Radioimmunoassays that demonstrate type-specific and group-specific antigenic reactivities for the major internal structural protein of murine mammary tumor viruses.

The 28,000-dalton (p28) major structural polypeptide of the mouse mammary tumor virus (MMTV) was isolated and used to develop a highly sensitive and specific radioimmunoassay. Under conditions of limiting antibody in competitive binding assays, as little as 50 pg of purified p28, as well as disrupted MMTV virions and mammary tumor extracts, competed specifically with 125I-labeled MMTV p28. The p28 polypeptide was further shown to contain both group-specific and type-specific antigenic determinants, thus also allowing for further differentiation of various MMTV strains.     

Type D primate retroviruses: a review.

The prototype virus of the type D retroviruses is the Mason-Pfizer monkey virus (MPMV). MPMV was originally isolated from a breast carcinoma of a female rhesus monkey (an Old World monkey). MPMV is of obvious importance in that it is the only retrovirus thus far isolated from a mammary tumor of a primate and has been shown to have transforming potential for primate cells in vitro. Subsequent to the isolation of MPMV viruses morphologically and immunologically indistinguishable from MPMV have been isolated from normal placenta and lactating mammary glands of other rhesus monkeys in captivity. Recently, viruses morphologically resembling MPMV have been isolated from a langur monkey (another Old World monkey) and from squirrel monkeys (a New World monkey). Based on nucleic acid hybridization studies, the latter 2 viruses represent endogenous viruses in their species of origin, whereas MPMV appears to be a horizontally related to the langur monkey isolate. Studies on the immunological relatedness of the type D retroviruses have demonstrated interspecies cross-reactivities between the major internal and external proteins of the viruses. Furthermore, these viruses also share cross-reactivity of their major external glycoproteins with those of the type C baboon endogenous virus. These interspecies reactivities can also be demonstrated in natural sera from both imported and laboratory-bred monkeys. The demonstration of these interspecies cross-reactivities shared by distantly related primate retroviruses provides a means for detecting determinants that are representative of all primate retroviruses presently known and yet to be isolated and may provide new assays for detection of a human retrovirus.     

Organotropism of the lymphoproliferative disease virus (LPDV) of turkeys

Turkey poults were inoculated with lymphoproliferative disease virus (LPDV) of turkey, and the organotropism of the inoculated virus was determined from the kinetics of virus expression and tumor formation. Molecular hybridization experiments, conducted to determine the level of viral RNA expression in the various organs of infected birds, established lymphoid tissues, including bone marrow, as the target for LPDV infection. Of these, bone marrow was the first to be infected but subsequently virus replication extended to the thymus, the spleen and the bursa Of Fabricius. The low level of LPDV expression in non-target organs probably stemmed from infiltration of infected lymphatic cells. The fact that the main organs for virus replication were not invariably the ones most clinically involved suggests that the target cells for virus infection need not necessarily be the targets for virus oncogenicity. A possible mode for LP[DV infection and transformation is proposed.     

Biochemical and immunological characterization of murine leukemia viruses that are paralysis-inducing in rats

The molecular size and pI of the viral structural proteins of four PVC viruses (PVC111, PVC211, PVC321 and PVC441) were compared by single or two-dimensional polyacrylamide gel electrophoresis. PVC111 had slightly larger p15E and gPr85 molecules (about 0.5 kilodalton) than did the other PVC viruses. On the other hand, the virion structural proteins p30, p15, p12E and p12 from all the viruses had the same molecular sizes and pIs. The gp70s and p10s from all the viruses showed the same molecular sizes. A monoclonal antibody to gp70 of PVC321 virus recognized the gp70s of all PVC viruses, but not the gp70s of four clones of the wild mouse ecotropic viruses, Friend murine leukemia viruses (F-MuLV), AKR ecotropic MuLV, dual-tropic F-MuLV or NZB endogenous xenotropic MuLV, revealing that these four PVC viruses are homologous with each other, but distinct from the known mouse retroviruses.     

