Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Jul 5;385(6704):105-112.
doi: 10.1126/science.adl1356. Epub 2024 Jul 4.

An intron endonuclease facilitates interference competition between coinfecting viruses

Affiliations

An intron endonuclease facilitates interference competition between coinfecting viruses

Erica A Birkholz et al. Science. .

Abstract

Introns containing homing endonucleases are widespread in nature and have long been assumed to be selfish elements that provide no benefit to the host organism. These genetic elements are common in viruses, but whether they confer a selective advantage is unclear. In this work, we studied intron-encoded homing endonuclease gp210 in bacteriophage ΦPA3 and found that it contributes to viral competition by interfering with the replication of a coinfecting phage, ΦKZ. We show that gp210 targets a specific sequence in ΦKZ, which prevents the assembly of progeny viruses. This work demonstrates how a homing endonuclease can be deployed in interference competition among viruses and provide a relative fitness advantage. Given the ubiquity of homing endonucleases, this selective advantage likely has widespread evolutionary implications in diverse plasmid and viral competition as well as virus-host interactions.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.. ΦPA3 gp210 is an HNH homing endonuclease that targets ΦKZ gp178.
(A) Efficiency of plating (EOP) of ΦKZ as measured by spot titer and normalized to the paired titer on GFPmut1. gp210-GFPmut1 causes a 99.9983% decrease in ΦKZ titer (ratio paired t-test, p=0.0011, N=3) while untagged gp210, not imported into the nucleus, causes an insignificant 27% decrease (ratio paired t-test, p=0.14, N=3). H82R mutation (gp210(H82R)-GFPmut1) fully rescues ΦKZ titer compared to gp210-GFPmut1 (ratio paired t-test, **p=0.0026, N=3). Error bars represent SEM, p values were calculated by ratio paired t-test. (B) Protein alignment of phage-encoded endonucleases related to gp210 (top is predicted secondary structure from Phyre2), including well-characterized homing endonuclease I-HmuI (bottom with confirmed secondary structure). Cylinders represent α-helices and arrows indicate β-strands. Blue highlights indicate conservation at each position, and pink highlights indicate catalytic residues. ClustalO alignment. (C) Protein alignment including the intron(−) version of the ΦPA3 RNAP (blue asterisk), after editing from the annotation in Genbank which can be found in Figure S1D. It is aligned with RpoB of MG1655 (E. coli) and PAO1 (P. aeruginosa) (black asterisks), 8 RNAPs encoded by chimalliviruses infecting different genera of hosts, and 4 RNAPs encoded by other Pseudomonas chimalliviruses. Blue highlights indicate conservation at each position, and pink highlights indicate catalytic residues. ClustalO alignment. The intron insertion site occupied in the ΦPA3 gene is indicated. Residue numeration above is based on the ΦPA3 sequence. (D) Efficiency of plating (EOP) of ΦKZ escape mutants relative to the wildtype ΦKZ on cells expressing gp210-GFPmut1. (E) Nucleotide alignment of intron(−) RpoB homologs in five Pseudomonas jumbo phages. The point mutation in the ΦKZ vRNAP gp178 at position 3215 (red box) is two bases upstream of the site that aligns with the intron insertion site (black dotted line) in ΦPA3. Red sequences code for the conserved aspartic acid residues. The exons of ΦPA3 are noted above. (F) EOP of ΦKZ on cells expressing GFPmut1 as a control, gp210-GFPmut1, or co-expressing gp210-GFPmut1 and one of three ΦKZ gp178 gene variants. The nucleotide sequences of these variants are shown in Figure S2F. Gp178(D1072A) is the mutant found in escaper phages. Gp178m17 has 17 silent