A single Glu(62)-to-Lys(62) mutation in the Mos residues of the R7Delta447Gag-tMos protein causes the mutant virus to induce brain lesions.

We previously reported that R7Delta447, a 2954-base-pair (bp) laboratory-generated Moloney murine sarcoma virus, induced subcutaneous tumors in about 14% of infected mice but did not induce brain lesions. We now report that R7Delta447K, a spontaneous mutant of R7Delta447, induced brain lesions as well as subcutaneous tumors in all injected mice. The genomes of the two viruses differ in a single base pair: the deduced Glu(62) of the Mos residue of the R7Delta447 Gag-tMos protein is changed to Lys(62). More R7Delta447 than R7Delta447K focus-forming units were detected in both NIH3T3 and mouse cerebral vascular endothelial (MCVE) cells. However, R7Delta447K transformed NIH3T3 and MCVE cells more acutely than did R7Delta447. A distinctive feature that distinguished the morphologic transformation of R7Delta447- and R7Delta447K-infected MCVE cells is the markedly prolonged spindle-shaped phase exhibited by R7Delta447-infected MCVE cells. In addition, R7Delta447K was more efficient in inducing the phosphorylation of ERK1/2 than R7Delta447 in both MCVE and NIH3T3 cells. Moreover morphologic transformation was inhibited, and levels of phosphorylated ERK1/2 were reduced when R7Delta447- or R7Delta447K-infected NIH3T3 or MCVE cells were grown in the presence of the MEK1/2-specific inhibitor PD98095. Thus, we have identified a key residue in the Gag-tMos protein that profoundly affects activation of the Mos/MEK/ERK pathway, virus and cell replication, morphologic transformation in vitro and pathogenicity in vivo.

Changes in the N- and C-terminal sequences of the murine R7 Gag-tMos protein affect brain lesion induction.

Our preliminary studies suggested that the novel gag-truncated mos (tmos) open reading frame (ORF) of R7, a spontaneous deletion mutant of Moloney murine sarcoma virus 124 (MoMuSV124), may be responsible for R7's unique ability to induce brain lesions in all R7-injected mice. However, when we replaced the gag-tmos ORF with either the MoMuSV124 or the homologous myeloproliferative sarcoma virus env-mos gene, we found that both recombinant viruses also induced brain lesions in all injected mice. Although these studies suggested that the critical determinants for brain lesion induction may reside in the tmos sequence common to all three viruses, they did not demonstrate if the N-terminus of Mos was dispensable for this activity. By inserting the FLAG sequence at the 3' end of the R7 gag-tmos ORF, we demonstrated that R7 does synthesize a Gag-tMos fusion protein. Using R7 gag deletion mutants with and without the FLAG sequence, we further demonstrated that (i) deletion of the entire gag sequence abolished R7's transforming activity; (ii) the ability of the virus to transform cultured NIH/3T3 cells was significantly reduced only when most of gag was deleted; (iii) the ability of the virus to induce brain lesions was inversely proportional to the extent of its gag deletions; and (iv) the insertion of FLAG at the Mos C-terminus did not reduce the in vitro transforming activity of the FLAG-tagged viruses but did reduce their ability to induce brain lesions. Thus, we have demonstrated that altering the N- or C-terminus of the R7 Gag-tMos fusion protein can affect disease manifestation.

Studies on the pathology, especially brain hemorrhage and angioendotheliomas, induced by two new mos-containing viruses.

