The prostate cancer-associated human retrovirus XMRV lacks direct transforming activity but can induce low rates of transformation in cultured cells.

The human retrovirus XMRV (xenotropic murine leukemia virus-related virus) is associated with prostate cancer, but a causal relationship has not been established. Here, we have used cultured fibroblast and epithelial cell lines to test the hypothesis that XMRV might have direct transforming activity but found only rare transformation events, suggestive of indirect transformation, even when the target cells expressed the human Xpr1 cell entry receptor for XMRV. Characterization of cells from three transformed foci showed that all were infected with and produced XMRV, and one produced a highly active transforming virus, presumably generated by recombination between XMRV and host cell nucleic acids. Given the sequence similarity of XMRV to mink cell focus-forming (MCF) viruses and the enhanced leukemogenic activity of the latter, we tested XMRV for related MCF-like cytopathic activities in cultured mink cells but found none. These results indicate that XMRV has no direct transforming activity but can activate endogenous oncogenes, resulting in cell transformation. As part of these experiments, we show that XMRV can infect and be produced at a high titer from human HT-1080 fibrosarcoma cells that express TRIM5alpha (Ref1), showing that XMRV is resistant to TRIM5alpha restriction. In addition, XMRV poorly infects NIH 3T3 cells expressing human Xpr1 but relatively efficiently infects BALB 3T3 cells expressing human Xpr1, showing that XMRV is a B-tropic virus and that its infectivity is regulated by the Fv1 mouse locus.

Mink epithelial cell killing by pathogenic murine leukemia viruses involves endoplasmic reticulum stress.

We previously demonstrated that mink cells undergo apoptosis after MCF13 murine leukemia virus (MLV) infection. In this study, we observed that virus-infected mink epithelial cells had significantly larger amounts of steady-state levels of MCF13 MLV envelope precursor protein (gPr80(env)) than did Mus dunni fibroblasts, which are resistant to virus-induced cytopathicity. Infection of mink cells with the noncytopathic NZB-9 MLV did not result in the accumulation of gPr80(env). MCF13 MLV infection of mink cells produced low cell surface expression of envelope glycoprotein and less efficient spread of infectious virus. Western blot analysis of mink epithelial cells infected with MCF13 MLV showed an increase in GRP78/BiP, which was not observed for either mink cells infected with NZB-9 MLV or M. dunni fibroblasts infected with MCF13 MLV. MCF13 MLV infection of mink cells also resulted in a significant upregulation of CHOP/GADD153. These results indicate that the accumulation of MCF13 MLV gPr80(env) triggers endoplasmic reticulum stress, which may mediate apoptosis in mink epithelial cells.

Differential cell killing by lymphomagenic murine leukemia viruses occurs independently of p53 activation and mitochondrial damage.

Upon inoculation into AKR mice, mink cell focus-forming murine leukemia virus (MCF MLV) accelerates thymic lymphoma formation. During the preleukemic phase of disease, we observed the induction of apoptosis in thymic lymphocytes. A similar induction of apoptosis was observed for cultured mink epithelial cells after MCF13 MLV infection. In this study, the relevance of viral pathogenicity to cell killing was determined by testing the susceptibility of various cell types from different species to lymphomagenic MLVs. We observed that the cytopathic effect of lymphomagenic MLVs was restricted to mink cells. Southern blot analysis of MLV-infected cells revealed an accumulation of the linear form of unintegrated viral DNA, particularly in mink cells after MCF13 MLV infection. Thus, a strong correlation was observed between viral superinfection, which results in the accumulation of high levels of unintegrated viral DNA, and cell killing. Immunoblot analysis for MCF13 MLV-infected mink epithelial cells did not show a significant change in total p53 levels or its phosphorylated form at Ser-15 compared with that in mock-treated cells. Moreover, a time course analysis for mink epithelial cells infected with MCF13 MLV did not reveal mitochondrial depolarization or a significant change in Bax levels. These results demonstrate that MCF13 MLV induces apoptosis preferentially in cells in which superinfection occurs, and the mechanism involved is independent of p53 activation and mitochondrial damage.

