The DUF1874 family was investigated for anti-CRISPR function because it is widespread in archaeal viruses and plasmids, bacteriophages and prophages

"Here, we investigated the DUF1874 protein, which is conserved and widespread in a variety of archaeal viruses and plasmids, bacteriophages and prophages (Extended Data Fig.1), for an Acr function."

The original authors interpret viral DUF1874 proteins as anti-CRISPR enzymes; cA4 cleavage is established, whereas family-wide physiological deployment is now disputed.

"Here we identify a new family of viral anti-CRISPR (Acr) enzymes that rapidly degrade cyclic tetra-adenylate (cA4). The viral ring nuclease AcrIII-1 is widely distributed in archaeal and bacterial viruses and in proviruses."

A previously unknown fold binds cA4 and a conserved active site cleaves it; the original authors infer an anti-CRISPR consequence whose native scope is disputed.

"The enzyme uses a previously unknown fold to bind cA4 specifically, and a conserved active site to rapidly cleave this signalling molecule, allowing viruses to neutralize the type III CRISPR defence system."

The original authors infer broad host range from messenger targeting; this is not a demonstration of effective counterdefence by every homolog in every host.

"The AcrIII-1 family has a broad host range, as it targets cA4 signalling molecules rather than specific CRISPR effector proteins."

Expressing this gene in a type III-B-only host rescues an unrelated duf1874-less virus from CRISPR immunity

"However, the same cells expressing the SIRV1 gp29 gene from a plasmid were readily infected, giving rise to plaque formation. These data are consistent with the hypothesis that SIRV1 gp29 is functioning as an Acr specific for type III CRISPR defence."

This protein was one of the two family members expressed and assayed

"we cloned and expressed two family members in E. coli: the SIRV1 gp29 protein and the YddF protein encoded by an integrative and conjugative element ICEBs1 from B. subtilis"

It degrades cyclic tetra-adenylate to linear di-adenylate with a 2',3'-cyclic phosphate

"Both proteins possess a potent ring nuclease activity, rapidly degrading cA4 to generate linear di-adenylate (ApA>P) with a cyclic 2’,3’ phosphate"

The observed single-turnover cleavage rate exceeds five per minute and exceeds that of the compared host ring nuclease; this is not a measured steady-state kcat.

"With a catalytic rate exceeding 5 min-1, the Acr enzyme is at least 60-fold more active than the cellular ring nuclease Crn1 from S. solfataricus."

The enzyme strongly prefers cyclic tetra-adenylate over the hexameric signal

"Both SIRV1 gp29 and YddF enzymes show a strong preference for cA4 over cA6"

The substrate binds at the dimer interface

"The complex reveals a molecule of cA4 bound at the dimer interface."

The fold is unrelated to the CARF domain, the only previously known cyclic oligoadenylate receptor

"The structure of AcrIII-1 is unrelated to the CARF domain, which is the only protein family thus far known to bind cOA"

The substrate complex was obtained with the inactive H47A variant at 1.55 Angstrom

"we co-crystallised an inactive variant (H47A) of SIRV1 gp29 with cA4 and solved the structure to 1.55 Å resolution"

Four residues are conserved across the family, including the catalytic histidine

"Conserved residues H47, R66, R85 and E88 are indicated by asterisks."

The sole structural deposition from this work is PDB 6SCF

"The structural coordinates and data have been deposited in the Protein Data Bank with deposition code 6SCF."

The phylogenetic query sequence is identified by RefSeq accession NP_666617

"AcrIII-1 homologs were collected by using gp29 (NP_666617) of SIRV1 as a query"