Structure and function of virion RNA polymerase of a crAss-like phage

Abstract

CrAss-like phages are a recently described family-level group of viruses that includes the most abundant virus in the human gut 1,2 . Genomes of all crAss-like phages encode a large virionpackaged protein 2,3 that contains a DFDxD sequence motif, which forms the catalytic site in cellular multisubunit RNA polymerases (RNAPs) 4 . Using Cellulophaga baltica crAss-like phage phi14:2 as a model system, we show that this protein is a novel DNA-dependent RNAP that is translocated into the host cell along with the phage DNA and transcribes early phage genes. We determined the crystal structure of this 2,180-residue enzyme in a self-inhibited, likely pre-virionpackaged state. This conformation is attained with the help of a Cleft-blocking domain that interacts with the active site motif and occupies the RNA-DNA hybrid binding grove. Structurally, phi14:2 RNAP is most similar to eukaryotic RNAPs involved in RNA interference 5,6 , although most of phi14:2 RNAP structure (nearly 1,600 residues) maps to a new region of protein folding space.Considering the structural similarity, we propose that eukaryal RNA interference polymerases take their origin in a phage, which parallels the emergence of the mitochondrial transcription apparatus 7 .

Analysis of metagenome-assembled viral genomes from the human gut reveals diverse putative CrAss-like phages with unique genomic features

“…The predicted RNAPs of crAss-like phages are extremely divergent in sequence from all known RNAPs and from each other, suggestive of unusually high evolutionary rates. The recent structural and functional study of the RNAP of C. baltica phage phi 14:2 confirms the involvement of this large protein in the transcription of the phage early genes and shows that it contains two double-psi beta-barrel (DPBB) domains within a single polypeptide, unlike cellular RNAPs in which the two DPBB domains belong to the two largest subunits 12 .…”

“…The gene complements of the crAss-like phages show a number of distinct features, in particular, a predicted complex transcription machinery centered around an RNA polymerase (RNAP) with an unusual structure related to the structure of eukaryotic RNA-dependent RNAPs involved in RNA interference. Recently, the prediction of this distinct RNAP has been validated by structural and functional analysis demonstrating that this RNAP is a virion component that is translocated into the host cell together with the phage DNA and is responsible for the transcription of the phage early genes 12 . A phylogenomic analysis of 250 genomes of crAss-like phages led to the proposal of 4 distinct subfamilies and 10 genera 13 .…”

Possible Implications of AlphaFold2 for Crystallographic Phasing by Molecular Replacement

“…Eight domains were classed as FM, and one as FM/TBM. There were two copies of the monomer in the asymmetric unit (PDB 6vr4; (Drobysheva et al, 2021)), related by a non-crystallographic two-fold. The assessment domains were used as models for molecular replacement.…”

Discovery, diversity and functional associations of crAss-like phages in human gut metagenomes from four Dutch cohorts

“…There are three major functional regions that can be distinguished in the crAss-like phage genome: a replication gene module, a transcription gene module and a capsid gene module (Koonin and Yutin, 2020; Yutin et al , 2018). Transcription of the three modules is initiated early (by a virion-packaged RNA polymerase (Holmfeldt et al, 2013; Shkoporov et al, 2018)), in the middle and late in the infection, respectively (Drobysheva et al, 2021; Shkoporov et al, 2021). The genomes of various crAss-like phages bear unique features, including reassignment of the stop codons TAG and TGA for amino acids and a high density of introns and inteins (Yutin et al , 2021).…”