Sequences responsible for the distinctive hemolytic potentials of Friend and Moloney murine leukemia viruses are dispersed but confined to the psi-gag-PR region.

Friend and Moloney murine leukemia viruses (F- and M-MuLV) induce distinct diseases in hematopoietic tissues following inoculation of newborn mice of susceptible strains. F-MuLV induces erythroleukemia preceded by severe early hemolytic anemia; M-MuLV induces thymomas and only very mild hemolysis. The major viral determinant of severe early hemolytic anemia residues in the env gene, but sequences located outside this gene can modulate this effect. By means of genetic chimeras of F- and M-MuLV, we have found that although they are confined to the 5' portion of the env gene intron, sequences that determine the distinctive hemolytic potentials of F- and M-MuLV are widely distributed over a region spanning the RNA encapsidation domain, the gag gene, and the portion of the pol gene encoding the viral protease. Within this large region, two fragments of M-MuLV, a 1.3-kb region encoding the matrix, pp12, and capsid proteins and a 0.8-kb region encoding the nucleocapsid and the viral protease, were capable, individually, of partially attenuating the capacity of F-MuLV for induction of severe early hemolytic anemia. In association, these two fragments conferred complete attenuation. Moreover, a second pair of adjacent fragments within this large region appeared to behave cooperatively to confer complete attenuation; a 0.36-kb region roughly corresponding to the encapsidation domain, although not detectably altering hemolytic potential on its own, deepened the attenuation conferred by the adjacent 1.3-kb region. Whether capable of inducing severe early hemolytic anemia or not and despite different efficiencies of induction of recombinant polytropic viruses, all chimeric viruses retained the erythroleukemogenicity of the F-MuLV parent.     

Molecular characterization of a neuropathogenic and nonerythroleukemogenic variant of Friend murine leukemia virus PVC-211.

PVC-211 murine leukemia virus (MuLV) is a replication-competent, ecotropic type C retrovirus that was isolated after passage of the Friend virus complex through F344 rats. Unlike viruses in the Friend virus complex, it does not cause erythroleukemia but causes a rapidly progressive hind limb paralysis when injected into newborn rats and mice. We have isolated an infectious DNA clone (clone 3d) of this virus which causes neurological disease in animals as efficiently as parental PVC-211 MuLV. The restriction map of clone 3d is very similar to that of the nonneuropathogenic, erythroleukemogenic Friend murine leukemia virus (F-MuLV), suggesting that PVC-211 MuLV is a variant of F-MuLV and that no major structural alteration was involved in its derivation. Studies with chimeric viruses between PVC-211 MuLV clone 3d and wild-type F-MuLV clone 57 indicate that at least one determinant for neuropathogenicity resides in the 2.1-kb XbaI-ClaI fragment containing the gp70 coding region of PVC-211 MuLV. Compared with nonneuropathogenic ecotropic MuLVs, the env gene of PVC-211 MuLV encodes four unique amino acids in the gp70 protein. Nucleotide sequence analysis also revealed a deletion in the U3 region of the long terminal repeat (LTR) of PVC-211 MuLV clone 3d compared with F-MuLV clone 57. In contrast to the env gene of PVC-211 MuLV, particular sequences within the U3 region of the viral LTR do not appear to be required for neuropathogenicity. However, the changes in the LTR of PVC-211 MuLV may be responsible for the failure of this virus to cause erythroleukemia, because chimeric viruses containing the U3 region of F-MuLV clone 57 were erythroleukemogenic whereas those with the U3 of PVC-211 MuLV clone 3d were not.     

Different abilities of Friend murine leukemia virus (MuLV) and Moloney MuLV to induce promonocytic leukemia are due to determinants in both psi-gag-PR and env regions.

