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The vertebrate immune system has evolved to respond vigorously to microbial infection but to ignore self-antigens. Evidence has emerged that B cell responses to viruses are initiated by immune recognition of ordered arrays of antigen on the viral surface. To test whether autoantibodies against a self-antigen can be induced by placing it in a context that mimics the ordered surface of a viral particle, a peptide representing an extracellular loop of the mouse chemokine receptor CCR5 was incorporated into an immunodominant site of the bovine papillomavirus virus L1 coat protein, which self-assembles into virus-like particles. Mice inoculated with chimeric L1-CCR5 particles generated autoantibodies that bound to native mouse CCR5, inhibited binding of its ligand RANTES, and blocked HIV-1 infection of an indicator cell line expressing a human-mouse CCR5 chimera. These results suggest a general method for inducing autoantibodies against self-antigens, with diverse potential basic research and clinical applications.

Papillomavirus-like particles (VLPs) are a promising prophylactic vaccine candidate to prevent human papillomavirus (HPV) infections and associated epithelial neoplasia. However, they are unlikely to have therapeutic effects because the virion capsid proteins are not detected in the proliferating cells of the infected epithelia or in cervical carcinomas. To increase the number of viral antigen targets for cell-mediated immune responses in a VLP-based vaccine, we have generated stable chimeric VLPs consisting of the L1 major capsid protein plus the entire E7 (11 kDa) or E2 (43 kDa) nonstructural papillomavirus protein fused to the L2 minor capsid protein. The chimeric VLPs are indistinguishable from the parental VLPs in their morphology and in their ability to agglutinate erythrocytes and elicit high titers of neutralizing antibodies. Protection from tumor challenge was tested in C57BL/6 mice by using the tumor cell line TC-1, which expresses HPV16 E7, but not the virion structural proteins. Injection of HPV16 L1/L2-HPV16 E7 chimeric VLPs, but not HPV16 L1/L2 VLPs, protected the mice from tumor challenge, even in the absence of adjuvant. The chimeric VLPs also induced protection against tumor challenge in major histocompatibility class II-deficient mice, but not in beta2-microglobulin or perforin knockout mice implying that protection was mediated by class I-restricted cytotoxic lymphocytes. These findings raise the possibility that VLPs may generally be efficient vehicles for generating cell-mediated immune responses and that, specifically, chimeric VLPs containing papillomavirus nonstructural proteins may increase the therapeutic potential of VLP-based prophylactic vaccines in humans.

Genital human papillomavirus (HPV) infection is a common sexually transmitted disease that at the present time is not effectively controlled or treated. Many infections are inapparent and transient. However, some HPV infections result in persistent lesions that in some cases undergo carcinogenic progression. A subset of genital HPVs, designated high-risk types, are preferentially associated with high-grade dysplasias and carcinomas. About 90% of cervical cancers contain high-risk HPV DNA, most often HPV16. Development of a subunit vaccine against high-risk genital HPVs is a desirable and, it appears, an increasingly feasible long-term goal. The viral E6 and E7 oncoproteins are selectively maintained and expressed in progressed HPV tumors and could potentially be targets for therapeutic vaccines. The L1 major virion structural proteins have recently been shown to self-assemble into virus-like particles when expressed in insect cells. These particles might serve as the basis for a prophylactic vaccine to prevent genital HPV infection.

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.

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 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.

The viral DNA sequences in mouse C127 cells transformed by bovine papillomavirus type 1 (BPV-1) virions, by full-length linear BPV-1 DNA, or by a defined transforming subgenomic DNA segment of BPV-1 were examined by reassociation kinetics and blot hybridization. In all cases, the transformed cells contained multiple copies of BPV-1 DNA, present exclusively as supercoiled or nicked circular extrachromosomal molecules or as a slowly migrating complex of circular viral DNA molecules. In the transformed cell lines established from cells transfected with full-length linear BPV-1 DNA, there was recircularization of the input DNA which in some cases resulted in the loss of the restriction site used in the linearization of the DNA. In the transformed cell lines established with the defined subgenomic segment there was circularization of the DNA accompanied by the acquisition of new sequences or duplication and rearrangement of the BPV-1 sequences. In contrast to other well-studied virus transformation systems, no integration of the BPV-1 genome into the host chromosome could be detected under conditions sensitive enough to detect 0.1-0.2 viral genome equivalent. It was concluded that maintenance of transformation may be mediated by nonintegrated viral DNA.

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.

When bovine papillomavirus (BPV) or its 7.9-kilobase full viral DNA genome induces focal transformation of mouse cells, the viral DNA is maintained in the transformed cells as multiple episomal copies. This transforming capacity and maintenance of the episomal state previously has been localized to a 69% subgenomic fragment of the viral DNA genome. We now have characterized further the BPV DNA sequences that can encode the transforming function. We first created a series of BPV DNA deletion mutants and correlated the location of the deletions with the capacity of the deleted viral DNAs to induce transformation of mouse cells. The results indicated that two discontinuous segments of the viral DNA were required for transformation. One segment, near the 5' end of the 69% transforming fragment, probably represents a control element of the viral DNA. The second segment, which lies within the 3' end of the 69% fragment, encodes transforming sequences of the viral DNA; ligation of a retroviral control element (the long terminal repeat DNA of Harvey murine sarcoma virus) to the 2.3-kilobase segment at the 3' end of the 69% fragment induces transformation of mouse cells. In contrast to mouse cells transformed by the full-length BPV DNA genome, the cells transformed by the deleted BPV DNA genomes contained few viral DNA copies; at least some copies appeared to be integrated. We conclude that different viral functions mediate cellular transformation and maintain the viral DNA in its episomal state.

