COMPENSATORY POINT MUTATIONS IN THE HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 GAG REGION THAT ARE DISTAL FROM DELETION MUTATIONS IN THE DIMERIZATION INITIATION SITE CAN RESTORE VIRAL REPLICATION

The dimerization initiation site (DIS), downstream of the long termina l repeat within the human immunodeficiency virus type 1 (HIV-1) genome , can form a stem-loop structure (SL1) that has been shown to be invol ved in the packaging of viral RNA. In order to further determine the r ole of this region in the virus life cycle, we deleted the 16 nucleoti des (nt) at positions +238 to +253 within SL1 to generate a construct termed BH10-LD3 and showed that this virus was impaired in viral RNA p ackaging, viral gene expression, and viral replication. Long-term cult ure of these mutated viruses in MT-2 cells, i.e., 18 passages, yielded revertant viruses that possessed infectivities similar to that of the wild type. Cloning and sequencing showed that these viruses retained the original 16-nt deletion but possessed two additional point mutatio ns, which were located within the p2 and NC regions of the Gag coding region, respectively, and which were therefore named MP2 and MNC. Site -directed mutagenesis studies revealed that both of these point mutati ons were necessary to compensate for the 16-nt deletion in BH10-LD3. A construct with both the 16-nt deletion and the MP2 mutation, i.e., LD 3-MP2, produced approximately five times more viral protein than BH10- LD3, while the MNC mutation, i.e., construct LD3-MNC, reversed the def ects in viral RNA packaging. We also deleted nt +261 to +274 within th e 3' end of SL1 and showed that the diminished infectivity of the muta ted virus, termed BH10-LD4, could also be restored by the MP2 and MNC point mutations. Therefore, compensatory mutations within the p2 and N C proteins, distal from deletions within the DIS region of the HIV gen ome, can restore HIV replication, viral gene expression, and viral RNA packaging to control levels.