Sarcoma Virus Pseudotype Research Articles

Subgroup J avian leukosis virus infection in turkeys: Induction of rapid onset tumours by acutely transforming virus strain 966

Avian leukosis virus subgroup J (ALV-J), isolated in the late 1980s, predominantly causes myelocytic myeloid leukosis in meat-type chickens. In the past 10 years, ALV-J infection has become very widespread, causing serious problems to the chicken meat industry. Previously, we have shown that turkey cells can be infected in vitro with Rous sarcoma virus pseudotypes of ALV-J. In this paper, we extend those observations to show that turkey monocyte cultures can be transformed in vitro with acutely transforming ALV-J strain 966. We also show that turkeys are experimentally susceptible to infection with ALV-J prototype strain HPRS—103. However, neoplastic lesions were not observed in these birds, probably due to the short experimental period of 10 weeks. When inoculated into 1-day-old turkey poults, acutely transforming ALV-J strain 966 induced tumours between 3 and 4 weeks after infection. Most of the birds showed tumours involving the liver, with histopathological lesions of myelocytomatosis. The demonstration of the spread of HPRS—103 by contact among turkeys, although observed only at low levels in the present study, stresses the importance of segregation of turkey and chicken breeding operations to avoid the spread of ALV-J infection.

Host range of Rous sarcoma virus pseudotype RSV(HPRS-103) in 12 avian species: support for a new avian retrovirus envelope subgroup, designated J.

The host ranges of the Rous sarcoma virus (RSV) pseudotype RSV(HPRS-103) of a novel avian leukosis virus (ALV), strain HPRS-103, and representative RSV pseudotypes of subgroups A to F, have been determined in embryo fibroblasts from 12 avian species. Domestic fowl, red jungle fowl, Sonnerat's jungle fowl and turkey were susceptible to infection by RSV(HPRS-103); ring-necked pheasant, Japanese green pheasant, golden pheasant, Japanese quail, guinea-fowl, Peking duck, Muscovy duck and goose were resistant. The host range pattern of RSV(HPRS-103) differs from those of viruses of subgroups A to G and I, and provides support for placing the HPRS-103 strain of ALV in a new envelope subgroup, designated J.

A novel subgroup of exogenous avian leukosis virus in chickens

An avian leukosis virus with a wide host range belonging to a new subgroup for chickens was isolated from meat-type chicken lines. The virus, of which HPRS-103 strain is the prototype, was of low oncogenicity in chickens but appeared to behave like an exogenous leukosis virus. Neutralizing antibodies to the virus were found in three of five meat-type chicken lines, but not in seven layer lines. The virus and its Rous sarcoma virus pseudotype did not replicate in, or transform, mammalian cells.

Interference grouping of murine leukemia viruses: A distinct receptor for the MCF-Recombinant viruses in mouse cells

Fourteen different isolates of mink cell focus-inducing (MCF) murine leukemia viruses (MuLV) were characterized with respect to viral interference on NIH/3T3 cells. I assayed viral interference by measuring the reduction in focus formation by murine sarcoma virus pseudotypes on cells chronically infected with MuLV. Although the MCFs interfered with each other in all combinations tested, there was no significant cross-interference between MCF and ecotropic MuLVs. These results imply that MCFs and ecotropic MuLVs enter NIH/3T3 cells via different receptors. Using a recombinant constructed in vitro by C. Holland and N. Hopkins, I also found that the receptor specificity of MuLV is determined by sequences to the 5′ side of 7.7 kbp (measured from the 5' end of the provirus). The distinct receptor specificity of MCF9 suggests that they may be recombinants in which the nonecotropic parent is not xenotropic MuLV, as originally proposed, but rather endogenous env sequences which are not part of nondefective viral genomes. The MCF receptor specificity may be encoded directly by these sequences. I also suggest that interference may have a profound effect on the dynamics of viral replication in vivo.