A virus-virus interaction circumvents the virus receptor requirement for infection by pathogenic retroviruses.

During ongoing C-type retrovirus infection, the probability of leukemia caused by insertional gene activation is markedly increased by the emergence of recombinant retroviruses that repeatedly infect host cells. The murine mink cell focus-inducing (MCF) viruses with this property have acquired characteristic changes in the N-terminal domain of their envelope glycoprotein that specify binding to a different receptor than the parental ecotropic virus. In this report, we show that MCF virus infection occurs through binding to this receptor (termed Syg1) and, remarkably, by a second mechanism that does not utilize the Syg1 receptor. By the latter route, the N-terminal domain of the ecotropic virus glycoprotein expressed on the cell surface in a complex with its receptor activates the fusion mechanism of the MCF virus in trans. The rate of MCF virus spread through a population of permissive human cells was increased by establishment of trans activation, indicating that Syg1 receptor-dependent and -independent pathways function in parallel. Also, trans activation shortened the interval between initial infection and onset of cell-cell fusion associated with repeated infection of the same cell. Our findings indicate that pathogenic retrovirus infection may be initiated by virus binding to cell receptors or to the virus envelope glycoprotein of other viruses expressed on the cell surface. Also, they support a broader principle: that cooperative virus-virus interactions, as well as virus-host interactions, shape the composition and properties of the retrovirus quasispecies.

Duplication of U3 sequences in the long terminal repeat of mink cell focus-inducing viruses generates redundancies of transcription factor binding sites important for the induction of thymomas.

The ability of mink cell focus-inducing (MCF) viruses to induce thymomas is determined, in part, by transcriptional enhancers in the U3 region of their long terminal repeats (LTRs). To elucidate sequence motifs important for enhancer function in vivo, we injected newborn mice with MCF 1dr (supF), a weakly pathogenic, molecularly tagged (supF) MCF virus containing only one copy of a sequence that is present as two copies (known as the directly repeated [DR] sequence) in the U3 region of MCF 247 and analyzed LTRs from supF-tagged proviruses in two resulting thymomas. Tagged proviruses integrated upstream and in the reverse transcriptional orientation relative to c-myc provided the focus of our studies. These proviruses are thought to contribute to thymoma induction by enhancer-mediated deregulation of c-myc expression. The U3 region in a tagged LTR in one thymoma was cloned and sequenced. Relative to MCF 1dr (supF), the cloned U3 region contained an insertion of 140 bp derived predominantly from the DR sequence of the injected virus. The inserted sequence contains predicted binding sites for transcription factors known to regulate the U3 regions of various murine leukemia viruses. Similar constellations of binding sites were duplicated in two proviral LTRs integrated upstream from c-myc in a second thymoma. We replaced the U3 sequences in an infectious molecular clone of MCF 247 with the cloned proviral U3 sequences from the first thymoma and generated an infectious chimeric virus, MCF ProEn. When injected into neonatal AKR mice, MCF ProEn was more pathogenic than the parental virus, MCF 1dr (supF), as evidenced by the more rapid onset and higher incidence of thymomas. Molecular analyses of the resultant thymomas indicated that the U3 region of MCF ProEn was genetically stable. These data suggest that the arrangement and/or redundancy of transcription factor binding sites generated by specific U3 sequence duplications are important to the biological events mediated by MCF proviruses integrated near c-myc that contribute to transformation.

Ikaros, a lymphoid-cell-specific transcription factor, contributes to the leukemogenic phenotype of a mink cell focus-inducing murine leukemia virus.