Moloney murine leukemia virus (M-MuLV) is capable of inducing promonocytic leukemia in 50% of adult BALB/c mice that have received peritoneal injections of pristane, but Friend MuLV strain 57 (F-MuLV) is nonleukemogenic under similar conditions. It was shown earlier that these differences could not be mapped to the U3 region of the virus long terminal repeat, indicating the probable influence of structural genes and/or R-U5 sequences. In this study, reciprocal chimeras containing exchanged structural genes and R-U5 sequences from these two closely related viruses were analyzed for differences in ability to induce disease. Results showed that two regions of F-MuLV, psi-gag-PR and env, when substituted for those of M-MuLV were dramatically disease attenuating. The 5'-most region, which is widely distributed, overlaps with the 5' end of the env intron and includes the RNA packaging region, psi, the entire gag coding region, and the viral protease coding region (PR) of pol. It was also found that reciprocal constructs having substitutions of both of these regions of M-MuLV in an F-MuLV background allowed full reestablishment of promonocytic leukemia. These leukemias were positive for c-myb rearrangements which are characteristic of M-MuLV-induced promonocytic leukemias. Neither region alone, however, was sufficient to produce disease with a greater incidence than 13%. Further studies demonstrated that the inability of viruses with psi, gag, PR, or env sequences from F-MuLV to induce leukemia in this model system was not due to their inability to replicate in hematopoietic tissue, to integrate into the c-myb locus early on after infection in vivo, or to express gag-myb mRNA characteristic of M-MuLV-induced preleukemic cells and acute leukemia.     

The envelope gene and long terminal repeat sequences contribute to the pathogenic phenotype of helper-independent Friend viruses.

Friend murine leukemia virus (F-MuLV) and Friend mink cell focus-inducing virus (Fr-MCF) are helper-independent murine retroviruses which induce a rapidly fatal erytholeukemia in NIH Swiss mice. Amphotropic clone 4070 (Ampho) is a murine retrovirus which does not cause leukemia in these animals. Mice inoculated with Ampho, an Fr-MCF/Ampho pseudotype, or F-MuLV developed leukemia in 0, 50, and 100% of animals, respectively. To identify the F-MuLV and Fr-MCF sequences responsible for leukemia, we constructed hybrid viral genomes between these viruses and Ampho, using subgenomic fragments of molecularly cloned viral DNA. Transfection of these hybrid viral DNAs into fibroblasts produces recombinant retroviruses. These new viruses are assayed in vivo for their ability to cause leukemia. Recombinant viruses constructed between the Ampho genome and the Fr-MCF envelope gene do not cause leukemia. Similarly, viruses constructed by using either the Fr-MCF long terminal repeat U3 region or the F-MuLV long terminal repeat U3 region and the remainder of the Ampho genome do not cause leukemia. However, if the Fr-MCF envelope gene plus the Fr-MCF U3 region are joined to Ampho, the resulting virus causes erythroleukemia in 14% of mice. Recombinant viruses made between the Fr-MCF envelope gene, the F-MuLV U3 region, and the remainder of the Ampho genome cause erythroleukemia in 38% of mice. This study demonstrates that both the envelope gene of Fr-MCF and the U3 regions of Fr-MCF and F-MuLV contain sequences which contribute to the leukemic phenotype of helper-independent Friend viruses.     

Hemolytic anemia and erythroleukemia, two distinct pathogenic effects of Friend MuLV: mapping of the effects to different regions of the viral genome

Two different pathogenic effects of the Friend ecotropic murine leukemia virus (F-MuLV) were distinguished by serial examinations of hematocrits and reticulocyte counts of IRW mice inoculated as newborns. F-MuLV induced hemolytic anemia with increased levels of erythropoiesis, which was detectable as early as 13 days of age, whereas blocked erythroid differentiation, associated with erythroleukemia, was apparent only after 30 days of age. Using strains of Friend-MuLV with different virulences, we constructed recombinant viruses that allowed us to map the hemolytic effect and the ability to induce rapid erythroleukemia to different regions of the viral genome. Moreover, the ability of the virus to induce rapid erythroleukemia appeared to be independent of the presence of severe early hemolytic anemia.     

Molecular cloning and characterization of an immunosuppressive and weakly oncogenic variant of Friend murine leukemia virus, FIS-2.