Recombinant virus 7 (R7), a spontaneous deletion mutant of SV7, which is itself a molecular clone of Moloney murine sarcoma virus 124 (MoMuSV 124), induces brain lesions and tumors of the subcutaneous tissue and spleen in all infected mice. In contrast, SV7 only induces tumors of the spleen and subcutaneous tissues. One of the genetic differences between R7 and SV7 is that R7 encodes a Gag-Mos protein whereas SV7 encodes an Env-Mos protein. To investigate whether the novel R7 gag-mos oncogene is required for brain lesion induction, two viruses (SV7d1 and SVM1) were constructed in which the R7 gag-mos sequences and the adjacent 53 bp of the 5' noncoding sequence were replaced by either the SV7 or myeloproliferative sarcoma virus (MPSV) env-mos oncogenes, respectively. Like R7, SV7d1 and SVM1 induced brain lesions and tumors in the spleen and subcutaneous tissues. A prominent component of R7-, SV7d1-, and SVM1-induced tumors of the brain, subcutaneous tissues, and spleen was the presence of abnormally enlarged cells with eccentric nuclei lining vessels, scattered singly or in small clusters. Their size, localization to the luminal surface of distended vessels, and binding to Bandeiraea simplicifolia (BS-1) lectin, an endothelial cell (EC) marker, suggest that they are most likely transformed ECs. Our findings therefore indicate that the induction of brain lesions is not limited to the expression of the R7 Gag-Mos protein. However, our findings also indicate that expression of the different forms of the Mos protein results in differences in the relative abundance of ECs in brain angioendotheliomas and subcutaneous and spleen tumors induced by these viruses.

Studies on the pathology, especially brain lesions, induced by R7, a spontaneous mutant of Moloney murine sarcoma virus 124.

We have recently isolated R7, a spontaneous Moloney murine sarcoma virus (MoMuSV) 124 variant. Molecular cloning and sequence analysis showed that, relative to MoMuSV 124, R7 has an extra repeat in each enhancer and a truncated mos gene in frame with the truncated gag coding sequence. This report presents a detailed study on the pathology induced by R7. R7 induced not only sarcomas with well developed angiomatous components but also brain lesions. Brain lesions were observed in all less-than-48-hour-old BALB/c mice inoculated with greater than 2 x 10(5) R7 focus-forming units (FFUs). R7 was detected in all brains examined by day 9 after inoculation, and brain lesions were observed in two of four mice examined by day 14 after inoculation. Light microscopy of brains revealed that approximately 15% of the lesions were unenclosed blood pools of varying sizes containing red blood cells and inflammatory cells spreading into surrounding brain tissues. The remainder of the brain lesions had tumor cells. These lesions ranged from a few enlarged vascular endothelial cells intermixed with blood cells to large circumscribed lesions consisting of well developed tangled masses of vessels surrounded by blood pools. Activated astrocytes surrounded and infiltrated the tumors. In addition, the thymus of R7-infected mice regressed significantly and precipitously due to apoptosis (especially of cortical thymocytes) at the end stage of the disease.

R7, a spontaneous mutant of Moloney murine sarcoma virus 124 with three direct repeats and an in-frame truncated gag-mos gene, induces brain lesions.

We have isolated Recombinant 7 (R7), a spontaneous mutant of SV7, a molecular clone of MoMuSV124. Like SV7, R7 induces subcutaneous fibrosarcomas, spleen tumors, and mesentery tumors infiltrated by proliferating vessels lined by transformed endothelial cells. However, it also induces brain lesions. We have molecularly cloned and sequenced the R7 proviral DNA and shown that the R7 genome consists of 3401 bp. It has three direct repeats in each enhancer. Its coding sequence consists of only 176 bp of p15, 263 bp of p30, a 7-bp insertion, and 853 bp of an N-terminally truncated mos gene. From the sequence of R7 we have deduced that the truncated mos sequence is in-frame with all of the gag sequence and the 7-bp insertion. The incorporation of the 3' end of the p15 sequence further suggests that the R7 Gag-Mos is myristylated. We have also shown that the molecularly cloned R7 virus transformed NIH/3T3 fibroblasts about sevenfold better than the parental SV7. We have also confirmed that molecularly cloned R7 induces the same disease phenotype as that induced by the nonmolecularly cloned R7.

Establishment and characterization of conditionally immortalized astrocytes to study their interaction with ts1, a neuropathogenic mutant of Moloney murine leukemia virus.