Mink cell focus-inducing (MCF) viruses induce T-cell lymphomas in AKR/J strain mice. MCF 247, the prototype of this group of nonacute murine leukemia viruses, transforms thymocytes, in part, by insertional mutagenesis and enhancer-mediated dysregulation of cellular proto-oncogenes. The unique 3' (U3) regions in the long terminal repeats of other murine leukemia viruses contain transcription factor binding sites known to be important for enhancer function and for the induction of T-cell lymphomas. Although transcription factor binding sites important for the biological properties of MCF 247 have not been identified, pathogenesis studies from our laboratory suggested to us that binding sites for Ikaros, a lymphoid-cell-restricted transcriptional regulator, affect the biological properties of MCF 247. In this report, we demonstrate that Ikaros binds to predicted sites in U3 sequences of MCF 247 and that site-directed mutations in these sites greatly diminish this binding in vitro. Consistent with these findings, ectopic expression of Ikaros in murine cells that do not normally express this protein significantly increases transcription from the viral promoter in transient gene expression assays. Moreover, site-directed mutations in specific Ikaros-binding sites reduce this activity in T-cell lines that express Ikaros endogenously. To determine whether the Ikaros-binding sites are functional in vivo, we inoculated newborn mice with a variant MCF virus containing a mutant Ikaros-binding site. The variant virus replicated in thymocytes less efficiently and induced lymphomas with a delayed onset compared to the wild-type virus. These data are consistent with the hypothesis that the Ikaros-binding sites in the U3 region of MCF 247 are functional and cooperate with other DNA elements for optimal enhancer function in vivo.

Role of the LTR region between the enhancer and promoter in mink cell focus-forming murine leukemia virus pathogenesis.

Long terminal repeat (LTR) sequences are important determinants of mink cell focus-forming (MCF) murine leukemia virus pathogenesis. These sequences include the enhancer and sequences between the enhancer and promoter (DEN). In a previous study we showed that a virus missing the DEN region in its LTR was severely attenuated in its ability to induce thymic lymphoma. In this study we observed that a virus with an LTR consisting of DEN but no enhancer sequences was pathogenic. We compared the pathogenicity of this DEN virus with other LTR mutant MCF13 viruses that contained a single enhancer (1R) or a single enhancer plus DEN (1R + DEN). All LTR mutant viruses generated thymic lymphoma, however, at a much lower incidence and with a longer latency compared with wild-type (WT) MCF13 virus. DEN virus replication in the thymus was the lowest compared with the 1R and 1R + DEN viruses. Viral replication in a different thymic subpopulation could not explain the decreased pathogenicity of the LTR mutant viruses compared with WT virus. However, lower levels of mutant virus replication in the thymus compared with WT during the preleukemic period may contribute to the attenuation of pathogenicity. The phenotype of tumors induced by the mutant viruses was similar and differed from tumors induced by WT virus by the presence of CD3(-)CD4(-)CD8(-) cells. Analysis of LTR sequences of infectious virus rescued from tumors induced by the 1R and 1R + DEN viruses showed that amplification of enhancer sequences had occurred during tumor development. The lack of DEN virus expression by tumor cells led us to propose that DEN sequences may play a role at an early step in tumorigenesis.

Generation of mink cell focus-inducing retroviruses: a model for understanding how viral-viral and viral-cellular interactions can result in biological consequences.

Using neoplastic cell lines as substrates for vaccine development could inadvertently result in viral-viral or viral-cellular interactions whose biological consequences are unclear. In this review, the generation of mink cell focus-inducing (MCF) retroviruses in the mouse is discussed as a model for understanding how viral-viral and viral-cellular interactions can result in the generation of new retroviruses with pathological consequences.

Lymphomas and high-level expression of murine leukemia viruses in CFW mice.

Historically, Swiss Webster mice of the CFW subline, both inbred and random-bred stocks, have been considered to have a low spontaneous occurrence of hematopoietic system tumors, and previous reports of infectious expression of murine leukemia viruses (MuLVs) have been rare and unremarkable. In marked contrast, in the present study of CFW mice from one source observed by two laboratories over a 2-year period, nearly 60% developed tumors, 85% of which were lymphomas, the majority of B-cell origin. All tumors tested expressed ecotropic MuLVs, and most expressed mink cell focus-inducing (MCF) MuLVs. Among normal mice of weanling to advanced age, over one-half were positive for ecotropic virus in tail or lymphoid tissues, and MCF virus was frequently present in lymphoid tissue, less often in tail. Patterns of ecotropic proviral integration indicated that natural infection occurred by both genetic and exogenous routes. Lymphomas were induced in NIH Swiss mice infected as neonates with tissue culture-propagated MuLVs isolated from normal and tumor tissue of CFW mice.