The FIS variant is a weakly leukemogenic, relatively strong immunosuppressive murine retrovirus which was isolated from the T helper cells of adult NMRI mice infected with Friend murine leukemia virus (F-MuLV) complex (FV). Unlike FV, it does not induce acute erythroleukemia but retains the immunosuppressive property of FV and induces suppression of the primary antibody response rapidly and persistently in adult mice. A previous study showed that the FIS variant contains two viral components, a replication-competent virus and a defective virus. In this study, we have biologically purified the FIS variant by end point dilution and we show that the replication-competent virus FIS-2 alone can induce immunosuppression as the parental FIS variant. Most newborn mice infected with FIS-2 developed erythroleukemia, but with an increased latency period compared with that of F-MuLV clone 57. In contrast, FIS-2 induced suppression of the primary antibody response and disease more rapidly than F-MuLV clone 57 in immunocompetent, adult mice. FIS-2 was further molecularly cloned and characterized. Restriction mapping and nucleotide sequence analysis of FIS-2 showed a high degree of homology between FIS-2 and F-MuLV clone 57, suggesting that FIS-2 is a variant of F-MuLV. The striking difference is the deletion of one of the tandem repeats in the FIS-2 long terminal repeat and the single point mutation in the binding sites for core-binding protein and FVa compared with the long terminal repeat of F-MuLV clone 57. Two single point mutations led to the appearance of two extra potential N glycosylation sites in the FIS-2 gag-encoded glycoprotein. Together, the results suggest that FIS-2 represents an interesting murine model to study retrovirus-induced immunosuppression on the basis of its unique combined property of low leukemogenicity and relatively strong and persistent immunosuppressive activity in adult mice.     

A nonstructural gag-encoded glycoprotein precursor is necessary for efficient spreading and pathogenesis of murine leukemia viruses.

In addition to the Gag-Pol and Env precursors whose translation initiates at AUG codons, murine, feline, and simian type C oncoviruses also express glycosylated Gag-Pol precursors (glycoGag), glycoGag translation is initiated at CUG codons located upstream of the Gag AUG initiation codon. In contrast to Gag, glycoGag is translocated into the endoplasmic reticulum and is absent from virions. Since glycoGag has been described to be dispensable ex vivo, we investigated the in vivo effects of a glycoGag- mutation in the Friend murine leukemia virus (F-MuLV). F-MuLV induces severe early hemolytic anemia and subsequent erythroleukemia within 2 months after inoculation of newborn mice. We obtained a glycoGag- F-MuLV, strain H5, by inserting an octanucleotide linker downstream of the CUG codon leading to the reading of a stop codon in all reading frames upstream of the Gag AUG. F-MuLV H5 did not induce severe early hemolytic anemia, and latency of erythroleukemia was significantly increased most likely because of an approximately 1-week delay in the in vivo spreading. Accordingly, induction of recombinant polytropic viruses was also significantly delayed. Close examination of ex vivo spreading kinetics also showed a slower dissemination of F-MuLV H5. Western blot (immunoblot) performed after inoculation of newborn mice with this glycoGag- virus indicated the emergence of new glycoGag+ viruses. PCR analyses with F-MuLV-specific primers demonstrated in vivo pseudoreversions restoring the glycoGag reading frame. Our results demonstrated that glycoGag expression is positively selected and essential for full spreading and pathogenic abilities.     

Deletion of a GC-rich region flanking the enhancer element within the long terminal repeat sequences alters the disease specificity of Moloney murine leukemia virus.