mutations that do not change amino acid sequence but are expected to interfere with gp210 nucleotide recognition. Gp178Δ504 has 504 nucleotides deleted (ratio paired t-test, **p=0.0034, N=5). (G) Sequences surrounding the insertion site of the intron containing gp210. ΦPA3 gp211/209: intron(−) version of the ΦPA3 vRNAP subunit. ΦKZ gp178: homologous vRNAP subunit. ΦKZ gp178 A3215C: gp178 with single nucleotide mutation. (H-K) Nuclease assay of purified gp210 incubated with plasmid DNA containing only the empty vector (H), the intron(−) ΦPA3 gene (I, gp211/209), ΦKZgp178 gene (J) or ΦKZgp178 gene with single nucleotide change A3215C (K). Gp210 concentration from left to right was 5.0, 2.5, 1.25, and 0 μM. Nt.BsaI is a reference digest for nicked plasmid (blue arrows, n). Supercoiled plasmid (sc) is indicated by black arrows.
Figure 2.
Figure 2.. Gp210-GFPmut1 causes a loss of ΦKZ progeny.
(A) Live fluorescence microscopy of ΦKZ infections stained with FM4–64 (magenta: membranes) and DAPI (blue: DNA) 70 minutes post infection (mpi). Infections proceeded in the presence of either GFPmut1, gp210-GFPmut1, or gp210(H82R)-GFPmut1 (green: GFP). DAPI stained capsids in phage bouquets are labeled by white arrowheads. Absence of DAPI stained capsids in cells expressing gp210-GFPmut1 noted with yellow arrowheads. DIC: Differential Interference Contrast. Scale bar is 1 μm. (B) Ratio of DAPI signal in the bouquet compared to the nucleus at 70 mpi. Unpaired t-test, ****p<0.0001. Error bars represent standard deviation. GFPmut1 n=220, gp210-GFPmut1 and gp210(H82R)-GFPmut1 n=200. (C) Line plots of DAPI signal intensity along a bisecting line at 70 mpi, average of n=50. * note that the y-axis for gp210-GFPmut1 is double that of the other panels. (D) EOP of ΦKZ progeny (logscale) collected from washed infections of cells expressing the indicated protein, measured by spot titer on cells without plasmid. Progeny grown in the presence of gp210-GFPmut1 plaque with an efficiency of 0.00001% of the progeny grown with GFPmut1 (ratio paired t-test, p=0.0005, N=5). Progeny produced with gp210(H82R)-GFPmut1 have a relative EOP of 190% (ratio paired t-test, p=0.56, N=5). Error bars represent SEM. (E) Number of capsids filled with DNA observed in tomograms of the control strain (no plasmid, N=5) or the strain expressing gp210-GFPmut1 (N=17) at 90 mpi. Unpaired t-test, ****p<0.0001.
Figure 3.
Figure 3.. Targeting of ΦPA3 gp210 to ΦKZ phage nucleus results in misassembly of ΦKZ major capsid protein.
(A, B) Cryo-FIB-ET of ΦKZ-infected P. aeruginosa cells at 90 mpi. (C, D) Cryo-FIB-ET of ΦKZ-infected P. aeruginosa cells expressing ΦPA3 gp210-GFPmut1 at 90 mpi. Magenta arrows point to a few properly assembled ΦKZ capsids and magenta dashed boxes indicate regions containing misassembled capsomer complexes. (E) Tomogram slices of a double-layered capsomer tube separated by 40 nm in the Z-direction. (F) Lattice plot of aligned subtomograms from the region depicted in (E). Subtomograms from the outer and inner layers are colored yellow and cyan, respectively. (G) AlphaFold2/ColabFold predicted structure of the ΦKZ major capsid protein (MCP) depicted as a cartoon model. The structure is annotated according to the HK97-fold features. (H) Exterior face and slabbed side views of the subtomogram reconstruction of the ΦKZ MCP from the tubular assemblies with a white oval indicating the 2-fold symmetry. (I) Same views as in (H) of the surface representation of the fitted model of the ΦKZ MCP protomer into the subtomogram reconstruction with the central hexamer protomers colored individually and surrounding hexamers colored white. Scale bars A-D: 250 nm, E: 100 nm.
Figure 4.
