Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus.

A mutant of Moloney murine leukemia virus (M-MuLV), pMOV-psi-, was constructed by deletion of about 350 nucleotides from an infectious proviral DNA clone between the putative env mRNA 5' splice site and the AUG that initiates the coding sequence for Pr65gag. Although the parent wild-type proviral clone, pMOV-psi+, quickly causes a high level of reverse-transcriptase-containing virus particles to be released from transfected NIH/3T3 cells, transfection of pMOV-psi- into these cells initially results in very little release. By 9 to 10 days after transfection, however, pMOV-psi- -transfected cells produce infectious virus. Thus pMOV-psi- has a defect that can be repaired in transfected NIH/3T3 cells, presumably by recombination with a sequence normally present in the cells. Cell lines with pMOV-psi- stably integrated into chromosomal DNA produce reverse-transcriptase-containing particles that lack detectable M-MuLV RNA but the cells efficiently complement replication-defective, packagable retroviruses. Thus pMOV-psi- has a defect in the packaging of genomic RNA into virions but can provide in trans the products necessary for virion production. The deletion in pMOV-psi- appears to define a site required in cis for packaging of MuLV RNA into virions. Cell lines carrying pMOV-psi- can be used to produce helper-free stocks of natural or synthetic defective retroviruses.