James N. Ihle

Polypeptides of mammalian oncornaviruses: III. Localization of p15 and reactivity with natural antibody

Abstract Antiserum to p15 of Friend murine leukemia virus (FLV) reacted with intact MuLVs in radioimmunoprecipitation (RIP) assays. The antigen-antibody complexes formed by this reaction were isolated and shown to contain p15 after analysis by SDS polyacrylamide gel electrophoresis. Natural antibody in mice to MuLV reacted in a similar fashion with p15, but in addition, also with the virus glycoproteins (gp71, gp45). Biological studies indicated that gp71 rather than p15 is principally involved in the virus-neutralization reaction. These and other studies indicate that p15 is localized on the virus surface and is recognized by natural antibodies in mice to MuLV.

Biological, immunological, and biochemical evidence that HIX virus is a recombinant between moloney leukemia virus and a murine xenotropic C type virus

Abstract Various parameters of HIX virus were examined to determine its origin and its relationship to other murine C type oncornaviruses. Envelope properties of HIX virus grown in cells of several species were subjected to analyses of host range, interference, and neutralization. Cloned amphotropic HIX virus was adapted to grow in human RD cells. After 6 months in culture, the resulting virus (HIX-RD) could enter mouse cells but essentially lost the capacity of propagating in mouse cells. Interference patterns of HIX and HIX-RD were identical to each other and unrelated to murine ecotropic MuLV interference. MSV(HIX) or MSV(HIX-RD) could not penetrate HIX-, HIX-RD-, or MuX-preinfected cells. However, infection with HIX exhibited a unique one-way interference in that MSV(MuX) could penetrate and transform HIX-preinfected cells. Neutralization of HIX and HIX-RD with relatively type-specific anti-gp70 sera showed that they resembled Moloney (M)-MuLV most closely. Significant neutralization was observed also with anti-Rauscher gp70 or BALB-2 MuX gp70 sera. Both HIX derivatives were acutely susceptible to inactivation with normal mouse sera, a characteristic of xenotropic viruses. Competition radioimmunoassays were performed to determine the antigenic relationship of HIX to other MuLV types. The highly type-specific phosphorylated p12 and the relatively type-specific gag region p15 of HIX were found to be identical to M-MuLV and less related to other murine C-type oncornaviruses. The examination of HIX gp70 with type-specific anti-M-MuLV or anti-C57L MuX gp70 sera showed that it was clearly different from either virus. Tryptic peptide maps of the gag region-p15 and p30 of HIX were identical to corresponding maps of M-MuLV proteins. The gp70 of HIX was unique and different from known eco-, xeno-, and amphotropic murine C type oncornaviruses. Based on known migration patterns of characteristic trypsin- and chymotrypsin-derived peptides of various eco-, and xenotropic MuLVs, it was concluded that gp70 of HIX was related to both MuX and M-MuLV. Tryptic fingerprint maps also revealed several significant differences between parental HIX and its HIX-RD variant. Comparative hybridizations of assorted high-molecular-weight (HMW) virus RNAs with complementary DNA from HIX virus showed that, with unfractionated probes, no significant differences could be seen between HIX and M-MuLV. Based on the above, HIX virus appears to contain predominantly M-MuLV-specific information except for its envelope gene which has been presumably derived from a recombinational event involving corresponding M-MuLV and MuX nucleotide sequences.

Inactivation of murine xenotropic oncornavirus by normal mouse sera is not immunoglobulin-mediated

Abstract Several normal mouse sera were examined for their neutralizing potential against various leukemia viruses. All sera inactivated murine xenotropic oncornavirus (MuX). One of the sera also neutralized AKR murine leukemia virus (MuLV), whereas none neutralized feline leukemia virus. The inactivation of MuX was not associated with mouse immunoglobulins. The active factor was heat stable, dialysable, and remained in the supernatant fraction after immunoglobulin precipitation by ammonium sulfate. The kinetics of inactivation of MuX by mouse sera were complex and distinct from known immunoglobulin neutralization. Specific antisera against isolated mouse immunoglobulins did not reduce the inactivating capacity of these sera for MuX, although the neutralization of AKR-MuLV by one of these same sera was found to be mediated by immunoglobulin G. The specificity of this inactivation is unknown in that absorption of mouse sera with Friend MuLV gp71, p15(E), or intact ecotropic virus failed to affect MuX inactivation, whereas absorption of mouse sera with normal or virus-producing mouse or cat cells was able to remove the inactivating potential.

Characterization of the immune response to the major glycoprotein (gp71) of Friend leukemia virus. II. Response in C57BL/6 mice.

