title: Viral DNA tethering domains complement replication-defective mutations in the p12 protein of MuLV Gag.
abstract: 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, LANA(1-32), 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.
authors: Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, NJ 08854, USA.
authors: William M
authors: WM
authors: Schneider
authors: Jonathon D
authors: JD
authors: Brzezinski
authors: Sriram
authors: S
authors: Aiyer
authors: Nirav
authors: N
authors: Malani
authors: Mercedes
authors: M
authors: Gyuricza
authors: Frederic D
authors: FD
authors: Bushman
authors: Monica J
authors: MJ
authors: Roth
mesh_terms: D000818:Animals; D000956:Antigens, Viral / Q000378:metabolism; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D002843:Chromatin / Q000378:metabolism; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000235:genetics*; D004285:Dogs; D036681:Fluorescence Recovery After Photobleaching; D015683:Gene Products, gag / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D009052:Leukemia Virus, Murine / Q000235:genetics*; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009687:Nuclear Proteins / Q000378:metabolism; D009856:Oncogene Proteins, Viral / Q000378:metabolism; D015202:Protein Engineering; D017422:Sequence Analysis, DNA; D015534:Trans-Activators / Q000378:metabolism; D016662:Virus Integration / Q000235:genetics*; D014779:Virus Replication / Q000235:genetics*
chemical_list: D000956:Antigens, Viral; D002843:Chromatin; D004279:DNA, Viral; C468293:Gag protein p12, moloney murine leukemia virus; D015683:Gene Products, gag; D009687:Nuclear Proteins; D009856:Oncogene Proteins, Viral; D015534:Trans-Activators; C109540:latency-associated nuclear antigen; C468298:oncogene protein E2, Human papillomavirus type 8; D049452:Green Fluorescent Proteins
keywords: gammaretroviral vectors; nuclear retention; retroviral integration
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