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.

Importance: 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.

FIG 1

Isoleucine activation of p12 tethering and rescue of viral titer. (A) The 84 residues of p12. M63 and I63 contain the Δ region (residues 49 to 64) that is deleted in the p12-Δ-G69R virus and replaced with the HPV-8 E2 peptide in the last two viruses in the list. Orange indicates phosphorylation sites detected by mass spectrometry (orange outline denotes sites detected only in specific samples), pink denotes the chromatin binding sequence previously defined in p12, blue denotes the sequence variations between viruses, and purple denotes the PM14 mutant backbone. (B) p12-GFP fusion proteins' tethering ability. Left panels, Hoechst stain (DNA); right panels, GFP. Scale bars, 10 μm. (C) Ratio of p12-GFP fusion protein intensity overlapping the mitotic chromatin versus that in the cytoplasm, as seen in panel B. Error bars represent ±1 standard error (SE); n >15. (D) lacZ infection titer of p12 serine 61 mutants in the wild-type p12 M63 context and with the p12 I63 suppressor mutant. Error bars represent ±1 SE; n = 3. (E) lacZ infection titer of all five p12 constructs assayed with mass spectrometry and their parent constructs p12-Δ_49–64 and p12-E2-Δ_49–64-PM14. Error bars represent ±1 SE; n = 3. All constructs with an asterisk (*) are statistically different from GFP (C) or p12-M63 (D, E) using Student's t test (α = 0.05).

FIG 2

Maturation and phosphorylation of MLV p12 mutants. (A to C) Viral pellets were labeled with ³⁵S (A) or ³²P (B, C), immunoprecipitated against p12, and then analyzed by SDS-PAGE and detected via ³⁵S (A), ³²P (B), and anti-p12 antibody (C). p12 and p12-containing precursor polypeptide sizes are marked on the left, while molecular mass standards (in kilodaltons) are marked on the right. (D) Viral pellets run via SDS-PAGE were analyzed by Western blotting with antiphosphotyrosine antibody. A duplicate gel was blotted for p12 (lower panel). Lanes: 1, no virus; 2, p12-M63; 3, p12-I63; 4, p12-Δ_49–64-G69R; 5, p12-E2-K5E-Δ_49–64-PM14; 6, p12-E2-ΔRRPSS-Δ_49–64-PM14.

FIG 3

Phosphorylation of the RRXS motif in the HPV-8 E2 peptide. MLV p12 chimeric viruses with the HPV-8 E2 phosphorylation motif insert and suppressor mutations were pelleted and run by SDS-PAGE on duplicate gels and were analyzed by Western blotting for phosphorylation (top) or for p12 (bottom). (A) Antiphosphoserine Western blot of viral pellets (rabbit anti-phosphoserine, catalog number 61-8100; Novex). (B) Rabbit anti-phospho-PKA substrate (RRXS*/T* motif) (catalog number 9624; Cell Signaling Technology) Western blot of viral pellets. Viruses containing the intact ₁₁RRXS₁₄ motif are indicated by a checkmark. p12 and p12-containing precursor polypeptide sizes are marked on the left, while molecular mass standards (in kilodaltons) are marked on the right.

FIG 4

LC-MS/MS identification of the p12 M63 serine 61 phosphorylation. (A) Peaks of nonphosphorylated (top) and phosphorylated (bottom) MLV p12 M63 ⁵⁵GEAPDPSPMAS⁶⁵ peptide. The orange serine was determined to be phosphorylated. (B) Fragmentation analysis of nonphosphorylated (top) and phosphorylated (bottom) ⁵⁵GEAPDPSPMAS⁶⁵ peptide. Ion peaks were labeled based on ion size calculations shown in panel D. Green labels indicate the full-length, unfragmented parent peptides. Ions with the characteristic 80-Da mass addition signaling phosphorylation are denoted with an asterisk (*). (C) Schematic of detected ions shown in panel B. Red “y” ions include the C terminus, blue “b” ions include the N terminus. (D) Monoisotopic ion mass calculations for the ⁵⁵GEAPDPSPMAS⁶⁵ peptide with (right) and without (left) the addition of the 79.96633-Da mass of the phosphorylation group on serine 61. Ions are 1⁺ charged, and the parent peptide is 2⁺ charged.

FIG 5

Tyrosine phosphorylation of p27 precursor protein. Viral samples collected from D17 and 293 cells, both expressing the mCAT receptor, were run by SDS-PAGE, and phosphorylation of tyrosine residues was detected via antiphosphotyrosine Western blotting. Viral precursor and mature protein sizes are indicated on the left, while molecular mass standards (in kilodaltons) are marked on the right. Bottom panel shows increased exposure to illuminate weak bands.

FIG 6

Activation of p12-M63-GFP chromatin binding. Full-length p12-GFP fusion proteins were compared to minimal binding motif constructs containing M63 versus I63. (A) Sequences of fusion proteins. (B) Quantification of GFP intensity overlapping the mitotic chromatin versus that in the cytoplasm; n > 15. Error bars are standard errors. Asterisks (*) indicate ratios that are statistically different from those of GFP using Student's t test (α = 0.001). Studies of GFP and p12_61–71-GFP-I63 were duplicated in a companion article in this issue (26) for comparison purposes.

FIG 7

Model for regulation of p12 chromatin tethering via S61 phosphorylation. (Left) MLV Gag precursor late domains recruit ESCRT proteins for budding. Proteolytic processing proceeds through the p27 (MA-p12) intermediate. PPPY late domain (yellow) phosphorylation is proposed to disrupt NEDD4 binding and allow repression of the chromatin binding motif (blue). (Center) After protease-mediated maturation, the capsid core forms, with the p12 N terminus (green) involved in proper core formation and stability and the p12 C terminus important for core uncoating. (Right) After reverse transcription and capsid core uncoating, the p12 N terminus (green) binds the PIC, containing capsid (light blue), integrase (purple), and BET (lime green), while the C terminus binds mitotic chromatin (represented by an individual nucleosome), to function in nuclear retention of the PIC. (Top right) Phosphorylation of S61 is proposed to disrupt binding of the p12 N-terminal PPPY motif to the p12 C-terminal chromatin binding motif, facilitating capsid core uncoating and relieving chromatin binding repression. (Bottom right) p12 I63, other C-terminal charge shifts, and p12 Δ are proposed to weaken the PPPY interaction with the p12 C-terminal chromatin tethering motif, rescuing the loss of the S61 phosphorylation switch.