1.1 × 10^-12 to 2.5 × 10^-12 cm/s with increasing pH from 6 to 8. At pH 7, ferricyanide permeability temperature dependency was found to fit an Arrhenius rate expression, increasing exponentially from 1.6 × 10^-12 to 5.8 × 10^-11 cm/s with increasing temperature from 25 to 70 °C, yielding a calculated energy barrier for permeation of 65 kJ/mol and a half-life for intravesicular ferricyanide loss as high as 2.4 weeks. Greater permeability observed at 15 °C relative to 25 °C is attributed to membrane defects present in the gel-phase. These results are consistent with diffusion of Fe(CN)6^3- across intact vesicle walls rather than a pore type mechanism and demonstrate the ability to tune retention of entrapped species by robust polymerized vesicles.

The upstream regulatory region (URR) of the bovine papillomavirus (BPV) genome contains an enhancer that is activated by a BPV E2 gene product. We have previously found that a bacterially derived E2 fusion protein specifically interacted with several fragments of URR DNA, suggesting that E2 may activate transcription by directly binding to the enhancer. Each of the bound fragments contains at least one copy of a conserved motif (ACCN6GGT). To determine if this motif is required and sufficient for specific E2 binding, we have now constructed a bacterial expression vector that encodes a full‐length E2 peptide and developed a refinement of the McKay DNA immunoprecipitation assay that allows the determination, to the nucleotide level, of the minimum sequence required for specific binding. The results show that the E2 recognition sequence is a single copy of this motif and that the variant ACCGN4CGGT is bound with greater affinity than the minimum ACCN6GGT motif. An oligonucleotide encoding the motif was able to inhibit E2‐dependent trans‐activation in a transient transfection assay, indicating that the virally encoded E2 also interacts with this sequence in mammalian cells. When present in two or more copies, but not in a single copy, the E2 binding element had intrinsic enhancer activity but only in cells expressing E2. The results indicate that the conserved motif alone is sufficient for E2‐mediated enhancement and that the binding of E2 to the motif is probably required for efficient enhancement. Since a single motif did not have a significant enhancer activity, it is likely that bound E2 molecules act cooperatively in activating transcription.

The electrochemical reduction of carbon dioxide was studied in methanol-based supporting electrolytes on various metal electrodes at ambient temperature and pressure. The ionophore of the catholyte was benzalkonium chloride, [RN(CH3)2CH2C6H5]+Cl−, where R=C8–C18, the chain length being distributed around C14. A divided H-type cell was used, the supporting electrolytes were 10−2moldm−3 benzalkonium chloride in double distilled methanol (catholyte) and a 10−1moldm−3 aqueous KHCO3 solution (anolyte). Nine different, high purity (>99.5%) metal electrodes were used: Ti, Fe, Co, Ni, Pt, Ag, Au, Zn and Sn. Carbon monoxide, methane and ethane were the main organic products. Silver, Au, Zn and Sn cathodes allowed for the best faradaic yields of CO production, the maximum amount of CO (71%, 185 mmol) being formed on the Ag electrode. Methane evolved on each of the nine tested electrodes, with current yields in the range from 0.2 to 3.0%. Ethane and ethylene were produced on the nickel electrode, with low faradaic efficiencies, 0.5 and 0.3%, respectively. No dimerization products were detected. This research can contribute to large-scale manufacturing of useful organic products from a readily available and cheap raw material: CO2-saturated methanol from industrial absorbers (the Rectisol process).

The most important methods for the electrochemical reduction of carbon dioxide on flat metallic cathodes have been systematically summarized using a novel classification approach. In contrast to the usual classification systems that were based solely on the products of electrolysis, the electroreduction procedures have been grouped according to both the nature of the cathode (sp or d group metal electrodes) and the solvent used for the supporting electrolyte (aqueous or nonaqueous solutions). The new classification system allows the identity of the electroreduction product to be better related to the nature of the metallic electrode and the supporting electrolyte. Similar reduction products are formed by each of the four possible combinations of electrodes and supporting electrolytes (sp group metals in aqueous and nonaqueous electrolytes, and d group metals in aqueous and nonaqueous electrolytes, respectively). The discussion has included both synthetic aspects and mechanistic considerations. Of special interest in this review is the discussion of procedures for the selective preparation of formic acid and for the manufacturing of hydrocarbons and/or alcohols using carbon dioxide as the carbon source.

Circular DNA intermediates of Harvey murine sarcoma virus (Ha-MuSV) have been cloned in lambda gtWES . lambda B and shown to be capable of transforming mouse NIH 3T3 cells [Hager, G. L., Chang, E. H., Chan, H. W., Garon, C. F., Israel, M. A., Martin, M. A., Scolnick, E. M. & Lowy, D. R. (1979) J. Virol. 31, 795-809]. By using the cloned Ha-MuSV DNA insert as a parental genome, we have constructed a series of insertion-deletion mutants by inserting an octomer containing the Sal I linker sequence (G-G-T-C-G-A-C-C) into various regions of the Ha-MuSV genome after partial digestion with Hae III. After ligation into lambda gtWES . lambda B-Sal I vector molecules, the mutant Ha-MuSV DNAs were cloned. Fourteen insertion-deletion mutants have been mapped by restriction enzyme digestion, and their biological activities have been correlated with the locations of mutations. The mutants whose lesion mapped within 3.0 kilobases (kb) frm the 3'-end of the Ha-MuSV genome retained full transforming ability. The mutants containing the Sal I linker insertion at 0.4 or 1.5 kb from the 5'-end also retained transforming ability, but the number of foci induced by the DNAs in transfection assays was greatly reduced. However, a mutant containing a deletion of 1.5 kb at the 5'-end and a mutant with a deletion of the sequences between 1.0 and 1.5 kb from the 5'-end completely lost their transforming potential. A model for the transforming region of Ha-MuSV is discussed. Furthermore, because Ha-MuSV sequences can be rescued from the mouse cells transformed by these mutants using Moloney murine leukemia virus as a helper virus, it implies that the in vitro modified DNAs may be converted into genuine mutant viruses.

A series of mutations encoding single-amino-acid substitutions within the v-rasH effector domain were constructed, and the ability of the mutants to induce focal transformation of NIH 3T3 cells was studied. The mutations, which spanned codons 32 to 40, were made by a "cassette" mutagenesis technique that involved replacing this portion of the v-rasH effector domain with a linker carrying two BspMI sites in opposite orientations. Since BspMI cleaves outside its recognition sequence, BspMI digestion of the plasmid completely removed the linker, creating a double-stranded gap whose missing ras sequences were reconstructed as an oligonucleotide cassette. Based upon the ability of the mutants to induce focal transformation of NIH 3T3 cells, a range of phenotypes from virtually full activity to none (null mutants) was seen. Three classes of codons were present in this segment: one which could not be altered, even conservatively, without a loss of function (codons 32 and 35); one which retained detectable biologic activity with conservative changes but which lost function with more drastic substitutions (codons 36 and 40); and one which retained function even with a nonconservative substitution (codon 39).