The cytopathic infection of primary astrocytes with ts1, a neuroimmunopathogenic mutant of Moloney murine leukemia virus (MuLV), has been correlated to intracellular accumulation of viral precursor envelope protein gPr80env. To further study this specific virus-astrocyte interaction in a homogenous population, several immortal astrocyte lines were established from neonatal FVB/N mice using the temperature-sensitive SV40 tsA58 T antigen. These cells expressed glial fibrillary acidic protein, vimentin and T antigen; appeared nontransformed; were star-shape with long processes. They were susceptible to ts1 infection and suffered a cytopathic effect similar to that caused by ts1 infection of primary astrocytes. This cytopathic effect was characterized by growth inhibition, loss of cell processes and syncytium formation. Some cells also rounded up, formed mini cells and became detached from the culture dish. As in primary astrocytes, the processing of gPr80env in the immortalized astrocytes was inefficient. Since the envelope proteins interact with the ecotropic MuLV receptor both intracellularly and on the cell surface and since the receptor has been shown to be an arginine transporter, we attempted to determine the effect of ts1 on arginine uptake by these cells. Our results showed that in both immortalized and primary astrocytes, ts1 infection reduced the uptake of arginine more than did wild-type virus infection. Since arginine localizes predominantly in astrocytes in the CNS and has diverse functions, the decrease of arginine uptake in ts1-infected astrocytes may alter the metabolism of these cells, leading to impairment of their functions.

Long-term cultivation and productive infection of primary thymocyte cultures by a thymocytopathic murine retrovirus.

ts1, a mutant of MoMuLV, selectively kills T cells and neurons in the infected host resulting in neuroimmunodegeneration. In the infected thymus there is an early increase in mitosis of thymocytes followed by rapid death, suggesting that thymocyte death may be induced by viral mitogenic activation. Studies on thymocytes obtained from ts1-infected mice indicated that the ts1-induced depletion of thymocytes is mediated through activation-induced death by apoptosis. To further investigate the interaction between ts1 and thymocytes, we have established long-term primary murine thymocyte cultures by placing the thymocytes together with thymic remnants in culture medium containing IL-2 and IL-7. These thymocytes retained their immature phenotype and we susceptible to infection by ts1 and its parental wild-type MoMuLV. ts1-infected thymocytes proliferated initially at accelerated rate but subsequently produced more infectious virus and died much faster than control or MoMuLV-infected thymocytes. These in vitro studies to some extent reflect our in vivo studies reported previously.

Cell types in the central nervous system infected by murine retroviruses: implications for the mechanisms of neurodegeneration.

Retroviruses are an important cause of neurologic disease in humans but the pathogenic mechanisms are poorly understood. To delineate pathogenic mechanisms in any neurologic disease in humans is extremely difficult and will continue to rely on the use of animal models. This review presents several murine models to study the pathogenic mechanisms of neurodegenerative disease which manifest noninflammatory spongiform lesions in the CNS. The cell types in the CNS infected by these murine retroviruses and their role in disease induction are discussed.

Murine retrovirus-induced depletion of T cells is mediated through activation-induced death by apoptosis.

ts1, a mutant of Moloney murine leukemia virus, causes neurologic disorders and acute immunodeficiency associated with the destruction of thymocytes and helper T cells. In this study, we examined whether apoptosis was involved in ts1-induced killings of T cells. Neonatal mice were inoculated with ts1, and 20 to 23 days postinoculation, when cytopathic effects on T cells normally appear, thymocytes and splenic lymphocytes were isolated and examined. Our results showed that several features of apoptosis were present in ts1-infected thymocytes and splenic lymphocytes. Apoptotic fragmented DNA, condensation of the chromatin, and enhanced cell death after stimulation with mitogens which was preventable with protein synthesis inhibitors, all of which are common features of apoptotic cell death, were observed in ts1-infected cells. Several other viruses, including human immunodeficiency virus, have been shown to cause apoptotic death of T cells. Here we show for the first time that a murine retrovirus which also induces immunodeficiency can cause apoptotic T-cell death. Future studies with this murine retrovirus may provide important results to help us better understand the mechanisms of retrovirus-induced apoptosis of T cells.

