Peter J. Fischinger

Properties of mouse leukemia viruses. I. Characterization of various antisera and serological identification of viral components.

Abstract Six antigenic components of murine leukemia viruses (MuLV) were identified by Ouchterlony tests after degradation of the viruses with Tween 80 and ether. A type-specific antigen (II v ), three gs-species (I, IIgs, and IV), and one gs-interspecies (V) antigen were recognized. The range of viral specificity of a further minor antigen (III) is not yet known. For five MuLV antisera, the reactivity spectra were evaluated by complement fixation, Ouchterlony, and neutralization tests. By Ouchterlony and, preferably, by complement fixation tests, it could be shown that the L cell-virus does not belong to either the FMR or the G type; it apparently is still another serotype of MuLV.

Immune response to immunostimulatory complexes (ISCOMs) prepared from human immunodeficiency virus type 1 (HIV-1) or the HIV-1 external envelope glycoprotein (gp120)

In mice, immunostimulatory complexes (ISCOMs) prepared from HIV-1 B external envelope glycoprotein (gp120) induced 10-fold higher antibody titres than gp120 emulsified in depot adjuvant, as measured by enzyme-linked immunosorbent assay (ELISA). Rhesus monkeys immunized with gp120 ISCOMs produced precipitating and virus neutralizing antibody titres equivalent to those seen in HIV-infected chimpanzees and humans. After multiple immunizations with HIV-1 B gp120 ISCOMs, a rhesus monkey developed a neutralizing response to the HIV-1 isolates RF and MN, but not to the CC isolate. Antisera from ISCOM-immunized rhesus monkeys recognized gp120 on the membranes of HIV-1 B-infected H9 cells, indicating the preservation of epitope structure in the ISCOMs matrix.

Properties of mouse leukemia viruses XVI. Suppression of spontaneous fatal leukemias in AKR mice by treatment with broadly reacting antibody against the viral glycoprotein gp 71

Abstract AKR mice were treated early in life with antibodies prepared in a goat against the major glycoprotein gp71 of Friend murine leukemia virus (FL V) and evaluated over a period of up to 2 years for various parameters associated with AKR thymoma/lymphoma. If both the mothers and offspring were given the antibody, suppression of AKR disease was observed to the degree that the 50% incidence of leukemia was delayed by about 1 year. A lesser effect was found if the treatment was initiated at an age of 3 days and application of the antibody to either mothers alone, or treatment of the mice at later times (39 days) was not successful. Thus, it was concluded that the critical period for treatment is between birth and the first few days of life. Postmortem examination of animals that were successfully treated during this early period revealed a significant proportion exhibiting a unique leukemic pattern. In addition, several died from nonleukemic neoplasms as well as other causes. Levels of virus or antiviral antibodies were also determined at various times in individual mice which had been segregated in family groups. The results demonstrated that overall, successful antibody treatment resulted in (1) suppression of virus and (2) development of significant levels of antiviral antibodies. In contrast, control mice exhibited the opposite pattern: high levels of virus and no detectable antibody. Moreover, it could be shown that antibody treatment reduced the incidence of MCF-like recombinant virus isolation. A hypothesis is presented which suggests that the main function of the administered antibody is to disturb a key event occurring during the early stages of life of the AKR mouse, which is of critical importance for the development of leukemia from 6 months of age onwards.

Viral Infection Across Species Barriers: Reversible Alteration of Murine Sarcoma Virus for Growth in Cat Cells

Infection of cat embryo cells by a centrifugally induced aggregate of murine sarcoma virus and feline leukemia virus gave rise to a defective, focus-forming virus which propagated in cat cells, but not in mouse cells. This virus, apparently enveloped with a feline leukemia virus coat, was later subjected to aggregation with murine leukemia virus, whereupon it regained the capacity for growth in mouse cells.

Neutralization of homologous and heterologous oncornaviruses by antisera against the p15(E) and gp71 polypeptides of Friend murine leukemia virus.