We used linker insertion-deletion mutagenesis to study the catalytic domain of the Harvey murine sarcoma virus v-rasH transforming protein, which is closely related to the cellular rasH protein. The mutants displayed a wide range of in vitro biological activity, from those that induced focal transformation of NIH 3T3 cells with approximately the same efficiency as the wild-type v-rasH gene to those that failed to induce any detectable morphologic changes. Correlation of transforming activity with the location of the mutations enabled us to identify three nonoverlapping segments within the catalytic domain that were dispensable for transformation and six other segments that were required for transformation. Segments that were necessary for guanosine nucleotide (GDP) binding corresponded to three of the segments that were essential for transformation; two of the three segments share strong sequence homology with other purine nucleotide-binding proteins. Loss of GDP binding was associated with apparent instability of the protein. Lesions in two of the three other required regions significantly reduced GDP binding, while small lesions in the last required region did not impair GDP binding or membrane localization. We speculate that this latter region interacts with the putative cellular target of ras. The results suggest that transforming ras proteins require membrane localization, guanosine nucleotide binding, and an additional undefined function that may represent interaction with their target.

Microinjection of monoclonal antibody Y13-259, which reacts with all known mammalian and yeast ras-encoded proteins, has previously been shown to prevent NIH 3T3 cells from entering the S phase (L. S. Mulcahy, M. R. Smith, and D. W. Stacey, Nature [London] 313:241-243, 1985). We have now found several transformation-competent mutant v-rasH genes whose protein products in transformed NIH 3T3 cells are not immunoprecipitated by this monoclonal antibody. These mutant proteins are, however, precipitated by a different anti-ras antibody. Each of these mutants lacks Met-72 of v-rasH. In contrast to the result for cells transformed by wild-type v-rasH, Y13-259 microinjection of NIH 3T3 cells transformed by these mutant ras genes did not prevent the cells from entering the S phase. These results imply that a transformation-competent ras gene can supply a normal essential function for NIH 3T3 cells. When the proteins encoded by the mutant ras genes were overproduced in Escherichia coli, several mutant proteins that lacked Met-72 failed to bind Y13-259 in a Western blot. However, a ras protein from a mutant lacking amino antibody, but a ras protein from a mutant lacking amino acids 72 to 84 did not. These results suggest that Y13-259 may bind to a higher ordered structure that has been restored in the mutant lacking amino acids 72 to 82.

To initiate an investigation of the initial step in papillomavirus infection, we have examined the interaction of bovine papillomavirus type 1 (BPV) virions with C127 cells by two assays, binding of radioiodinated BPV virions to cell monolayers and BPV-induced focal transformation. Under physiological conditions, the labeled virions bound to the cell surface in a dose-dependent manner within 1 h. Antibody studies indicated that the interaction was specific and related to infectivity: polyclonal sera raised to BPV virions or to baculovirus-expressed BPV L1 virus-like particles (VLPs) inhibited BPV binding and focal transformation, while sera to denatured BPV virions, to denatured BPV L1, or to human papillomavirus type 16 (HPV-16) VLPs were not inhibitory. An exception was that antisera to BPV L2 were neutralizing but did not inhibit binding. Unlabeled BPV virions and BPV VLPs competed with binding to the cell surface in a concentration-dependent manner. Binding to the cell surface appeared to depend primarily on L1, since BPV VLPs composed of L1 alone or of L1/L2 were equally effective in inhibiting binding and focal transformation. VLPs of HPV-16 also inhibited BPV binding and BPV transformation of C127 cells, suggesting that they interact with the same cell surface molecule(s) as BPV virions. Radiolabeled BPV bound specifically to several mammalian cell lines of fibroblastic and epithelial origin, as well as to a human schwannoma and melanoma lines, although some lines bound up to 10 times as many counts as others. Radiolabeled HPV-16 VLPs bound to both human keratinocytes and mouse C127 cells. The results suggest that papillomaviruses bind a widely expressed and evolutionarily conserved cell surface receptor.

We report a system for generating infectious papillomaviruses in vitro that facilitates the analysis of papillomavirus assembly, infectivity, and serologic relatedness. Cultured hamster BPHE-1 cells harboring autonomously replicating bovine papillomavirus type 1 (BPV1) genomes were infected with recombinant Semliki Forest viruses that express the structural proteins of BPV1. When plated on C127 cells, extracts from cells expressing L1 and L2 together induced numerous transformed foci that could be specifically prevented by BPV neutralizing antibodies, demonstrating that BPV infection was responsible for the focal transformation. Extracts from BPHE-1 cells expressing L1 or L2 separately were not infectious. Although Semliki Forest virus-expressed L1 self-assembled into virus-like particles (VLPs), viral DNA was detected in particles only when L2 was coexpressed with L1, indicating that genome encapsidation requires L2. Expression of human papillomavirus type 16 (HPV16) L1 and L2 together in BPHE-1 cells also yielded infectious virus. These pseudotyped virions were neutralized by antiserum to HPV16 VLPs derived from European (114/K) or African (Z-1194) HPV16 variants but not by antisera to BPV VLPs, to a poorly assembling mutant HPV16 L1 protein, or to VLPs of closely related genital HPV types. Extracts from BPHE-1 cells coexpressing BPV L1 and HPV16 L2 or HPV16 L1 and BPV L2 were not infectious. We conclude that (i) mouse C127 cells express the cell surface receptor for HPV16 and are able to uncoat HPV16 capsids; (ii) if a papillomavirus DNA packaging signal exists, then it is conserved between the BPV and HPV16 genomes; (iii) functional L1-L2 interaction exhibits type specificity; and (iv) protection by HPV virus-like particle vaccines is likely to be type specific.

Detailed restriction endonuclease maps were developed for Harvey murine sarcoma virus (Ha-MuSV) DNA (clone H-1), molecularly closed at its unique EcoRI site in pBR322, for three nonoverlapping subgenomic HindIII clones which together span the entire H-1 clone and for a molecularly cloned DNA copy of a portion of rat 30S RNA (which represents the majority of the rat genetic sequences in Ha-MuSV). Molecular hybridization of the 30S clone to small restriction fragments of clone H-1 revealed a 0.9-to-1.0-kilobase pair region in the 5' half of the Ha-MuSV genome not homologous to the 30S clone, although the 30S clone did contain related sequences in Ha-MuSV on both sides of this nonhomologous region. By using cloned sequences from a segment of the Ha-MuSV nonhomology region as a probe for hybridization to Southern blots of DNA from rat, mouse, bat, and chicken cells, one to three bands were detected in DNA of each species. By contrast, the 30S clone DNA was highly related to many sequences in rat DNA, partially related to fewer mouse DNA sequences, and homologous only to one to three bands in bat and chicken DNA. Earlier work had shown that the 5' half of the Ha-MuSV genome coded for transformation and for the viral p21 protein (Chang et al., J. Virol. 35: 76--92, 1980; Wei et al., Proc. Natl. Acad. Sci. U.S.A., in press). We used two subgenomic HindIII clones whose shared HindIII site mapped within the 5' region of clone H-1 nonhomologous to the 30S clone to test whether the nonhomologous segment might encode the transforming and p21 functions. Although neither of the subgenomic HindIII fragments by themselves induced transformation, ligation of these two nontransforming DNAs to each other did restore p21-mediated transformation. A conclusion consistent with these results is that a region in the 5' half of the Ha-MuSV genome evolutionarily distinct from and not present in rat 30S RNA is essential for transformation and for p21 encoding.