Common surface receptors on both mouse and rat cells distinguish different classes of mouse mammary tumor viruses

Cell surface receptors which bind mouse mammary tumor virus (MMTV) were detected on mouse and rat cells. Virus binding was quantitated by measuring 125T-protein A binding to immune complexes composed of a C3H MMTV gp52 type-specific monoclonal antibody and receptor-bound MMTV. C3H MMTV binding to normal mouse mammary epithelial cells (NMuMG) was dose dependent and was ≥50% inhibited by GR MMTV, but not by endogenous C3Hf MMTV or Gross murine leukemia virus. These results were confirmed in [ 3H]leucine MMTV binding inhibition assays in which GR MMTV and C3H MMTV blocked C3H [ 3H]MMTV binding while C3Hf MMTV did not block. The affinities of C3H [ 3H]MMTV binding to receptors on NMuMG, NIH Swiss (SLP), and Fischer rat embryo (FRE) cells were identical by Scatchard analysis. C3Hf [ 3H]MMTV also bound these cells, but with an affinity approximately 10 times weaker than the C3H [ 3H]MMTV binding. Interference assays using a Kirsten sarcoma virus (C3H MMTV) pseudotype confirmed the importance of C3H MMTV specific binding to viral penetration and expression. Pretreatment of SLP or FRE cells with 100 μg of C3H MMTV or GR MMTV inhibited focus formation by ≥50% while C3Hf MMTV and RIII MMTV did not inhibit. Therefore, the functional C3H MMTV cell receptors on mouse and rat cells were related and were able to distinguish C3H MMTV and GR MMTV from C3Hf MMTV and RIII MMTV. These comparable receptors may represent evolutionarily conserved surface components of murine cells.

Lack of focus formation of S+L- mink cells by the ecotropic murine leukemia viral genome.

Infection of mink S+L- cells with ecotropic murine leukemia virus, achieved by phenotypic mixing with xenotropic virus, did not result in the induction of transformed foci. Also, clonal line of S+L- mink cells, chronically infected with ecotropic murine leukemia virus, which produce both the ecotropic virus and its murine sarcoma virus pseudotype are morphologically indistinguishable from normal S+L- mink cells. Neutralizing antiserum added to S+L- mink cells inoculated with xenotropic virus 24 h earlier prevented the formation of foci of transformed cells. Together, these observations indicate that focus formation in S+L- cells requires a regional spread of infection, with the insertion of additional murine sarcoma virus genomes and resultant transformation occurring from a gene dosage effect.

C3H/HeN mammary tumor-bearing mice develop type-specific neutralizing antibodies and group-specific precipitating antibodies for the mouse mammary tumor virus.

The development of mouse mammary tumor virus (MMTV)-neutralizing antibodies in various strains of mice was measured by their ability to neutralize the focus-forming capacity of a Kirsten sarcoma virus (C3H MMTV) pseudotype containing the MMTV envelope glycoprotein gp52. C3H/HeN, but not GR/N and RIII, mammary tumor-bearing mice were found to develop neutralizing antibodies to this pseudotype. In addition, non-tumor-bearing C3H/HeN, GR/N, RIII, NIH Swiss, C57BL/6, and BALB/c mice and 13 feral mice were also negative for neutralizing antibodies. The neutralization was immunoglobulin G mediated, and the antibodies of C3H/HeN mammary tumor-bearing mice were type specific and capable of distinguishing C3H and GR/N MMTVs from RIII and C3H/HeNf MMTVs. Precipitating antibodies were detected in sera from RIII and GR/N tumor-bearing mice, GR/N non-tumor-bearing mice, and six of the feral mice, although these same sera did not neutralize the Kirsten sarcoma virus (C3H MMTV) pseudotype. The results of this study and of a previous study demonstrate that C3H/HeN mammary tumor-bearing mice develop three functionally distinct antibody populations: (i) group-specific virus-precipitating antibodies; (ii) type-specific virus-neutralizing antibodies; and (iii) type-specific cytotoxic antibodies.

Analysis of proteins of mouse sarcoma pseudotype viruses: type-specific radioimmunoassay for ecotropic virus p30's.

