Synthesis of virus-specific high-mobility DNA after temperature upshift of SC-1 cells chronically infected with moloney murine leukemia virus mutant ts1.

Premature termination products of reverse transcription that consist physically of viral minus-sense single-stranded DNA that is shorter than one long terminal repeat and partial DNA duplexes are dramatically increased in the central nervous system (CNS) of FVB/N mice that are infected by ts1, a temperature-sensitive mutant of Moloney murine leukemia virus. Due to their migration in agarose gels, these incomplete physical forms of DNA have been designated high-mobility (HM) DNA. In non-CNS tissues, the level of HM DNA is either low or not detectable. In order to determine the conditions that are necessary for the synthesis of HM DNA in vivo, we have characterized the physical forms of HM DNA that were synthesized in vitro in chronically infected SC-1 cells after temperature upshift. At the permissive temperature of 34 degrees C, the chronically infected SC-1 cells did not synthesize HM DNA. After temperature upshift of the cultured cells from 34 to 37 degrees C, the chronically infected SC-1 cells developed extremely high levels of HM DNA. Following temperature downshift of the cultured cells from 37 to 34 degrees C, a decrease in the level of HM DNA and an increase in the level of unintegrated linear proviral DNA occurred simultaneously. These results suggested that the accumulation of HM DNA both in vitro and in vivo may be the result of superinfection.

Zidovudine [AZT] myotoxicity: quantitative separation of AZT effects on proliferation and differentiation of muscle cells in vitro. Lack of myotoxicity potentiation by retrovirus.

We compared quantitatively the myotoxicity of 3'-azido-2',3'-dideoxythymidine (AZT) against uninfected and ts1 retrovirus infected mouse skeletal muscle (ATCC CRL 1772) cells at different stages of maturation in vitro. The AZT half inhibitory concentration (IC50) for myoblast proliferation was determined for uninfected myoblasts and parallel cultures infected with ts1 virus. The AZT IC50 for muscle cell differentiation was determined in cultures where myoblasts were grown to confluence and then changed to the fusion medium to which AZT was added at increasing concentrations. Creatine kinase activity was used as a marker of muscle cell differentiation and was determined in homogenates after 7 days. Total cellular mitochondrial DNA was analyzed by Southern blotting. The estimated AZT IC50 for muscle cell proliferation (2-5 microM) was significantly less than the AZT IC50 for muscle cell differentiation (100 microM). Infection with ts1 retrovirus did not significantly shift the IC50 for either proliferation or differentiation of muscle cells. Toxic concentrations of AZT did not cause selective depletion of mitochondrial DNA. The myotoxic effects of AZT on myoblast proliferation and muscle cell differentiation in vitro were quantitatively different and were not changed by productive ts1 retrovirus infection of muscle cells. These results suggest that AZT may impair muscle fiber regeneration in the course of retrovirus associated myopathy. The mechanism of AZT myotoxicity was not explained by alterations in total mitochondrial DNA content.

ts1-Induced spongiform encephalomyelopathy: physical forms of high-mobility DNA in spinal cord tissues of paralyzed mice are products of premature termination of reverse transcription.

ts1 is a temperature-sensitive mutant of Moloney murine leukemia virus that causes hind-limb paralysis in mice. In tissues of the central nervous systems of paralyzed moribund FVB/N mice, a major component of the unintegrated viral DNA of ts1 consists of highly mobile physical forms of viral-specific DNA (HM DNA). Previous studies with ecotropic virus-specific polarity probes showed that the gp70-coding region of the env gene in the HM DNA was minus-sense single-stranded DNA. The physical forms of the HM DNA have now been characterized in more detail with additional ecotropic virus-specific probes that hybridized to the p15E-coding region of the env gene and two locations within the U3 region of the long terminal repeat. Two major classes of HM DNA were found: class I molecules consist of short minus-sense single-stranded DNA; class II molecules are partial DNA duplexes that are longer than the class I molecules. The two classes of HM DNA molecules are intermediate products of reverse transcription of the viral RNA of ts1. Since tissues that are infected with cytopathic retroviruses may contain high levels of unintegrated viral DNA, the HM DNA may have a role in inducing neurodegeneration in the central nervous systems of mice that are infected with ts1.