Characterization of a continuous lymphocyte cell line derived from BALB/c mice inoculated with a recombinant Moloney murine leukemia virus-TB.

Neonatal BALB/c mice were inoculated (ip) with a recombinant Moloney murine leukemia virus-TB. Majority of the inoculated mice developed lymphoma within 5-7 months post infection. The cells from splenic lymphomas were cultured and 3 continuous cell lines (GP1, GP2 and GP3) developed. GP1 was single cell cloned and characterized. Based on Thy 1.2 (98.4%) phenotypic marker, the cell line was categorized as T cell line. The percent positivity for different cell surface markers on analysis with FACS was 98.4, 4.8, 5.5, 2.2, 1.8, 1.2 and 9.5 for Thy 1.2, mu, L3T4, Lyt2, Ia, IL2R and PNA receptor, respectively. A total of 16.5% GP1 cells was also positive for Moloney murine leukemia virus envelope protein (gp 70). Incomplete retrovirus like particles were demonstrated in the cytoplasm of GP1 cells by electron microscopy. The cell line on inoculation(ip) in neonatal BALB/c mice produced lymphomic lesions in almost all the vital organs of the mice.

Virulence in lymphomagenesis is modulated by alterations in proteins binding to CORE and GRE-LVa elements of the MoMuLV enhancer.

Changing nucleotide 3 of the CORE consensus from T to C has no effect on the binding of a 42 kDa protein, which has little affinity for the CCAAT binding site. Changing nucleotide 6 of the CORE consensus from T to C significantly reduces binding of the 42 kDa protein. Studies on the pathology induced by various MoMuLV mutants with CORE mutations showed that the CORE elements are important in modulating virulence in T-cell lymphomagenesis in BALB/c mice. However, disease specificity appears to be influenced as much by the host as it is by the virus. LVa is preferentially bound to the GRE-LVa sites. Deletion of nucleotide 9 and changing nucleotides 10 and 11 from GG to AA in the GRE-LVa element does not disrupt binding of LVa. Changing nucleotide 10 from G to A and nucleotide 13 from A to T in the GRE-LVa element does not disrupt binding of GR but allows binding of a novel protein which displaces or abolishes binding of LVa. These five nucleotide changes alone do not alter disease specificity and had a minimal effect on virulence in T-cell lymphomagenesis in BALB/c mice. Additionally, changing nucleotide 3 in CORE(a) and nucleotide 6 in CORE(b) does not alter disease specificity but has a small additional effect on virulence in T-cell lymphomagenesis.

SV7, a molecular clone of Moloney murine sarcoma virus 349, transforms vascular endothelial cells.

SV7, a progeny of Moloney murine sarcoma virus 349 cells, was molecularly cloned. SV7 induced sarcomas consisting of vascular and fibrous components. The large blood-filled vascular dilatations appeared grossly as dark red spots in the tumors and constituted up to 50% of the tumor volume. These vascular structures, ranging from small capillaries to cavernous vascular dilatations, were lined by one to several layers of neoplastic endothelial cells. Thick papillary outgrowths of the neoplastic endothelium extended into and often occluded the vessel lumens. The fibrous component consisted mostly of spindle cells and granulocytes, which provided the stroma for the vascular structures. The vascular and fibrous components appeared to have arisen independently. Lymphopenia accompanied by myeloid metaplasia was observed in the spleen of both SV7- and myeloproliferative sarcoma virus (MPSV)-infected mice. The blood of SV7-infected mice had a much higher level of circulating granulocytes than did that of MPSV-infected mice. The latter manifested a more advanced myeloid metaplasia, characterized by aggregates of myelomonocytic blast cells in the spleen.