Moloney murine leukemia virus (M-MuLV) is a replication-competent retrovirus which induces T-lymphoblastic lymphoma 2 to 4 months after inoculation. Enhancer sequences in the U3 region of the M-MuLV long terminal repeat, primarily the 75-bp tandem repeats, strongly influence the disease specificity and latency of M-MuLV. We investigated the role of GC-rich sequences downstream of the tandem repeats in the disease specificity of M-MuLV. A recombinant M-MuLV lacking 23 bases of a GC-rich sequence (-174 to -151), Delta 27A M-MuLV, was tested for pathogenesis in neonatal NIH Swiss mice. Delta 27A M-MuLV induced disease with a longer latency than did M-MuLV (7 versus 3 months) in greater than 85% of inoculated mice. More interestingly, this virus showed an expanded repertoire of hematopoietic diseases. Molecular analyses and histopathologic examinations indicated that while 39% of mice inoculated with Delta 27A M-MuLV developed T-cell lymphoblastic lymphoma typical of wild-type M-MuLV, the majority developed acute myeloid leukemia, erythroleukemia, or B-cell lymphoma. Viral DNA corresponding to Delta 27A M-MuLV was detectable in most of the tumors analyzed. These findings indicate that the GC-rich region significantly influences the disease specificity and latency of M-MuLV.     

Protection against retroviral diseases after vaccination is conferred by interference to superinfection with attenuated murine leukemia viruses.

Cell cultures expressing a retroviral envelope are relatively resistant to superinfection by retroviruses which bear envelopes using the same receptor. We tested whether this phenomenon, known as interference to superinfection, might confer protection against retroviral diseases. Newborn mice first inoculated with the attenuated strain B3 of Friend murine leukemia virus (F-MuLV) were protected against severe early hemolytic anemia and nonacute anemiant erythroleukemia induced by the virulent strain 57 of F-MuLV. Vaccinated animals were also protected as adults against acute polycythemic erythroleukemia induced upon inoculation with the viral complex containing the defective spleen focus-forming virus and F-MuLV 57 as helper virus. Animals were inoculated as newborns, which is known to induce immune tolerance in mice, and the rapid kinetics of protection, incompatible with the delay necessary for the immune response to develop, indicated that protection was not due to an immune mechanism but rather was due to the rapid and long-lasting phenomenon of interference. This result was confirmed by combining parental and envelope chimeric MuLV from different interference groups as vaccinal and challenge viruses. Although efficient protection could be provided by vaccination by interference, we observed that attenuated replication-competent retroviruses from heterologous interference groups might exert deleterious synergistic effects.     

Contribution of the gag and pol sequences to the leukemogenicity of Friend murine leukemia virus.

Friend murine leukemia virus (F-MuLV) is a highly leukemogenic replication-competent murine retrovirus. Both the F-MuLV envelope gene and the long terminal repeat (LTR) contribute to its pathogenic phenotype (A. Oliff, K. Signorelli, and L. Collins, J. Virol. 51:788-794, 1984). To determine whether the F-MuLV gag and pol genes also possess sequences that affect leukemogenicity, we generated recombinant viruses between the F-MuLV gag and pol genes and two other murine retroviruses, amphotrophic clone 4070 (Ampho) and Friend mink cell focus-inducing virus (Fr-MCF). The F-MuLV gag and pol genes were molecularly cloned on a 5.8-kilobase-pair DNA fragment. This 5.8-kilobase-pair F-MuLV DNA was joined to the Ampho envelope gene and LTR creating a hybrid viral DNA, F/A E+L. A second hybrid viral DNA, F/Fr ENV, was made by joining the 5.8-kilobase-pair F-MuLV DNA to the Fr-MCF envelope gene plus the F-MuLV LTR. F/A E+L and F/Fr ENV DNAs generated recombinant viruses upon transfection into NIH 3T3 cells. F/A E+L virus (F-MuLV gag and pol, Ampho env and LTR) induced leukemia in 20% of NIH Swiss mice after 6 months. Ampho-infected mice did not develop leukemia. F/Fr ENV virus (F-MuLV gag and pol, Fr-MCV env, F-MuLV LTR) induced leukemia in 46% of mice after 3 months. Recombinant viruses containing the Ampho gag and pol, Fr-MCF env, and F-MuLV LTR caused leukemia in 38% of mice after 6 months. We conclude that the F-MuLV gag and pol genes contain sequences that contribute to the pathogenicity of murine retroviruses. These sequences can convert a nonpathogenic virus into a leukemia-causing virus or increase the pathogenicity of viruses that are already leukemogenic.     

Tissue-specific replication of Friend and Moloney murine leukemia viruses in infected mice.