Murine sarcoma virus pseudotypes were prepared by infection of nonproducer cells (A1-2), which were transformed by the Gazdar strain of mouse sarcoma virus, with Gross (N-tropic), WN1802B (B-tropic), or Moloney (NB-tropic) viruses. The respective host range pseudotype sarcoma viruses were defined by the titration characteristics on cells with the appropriate Fv-1 genotype. Proteins from virus progeny were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Bands present in both the 65,000- and the 10,000- to 20,000- molecular-weight regions of the gel distinguished the pseudotype viruses from their respective helpers. Furthermore, two protein bands were noted in the p30 region of murine sarcoma virus (Gross), one corresponding to Gross virus p30, and another of slightly slower mobility. However, since the mobility of the putative sarcoma p30 is nearly indentical to that of WN1802B, its presence could not be established by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Type-specific radioimmunoassays for Gross virus p30 and for WN1802B p30 were applied for analysis of pseudotype preparations, and among several ecotropic viruses tested, only the homologous virus scored in the respective assay. By use of these assays, pseudotype viruses were found to contain only 8 to 48% helper-specific p30's; the remainder is presumably derived from the sarcoma virus.

Prevention of spontaneous leukemia in mice by oncornavirus immunization

C57L, NIH, and SWR mice were immunized with inactivated Gross leukemia virus (GLV) and then mated with AKR males. Their F1 offspring were then immunized with the murine sarcoma virus pseudotype of GLV, MSV(GLV). The concentrations of infectious ecotropic AKR virus in tail extracts of immunized mice were 100- to 1,000-fold lower than in non-immunized controls when tested at 30--40 days of age. Although viral titers increased slightly with time, the titers remained at least one log10 lower in the immunized mice than in non-immunized F1 control mice at all times tested. The reduction in the level of expression of endogenous ecotropic virus showed a highly significant positive correlation with the reduction in incidence of spontaneous leukemia in these mice. These data thus show that successful immunoprevention of leukemia in mice can be achieved with viral vaccines.

Enhanced Oncornavirus Expression in Marek's Disease Tumors From Specific-Pathogen-Free Chickens<xref ref-type="fn" rid="fn2">2</xref>

An oncornavirus was recovered from cell cultures of kidney tumors from specific-pathogen-free chickens inoculated with Marek's disease herpesvirus (MDHV). The MDHV inoculum was free of infectious avian leukosis virus (ALV). Direct examination of a variety of tissues from MDHV-inoculated chickens demonstrated increased levels of ALV-specific RNA compared to tissues from diluent-inoculated (control) chickens. DNA from cultured kidney tumor cells annealed to an ALV complementary DNA probe at the same rate and exhibited the same extent of homology as DNA from cultured control kidney cells. This finding indicated the absence of exogenous ALV proviral sequences. As with vertically transmitted endogenous ALV of subgroup E, the oncornavirus recovered from kidney tumor cell cultures failed to replicate in chicken embryo fibroblast (CEF) cultures of the C/E phenotype, but did replicate in turkey embryo fibroblasts (TEF), which are permissive for replication of endogenous ALV of subgroup E. These oncornavirus particles served as a helper virus to form Rous sarcoma virus pseudotypes, which produced foci in TEF cultures but not in CEF cultures of the C/E phenotype. Whether enhanced expression of endogenous oncornavirus contributes to MDHV-induced tumorigenesis is not known.

Sarcoma virus-specific phosphoproteins are packaged in “rescued” type C virions

Phosphorylated proteins in pseudotype virions containing various mammalian type C sarcoma viruses have been analyzed. Sarcoma virus pseudotypes obtained by acute infection of nonproductively transformed cell lines with a variety of helper viruses were analyzed by denaturing gel filtration. Each sarcoma virus appears to specify at least one major size class of phosphoprotein, present in the pseudotype virions, that is not present in the helper virions and is independent of the cell line or the helper virus used.