Construction and characterization of expression systems for the env gene of ts1, a mutant of Moloney murine leukemia virus-TB.

A temperature-sensitive mutant of the Moloney murine leukemia virus-TB, ts1, causes hindlimb paralysis and immunodeficiency in mice. At the restrictive temperature, the envelope precursor polyprotein, gPr80env, is inefficiently processed intracellularly, and this is associated with the neurovirulence of ts1. To test the hypothesis that expression of the envelope proteins of ts1 alone without infectious virus production can induce paralysis, it is necessary to use either transmissible retroviral expression vectors or microinjection of eukaryotic gene expression plasmid to introduce the env gene of ts1 into germlines of mice. In this study, we have constructed three retrovirus vectors and three gene expression plasmids, all of which contain the env gene of ts1. By comparing the different expression systems, we found that one construct, pts1-env(F) can express the envelope proteins at a level comparable to the level expressed in ts1-infected cells. Furthermore, the expressed envelope proteins of pts1-env(F)-transfected cells possess the phenotypes of the proteins expressed by the env gene of ts1.

The Moloney murine leukemia virus enhancer and its flanking sequences collaborate to determine virulence in T-cell lymphomagenesis.

A panel of recombinant virus genomes was constructed by exchanging homologous genome fragments between the potent T-cell lymphoma inducer Moloney murine leukemia virus (MoMuLV) and its closely related but significantly less virulent relative MoMuLV-TB. Testing of these recombinant viruses in BALB/c mice established that only nucleotide changes within the Clal(-590)-Kpnl(36) fragment altered virulence. Fine analysis of this fragment showed that while mutations within the enhancer of MoMuLV-TB attenuated the latency period most, mutations within the MoMuLV-TB fragments flanking the enhancer also helped reduce the virulence of MoMuLV. The present study also suggests that the small difference in the relative number of lymphomas that developed primarily in the spleens of MoMuLV- or MoMuLV-TB-infected mice may correlate with nucleotide differences between the Clal-Kpnl fragments of the two viruses. However, the significantly greater proportion of premature death observed in MoMuLV-TB-relative to MoMuLV-infected mice could not be correlated with nucleotide differences in a specific genome fragment.

Site-directed mutagenesis of the codon for Ile-25 in gPr80env alters the neurovirulence of ts1, a mutant of Moloney murine leukemia virus TB.

ts1, a spontaneous temperature-sensitive mutant of Moloney murine leukemia virus TB, causes hind-limb paralysis in mice. A Val-25----Ile substitution in gPr80env is responsible for temperature sensitivity, inefficient processing of gPr80env, and neurovirulence. In this study, the Ile-25 in gPr80env was replaced with Thr, Ala, Leu, Gly, and Glu by site-directed mutagenesis of the codon for Ile-25 to generate a new set of mutant viruses, i.e., ts1-T, -A, -L, -G, and -E, respectively. The phenotypic characteristics of these mutant viruses differed from those of ts1. For each mutant, the degree of temperature sensitivity was correlated with the degree of inefficient processing of gPr80env, and the following rank order was observed for both parameters: ts1-E greater than ts1-G greater than ts1-L greater than ts1-A greater than ts1 greater than ts1-T. In FVB/N mice, mutant viruses of low and intermediate temperature sensitivity and inefficiency in processing of gPr80env were neurovirulent and consistently caused mutant-specific disease profiles: ts1-T caused severe whole-body tremor, ts1-A generally caused hind-limb paralysis, and ts1-L generally caused a delayed-onset paraparesis. By 150 days postinfection, FVB/N mice that were infected with ts1-G and -E, mutants of high temperature sensitivity and inefficiency in processing of gPr80env, had lymphoid leukemia instead of a neurological disease. These results suggest that the dynamics of gPr80env processing are important in determining the neurovirulent phenotype in vivo.

Moloney murine sarcoma virus 349 induces Kaposi's sarcomalike lesions in Balb/c mice.

