Tissue distribution and timing of appearance of polytropic envelope recombinants during infection with SL3-3 murine leukemia virus or its weakly pathogenic SL3DeltaMyb5 mutant.

A time course analysis was performed to identify the sites of formation and timing of appearance of polytropic recombinant viruses following infection of NIH/Swiss mice with the murine retrovirus SL3-3 murine leukemia virus (SL3) or with a weakly pathogenic mutant termed SL3DeltaMyb5. The results indicated that (i) polytropic recombinant viruses occur initially in the thymus of SL3-infected animals, (ii) the timing of appearance of polytropic recombinants in bone marrow is not consistent with their participation in the previously reported formation of transplantable tumor-forming cells at 3 to 4 week postinoculation, and (iii) the efficient generation of recombinant virus is correlated with efficient tumor induction.

The FeLV-945 LTR confers a replicative advantage dependent on the presence of a tandem triplication.

Feline leukemia virus (FeLV), like other naturally occurring retroviruses, is characterized by a high degree of genetic diversity. FeLV-945 is a natural isolate derived from non-B-cell non-T-cell lymphomas classified anatomically as multicentric. FeLV-945 exhibits a unique structural motif in the LTR composed of a 21-bp tandem triplication downstream of a single copy of enhancer. The unique FeLV-945 LTR is precisely conserved among eight independent multicentric lymphomas collected in a geographic cluster. Previous studies using reporter gene constructs predict that the FeLV-945 LTR would confer a replicative advantage on the virus that contains it, particularly in primitive hematopoietic cells. Such an advantage may account for the precise conservation of the unique LTR sequence. To test that prediction, a set of recombinant, infectious FeLVs was developed that are isogenic other than the presence of the FeLV-945 LTR or mutations of it. Replication assays show that the FeLV-945 LTR confers a distinct growth advantage in K-562, FEA, and 3201 cells and implicate the 21-bp triplication in that function. Replacement of two copies of the triplicated element with random sequence greatly diminished the replicative capacity, thus implicating the triplicated sequence itself in LTR function. The 21-bp triplication was shown to contain specific nuclear protein binding sites, which may account for the selective pressure to conserve the sequence.

Tumorigenic potential of a recombinant retrovirus containing sequences from Moloney murine leukemia virus and feline leukemia virus.

A recombinant retrovirus, termed MoFe2-MuLV, was constructed in which the U3 region of T-lymphomagenic Moloney murine leukemia virus (Mo-MuLV) was replaced by that of FeLV-945, a provirus of unique long terminal repeat (LTR) structure identified only in non-T-cell, non-B-cell lymphomas of the domestic cat. The LTR of FeLV-945 is unusual in that it contains only a single copy of the transcriptional enhancer followed 25 bp downstream by a 21-bp sequence in triplicate in tandem. Infectivity of MoFe2-MuLV was demonstrated in vitro in SC-1 cells and in vivo in neonatal NIH-Swiss mice. Tumors occurred in MoFe2-MuLV-infected animals following a latency period of 4 to 10 months (average, 6 months). The results of Southern blot analysis of the T-cell receptor beta locus demonstrated that all tumors were lymphomas of T-cell origin. MoFe2-MuLV LTRs were amplified by PCR from tumor DNA and were characterized by nucleotide sequence analysis. LTRs from the tumors that occurred with relatively shorter latency predominantly retained the original MoFe2-MuLV sequence intact and unaltered. Tumors that occurred with relatively longer latency contained LTRs that also retained the 21-bp sequence triplication characteristic of the original virus but had acquired various duplications of enhancer sequences. The repeated identification of enhancer duplications in late-appearing tumors suggests that the duplication affords a selective advantage, although apparently not in the efficient induction of T-cell lymphoma. Proto-oncogenes known to be targets of insertional mutagenesis in the majority of Mo-MuLV-induced tumors or in feline non-T-cell, non-B-cell lymphomas were shown not to be rearranged in any tumor examined. Mink cell focus-inducing (MCF) proviral DNA was readily detectable in some, but not all, tumors. The presence or absence of MCF did not correlate with the kinetics of tumor induction. These studies indicate that the single-enhancer, triplication-containing FeLV LTR, typical of non-T-cell, non-B-cell lymphomas in cats, is competent in the induction of T-cell lymphoma in mice. The findings suggest that the mechanism of MoFe2-MuLV-mediated lymphomagenesis may differ from that of Mo-MuLV-mediated disease, considering the possible involvement of novel oncogenes and the variable presence of MCF recombinants.

Cooperating events in lymphomagenesis mediated by feline leukemia virus.

