Sequence-specific and/or stereospecific constraints of the U3 enhancer elements of MCF 247-W are important for pathogenicity.

The oncogenic potential of many nonacute retroviruses is dependent on the duplication of the enhancer sequences present in the unique 3' (U3) region of the long terminal repeat (LTR). In a molecular clone (MCF 247-W) of the murine leukemia virus MCF 247, a leukemogenic mink cell focus-inducing (MCF) virus, the U3 enhancer sequences are tandemly repeated in the LTR. We mutated the enhancer region of MCF 247-W to test the hypothesis that the duplicated enhancer sequences of this virus have a sequence-specific and/or a stereospecific role in enhancer function required for transformation. In one virus, we inserted 14 nucleotide bp into the novel sequence generated at the junction of the two enhancers to generate an MCF virus with an interrupted enhancer region. In the second virus, only one copy of the enhancer sequences was present. This second virus also lacked the junction sequence present between the two enhancers of MCF 247-W. Both viruses were less leukemogenic and had a longer mean latency period than MCF 247-W. These data indicate that the sequence generated at the junction of the two enhancers and/or the stereospecific arrangement of the two enhancer elements are required for the full oncogenic potential of MCF 247-W. We analyzed proviral LTRs within the c-myc locus in tumor DNAs from mice injected with the MCF virus with the interrupted enhancer region. Some of the proviral LTRs integrated upstream of c-myc contain enhancer regions that are larger than those of the injected virus. These results are consistent with the suggestion that the virus with an interrupted enhancer changes in vivo to perform its role in the transformation of T cells.

Viral determinants of HIV-1 sufficient to extend tropism to macrophages are distinct from the determinants that control the cytopathic phenotype in HL-60 cells.

DESIGN: Infection of the human promyelocytic cell line HL-60 with NL4-3, a molecularly cloned HIV-1 strain that productively infects T cells, results in adaptation of the virus and production of a variant, NL4-3(M). Unlike NL4-3, NL4-3(M) has a rapid cytopathic effect in HL-60 and other myeloid cell lines. OBJECTIVE: To demonstrate that the tropism of NL4-3(M) is extended to primary monocyte-derived macrophages (MDM), and to determine whether the envelope gene, env, of NL4-3(M) is responsible for cytopathicity in HL-60 cells and replication in MDM. METHODS: A chimeric virus (NL4-3envA) containing the majority of env of NL4-3(M) was generated, and tested for virus replication and cytopathic effect in H9 and HL-60 cells, as well as for virus replication in primary MDM. To assess virus replication, the cultures were analyzed for expression of viral envelope glycoproteins on the infected cells and production of extracellular HIV-1 p24 antigen. Cytopathic effect on HL-60 cells was evaluated by monitoring the viabilities of the cultures. In addition, the majority of env of NL4-3envA was sequenced. RESULTS: The biological phenotypes of NL4-3, NL4-3(M), and NL4-3envA are distinctly different. Although both NL4-3(M) and NL4-3envA replicate in MDM, only NL4-3(M) is rapidly cytopathic in HL-60 cells. Nine amino-acid changes were identified within the envelope glycoproteins of NL4-3envA compared with NL4-3. CONCLUSIONS: The viral determinants of NL4-3(M) sufficient to extend the tropism of this virus to MDM reside, in part, in env. These genetic determinants are distinct from the viral determinants that control the cytopathic phenotype of this virus in HL-60 cells.

Gene transfer to human cells using retrovirus vectors produced by a new polytropic packaging cell line.

We report here the construction of a new packaging cell line, called MPAC, that packages defective retroviral vectors in viral particles with envelope proteins derived from a Moloney mink cell focus-inducing (MCF) polytropic virus. We characterized the tropism of MPAC-packaged retroviral vectors and show that some human cell lines can be infected with these vectors while others cannot. In addition, we show that some human cells fully support MCF virus replication while others either partially or fully restrict MCF virus replication.

A direct demonstration of recombination between an injected virus and endogenous viral sequences, resulting in the generation of mink cell focus-inducing viruses in AKR mice.