Active immunization against murine radiation‐induced leukemia using a sarcoma virus pseudotype

Immunization of C57BL/Ka mice against the radiation leukemia virus (Rad LV) markedly reduced their susceptibility to development of leukemia after X-radiation. The active immunogen used consisted of a sarcoma virus pseudotype of Rad LV which induces regressing sarcomas in young adult mice. These immunized animals were challenged with four weekly 160-rad X-ray exposures. The appearance of lymphomas and leukemias resulting from such irradiation was delayed and their incidence significantly decreased. These results indicate that oncornavirus immunization may be used to reduce the incidence of murine leukemias which are triggered by a physical agent.

Serological characterization of a putative human C-type oncornavirus by means of the Sepharose bead immunofluorescence assay

A putative human C-type oncornavirus isolate was found to be closely related to the simian sarcoma virus by means of the Sepharose bead immunofluorescence assay. Sepharose-beads coupled with antisera directed against purified major internal proteins of C-type oncornaviruses were incubated first with tissue culture supernatants and thereafter with antisera conjugated with fluorescein isothiocyanate. The fluorescence of individual beads was then measured microfluorometrically. This technique is a highly sensitive and reproducible assay for the detection of viral proteins. The SKA 21-3 culture of rabbit cornea cells, transformed by a murine sarcoma virus pseudotype with a presumably human derived C-type oncornavirus as helper proved to produce abundant amounts of proteins related to the p 28 of simian sarcoma virus but not to the p 30 of murine leukemia virus. When cells from a tumorous lymph node induced in a rat by the SKA 21-3 virus were cocultivated with a canine thymus cell line, A 7573, high concentrations of viral proteins were detected in the supernatant after a few passages. The same result was obtained when a leukemic lymph node from a rat inoculated with the presumed human virus, free of the murine sarcoma virus, was cocultivated with the human A 204 line.

Infection of bone marrow cells in vitro with FLV: Effects on stem cell proliferation, differentiation and leukemogenic capacity

Long-term cultures of proliferating hematopoietic stem cells derived from bone marrow permit the study of the interaction between murine leukemia virus (MuLV) infection and the proliferation and differentiation of stem cells. We have used this system to analyze the replication of different biological variants of MuLV in bone marrow cells; the effect of MuLV infection upon pluripotent stem cell (CFU-S) proliferation; and the effect of MuLV on differentiation of CFU-S along different hematopoietic pathways. Two MuLV variants were studied in detail: the Moloney strain of lymphatic leukemia virus (Mol-MuLV) and the erythroleukemic Friend virus complex (FLV) consisting of the lymphoid leukemia helper virus and the defective spleen focus-forming virus (SFFV). Mol-MuLV and its sarcoma virus pseudotype, MSV(Mol-MuLV), replicate efficiently in the bone marrow cultures; however, CFU-S are lost more readily than in uninfected cultures, and the cultures are soon represented by a majority population of mononuclear macrophages. On the other hand, infection with FLV produces a prolonged survival of the spleen colony-forming cells, CFU-S, and CFU-C (the committed granulocytic precursor cells). Production of erythroleukemogenic SFFV is maintained in these cultures for more than 40 weeks. No erythroblastic differentiation was observed in vitro, however, neither erythroblast precursor cells (CFU-E) nor hemoglobin-producing cells could be detected. This suggests that the target cell for FLV is an earlier precursor cell.

Formation of reticuloendotheliosis virus pseudotypes of Rous sarcoma virus

Superinfection of chicken embryo fibroblasts transformed by the defective Bryan strain of Rous sarcoma virus (BH-RSV) with two different reticuloendotheliosis viruses (REVs), REV strain T (REV-T) or spleen necrosis virus (SNV), resulted in the production of infectious sarcoma virus pseudotypes. These pseudotypes were neutralized by antiserum prepared against SNV and were unable to infect chicken cells preinfected with either REV-T or SNV. These results suggest that defective BH-RSV is able to use the glycoprotein from REV to form infectious pseudotypes. On the other hand, neither REV-T nor SNV was able to supply a functional reverse transcriptase to the polymerase-negative mutant BH-RSValpha, nor was REV-T or SNV able to complement the defect in the internal protein gene of the temperature-sensitive avian sarcoma virus mutant NY45.

