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 ( 61 SPMASRLRGRR 71 ) 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.T he murine leukemia virus (MLV) (genus Gammaretrovirus, family Retroviridae) Gag protein p12 is a multifunctional protein required in both early and late stages of viral replication. During the late stages of viral assembly, new virions must bud off their host cells, requiring membrane scission. The MLV p12 encodes a late-assembly (L) domain ( 31 PPPY 34 ) (1, 2), reported to interact with the host vesicular trafficking machinery, specifically the E3 ubiquitin ligases in the NEDD4 family, to facilitate Gag ubiquitylation and subsequent budding (3). In addition, the 25 DLL 27 motif in p12 has been reported to recruit clathrin to budding virions, which is required for correct particle morphogenesis and infectivity (4).In infected cells, two domains required for early replication were identified by mutational analysis: within the p12 N terminus at residues 10 to 24 (mutants PM5 to PM8) and within the p12 C terminus at residues 60 to 74 (mutants PM13 to PM15) (1, 5). The N terminus of p12 interacts with capsid (CA), ...

Repression of the Chromatin-Tethering Domain of Murine Leukemia Virus p12

“…5C). Tethering is also observed using the corresponding p12-M63-GFP minimal tethering domain (p12 61-71 -M63-GFP) (31). These results (i) show that the p12 C-terminal motif, from S61 through R71, is necessary and sufficient for the chromatin binding function of MLV p12 and (ii) uncovered that an N-terminal region is involved in suppressing this chromatin binding.…”

“…Details shown in Fig. 6 relating to the regulation of late viral functions and the role of phosphorylation are expanded in an accompanying article (31). The model is based on structural studies of the Rous sarcoma virus (RSV) Gag (Fig.…”

A common binding motif in the ET domain of BRD3 forms polymorphic structural interfaces with host and viral proteins

“…Interestingly, the NSD3 ET binding motif is flanked by lowcomplexity regions [DisMeta, (Huang et al, 2014)], including a Pro-rich sequence N-terminal to the ETBM (Supplementary Figure S4), that are predicted to be intrinsically-disordered. Recently it was proposed that BET proteins interact with MLV IN after p12-mediated nuclear retention of the viral PIC (Brzezinski et al, 2016a, Brzezinski et al, 2016b, Borrenberghs et al, 2019. Using FRET-based studies, the interaction of BET proteins with MLV IN was shown to cause alteration in the quaternary structure of IN within the PIC (Borrenberghs et al, 2019).…”

Murine leukemia virus p12 tethers the capsid-containing pre-integration complex to chromatin by binding directly to host nucleosomes in mitosis

“…It is not known whether p12 directly binds chromatin or if it interacts with another chromatin binding factor. Notably, fluorescence microscopy revealed that recombinant Mo-MLV p12 did not localise to mitotic chromatin in mammalian cells [ 19 ]. One possibility is that another viral protein is required for chromatin tethering.…”

“…Alternatively, the inability of recombinant Mo-MLV p12 to bind chromatin could be due to the absence of an essential post-translational modification. Previous studies have identified p12 as the main phosphorylated protein in Mo-MLV with the majority of phosphorylation occurring on serine-61 within the CTD [ 19 , 23 – 25 ]. In contrast with viral p12, recombinant Mo-MLV p12 was found to be non-phosphorylated in mammalian cells [ 19 ].…”