Viral DNA tethering domains complement replication-defective mutations in the p12 protein of MuLV Gag

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 110; no. 23; pp. 9487 - 9492
• Main Authors: Schneider, William M., Brzezinski, Jonathon D., Aiyer, Sriram, Malani, Nirav, Gyuricza, Mercedes, Bushman, Frederic D., Roth, Monica J.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 04.06.2013; National Acad Sciences
• Subjects: Animals; Antigens; Antigens, Viral - metabolism; Base Sequence; Biological Sciences; Blotting, Western; Cell Line; Chromatin; Chromatin - metabolism; Chromosomes; Cloning, Molecular; Complementation; Deoxyribonucleic acid; DNA; DNA, Viral - genetics; Dogs; Fluorescence Recovery After Photobleaching; Gene Products, gag - genetics; Genetic mutation; Green Fluorescent Proteins - metabolism; Humans; Immunoprecipitation; Infections; Kaposis sarcoma; Leukemia; Leukemia Virus, Murine - genetics; Mathematical functions; Microscopy, Confocal; Molecular Sequence Data; Mutation; Mutation - genetics; Nuclear Proteins - metabolism; Oncogene Proteins, Viral - metabolism; Protein Engineering; Proteins; Sequence Analysis, DNA; Trans-Activators - metabolism; Viral DNA; Virus Integration - genetics; Virus Replication - genetics; Viruses; gammaretroviral vectors; retroviral integration; nuclear retention
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1221736110

The p12 protein of murine leukemia virus (MuLV) group-specific antigen (Gag) is associated with the preintegration complex, and mutants of p12 (PM14) show defects in nuclear entry or retention. Here we show that p12 proteins engineered to encode peptide sequences derived from known viral tethering proteins can direct chromatin binding during the early phase of viral replication and rescue a lethal p12-PM14 mutant. Peptides studied included segments of Kaposi sarcoma herpesvirus latency-associated nuclear antigen (LANA)₁₋₂₃, human papillomavirus 8 E2, and prototype foamy virus chromatin-binding sequences. Amino acid substitutions in Kaposi sarcoma herpesvirus LANA and prototype foamy virus chromatin-binding sequences that blocked nucleosome association failed to rescue MuLV p12-PM14. Rescue by a larger LANA peptide, LANA₁₋₃₂, required second-site mutations that are predicted to reduce peptide binding affinity to chromosomes, suggesting that excessively high binding affinity interfered with Gag/p12 function. This is supported by confocal microscopy of chimeric p12-GFP fusion constructs showing the reverted proteins had weaker association to condensed mitotic chromosomes. Analysis of the integration-site selection of these chimeric viruses showed no significant change in integration profile compared with wild-type MuLV, suggesting release of the tethered p12 post mitosis, before viral integration.