We have studied the replication of ecotropic murine leukemia viruses (MuLV) in the spleens and thymuses of mice infected with the lymphocytic leukemia-inducing virus Moloney MuLV (M-MuLV), with the erythroleukemia-inducing virus Friend MuLV (F-MuLV), or with in vitro-constructed recombinants between these viruses in which the long terminal repeat (LTR) sequences have been exchanged. At 1 week after infection both the parents and the LTR recombinants replicated predominantly in the spleens with only low levels of replication in the thymus. At 2 weeks after infection, the patterns of replication in the spleens and thymuses were strongly influenced by the type of LTR. Viruses containing the M-MuLV LTR exhibited a remarkable elevation in thymus titers which frequently exceeded the spleen titers, whereas viruses containing the F-MuLV LTR replicated predominantly in the spleen. In older preleukemic mice (5 to 8 weeks of age) the structural genes of M-MuLV or F-MuLV predominantly influenced the patterns of replication. Viruses containing the structural genes of M-MuLV replicated efficiently in both the spleen and thymus, whereas viruses containing the structural genes of F-MuLV replicated predominantly in the spleen. In leukemic mice infected with the recombinant containing F-MuLV structural genes and the M-MuLV LTR, high levels of virus replication were observed in splenic tumors but not in thymic tumors. This phenotypic difference suggested that tumors of the spleen and thymus may have originated by the independent transformation of different cell types. Quantification of polytropic MulVs in late-preleukemic mice infected with each of the ecotropic MuLVs indicated that the level of polytropic MuLV replication closely paralleled the level of replication of the ecotropic MuLVs in all instances. These studies indicated that determinants of tissue tropism are contained in both the LTR and structural gene sequences of F-MuLV and M-MuLV and that high levels of ecotropic or polytropic MuLV replication, per se, are not sufficient for leukemia induction. Our results further suggested that leukemia induction requires a high level of virus replication in the target organ only transiently during an early preleukemic stage of disease.     

Subgenomic fragment of molecular cloned Friend murine leukemia virus DNA contains the gene(s) responsible for Friend murine leukemia virus-induced disease.

Friend murine leukemia virus (G-MuLV) is a helper-independent, type C retrovirus isolated from stocks of Friend virus complex (spleen focus-forming virus plus MuLV). In cell culture, F-MuLV has an ecotropic and NB-tropic host range and causes XC cells to fuse. When injected into newborn NIH Swiss mice, F-MuLV produces hepatosplenomegaly, severe anemia, and numerous circulating hematopoietic precursors in the peripheral blood with normal thymus and lymph nodes after 3 to 6 weeks. Recently, we molecularly cloned an 8.5-kilobase pair (kbp) form of F-MuLV DNA from which we could recover the pathogenic F-MuLV virus by DNA transfection of NIH 3T3 cells. From this molecularly cloned F-MuLV DNA, we have now subcloned in pBR322 a 4.1-kbp HindIII fragment which contains in continuity 3.0 kbp from the 3' terminus (env and c region), 0.6 kbp of the terminal repeat sequences, and 0.5 kbp from the 5'terminus of the viral RNA (genome). NIH 3T3 fibroblasts were transfected with this DNA fragment an then infected with the wild mouse amphotropic retrovirus (cl 1504-A). In cell culture, 1504-A is a helper-independent type C virus which has an N-tropic host range and does not cause fusion of XC cells. When injected into newborn NIH Swiss mice, 1504-A does not produce splenomegaly or thymic enlargement in mice held for up to 8 months. The transfection with the F-MuLV fragment and the infection with 1504-A consistently yielded virus preparations that were XC positive. From such virus stocks we were able to isolate both helper-independent and replication-defective XC-positive viruses. The helper-independent virus was shown to be a recombinant virus since it contains a gp70 molecule derived at least in part from F-MuLV and a specific gag precursor derived from 1504-A as determined by radioactive immune precipitation assays. When injected into newborn Swiss mice, the recombinant helper-independent virus caused hepatosplenomegaly in approximately 50% of the mice in 6 to 8 weeks. The histology of the diseased splenic tissue was indistinguishable from that seen in the disease caused by the whole F-MuLV. The replication-defective virus could be pseudotyped with new 1504-A virus, and this viral complex also caused the F-MuLV disease picture when the complex was injected into newborn Swiss mice. We conclude that the genetic information responsible for the pathogenicity of F-MuLV is contained within the 4.1-kbp DNA fragment, which includes env gene sequences, the terminal repeat sequences, and the c region sequences of the F-MuLV genome.     

