title: Phosphorylation Requirement of Murine Leukemia Virus p12.
abstract: The p12 protein of murine leukemia virus (MLV) Gag is associated with the preintegration complex (PIC), and mutants of p12 (PM14) exhibit defects in nuclear entry/retention. Mutants of the phosphorylated serine 61 also have been reported to have defects in the early life cycle. Here we show that a phosphorylated peptide motif derived from human papillomavirus 8 (HPV-8), the E2 hinge region including residues 240 to 255, can functionally replace the main phosphorylated motif of MLV p12 and can rescue the viral titer of a strain with the lethal p12-PM14 mutation. Complementation with the HPV-8 E2 hinge motif generated multiple second-site mutations in live viral passage assays. Additional p12 phosphorylation sites were detected, including the late domain of p12 (PPPY) as well as the late domain/protease cleavage site of matrix (LYPAL), by mass spectrometry and Western blotting. Chromatin binding of p12-green fluorescent protein (GFP) fusion protein and functional complementation of p12-PM14 occurred in a manner independent of the E2 hinge region phosphorylation. Replacement of serine 61 by alanine within the minimal tethering domain (61SPMASRLRGRR71) maintained tethering, but in the context of the full-length p12, mutants with substitutions in S61 remained untethered and lost infectivity, indicating phosphorylation of p12 serine 61 functions to temporally regulate early and late p12 functions.
  
  
  OBJECTIVE
  The p12 protein, required for both early and late viral functions, is the predominant phosphorylated viral protein of Moloney MLV and is required for virus viability. Our studies indicate that the N terminus of p12 represses the early function of the chromatin binding domain and that deletion of the N terminus activates chromatin binding in the wild-type Moloney MLV p12 protein. Mass spectrometry and mutagenesis studies suggest that phosphorylation of both the repression domain and the chromatin binding domain acts to temporally regulate this process at the appropriate stages during infection.
authors.affiliation: Rutgers-Robert Wood Johnson Medical School, Department of Pharmacology, Piscataway, New Jersey, USA.
authors.forename: Jonathon D
authors.identifier: http://orcid.org/0000-0001-8669-2754
authors.initials: JD
authors.lastname: Brzezinski
authors.affiliation: Rutgers-Robert Wood Johnson Medical School, Department of Pharmacology, Piscataway, New Jersey, USA.
authors.forename: Roland
authors.identifier: http://orcid.org/0000-0003-1093-0378
authors.initials: R
authors.lastname: Felkner
authors.affiliation: The School of Arts and Sciences, Rutgers University, New Brunswick, New Jersey, USA.
authors.forename: Apexa
authors.identifier: http://orcid.org/0000-0001-8343-598X
authors.initials: A
authors.lastname: Modi
authors.affiliation: The School of Arts and Sciences, Rutgers University, New Brunswick, New Jersey, USA.
authors.forename: Mengdan
authors.identifier: http://orcid.org/0000-0003-2057-723X
authors.initials: M
authors.lastname: Liu
authors.affiliation: Rutgers-Robert Wood Johnson Medical School, Department of Pharmacology, Piscataway, New Jersey, USA roth@rwjms.rutgers.edu.
authors.forename: Monica J
authors.identifier: http://orcid.org/0000-0002-2219-0402
authors.initials: MJ
authors.lastname: Roth
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002843:Chromatin / Q000737:chemistry / Q000378:metabolism* / Q000821:virology; D005786:Gene Expression Regulation; D015683:Gene Products, gag / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005816:Genetic Complementation Test; D057809:HEK293 Cells; D054884:Host-Pathogen Interactions; D006801:Humans; D051379:Mice; D008938:Mitosis; D008979:Moloney murine leukemia virus / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D009154:Mutation; D027383:Papillomaviridae / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000378:metabolism*; D015398:Signal Transduction; D014771:Virion / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D019065:Virus Assembly; D014779:Virus Replication
chemical_list: D002843:Chromatin; C468293:Gag protein p12, moloney murine leukemia virus; D015683:Gene Products, gag; D011993:Recombinant Fusion Proteins; D012694:Serine