Deoxyglucose uptake by mouse astrocytes: effects of temperature and retrovirus infection.

Deoxyglucose uptake by FVB/N mouse astrocytes was studied before and after infection by ts1 retrovirus which causes a neurodegenerative disease in mice similar to HIV-1 encephalopathy in man. The Michaelis-Menten kinetic parameters, Km and Vmax, of 2-deoxy-D-glucose uptake by brain and cerebellar astrocytes were measured following culture at 34 degrees C where ts1 retrovirus replicates optimally, and at 37 degrees C. Compared to astrocytes cultured at 37 degrees C, astrocytes cultured at 34 degrees C had increased Km and decreased deoxyglucose uptake despite increased or unchanged Vmax. Following ts1 retrovirus infection, brain astrocyte deoxyglucose uptake doubled [132%] associated with decreased Km but unchanged Vmax, whereas cerebellar astrocyte deoxyglucose uptake doubled [102%] associated with increased Vmax but unchanged Km. These observations of altered deoxyglucose uptake kinetic parameters following retrovirus infection indicate different neurochemical mechanisms for the regional variation in deoxyglucose uptake observed following retrovirus infection of the CNS in vivo.

Development of physical forms of unintegrated retroviral DNA in mouse spinal cord tissue during ts1-induced spongiform encephalomyelopathy: elevated levels of a novel single-stranded form in paralyzed mice.

ts1 is a murine leukemia virus that causes rapidly evolving hindlimb paralysis in susceptible strains of mice. Following perinatal infection, three physical forms of unintegrated viral DNA were detected in the spinal cord by Southern blot hybridization. Linear and supercoiled closed-circle viral double-stranded DNAs were detected in both the central nervous system and non-central nervous system tissues. An elevated level of a novel minus-sense single-stranded form of viral DNA, which had a very high mobility in agarose gels, was correlated with the onset of symptoms of paralysis. As the severity of paralysis progressed, the level of this single-stranded form increased rapidly, with the highest level in the spinal cords of moribund mice. Since the virulence of a number of cytopathic retroviruses has been associated with the presence of increased amounts of unintegrated viral DNA in the tissues of the infected hosts, this novel form of highly mobile unintegrated single-stranded DNA may have a role in the neuropathogenesis of ts1.

Murine leukemia virus induced central nervous system diseases.

The ts1 mutant of Moloney murine leukemia virus TB (MoMuLV-TB) causes a degenerative neurologic and immunologic disease in mice characterized by development of spongiform encephalomyelopathy that results in hind-limb paralysis, marked thymic atrophy associated with immunodeficiency, and generalized body wasting. T cells, particularly CD4+ helper T cells, play a key role in the pathogenesis of the disease induced by ts1. Therefore, ts1 is unique among the described murine retroviruses in its ability to afflict both the central nervous system (CNS) and the T-cell compartment of the immune system in the same host. This particular ability to cause degenerative diseases involving both the CNS and immune system is shared by the lentiviruses responsible for development of the acquired immunodeficiency syndromes of humans and macaques. Our goal has been to elucidate the specific cellular and molecular mechanisms that underlie this neuro- and immunopathogenicity of ts1. We have previously reported that the primary neuropathogenic determinant of ts1 maps to a single amino acid substitution, Val-25-Ile, in the precursor envelope protein gPr80env. Further, at the restrictive temperature, the Val-25-Ile substitution did not prevent oligomerization of the gPr80env proteins; however, the structure of the oligomer was incompetent for transport from the ER to the Golgi. These findings suggest that the cytopathic effect of ts1 in neural cells might be due to accumulation of the gPr80env oligomers in the ER. Since glial cells are targets of ts1 infection in vivo, primary astrocytic cultures were established and the cytopathic effect of ts1 and MoMuLV-TB on these cells assessed. Both viruses replicate well in astrocytes and their replication is cytopathic, albeit to different degrees. The ts1 mutant appears to produce greater cell killing than the wild-type virus. Furthermore, it was found that the rate of processing of gPr80env of ts1 in astrocytes is slower than that of MoMuLV-TB. Therefore, the inefficient transport and processing of gPr80env of ts1 appears to correlate with its cytopathic effect in these cells. Electron microscopic studies of the ts1-infected astrocytes revealed large numbers of aberrant particles in the ER. The in vitro cytopathic effect of ts1 on astrocytes may reflect what happens in vivo. An indirect mechanism of neuronal-cell killing by ts1 is proposed.