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.

Genetic and immunological parameters governing in vivo susceptibility/resistance to retrovirally induced murine malignant histiocytosis.

Malignant histiocytosis sarcoma virus (MHSV) arose as a recombinant of c-Harvey-ras murine sarcoma virus (Ha-MuSV) and Friend mink cell focus-forming virus (F-MCFV). It is a defective acute transforming retrovirus that, along with Friend murine leukemia helper virus (F-MuLV), induces malignant histiocytosis (MH) in susceptible adult mice. We have assessed the in vivo susceptibility to MHSV in inbred homozygous, F1 hybrid, congenic, and recombinant inbred (RI) mice. We have shown that: (1) in vivo resistance to MHSV is multigenic, regulated by MHC and non-MHC genes in a different fashion than with F-MCFV, F-MuLV, or Ha-MuSV; (2) using BXD RI mice, the resistance phenotype is linked with 95.8% probability to two linked loci, Pmv-9 and Iapls3-14, on chromosome 13 (homologous to the area of human chromosome 5 for which a chromosomal break point at position 5q35 is associated with human MH); and (3) CD4+ T cells are critical for MHSV resistance.

Neurologic disease induced by polytropic murine retroviruses: neurovirulence determined by efficiency of spread to microglial cells.

Several murine leukemia viruses (MuLV) induce neurologic disease in susceptible mice. To identify features of central nervous system (CNS) infection that correlate with neurovirulence, we compared two neurovirulent MuLV, Fr98 and Fr98/SE, with a nonneurovirulent MuLV, Fr54. All three viruses utilize the polytropic receptor and are coisogenic, each containing a different envelope gene within a common genetic background. Both Fr98 and Fr98/SE induce a clinical neurologic disease characterized by hyperexcitability and ataxia yet differ in incubation period, 16 to 30 and 30 to 60 days, respectively. Fr54 infects the CNS but fails to induce clinical signs of neurologic disease. In this study, we compared the histopathology, regional virus distribution, and cell tropism in the brain, as well as the relative CNS viral burdens. All three viruses induced similar histopathologic effects, characterized by intense reactive astrogliosis and microglial activation associated with minimal vacuolar degeneration. The infected target cells for each virus consisted primarily of endothelial and microglial cells, with rare oligodendrocytes. Infection localized predominantly in white matter tracts of the cerebellum, internal capsule, and corpus callosum. The only feature that correlated with relative neurovirulence was viral burden as measured by both viral CA protein expression in cerebellar homogenates and quantification of infected cells. Interestingly, Fr54 (nonneurovirulent) and Fr98/SE (slow disease) had similar viral burdens at 3 weeks postinoculation, suggesting that they entered the brain with comparable efficiencies. However, spread of Fr98/SE within the brain thereafter exceeded that of Fr54, reaching levels of viral burden comparable to that seen for Fr98 (rapid disease) at 3 weeks. These results suggest that the determinants of neurovirulence in the envelope gene may influence the efficiency of virus spread within the brain and that a critical number of infected cells may be required for induction of clinical neurologic disease.

Identification of nucleotide sequences that regulate transcription of the MCF13 murine leukemia virus long terminal repeat in activated T cells.

The region downstream of the enhancer (DEN) of the long terminal repeat of the mink cell focus-forming murine leukemia virus is important for viral pathogenicity. Another important activity of DEN is its control of transcription in activated T cells, and we have determined that an NF-kappaB site is critical for this activity.

Leukemogenesis by Moloney murine leukemia virus: a multistep process.

Moloney murine leukemia virus is a prototypical simple retrovirus that has been an extremely useful model for leukemogenesis. Important steps in leukemogenesis include proviral activation of cellular proto-oncogenes, generation of mink cell focus-inducing recombinants, and early (preleukemic) virus-induced changes in hematopoiesis.

