This project aims to elucidate the molecular basis for differences in murine leukemia virus (MuLV) in vitro infective host range, in the hope that the consequent knowledge will facilitate an understanding of differences in in vivo pathogenicity and the host elements responsible for restriction of retroviral replication. As a model system, we have been examining an ecotropic MuLV isolate (RF.AMLV) which, along with the ecotropic Moloney MuLV (MoMuLV eco), is greatly restricted for in vitro infection of a Mus dunni mouse fibroblast cell line. This is quite unusual, since the Mus dunni cell line is highly sensitive to infection by most other ecotropic MuLV. Serial passage of the RF.AMLV stock through the dunni cell line gave rise to a virus preparation with altered in vitro tropism. The molecular basis of this alteration was identified by DNA sequence analysis, which revealed that for over 2.1 kbp of the envelope (env) gene sequence, only one nucleotide base change distinguished the dunni cell-tropic preparation from the original virus stock. This mutation encodes a single amino acid difference in the envelope protein domain responsible for determination of MuLV tropism. Demonstration that this single amino acid change is sufficient to account for the observed changes in ecotropic host range was accomplished by cloning the mutant env sequence into a MoMuLV backbone; transfection of the resultant recombinant retrovirus gave rise to viral stocks exhibiting the mutant phenotype (i.e. altered ecotropic host range). Additionally, parental or mutant envelope proteins expressed from the same transfected cell type manifested the corresponding phenotype, eliminating any possibility that host cell determinants were solely responsible for the acquisition of the altered infectiousness. Analysis of similar ecotropic host range variants generated in the course of retrovirus infection in vivo is underway to ascertain whether similar or identical amino acid alterations are responsible for these examples as well. Structure/function analyses have been begun to elucidate the biochemical basis for the host range selectivity of the parental and mutant envelope protein-bearing viruses.