Friend murine leukemia virus (G-MuLV) is a helper-independent, type C retrovirus isolated from stocks of Friend virus complex (spleen focus-forming virus plus MuLV). In cell culture, F-MuLV has an ecotropic and NB-tropic host range and causes XC cells to fuse. When injected into newborn NIH Swiss mice, F-MuLV produces hepatosplenomegaly, severe anemia, and numerous circulating hematopoietic precursors in the peripheral blood with normal thymus and lymph nodes after 3 to 6 weeks. Recently, we molecularly cloned an 8.5-kilobase pair (kbp) form of F-MuLV DNA from which we could recover the pathogenic F-MuLV virus by DNA transfection of NIH 3T3 cells. From this molecularly cloned F-MuLV DNA, we have now subcloned in pBR322 a 4.1-kbp HindIII fragment which contains in continuity 3.0 kbp from the 3' terminus (env and c region), 0.6 kbp of the terminal repeat sequences, and 0.5 kbp from the 5'terminus of the viral RNA (genome). NIH 3T3 fibroblasts were transfected with this DNA fragment an then infected with the wild mouse amphotropic retrovirus (cl 1504-A). In cell culture, 1504-A is a helper-independent type C virus which has an N-tropic host range and does not cause fusion of XC cells. When injected into newborn NIH Swiss mice, 1504-A does not produce splenomegaly or thymic enlargement in mice held for up to 8 months. The transfection with the F-MuLV fragment and the infection with 1504-A consistently yielded virus preparations that were XC positive. From such virus stocks we were able to isolate both helper-independent and replication-defective XC-positive viruses. The helper-independent virus was shown to be a recombinant virus since it contains a gp70 molecule derived at least in part from F-MuLV and a specific gag precursor derived from 1504-A as determined by radioactive immune precipitation assays. When injected into newborn Swiss mice, the recombinant helper-independent virus caused hepatosplenomegaly in approximately 50% of the mice in 6 to 8 weeks. The histology of the diseased splenic tissue was indistinguishable from that seen in the disease caused by the whole F-MuLV. The replication-defective virus could be pseudotyped with new 1504-A virus, and this viral complex also caused the F-MuLV disease picture when the complex was injected into newborn Swiss mice. We conclude that the genetic information responsible for the pathogenicity of F-MuLV is contained within the 4.1-kbp DNA fragment, which includes env gene sequences, the terminal repeat sequences, and the c region sequences of the F-MuLV genome.

Genetic evidence suggests that the bovine papilloma virus type 1 (BPV) E2 open reading frame may encode at least two gene products involved in the regulation of viral gene expression. One, which is probably the full-length product, trans-activates transcription via an enhancer in the viral regulatory region. A second, containing sequences from the 3' end of the open reading frame, inhibits the trans-activating activity of the first product. We now report the identification and initial characterization of three E2-encoded proteins, with mobilities corresponding to 48, 31, and 28 kDa in cells transformed by the wild-type BPV. Pulse-chase experiments indicated that the 48-kDa protein had the longest half-life (40 min), but there was no indication that one species was the precursor of another. The 48-kDa species corresponds to the full-length trans-activating protein. The two smaller species contain only carboxyl-terminal determinants, and either or both could represent inhibitory E2 proteins. Subcellular fractionation localized all three E2 proteins to the nucleus. Consistent with the low rate of viral transcription in BPV-transformed cells, the 31-kDa presumptive repressor species was more abundant than the 48-kDa species.

Infection by certain human papillomavirus types is regarded as the major risk factor in the development of cervical cancer, one of the most common cancers of women worldwide. Analysis of the immunogenic and structural features of papillomavirus virions has been hampered by the inability to efficiently propagate the viruses in cultured cells. For instance, it has not been established whether the major capsid protein L1 alone is sufficient for virus particle assembly. In addition, it is not known whether L1, L2 (the minor capsid protein), or both present the immunodominant epitopes required for induction of high-titer neutralizing antibodies. We have expressed the L1 major capsid proteins of bovine papillomavirus type 1 and human papillomavirus type 16 in insect cells via a baculovirus vector and analyzed their conformation and immunogenicity. The L1 proteins were expressed at high levels and assembled into structures that closely resembled papillomavirus virions. The self-assembled bovine papillomavirus L1, in contrast to L1 extracted from recombinant bacteria or denatured virions, also mimicked intact bovine papillomavirus virions in being able to induce high-titer neutralizing rabbit antisera. These results indicate that L1 protein has the intrinsic capacity to assemble into empty capsid-like structures whose immunogenicity is similar to infectious virions. This type of L1 preparation might be considered as a candidate for a serological test to measure antibodies to conformational virion epitopes and for a vaccine to prevent papillomavirus infection.

The effect of mouse L-cell interferon on bovine papillomavirus type 1 (BPV-1) transformation of murine cells was examined. Mouse interferon reduced the level of BPV-1-induced transformation of mouse C127 cells by 95%. Long-term treatment of established BPV-1-transformed mouse cell clones with mouse L-cell interferon led to a decrease in the average number of the plasmid viral genomes present in these cells to 1/3 to 1/8. Although revertant lines could not be isolated from these lines in the absence of treatment with interferon, flat revertants were easily selected from two independent clonal transformed lines carried for 60 generations in the continued presence of 200 units of interferon per ml. These flat revertants had the biological characteristics of nontransformed C127 cells and could be retransformed by BPV-1. Southern blot hybridization failed to detect BPV-1 DNA in any of eight independent revertant lines examined under conditions that could detect 0.2 copies per cell. We conclude that interferon treatment has resulted in a selective reduction of the amount of extrachromosomal BPV-1 DNA in transformed cells and has cured some treated cells completely of their viral DNA.

Transfer of neor and bovine papillomavirus type 1 (BPV1) DNA into rat embryo fibroblasts led to colony formation in G418-containing medium, with no detectable background in controls with neor DNA alone. More than 50% of the drug-resistant clones could be further propagated in culture. The genetic functions of BPV1 involved in colony formation and in long-term immortalization were investigated by both translation termination mutations in the full-length genome, which inactivate individual open reading frames, and constructs in which these open reading frames were separately expressed under control of long terminal repeat promoter enhancers. Expression of either open reading frame E2 or E5 was sufficient for formation of a drug-resistant colony, but long-term growth in culture required that of E6. No significant cooperative effect was observed upon cotransfection of BPV1 and ras oncogene DNAs. Expression of the early region of the human papillomavirus type 16 also led to immortalization of rat embryo fibroblast cells in the same assay, and, unlike what was previously reported in baby rat kidney cells, it required neither activation by a heterologous promoter, nor a cooperating ras oncogene.