Cytotoxic T lymphocyte responses to the envelope proteins of endogenous ecotropic and mink cytopathic focus-forming murine leukemia viruses in H-2b mice.

To study the possible role of immune selection in the in vivo generation of pathogenic recombinant murine leukemia viruses (MuLV), we have constructed recombinant vaccinia viruses (rVV) expressing the envelope genes of three MuLV: AKR623, MCF247, and MCF13. rVV expressing either AKR623 or MCF247 env could prime H-2b mice for anti-AKR/Gross virus CTL responses, and stimulate the in vitro generation of CTL from the spleens of mice immunized with an AKR/Gross virus-positive lymphoma. MC57 (H-2b) cells infected with either 623EnvVac or 247EnvVac could serve as targets for ARK/Gross virus-specific CTL. Cells infected with the rVV expressing MCF13 env, however, were lysed much less efficiently by these CTL. 13EnvVac was also ineffective in priming or stimulating retrovirus-specific CTL. Finally, experiments with synthetic peptides and minigenes suggested that the reduced immunogenicity of the MCF13 envelope protein likely resulted from a single amino acid substitution within an immunodominant epitope of the p15E (TM) protein. The region of MCF13 env that encodes this epitope is derived from an endogenous xenotropic virus, while the allelic sequences in MCF247 are of ecotropic virus origin. These results suggest the potential for recombination within the MuLV envelope gene to allow escape from host cellular immune responses.

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.

Oncogenicity of AKR mink cell focus-inducing murine leukemia virus correlates with induction of chronic phosphatidylinositol signal transduction.

Naturally occurring recombinant murine leukemia viruses (MuLVs), termed mink cell focus-inducing (MCF) viruses, are the proximal leukemogens in spontaneous thymic lymphomas of AKR mice. The mechanism by which these viruses transform lymphocytes is not clear. Previous studies have implicated either integrational activation of proto-oncogenes, chronic autocrine immune stimulation, and/or autocrine stimulation of growth factor receptors (e.g., interleukin 2 receptors) via binding of the viral env glycoprotein (gp70) to these receptors. Any one of these events could also involve activation of second messenger signaling pathways in the cell. We examined whether infection with oncogenic AKR-247 MCF MuLV induced transmembrane signaling cascades in thymocytes of AKR mice. Cyclic AMP levels were not changed, but there was enhanced turnover of phosphatidylinositol phosphates, with concomitant increases in diacyglycerol and inositol 1,4,5-triphosphate. Thus, phospholipase C activity was increased. Protein kinase C activity was also elevated in comparison to that in uninfected thymocytes. The above events occurred in parallel with MCF expression in the thymus and were chronically maintained thereafter. No changes in phospholipid turnover occurred in an organ which did not replicate the MCF virus (spleen) or in thymocytes of AKR mice infected with a thymotropic, nononcogenic MCF virus (AKV-1-C36). Therefore, only the oncogenic MCF virus induced phosphatidylinositol signal transduction. Flow cytometric comparison of cell surface gp70 revealed that AKR-247 MCF virus-infected thymocytes expressed more MCF virus gp70 than did thymocytes from AKV-1-C36 MCF virus-infected mice, suggesting that certain threshold quantities of MCF virus env glycoproteins may be involved in this signaling. This type of signal transduction is not induced by stimulation of the interleukin 2 receptor but is involved in certain oncogene systems (e.g., ras and fms). Its chronic induction by oncogenic MCF MuLV may thus initiate thymocyte transformation.

Termination of the B cell lymphoma dormant state in thymectomized AKR mice.