Moloney murine sarcoma virus (MoMuSV349) is produced by MuSV349 cells in at least eight-fold excess over the replication-competent helper virus. Less than 48-hours-old Balb/c mice inoculated intraperitoneally with supernatant from MuSV349 cells containing approximately 10(4) MuSV349 infectious units developed clinical symptoms, including severe generalized wasting, 15 to 20 days after inoculation. These infected mice became moribund 35 to 45 days after inoculation. Gross examination of the bodies revealed the presence of cutaneous and subcutaneous 0.2-cm to 1.5-cm macules, plaques, or nodules located predominantly on the ventral abdomen and legs. Nodules also were found in the spleen, liver, ovaries, testes, meninges, nerves, and skin. The nodules were semisoft, cystic, or solid and some expressed variable amounts of blood. Histologic examination of the macules, plaques, and nodules showed spindlelike cells intermingled with tortous, jagged vascular channels lined by plump and normal endothelial cells and unlined slitlike spaces filled with erythrocytes. These angiomatous lesions were infiltrated extensively with neutrophils, lymphocytes, macrophages, and some plasma cells. In some cases the lesions also included foci of densely packed eosinophils. These angiomatous lesions are clearly distinguishable from the fibrosarcomas induced by the myeloproliferative sarcoma virus (MPSV), and resemble the sarcomas induced in mice by Gz-MSV and Balb MSV, the sarcomas induced in rats by MPSV and Ha-MSV, and the acute generalized form of Kaposi's sarcoma (KS) associated with acquired immune deficiency syndrome (AIDS) in humans. Electron microscopy also revealed the presence of numerous extracellular type C virions and virions budding from the plasma membrane of endothelial and spindlelike cells. Erythrophagocytosis by the endothelial and spindlelike cells was demonstrated by light and electron microscopy. The widely disseminated lesions appear to have developed simultaneously as a consequence of viremia rather than metastasis.

A Val-25-to-Ile substitution in the envelope precursor polyprotein, gPr80env, is responsible for the temperature sensitivity, inefficient processing of gPr80env, and neurovirulence of ts1, a mutant of Moloney murine leukemia virus TB.

ts1 is a neurovirulent spontaneous temperature-sensitive mutant of Moloney murine leukemia virus TB which causes hindlimb paralysis in mice. Previously, it had been shown that the temperature-sensitive defect resided in the env gene. At the restrictive temperature, the envelope precursor polyprotein, gPr80env, is inefficiently processed intracellularly into two cleavage products, gp70 and Prp15E. This inefficient processing of gPr80env is correlated with neurovirulence. In this study, it was shown that a single amino acid substitution, Val-25----Ile in gPr80env, is responsible for the temperature sensitivity, inefficient processing of gPr80env at the restrictive temperature, and neurovirulence of ts1. At the restrictive temperature, a steady-state level of nonprocessed, endoglycosidase H-sensitive gPr80env remained in the endoplasmic reticulum of cells infected by ts1, but no endoglycosidase H-resistant gPr80env and only trace amounts of gp70 were detected in the infected cells. Since the host cell-encoded processing protease resides in the cis cisternae of the Golgi apparatus, inefficient processing of gPr80env at the restrictive temperature is most likely due to inefficient transport of gPr80env from the endoplasmic reticulum to the cis cisternae of the Golgi apparatus rather than due to misfolded gPr80env being a poor substrate for the processing protease at the restrictive temperature.

A single mutation in one of the CORE elements of Moloney murine leukemia virus reduced binding of a 42-kDa T lymphoma cell nuclear factor but did not affect lymphomagenesis.