By using molecularly cloned ecotropic AKR murine leukemia virus (MuLV) DNA, a 400-base-pair ecotropic type-specific segment in the env region has been identified. This DNA segment and other defined viral subgenomic fragments have been used as 32P-labeled probes to identify and analyze the structure of integrated ecotropic viral DNA sequences in uninfected mouse genomes. Those mice from which endogenous ecotropic MuLV of the AKR type have been isolated contained at least one virtually complete linear copy of the viral genome. Strains from which ecotropic MuLV has not been isolated lacked ecotropic-specific sequences. All inbred mouse strains tested also contained MuLV DNAs of genomic length whose restriction endonuclease digestion pattern was characteristic of xenotropic viruses.

Human papillomavirus type 16 (HPV16) E6 and E7 are selectively retained and expressed in HPV16-associated human genital tumors. E6 is active in several cell culture assays, including transformation of NIH 3T3 cells, trans activation of the adenovirus E2 promoter, and cooperation with E7 to immortalize normal human keratinocytes. Biochemically, the HPV16 E6 protein has been shown to bind to tumor suppressor protein p53 in vitro and induce its degradation in a rabbit reticulocyte lysate. To examine the relationship between the various biological activities of E6 and inactivation of p53, we tested the abilities of dominant negative mutants of p53 to substitute functionally for E6 in the three cell culture assays. While wild-type p53 inhibited keratinocyte proliferation, both mouse and human mutant p53s, in conjunction with E7, increased proliferation of the keratinocytes, resulting in generation of immortalized lines. However, in contrast to E6, mutant p53 was unable to induce transformation or trans activate the adenovirus E2 promoter in NIH 3T3 cells. These results suggest that inactivation of wild-type p53 is necessary for HPV-induced immortalization of human keratinocytes and that different or additional activities are required for E6-dependent transformation and trans activation of NIH 3T3 cells.

Dexamethasone (3 X 10(-10) to 3 X 10(-6) M) induced foci of morphologically transformed cells in a small proportion of a mink cell line that contains the Moloney murine sarcoma viral genome (S+L-). The induction was glucocorticoid specific, since other steroids with glucocorticoid activity (prednisolone, cortisol, and aldosterone) induced foci with an efficiency that paralleled their glucocorticoid activity, and steroids lacking glucocorticoid activity (17B-estradiol, testosterone, and progesterone) failed to induce foci. Viral antigen, as measured by specific immunofluorescence, was localized to the foci. The induction of foci by dexamethasone (3 X 10(-7)) was accompanied by an approximately 10-fold increase in intracellular Moloney murine sarcoma virus-specific RNA and viral p30 antigen. Removal of dexamethasone was followed by the disappearance of foci and a decrease in viral RNA and p30. In this cell system, therefore, glucocorticoids can affect the intracellular levels of type C viral RNA and protein.

A novel species of 30S RNA has been detected in a variety of mouse cell lines. The 30S RNA is specifically packaged by helper-independent type C viruses propagated in such cells. Nucleic acid hybridization detects no homology between the 30SRNA and the genomic RNA of helper-independent mouse type C viruses. The properties of the 30S RNA suggest that it is a defective endogenous mouse type C virus and that it is analogous to a previously described class of defective endogenous rat type C virus, which has been shown previously to be the progenitor of Kirsten and Harvey murine sarcoma viruses.

The genomes of murine leukemia viruses (MuLV) isolated from wild mice have been studied. Detailed restriction endonuclease maps of the 8.8-kilobase (kb) unintegrated linear viral DNAs were derived for five ecotropic and five amphotropic MuLV's from California field mice, for Friend MuLV, and for one ecotropic and one xenotropic MuLV from Mus musculus castaneus. In general, the California MuLV's were similar in their leftward 6 kb (corresponding to the leftward long terminal repeat [LTR], gag, and pol) and rightward 1 kb (7.8 to 8.8 kb, corresponding to p15E and the rightward LTR). For the region spanning 6.0 to 7.7 kb (which includes the sequences that encode gp70) the amphotropic MuLV's shared few enzyme sites with the ecotropic MuLV's, although the California ecotropic MuLV's were highly related to each other in this region, as were the amphotropic MuLV's. Cross-hybridization studies between amphotropic and California ecotropic MuLV DNAs indicated that they were not homologous in the region 6.3 to 7.6 kb; the California ecotropic viral DNAs cross-hybridized in this region to AKR ecotropic MuLV. When the California viral DNAs were compared with AKR ecotropic viral DNA, many differences in enzyme sites were noted throughout the genome. The U3 regions of the wild mouse LTRs showed partial homology to this region in AKR MuLV. The LTR of Moloney MuLV was highly related to that of the California MuLV's, whereas the LTR of Friend MuLV appeared to be a recombinant between the two types of LTRs. The M. musculus castaneus isolates were most closely related to ecotropic and xenotropic MuLV's isolated from inbred mice. One amphotropic MuLV DNA was cloned from supercoiled viral DNA at its unique EcoRI site in pBR322. Viral DNAs with one and two LTRs were isolated. After digestion with EcoRI, DNAs of both types were infectious. It is concluded that ecotropic and amphotropic MuLV's differ primarily in the region which encodes gp70.

The NF1 gene, which is altered in patients with type 1 neurofibromatosis, encodes neurofibromin, a protein whose GTPase-activating function can negatively regulate GTP-Ras by accelerating its conversion to inactive GDP-Ras. In schwannoma cell lines from patients with neurofibromatosis, loss of neurofibromin was previously shown to be associated with impaired regulation of GTP-Ras. Our analysis of other neural crest-derived tumor cell lines has shown that some melanoma and neuroblastoma cell lines established from tumors occurring in patients without neurofibromatosis contain reduced or undetectable levels of neurofibromin, with concomitant genetic abnormalities of the NF1 locus. In contrast to the schwannoma cell lines, GTP-Ras was appropriately regulated in the melanoma and neuroblastoma lines that were deficient in neurofibromin, even when c-H-ras was overexpressed in the lines. These results demonstrate that some neural crest tumors not associated with neurofibromatosis have acquired somatically inactivated NF1 genes and suggest a tumor-suppressor function for neurofibromin that is independent of Ras GTPase activation.

We have generated four mouse monoclonal antibodies (MAbs) to bovine papillomavirus virions that bound type-specific, adjacent, and conformationally dependent epitopes on the L1 major capsid protein. All four MAbs were neutralizing at ratios of 1 MAb molecule per 5 to 25 L1 molecules, but only three effectively blocked binding of the virus to the cell surface. Therefore, antibodies can prevent papillomavirus infection by at least two mechanisms: inhibition of cell surface receptor binding and a subsequent step in the infectious pathway. The neutralizing epitopes of the bovine papillomavirus L2 minor capsid protein were mapped to the N-terminal half of L2 by blocking the neutralizing activity of full-length L2 antiserum with bacterially expressed peptides of L2. In addition, rabbit antiserum raised against amino acids 45 to 173 of L2 had a neutralizing titer of 1,000, confirming that at least part of the N terminus of L2 is exposed on the virion surface.

