Miriam Lieberman

Endoproteolytic cleavage of gp160 is required for the activation of human immunodeficiency virus

The envelope protein of human immunodeficiency virus (HIV) is synthesized as a polyprotein (gp160) and cleaved intracellularly to a gp120-gp41 heterodimer. In this study, the tryptic-like endoproteolytic cleavage site was removed by site-directed mutagenesis and replaced with a chymotryptic-like site. The resultant mutant, RIP7/mut10, was found to be indistinguishable from wild-type HIV when analyzed at the level of proviral replication, RNA processing, protein expression, and viral assembly. However, the gp160 polyprotein was not cleaved and the mutated virions were biologically inactive, until and unless they were exposed to limiting concentrations of chymotrypsin. As is the case for other enveloped mammalian viruses, endoproteolytic cleavage of the HIV envelope protein and release of a unique hydrophobic domain appear to be necessary for the full expression of viral infectivity.

Leukemogenic Activity of Filtrates from Radiation-Induced Lymphoid Tumors of Mice

Cell-free filtrates of x-ray-induced lymphoid tumors of strain C57BL/ Ka mice have elicited, on injection into newborn isologous hosts, a lymphoma incidence of 15 to 19 percent. In control mice of the same subline, the incidence of spontaneous lymphoma is about 1 percent. No leukemogenic activity could be detected in filtrates from thymi harvested at 2 to 32 days following completion of x-ray treatment. Activity was evident at 64 days and was perhaps somewhat greater at 128 days. Serial cell-free passage of filtrates in newborn F1 hybrid mice resulted in a marked increase in lymphoma incidence (69 percent), coupled with a shortening of the median latency. Supplementary x-irradiation failed to enhance the activity of filtrates after neonatal injection.

Physicochemical, biological and serological properties of a leukemogenic virus isolated from cultured radl-V-induced lymphomas of C57BL/Ka mice

Abstract RadLV/VL 3 , a virus spontaneously and continuously produced at high titer by BL/VL 3 cells, a line established in culture from a RadLV-induced C57BL/Ka mouse thymic lymphoma, has been extensively characterized. Evidence is presented indicating that it is a C-type RNA virus with highly thymotropic and leukemogenic biologic properties identical with those of its RadLV parent. It was not significantly neutralized by antibodies prepared against other ecotropic or xenotropic murine C-type viruses, used individually or in combination, whereas these antibodies effectively neutralized MCF-247 virus. However, antisera prepared against RadLV/VL 3 in rabbits and mice readily neutralized RadLV/VL 3 . Curiously, the rabbit antiserum also had strong neutralizing activity against three other C-type endogenous viruses of the C57BL/Ka strain, BL/Ka(B), BL/Ka(N), and BL/Ka(X), all of which are fibrotropic (replicate readily in fibroblasts of appropriate tropism) and lack thymotropic and leukemogenic activity. In interference assays, these viruses and RadLV/VL 3 exhibited little or no capacity for mutual or self-interference, nor did RadLV/VL 3 interfere with the replication of MCF-247 virus in mink cells. However, the addition to cultures of increasing concentrations of the purified envelope glycoprotein (gp71) of RadLV/VL 3 , though devoid of effect on BL/Ka(B) and BL/Ka(N), strongly inhibited the replication of RadLV and RadLV/VL 3 both in vitro and in vivo , and partially inhibited the xenotropic replication of BL/Ka(X). Competition radioimmunoassays with purified virion polypeptides demonstrated that the gp71 of RadLV/VL 3 is distinctively different from that of all other viruses tested; that the p12s of RadLV/VL 3 and BL/Ka(B) are similar or identical, whereas that of BL/Ka(N) competes in the AKR-MuLV p12 assay; and that the p30s of all of the viruses studied are indistinguishable by this method. Finally, RadLV/VL 3 failed to elicit cytopathic changes or transformed cell foci after serial passage in mink lung cells, even after co-infection with the xenotropic BL/Ka(X) virus, indicating that it is apparently devoid of MCF activity. Thus, MCF activity is apparently not a prerequisite for thymotropic and leukemogenic activity, at least in the C57BL/Ka strain.

Inhibition of radiogenic lymphoma development in mice by interferon.

Summary Long-term administration of interferon resulted in the partial suppression of radiation-induced lymphosarcoma development in strain C57BL mice. The inducer of interferon, polyinosinic acid-polycytidylic acid, was without effect under the same conditions. Interferon also protected C57BL mice against the induction of these tumors by a virus, RadLV, but did not modify the death rate following injection of tumor cells.

