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

Murine leukemia virus (MLV) p12, encoded within Gag, binds the viral preintegration complex (PIC) to the mitotic chromatin. This acts to anchor the viral PIC in the nucleus as the nuclear envelope re-forms postmitosis. Mutations within the p12 C terminus (p12 PM13 to PM15) block early stages in viral replication. Within the p12 PM13 region (p12 60PSPMA65), our studies indicated that chromatin tethering was not detected when the wild-type (WT) p12 protein (M63) was expressed as a green fluorescent protein (GFP) fusion; however, constructs bearing p12-I63 were tethered. N-terminal truncations of the activated p12-I63-GFP indicated that tethering increased further upon deletion of p12 25DLLTEDPPPY34, which includes the late domain required for viral assembly. The p12 PM15 sequence (p12 70RREPP74) is critical for wild-type viral viability; however, virions bearing the PM15 mutation (p12 70AAAAA74) with a second M63I mutant were viable, with a titer 18-fold lower than that of the WT. The p12 M63I mutation amplified chromatin tethering and compensated for the loss of chromatin binding of p12 PM15. Rescue of the p12-M63-PM15 nonviable mutant with prototype foamy virus (PFV) and Kaposi's sarcoma herpesvirus (KSHV) tethering sequences confirmed the function of p1270-74 in chromatin binding. Minimally, full-strength tethering was seen with only p12 61SPIASRLRGRR71 fused to GFP. These results indicate that the p12 C terminus alone is sufficient for chromatin binding and that the presence of the p12 25DLLTEDPPPY34 motif in the N terminus suppresses the ability to tether. IMPORTANCE: This study defines a regulatory mechanism controlling the differential roles of the MLV p12 protein in early and late replication. During viral assembly and egress, the late domain within the p12 N terminus functions to bind host vesicle release factors. During viral entry, the C terminus of p12 is required for tethering to host mitotic chromosomes. Our studies indicate that the p12 domain including the PPPY late sequence temporally represses the p12 chromatin tethering motif. Maximal p12 tethering was identified with only an 11-amino-acid minimal chromatin tethering motif encoded at p1261-71 Within this region, the p12-M63I substitution switches p12 into a tethering-competent state, partially rescuing the p12-PM15 tethering mutant. A model for how this conformational change regulates early versus late functions is presented.

Phosphorylation Requirement of Murine Leukemia Virus p12.

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.

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

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.