A 2.4-kilobase-pair fragment of the Friend murine leukemia virus genome contains the sequences responsible for friend murine leukemia virus-induced erythroleukemia.

Friend murine leukemia virus (F-MuLV) is a replication-competent, ecotropic, NB-tropic retrovirus which produces a rapidly fatal erythroleukemia in susceptible strains of mice. We previously molecularly cloned the entire F-MuLV genome. Transfection of this cloned DNA into NIH 3T3 mouse fibroblasts produces a virus with the same leukemia-inducing characteristics as F-MuLV. To identify which portion of the F-MuLV genome is responsible for causing leukemia, we made recombinant viruses between subgenomic fragments of F-MuLV DNA and another retrovirus--Amphotroph clone 4070. Amphotroph clone 4070 is a replication-competent, amphotrophic, N-tropic virus which does not produce any detectable malignancy in mice. A 2.4-kilobase-pair fragment of F-MuLV DNA was isolated. This DNA fragment encompassed approximately 700 base pairs from the 3' end of the F-MuLV pol gene and 1.7 kilobase pairs of the env gene including all of gp70 and the N-terminal four-fifths of p15E. A molecularly cloned fragment of Amphotroph DNA was ligated to the 2.4-kilobase-pair F-MuLV DNA, and an 8.3-kilobase-pair hybrid F-MuLV-Amphotroph DNA was subcloned into a new plasmid (p5a25-H). Transfection of p5a25-H DNA into fibroblasts resulted in the production of a replication-competent, ecotropic, N-tropic retrovirus--5a25-H virus. Inoculation of this virus into newborn NIH Swiss mice caused leukemia within 4 to 6 months. The disease caused by 5a25-H was pathologically and histologically indistinguishable from the disease caused by F-MuLV. We conclude that the F-MuLV sequences needed to cause disease are contained in these 2.4 kilobase pairs of DNA.     

Analysis of the Disease Potential of a Recombinant Retrovirus Containing Friend Murine Leukemia Virus Sequences and a Unique Long Terminal Repeat from Feline Leukemia Virus

We have molecularly cloned a feline leukemia virus (FeLV) (clone 33) from a domestic cat with acute myeloid leukemia (AML). The long terminal repeat (LTR) of this virus, like the LTRs present in FeLV proviruses from other cats with AML, contains an unusual structure in its U3 region upstream of the enhancer (URE) consisting of three tandem direct repeats of 47 bp. To test the disease potential and specificity of this unique FeLV LTR, we replaced the U3 region of the LTR of the erythroleukemia-inducing Friend murine leukemia virus (F-MuLV) with that of FeLV clone 33. When the resulting virus, F33V, was injected into newborn mice, almost all of the mice eventually developed hematopoietic malignancies, with a significant percentage being in the myeloid lineage. This is in contrast to mice injected with an F-MuLV recombinant containing the U3 region of another FeLV that lacks repetitive URE sequences, none of which developed myeloid malignancies. Examination of tumor proviruses from F33V-infected mice failed to detect any changes in FeLV U3 sequences other than that in the URE. Like F-MuLV-infected mice, those infected with the F-MuLV/FeLV recombinants were able to generate and replicate mink cell focus-inducing viruses. Our studies are consistent with the idea that the presence of repetitive sequences upstream of the enhancer in the LTR of FeLV may favor the activation of this promoter in myeloid cells and contribute to the development of malignancies in this hematopoietic lineage.     