We analyzed viral recombination events that occur during the preleukemic period in AKR mice. We tagged a molecular chimera between the nonleukemogenic virus Akv and the leukemogenic mink cell focus-inducing (MCF) virus MCF 247 with an amber suppressor tRNA gene, supF. We injected the supF-tagged chimeric virus that contains all of the genes of MCF 247 except the envelope gene, which in turn is derived from Akv, into newborn AKR mice to evaluate its pathogenic potential. Approximately the same percentage of animals developed leukemia with similar latent periods when injected with either the tagged or nontagged virus. DNA from tumors induced in AKR mice by the tagged chimeric virus was analyzed by Southern blotting with the supF gene as a probe. One set of tumors contained the injected supF-tagged virus. Two kinds of supF-tagged proviruses were found in a second set of tumors. One group of supF-tagged viruses had a restriction map consistent with that of the injected virus, while the other group of proviruses had restriction maps that suggested that the proviruses had acquired an MCF virus-like envelope gene by recombination with endogenous viral sequences. These results demonstrate that injected viruses recombine in vivo with endogenous viral sequences. Furthermore, the progression to leukemia was accelerated in mice that develop tumors containing proviruses with an MCF virus env gene, emphasizing the importance of the role of the MCF virus env gene product in transformation.

Transplantable Marek's disease lymphomas. IV. Differences in lethality of lymphoma cell lines determined by route of inoculation.

Mortality caused by inoculation of cells from four Marek's disease herpes virus-induced transplantable lymphomas was studied in two related inbred lines of chickens. While i.m. inoculation of 10(4) cells from each lymphoma generally caused death of all syngeneic recipients by 18 days post-inoculation, one of the lymphomas (UG2) was unique in that the same number of cells, when inoculated i.v., caused less than 20% mortality by that time. Lethality induced by cells from the other three lymphomas, when inoculated i.v., was as high or higher than when inoculated i.m. Mortality after intra-abdominal or s.c. inoculation of cells from all four lymphomas was similar to that after i.m. inoculations. Chickens inoculated with syngeneic UG2 cells by the i.v. route were significantly protected against a subsequent i.m. challenge with the same or other syngeneic lymphoma cells. However, UG2 lymphoma cells were highly lethal when inoculated i.v. into birds previously treated with the antimacrophage agent carageenan or immunosuppressed by neonatal treatment with cyclophosphamide. Thus, UG2 cells are distinctive in that, when inoculated i.v., they do not cause death of syngeneic hosts but instead induce resistance to a lethal challenge.

Transplantable Marek's disease lymphomas. III. Induction of MHC-restricted tumor immunity by lymphoblastoid cells in F1 hosts.

F1 chickens, which were a cross between birds of the related inbred lines G-B1 and G-B2, were immunized with in vitro cultured virus-non-producer lymphoblastoid cells that were either syngeneic or allogeneic with the challenge tumor cells. The lymphoblastoid cells were derived from Marek's disease herpesvirus (MDV)-induced transplantable tumors. Previous findings showed that such immunization of G-B1 and G-B2 chickens prevented early mortality caused by the tumors. Lymphoblastoid cells syngeneic with the tumors were more effective than allogeneic cells, suggesting that an MHC-restricted immune response was induced, or, alternatively, that immune elimination of allogeneic cells prevented them from initiating strong immunity to MDV-associated tumor antigens. Immune elimination of the immunizing cells should not occur in F1 birds heterozygous for the 2 different parental MHC haplotypes (B6 and B13). Early mortality among F1 chickens immunized with lymphoblastoid cells that were syngeneic with the challenge tumor cells was significantly lower than for non-immunized control chickens or for birds immunized with lymphoblastoid cells that were allogeneic with the challenge tumor cells. Our results suggest that MDV-induced tumor antigens may be recognized by the host as altered-self MHC antigens.

Genetic interaction between non-MHC T- and B-cell alloantigens in response to Rous sarcomas in chickens.

Chickens of Regional Poultry Research Laboratory (RPRL) inbred line 6(3) regress sarcomas induced by Bryan high-titer Rous sarcoma virus to a greater extent than chickens of line RPRL 100, although these lines are identical for the major histocompatibility B complex. They differ, however, at three independent autosomal loci: Ly-4 and Th-1 determine the surface alloantigens of partly overlapping subsets of T lymphocytes, and Bu-1 determines a surface alloantigen of B lymphocytes. The association of genotypes at these loci with quantitative variation in their ability to regress Rous sarcomas was tested in segregating F4 generation progeny derived from crosses of lines 100 and 6(3). The Ly-4 and Bu-1 genotypes showed association with Rous sarcoma regression, but the Th-1 genotype did not. Chickens of the Ly-4a/Ly-4a, Bu-1b/Bu-1b and Ly-4b/Ly-4b, Bu-1a/Bu-1a genotypes had a significantly higher regressor ability than the other two double homozygous genotypes. These results indicate that higher regression is associated with (1) interaction between the Ly-4 and Bu-1 loci, and (2) complementation between either the line 6 Ly-4a allele and the line 100 Bu-1b allele, or the line 100 Ly-4b allele and the line 6 Bu-1a allele.