We have compared aspects of the mouse sos1 (msos1) and msos2 genes, which encode widely expressed, closely related Ras-specific exchange factors. Although an msos1 plasmid did not induce phenotypic changes in NIH 3T3 cells, addition of a 15-codon myristoylation signal to its 5' end enabled the resulting plasmid, myr-sos1, to induce approximately one-half as many foci of transformed cells as a v-H-ras control. By contrast, an isogenic myr-sos2 plasmid, which was made by fusing the first 102 codons from myr-sos1 at homologous sequences to an intact msos2 cDNA, did not induce focal transformation directly, although it could form foci in cooperation with c-H-ras. Pulse-chase experiments indicated that the half-life of Sos¿1 in NIH 3T3 cells was greater than 18 h, while that of Sos2 was less than 3 h. While in vitro-translated Sos1 was stable in a rabbit reticulocyte lysate, Sos2 was degraded in the lysate, as were each of two reciprocal chimeric Sos1-Sos2 proteins, albeit at a slower rate. In the lysate, Sos2 and the two chimeric proteins could be stabilized by ATPgammaS. Unlike Sos1, Sos2 was specifically immunoprecipitated by antiubiquitin antibodies. In a myristoylated version, the chimeric gene encoding Sos2 at its C terminus made a stable protein in NIH 3T3 cells and induced focal transformation almost as efficiently as myr-msos1, while the myristoylated protein encoded by the other chimera was unstable and defective in the transformation assay. We conclude that mSos2 is much less stable than mSos1 and is degraded by a ubiquitin-dependent process. A second mSos2 degradation signal, mapped to the C terminus in the reticulocyte lysate, does not seem to function under the growth conditions of the NIH 3T3 cells.

DNA of the AKR mouse contains a set of murine leukemia virus sequences that are not present in DNA of the NIH Swiss mouse. NIH mice partially congenic for the AKR murine-leukemia-virus-inducing locus Akv-1 contain this set of sequences, and, in a three-point cross segregating for Akv-1 on an NIH background, the sequences segregated with Akv-1. It is concluded that the Akv-1 locus contains viral sequences.

DNA from virus-negative AKR mouse embryo cells is infectious for NIH 3T3 cells if the transfected cells are treated with 5-iododeoxyuridine (IdUrd) either before or after addition of the DNA. The virus isolated after transfection of the AKR DNA is an ecotropic murine leukemia virus (MuLV) indistinguishable from endogenous AKR MuLV. The AKR DNA is not infectious in the absence of IdUrd treatment of the recipient cells. DNA from AKR cells treated with IdUrd before DNA isolation is not infectious unless the recipient cells have been treated with IdUrd. Transfected DNA from NIH mouse cells is not infectious even when the recipient cells have been treated with IdUrd. DNA from cells chronically infected with AKR MuLV or Rauscher MuLV is infectious with or without IdUrd treatment of the recipient cells, but the infectivity is not enhanced by IdUrd. The results indicate that the endogenous AKR MuLV DNA genome is potentially infectious. The data are consistent with an IdUrd-sensitive restriction in the recipient NIH 3T3 cells that prevents viral replication from endogenous AKR MuLV DNA but does not prevent viral replication from MuLV DNA of productively infected cells.

The spleen focus-forming virus (SFFV), a replication-defective murine leukemia virus that causes the rapid transformation of certain hematopoietic target cells, has acquired specific xenotropic viral genetic information not contained in Friend helper virus. In the current studies, it is shown that a cDNA that represents a xenotropic virus portion of SFFV detects genetic sequences derived from the env gene region of murine xenotropic virus. The significance of the acquisition of these xenotropic viral sequences by SFFV is discussed with regard to their possible role in the rapid leukemogenicity of SFFV, and an analogy is drawn between the formation of SFFV and the formation of the Kirsten and Harvey sarcoma viruses.

E2F-1 is a member of a family of transcription factors implicated in the activation of genes required for the progression through the S phase of the cell cycle. We have examined the biological activities of E2F-1 with short-term colony-forming assays and long-term immortalization assays. High levels of E2F-1, produced by transfection of the E2F-1 cDNA under the control of a strong promoter, reduced colony formation in normal human foreskin keratinocytes (NHFKs). This inhibition could not be overcome by wild-type human papillomavirus type 16 (HPV16) E6 and E7, two proteins which cooperate to immortalize NHFKs, or by a transdominant p53 mutant. High levels of E2F-1 also inhibited growth of primary and established fibroblasts. The growth-inhibitory activity required the DNA binding function of E2F-1 but not its transactivation or pRB binding activities. A positive role for lower levels of E2F-1 in NHFK immortalization was established by examining the ability of E2F-1 to complement HPV16 E7 mutants that were unable to cooperate with HPV16 E6 to immortalize NHFKs. Although E2F-1 was unable by itself to cooperate with E6, it did, in conjunction with E6, complement a p24GLY mutant of E7 that is defective for immortalization and binding of pRB and pRB-related proteins. By contrast, E2F-1 was unable to complement two other E7 mutants, p2PRO and p31/32ARG/PRO, which are also defective in the immortalization assay, although their proteins display wild-type binding of pRB in vitro. Since the binding of E7 to pRB results in disruption of pRB-E2F interaction and release of transcriptionally active E2F, the data support the hypothesis that binding of pRB by E7 and the consequence increase in E2F, the data support the hypothesis that binding of pRB by E7 and the consequence increase in E3F activity are important but not sufficient for E7-induced keratinocyte immortalization.

Morphological transformation of NIH 3T3 cells was observed following coexpression of a portion of the ras GTPase-activating protein (GAP) comprising the amino terminus (GAP-N) and a mutant of v-src (MDSRC) lacking the membrane-localizing sequence. Cells expressing either of these genes alone remained nontransformed. Coexpression of GAP-N with MDSRC did not alter the subcellular localization, kinase activity, or pattern of cellular substrates phosphorylated by the MDSRC product. In contrast to SHC, phospholipase C-gamma 1, and the p85 alpha phosphatidylinositol 3'-kinase subunit, the endogenous GAP product (p120GAP) was highly tyrosine-phosphorylated only in cells transformed by wild-type v-src. Furthermore, for transformation induced by wild-type v-src as well as by coexpression of MDSRC and GAP-N, a strict correlation was observed between cell transformation, elevated tyrosine phosphorylation of p62, p190, and a novel protein of 150 kDa, and complex formation between these proteins and p120GAP. As with cells transformed by wild-type v-src, the MDSRC plus GAP-N transformants remained dependent on endogenous Ras. The results suggest that tyrosine phosphorylation and complex formation involving p120GAP represent critical elements of cell transformation by v-src and that complementation of the cytosolic v-src mutant by GAP-N results, at least in part, from the formation of these complexes.

The bovine papillomavirus E5 gene encodes an oncoprotein that can independently transform rodent fibroblasts. This small 44-amino-acid protein is thought to function through the activation of growth factor receptors. E5 activation of the epidermal growth factor receptor results in an increase in the number of activated receptors at the cell surface. This finding suggests that E5 may act by inhibiting the normal down regulation of activated epidermal growth factor receptor via coated pit-mediated endocytosis. We have constructed a fusion protein consisting of glutathione S-transferase and the conserved C-terminal domain of E5 (GST-E5) in order to identify E5-associated cellular proteins that may be involved in its transforming activity. We have identified a 125-kDa cellular protein with a strong associated serine kinase activity that specifically associated with GST-E5 in the reduced form but not with GST-E5 fusions that contained changes in several conserved amino acids. Microsequence and biochemical analyses suggest that p125 is a novel member of the alpha-adaptin family. Since alpha-adaptins have previously been shown to be involved in coated pit-mediated cell surface receptor endocytosis and down regulation, these results suggest that p125 may be an alpha-adaptin-like molecule involved in growth factor receptor down regulation and that E5 may act by inhibiting its activity.