The genetic determinant responsible for virulence in Moloney murine leukemia virus (MoMuLV) induced T-cell lymphomagenesis has recently been mapped [J Virol 63:471-480, 1989] by homologous genomic fragment exchange between MoMuLV and MoMuLV-TB to the Clal/Xbal at the 3' end of the genome. This region of MoMuLV and MoMuLV-TB differs in 11 nucleotides. Of these 11 nucleotide differences, 9 are distributed within the two CORE, the two distal NF1, and the two GRE/LVa elements of the enhancer. Since both the CORE binding sites of MoMuLV-TB are mutated with respect to those of MoMuLV, we compared nuclear proteins of a thymus-bone marrow cell line and a T-lymphoma cell line (EMT), which bind to the wild-type and mutant CORE binding sites. Using both the bandshift assay and southwestern analysis with labeled synthetic deoxyoligonucleotides, we showed that a 42-kDa protein from TB and EMT cells bound specifically to the MoMuLV CORE element. The T----C transversion of nucleotide 6 of the CORE consensus, TGTGGT/CTAA, significantly reduced binding of the 42-kDa TB and EMT cell factors. However, the transversion of nucleotide 3 from T----C had little effect on the binding of the 42-kDa protein to the CORE element. In addition, the 42-kDa protein bound weakly to the CCAAT element of MoMuLV. A recombinant virus, NwtTB-6, was generated by introducing the two CORE mutations of MoMuLV-TB into the MoMuLV genome. Although the latency period of NwtTB-6 in the induction of lymphoma was not significantly different from that of MoMuLV, preliminary findings suggest that the lymphoma induced by NwtTB-6 may be more widely distributed.

ts1, a mutant of Moloney murine leukemia virus-TB, causes both immunodeficiency and neurologic disorders in BALB/c mice.

BALB/c mice infected with ts1, a mutant of Moloney murine leukemia virus-TB, develop generalized body wasting, profound neurologic disorders, severe thymic atrophy and lymphopenia due to destruction of T lymphocytes and drastic immunodeficiency. ts1 was found not only able to infect T lymphocytes but also to impair their function. In addition, ts1 also infects and induces syncyntia formation in macrophages. The genetic determinant(s) responsible for ts1's ability to induce immunodeficiency has been localized to the env gene.

The reduced virulence of the thymotropic Moloney murine leukemia virus derivative MoMuLV-TB is mapped to 11 mutations within the U3 region of the long terminal repeat.

Chimeric constructs were generated by exchanging genomic fragments between the potent T-cell lymphoma inducer Moloney murine leukemia virus (MoMuLV) and its derivative MoMuLV-TB, which induces T-cell lymphoma after a relatively longer latent period. Analysis of the T-cell lymphoma-inducing potential of the hybrid viruses that were obtained localized the primary determinant critical to efficient T-cell lymphoma induction to the MoMuLV ClaI-XbaI fragment which comprises 48 nucleotides (nt) of p15E, p2E, the 3'-noncoding sequence, and 298 nt of U3. The 438-base-pair ClaI-XbaI fragments of MoMuLV and MoMuLV-TB differed in only 11 nt. Nine mutations were found within the enhancer. These mutations occurred within the two CORE, the two GRE-LVa, and two of the four NF1 nuclear factor-binding motifs. MoMuLV-TB replicated better than MoMuLV in thymus-bone marrow (TB) cells, a cultured cell line of lymphoid origin. In addition, MoMuLV-TB and NwtTB-2, a recombinant virus with the ClaI-SmaI fragment of MoMuLV-TB in a MoMuLV background, replicated in thymocytes as efficiently as did MoMuLV or TBNwt-2, the reciprocal recombinant virus, with the ClaI-SmaI fragment of MoMuLV in a MoMuLV-TB background. Like NwtTB-4, a recombinant virus with the ClaI-XbaI fragment of MoMuLV-TB in a MoMuLV background, NwtTB-2 induced lymphoma after a long latent period. The finding given above suggests that thymotropism is not the only factor that determines the T-cell lymphoma-inducing potential of MoMuLV. It appears likely that mutations in one or more of the MoMuLV-TB nuclear factor-binding motifs may have altered the interaction of the enhancer with specific nuclear factors; this, in turn, may affect the T-cell lymphoma-inducing potential of MoMuLV-TB.