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 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 p12_70-74 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 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.

FIG 1

Sequences and viral titers of the MLV p12 PM15 mutants with alternate tethering domains. (A) MLV p12 wild-type protein sequence, alternate viral motif insert site at G49 (triangle), and the PM13 (purple), PM14 (red), and PM15 (green) mutation sites. (B) Alternate chromatin tethering domains inserted at G49 of p12. (C) Viral titer of each virus in D17 cells, measured by LacZ staining units (LSU) per nanogram of capsid (n = 3). All viruses were pseudotyped with VSV-G Env except the control, “No VSV-G.” Viral titers with asterisks (*) are significantly above the mutant (p12-M63-PM15) titer, using Student's t test with a Bonferroni correction for multiple tests (α = 0.01).

FIG 2

Isoleucine 63 rescue of the MLV p12 PM15 mutant. (A) MLV p12 C-terminal sequence with I63 and I63-PM15-suppressor-mutation-E56K notated. (B) Viral titer of each virus in D17 cells, measured as percent wild-type (p12-M63) LacZ staining units (LSU) per nanogram of capsid infected (n = 3). Viral titers with asterisks (*) are significantly below the wild-type titer (p12-M63, 7.6 × 10⁴ LSU), using Student's t test with a Bonferroni correction for multiple tests (α = 0.01). No VSV-G titer was <0.001% of the wild-type titer. (C) Viral reverse transcription of PM15 mutants was quantified via qPCR measurement of plus strand extension DNA products present 4 h postinfection (hpi). Samples were normalized to RPPH1 gene levels and then to wild-type (p12-M63) levels to generate ΔΔC_T values (y axis). Error bars are standard errors (n = 3). (D) Confocal images of p12-GFP fusion proteins, imaged during mitosis. (Left panels) Hoechst staining of DNA; (right panels) GFP. Scale bar, 10 μm. (E) Ratio of p12-GFP fusion protein intensity overlapping the mitotic chromatin to that in the cytoplasm, as seen in panel D. n ≥ 10. The asterisk (*) indicates a statistical difference from GFP using Student's t test with a Bonferroni correction for multiple tests (α = 0.01). Experiments whose results are shown in Fig. 2 to 4 are contemporaneous; images of the GFP, p12-I63, and p12-I63-PM15 are duplicated for comparison purposes.

FIG 3

PFV complementation of MLV p12 PM15. (A) MLV p12 C-terminal sequence with PFV insert. Modified residues are in red. (B) Viral titer of each virus in D17 cells, measured as percent wild-type (p12-M63) LacZ staining units (LSU) per nanogram of capsid (n = 3). Viral titers with asterisks (*) are significantly below the wild-type titer (p12-M63, 7.6 × 10⁴ LSU), using Student's t test with a Bonferroni correction for multiple tests (α = 0.01). No VSV-G titer was <0.001% of the wild-type level. (C) Confocal images of p12-GFP fusion proteins with the PFV insert, imaged during mitosis. (Left panels) Hoechst staining of DNA; (right panels) GFP. Scale bar, 10 μm. (D) Ratio of p12-GFP fusion protein intensity overlapping the mitotic chromatin to that in the cytoplasm, as seen in panel C. n ≥ 10. An asterisk (*) indicates a statistical difference from GFP using Student's t test with a Bonferroni correction for multiple tests (α = 0.01). Experiments whose results are shown in Fig. 2 to 4 are contemporaneous; images of the GFP, p12-I63, and p12-I63-PM15 are duplicated for comparison purposes.

FIG 4

KSHV LANA complementation of the MLV p12 PM15 mutant. (A) MLV p12 C-terminal sequence with KSHV LANA_1–32 and LANA_1–23 histone binding motif sequences. Modified residues are in red. (B) Confocal images of p12-GFP fusion proteins with the LANA inserts, imaged during mitosis. (Left panels) Hoechst staining of DNA; (right panels) GFP. Scale bar, 10 μm. (C) Ratio of p12-GFP fusion protein intensity overlapping the mitotic chromatin to that in the cytoplasm, as seen in panel B. n ≥ 10. An asterisk (*) indicates a statistical difference from GFP using Student's t test with a Bonferroni correction for multiple tests (α = 0.01). Experiments whose results are shown in Fig. 2 to 4 are contemporaneous; images of the GFP, p12-I63, and p12-I63-PM15 are duplicated for comparison purposes.

FIG 5

GFP fusion protein tethering of MLV p12-I63 protein truncations. (A) Diagram of p12-GFP fusion protein truncation sequences. Late domain and PM13 to PM15 positions are indicated. All truncations run to the C terminus of p12 (amino acid 84), except p12_61–71. (B) Confocal images of p12-GFP fusion protein truncations, imaged during mitosis. (Left panels) Hoechst staining of DNA; (right panels) GFP. Scale bar, 10 μm. (C) Ratio of p12-GFP fusion protein intensity overlapping the mitotic chromatin to that in the cytoplasm, as seen in panel B. n ≥ 10. All constructs with an asterisk (*) are statistically different from GFP using Student's t test (α = 0.001, with a Bonferroni correction for multiple tests). The experiment whose results are shown in the blue box represents an independent assay.

FIG 6

Comprehensive model of p12 early functions. (A) Structural comparison between RSV gag p2/p10 proteins and MLV gag p12 protein. The positions of the PPPY late domain and the C-terminal (putative) α-helices are indicated. (B and C, left to right) Alignment of viral proteins during budding and in immature virions. MLV p12-CA interactions are based on the RSV structure (33) (left). After protease processing of the Gag polyprotein, mature p12 makes N- to C-terminal contacts to induce the correct p12 conformation for assembly and stabilization of the capsid core (center). During mitosis, the N- to C-terminal contact must be disrupted to facilitate capsid core uncoating and free the C terminus to tether the PIC to the mitotic chromatin, for nuclear retention (right). p12 and capsid interactions are shown from the top down (B) and from the side (C).