We have described the isolation of a replication-defective murine leukemia virus from a culture of AKR lymphoma cells [Rein et al., Nature (London) 282:753-754, 1979]. To facilitate the characterization of this murine leukemia virus, we transmitted it to mink cells and analyzed its genome by restriction mapping of the mink cellular DNA. This genome resembled the Akv genome quite closely, but it had an additional KpnI cleavage site at 1.3 kilobase pairs from the 5' end of the provirus and a small (approximately 50-base-pair) deletion between 1.8 and 3.0 kilobase pairs from the 5' end. When we tested these mink cells by immune precipitation or by competition radioimmunoassay, we found that they synthesized gPr82env, but contained no detectable gag or pol proteins. It seems likely that the KpnI cleavage site at 1.3 kilobase pairs reflects an abnormal sequence at or near the beginning of the gag gene, which prevents gag or pol translation by introducing a frameshift or termination codon into this region.

The nucleotide sequence of the long terminal repeat (LTR) of three murine retroviral DNAs has been determined. The data indicate that the U5 region (sequences originating from the 5' end of the genome) of various LTRs is more conserved than the U3 region (sequences from the 3' end of the genome). The location and sequence of the control elements such as the 5' cap, "TATA-like" sequences, "CCAAT-box," and presumptive polyadenylic acid addition signal AATAAA in the various LTRs are nearly identical. Some murine retroviral DNAs contain a duplication of sequences within the LTR ranging in size from 58 to 100 base pairs. A variant of molecularly cloned Moloney murine sarcoma virus DNA in which one of the two LTRs integrated into the viral DNA was also analyzed. A 4-base-pair duplication was generated at the site of integration of LTR in the viral DNA. The host-viral junction of two molecularly cloned AKR-murine leukemia virus DNAs (clones 623 and 614) was determined. In the case of AKR-623 DNA, a 3- or 4-base-pair direct repeat of cellular sequences flanking the viral DNA was observed. However, AKR-614 DNA contained a 5-base-pair repeat of cellular sequences. The nucleotide sequence of the preintegration site of AKR-623 DNA revealed that the cellular sequences duplicated during integration are present only once. Finally, a striking homology between the sequences flanking the preintegration site and viral LTRs was observed.

The Raf-1 serine/threonine protein kinase plays a central role in many of the mitogenic signaling pathways regulating cell growth and differentiation. The regulation of Raf-1 is complex, and involves protein-protein interactions as well as changes in the phosphorylation state of Raf-1 that are accompanied by alterations in its electrophoretic mobility. We have previously shown that a 33-kDa COOH-terminal, kinase-inactive fragment of Raf-1 underwent a mobility shift in response to the stimulation of cells with serum or phorbol esters. Here we demonstrate that treatment of NIH 3T3 cells or Sf9 cells with hydrogen peroxide (H2O2) also induces the mobility shift of the kinase-inactive Raf-1 fragment. A series of deletion mutants of the Raf-1 COOH terminus were analyzed, and the region required for the mobility shift was localized to a 78-amino acid fragment (residues 566-643). Metabolic labeling revealed that the slower migrating forms of the 33-kDa and of the smaller fragment contained phosphorus. Mutation of a previously characterized phosphorylation site, serine 621, to alanine prevented the mobility shift as well as phosphate incorporation or Src and Ras-dependent kinase activation in Sf9 cells when this mutation was engineered into the full-length Raf-1. Mutation of 621 to aspartate yielded a protein that existed in both the shifted and unshifted forms, demonstrating that a negative charge at 621 was necessary, but not sufficient, for the mobility shift to occur; however, its full-length form was still resistant to activation in the Sf9 system. Additional mutation of nearby serine 624 to alanine blocked the shift, implicating this residue as the site of the second of a two-step modification process leading to the slower migrating form. Co-expression of the 33-kDa fragment with an activated form of mitogen-activated protein kinase kinase in NIH 3T3 led to the appearance of the shifted form in a serum-independent manner. These results demonstrate that a mitogen-activated protein kinase kinase-induced event involving modification of serines 621 and 624 leads to the mobility shift of Raf-1.

The lack of an endonuclease EcoRI site in the AKR murine leukemia virus (MuLV) DNA genome was utilized to molecularly clone, in Charon 4A lambda DNA, integrated infectious AKR MuLV DNA isolated from productively infected mouse cells. Three lambda-mouse recombinants (clones 614, 621, and 623) were selected by virtue of their reactivity with AKR MuLV [32P]cDNA. Clones 614 and 623 contained the complete AKR MuLV DNA flanked by nonviral cell sequences of which no more than 100 base pairs beyond the viral DNA appear to be shared. DNAs from both clones 614 and 623 were highly infectious for mouse cells and yielded N-tropic ecotropic MuLV; the specific infectivity of the DNA and the titer of the derived virus was more than 10-fold higher with 623. Clone 621 contained only some viral DNA and was not infectious under similar conditions.

We have determined that three type-specific and conformationally dependent monoclonal antibodies, H16.E70, H16.U4, and H16.V5, neutralize pseudotype human papillomavirus type 16 (HPV16) virions in vitro. H16.U4 and H16.V5 neutralized pseudotype virions derived from the German HPV16 variant 114K and the Zairian variant Z-1194 with equal efficiency. In contrast, neutralization of Z-1194 pseudotype virions by H16.E70 was two orders of magnitude weaker than neutralization of 114K pseudotype virions. This difference correlated with enzyme-linked immunosorbent assay reactivity of H16.E70 to L1 virus-like particles of the two variants. A substitution at residue 282 of L1 was responsible for this differential reactivity, suggesting that this residue constitutes part of the H16.E70 epitope.

To assess the potential for cross-protection among genital human papillomavirus (HPV) types in virus-like particle (VLP)-based vaccinations, inhibition of HPV VLP-mediated hemagglutination by rabbit antisera raised against HPV type 6b (HPV-6b), HPV-11, HPV-16, HPV-18, HPV-31, HPV-33, and HPV-45 was analyzed. Only highly homologous types (HPV-6b and HPV-11, and HPV-18 and HPV-45) exhibited detectable serological cross-reaction for the class of antibodies that inhibit virion-to-cell surface binding. However, analysis of neutralizing monoclonal antibodies to several animal and human papillomaviruses indicated that over half of these antibodies do not prevent cell surface binding, but these latter antibodies do not appear to be more cross-reactive in enzyme-linked immunosorbent assays than those that mediate inhibition of hemagglutination. The data strongly suggest that while there may be limited cross-protection between highly (>85% L1 amino acid identity) homologous types, protection by HPV VLP-based vaccines will be predominantly type specific.