Abstract C57BL/6 mice responded to immunization with purified gp71 of Friend murine leukemia virus by mounting both humoral and cell-mediated responses. As measured by a number of tests, the responses were generally stronger than those obtained in BALB/c mice. However, in contrast to the BALB/c mice, immunization of C57BL/6 mice with gp71 did not result in the development of cytotoxic lymphocytes, although spleen lymphocytes were capable of undergoing blastogenesis when incubated with purified gp71. As in the BALB/c mice, the humoral response was type-specific. A unique feature of the response in gp71-immunized C57BL/6 mice was the accelerated activation of the endogenous virus as measured by the development of an immune response to its distinct envelope antigens. This resulted in the production of three distinguishable antibody populations: (1) type-specific antibodies to FLV gp71; (2) type-specific antibodies reacting with AKR gp71 (AKR virus being related to the endogenous virus of C57BL/6 mice); and (3) antibodies directed against p15 (E) which reacted both with AKR and Friend-Moloney-Rauscher viruses and are therefore considered group-specific in the murine system. The possible significance of the activation of the endogenous virus subsequent to gp71 immunization of C57BL/6 mice is discussed.

Oncogenic and immunogenic potential of cloned HIX virus in mice and cats

Many naturally occurring and laboratory derived strains of murine leukemia viruses (MuLV) have been isolated. These have initially been ecotropic in host range in that they grew in mouse cells and produced lymphoid neoplasms in mice or rats (Gross, 1951, Friend, 1957, Moloney, 1960). Other classes of murine retroviruses have also been isolated from mouse cells which share a great deal of nucleic acid homology and antigenic determinants with MuLV; these have been labeled according to host range into xenotropic if they were restricted from growth in mouse cells or amphotropic if they had a host range encompassing both mouse cells and cells of other species (Levy, 1973, Aaronson and Stephenson, 1973; Benveniste et al., 1974; Hartley et al., 1976). Xenotropic viruses are not known to be oncogenic. Recently several unusual variants of MuLV type viruses with amphotropic properties have been isolated which appear to be hybrids (recombinants) of MuLVs and murine xenotropic virus (MuX) (Fischinger et al., 1975, Hartley et al., 1977). One of the isolates, MCF, was isolated from AKR lymphomas and was implicated as possibly the oncogenic virus causative of the AKR mouse lymphoma (Hartley et al., 1977). Our isolate, named HIX (hybrid of IC-strain of Moloney MuLV and MuX), has been tested now for almost two years for oncogenicity in several species because of its amphotropic host range. It was of interest to determine whether HIX virus, cloned by three cycles of limiting dilution isolation, was able to cause neoplasms in various species. The present report shows the induction of lymphomas by HIX virus in mice as well as the reisolation of only HIX virus in pure culture from these tumors. Despite growth of HIX to high titers in cat embryo cells, HIX virus inoculated newborn kittens have not shown any signs of disease.

Characterization of the immune response to the major glycoprotein (gp71) of Friend leukemia virus. III. Influence on endogenous MuLV-mediated pathogenesis.

The response of AKR mice to immunization with purified gp71 of Friend murine leukemia virus (FLV)9 was characterized. A humoral response was detectable by radioimmune precipitation assay with FLV or radioimmunoassay with FLV gp71. In contrast, no reactivity with either the endogenous AKR virus or AKR gp71 was detectable. The humoral immune response to FLV gp71 was also detectable in complement-dependent cytotoxicity assays and again appeared type-specific for FLV-producing cell lines and could be specifically blocked by absorption with FLV gp71. In contrast, no specific neutralization of FLV was detectable with immune sera. Cell-mediated reactivity was examined by blastogenesis assays with FLV gp71 and in vitro cytotoxicity assays. Although no consistent cell-mediated cytotoxicity was demonstrable in vitro, speen cells from FLV gp71-immunized mice did undergo blastogensis when incubated with purified gp71. The ability of FLV gp71 immunization to protect AKR mice from spontaneous thymomas and irradiated C57B1/6 mice from radiation-induced thymomas was also examined. The results demonstrate that FLV gp71 immunization has no clear influence on induction of radiation thymomas in C57Bl/6 mice. In contrast, FLV gp71 immunization greatly increased the rate of mortality of AKR mice from thymic lymphomas without altering the time of onset of disease-related deaths. The possible mechanisms behind this enhancement of thymomas by FLV gp71 immunization, including the activation of the endogenous AKR virus, are considered.

Further characterization of the oncornavirus inactivating factor in normal mouse serum.