Comparative studies of purified subviral component vaccines and formalin-treated mammary tumor viruses from four inbred strains of mice

Formalin-treated virus vaccines were prepared from purified murine mammary tumor viruses (MuMTV) from 4 inbred strains of mice: RIII/Imr, GR/Imr, C3H/Imr, and A/Imr. In addition, subviral components were isolated from these 4 strains and purified to homogeneity. The inactivated viruses, their major envelope glycoproteins (gp50-gp55), and their major internal core protein (p28) were emulsified in complete Freund's adjuvant and used as vaccines for prevention of mammary tumors in mice. All 4 Formalin-treated virus vaccines reduced significantly the incidence of mammary tumors in "virus-free" C57BL and BALB/c mice when inoculated prior to challenge with live MuMTV. The RIII-, GR-, and A-MuMTV strains showed extensive heterologous cross-protection, whereas the C3H-MuMTV strain showed significant protection only against C3H- and A-MuMTV challenge. The major viral glycoproteins gp50-gp55 reduced significantly the tumor incidence when mice were challenged with isologous infectious virus after immunization, although these glycoproteins showed different degrees of cross-protection than did the same virus strains used as "intact" but Formalin-treated preparations. RIII-gp55 and GR-gp55 cross-protected against each other but not against challenge with C3H- and A-MuMTV strains; the A-gp50 protected against challenge with A- and RIII-MuMTV strains; C3H-gp55 demonstrated limited activity against C3H-MuMTV challenge only. The internal viral core proteins (p28) were ineffective in all systems studied. The same vaccines were tested in MuMTV-positive, high-tumor-incidence strains from which they were derived. At best, the appearance of spontaneous tumors was delayed in a few experimental sets; eventually, all mice developed mammary tumors. The foster-nursed C3HfC57BL strain of mice, which is not exposed to exogenous MuMTV during suckling and which develops mammary tumors after activation of the endogenous virus genome later in life, was responsive only when the heterologous GR-MuMTV Formalin-treated vaccine was used. The association between the ability of virus vaccines to protect a mouse strain and the degree of natural virus expression in that strain is discussed.     

Detection, quantitation, and characterization of the major internal virion antigen of the bovine leukemia virus by radioimmunoassay.

The major internal polypeptide of the bovine leukemia virus (BLV) was purified to homogeneity with the use of gel filtration and affinity chromatography. Like previous results, the protein had a molecular weight of 25,000 daltons as determined by electrophoresis in polyacrylamide gels with sodium dodecyl sulfate. More than 90% of the 125I-labeled protein was precipitated by bovine sera that reacted in immunofluorescence tests with acetone-fixed BLV-infected cells. In contrast, minimal precipitation (less than 5%) was observed with sera from 36 cattle in leukemia-free herds; these sera, negative by immunofluorescence, included six samples that had high titers of antibodies to the foamy-like bovine syncytia virus (BSV). Antisera prepared against several other oncornaviruses or the Mason-Pfizer monkey virus (M-PMV) did not bind the BLV p25 protein. Conversely, the labeled p30 polypeptides of several oncornaviruses tested did not react with bovine sera that had high titers of antibodies to BLV p25. Competitive radioimmunoassay(s) (RIA) also failed to detect cross-reactions between BLV p25 protein and the internal polypeptides of other mammalian and avian oncornaviruses, M-PMV, or foamy-like BSV. The RIA for BLV p25 antigen was also highly sensitive and specific for the detection and quantitation of the antigen in virus preparations and cell homogenates.     

Competition radioimmunoassay for mason-pfizer monkey virus: comparison with recent isolates.

The major core protein of Mason-Pfizer monkey virus was purified by DEAE ion exchange column chromatography and shown to be 27,000 daltons (p27). Following the characterization of monospecific antisera prepared against p27, a radioimmunoassay was developed with these reagents and competition experiments were done with come of the recent M-PMY-like isolates as well as with other oncornaviruses. Results suggest that three of the viruses tested, AO, X-381 and FTP-1, are similar to M-PMV while J-96 virus is related, but not identical, to M-PMV. It is also shown that competition RIA can be used successfully to detect the presence of viral proteins in tissue homogenates and cell extracts.     