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.

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.

Harvey and Kirsten murine sarcoma viruses each encode a structurally and functionally related 21-kilodalton protein (p21), which is the transforming protein of each virus. Using probes from the 0.9-kilobase (kb) p21-coding region of each virus (called v-Ha-ras and v-Ki-ras, respectively), we have molecularly cloned from normal human genomic DNA the sequences that hybridize to these probes. Four evolutionarily divergent restriction endonuclease fragments were isolated. Two hybridized preferentially to v-Ha-ras and were designated human c-Ha-ras1 and c-Ha-ras2; two hybridized preferentially to v-Ki-ras and were called c-Ki-ras1 and c-Ki-ras2. Human c-Ha-ras1 contained 0.9 kb of sequence homologous with v-Ha-ras interspersed with three intervening sequences; this gene was closely related to a previously cloned rat c-Ha-ras gene that also contained intervening sequences. Human c-Ha-ras2 was more divergent from v-Ha-ras and also hybridized poorly to human c-Ha-ras1. One c-Ki-ras gene contained 0.9 kb homologous to v-Ki-ras and had one intervening sequence, whereas the other contained only 0.3 kb homologous to v-Ki-ras. The results indicated that human DNA contains several copies of the c-ras family and that c-Ha-ras1 (with intervening sequences) was more highly conserved evolutionarily than was c-Ha-ras2.

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.

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.

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.

The AKR mouse has a high titer of murine leukemia virus early in life, and virus-negative cells derived from embryos of this mouse strain can be activated to yield murine leukemia virus by treatment with 5-iododeoxyuridine. In contrast to this high-virus strain, the NIH Swiss mouse has a low incidence of leukemia and no murine leukemia virus has been isolated from it (virus-negative). We have investigated this difference between AKR and NIH mice by examining the sequences specific for murine leukemia virus in nucleic acids of these mice. A single-stranded viral-DNA probe synthesized in vitro using murine-leukemia-virus from the AKR mouse contains at least 87% of the sequences present in the 70S viral RNA; most of these sequences are in proportions similar to their content in the 70S RNA. Using this probe in nucleic acid hybridization experiments, we have shown that NIH-mouse-cell DNA and AKR-mouse-cell DNA differ with respect to sequences specific for AKR murine-leukemia-virus: NIH-mouse-cell DNA lacks some of the virus-specific sequences present in AKR-mouse-cell DNA, and there are two distinct sets of virus-specific sequences in AKR-mouse-cell DNA, whereas there is only one set in NIH-mouse-cell DNA.RNA from virus-negative AKR-mouse cells grown in tissue culture contains some, but not all, virus-specific RNA sequences; however, within 48 hr after initiating treatment of these cells with 5-iododeoxyuridine, the complete viral genome is represented in cellular RNA.

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.

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.

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.

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.

Studies with a single-stranded DNA probe complementary to the RNA of mouse-tropic AKR murine leukemia virus indicate that the complete genome of the AKR-type murine leukemia virus is present in the DNA of high- and low-virus-yielding mouse strains, while DNA of non-virus-yielding strains contains only a part of the genome. Furthermore, in those strains where the genome is complete, two populations of virus-specific DNA sequences can be identified (more abundant and less abundant species) according to their rate of association with the probe. Low-virus-yielding mouse strains contain fewer copies of the less abundant species and, consequently, fewer complete viral genomes than do high-virus-yielding strains. Thus, in the ten strains tested, there is a good correlation between completeness of the genome of AKR-type murine leukemia virus in cellular DNA and the capacity of the cells to release infectious AKR-type murine leukemia virus. Moreover, the number of complete viral genomes correlates with the frequency of infectious virus production by virus-positive strains. DNA from wild Mus musculus also contained viral sequences, the sample tested showing reassociation kinetics identical to the non-virus-producing strains.

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.

Poly(1-vinyluracil) and poly(9-vinyladenine), as well as the corresponding polynucleotides poly(uridylate) and poly(adenylate), inhibit acute murine leukemia virus infection in mouse-embryo cells, but they do not significantly inhibit the replication of Sindbis and vesicular stomatitis viruses. The polymers were most effective as inhibitors when added during an early stage of virus replication. Effects of vinyl polymers on the RNA-dependent DNA polymerase from the virions of murine leukemia virus were also observed.

Genetic information of murine leukemia viruses is known to be present in essentially all mice. Delineation of the biological effects of the expression of this genetic information is complicated by the difficulty in determining whether the presence of a viral antigen in a tissue is the result of gene activation that occurs independently in a large number of cells, proliferation of antigen-positive cells, or of spread of infectious virus from a few cells. This problem is further complicated by the difficulty in determining whether activation of virus genetic material is the cause or result of a biological phenomenon, such as malignancy. Further understanding of the activation of virus genes in normal cells is of great importance in answering these questions. In vitro techniques were used for the study of induction of virus. Virus-negative cell lines were established from embryos of AKR mice, which in vivo produce high titers of infectious murine leukemia virus. It was found that 5-iododeoxyuridine and 5-bromodeoxyuridine were potent inducers of virus synthesis in all clones and subclones of the AKR cells tested. In addition to establishing that the complete viral genome is a heritable component of all cells of the AKR mouse, these studies of virus activation provide novel approaches to a number of important problems in the biology of leukemia viruses.