The Conserved Coronavirus Macrodomain Promotes Virulence and Suppresses the Innate Immune Response during Severe Acute Respiratory Syndrome Coronavirus Infection

Abstract

ADP-ribosylation is a common posttranslational modification that may have antiviral properties and impact innate immunity. To regulate this activity, macrodomain proteins enzymatically remove covalently attached ADP-ribose from protein targets. All members of the Coronavirinae, a subfamily of positive-sense RNA viruses, contain a highly conserved macrodomain within nonstructural protein 3 (nsp3). However, its function or targets during infection remain unknown. We identified several macrodomain mutations that greatly reduced nsp3’s de-ADP-ribosylation activity in vitro. Next, we created recombinant severe acute respiratory syndrome coronavirus (SARS-CoV) strains with these mutations. These mutations led to virus attenuation and a modest reduction of viral loads in infected mice, despite normal replication in cell culture. Further, macrodomain mutant virus elicited an early, enhanced interferon (IFN), interferon-stimulated gene (ISG), and proinflammatory cytokine response in mice and in a human bronchial epithelial cell line. Using a coinfection assay, we found that inclusion of mutant virus in the inoculum protected mice from an otherwise lethal SARS-CoV infection without reducing virus loads, indicating that the changes in innate immune response were physiologically significant. In conclusion, we have established a novel function for the SARS-CoV macrodomain that implicates ADP-ribose in the regulation of the innate immune response and helps to demonstrate why this domain is conserved in CoVs.

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

“…Our group has shown that the X domain (Mac1) is dispensable for RNA replication in the context of a SARS-CoV replicon (Kusov et al, 2015). Recently, evidence accumulated showing that the X domain plays a role in counteracting the host innate immune response (Eriksson et al, 2008;Kuri et al, 2011;Fehr et al, , 2016.…”

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 .…”

“…In coronaviruses, the MacroD‐type macrodomain is a part of the multidomain nonstructural protein 3 (nsP3). It has been shown that this macrodomain promotes virulence and suppresses the innate immune response during severe acute respiratory syndrome (SARS) coronavirus infection . In addition to the MacroD‐type of macrodomains, a highly diverged macrodomain SUD‐M was found as a part of the nsP3 in SARS coronaviruses.…”

“…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 .…”

ADP-Ribosylation Reactions in Animals, Plants, and Bacteria

“…Furthermore, an ADP-ribose-1'-phosphate phosphatase mutant of the nsp3 Macrodomain of murine coronavirus MHV-A59 showed reduced replication in the liver and impaired development of liver pathology by inhibiting the expression of inflammatory cytokines [23]. The viral nsp3 Macrodomain of Severe Acute Respiratory Syndrome (SARS) coronavirus has been demonstrated to modulate and to facilitate escape from the immune response [24]. Again, a Macrodomain mutant that interferes with ADP-ribose-1'-phosphate phosphatase activity showed increased sensitivity to the IFNα-mediated antiviral response [25].…”