Macro domains are found in organisms from all kingdoms of life, and it is thus assumed that they play fundamental and perhaps universal roles in the perpetuation or expression of genetic information. Although the sequence signatures of these domains were detected a while ago, their function remained elusive until a genome-wide screen of yeast proteins showed that the yeast macro domain protein Poa1p could carry out a specialized ADP-ribose phosphoesterase activity (22
). Poa1p is not closely related to any known phosphatase, and it is quite specific for ADP-ribose 1"-phosphate, a by-product of tRNA splicing pathway, with a turnover constant of 1.7 min−1
). Such a value is rather low for an enzyme putatively involved in regulating the concentration of a metabolite, as the enzyme might be unable to cope efficiently with abrupt metabolite concentration variations. Subsequently, the crystal structure of an archaeal macro domain revealed that it shares structural features with P-loop-containing nucleotide triphosphate hydrolases (2
Specific ADP-ribose 1"-phosphatase activity has been recently described for three positive-strand RNA viruses of the Coronavirus
), also with a modest turnover constant of 5 to 20 min−1
. Our present study confirms that viral macro domains are indeed able to hydrolyze ADP-ribose 1"-phosphate. However, in our opinion, the available evidence does not sufficiently support a scenario in which this phosphatase activity would represent the sole biologically relevant function of the macro domain in vivo. In line with this idea, the enzymatic activity of the coronavirus macro domain is dispensable for viral RNA synthesis and virus reproduction under cell culture conditions (30
). There are several lines of evidence that suggest functions other than ADP-ribose 1"-phosphatase for viral macro domains. First, viral (and cellular) macro domains are not closely related to other known phosphatases. The method of the discovery of the activity might well have been misleading. The phosphatase activity was discovered by a genome-wide search for this particular activity and not by a phenotype-guided search. Deletion of the POA1
gene in yeast had no detectable phenotype under various growth conditions, suggesting a role in a temporary or minor pathway (37
). The macro domain-containing viruses replicate in the cytoplasm of the infected cell and do not appear to use nuclear tRNA splicing pathways that generate ADP-ribose 1"-phosphate, the putative substrate of macro domains. To our knowledge, there is nothing to suggest that such a substrate would be generated during RNA virus replication.
Second, even key amino acids close to the putative catalytic center are not conserved, suggesting different specific activities for the large variety of viral and cellular macro domains. We are left with questions, such as why should a conserved catalytic activity thought to be involved in metabolic regulation be based on nonconserved catalytic residues? Why do the catalytic efficiencies observed for these structurally and evolutionary conserved macro domains differ so widely?
Third, in animal cells, macro domains are often found associated physically with PARP, an enzyme involved in poly(ADP-ribose) metabolism. The recent demonstration that the archaeal macro domain AF1521 is an ADP-ribose and poly(ADP-ribose) binding module is in full agreement with our corresponding proposition for such a function for viral macro domains (17
). In the case of AF1521, ADP-ribose is the best inhibitor (50% inhibitory concentration of ~30 μM) of the ADP-ribose 1"-phosphatase activity and the KD
for ADP-ribose is around 126 nM, i.e., ~200-fold lower than that of the SARS-CoV macro domain studied here (KD
~24 μM). The discovery of poly(ADP-ribose) binding (17
) is important in that it casts a new light on the possible role of macro domains also in the viral world. Our work shows that the ADP-ribose binding site adopts two conformations, in which the ADP-ribose (putative product) clearly fits much better than ADP-ribose 1"-phosphate (putative substrate). Altogether, we favor the hypothesis that the hydrolysis of ADP-ribose 1"-phosphate might well be one result of evolution, whereas poly(ADP-ribose) binding might be another.
These considerations prompted us to assay poly(ADP-ribose) binding by several viral macro domains. Indeed, they were subsequently found to be robust poly(ADP-ribose) binding modules and, interestingly, the poorly active SFV ADP-ribose 1"-phosphatase turned out to be the most efficient free and PARP-1-linked poly(ADP-ribose) binding module relative to its fellow coronavirus and HEV counterparts (Fig. ). The structural model of bound di-ADP-ribose indicates that such a unit could bind to a groove present on the surface of the macro domain, lined with conserved amino acid residues in coronaviruses (Fig. ).
There are a number of questions concerning the role of these macro domains during the course of a viral infection. In alphaviruses, involvement in the cell response to infection has also been proposed (8
). The macro domain-containing viruses have a cytoplasmic replication cycle, and their macro domain proteins do not appear to enter into the nucleus (reviewed in reference 34
), raising the question of whether or not the nuclear PARP-1 activity is relevant for their functions. The alphavirus Sindbis virus was one of the first viruses shown to induce cell apoptosis during fusion and entry. Membrane depolarization induces a cascade of events involving many partners such as nuclear factor κB and various phospholipases leading to NAD+
and ATP depletion, activation of the nuclear PARP-1, and eventual cell death (reference 26
and references therein). Thus, the infected cell sends signals to the nucleus to regulate protein function by poly(ADP-ribosyl)ation in response to the infection. Even though PARP-1 is a nuclear enzyme, cross talk with the mitochondria has been demonstrated (reference 36
and references therein). Thus, the presence of the macro domain in cytoplasmic viral proteins raises the question about the existence of a cytoplasmic poly(ADP-ribosyl)ated messenger. One possibility is that this poly(ADP-ribosyl)ated messenger would be exported from the nucleus and intercepted by the viral macro domain, perhaps to stop ATP depletion and maintain the nucleotide pools required for active viral RNA synthesis (26
). There are 17 members of the PARP family in the human genome, and the majority remains completely uncharacterized with respect to function and localization (28
). Of the characterized PARPs, at least vault PARPs (VPARP, also known as PARP-4) and tankyrases are partially localized in the cytoplasm (27
). Interestingly, one of the PARP family members was originally identified as the zinc finger antiviral protein (11
). It is also active against alphaviruses (3
), but its putative PARP activity remains uncharacterized. It is an intriguing possibility that one or several of the novel PARPs might be located in the cytoplasm and be the target of viral macro domains, suggesting that the poly(ADP-ribose) binding function identified here may lead to the discovery of novel cell-virus interaction pathways.