Virus-like particles (VLPs) composed of L1 derived from bovine papillomavirus type 1 (BPV-1), several human papillomavirus types, or cottontail rabbit papillomavirus (CRPV) agglutinated mouse but not human or rat erythrocytes. Treatment of mouse erythrocytes with trypsin prevented hemagglutination (HA) by BPV-1. Sera from rabbits immunized with native CRPV VLPs, which protect against experimental CRPV infection, exhibited high titers of antibodies that inhibited CRPV VLP HA activity, while sera from rabbits immunized with denatured CRPV VLPs or native BPV VLPs, which do not protect against CRPV infection, were not inhibitory. Testing for HA inhibition is a rapid and simple method for examining the serological relatedness of papillomaviruses and measuring protective antibody titers after VLP vaccination.

We tested the ability of vaccination with virus-like particles (VLPs) to protect domestic rabbits against papillomas induced by the cottontail rabbit papillomavirus (CRPV). A recombinant baculovirus system that expressed only the L1 major papillomavirus structural protein or L1 plus the minor L2 protein was used in insect cells as the source of VLPs. Groups of 10 rabbits were immunized with native or denatured VLPs from CRPV or type 1 bovine papillomavirus by using Freund's adjuvant. Alum was used as the adjuvant for an additional group immunized with CRPV L1-L2 VLPs. Animals were challenged with 5 x 10(10) and 2 x 10(11) particles on opposing flanks. No protection was seen in rabbits immunized with native or denatured bovine papillomavirus L1-L2 or with denatured CRPV L1-L2. In these groups, the lower and higher challenge doses resulted in 27 of 30 animals with extensive papillomas, with each of the remaining animals having a smaller number of persistent papillomas. Progression to carcinoma developed in 20 rabbits. Animals inoculated with native CRPV VLPs composed of L1 alone or L1-L2 developed many fewer lesions; the lower and higher challenge doses resulted in 17 of 29 and 5 of 29 rabbits, respectively, with no lesions, and the remainder developed only one to eight papillomas, which all regressed except for those on 1 rabbit. None developed cancer within 1 year of infection. Rabbits vaccinated with native CRPV VLPs developed high-titer antibodies in an enzyme-linked immunosorbent assay based on native VLPs, and passive transfer of serum or immunoglobulin G from rabbits immunized with CRPV VLPs protected against CRPV challenge. We conclude that native VLPs can induce antibody-mediated, type-specific protection against experimental papillomavirus infection.

The L1 genes of two human papillomavirus type 16 (HPV16) isolates derived from condylomata acuminata were used to express the L1 major capsid protein in insect cells via recombinant baculoviruses. Both L1 major capsid proteins self-assembled into virus-like particles (VLP) with high efficiency and could be purified in preparative amounts on density gradients. The yield of VLP was 3 orders of magnitude higher than what has been obtained previously, using L1 derived from the prototype HPV16. DNA sequence comparison identified a single nonconserved amino acid change to be responsible for the inefficient self-assembly of the prototype L1. VLP were also obtained by expressing L1 of HPV6, HPV11, and cottontail rabbit papillomavirus, indicating that L1 from a variety of papillomaviruses has the intrinsic capacity to self-assemble into VLP. Coexpression of HPV16 L1 plus L2 by using a baculovirus double-expression vector also resulted in efficient self-assembly of VLP, and the average particle yield increased about fourfold in comparison to when L1 only was expressed. Coimmunoprecipitation of L1 and L2 and cosedimentation of the two proteins in a sucrose gradient demonstrated that L2 was incorporated into the particles. The ability to generate preparative amounts of HPV16 L1 and L1-L2 VLP may have implications for the development of a serological assay to detect anti-HPV16 virion immune responses to conformational epitopes and for immunoprophylaxis against HPV16 infection.

The bovine papillomavirus E5 transforming protein appears to activate both the epidermal growth factor receptor (EGF-R) and the platelet-derived growth factor receptor (PDGF-R) by a ligand-independent mechanism. To further investigate the ability of E5 to activate receptors of different classes and to determine whether this stimulation occurs through the extracellular domain required for ligand activation, we constructed chimeric genes encoding PDGF-R and EGF-R by interchanging the extracellular, membrane, and cytoplasmic coding domains. Chimeras were transfected into NIH 3T3 and CHO(LR73) cells. All chimeras expressed stable protein which, upon addition of the appropriate ligand, could be activated as assayed by tyrosine autophosphorylation and biological transformation. Cotransfection of E5 with the wild-type and chimeric receptors resulted in the ligand-independent activation of receptors, provided that a receptor contained either the transmembrane domain of the PDGF-R or the cytoplasmic domain of the EGF-R. Chimeric receptors that contained both of these domains exhibited the highest level of E5-induced biochemical and biological stimulation. These results imply that E5 activates the PDGF-R and EGR-R by two distinct mechanisms, neither of which specifically involves the extracellular domain of the receptor. Consistent with the biochemical and biological activation data, coimmunoprecipitation studies demonstrated that E5 formed a complex with any chimera that contained a PDGF-R transmembrane domain or an EGF-R cytoplasmic domain, with those chimeras containing both domains demonstrating the greatest efficiency of complex formation. These results suggest that although different domains of the PDGF-R and EGF-R are required for E5 activation, both receptors are activated directly by formation of an E5-containing complex.

The wild-type E6 and E7 genes of human papillomavirus type 16 (HPV16) can cooperate to immortalize normal human keratinocytes in culture. The E6 open reading frame of HPV16 and other HPV types highly associated with cervical cancer has the potential of encoding both full-length E6 and two truncated E6* proteins, the latter being generated via splicing within the E6 open reading frame portion of the E6-E7 polycistronic transcript. Those types, such as HPV6, that are infrequently associated with cervical carcinoma lack the splice site and encode only a full-length E6. We have now found that, in addition to cooperating with E7 to immortalize keratinocytes, HPV16 E6 can induce anchorage-independent growth in NIH 3T3 cells and trans-activate the adenovirus E2 promoter. HPV6 E6 was also able to trans-activate the adenovirus E2 promoter, although it was inactive in both cell transformation assays. An HPV16 splice site mutant which expressed only the full-length HPV16 E6 was active in all three assays, indicating that the E6* proteins are not required for these activities. The plasmid which encodes the E6* proteins was inactive and did not potentiate the activity of the HPV16 splice site mutant. The mutation that prevented splicing in E6-E7 mRNA severely reduced the level of E7 protein and increased E6 protein. Taken together, the results suggest that the primary function of the splice within E6 is to facilitate the translation of E7 and reduce translation of full-length E6, rather than to generate biologically active E6* proteins.

Papillomavirus proteins E6 and E7 have Cys-X-X-Cys repeats which have been suggested to mediate zinc binding. We have developed a modification of an assay that detects zinc binding to proteins immobilized on filters. Using well-characterized metalloproteins, we show that, under reducing conditions, this assay distinguishes proteins that coordinate zinc through cysteine residues from those that bind the metal through other amino acids. Under these conditions, E6 and E7 polypeptides of human papillomavirus type 18 and bovine papillomavirus type 1 exhibited high-affinity zinc binding. Our results suggest that E6 and E7 are metalloproteins and may coordinate the metal ions through cysteine residues.

