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ADP-Ribosylhydrolase Activity of Chikungunya Virus Macrodomain Is Critical for Virus Replication and Virulence

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ADP-Ribosylhydrolase Activity of Chikungunya Virus Macrodomain Is Critical for Virus Replication and Virulence ADP-ribosylhydrolase activity of Chikungunya virus macrodomain is critical for virus replication and virulence Robert Lyle McPhersona,1, Rachy Abrahamb,1, Easwaran Sreekumarb,1,2, Shao-En Ongc, Shang-Jung Chenga, Victoria K. Baxterb,d, Hans A. V. Kistemakere, Dmitri V. Filippove, Diane E. Griffinb,3, and Anthony K. L. Leunga,f,3 aDepartment of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205; bW. Harry Feinstone Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205; cDepartment of Pharmacology, University of Washington, Seattle, WA 98195; dDepartment of Molecular and Comparative Pathobiology, School of Medicine, Johns Hopkins University, Baltimore, MD 21205; eDepartment of Bio-organic Synthesis, Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands; and fDepartment of Oncology, School of Medicine, Johns Hopkins University, Baltimore, MD 21205 Contributed by Diane E. Griffin, January 5, 2017 (sent for review October 20, 2016; reviewed by William Lee Kraus and Susan R. Weiss) Chikungunya virus (CHIKV), an Old World alphavirus, is trans- N-terminal domain of nsP2 has helicase, ATPase, GTPase, mitted to humans by infected mosquitoes and causes acute rash methyltransferase, and 5′ triphosphatase activity. nsP4 is the and arthritis, occasionally complicated by neurologic disease and RNA-dependent RNA polymerase (4). chronic arthritis. One determinant of alphavirus virulence is non- The function of nsP3 has been more enigmatic. nsP3 is found structural protein 3 (nsP3) that contains a highly conserved MacroD- in replication complexes and cytoplasmic nonmembranous type macrodomain at the N terminus, but the roles of nsP3 and the granules. It induces the membrane remodeling necessary for the macrodomain in virulence have not been defined. Macrodomain is a formation of cytoplasmic vacuoles and regulates RNA synthesis conserved protein fold found in several plus-strand RNA viruses that in a cell type-dependent manner (5). In vivo, nsP3 is a determinant binds to the small molecule ADP-ribose. Prototype MacroD-type of neurovirulence in mice (6–10). nsP3 has three major domains, a macrodomains also hydrolyze derivative linkages on the distal ribose highly conserved N-terminal macrodomain, a central zinc-binding ring. Here, we demonstrated that the CHIKV nsP3 macrodomain is region, and a poorly conserved, unstructured, acidic, and highly MICROBIOLOGY able to hydrolyze ADP-ribose groups from mono(ADP-ribosyl)ated phosphorylated C-terminal domain. The C-terminal domain inter- proteins. Using mass spectrometry, we unambiguously defined its acts with and regulates the function of many cellular proteins to substrate specificity as mono(ADP-ribosyl)ated aspartate and impact signaling, viral replication, and stress granule assembly glutamate but not lysine residues. Mutant viruses lacking hydro- (11–15). The roles of the N-terminal macrodomain and central lase activity were unable to replicate in mammalian BHK-21 cells or zinc-binding region are less well characterized. Aedes albopictus mosquito cells and rapidly reverted catalytically Macrodomains are a conserved protein fold observed from inactivating mutations. Mutants with reduced enzymatic activity archaea to higher eukaryotes characterized by their binding to had slower replication in mammalian neuronal cells and reduced the small molecule ADP-ribose and its derivatives (16–18). virulence in 2-day-old mice. Therefore, nsP3 mono(ADP-ribosyl)hy- Macrodomains are part of the nsPs in a subset of positive-strand drolase activity is critical for CHIKV replication in both vertebrate hosts and insect vectors, and for virulence in mice. Significance macrodomain | ADP-ribosylation | mass spectrometry | virulence | viral replication Viral outbreaks can be sudden and result in devastating social and economic consequences. Chikungunya virus (CHIKV) out- breaks of rash, arthritis, and neurologic disease have occurred lphaviruses are members of the Togaviridae family of ico- in Africa, Asia, Europe, and the Americas. There are no licensed sahedral, enveloped, message-sense RNA viruses that are A vaccines to prevent or drugs to treat CHIKV infection, and the maintained in a natural cycle between vertebrate and in- mechanisms of disease generation are poorly understood. vertebrate hosts. Alphavirus infection can result in human dis- Here, we identify a macrodomain-containing CHIKV protein ease with viruses originating from the “Old World” primarily “ ” with enzymatic ADP-ribosylhydrolase activity that is critical for causing rash and arthritis and New World causing encephalitis. virulence in mammals and define the substrate specificity of However, recent strains of Chikungunya virus (CHIKV), an “Old ” this enzyme. Given the conservation of viral macrodomains in World alphavirus, cause neurologic disease as well as rash and all alphaviruses and coronaviruses, our study may guide the arthritis (1). CHIKV originates from sub-Saharan Africa but has design of small molecule chemical compounds that block hy- spread to Southeast Asia, Europe, and more recently the Americas, drolase activity for treatment of a range of virus infections. where its transmission by Aedes aegypti and Aedes albopictus vectors has the potential to cause infection in tens of millions of people (1). Author contributions: R.L.M., R.A., E.S., D.E.G., and A.K.L.L. designed research; R.L.M., There are no approved therapeutics for CHIKV partly because of a R.A., E.S., S.