Murine xenotropic type C viruses III. Phenotypic mixing with avian leukosis and sarcoma viruses

Phenotypic mixing has been demonstrated between murine and avian type C viruses. Pseudotypes of Rous sarcoma virus (RSV) have been obtained which carry envelope determinants derived from xenotropic or ecotropic murine leukemia viruses (MuLV). Mouse sarcoma virus (MSV) pseudotypes have been constructed with envelope characteristics of subgroup C and E avian leukosis viruses. RSV pseudotypes produced with MuLV can infect mammalian cells, and MSV pseudotypes with avian leukosis virus infect chicken cells. The expression of transformation by these sarcoma genomes seems suppressed in certain of the heterologous hosts. Exogenous infection of mammalian cells by RSV(MuLV) results in not only transformation of the heterologous host but also the production of infectious progeny virus. The extent of this virus production appears determined by the ability of the helper virus to replicate in the host cell.

Autogenous Humoral Immunity to Baboon Xenotropic Endogenous Type C Virus 2

A radioimmune precipitation assay was used to evaluate the humoral immune response of baboons (Papio cynocephalus) to their endogenous, xenotropic, type C (M7) virus. Using 125I or [3H]leucine-labeled intact virus preparation, we demonstrated that baboons possess naturally occurring virus-binding antibodies to this virus. Antibody appeared directed against envelope and not internal viral proteins. Immunoglobulins responsible for virus-binding were predominant in the IgG and IgA serum fractions, whereas IgM participated to a minor degree. The biologic activities of these immunoglobulins were investigated by means of a focus reduction assay for virus neutralization with a murine sarcoma virus (M7) pseudotype. We determined that, despite the presence of considerable virus binding, none of the normal baboon sera tested possessed neutralizing activity for M7 virus.

Avian tumor virus interactions with chicken fibroblast plasma membranes

A method is described which will rapidly measure the binding of avian tumor viruses (ATV) to plasma membrane receptors. With this procedure it may be shown that Rous sarcoma virus pseudotypes bind to protease-labile, heat-stable structures on the surface of chicken embryo fibroblast (CEF) plasma membranes. The binding sites for ATV subgroups A and B appear distinct, and membranes from genetically resistant CEF bind as well those of sensitive CEF.

Adaptation of N-Tropic Friend Leukaemia Virus and its Murine Sarcoma Virus Pseudotype to Non-permissive B-Type C57BL/6 Mouse Cell Line

N-tropic Friend leukaemia virus (FLV) was serially passaged in a B-type C57BL/6 mouse cell line, YH-7. After a single passage, the infectious efficiency of FLV in the restrictive YH-7 cells was significantly increased. This adapted character of FLV could be reversed by a single passage in permissive N-type MLg cells. The true host range conversion from N to NB was accomplished after eleven to twelve passages in YH-7 cells. In contrast, the host range conversion of N-tropic murine sarcoma pseudotype, MuSV(FLV), took place after two passages.

Murine sarcoma virus pseudotypes acquire a determinant specifying N or B tropism from leukaemia virus during rescue

SEVERAL classes of murine leukaemia viruses (MuLV) are recognised according to their ability to induce XC plaque formation with high or low efficiency after infection of various mouse cell lines1,2. N- and B-tropic MuLVs, for example, can be distinguished on the basis of their relative ability to infect N-type or B-type mouse cells, the cells themselves differing at a single genetic locus, Fv-1 (ref. 3). Fv-1 restriction is not absolute1, is dominant over susceptibility in F1 hybrids4, and may be mediated by a specific intracellular inhibitor5,6. Studies using vesicular stomatitis virus (VSV) pseudotypes made with N- or B-tropic MuLV have shown that Fv-1 restriction occurs after virus penetration7,8, although the mechanism of inhibition is not known. A clear difference exists between Fv-1 restriction of MuLV and the host range restriction of avian leukaemia virus subgroups, in which host range restriction occurs at the cell surface9,10. Another class of MuLVs, NB-tropic, does not seem to be sensitive to Fv-1-mediated restriction1.