Rescue of multiple viral functions by a second-site suppressor of a human immunodeficiency virus type 1 nucleocapsid mutation.
Human immunodeficiency type 1 (HIV-1) bearing the nucleocapsid (NC) mutation R10A/K11A is replication defective. After serial passage of the mutant virus in tissue culture, we isolated a revertant that retained the original mutation. It had acquired, in addition, a new mutation (E21K) that was formally demonstrated to be sufficient for restoration of viral replication. Detailed analysis of the replication defect of R10A/K11A revealed a threefold reduction in virion yield and a fivefold reduction in packaging of viral genomic RNA. Real-time PCR was then used to quantitate viral DNA synthesis following infection of Jurkat T cells. After adjustment for the assembly and packaging defects, a minor (twofold) reduction in synthesis of either strong-stop, full-length linear DNA or 2-LTR circles was observed with R10A/K11A virions, indicating that reverse transcription and nuclear transport of the viral genome were largely intact. However, after adjustment for the amounts of full-length or 2-LTR circles produced, R10A/K11A virions were at least 10-fold less infectious than wild type, indicating that viral DNA produced by the R10A/K11A mutant failed to integrate. Each of the above-mentioned defects was corrected by introduction of the second-site compensatory mutation E21K. These results demonstrate that the replication defect of mutant R10A/K11A can be explained by impairment at multiple steps in the viral life cycle, most important among them being integration and RNA packaging. The E21K mutation is predicted to restore positive charge to the face of the R10A/K11A mutant NC protein that interacts with the HIV-1 SL3 RNA stem-loop, emphasizing the importance of NC basic residues for HIV-1 replication.
Cimarelli
A
A
Department of Microbiology, Columbia University College of Physicians and Surgeons, New York, New York 10032, USA.
Sandin
S
S
Höglund
S
S
Luban
J
J
D000595:Amino Acid Sequence; D002461:Cell Line, Transformed; D004279:DNA, Viral / Q000096:biosynthesis; D015497:HIV-1 / Q000235:genetics / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D019169:Jurkat Cells; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D019590:Nucleocapsid Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D010641:Phenotype; D016133:Polymerase Chain Reaction / Q000379:methods; D011487:Protein Conformation; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism; D014158:Transcription, Genetic; D014771:Virion / Q000502:physiology / Q000648:ultrastructure; D019065:Virus Assembly; D014779:Virus Replication
D004279:DNA, Viral; D019590:Nucleocapsid Proteins; D012367:RNA, Viral