Identification of genetic determinants that regulate tumorigenicity of Friend murine leukemia virus in rats

A neuropathogenic variant of Friend murine leukemia virus (FrMLV), clone A8, has been shown to cause thymoma and infiltration of leukemic cells to organs at 7-8 weeks post-infection in rats with a more rapid progression than clone 57. We have previously reported that the determinant for induction of aggressive leukemia in rats is located in the ClaI-AatII fragment containing the long terminal repeat (LTR) and the 5' half of the 5' leader sequence of A8 virus. Further studies of chimeric viruses restricted the determinant for the induction of thymoma to only the 0.6-kb ClaI-KpnI fragment of A8. This fragment contains a 0.1 kb region of the 3' terminus of the env gene, the intergenic region, the U3, and the 5' half of the R region in the LTR. Major differences in the fragment between A8 and 57 viruses were found in the U3 region, especially in the enhancer motifs. These results indicate that the enhancer region of A8-LTR contributes to the manifestation of thymoma with rapid progression in rats.     

Viral determinants that control the neuropathogenicity of PVC-211 murine leukemia virus in vivo determine brain capillary endothelial cell tropism of the virus in vitro.

PVC-211 murine leukemia virus (MuLV) is a neuropathogenic, weakly leukemogenic variant of the nonneuropathogenic, highly leukemogenic Friend MuLV (F-MuLV). Chimeric viruses constructed from PVC-211 MuLV clone 3d and F-MuLV clone 57 indicate that the env gene of PVC-211 MuLV contains the determinant(s) responsible for pathological changes in the central nervous system. However, sequences within the 5' one-third (AatII-EcoRI region) of the PVC-211 MuLV genome, which include the 5' leader sequence, the gag gene, and the 5' quarter of the pol gene, are also needed in conjunction with the env gene determinant(s) to cause clinically evident neurological disease in the majority of virus-infected animals after a short latency. In the presence of the AatII-EcoRI region of the PVC-211 MuLV genome, the PVC-211 MuLV env gene sequences encoding the amino-terminal half of the SU protein, which contains the receptor-binding region of the protein, were sufficient to cause rapidly progressive neurological disease. When PVC-211 MuLV, F-MuLV, and various chimeric viruses were tested for their ability to replicate in cultured brain capillary endothelial cells (BCEC), the primary site of PVC-211 MuLV replication within the central nervous system, there was a direct correlation between the replication efficiency of a virus in BCEC in vitro and its ability to cause neurological disease in vivo. This observation indicates that the sequences in PVC-211 MuLV that render it neuropathogenic affect its replication in BCEC and suggests that rapid and efficient replication of the virus in BCEC is crucial for the pathological changes in the central nervous system that result in development of neurological disease.     

Recombinant mink cell focus-inducing virus and long terminal repeat alterations accompany the increased leukemogenicity of the Mo+PyF101 variant of Moloney murine leukemia virus after intraperitoneal inoculation.

We recently showed that different routes of inoculation affect the leukemogenicity of the Mo+PyF101 variant of Moloney murine leukemia virus (M-MuLV). Intraperitoneal (i.p.) inoculation of neonatal mice with Mo+PyF101 M-MuLV greatly enhanced its leukemogenicity compared with subcutaneous (s.c.) inoculation. We previously also suggested that the leukemogenicity defect of Mo+PyF101 M-MuLV when inoculated s.c. may result from the inability of this virus to form env gene recombinant (mink cell focus-inducing [MCF]) virus. In this study, virus present in end-stage tumors and in preleukemic animals inoculated i.p. by Mo+PyF101 M-MuLV was characterized. In contrast to s.c. inoculation, all tumors from i.p.-inoculated mice contained high levels of recombinant MCF virus. Furthermore, Southern blot analyses demonstrated that the majority of the tumors contained altered Mo+PyF101 M-MuLV long terminal repeats. The U3 regions from several tumors with altered long terminal repeats were cloned by PCR amplification. Sequence analyses indicated that the M-MuLV 75-bp tandem repeat in the enhancer region was triplicated. This amplification was also previously observed in mice infected s.c. with a pseudotypic mixture of Mo+PyF101 M-MuLV and Mo+PyF101 MCF virus. The enhancer triplication was an early event, and it occurred within 2 weeks postinfection. Recombinant MCF viruses were not detected by Southern blot analyses until 4 weeks postinfection. Thus, the M-MuLV enhancer triplication event was initially important for efficient propagation of ecotropic Mo+PyF101 M-MuLV. The increased leukemogenicity following i.p. inoculation could be explained if the triplication enhances Mo+PyF101 M-MuLV replication in the bone marrow and bone marrow infection is required for recombinant MCF virus formation.     

