Mono-ADP-ribosylation by ARTD10 restricts Chikungunya virus replication by interfering with the proteolytic activity of nsP2

Abstract

24 A subset of intracellular mono-ADP-ribosyltransferases diphtheria toxin-like (ARTDs, aka 25 mono-PARPs) is induced by type I interferons. Some of these mono-ARTDs feature antiviral 26 activity while certain RNA viruses, including Chikungunya virus (CHIKV), encode mono-ADP-27 ribosylhydrolases, suggesting a role for mono-ADP-ribosylation (MARylation) in host-virus 28 conflicts. CHIKV expresses four non-structural proteins (nsP1-nsP4), with nsP3 containing a 29 macrodomain that hydrolyzes and thereby reverses protein MARylation in vitro and in cells. 30 This de-MARylation activity is essential as hydrolase inactivating mutations result in 31 replication defective virus. However, the substrates of MARylation during CHIKV infection 32 are unknown and thus it is unclear how the macrodomain contributes to virus replication 33 and how mono-ARTD-dependent MARylation confers antiviral immunity. We identified 34 ARTD10 and ARTD12 as restriction factors for CHIKV replication in a catalytic activity-35 dependent manner. CHIKV replication requires processing of the non-structural polyprotein 36 nsP1-4 by the nsP2-encoded protease and the assembly of the four individual nsPs into a 37 functional replication complex. Expression of ARTD10 and ARTD12 resulted in a reduction 38 of processed nsPs. Similarly, MAR hydrolase inactive CHIKV replicon mutants revealed a 39 decrease in processed nsPs, comparable to an nsP2 protease defective mutant. This 40 suggested that the macrodomain contributes to nsP2 protease activity. In support, a 41 hydrolase-deficient virus was complemented by a protease-deficient virus. We 42 hypothesized that MARylation regulates the proteolytic function of nsP2. Indeed, we found 43 that nsP2 is MARylated by ARTD10. This inhibited nsP2 protease activity, thereby preventing 44 polyprotein processing and consequently virus replication. This inhibition was antagonized 45 by the MAR hydrolase activity of nsP3. Together, our findings provide a mechanistic 46 explanation for the need of the viral MAR hydrolase for efficient replication of CHIKV. 47 Author Summary 48 Infectious diseases still pose major health threats. Especially fast evolving viruses find ever 49 new strategies to manipulate the immune response. With climate warming and increased 50 human mobility vector-borne pathogens like Chikungunya virus (CHIKV) spread and cause 51 world-wide epidemics. Beyond the acute phase, CHIKV patients regularly suffer from chronic 52 rheumatism. This entails a decline in life quality and an economic burden. To date no drugs 53 are approved and the mode of pathogenesis remains elusive. Here we describe a mechanistic 54 function of the CHIKV nsP3 macrodomain. We found that the viral nsP2 is mono-ADP-55 ribosylated interfering with its auto-proteolytic function. The nsP3 macrodomain removes this 56 modification and restores the protease activity that is essential for replication. Because 57 macrodomains are highly conserved they might represent broad antiviral targets. 58 59 Introduction 60 Upon viral infection host cel...

The Viral Macrodomain Counters Host Antiviral ADP-Ribosylation

“…More recently, it was shown that mutations V33E and D10A of the CHIKV macrodomain, which likely impair both ADP-ribose binding and hydrolysis [9,25], affected polyprotein processing by the nsP2 protease. In vitro, it was shown that the protease could be ADP-ribosylated by PARP10, that protease activity was altered by ADP-ribosylation, and that the macrodomain could reverse this modification [33]. It will be of great interest to determine if nsP2 is ADP-ribosylated during infection.…”

ADP-ribose and analogues bound to the deMARylating macrodomain from the bat coronavirus HKU4

“…For example, MARylation of G3BP1, a major SG component, was lost upon overexpression of CHIKV nsp3 (71). Other potential substrates are viral nonstructural proteins, such as the CHIKV nsp2 viral protease, which is MARylated by PARP10 and activated by deMARylation (72).…”

ADP-ribosylation of RNA and DNA: fromin vitrocharacterization toin vivofunction

“…In addition, PARP inhibitors enhance the replication of SARS-CoV and decrease interferon production during infection with a macrodomain mutant virus ( 140 ). Similarly, for CHIKV, the ADP-ribosylhydrolase activity appears essential for viral replication and virulence ( 141 , 142 ). Therefore, the viral macrodomains are thought to play essential roles in infectivity.…”

“…The NAD + cap blocks efficient translation of RNAs ( 80 ) and the structurally similar ADP-ribose cap could thus impair the translation of viral RNA. A recent study showed that PARP10 overexpression leads to decreased levels of processed viral non-structural proteins in cells ( 142 ), which could be due to impaired processing of the polyprotein as suggested, or due to lower protein translation from a potentially ADP-ribosylated viral RNA. Thus, viral macrodomains may have evolved to antagonize MARylation executed by PARPs that are responsive to interferon signalling.…”