Abstract Antisera prepared in rabbit or goat against the purified polypeptides of Friend murine leukemia virus (F-MuLV) were tested in neutralization tests against homologous F-MuLV, Gross (G−) MuLV, murine xenotropic virus (MuX), and feline leukemia virus (FeLV). Antisera directed against p10 and p30 did not neutralize; anti gp71 sera were strongly neutralizing for F-MuLV, MuX, and G-MuLV. The last virus was somewhat less sensitive. Heated anti gp71 sera were as active against the murine viruses as the unheated sera. FeLV was neutralized by unheated, fresh anti gp71 sera, albeit somewhat more weakly than mouse viruses. Although heating of the anti gp71 serum removed its neutralizing potency for FeLV, it could be generally restored by the addition of complement. Neutralization of two other nonmurine oncornaviruses by anti gp71 serum resembled that of FeLV. Neutralization kinetic curves and multiplicity curves were done with anti gp71 serum and F-MuLV, G-MuLV, and MuX. These resembled analogous curves obtained with other animal viruses. When the anti gp71 serum was fractionated, the 7 S IgG fraction contained essentially all the neutralizing antibody. Anti p15(E) serum did not neutralize homologous F-MuLV or the other ecotropic G-MuLV. It was able to neutralize MuX and FeLV, but only in the presence of complement. The slopes of the kinetic curves obtained with MuX and the anti p15(E) serum showed a dependency on both the antibody concentration and complement. The combination of the anti p15(E) and anti gp71 sera with and without complement generally showed simple additive effects except that a combination of the heated sera could neutralize FeLV in the absence of added complement. The specificity of neutralizing antibody was examined by reciprocal cross-absorptions of sera with respective purified antigens. The gp71 polypeptide completely absorbed the anti gp71 neutralizing antibody for MuX and FeLV, whereas the p15(E) antigen was without effect. Reciprocally the p15(E) antigen removed neutralizing antibody for MuX and FeLV from anti p15(E) sera while the gp71 polypeptide was inactive. Purified F-MuLV absorbed the neutralizing activity of both antisera confirming that gp71 and p15(E) are surface antigens. As a corollary, some complex anti FeLV sera could neutralize MuX as well as FeLV. This activity for MuX was heat-stable and could be specifically abrogated by F-MuLV gp71 antigen but not the p15(E) antigen. These data demonstrated that potent and functional neutralizing antibody could be elicited against the group- and interspecies-specific determinants of two oncornavirus surface components.

Expression of Murine Leukemia Virus Structural Antigens on the Surface of Chemically Induced Murine Sarcomas

Cultured cells of different chemically-induced C57BL/6N murine sarcomas produced variable amounts of infectious murine leukemia virus (MuLV) and contained proportional amounts of MuLV structural components as determined by radioimmunoassay. Monospecific antisera directed against the major MuLV glycoprotein (gp71), the major internal antigen (p30), and the ribonucleoprotein (p10) were capable of mediating tumor cell lysis in the presence of complement, suggesting that these viral structural components were localized at least in part to the cell surface. Membrane immunofluorescence studies with MuLV p30 antiserum confirmed surface localization. Addition of MuLV p30 polypeptide to normal cells and tumor cells enhanced the cytotoxicity of MuLV p30 antiserum. Studies are presented which suggest that the presence of MuLV structural components on cell surfaces can be independent of virus production and cellular transformation.

Properties of mouse leukemia viruses: II. Isolation of viral components

Abstract Three viral substructures were isolated from Tween-ether degraded murine leukemia viruses (MuLV) by Sephadex G-150 chromatography and density gradient centrifugation: a material containing the antigenic determinants IIgs (gs-spec.) and IIv (type specific), a substructure termed component x, and antigen V (gs-inter-spec.). The antigenic determinants IIgs and IIv could not be separated by various methods and are apparently associated with some lipid. This material absorbed neutralizing antibody and possessed hemagglutinating activity when isolated from Friend and Rauscher virus. Component x was demonstrable only by complement fixation tests with complex MuLV antisera. Whether it represents a group- or a type-specific viral antigen could not be decided; however, it did not absorb neutralizing antibody. In the electron microscope, component x preparations consisted of disks with a diameter of about 100 A and filaments formed by aggregation of the disks. Antigen V was isolated from Gross virus. In Ouchterlony tests it demonstrated a reaction of identity with a component of feline leukemia virus. MuLV-antigen I (gs-spec.) could be removed by treatment of the murine viruses with neuraminidase and phospholipase C or less efficiently by treatment with phospholipase C alone. Some of the isolated materials were analyzed by acrylamide gel electrophoresis. The significance of the various components is discussed.

Titration Patterns of a Murine Sarcoma-Leukemia Virus Complex: Evidence for Existence of Competent Sarcoma Virions

Stocks of inurine sarcoma virus show titration patterns ranging from one-to two-hit kinetics. The comparison of various titrations of this virus, both with and without added helper virus, to theoretical model systems composed of defined constituents, suggests the existence of a sarcoma virus that does not need coinfectinig murine leukemia virus to be manifested as a focus-forming unit. The behavior of such nondefective particles is compatible with a postulated leukemia-sarcoma virus hybrid.

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.

Reversion of Murine Sarcoma Virus Transformed Mouse Cells: Variants without a Rescuable Sarcoma Virus

Murine sarcoma virus transformed mouse 3T3 cells, which are negative for murine leukemia virus and which yield sarcoma virus after superinfection with murine leukenmia virus, spotaneously give rise to flat variants front which murine sarcoma virus can no longer be rescued. The revertants support leukemia viruis growth and show an enhanced sensitivity to murine sarcoma superinfection and, like normal cells, do not release RNA-dependent DNA polymerase activity. Because revertants could be obtained with high frequency from progeny of single transformed cells, each cell that containts the sarconma virus genome seems to have the capacity to suppress or eliminate an RNA tumor virus native to its species of origin.