Feline leukemia virus (FeLV)-mediated lymphomagenesis in the domestic cat has been examined as a model of lymphoid malignancy in a naturally outbreeding population. The pathogenesis of two distinct, naturally occurring types of FeLV-induced tumors has been investigated: (1) a thymic lymphoma of T-cell origin, typical of FeLV-induced lymphoma, and (2) an extrathymic, extranodal lymphoma of non-B non-T-cell origin. The genetic features of these tumors are clearly distinguishable, and include determinants encoded both by the virus and the host. Virally encoded determinants of pathogenesis include the long terminal repeat (LTR) and the envelope SU protein. Cellular determinants include the involvement of a set of proto-oncogenes, and other factors characteristic of the specific cell type of origin of the tumor. Functional studies are aimed at evaluating the action and interaction of these genetic determinants in the pathogenesis of lymphoma in an animal model system.

Genetic determinants of feline leukemia virus-induced multicentric lymphomas.

Three discrete forms of feline leukemia virus (FeLV)-associated lymphoma have been described clinically: (1) thymic, (2) alimentary, and (3) multicentric. The most common and best-characterized lymphomas are of T-cell origin, generally occurring in the thymus. These tumors typically contain mature T-cells, involve the activation of a distinctive set of proto-oncogenes, and contain FeLV proviruses whose long terminal repeat (LTR) sequences contain tandemly repeated enhancers. Previous studies of a small group of extrathymic, multicentric lymphomas implicated a different set of genetic determinants. The present study expands those observations by examining the lineage of origin, the involvement of proto-oncogenes, and the structure of LTR and env gene sequences in a set of 11 natural, extrathymic lymphomas of the multicentric type. A pattern of genetic events associated with FeLV-positive multicentric lymphomas emerges from this analysis that is clearly distinct from the pattern associated with thymic lymphomas. The tumors do not contain T-cells or B-cells, as evidenced by the germ line organization of TCR beta and IgH loci. Proto-oncogenes strongly implicated in T-cell lymphomagenesis are not involved in these tumors. Rather, a distinct set of proto-oncogenes may be involved. Most striking is the repeated occurrence of an FeLV isolate whose LTR and env gene bear unique sequence elements.

Function of a unique sequence motif in the long terminal repeat of feline leukemia virus isolated from an unusual set of naturally occurring tumors.

Feline leukemia virus (FeLV) proviruses have been characterized from naturally occurring non-B-cell, non-T-cell tumors occurring in the spleens of infected cats. These proviruses exhibit a unique sequence motif in the long terminal repeat (LTR), namely, a 21-bp tandem triplication beginning 25 bp downstream of the enhancer. The repeated finding of the triplication-containing LTR in non-B-cell, non-T-cell lymphomas of the spleen suggests that the unique LTR is an essential participant in the development of tumors of this particular phenotype. The nucleotide sequence of the triplication-containing LTR most closely resembles that of FeLV subgroup C. Studies performed to measure the ability of the triplication-containing LTR to modulate gene expression indicate that the 21-bp triplication provides transcriptional enhancer function to the LTR that contains it and that it substitutes at least in part for the duplication of the enhancer. The 21-bp triplication confers a bona fide enhancer function upon LTR-directed reporter gene expression; however, the possibility of a spacer function was not eliminated. The studies demonstrate further that the triplication-containing LTR acts preferentially in a cell-type-specific manner, i.e., it is 12-fold more active in K-562 cells than is an LTR lacking the triplication. A recombinant, infectious FeLV bearing the 21-bp triplication in U3 was constructed. Cells infected with the recombinant were shown to accumulate higher levels of viral RNA transcripts and virus particles in culture supernatants than did cells infected with the parental type. The triplication-containing LTR is implicated in the induction of tumors of a particular phenotype, perhaps through transcriptional regulation of the virus and/or adjacent cellular genes, in the appropriate target cell.

Coincident involvement of flvi-2, c-myc, and novel env genes in natural and experimental lymphosarcomas induced by feline leukemia virus.