AKR mice are highly susceptible to spontaneous T cell lymphomagenesis and thymus removal at the age of 1 to 3 mo greatly reduces its development. Twelve-mo-old AKR mice thymectomized at young age were shown previously to carry potential lymphoma cells that could be triggered to develop into B cell lymphomas (80 to 100%) after removal from their host "restrictive" environment into young histocompatible hosts. Additional attempts were made to terminate the potential lymphoma cell dormant state in 12-mo-old thymectomized AKR mice. Replenishment of some deficiencies caused by thymectomy at a young age, including a s.c. syngeneic thymus graft or a single injection of the dual tropic recombinant virus isolates DTV-71 or MCF-247 into 12-mo-old thymectomized AKR mice resulted in Ly-1+ pre-B or B cell lymphoma development in 80 to 98% of these treated mice. In vivo elimination of T cell subsets by administration of cyclosporin A or by mAb expressed on Th cells (anti-CD4) or cytotoxic T cells (anti-CD8) stimulated the progression of dormant potential lymphoma cells towards B cell lymphoma development. The most striking results were observed after administration of anti-CD8 mAb: 90 to 100% of these treated mice developed Ly-1+ B cell lymphomas within 80 days. The effect of rIL-2 on dormant PLC was also tested. Administration of rIL-2 to 12-mo-old thymectomized mice terminated tumor dormancy in 94% of the treated mice within 66 days. Tests of the resulting B lymphomas for dual tropic recombinant virus/mink cell focus-inducing virus infection indicated that the breakdown of tumor dormancy did not result from development of pathogenic class I mink cell focus-inducing viruses. These results suggest that T cell subsets and/or their products are involved in the proliferation arrest of potential lymphoma cells present in thymectomized AKR mice.

Calorie restriction suppresses subgenomic mink cytopathic focus-forming murine leukemia virus transcription and frequency of genomic expression while impairing lymphoma formation.

Calorie restriction suppresses mammary proviral mRNA expression and protooncogene activation in breast tumor-prone C3H/Ou mice while inhibiting tumor formation. To determine whether the beneficial effects of chronic energy-intake restriction (CEIR) can be extended to an organ site of retrovirus-induced tumorigenesis where the dynamics of growth and sexual maturity are not paramount as they are in breast tissue, calorie restriction of 40% was imposed on thymic lymphoma-prone AKR mice when 4 weeks old. Recombination between various murine leukemia virus (MuLV) mRNAs, resulting in the generation of an 8.4-kilobase genomic-length transcript with mink cytopathic focus-forming (MCF) characteristics, is considered the proximal retroviral event in AKR lymphomagenesis. Thymic expression of subgenomic MCF MuLV mRNA was uniformly suppressed among 6- and 8-week-old CEIR mice (P less than 0.02). This suppression of MuLV transcription preceded a 25% reduction in the appearance of genomic-length MCF transcripts among CEIR mice and a 28% reduction in cumulative lymphoma mortality. The latency to median tumor incidence was extended greater than 3 months by calorie restriction, and median lifespan was extended approximately 50%. Survival curves for the full-fed and CEIR dietary cohorts were found to be significantly different (P less than 0.0001), with full-fed mice experiencing a 3 times greater risk of lymphoma mortality. These findings extend the known range of pathologic states influenced by CEIR in inbred mice and show that retroviral mechanisms involved in generation of lymphoid malignancy can be significantly impaired by calorie restriction.

Construction and properties of an "artificial" spleen focus-forming virus.

The replication-defective Friend spleen focus-forming virus (F-SFFV) induces acute erythroblastosis in adult mice. The envelope-related (env) gene and LTR are the only functional elements of the viral genome. The env-coded glycoprotein gp55 has been shown to be responsible for target cell specificity and for the short latency of the disease caused by SFFV. This molecule closely resembles the env coded proteins gp70 + p15E of mink cell focus inducing viruses (MCFV). The only substantial differences between these two env genes are a large deletion spanning 585 nucleotides in the middle of the F-SFFV gene and a frameshift mutation near the 3' end leading to a modified and shortened membrane anchor in the mature protein. To determine if the large deletion and/or the frameshift mutation are capable of changing the properties of a nonpathogenic MCFV into those of an acutely pathogenic SFFV we introduced these changes into the env gene of an MCFV. The results show that the mutated MCFV is as acutely pathogenic as F-SFFV. We therefore conclude that the modified membrane anchor of gp55 and the change caused by the large deletion are the essential determinants of the high pathogenicity of SFFV.