The conserved macrodomains of the non-structural proteins of Chikungunya virus and other pathogenic positive strand RNA viruses function as mono-ADP-ribosylhydrolases

Abstract

Human pathogenic positive single strand RNA ((+)ssRNA) viruses, including Chikungunya virus, pose severe health problems as for many neither efficient vaccines nor therapeutic strategies exist. To interfere with propagation, viral enzymatic activities are considered potential targets. Here we addressed the function of the viral macrodomains, conserved folds of non-structural proteins of many (+)ssRNA viruses. Macrodomains are closely associated with ADP-ribose function and metabolism. ADP-ribosylation is a post-translational modification controlling various cellular processes, including DNA repair, transcription and stress response. We found that the viral macrodomains possess broad hydrolase activity towards mono-ADP-ribosylated substrates of the mono-ADP-ribosyltransferases ARTD7, ARTD8 and ARTD10 (aka PARP15, PARP14 and PARP10, respectively), reverting this post-translational modification both in vitro and in cells. In contrast, the viral macrodomains possess only weak activity towards poly-ADP-ribose chains synthesized by ARTD1 (aka PARP1). Unlike poly-ADP-ribosylglycohydrolase, which hydrolyzes poly-ADP-ribose chains to individual ADP-ribose units but cannot cleave the amino acid side chain - ADP-ribose bond, the different viral macrodomains release poly-ADP-ribose chains with distinct efficiency. Mutational and structural analyses identified key amino acids for hydrolase activity of the Chikungunya viral macrodomain. Moreover, ARTD8 and ARTD10 are induced by innate immune mechanisms, suggesting that the control of mono-ADP-ribosylation is part of a host-pathogen conflict.

Nsp3 of coronaviruses: Structures and functions of a large multi-domain protein

“…Recently, it was demonstrated that macrodomains from several +ssRNA viruses (such as HEV, SARS-CoV, HCoV 229E, Venezuelan equine encephalitis virus (VEEV), and Chikungunya virus (CHIKV)) act as hydrolases removing mono-and/or poly(ADP-ribose) from mono-or poly(ADP-ribosyl)ated proteins, activities designated as de-mono-ADP-ribosylation (de-MARylation) and de-poly-ADP-ribosylation (de-PARylation), respectively (Li et al, 2016a;Fehr et al, 2016;Eckei et al, 2017;McPherson et al, 2017). The weak ADRP activity described for the X domain in the literature is most probably just a nonphysiological side reaction of de-MARylation and/or de-PARylation.…”

ADP‐ribosylation: new facets of an ancient modification

“…Some macrodomains have also evolved enzymatic activity and are capable of hydrolysing ADP‐ribosylation (see below) . As a consequence, macrodomain‐containing proteins are involved in a diverse set of cellular functions, such as chromatin remodelling and DNA‐damage repair, oxidative stress response, metabolic processes and pathogenic mechanisms . In addition to macrodomain, several other widely distributed domains have been described as readers for ADP‐ribosylation, such as the PAR‐binding zinc finger (PBZ) , the WWE (named after three of its conserved residues) , the oligonucleotide/oligosaccharide‐binding (OB) domain and the PAR‐binding motifs (PBM) which is abundant in DNA‐damage repair proteins .…”

“…This unique macrodomain binds nucleic acids, preferentially RNA, and is crucial for viral genome replication/transcription . Numerous other examples show that viral macrodomains affect virus replication and interferon‐response in humans . Viral macrodomains may act against mammalian PARPs that are known to possess antiviral activity .…”

“…Later on, homologous transferases and modification‐reversing hydrolytic enzymes have been discovered in organisms from all kingdoms of life . Moreover, many recent observations show how viral genomes evolved the genetic tools that enable them to modulate ADP‐ribosylation signalling of infected cells . ADP‐ribosylation seems to be particularly prominent in the highest organisms and it is best studied for the poly(ADP‐ribose) polymerase (PARP) superfamily of ART enzymes .…”

Quantitative Determination of MAR Hydrolase Residue Specificity In Vitro by Tandem Mass Spectrometry

“…Our lab and others developed a technique that uses phosphodiesterases to cleave protein-conjugated ADP-ribose to a phosphoribose tag that can be enriched and identified using established phosphoproteomic techniques 3,5,21–23,26–28 . Recently we have used this technique to confidently identify the sites of modification of the MARylated catalytic domain of PARP10 (PARP10 CD ) 15,27 , a MARylated substrate commonly used for in vitro MAR hydrolase assays 11–16 . We then quantified the intensity of these sites in samples incubated with or without our MAR hydrolase of choice (e.g., Chikungunya viral macrodomain) to unambiguously determine the residue specificity of the MAR hydrolase activity.…”