nsp3bADRP

The SARS ADRP readily hydrolyzes the 1" phosphate group from Appr-1"-p in vitro demonstrating that it is an active enzyme . Another group validated this finding: both the SARS ADRP and the human coronavirus (HCoV)-229E counterpart were shown to dephosphorylate Appr-1"-p to ADP-ribose in a highly specific manner, the enzyme having no detectable activity on several other nucleoside phosphates. The crystal structure of the ADRP domain has been determined by us (the apo form) and by Egloff and co-workers in complex with ADP-ribose. They show that the putative active site and substrate-binding residues are conserved across its three structural homologues, yeast Ymx7, Archeoglobus fulgidus AF1521 and Er58 from E. coli. A notable exception is that proposed active site residue Asp90 in Ymx7 is an alanine in both the SARS ADRP (Ala50) and AF1521 (Ala44) One of its sequence homologue, yeast YBR022W, is a known phosphatase that acts on ADP ribose-1"-phosphate (Appr-1"-p). We proposed that the histidine in both enzymes proximal to the terminal 1″ phosphate of the substrate (His45 in ADRP and His39 in AF1521), might therefore be involved in catalysis. Alternatively, we suggested that the predominant nucleophile in the catalytic site may actually be an Asp or Glu in the conformationally flexible loop 101NAGEDIQ107 in SARS and the corresponding region in other coronaviral ADRPs . The former proposition was verified by site directed mutagenesis data in the HCoV-229E ADRP, which showed that residues Asn37, Asn40, His45, Gly44 and Gly48 are part of the active site in the SARS ADRP.

In coronaviruses, an early post infection event is the transcription of a nested set of sub-genomic mRNAs. Each sub-genomic mRNA contains a short 5'-terminal ‘leader’ sequence derived from the 5' end of the genome. The fusion of the two noncontiguous RNA segments is a poorly understood process. It is thought to be achieved by a discontinuous step in the synthesis of the minus-strand and involves transcription regulatory sequences. In eukaryotes, pre-tRNA splicing is initiated by cleavage at the splice site by an endonuclease. The resulting tRNA fragments are then ligated to yield mature tRNA that retains the 2’ phosphomonoester group at the splice site. Using NAD as an acceptor, a phosphotransferase removes the 2' phosphate to yield ADP-ribose-1"-2" cyclic phosphate (Appr>p). A cyclic phosphodiesterase then hydrolyzes Appr>p to yield Appr-1"-p. Finally, a phosphatase converts Appr-1"-p into ADP-ribose and releasing inorganic phosphate. Therefore the role of an ADRP in the coronavirus life cycle may closely parallel that in the eukaryotic tRNA splicing pathway. While the equivalent for the cyclic phosphodiesterase appears absent in the SARS proteome, the Appr-1"-p phosphatase (SARS ADRP) and an endonuclease (nsp15) are present. Characterization of an Appr-1"-pase-deficient HCoV-229E mutant revealed no significant effects on viral RNA synthesis and virus titer.

Recently, Egloff et al., have suggested that ADRP may primarily be a poly(ADP-ribose) binding (PAR-binding) module. PARylation occurs in compromised cells to trigger apoptosis. PAR polymerases (PARPs) are responsible for so tagging proteins. PARP is activated on recognizing nicked DNA, and it helps in DNA repair. It auto-PARylates itself, and in case of extreme DNA damage, gets overactivated and depletes the cell of its nucleotide pool. If ADRP binds PAR, then it can bind proteins that are PARylated, including PARP. Indeed, binding the latter may be most beneficial, since it can tether down this protein, slow down apoptosis, and prevent nucleotide depletion, prolonging viral replication and transcription in the infected cell.

The existence of the ADRP domain in all CoV nsp3s (as well as in several other viruses) argues for its critical role in the viral life cycle. Its function as an ADRP in recycling organic phosphate appears to be a dispensable function and does not correlate with the conservation of this domain. It appears that its role as an ADRP may be secondary to a more important role – such as its proposed role as a PAR-binding module. If so, the role of PAR-binding in the viral life cycle needs to be delineated by further experimental studies.

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