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 terminal repeat within the human immunodeficiency virus type 1 (HIV-1) genome, can form a stem-loop structure (SL1) that has been shown to be involved in the packaging of viral RNA. In order to further determine the role of this region in the virus life cycle, we deleted the 16 nucleotides (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 packaging, viral gene expression, and viral replication. Long-term culture 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 mutations, 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 mutations 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., LD3-MP2, produced approximately five times more viral protein than BH10-LD3, while the MNC mutation, i.e., construct LD3-MNC, reversed the defects in viral RNA packaging. We also deleted nt +261 to +274 within the 3' end of SL1 and showed that the diminished infectivity of the mutated virus, termed BH10-LD4, could also be restored by the MP2 and MNC point mutations. Therefore, compensatory mutations within the p2 and NC proteins, distal from deletions within the DIS region of the HIV genome, can restore HIV replication, viral gene expression, and viral RNA packaging to control levels.
Liang
C
C
McGill University AIDS Centre, Lady Davis Institute-Jewish General Hospital, Montreal, Quebec, Canada H3T 1E2.
Rong
L
L
Laughrea
M
M
Kleiman
L
L
Wainberg
M A
MA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D019556:COS Cells; D002213:Capsid / Q000235:genetics*; D036022:Capsid Proteins; D018929:Cell Culture Techniques; D002461:Cell Line, Transformed; D004279:DNA, Viral; D019281:Dimerization; D015683:Gene Products, gag / Q000235:genetics*; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics* / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics*; D017354:Point Mutation; D012367:RNA, Viral; D017384:Sequence Deletion; D013997:Time Factors; D014764:Viral Proteins; D019065:Virus Assembly; D014779:Virus Replication; D054301:gag Gene Products, Human Immunodeficiency Virus
D036022:Capsid Proteins; D004279:DNA, Viral; D015683:Gene Products, gag; C071884:NCP7 protein, Human immunodeficiency virus 1; D010446:Peptide Fragments; D012367:RNA, Viral; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; C109809:p2 gag peptide, Human immunodeficiency virus 1