High-frequency C-type virus induction by inhibitors of protein synthesis.

When inhibitors of protein synthesis are added to BALB/c mouse cells in culture, induction of naturally integrated C-type RNA virus occurs in a high percentage of cells. The action of protein synthesis inhibitors differs from that of halogenated pyrimidines, another class of virus inducers, in their effects on biologically distinguishable viruses. The use of such inhibitors to study integrated virus expression provides a means for studying gene regulation in mammalian cells.

Molecular dissection of transcriptional control elements within the long terminal repeat of the retrovirus.

The retroviral long terminal repeat (LTR) contains transcriptional control elements that affect viral gene expression. By deletion mutagenesis of the genome of the cloned Abelson murine leukemia virus, regulatory signals could be mapped to at least three domains within the LTR. A defective 5' LTR that did not sustain transforming gene function was complemented by an intact LTR positioned at the 3' end of the genome. This versatility of the retroviral genome with respect to its transcriptional control elements appears to provide a strong selective advantage for viral gene expression.

Endogenous C-type viruses of BALB-c cells: frequencies of spontaneous and chemical induction.

A sensitive biological assay has been developed for studying the frequency of virus induction from mouse cells containing poorly infectious endogenous C-type viruses. A low spontaneous frequency of virus activation of one but not a second endogenous virus of BALB/c cells has been demonstrated. The resistance of BALB/c cells to superinfection by the more inducible virus appears to be absolute, whereas resistance to the other virus is only relative. Thus, the evidence indicates that the BALB/c cell exerts differential effects at two levels of regulation on the expression of its biologically distinguishable endogenous C-type viruses.

Analysis of the primary translational product and integrated DNA of a new feline sarcoma virus, GR-FeSV.

The Gardner-Rasheed strain of feline sarcoma virus (GR-FeSV), is a recent isolate of a naturally occurring cat sarcoma. The primary translational product of GR-FeSV (GR P70) was shown to be a phosphoprotein with associated tyrosine-specific protein kinase activity. The relationship between the GR-FeSV provirus and once genes of other transforming retroviruses known to code for tyrosine kinases was examined by molecular hybridization. Probes repesenting onc genes of Snyder-Theilen and McDonough strains of feline sarcoma virus, Rous sarcoma virus, and Abelson murine leukemia virus did not detectably hybridize integrated GR-FeSV. These findings suggest that GR-FeSV contains a distinct tyrosine kinase-coding onc gene.

Different lymphoid cell targets by transformation by replication-competent Moloney and Rauscher mouse leukemia viruses.

Mouse leukemia viruses (MuLV) have been reported to induce tumors involving cells within the T lymphocyte lineage. In the present study, striking differences were demonstrated in the target cells for in vivo transformation by two clonal replication-competent type C viruses, Moloney- and Rauscher-MuLV. Moloney-MuLV-induced tumors and lymphoma cell lines exhibited Thy.1 antigen in the absence of detectable Fc or C3 receptors, indicating their T cell origin. Rauscher-MuLV primary tumors and lymphoma cell lines of the same mouse strain, however, invariably exhibited Fc receptors in the absence of Thy.1 antigen, suggesting that these tumors were of the B lymphoid lineage. The pattern of immunoglobulin synthesis by individual Rauscher-MuLV tumor cell lines was determined by both biosynthetic and radioimmunologic techniques. Rauscher-MuLV lymphoma lines invariably expressed immunoglobulin heavy (mu) chain in the absence of detectable light (kappa or lambda) chains. These findings establish that the target of neoplastic transformation in response to Rauscher-MuLV is an immature cell within the B lymphoid lineage. The demonstration of different target cells for transformation by well characterized clonal strains of mouse leukemia virus should aid in elucidating the mechanisms by which these viruses induce malignancy.

Induction of type-C RNA virus by cycloheximide: increased expression of virus-specific RNA.

