A mutant murine leukemia virus with a single missense codon in pol is defective in a function affecting integration.

Abstract

Site-directed mutagenesis of cloned Moloney murine leukemia virus (MuLV) DNA was used to define a function encoded in the 3'' region of the viral pol gene and required for efficient integration of viral DNA. One mutant, MuLV-SF1, contained a single base substitution (C to T at base 495) that resulted in an arginine to cysteine change in a region highly conserved among retroviruses. Mutant DNA, introduced into rat cells by cotransfection with a herpes simplex virus thymidine kinase gene (HSV tk), directed production of virus particles with reverse transcriptase activity. Infection of cells with these particles led to synthesis of full-length linear and circular forms of unintegrated viral full-length linear and circular forms of unintegrated viral DNA; integrated viral DNA was decreased at least by a factor of 10 when examined by DNA hybridization, and the mutant particles were less efficient than wild-type virus at establishing an infection by a factor of at least 300. Pseudotypes formed with the proteins of MuLV-SF1 and the genome of a replication defective marker MuLV, carrying the HSV tk gene, were less effective by at least a factor of 100 in producing tk+ colonies than pseudotypes formed with proteins encoded by wild-type virus. When the MuLV-SF1 pseudotypes did produce tk+ cells, most of the proviruses were integrated aberrantly. Evidently, the MuLV-SF1 pol gene is defective for a function that is required for normal integrative recombination and dissociable from DNA synthesis.