tsl 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, gp7O and Prpl5E. This inefficient processing of gPr8Oe"v is correlated with neurovirulence. In this study, it was shown that a single amino acid substitution, Val-25-Ile in gPr80"v, is responsible for the temperature sensitivity, inefficient processing of gPr8(W'v at the restrictive temperature, and neurovirulence of tsl. At the restrictive temperature, a steady-state level of nonprocessed, endoglycosidase H-sensitive gPr8Oenv remained in the endoplasmic reticulum of cells infected by tsl, but no endoglycosidase H-resistant gPr8Oe"v 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 gPr80e"V at the restrictive temperature is. most likely due to inefficient transport of gPr8 env from the endoplasmic reticulum to the cis cisternae of the Golgi apparatus rather than due to misfolded gPr80rnV being a poor substrate for the processing protease at the restrictive temperature.

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, a temperature-sensitive mutant of Moloney murine leukemia virus TB, induces hind-limb paralysis in mice. The DNA of both the tsl 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 tsl envelope protein have been identified which may be responsible for the tsl phenotype, which includes temperature sensitivity, nonprocessing of Pr8Oenv, and neurovirulence.

tsl and tS7, two temperature-sensitive mutants of Moloney murine leukemia virus strain TB induce hind-limb paralysis in 100% of CFW/D mice injected. These two paralytogenic mutants also share a defect in their inability to process the env precursor protein, Pr8Oe"v, at the restrictive temperature. To identify the mutation(s) in the genomes of the paralytogenic mutants which cause the inability to process Pr8Oe"v efficiently and confer the ability to cause hind-limb paralysis instead of lymphoma, we constructed chimeric genomes between tsl and Moloney murine leukemia virus or the TB strain of the virus. We identified a 3.9-kilobase-pair HindIII-PstI sequence from nucleotides 4895 through 8264 and 1 through 567 of tsl, comprising the 3' end of the pol and all of the env genes, the long terminal repeat, and the 5' noncoding sequence, as being responsible for the temperature sensitivity, the inefficiency in processing Pr8Oe"v, and the induction of paralysis. We extended these findings by demonstrating that the 1.6-kilobase-pair pol-gp7O HindIII-BamHI DNA sequence from nucleotides 4895 through 6537 of tsl within the 3.9-kilobase-pair HindIII-PstI fragment is necessary for tsl to induce paralysis. In addition, we showed that this 1.6-kilobase-pair fragment also controls the processing of Pr8Oe"v and the temperature sensitivity of tsl. * Corresponding author. for further investigation into the molecular mechanism of this retrovirus-induced paralysis. To identify the mutation(s) in the genomes of the paralytogenic mutants which confer temperature sensitivity, inefficient processing of Pr8Oehz', and the ability to cause hind-limb

To better understand the molecular mechanism involved in retrovirus tsl-induced paralytic disease in mice, we constructed a panel of recombinant viruses between tsl and the wild-type viruses Moloney murine leukemia virus (MoMuLV) and MoMuLV-TB, a strain of MoMuLV. These recombinant viruses were constructed in an attempt to identify the sequence(s) in the genome of tsl which contains the critical mutation(s) responsible for the neurovirulence of tsl. Two functionally distinct sequences in the genome of tsl were found to be responsible for its paralytogenic ability. One of these sequences, the 0.77-kilobase-pair XbaI-BamHI (nucleotides 5765 to 6537) fragment which encodes the 5' half of gp7O and 11 base pairs upstream of the env gene coding sequence, determines the inability of tsl to process Pr8Oenv. The other sequence, the 2.30-kilobase-pair BamHI-PstI (nucleotides 538 to 8264 and 1 to 567) fragment, which comprises nearly two-thirds of the env gene, the long terminal repeat, and the 5' noncoding sequence, determines the enhanced neurotropism of tsl. Replacement of any one of these two regions with the homologous region from either one of the two wild-type viruses resulted in recombinant viruses which either totally failed to induce paralysis or induced a greatly attenuated form of paresis in some of the infected mice.