The flvi-2 locus is a target of insertional mutagenesis in thymic lymphosarcomas induced by feline leukemia virus (FeLV). flvi-2 encodes the gene bmi-1, whose product is implicated as a myc-collaborator in the induction of B- and T-cell lymphoma. We have examined the involvement of flvi-2 and myc in natural and experimentally induced FeLV-positive feline lymphosarcomas which are heterogeneous in anatomical origin, geographic origin, and strain of FeLV involved. We further compared these findings with previous reports of novel FeLV env genes in the same tumors. The results show that proviral insertion at flvi-2 occurs commonly in natural and experimental feline thymic lymphosarcomas of diverse origins [52% overall], and that alterations in c-myc commonly accompany insertional mutagenesis of flvi-2 [54% overall]. However, 46% of tumors with flvi-2 insertions apparently lack involvement of c-myc. These observations support the hypothesis that interruption of flvi-2 may be an early event in a multistep cascade, one possibility for completion of which is activation of c-myc. Interruption of flvi-2 was not observed in nonthymic lymphosarcomas of alimentary or multicentric origin, although c-myc may be involved. A proportion of both thymic and nonthymic tumors have been shown previously to contain FeLV proviruses with recombinant or mutant env genes. Our findings strongly implicate the insertional mutagenesis of flvi-2, the activation of c-myc, and the emergence of novel env genes in FeLV-mediated lymphomagenesis, particularly in the induction of thymic lymphosarcoma. The data show that these events may overlap, but do not necessarily occur concurrently.

flvi-2, a target of retroviral insertional mutagenesis in feline thymic lymphosarcomas, encodes bmi-1.

LC-FeLV is a myc-containing strain of feline leukemia virus which induces thymic lymphosarcoma in the domestic cat with short latency. A locus in feline DNA, termed flvi-2, is commonly interrupted in naturally occurring and experimentally induced thymic lymphosarcomas containing LC-FeLV; thus, interruption of a gene encoded by flvi-2 may cooperate with the myc oncogene in the induction of T-cell tumors by LC-FeLV. Clones homologous to flvi-2 have been isolated from a normal human thymus cDNA library. Nucleotide sequence analysis of the cDNA clones demonstrates that flvi-2 encodes bmi-1, a gene previously identified as a target for MoMuLV integration and as a myc-collaborator in retrovirally-induced B-cell lymphomas in E mu-myc transgenic mice. In feline thymic lymphomas, retroviral integrations occur downstream of the gene, and result in enhanced expression of a bmi-1 transcript of normal size. These findings demonstrate the interruption of bmi-1 in natural as well as experimentally induced tumors, implicate the activation of bmi-1 in the induction of T-cell as well as B-cell lymphoma, and support the premise that bmi-1 functions as a myc collaborator.

Insertional mutagenesis of flvi-2 in tumors induced by infection with LC-FeLV, a myc-containing strain of feline leukemia virus.

LC-FeLV is a myc-containing strain of feline leukemia virus (FeLV) which exhibits only partial transforming activity in vitro and in vivo. LC-FeLV infection in kittens may induce, but does not necessarily induce, thymic lymphosarcoma in viremic animals after a short latency. These observations suggest that infection with LC-FeLV is not sufficient to induce complete transformation and that another genetic event(s) is required. One possibility for such an event is that the integrating provirus acts as an insertional mutagen and thereby disrupts the structure or function of another proto-oncogene. Using a strategy of transposon tagging, this possibility was examined in eight feline T-cell lymphosarcomas, including four induced by experimental infection with LC-FeLV, three induced by natural infection with FeLV, and one FeLV-negative tumor. The analysis demonstrated one locus, termed flvi-2, to be structurally altered in six of the tumors examined, including three induced by LC-FeLV and three in which no activated myc oncogene is apparent. Inverse polymerase chain reaction was used to demonstrate the presence and transcriptional orientation of proviruses integrated at flvi-2 in five of these tumors. The flvi-2 locus does not hybridize to cloned probes representing 21 previously identified proto-oncogenes or common domains of retroviral integration. Thus, the data suggest that interruption of the flvi-2 locus cooperates with the myc oncogene in the induction of T-cell lymphomas by LC-FeLV; indeed, the observations indicate that the insertional mutagenesis of flvi-2 plays a role in T-cell lymphomagenesis even in the absence of feline v-myc.

An unusual retrovirus-like sequence identified in human DNA.

The human genome contains many different types of endogenous proviruses and retrovirus-like elements. An unusual element of this kind has been isolated from human DNA on the basis of its relatedness to the integrase-coding domain of the pol gene of feline leukaemia virus (FeLV). The element, termed Hs5, is related to FeLV only over a short region of 81 nucleotides predicted to encode the carboxyl terminus of the FeLV integrase protein, p46pol. The region of relatedness between Hs5 and FeLV identifies a short conserved amino acid stretch which is shared among distantly related retroviruses. The conservation of this sequence, its position, and predicted secondary structure suggest that it may represent a conserved substrate binding site or active site of the integrase enzyme. Nucleotide sequence analysis of Hs5 reveals that it is not an intact retrovirus, but contains only the 3' terminus of pol and a defective env gene without apparent long terminal repeat; Hs5 is unusual among human endogenous retrovirus-like elements in this respect.

