Viral Macro Domains Reverse Protein ADP-Ribosylation

Abstract

ADP-ribosylation is a posttranslational protein modification in which ADP-ribose is transferred from NAD(+) to specific acceptors to regulate a wide variety of cellular processes. The macro domain is an ancient and highly evolutionarily conserved protein domain widely distributed throughout all kingdoms of life, including viruses. The human TARG1/C6orf130, MacroD1, and MacroD2 proteins can reverse ADP-ribosylation by acting on ADP-ribosylated substrates through the hydrolytic activity of their macro domains. Here, we report that the macro domain from hepatitis E virus (HEV) serves as an ADP-ribose-protein hydrolase for mono-ADP-ribose (MAR) and poly(ADP-ribose) (PAR) chain removal (de-MARylation and de-PARylation, respectively) from mono- and poly(ADP)-ribosylated proteins, respectively. The presence of the HEV helicase in cis dramatically increases the binding of the macro domain to poly(ADP-ribose) and stimulates the de-PARylation activity. Abrogation of the latter dramatically decreases replication of an HEV subgenomic replicon. The de-MARylation activity is present in all three pathogenic positive-sense, single-stranded RNA [(+)ssRNA] virus families which carry a macro domain: Coronaviridae (severe acute respiratory syndrome coronavirus and human coronavirus 229E), Togaviridae (Venezuelan equine encephalitis virus), and Hepeviridae (HEV), indicating that it might be a significant tropism and/or pathogenic determinant.

Importance: Protein ADP-ribosylation is a covalent posttranslational modification regulating cellular protein activities in a dynamic fashion to modulate and coordinate a variety of cellular processes. Three viral families, Coronaviridae, Togaviridae, and Hepeviridae, possess macro domains embedded in their polyproteins. Here, we show that viral macro domains reverse cellular ADP-ribosylation, potentially cutting the signal of a viral infection in the cell. Various poly(ADP-ribose) polymerases which are notorious guardians of cellular integrity are demodified by macro domains from members of these virus families. In the case of hepatitis E virus, the adjacent viral helicase domain dramatically increases the binding of the macro domain to PAR and simulates the demodification activity.

FIG 1

Proteins relevant to this study. Predicted macro domain (amino acids [aa] 789 to 902) and Hel domain (amino acids 974 to 1185) inside ORF1 of the HEV genome (MeT, methyltransferase; Y, Y domain; PCP, papain-like cysteine protease; V, hypervariable proline-rich hinge; macro: macro domain; Hel, helicase; RdRP, RNA-dependent RNA polymerase). Rectangles show the produced proteins, as indicated. (B) Coomassie blue-stained SDS-PAGE gel showing the purified macro domain (macro-HEV), Hel (Hel-His-Trx HEV), macro-Hel, VEEV macro domain, SARS-CoV macro domain, Tetrahymena thermophila PARG, PARP5a catalytic domain (PARP5 human), and PARP15 catalytic domain (PARP15 human). (C) Structure-based sequence alignment of selected macro domains. Red rectangles, confirmed A1″Pase (I) or de-MARylation (II) activity; gray rectangles, no activity; blank rectangle, not tested. Activity data are based on the results of this study and previously published data (2). Macro domains carrying both A1″Pase and de-MARylation activities are further divided into MacroD2- and c6orf130-like subgroups, based on sequence conservation and catalytic mechanisms. Asterisks indicate important amino acids for PAR binding and/or de-MARylation activity of the MacroD2-like subgroup. E. coli, Escherichia coli; Trx, thioredoxin.

FIG 2

PAR binding studies. ³²P-labeled PARylated PARP5a or free PAR was incubated with decreasing concentrations of proteins, dot blotted, and imaged. (A) Binding of the macro domain, Hel, and macro-Hel of HEV to PARylated PARP5a and free PAR. (B) Effects of mutations on the binding of macro-Hel to free PAR. (C) Binding of the HEV macro domain, macro-Hel (HEV), and the VEEV macro domain to free PAR. (D) Poly(ADP-ribose) competitively inhibits the binding of Cy3-labeled RNA to macro-Hel. WT, wild type.

FIG 3

Effects of HEV macro-Hel and other macro domains on PARylated PARP5 and PARP 1 and on MARylated PARP15 and PARP10. SDS–15% PAGE (upper) and 7 M urea–20% PAGE (lower) autoradiography showing the following: de-PARylation of the PARP5a catalytic domain (PARP5cd) by T. thermophila PARG, the macro domain, Hel (Hel-His-Trx), macro-Hel wild type and mutants G50A, G48S-G49S-G50A, and G48S-G49S (A); de-MARylation of the PARP15 catalytic domain by the human PARG, the macro domain, Hel, macro-Hel as well as by the VEEV and SARS macro domains on mono-ADP-ribosylated. Identification of ADP-ribose was based on comigration with cold ADP-ribose detected using UV shadowing. (C) De-MARylation of the PARP10 catalytic domain by different macro domains as observed by SDS-PAGE with Coomassie blue staining (CB) or autoradiography (³²P). (D) De-PARylation of PARP1 by different macro domains as observed by SDS-PAGE with Coomassie blue staining (CB) or autoradiography (³²P). Quantifications of ³²P signal removed are shown above. Error bars indicate standard deviations (n = 2). ADPr, ADP-ribose.

FIG 4

Time course of macro domain and macro-Hel activity on mono-ADP-ribosylated PARP15 (A) and poly(ADP)-ribosylated PARP5a (B) catalytic domains. The auto-ADP-ribosylated PARP5a or -15 catalytic domain was incubated with 100 nM macro domain or macro-Hel at 30°C for 1 h or 2 h. PSL-BG, photo-stimulated luminescence counts minus background counts.

FIG 5

Effect on HEV replication of mutations within the macro-Hel domain. Huh7 cells were transfected with HEV genotype 1 Sar55/S17/luc wild-type (WT) or capped mutant replicon RNA. Luciferase activity was measured after 72 h of incubation at 35°C.

FIG 6

The mode of ADP-ribose coordination in the VEEV macro domain pocket and proposed catalytic mechanism for de-MARylation activity. (A) Superimposition of VEEV macro domain (Protein Data Bank [PDB] accession number 3GQO) with human MacroD2 (PDB accession number 4IQY). (B) Close-up view of the 2.6Å-resolution X-ray structure of the VEEV macro domain in complex with ADP-ribose. ADP-ribose is shown with the corresponding electron density map (light gray). Hydrogen bonds between protein and ADP-ribose are indicated by dashed lines (dark gray). The H₂O molecule is coordinated by hydrogen bonds to the 1″-OH, and oxygen from the α-phosphate is shown. (C) Proposed scheme for a catalytic mechanism (see the text for details). The 2mF_o − DF_c electron density map is contoured at 1.0 σ. The figure was generated using PyMOL.