Alteration from T- to B-cell tropism reduces thymic atrophy and cytocidal effects in thymocytes but not neurovirulence induced by ts1, a mutant of Moloney murine leukemia virus TB.

The ts1 mutant of Moloney murine leukemia virus TB causes degenerative neurologic and immunologic disease in mice, characterized by development of spongiform encephalomyelopathy resulting in hindlimb paralysis, marked thymic atrophy associated with immunodeficiency, and generalized body wasting. To investigate the pathogenesis of the thymic atrophy caused by ts1, we constructed a chimeric virus, ts1-Cas(NS), in which a major portion of the U3 region of the long terminal repeat of ts1, a T-lymphotropic and neurovirulent murine leukemia virus, was replaced by the corresponding U3 region of Cas-Br-E, a B-lymphotropic and neurovirulent murine leukemia virus. In FVB/N mice, ts1-Cas(NS) induced paralytic and wasting disease with incidence, severity, and latency similar to that induced by ts1, but it failed to cause thymic atrophy as severe as that observed in ts1-infected mice. Furthermore, thymocytes cultured from ts1-Cas(NS)-infected mice died at a much slower rate than those of ts1-infected mice. The U3 substitution in ts1-Cas(NS) specifically diminished the ability of the virus to replicate in the thymus, whereas viral replication in the spinal cord was not significantly affected; thus, neurovirulence was not changed. The correlation of reduced thymic atrophy with decreased thymic viral titers and the decreased ability of ts1-Cas(NS) to cause thymocyte death in mice suggest strongly that the marked thymic atrophy in ts1-infected mice is not an indirect effect occurring secondary to neurodegenerative and wasting disease but is a direct cytopathic effect of high-level viral replication in the thymus.

High susceptibility of FVB/N mice to the paralytic disease induced by ts1, a mutant of Moloney murine leukemia virus TB.

The ts1 mutant of Moloney murine leukemia virus TB causes a degenerative neurologic and immunologic disease in susceptible strains of mice. This disease syndrome is characterized by development of spongiform encephalomyelopathy resulting in hindlimb paralysis, generalized bodywasting, and marked thymic atrophy associated with immune deficiency. The viral genetic determinants responsible for hindlimb paralysis in BALB/c and CFW/D mice have been localized to two point mutations in the env gene: one results in a Val-25----IIe substitution in the envelope precursor polyprotein gPr80env and the other, in an Arg-430----Lys substitution in the gp70. In this report we present studies showing that FVB/N mice were highly susceptible to ts1 and exhibited the shortest and most uniform latency period of all the murine strains tested. In addition, we have found that, unlike in CFW/D and BALB/c mice, only the Val-25----IIe substitution in the gPr80env is required to induce hindlimb paralysis in FVB/N mice. Our studies show that there was enhanced replication of ts1 in all tissues of FVB/N mice and that the virus titer in the spinal cord was more than 10-fold higher in FVB/N than in BALB/c mice by 30 days postinoculation, when the clinical signs of paralysis became evident in FVB/N mice. Apparently, other host factors that do not require the Arg-430----Lys substitution allowed high levels of viral replication within the central nervous system of FVB/N mice. These results, together with the finding that 100% of FVB/N mice that were inoculated with ts1 at 5 days of age developed hindlimb paralysis at 30-60 days postinoculation, whereas only 33% of 5-day-old BALB/c mice developed hindlimb paralysis with a much longer latency period, suggest that subtle virus-host interactions determine the incidence, the latency period, and the severity of the disease caused by 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.

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.

Two subfamilies of murine retrotransposon ETn sequences.

