Construction and analysis of deletion mutations in the pol gene of Moloney murine leukemia virus: a new viral function required for productive infection.

We have used in vitro mutagenesis to explore the functions of the gene products encoded by the pol gene of Moloney murine leukemia virus (M-MuLV). Deletions were constructed at a variety of positions in the gene, and the altered DNA copies of the viral genome were introduced into mouse cells by cotransformation. The mutants could be divided into two classes depending on the phenotype and map position of the deletion within the pol gene. Mutants with deletions mapping in the 5' portion of the gene were found to be completely deficient in reverse transcriptase activity. Mutants mapping in the 3' portion of the gene, however, assembled and released virions with normal levels of reverse transcriptase and RNAase H activities. When applied to permissive cells, these virions directed the synthesis of all three forms of unintegrated viral DNA: full-length, double-stranded linear DNA and the two circular forms with one and two copies of the long terminal repeat sequences. The infection was arrested at this point and the infected cells did not become producers of virus. Thus the 3' portion of the pol gene encodes a polypeptide with a function distinct from that of reverse transcriptase, which is not required for synthesis of viral DNA but is essential for establishment of that DNA in a stable, active form in the infected cell. We suggest that this function may be the integration of the proviral DNA.