Abstract The nature of the oncornavirus inactivating factor (OIF) contained in the normal sera of certain mouse strains was investigated by various biochemical and biological techniques. The OIF copurified quantitatively with the lipoprotein fraction of STU mouse serum subjected to flotation through sucrose density gradients. The buoyant material consisted of a heterogenous population of spherical particles (200–1200 A) which had a density 70%) lipid moiety. All of these properties are characteristic of serum very low-density lipoproteins (VLDL) and, therefore, suggest that OIF is contained in the VLDL fraction of normal mouse serum. In contrast to whole serum which specifically inactivated xenotropic and polytropic murine oncornaviruses, the isolated VLDL fraction was also able to inactivate murine ecotropic viruses and feline leukemia virus. Fractionation of STU lipoproteins or whole serum by extraction with ether:ethanol (3:1) removed the OIF into the organic solvent. This factor extract inactivated the same spectrum of viruses as serum VLDL. However, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of unlabeled and radioiodinated factor extracts revealed no detectable protein components. Thus, the inactivating capacity of normal serum apparently can be mediated by lipid micelles devoid of protein. NIH Swiss mouse sera, previously reported not to have OIF, did harbor high titers of factor in an inactive form which displayed activity upon flotation or after extraction with ether: ethanol. However, this phenomenon was specific for NIH mouse sera, since VLDL and ether:ethanol extracted fractions of sera from a number of mammalian species were not found to be active against xenotropic or recombinant murine leukemia viruses (MuLVs). In view of the potential role of recombinant MuLVs in murine leukemia, sera of individual AKR mice were serially examined for a diminution of OIF in the preleukemia or leukemic stages of the disease. Also, serial bleedings were tested from individual C57B1 mice prior to and after irradiation and then monthly until the development of radiation-induced leukemia. In both cases, no apparent significant reduction of OIF titer was observed.

B-MuX: a unique murine C-type virus containing the "env" gene of xenotropic viruses and the "gag" gene of the ecotropic virus.

Abstract A unique C-type virus, B-MuX, was isolated from nonproducer, BALB/c-transformed fibroblasts after IdUrd induction. B-MuX has the envelope gp70 properties of the endogenous BALB:virus-2 genome as determined by neutralization, interference, host-range characteristics and competition radioimmunoassays. Tryptic peptide analysis of gp70 from B-MuX revealed that B-MuX gp70 contains peptides in common to xenotropic virus gp70s and overall was very similar in structure to the xenotropic BALB: virus-2 gp70. In contrast, the gag gene products are serologically identical with the p15 and p12 of the endogenous, ecotropic virus. The p30 of B-MuX is structurally similar to that of the endogenous, ecotropic virus, and differs from the p30 of BALB/c xenotropic viruses as determined by tryptic peptide analysis. The results suggest that either B-MuX is a new type of endogenous, xenotropic virus or a recombinant between the endogenous xenotropic and ecotropic viruses. The unaltered xenotropic behavior of B-MuX demonstrates that the gag gene region does not contribute to this virological property.

Properties of mouse leukemia viruses XIX. Effective antibody therapy of AKR leukemia occurs independently of virus neutralization and produces long-term changes in the virus status of the thymus

Administration of high-titered goat anti-FLV gp71 IgG to AKR mice during a narrow neonatal therapy window suppresses the early development of MuLV infectious cell centers (ICC) in spleen, thymus, and bone marrow. By 4-5 weeks of age ICC appear in spleen and marrow cell populations, rapidly increasing to plateau levels within 2 weeks in the absence of viremia. The thymus of treated animals remains devoid of detectable ICC throughout the 4-month period of testing. This pattern of ICC suppression occurs independently of virus neutralization as shown by the inability of F(ab)2 preparations, which retained neutralizing activity, to prevent early establishment of ICC. Immune IgG significantly decreases, but does not eliminate gp71 expression in all tissues tested. In control animals, ICC reside within a minor subpopulation of cortical, thymic T cells, whereas peripheral (i.e., splenic) ICC are totally devoid of conventional T cell, B cell, and macrophage phenotypic markers. Although thymocytes appear to be a major target of this antibody therapy, T-cell reactivity is not compromised.

Properties of mouse leukemia viruses XVII. Factors required for successful treatment of spontaneous AKR leukemia by antibodies against gp71

Abstract AKR mice were treated with heterologous anti-gp71 antibodies under various conditions in order to establish the optimal criteria for effective suppression of leukemia development. The strongest effect was observed when mice were treated at birth; and when this regimen was used, prior treatment of the mothers did not provide additional protection. If treatment was delayed until Day 3 ( Schwarz et al. , 1979 ), the beneficial effect of the serum diminished sharply, emphasizing the presence of a narrow window very early in the life of the AKR mouse when antibody must be present in order to have an effect on subsequent leukemia development. A number of parameters were examined in the experimental mice and as in our previous study ( Schwarz et al. , 1979 ), suppression of leukemia, which occurred in 68% of the animals, correlated with elimination of viremia and appearance of natural antiviral antibodies. Interestingly, our results suggest that antibody therapy is primarily effective against the thymic form of the disease. The availability of nonviremic, antibody-positive animals afforded us the opporounity to examine if these characteristics could be transmitted to the offspring. From selected mating crosses, we successfully derived both F1 and F2 generations of AKR mice which possessed high titers of antiviral antibodies and were nonviremic at 21–28 weeks of age. It appeared that a maternal effect may be responsible for this phenomenon. The implications of these findings are discussed in relation to the development of AKR leukemogenesis.