The E1 open reading frame of bovine papillomavirus type 1 (BPV) has been shown previously to encode trans-acting functions, M and R, that are involved in extrachromosomal replication of the viral genome. We have determined that several E1 mutants mapping in both the M and R regions and a single mutant of the upstream regulatory region have a higher transforming activity on mouse C127 cells than the wild-type genome does. A representative mutant in M, a mutant in R, and the upstream regulatory region mutant were complemented in trans by the wild-type genome, but the two E1 mutants did not complement each other, suggesting that they affect the same inhibitory function. A long terminal repeat-activated clone constructed to express the intact E1 open reading frame reversed the high-transformation phenotype of the mutants. In contrast to the high-copy-number autonomous replication of the wild-type genome, the genomes of the E1 mutants were, as previously described for other E1 mutants, integrated at lower copy numbers in the transformed cells. Relative to the viral genome copy number, both the E1 M and R mutant transformed cells contained an average of 10-fold more BPV-specific transcripts than did the wild-type transformed cells. Cycloheximide treatment of the cells transformed by the E1 mutants did not lead to the rapid 10-fold increase in the accumulation of viral transcripts observed with the wild-type genome. These results suggest either that integration of the BPV genome makes it unresponsive to a labile repressor or that an E1 gene product, containing both M and R sequences, is a repressor of BPV transcription.

The rat-derived Harvey murine sarcoma virus (Ha-MuSV) contains a transduced ras oncogene activated by two missense mutations and flanked by rat retroviruslike VL30 sequences. Ha-MuSV induces focal transformation of mouse NIH 3T3 cells in vitro and tumors (fibrosarcomas and splenic erythroleukemias) in newborn mice. We have used these two assays to study the contribution of coding and noncoding viral sequences to the biological activity of Ha-MuSV. A good correlation was found between the in vitro and in vivo assays. In several different isogenic Ha-MuSV variants, those with a rasH gene that had one or both of the Ha-MuSV missense mutations were much more active biologically than the corresponding proto-oncogene. A Ha-MuSV variant that encoded the proto-oncogene protein induced lymphoid leukemias (with thymomas), with a relatively long latent period, rather than the fibrosarcomas and erythroleukemias characteristic of Ha-MuSV with one or both missense mutations. A VL30-derived segment with enhancer activity was identified downstream from v-rasH. A mutant Ha-MuSV from which this 3' noncoding segment was deleted expressed lower levels of the wild-type viral protein, displayed impaired transforming activity in vitro, and induced lymphoid leukemias (with thymomas). 5' noncoding rat c-rasH sequences were found to increase the biological activity of the virus when substituted for the corresponding segment of v-rasH. We conclude that (i) the biological activity of Ha-MuSV can be influence significantly by noncoding sequences located outside the long terminal repeat as well as by coding sequences, (ii) VL30 sequences positively regulate the expression of v-rasH, (iii) relatively low biological levels of ras, whether resulting from low-level expression of wild type v-rasH or high-levels of ras proto-oncogene protein, induce a type of tumor that differs from tumors induced by high biological levels of ras, and (iv) the in vivo pathogenicity of the Ha-MuSV variants correlated with their transforming activity on NIH 3T3 cells.

We have previously shown that the early region of the bovine papillomavirus type 1 genome contains two nonoverlapping segments that can independently induce the morphological transformation of cultured cells. The transforming gene from the 5' end of the early region is encoded by the E6 open reading frame. The second transforming segment was previously localized to a 2.3-kilobase fragment (2.3T) from the 3' end of the early region. To determine which of the four open reading frames (E2, E3, E4, and E5) located within 2.3T encodes a transforming gene, we have now introduced a series of insertion and deletion mutations into a clone (pHLB1) in which 2.3T is activated by the Harvey viral long terminal repeat, and we tested the mutants for their ability to induce focal transformation. Our results indicate that the E5 open reading frame, which could encode a low-molecular-weight hydrophobic peptide, is required for pHLB1-induced transformation of NIH 3T3 cells, but that the E2, E3, and E4 open reading frames are not.

Bovine papillomavirus type 1 (BPV-1) has been used as a model for studying papillomavirus genetics because BPV-1 virions or BPV-1 genomic viral DNA efficiently induce morphologic transformation of certain cultured cells. Previous studies of BPV-1-induced transformation have found that a cloned 5.4-kilobase (kb) fragment (69T) of the genome is transforming and that a 2.3-kb segment from the 3' end of this fragment is also transforming if activated by a retroviral regulatory element (the long terminal repeat). We now report that 69T contains another transforming segment near its 5' end that can also be activated by a long terminal repeat. Since this second segment does not overlap the 3' transforming segment, we conclude that BPV-1 encodes at least two genes that can independently transform cultured cells. Mutational analysis of the 5' transforming segment suggests that the transforming gene of this segment lies within the E6 open reading frame. The two transforming segments differ in their biological activity in that the E6-containing fragment can transform C127 mouse cells but not NIH3T3 mouse cells, whereas the 3' fragment can transform both cell lines.

The neurofibromatosis type 1 (NF1) gene responsible for von Recklinghausen neurofibromatosis is related to regulators of ras proteins, and a portion of NF1 that is homologous to the ras GTPase-activating protein (GAP) encodes a similar GTPase-stimulating activity. We have raised rabbit antisera to a bacterially synthesized 48-kDa peptide corresponding to the GAP-related domain of NF1 (NF1-GRD). These antisera immunoprecipitated the NF1-GRD peptide, and one of them specifically inhibited the GTPase-stimulating activity of NF1-GRD. The sera specifically detected a 280-kDa protein in lysates of mouse NIH 3T3 and human HeLa cells. This protein corresponds to the NF1 gene product, as shown by several criteria, including partial proteolysis. Subcellular fractionation revealed that while GAP is predominantly cytoplasmic, all of the NF1 was recovered in a pellet (100,000 x g) fraction. NF1 was present in a large molecular mass complex in fibroblast and Schwannoma cell lines and appears to associate with a very large (400-500 kDa) protein in both cell types. The relevance of these findings to cellular regulation of p21ras is discussed.

Harvey murine sarcoma virus (Ha-MuSV) is a mouse-rat recombinant retrovirus that encodes a protein designated p21, required for virally induced transformation. Using a radiolabeled DNA fragment from the p21 coding region, we have detected homologous DNA sequences in the normal DNA of rats and of several other vertebrate species. Moreover, many tested cells from these species contain low levels of a p21 protein that is highly related to viral 21. Now we report two independent fragments from normal rat DNA containing sequences (sarc) homologous to the Ha-MuSV transforming region that were cloned in the bacteriophage vector Charon 4A. Sarc sequences in the one fragment are completely colinear with the viral sequences and share apparently all restriction endonuclease sites. Sarc sequences in the second fragment have several sets of intervening sequences and lack some restriction endonuclease sites found in the viral transforming region. Despite the presence of these intervening sequences in the second sarc fragment, we have been able to ligate this sarc fragment to the long terminal repeat sequence of HaMuSV and to induce cellular transformation and high levels of p21 expression upon transfection of this DNA to NIH 3T3 mouse cells. These results suggest that elevated levels of p21, normally expressed at low levels in a variety of cells, can induce cellular transformation.