GROWTH DOMINANCE OF A REVERTANT VIRUS GENERATED DURING IN-VITRO SERIAL PASSAGE OF NEF FRAMESHIFT MUTANT OF HIV-1

We prepared a series of nef mutant HIV-1 with a frameshift mutation at the Xho I site by up to 50 serial transfers into MT-4 cells. Here, we studied revertants. Immunofluorescrnce using an anti-Nef monoclonal a ntibody revealed that cells first became Nef antigen-positive at the 2 3rd passage. The percentage of Nef antigen-positive cells gradually in creased and reached almost 100% by the 27th passage. The sequence of t he provirus in the cells supported the generation of a revertant. This revertant mutated at the site immediately after the initially introdu ced frameshift mutation. This resulted in the substitution of only thr ee amino acids and the insertion of two, which restored the proline-ri ch domain, a conserved region believed essential to viral replication, at the middle of Nef. Thus, the growth dominance of the revertant vir us, compared with the original nef mutant, was directly demonstrated i n vitro using serial passages consisting of mixed HIV-1 populations in a single cell. (C) 1996 Academic Press, Inc.