Mouse cells contain the genetic information for multiple endogenous type-C RNA viruses. The mechanisms by which the cell controls expression of these naturally integrated viruses are not yet known. Recently, chemicals that inhibit protein synthesis have been shown to induce a specific type-C virus at high frequency from BALB/c mouse embryo cells. In the present studies, virus activation in response to a representative translational inhibitor, cycloheximide, is demonstrated to be transient, with virus release primarily occurring within the first 12-24 hr following drug exposure. Analysis of virus-specific RNA in cells by molecular hybridization revealed an absolute increase in viral RNA concentration in cycloheximide-treated cells. This was blocked by simultaneous exposure of the cells to actinomycin D. Further, inhibition of RNA synthesis during but not subsequent to cycloheximide exposure prevented virus activation. These findings show that virus induction by cycloheximide requires de novo RNA synthesis during but not after drug exposure and suggest that the required RNA species may be that of the virus itself. The present results are consistent with the hypothesis that translational inhibitors prevent synthesis of a labile protein whose normal action is to inhibit viral RNA transcription or to cause degradation of viral RNA.

Immunoglobulin synthesis and gene rearrangements in lymphoid cells transformed by replication-competent Rauscher murine leukemia virus: transformation of B cells at various stages of differentiation.

Lymphoid cells transformed by Rauscher murine leukemia virus (R-MuLV) belonged to the B cell lineages. One group of cells exhibited Fc receptors but completely lacked immunoglobulin mu heavy and kappa light chains. The majority of the cells resemble pre-B type. They displayed mu chains but kappa chains were completely absent. Very rarely certain cells synthesized both mu and kappa chains. Based on the presence of Fc receptors and IgM synthesis the cells transformed by R-MuLV belonged to three B cell developmental stages. These cells were tested for immunoglobulin gene rearrangements using JH and CK probes. DNA from cell lines without any detectable levels of IgM mu exhibited embryonic as well as rearranged JH genes, whereas cells expressing IgM possess, in addition, productive and non-productive light chain gene rearrangements. The most terminally differentiated cell possesses JH and CK rearrangement associated with the synthesis of mu and kappa chains. Presumably the cells with rearranged JH and CK genes without immunoglobulin synthesis represent a developmental transition. We conclude that cells transformed by R-MuLV belonged to five step-wise compartments of B cell development. Our findings implicate definite sequential events of immunoglobulin gene rearrangement and expression during B cell development.

Acquisition of transforming properties by alternative point mutations within c-bas/has human proto-oncogene.

The transforming gene of a human lung carcinoma-derived cell line, Hs242, has been cloned in biologically active form, and identified as c-bas/has (otherwise known as c-Ha-ras). The genetic lesion responsible for the transforming activity of the Hs242 oncogene has been localized to a point mutation in the second exon which results in the substitution of leucine for glutamine as amino acid 61 of the predicted protein. No changes were observed in the first exon, the region of c-bas/has in which a point mutation is responsible for activation of the T24 and EJ bladder carcinoma oncogenes.

Abelson murine leukemia virus: structural requirements for transforming gene function.

The integrated Abelson murine leukemia virus (A-MuLV) genome cloned in bacteriophage lambda gtWES.lambda B was used to localize viral genetic sequences required for transformation. Comparison of the biological activity of cloned A-MuLV genomic and subgenomic fragments showed that subgenomic clones that lacked the 5' long terminal repeat and adjoining sequences (300 base pairs downstream of the repeat) were not biologically active. In contrast, subgenomic clones that lacked the 3' long terminal repeat and as much as 1.3 kilobase pairs of the A-MuLV cell-derived abl gene were as efficient as wild-type viral DNA in transformation. The A-MuLV-encoded polyprotein P120 and its associated protein kinase activity were detected in transformants obtained by transfection with Cla I, BamHI, and HindIII subgenomic clones. In contrast, individual transformants obtained with subgenomic Sal I clones expressed A-MuLV proteins ranging in size from 82,000 to 95,000 daltons. Each demonstrated an associated protein kinase activity. These results provide direct genetic evidence that only the proximal 40% of abl with its associated 5' helper viral sequences is required for fibroblast transformation.

Activation of an endogenous mouse type C virus by ultraviolet-irradiated herpes simplex virus types 1 and 2.

Infection of BALB/c mouse cells with UV-irradiated herpes simplex virus (HSV) types 1 and 2 resulted in activation of a xenotropic type C virus detected by infectious center formation in permissive rat cells. The levels of type C virus activated by HSV were related to the UV dose and the multiplicity of infection used. The ability of HSV to activate type C virus was eliminated by heat-inactivation and by neutralization with specific antiserum against HSV, but was not affected by purification or treatment with DNase and RNase. Maximum levels of type C virus in the cells and medium were observed within 1 day after HSV infection, and the levels returned to control cell values within 3-4 days. The possible significance of these findings with respect to the putative oncogenic potential of HSV is discussed.