The multifaceted retrovirus

I have attempted to illustrate the many different properties of retroviruses and their presence in a wide variety of animal species including humans. Since the turn of this century, progress in the field of retrovirology has been noteworthy and many new and important scientific observations have been made (Table 7). Along the way, certain dogmas were replaced with new tenets. The recent recognition of retroviruses associated with human cancer and immunodeficiency places them into consideration as potential agents responsible for other human diseases such as autoimmunity and multiple sclerosis. Not only can retroviruses be oncogenic or cytopathic agents but they can also exist highly conserved as endogenous genes in the chromosomal DNA of many different species and not cause disease. In fact, this latter group appears to be predominant, suggesting their role in normal developmental processes and as progenitors of the pathogenic types. The virus-like genomes recognized in Drosophila and other lower animal species could be examples of this fact and may represent important biological entities throughout nature. The genetic material of retroviruses resembles transposons and may reflect the ability of these viruses to be passed within the host and to affect the evolutionary pathway. They could, as transposable elements, be transmitted as well to many different animal species. By their ability to move within the genetic machinery of the cell, these viruses could influence development in animals through promotion, enhancement, or suppression of specific cellular genes. This idea has been proposed for the noninfectious type A particles that have been observed to show these effects in cultured cells. One important observation is that the effect of retroviruses on cells has a varied pattern which may be emphasized by one group (e.g., vacuolization by foamy virus) or shared by other groups (e.g.., syncytial cell formation by type C and type D oncovirinae, spumavirinae, and lentivirinae) (Table 6). Moreover, the heterogeneity of the lentiviruses and the transduction of normal cellular genes by many of the oncogenic viruses indicate the changes that can occur as retroviruses infect and replicate within the cell. The overview is very informative. Virus-cell interaction can lead to biological expressions that depend on the phenotype of the cell and the viral genetic structure. Throughout its existence in nature the retrovirus has been evolving, conserving certain features while developing new ones with different properties; it clearly emerges as a multifaceted agent.     

Defective DNA repair in cells with human T-cell leukemia/bovine leukemia viruses: role of tax gene.

BACKGROUND: Human T-cell leukemia virus (HTLV)/bovine leukemia virus (BLV) group retroviruses, which cause hematopoietic cancers, encode a unique protein, Tax, involved in the transformation of infected cells. Our purpose was to determine whether the mechanism by which Tax protein induces transformation in HTLV- or BLV-infected cells involves DNA damage. METHODS: We used a micronucleus assay to measure chromosomal damage and alkali denaturation analysis to test host-cell DNA integrity in cells infected with HTLV, BLV, or simian T-lymphotropic virus or in cells transfected with the tax gene of HTLV or BLV. Controls included uninfected cells and cells infected with other oncogenic retroviruses or oncogenic DNA viruses. We used a plasmid reactivation assay to examine whether the damage might be due to the inhibition of DNA repair. To ascertain which of several repair pathways might be inhibited, chemical methods were used to selectively introduce lesions repaired by specific pathways into the reporter plasmid. RESULTS: The presence of Tax was associated with DNA damage. HTLV- or BLV-infected or tax-transfected cells showed normal ability to repair damage induced by deoxyribonuclease I or psoralen but markedly decreased ability to repair damage induced by UV light, quercetin, or hydrogen peroxide. CONCLUSIONS: These data suggest that the DNA repair pathway most inhibited by Tax is base-excision repair of oxidative damage. To our knowledge, this is the first report demonstrating inhibition of DNA repair by any retrovirus and suggests that this inhibition of DNA repair may contribute to the mechanism of cell transformation by the HTLV/BLV group of viruses.     

Improved gene transfer selectivity to hepatocarcinoma cells by retrovirus vector displaying single-chain variable fragment antibody against c-Met

Engineered retroviruses are widely used vectors for cancer gene therapy approaches. However, the ability to target cells of therapeutic interest while controlling the expression of the transferred genes would improve both the efficiency and the safety of viral vectors. In this study, we investigated the ability of a retroviral amphotropic envelope displaying single-chain variable-fragment (scFv) directed against the c-Met receptor, to target the entry of recombinant retroviruses to human hepatocarcinoma cells. Four single-chain antibody fragments directed against the c-Met receptor were generated and inserted into the viral envelope protein as an N-terminal fusion. The modified envelopes were incorporated into virus particles and one of the chimeric viruses, 3D6-Env, transduced preferentially human hepatoma cells rather than proliferating human hepatocytes. In another construct, the urokinase cleavage site was inserted between the scFv moiety and the envelope. Chimeric scFv-urokinase-Env viruses transduced hepatoma cells with a similar efficiency to that of the control virus and their infectivity in human hepatocytes remained low. These results indicate that amphotropic retroviruses with engineered envelopes to display scFv directed against the c-Met receptor can efficiently and selectively deliver genes into hepatoma cells.     

