DIFFERENCES IN MUTAGENESIS DURING MINUS STRAND, PLUS STRAND AND STRAND TRANSFER (RECOMBINATION) SYNTHESIS OF THE HIV-1 NEF GENE IN VITRO

摘要

We have developed an HIV nef-Escherichia coil lacZ fusion system in vitro that allows the detection of low frequency mutations, including frameshifts, deletions and insertions. A portion of the net gene that encompasses a hypervariable region was fused in-frame with a downstream lacZ alpha peptide coding region. The resulting lacZ alpha peptide fusion protein remained functional. Any frameshift mutations in the net insert would put the downstream lacZ a peptide gene out of frame, eliminating a complementation. With this system we compared the error rates of frameshift mutations that arise during DNA-directed and RNA-directed DNA synthesis. Results showed that DNA-directed and RNA-directed DNA synthesis did not contribute equally to the generation of mutations. DNA-directed DNA synthesis generated frameshift mutations at a frequency similar to 10-fold higher than those arising from RNA-directed DNA synthesis. RNA-directed DNA synthesis in the presence of acceptor templates showed an increase in mutation rate and differences in the mutation spectrum. The enhancement of mutation rate was caused by the appearance of mutations at three new locations that correlated with likely recombination sites. Results indicate that recombination is another source of mutations during viral replication.