H-2 antigen expression: Loss in vitro, restoration in vivo, and correlation with cell-mediated cytotoxicity in a mouse lymphoma cell line

The susceptibility to cell-mediated cytolysis of cells of the recently developed C57BL/Ka(H-2b) lymphoma cell line, BL/VL3, was investigated in allogeneic assays with thymus-dependent lymphocytes (T cells). Compared to EL4, the widely used C57BL/6(H-2b) lymphoma cell line, BL/VL3 cells were found to be insensitive to T-cell-mediated lysis as detected by the use of51Crrelease methods. When used as immunogens in alloreactive combinations with BALB/c(H-2d) splenocytes as responder cells, BL/VL3 cells failed to elicit sensitization. Serological tests showed that this cell line had profoundly reduced levels of H-2b antigens on its surface. When BL/VL3 cells were reinjected into C57BL/Ka and BALB/c mice, full recovery of H-2b antigen expression at the cell surface was observed in both syngeneic and allogeneic hosts after only 11 days of in vivo growth. Concomitantly, they acquired the ability to induce cytotoxic responses in allogeneic T cells and became susceptible to their lytic activity. The expression of H-2 antigens on the surface of BL/VL3 cells is a reversibly modulated function that depends on in vivo growth conditions and is lost in vitro in the absence of immunoselective pressure.

The Scid-hu Mouse: Current Status and Potential Applications

The detailed analysis of physiology and pathophysiology in man is a difficult and frequently impossible task. In many instances, volunteers will not serve as experimental subjects; multiple variables cannot be simultaneously explored; and placebo-controlled trials are confounded by intervening social dynamics. Animals, used instead as surrogates for man, then only provide approximate models of uncertain relevance. For most diseases affecting man, even animal models are unavailable, precisely because these diseases affect only man.

In Vivo interaction between RNA viruses isolated from the C57BL/Ka strain of mice

Abstract Virus replication can be initiated in the C57BL/Ka thymus by inoculation of three virus preparations isolated from mice of this strain, designated RadLV, RadLV-LTC, and BL/Ka(6), but not by three others, designated BL/Ka(B), BL/Ka(N), and BL/Ka(X). Coinfection by various mixtures of the three nonthymotropic viruses does not result in productive infection of the thymus. The addition of these nonthymotropic viruses to serial dilutions of RadLV, RadLV-LTC, or BL/Ka(6) does not significantly alter the replication of the wild-type RadLV preparations tested, whereas it significantly facilitates the replication of RadLV-LTC and BL/Ka(6). It is postulated that RadLV-LTC and BL/Ka(6) contain an attenuated or defective thymotropic and leukemogenic particle, the intrathymic replication of which is facilitated by coinfection with the nonthymotropic viruses.

Rescue of a thymotropic, leukemogenic C-type virus from cultured, nonproducer lymphoma cells of strain C57BL/Ka mice☆

Abstract A permanent cell line, BL/RL12-NP, derived from a radiation-induced C57BL/Ka mouse lymphoid tumor, has remained devoid of MuLV expression, except for the rare, sporadic initiation of virus production in some cultures. It can, however, be stably infected by the radiation leukemia virus (RadLV), and the progeny virus population retains the biological and serological properties of the parental RadLV. The cells can also be infected by a B-ecotropic, nonthymotropic, nonleukemogenic C57BL/Ka virus isolate, BL/Ka (B). In the latter situation, the emerging virus particles may exhibit thymotropic and leukemogenic (T+L+) attributes similar to those of RadLV, while retaining at least some of the envelope determinants of BL/Ka (B). These observations suggest that, following productive infection by a nonleukemogenic helper virus, oncogenic sequences endogenous to the non-producer lymphoma cells may be packaged in infectious progeny virions. The data are interpreted as providing strong support for the existence, in radiogenic lymphomas, of defective T+L+ sequences, designated RadLV-O. Possible mechanisms whereby RadLV-O is later expressed as an infectious leukemogenic virus are discussed.

Rapid in vitro assay for thymotropic, leukemogenic murine C-type RNA viruses

Abstract A procedure for the in vitro quantitation of thymotropic and leukemogenic murine C-type RNA viruses is described. When serial dilutions of murine leukemia viruses from mice of the C57BL/Ka strain were plated on a nonproducer lymphoma cell line derived from a radiation-induced lymphoma of this strain, end-point titers were obtained that correlated well with those obtained by titration methods in vivo .

Patterns of thymocyte differentiation markers on virus and radiation induced lymphomas of C57BL/Ka mice

To better understand the biology of tumorigenesis in virus and radiation lymphomas of C57Bl/Ka mice, we have examined the cell surface phenotypes of a large series of primary tumors induced by both agents. Data derived using flow cytometry and recently available monoclonal antibodies to thymocyte differentiation antigens supports three major conclusions. First, tumor cell populations are unimodal for staining with most antibodies and are probably of clonal origin. Second, many, but not all, tumor cells show surface phenotypes similar to those of previously defined subpopulations of normal thymocytes. Third, at the cell surface level, no major differences between virus- and radiation-induced lymphomas can be discerned. Our data thus further define the relationship between thymomas induced by these two agents.