-E.O., S.-J.C., and V.K.B. performed research; H.A.V.K. and D.V.F. contributed poor understanding of the basic molecular underpinnings of its in- new reagents/analytic tools; R.L.M., R.A., E.S., S.-E.O., D.E.G., and A.K.L.L. analyzed data; and R.L.M., D.E.G., and A.K.L.L. wrote the paper. fection and replication cycle. ′ Reviewers: W.L.K., The University of Texas Southwestern Medical Center at Dallas; and The 5 ORF of the alphaviral genome encodes four nonstructural S.R.W., University of Pennsylvania. proteins (nsPs) that function in replication of the viral RNA and The authors declare no conflict of interest. production of the subgenomic RNA (2, 3). nsPs are translated as 1R.L.M., R.A., and E.S. contributed equally to this work. two polyproteins (nsP123 and nsP1234) that form replication 2Present address: Viral Disease Biology Program, Rajiv Gandhi Centre for Biotechnology, complexes tethered to cytoplasmic vacuoles formed from modified Thiruvananthapuram, Kerala 695014, India. endosomal membranes. The polyproteins are processed into indi- 3 To whom correspondence may be addressed. Email: [email protected] or dgriffi6@ vidual proteins by a papain-like protease in the C-terminal portion jhmi.edu. of nsP2. nsP1 has methyl and guanylytransferase activities, is pal- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. mitoylated, and binds the replication complex to membranes. The 1073/pnas.1621485114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1621485114 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 RNA viruses, including alphaviruses, coronaviruses, and hepa- A MD B titis E virus (16–18). Study of viral macrodomain function has provided conflicting results with respect to their importance for Input +Buffer+MacroD2 +nsP3 Time (min) 0125 10 30 60 120 75 MacroD2 +Buffer virus replication. In general, viruses with mutations in the ADP- 50 – CD ribose binding site of the macrodomain replicate well in most 37 +MacroD2 tissue culture cells, but often exhibit attenuated replication in 25 PARP10CD – MD PARP10 differentiated cells and decreased virulence in vivo (10, 19 23). 20 +nsP3 – For instance, mutations of conserved residues in the ADP-ribose nsP3MD Autoradiograph 15 100 binding region of the Sindbis virus macrodomain impair replica- Total Protein tion and RNA synthesis in neurons and attenuate neurovirulence in mice (10). Similarly, mutation of the macrodomain of coronavirus 50 +Buffer 32 PP-ARP10 CD +MacroD2 nsP3 at a conserved site attenuates virulence and replication in +nsP3MD mice and affects induction of and sensitivity to IFN and in- P-signal removed 32 % 0 flammatory cytokines (19, 20, 22, 23). 100 0 40 80 120 Viral macrodomains are of the MacroD subfamily, which in- Time (min) 80 cludes archaeal Af1521 and human MacroD2. Recently, our C – nsP3MD group and others have found that some MacroD-type macro- 1.5 + nsP3MD 60 domains bind and hydrolyze ADP-ribose monomers conjugated 1.0 to the side chains of protein residues, a posttranslational modi- 40 fication known as ADP-ribosylation (23–27). ADP-ribose groups P-signal removed 0.5 32 20 can be attached singly as mono(ADP-ribose) (MAR) or in branched % Relative Intensity 0.0 or linear polymeric chains as poly(ADP-ribose) (PAR) by the enzy- 0 VVR matically active members of a family of 17 ADP-ribosyltransferases, MD E Input TLDAAR GVYFAR commonly known as poly(ADP-ribose) polymerases (PARPs) +Buffer +nsP3 +MacroD2 NTGEFQEVVR AFYD (28–30). ADP-ribosylation involves the transfer of ADP-ribose PSGLPGRSPDT D + LAE LAENTGEFQ NATVYGK from NAD onto a range of amino acids, including aspartate VSHPLLQQQYELYR (D), glutamate (E), and lysine (K) (30–32). Until recently, it has ASPD not been possible to identify the site of ADP-ribosylation by Fig. 1. CHIKV nsP3MD removes ADP-ribose from MARylated PARP10CD. mass spectrometry (MS) (reviewed in refs. 31 and 33). Sequence (A and B) Total protein stain, autoradiography, and quantification of alignment of the CHIKV macrodomain with the known human 32P-MARylated PARP10CD demodification reactions by MacroD2 or CHIKV MD MAR hydrolase MacroD2 revealed that the residues required for nsP3 at 1 h time-point (A) and over a time course of 2 h (B). (C) Quanti- catalysis are conserved (refs. 25 and 26 and SI Appendix, Fig. S1). fication of LC-MS extracted ion chromatograms for phosphoribosylated PARP10CD peptides, where modified residues are denoted in red. In this study, we determined that the CHIKV macrodomain possesses MAR hydrolase activity and unambiguously identified its substrate specificity by MS.
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