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. 2024 Mar 27;16(4):513.
doi: 10.3390/v16040513.

Genome Analysis of Epsilon CrAss-like Phages

Igor V Babkin  1 , Artem Y Tikunov  1 , Ivan K Baykov  1   2 , Vera V Morozova  1 , Nina V Tikunova  1
Affiliations

Genome Analysis of Epsilon CrAss-like Phages

Igor V Babkin et al. Viruses. .

Abstract

CrAss-like phages play an important role in maintaining ecological balance in the human intestinal microbiome. However, their genetic diversity and lifestyle are still insufficiently studied. In this study, a novel CrAssE-Sib phage genome belonging to the epsilon crAss-like phage genomes was found. Comparative analysis indicated that epsilon crAss-like phages are divided into two putative genera, which were proposed to be named Epsilonunovirus and Epsilonduovirus; CrAssE-Sib belongs to the former. The crAssE-Sib genome contains a diversity-generating retroelement (DGR) cassette with all essential elements, including the reverse transcriptase (RT) and receptor binding protein (RBP) genes. However, this RT contains the GxxxSP motif in its fourth domain instead of the usual GxxxSQ motif found in all known phage and bacterial DGRs. RBP encoded by CrAssE-Sib and other Epsilonunoviruses has an unusual structure, and no similar phage proteins were found. In addition, crAssE-Sib and other Epsilonunoviruses encode conserved prophage repressor and anti-repressors that could be involved in lysogenic-to-lytic cycle switches. Notably, DNA primase sequences of epsilon crAss-like phages are not included in the monophyletic group formed by the DNA primases of all other crAss-like phages. Therefore, epsilon crAss-like phage substantially differ from other crAss-like phages, indicating the need to classify these phages into a separate family.

Keywords: crAss-like phages; diversity-generating retroelements; genome; repressors/anti-repressors; reverse transcriptase; virus taxonomy.

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Conflict of interest statement

All co-authors have seen and agree with the contents of the manuscript and the order of authors, and there is no financial interest to report. All co-authors declare that they have no conflicts of interest.

Figures

Figure 1
Figure 1
The whole genome map of crAssE-Sib phage. ORFs are colored according to their proposed function: DNA replication—blue; head assembly and structural—green; transcription—black; reverse transcriptase—red; other ORFs are colored with magenta.
Figure 2
Figure 2
ViPTree analysis of the crAssE-Sib phage. Dendrogram plotted by ViPTree version 3.7 using crAssE-Sib and phages with SG values > 0.001. The crAssE-Sib phage is marked with a blue asterisk; phi14:2 phage is marked with a black triangle; phage sequences that were downloaded from the NCBI GenBank manually are marked with red phylogenetic branches.
Figure 3
Figure 3
Bidirectional clustering heatmap visualizing VIRIDIC-generated similarity matrix for crAssE-Sib and related phage genomes. The crAssE-Sib phage is marked with a blue asterisk.
Figure 4
Figure 4
Comparative genome alignment of the crAssE-Sib and close phage genomes. Analysis was performed using VipTree software v. 3.7. The percentage of sequence similarity is indicated in color; the color scale is shown at the top.
Figure 5
Figure 5
Maximum likelihood phylogenetic tree of the crAssE-Sib terminase large subunit generated using IQ-tree software v. 2.0.5. The crAssE-Sib phage is marked with a black circle. Nodes with 95% statistical significance calculated from 1000 ultrafast bootstrap (UFBOOT) replicates are marked with asterisks. The scale bar represents the number of substitutions per site.
Figure 6
Figure 6
Maximum likelihood phylogenetic tree of the crAssE-Sib primase generated using IQ-tree software. The crAssE-Sib phage is marked with a black circle. Nodes with 95% statistical significance calculated from 1000 ultrafast bootstrap (UFBOOT) replicates are marked with asterisks. The scale bar represents the number of substitutions per site.
Figure 7
Figure 7
Maximum likelihood phylogenetic tree of the crAssE-Sib RNA polymerase generated using IQ-tree software. The crAssE-Sib phage is marked with a black circle. Nodes with 95% statistical significance calculated from 1000 ultrafast bootstrap (UFBOOT) replicates are marked with asterisks. The scale bar represents the number of substitutions per site.
Figure 8
Figure 8
Schematic structure of DGR cassettes found in the genomes of crAssE-Sib and other members of the Epsilonunovirus genus. Schematic structure of DGR cassettes found in the genomes of crAssE-Sib and other members of the Epsilonunovirus genus. DGRs contain the receptor binding protein (RBP—green arrow) and reverse transcriptase (RT—red arrow) genes as well as template repeat (TR—light blue) with IMH* sequence (orange) and variable repeat (VR—blue) with initiation of the mutagenic homing (IMH—yellow) sequence. RT is fragmented or absent in many Epsilonunoviruses. Empty arrows indicate hypothetical proteins.
Figure 9
Figure 9
Multiple sequence alignment representations of aa sequences of the VRs region of crAssE-Sib and related phages.
Figure 10
Figure 10
Comparison between 3D model of crAssE-Sib receptor binding protein (RBP) and experimental structures of TaqVP protein from Thermus aquaticus (pdb id 5VF4) and Mtd protein of the BPP-1 phage (pdb id 1YU0). VR regions are in green; magenta alpha helices show similar orientation of the molecules. All molecules are on the same scale.
Figure 11
Figure 11
Maximum likelihood phylogenetic tree of the crAssE-Sib RT generated using IQ-tree software. The crAssE-Sib phage is marked with a black circle. Nodes with 95% statistical significance are marked with asterisks calculated from 1000 ultrafast bootstrap (UFBOOT) replicates. The scale bar represents the number of substitutions per site.
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