Figure 4.. Gp210 provides a competitive advantage in phage co-infections.
(A) Titer (PFU/ml) of ΦPA3 (starting MOI 10) or ΦKZ (starting MOI 0.1) after one round of competition with either wildtype ΦPA3 (ΦPA3) or ΦPA3 lacking gp210 (ΦPA3Δ210; Figure S6). Two experiments were conducted with 5 independent replicates each. ΦKZ has significantly higher titers after one round of competition with ΦPA3Δ210 compared to wildtype ΦPA3 (unpaired t-test, *p=0.014, ***p=0.0003). (B) Fold change of titers from (A) of ΦPA3Δ210 relative to ΦPA3 when in competition with ΦKZ (left) or ΦKZ in competition with ΦPA3Δ210 compared to ΦKZ in competition with ΦPA3 (right) (unpaired t-test, ****p<0.0001). Fold-change was determined by dividing each of the experimental titers (ΦPA3Δ210(ΦKZ), ΦKZ(ΦPA3Δ210)) by the mean of the control titers (ΦPA3(ΦKZ), ΦKZ(ΦPA3)) for each experiment. (C) Fold change of titers of ΦPA3Δ210 relative to ΦPA3 when in competition with ΦKZ (left) or ΦKZ in competition with ΦPA3Δ210 compared to ΦKZ in competition with ΦPA3 (right) when both phage are present at an MOI of 0.01 (unpaired t-test, p=0.24). Fold-change was determined by dividing each of the experimental titers (ΦPA3Δ210(ΦKZ), ΦKZ(ΦPA3Δ210)) by the mean of the control titers (ΦPA3(ΦKZ), ΦKZ(ΦPA3)) for each experiment. (D) ΦKZgp104-mCherry (magenta) and ΦPA3gp108-sfGFP (green) were co-expressed in P. aeruginosa and infected with both ΦKZ and ΦPA3 (top panels: DAPI staining left, right mCherry and sfGFP right), ΦKZ alone (bottom panels, DAPI left, mCherry and sfGFP right)), or ΦPA3 alone (bottom panels, DAPI left, mCherry and sfGFP right). In the top right image, an example of a phage nucleus importing both proteins (co-import) is indicated by a white star, a co-infection with two separate nuclei importing either the ΦKZ or the ΦPA3 protein is shown by a white arrow, two nuclei importing only the ΦKZ protein is marked by a white arrowhead, and a nucleus importing only the ΦPA3 protein is pointed out by a yellow arrowhead. DIC is shown in gray. Scale bars are 1 μm. (E) Quantitation of fields from the experiment in (D). “Co-infections” is when import of both proteins into the same or distinct nuclei is observed, “Distinct import” is when the nuclear proteins were not colocalized, and “Co-import” is when both proteins were colocalized in the same nucleus. (F) gp210 (green pacman) tagged with GFPmut1 is artificially imported into the ΦKZ nucleus (red shell, blue fill) where it cuts ΦKZ DNA in the vRNAP gene of gp178, dependent upon adenosine 3215, inhibiting ΦKZ bouquet formation. (G) When separate nuclei are formed by ΦKZ (red) and ΦPA3 (green), ΦPA3 expresses gp210 which is translated in the cytosol and encounters the ΦKZ nuclear shell. ΦKZ excludes gp210 allowing it to replicate simultaneously with ΦPA3. (H) Hypothesis of the effects of gp210 on a hybrid nucleus; ~50% of co-infections (E). In a hybrid nucleus, gp210 would be imported and cut the ΦKZ vRNAP gene gp178. Recombination efficiency is reduced with ~60% identity between the vRNAP alleles, leading to a competitive advantage for ΦPA3.

Update of

References

    1. Cech TR, Self-splicing of group I introns. Annu. Rev. Biochem 59, 543–568 (1990). - PubMed
    1. Yamada T, Tamura K, Aimi T, Songsri P, Self-splicing group I introns in eukaryotic viruses. Nucleic Acids Res. 22, 2532–2537 (1994). - PMC - PubMed
    1. Edgell DR, Gibb EA, Belfort M, Mobile DNA elements in T4 and related phages. Virol. J 7, 290 (2010). - PMC - PubMed
    1. Nawrocki EP, Jones TA, Eddy SR, Group I introns are widespread in archaea. Nucleic Acids Res. 46, 7970–7976 (2018). - PMC - PubMed
    1. Edgell DR, Selfish DNA: homing endonucleases find a home. Curr. Biol 19, R115–7 (2009). - PubMed

Publication types