Isoforms of the human ets-1 protein: generation by alternative splicing and differential phosphorylation

The ets-1 gene belongs to the ets gene family (ets-1, ets-2, erg, and elk) and is homologous to the v-ets oncogene found in the avian leukemia virus E26. The ets-1 gene products were characterized using a specific monoclonal antibody developed against a bacterially expressed v-ets protein. The ets-1 gene product in the human T-cell line CEM was found to consist of at least six species: four major species with apparent molecular weights of 51 kDa (p51), 48 kDa (p48), 42 kDa (p42), and 39 kDa (p39); and two minor species of 52 kDa (pp52) and 49 kDa (pp49), which are demonstrated to be the phosphorylated forms of p51 and p48, respectively. All of the ets-1 proteins are related to each other and are considered products of the ets-1 gene. Subcellular localization showed that the pp52 and p51 are found mainly in the cytoplasm, while p48 and p39 are found mainly in the nucleus. Specific antibodies against various exons of ets-1 showed that both p42 and p39 lack a region corresponding to exon VII. Polymerase chain reaction analyses revealed the presence of an additional RNA product that corresponds to mRNA lacking exon VII. These results suggest that the human ets-1 gene encodes multiple proteins that are generated by at least two distinct mechanisms: alternative splicing of mRNA and protein phosphorylation.     

Viral carcinogenesis: revelation of molecular mechanisms and etiology of human disease

The RNA and DNA tumor viruses have made fundamental contributions to two major areas of cancer research. Viruses were vital, first, to the discovery and analysis of cellular growth control pathways and the synthesis of current concepts of cancer biology and, second, to the recognition of the etiology of some human cancers. Transforming retroviruses carry oncogenes derived from cellular genes that are involved in mitogenic signalling and growth control. DNA tumor viruses encode oncogenes of viral origin that are essential for viral replication and cell transformation; viral oncoproteins complex with cellular proteins to stimulate cell cycle progression and led to the discovery of tumor suppressors. Viral systems support the concept that cancer development occurs by the accumulation of multiple cooperating events. Viruses are now accepted as bona fide etiologic factors of human cancer; these include hepatitis B virus, Epstein-Barr virus, human papillomaviruses, human T-cell leukemia virus type I and hepatitis C virus, plus several candidate human cancer viruses. It is estimated that 15% of all human tumors worldwide are caused by viruses. The infectious nature of viruses distinguishes them from all other cancer-causing factors; tumor viruses establish long-term persistent infections in humans, with cancer an accidental side effect of viral replication strategies. Viruses are usually not complete carcinogens, and the known human cancer viruses display different roles in transformation. Many years may pass between initial infection and tumor appearance and most infected individuals do not develop cancer, although immunocompromised individuals are at elevated risk of viral-associated cancers. Variable factors that influence viral carcinogenesis are reviewed, including possible synergy between viruses and environmental cofactors. The difficulties in establishing an etiologic role for a virus in human cancer are discussed, as well as the different approaches that proved viral links to cancer. Future directions for tumor virus studies are considered.     

Immunologic studies of the low molecular weight DNA binding protein of murine oncornaviruses.

A low molecular weight, highly basic DNA-binding protein was purified from several oncornaviruses by the sequential procedures of gel filtration in guanidine-hydrochloride, DEAE-cellulose chromatography and affinity chromatography on single-stranded DNA sepharose. The binding protein from Rauscher and woolly monkey type-C viruses was the fastest migrating of the virion proteins in SDS-polyacrylamide gels and thus is designated p10 according to previous convention although our estimates of molecular weight were 8-9,000 daltons. The binding protein from these two viruses was resolved into two bands by acid-urea electrophoresis although only a single NH2 terminal amino acid was detected (S. Oroszlan, personal communication), thus indicating charge heterogeneity. Antibody to Rauscher virus p10 species-specific in gel diffusion and complement-fixation tests and did not exhibit cross-reactivity with other virion proteins. A DNA-binding protein was also detected in preparations of mouse mammary tumor virus. This purified protein had an apparent molecular weight of 12,500, was the second fastest migrating component in the virus preparations, and was antigenically unrelated to the mouse type-C virus p10.     

Type-C virus structural antigens on the surface of the infected cell as determined by a humoral cytotoxicity assay.

