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ts1 is a highly neuropathogenic and lymphocytopathic mutant of Moloney murine leukemia virus TB (MoMuLV-TB). We previously reported that the primary neuropathogenic determinant of ts1 maps to a single amino acid substitution, Val-25-->Ile, in precursor envelope protein gPr80env. This Val-25-->Ile substitution apparently renders gPr80env inefficient for transport from the endoplasmic reticulum to the Golgi apparatus. These findings suggest that the cytopathic effect of ts1 in neural cells might be due to the accumulation of gPr80env in the endoplasmic reticulum. Since endothelial and glial cells are targets of ts1 infection in the central nervous system, we established primary endothelial and astrocyte cultures to investigate the mechanism of cell killing caused by ts1. A continuous cell line, TB, was used as a control. Our results showed that both ts1 and MoMuLV-TB replicated and induced a cytopathic effect in astrocyte cultures, albeit to different degrees; ts1 appeared to be more lethal than MoMuLV-TB. On the other hand, ts1 and MoMuLV-TB infections of endothelial or TB cells were not cytopathic. The cytopathic effect in infected astrocytes correlated with the inefficiency of gPr80env transport and the intracellular accumulation of gPr80env as well as aberrant virus particles.

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.

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.

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.

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.