Early transposon (ETn) elements are 5.7-kb retrotransposons found in the murine genome. We have sequenced large portions of two ETn elements that have apparently transposed within the DNA of a murine myeloma cell line, P3.26Bu4. One of the transposed ETn elements has 5' and 3' long terminal repeats (LTRs) that are exact duplicates of each other and has a 6-bp target site duplication. These results suggest that this element, which inserted into an immunoglobulin gamma 1 switch region, moved by a retrotransposition process. Our nucleotide sequences confirm that individual ETn elements are very similar to one another and lack open reading frames. However, the ETn sequences reported here and those previously described differ significantly near their 5' LTRs, including 200 bp of weak similarity and 240 bp of complete disparity. Southern hybridization analysis suggests that both subfamilies of ETn sequences are represented many times in the mouse genome. The possibility that the disparate sequences have a role in transposition by ETn elements is discussed.

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.

Identification of point mutations in the envelope gene of Moloney murine leukemia virus TB temperature-sensitive paralytogenic mutant ts1: molecular determinants for neurovirulence.

ts1, a temperature-sensitive mutant of Moloney murine leukemia virus TB, induces hind-limb paralysis in mice. The DNA of both the ts1 and Moloney murine leukemia virus TB env genes has been sequenced, and the encoded amino acid sequences have been deduced from the DNA sequences. Four amino acids in the ts1 envelope protein have been identified which may be responsible for the ts1 phenotype, which includes temperature sensitivity, nonprocessing of Pr80env, and neurovirulence.

Interruption of two immunoglobulin heavy-chain switch regions in murine plasmacytoma P3.26Bu4 by insertion of retroviruslike element ETn.

A number of moderately reiterated murine genetic elements have been shown to have structures like those of retroviral proviruses. These elements are thought to be transposons, although little evidence of their transposability exists. Two members of one of these families of reiterated elements, the ETn family, have inserted into separate immunoglobulin heavy-chain switch regions in the plasmacytoma P3.26Bu4. Switch regions are those DNA segments associated with each immunoglobulin heavy-chain gene in which the somatic recombinations that accompany the heavy-chain switch occur. This role in somatic recombination may be relevant to the ETn insertions into the switch regions in P3.26Bu4 DNA. P3.26Bu4 and a number of other B-lineage cells contain ETn transcripts.

Complete nucleotide sequence of the murine gamma 3 switch region and analysis of switch recombination sites in two gamma 3-expressing hybridomas.

The heavy chain isotype switch is mediated by a DNA rearrangement between a donor switch region (usually mu) and a recipient switch region (gamma, epsilon, or alpha). Switch regions lie upstream of the appropriate heavy chain constant region gene and are composed of simple sequences repeated in tandem. It is not known to what extent the tandemly repeated sequences are important to the heavy chain switch recombination, and to what extent other features of switch region sequences might contribute to the switch process. We studied switches to the gamma 3 isotype by sequencing the entire gamma 3 switch region. This switch region is composed of forty-four 49 base pair units repeated in tandem. These repeated units share modest homology with the mu switch region repeated elements. Evolution of the gamma 3 switch region seems to involve insertions and deletions of the 49mer elements. We also molecularly cloned rearranged switch regions from two gamma 3-expressing hybridomas and determined the DNA sequences at the mu-gamma 3 recombination sites. We located these switch recombination sites within the germ-line gamma 3 switch region, as well as switch recombination sites from two myelomas. All four sites are found in the 5' one-third of the gamma 3 switch region. We discuss some additional trends in the sequence data near these four recombination sites.

Methylation of plasmacytoma c-myc genes.

The chromosomal translocation associated with many tumors of immunoglobulin-producing cells frequently results in the joining of the immunoglobulin heavy-chain locus and the c-myc oncogene. This translocation of c-myc has profound structural and functional consequences for the oncogene, including loss of the 5' end of the gene and transcriptional deregulation. We report in this communication that translocation results in a new methylation pattern of c-myc. In normal kidney and liver tissue, the c-myc gene is methylated at its 3' end. The translocated gene in plasmacytoma DNA is extensively demethylated. On the other hand, the nonrearranged c-myc gene in plasmacytoma DNA (which is transcriptionally silent) is extensively methylated. In addition, we confirm the nucleotide sequence (with 19 discrepancies out of 1400 bp) 5' to the murine c-myc gene, as reported by Corcoran et al. [Cell 40 (1985) 71-79].