Antisera prepared against the major structural polypeptides of Rauscher leukemia virus including gp70, p12, p30, p10 and p15 (E) were tested in a complement-dependent cytotoxicity assay against cell lines infected with Rauscher murine leukemia virus and with the three known classes of endogenous murine leukemia viruses. gp70 antisera and p12 antisera were active in cytotoxicity showing predominantly type-specificity to R-MuLV³, but were shown to cross-react in varying degrees to the three classes of endogenous type-C virus(es). This type-specific and group-specific activity was confirmed by absorption experiments with both infected cells and concentrated intact and disrupted virus. Immunoprecipitation of highly purified, labelled proteins of the murine type-C viruses with these antisera yielded a pattern of reactivity that correlated very well with those obtained by humoral cytotoxicity and, further, defined the polypeptide-specific reactivities of these antisera. Cytotoxic activity obtained with p12 antisera was not well correlated with the intact virus content or the virus-producing ability of different R-MuLV shedding mouse cells whereas activity with gp70 antisera was. Cytotoxic activity was not observed with p10 or p30 antisera and virus-specific cytotoxic activity was not observed with p15(E). Immunization of three strains of mice differing in their endogenous type-C virus expression with inactivated, disrupted R-MuLV yielded antisera active in radioimmuno-assays to the homologous R-MuLV proteins which were found to be immunologically active on the surface of the virus-infected cell (gp70 and p12), as well as to the other type-C viral proteins, p30, p15 and p10. The natural host's ability to respond immunologically to these viral proteins and the in vitro cytolysis of virus-positive cells with antisera of this specific reactivity led to the conclusion that these viral proteins may play an important role as potential transplantation antigens in naturally occurring tumors.     

Ultrastructural comparison of Oncovirinae (type C), Spumavirinae, and Lentivirinae: three subfamilies of Retroviridae found in farm animals

The successive steps of maturation of seven retroviruses from five species of farm animals and one retrovirus from a mouse were compared in cell cultures. The viruses included three type C oncoviruses, one spumavirus, and three lentiviruses. Although members of the 3 subfamilies shared some gross morphologic features such as budding on plasma membranes, core, and surface projections, differences were noted in the ultrastructural detail of these features. Type C oncoviruses did not show any structural differentiation in identifiable form in the cytoplasm as opposed to characteristic features observed in the spumavirus and lentivirus subfamilies, respectively. Budding viruses were distinct among the 3 subfamilies. The type C bovine leukemia virus budding on vacuole membranes differed from the two other type C viruses by lacking an electron-lucent intermediate layer as did the lentiviruses. Differentiation between type C oncoviruses and lentiviruses could be confusing because of the similarity of the fully mature virions appearing in the intercellular space. However, each subfamily of retroviruses can be readily differentiated from one another when each morphologic stage of virus replication is examined by electron microscopy.     

Conventional and immunocolloidal gold electron microscopy of eight simian retroviruses closely related to human T-cell leukemia virus type I

Simian retroviruses closely related to human T-cell leukemia virus type I (HTLV-I) were isolated from 8 species, examined by both conventional and thin section immunocolloidal gold electron microscopy, and compared with HTLV-I. Mature forms of simian viruses were found in extracellular aggregates and within cytoplasmic vacuoles. They were morphologically similar to each other and to HTLV-I. They consisted of a seemingly smooth envelope and a centrally located nucleoid. Their size varied considerably among species and also within the same species; this is characteristic of this group of retroviruses. No budding particles of simian viruses were observed. Thin section immunocolloidal gold electron microscopy using various human and simian sera showed that simian viruses were antigenically related to each other and to HTLV-I. One drawback of this otherwise very useful technique was the difficulty in identifying virions because of the poor preservation of their fine structure by fixation with glutaraldehyde alone. This was overcome by using materials prepared for conventional electron microscopy, in which virions showed weak but specific reactions with gold particles after deosmification and antigen restoration with sodium metaperiodate.     

Leukemogenicity of clonal isolates of murine leukemia viruses.

The leukemogenicity of three types of cloned, in vitro grown murine retroviruses was studied. Two Moloney virus clones caused leukemia, as did five clones of the B-tropic endogenous virus of BALB/c mice. Neither of two clones of N-tropic BALB/c virus caused leukemia in Fv-1n/n mice, and the viruses were not recoverable from the animals. The ability to induce leukemia therefore appeared to reside in the genome of at least certain nondefective murine retroviruses.