Sequence analysis of murine leukemia virus envelope gene from inoculated mice

Introduction of amino acids substitutions in murine leukemia virus genome is a powerful method to determine the relative importance of various viral factors in pathogenesis. However, introduction of such amino acids substitution could result in viruses at a selective disadvantage, and eventual selection of revertants. It is thus essential to verify if the mutation is maintained stably in replicating virus and in infected tumor cells. In the present study, viral nucleic acid sequences from diseased animals were determined using different approaches. Small blood samples were found adequate for direct RNA extraction and reverse transcriptase-PCR amplification followed by automated DNA sequencing. Alternatively, replication-competent viruses were recovered specifically by applying blood samples onto permissive cells; viral RNA is then extracted from tissue culture medium and similarly sequenced. Tissue samples were also used to amplify viral sequences from tumors DNA while small pieces of tumors tissues were applied onto permissive cells to isolate replicating viruses. The combined experimental approach was used to show sequence conservation using a mutant altered in the intracytoplasmic region of viral envelope glycoprotein. No difference was observed between viruses recovered directly from the animal and those amplified onto cultured cells.