A simple method of examining retroviral species produced by infected cells.

A simple and rapid method to examine the retroviral species present in the culture supernatant of productively infected cells is described. The method involves purification of viral genomic RNA directly from the culture supernatant and examination of that RNA by Northern or dot blot analysis. The method provides qualitative and quantitative information about the RNA species present and is particularly valuable for the detection of genetic variants in the population.

Transforming potential of a myc-containing variant of feline leukemia virus in vitro in early-passage feline cells.

We studied a naturally occurring variant of feline leukemia virus (FeLV) in which the oncogene myc has substituted for a portion of the viral structural genes (myc-FeLV). myc-FeLV was rescued by replication in the presence of FeLV as helper, and its biological activity was examined in early-passage feline cells in vitro. Infection of leukocytes from peripheral blood, spleen, or thymus, or of kitten fibroblasts did not immortalize these cells or alter them morphologically. Northern blot (RNA blot) analysis of virion RNA prepared from the supernatant of infected cells demonstrated the 8.2-kilobase genome of FeLV, but did not demonstrate the 5.0-kilobase genome of myc-FeLV. Apparently, the myc-FeLV genome was lost in the absence of the selective pressure of transformation. In contrast, infection of embryonic fibroblasts with myc-FeLV(FeLV) rendered these cells capable of greatly increased, if not infinite, proliferative potential. The cells were morphologically altered compared with controls and were only loosely adherent to the substrate. The cells failed to proliferate in semisolid medium and did not form tumors when inoculated subcutaneously into athymic mice. Blot analyses demonstrated the presence and expression of integrated proviral DNAs of both FeLV and myc-FeLV in these cells. They appear, then, to represent cells partially transformed by infection with myc-FeLV(FeLV). The action of feline v-myc in early-passage cells in vitro was compared to that of avian v-myc.

Absence of alpha-glycerophosphate dehydrogenase in axenically grown Entamoeba histolytica.

We conclude that there is no evidence for the presence of alpha-glycerophosphate dehydrogenase among the cytoplasmic enzymes of Entamoeba histolytica, and that a contrary finding was probably caused by the action of a different amebal enzyme on unrecognized contamination in the lithium salt of the substrate used in that investigation.

The partial purification and characterization of adenosine kinase from Entamoeba histolytica.

Axenically grown Entamoeba histolytica was found to contain adenosine kinase. This organism lacks de novo purine biosynthetic pathways. Adenosine kinase provides the amoeba with a method for salvaging adenosine from ingested nucleosides or from degraded nucleotides. Adenosine kinase was purified 64-fold, by chromatography on Sephacryl S-200, DEAE-cellulose, and (C-8)-adenosine-agarose. The latter separated it from amebal adenylate kinase. Adenosine kinase has a molecular weight of 38,000 and requires glycerol for stability. It utilizes adenosine triphosphate to phosphorylate adenosine, and 7-deazaadenosine (tubercidin), but adenine 9-beta-D-arabinofuranoside (ara-A) is not detectably phosphorylated. It requires Mg++ as a cofactor.

Separation and characterization of two UTP-utilizing hexose phosphate uridylyltransferases from Entamoeba histolytica.

Two UTP-utilizing uridylyltransferases which react with both glucose 1-phosphate and galactose 1-phosphate were isolated from cell-free extracts of Entamoeba histolytica. The more specific of these enzymes, glucose-1-phosphate uridylyltransferase, acts preferentially on glucose 1-phosphate, having a maximum velocity 20-fold greater with this substrate than with galactose 1-phosphate. It was purified 200 fold with a 25% yield and has a molecular weight of 45 000. This enzyme requires a reducing agent for stability. The less specific transferase reacts with both hexose phosphates, having a maximum velocity of 1.35 times greater with galactose 1-phosphate. It was purified 1000 fold with a 20% yield, and has a molecular weight of 40 000. The common Leloir enzyme, UDP glucose-hexose-1-phosphate uridylytransferase (EC 2.7.7.12), was not found in this organism. To avoid confusion with the Leloir enzyme our experience suggests that the less specific enzyme, which is presently referred to in the literature as galactose-1-phosphate uridylyltransferase (EC 2.7.7.10), should be named UTP:hexose-1-phosphate uridylyltransferase (EC 2.7.7.?). The more specific enzyme (EC 2.7.7.9) should be more clearly named UTP:glucose-1-phosphate uridylyltransferase.