Escape from in vivo restriction of Moloney mink cell focus-inducing viruses driven by the Mo+PyF101 long terminal repeat (LTR) by LTR alterations.

Mo+PyF101 M-MuLV is a variant Moloney murine leukemia virus containing polyomavirus F101 enhancers inserted just downstream from the M-MuLV enhancers in the long terminal repeat (LTR). The protein coding sequences for this virus are identical to those of M-MuLV. Mo+PyF101 M-MuLV induces T-cell disease with a much lower incidence and longer latency than wild-type M-MuLV. We have previously shown that Mo+PyF101 M-MuLV is defective in preleukemic events induced by wild-type M-MuLV, including splenic hematopoietic hyperplasia, bone marrow depletion, and generation of recombinant mink cell focus-inducing viruses (MCFs). We also showed that an M-MCF virus driven by the Mo+PyF101 LTR is infectious in vitro but does not propagate in mice. However, in these experiments, when a pseudotypic mixture of Mo+PyF101 M-MuLV and Mo+PyF101 MCF was inoculated into newborn NIH Swiss mice, they died of T-cell leukemia at times almost equivalent to those induced by wild-type M-MuLV. Tumor DNAs from Mo+PyF101 M-MuLV-Mo+PyF101 MCF-inoculated mice were examined by Southern blot analysis. The predominant forms of Mo+PyF101 MCF proviruses in these tumors contained added sequences in the U3 region of the LTR. The U3 regions of representative tumor-derived variant Mo+PyF101 MCFs were cloned by polymerase chain reaction amplification, and sequencing indicated that they had acquired an additional copy of the M-MuLV 75-bp tandem repeat in the enhancer region. NIH 3T3 cell lines infected with altered viruses were obtained from representative Mo+PyF101 M-MuLV-Mo+PyF101 MCF-induced tumors, and mice were inoculated with the recovered viruses. Leukemogenicity was approximately equivalent to that in the original Mo+PyF101 M-MuLV-Mo+PyF101 MCF viral stock. Southern blot analysis on the resulting tumors now predominantly revealed loss of the polyomavirus sequences. These results suggest that the suppressive effects of the PyF101 sequences on M-MuLV-induced disease and potentially on MCF propagation were overcome in two ways: by triplication of the M-MuLV direct repeats and by loss of the polyomavirus sequences.     

Molecular analysis of the envelope gene and long terminal repeat of Friend mink cell focus-inducing virus: implications for the functions of these sequences.

We sequenced the envelope (env) gene and 3' long terminal repeat of a Friend mink cell focus-inducing virus (F-MCFV). We also sequenced the gp70 coding regions for two cDNA clones of another F-MCFV. The deduced amino acid sequence of the env gene products of both F-MCFVs were compared to the corresponding sequences of other MCFVs and of ecotropic viruses. The env polypeptides of the different viruses showed long stretches of homology in the carboxy-terminal half of gp70 and in p15env ("constant region"). The amino-terminal half of gp70 was very similar in all MCFVs, but showed extensive variations relative to the ecotropic viruses ("differential region"). This differential region in all MCFVs is of endogeneous origin. We show evidence that this region carries determinants for ecotropic or polytropic host range. No indication could be found that the env gene products determine the histological type of disease caused by particular MCFVs. When the long terminal repeats of F-MCFV and Friend murine leukemia virus were compared with those of other viruses causing either lymphatic leukemia or erythroleukemia, several nucleotides were localized which might determine the histological type of disease caused by these viruses.