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

Abstract

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 60 PSPMA 65 ), 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 25 DLLTEDPPPY 34 , which includes the late domain required for viral assembly. The p12 PM15 sequence (p12 70 RREPP 74 ) is critical for wild-type viral viability; however, virions bearing the PM15 mutation (p12 70 AAAAA 74 ) 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 p12 70 -74 in chromatin binding. Minimally, full-strength tethering was seen with only p12 61 SPIASRLRGRR 71 fused to GFP. These results indicate that the p12 C terminus alone is sufficient for chromatin binding and that the presence of the p12 25 DLLTEDPPPY 34 motif in the N terminus suppresses the ability to tether. IMPORTANCEThis 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 p12 61-71 . 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.T he p12 structural protein of murine leukemia virus (MLV) was recently identified as a critical chromatin tether for the early viral life cycle. MLV requires cells to undergo mitosis to gain nuclear entry and requires a mechanism to remain nuclear after mitosis is complete, since integration occurs after exit from mitosis (1, 2). p12 accomplishes this by binding the viral preintegration complex (PIC) with its N terminus (3) and nuclear chromatin with its C terminus (2-4). This p12 chromatin binding motif was shown to not affect MLV integration targeting to transcriptional start sites (4), and inte...

Phosphorylation Requirement of Murine Leukemia Virus p12

“…In addition to the PPPY motif's function in late domain budding, recent findings show that the p12 N-terminal 25 DLLTEDPP PY 34 motif is a repressor of the p12 C-terminal chromatin binding motif, suggesting that these two motifs are binding during infection (26). This dual function for the PPPY motif is supported by previous research indicating that it had both early and late defects in replication (1).…”

“…Phosphorylation of p12 can act directly through activating a tethering competent conformation or indirectly through releasing a repression domain. Deletion studies of p12 indicated that the N terminus of p12 acts as a repression domain of tethering (26). The role of phosphorylation to regulate tethering was examined with the minimal tethering domain 61 SPMASRLRGRR 71 -GFP fusion protein (Fig.…”

“…Interestingly, these motifs were able to rescue the budding of the p12 PPPY deletion even when placed into the matrix or nucleocapsid proteins; however, these mutants blocked early viral infection at a stage prior to reaching the nucleus, suggesting a second role for the p12 PPPY domain (25). Recent p12 truncation analysis unveiled the region including the PPPY motif to negatively regulate the C-terminal chromatin binding motif (26).…”

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).…”

“…During MLV infection, the viral integrase associates with the reversetranscribed viral DNA as part of a nucleoprotein complex called the pre-integration complex (PIC), which also contains other viral proteins including p12 and capsid (CA), along with host proteins including barrier-to-autointegration factor (BAF) (Prizan-Ravid et al, 2010, Fassati and Goff, 1999, Lee and Craigie, 1998. The p12 protein tethers the PIC to mitotic chromatin and ensures nuclear retention through chromatin tethering (Brzezinski et al, 2016b, Elis et al, 2012, Schneider et al, 2013, Wight et al, 2012. However, subsequent interaction with BET proteins is an important determinant for genomic targeting of the PIC towards transcription start sites (TSS) and CpG islands (Wu et al, 2003).…”