A phage parasite deploys a nicking nuclease effector to inhibit viral host replication

Abstract

PLEs (phage-inducible chromosomal island-like elements) are phage parasites integrated into the chromosome of epidemic Vibrio cholerae. In response to infection by its viral host ICP1, PLE excises, replicates and hijacks ICP1 structural components for transduction. Through an unknown mechanism, PLE prevents ICP1 from transitioning to rolling circle replication (RCR), a prerequisite for efficient packaging of the viral genome. Here, we characterize a PLE-encoded nuclease, NixI, that blocks phage development likely by nicking ICP1’s genome as it transitions to RCR. NixI-dependent cleavage sites appear in ICP1’s genome during infection of PLE(+) V. cholerae. Purified NixI demonstrates in vitro nuclease activity specifically for sites in ICP1’s genome and we identify a motif that is necessary for NixI-mediated cleavage. Importantly, NixI is sufficient to limit ICP1 genome replication and eliminate progeny production, representing the most inhibitory PLE-encoded mechanism revealed to date. We identify distant NixI homologs in an expanded family of putative phage parasites in vibrios that lack nucleotide homology to PLEs but nonetheless share genomic synteny with PLEs. More generally, our results reveal a previously unknown mechanism deployed by phage parasites to limit packaging of their viral hosts’ genome and highlight the prominent role of nuclease effectors as weapons in the arms race between antagonizing genomes.

Evolutionary Sweeps of Subviral Parasites and Their Phage Host Bring Unique Parasite Variants and Disappearance of a Phage CRISPR-Cas System

“…Some of the newly discovered PLE variants, however, only appeared for a short time, such as PLE6, which was detected once in Bangladesh in 1987, and PLE8 in Vietnam in 1995 and 2004. We also detected one possible phage satellite with partial genetic relationship to PLEs, as has been described in non-cholera vibrios recently ( 25 ); however, its sequence could not be fully resolved from available data. It is highly likely that we still have not captured the full extent of PLE diversity and variant flux over time, as V. cholerae isolate sampling was sporadic and limited until recent decades (with over half of all isolates collected in only the last 9 years).…”

“…Annotation and sequence comparison of open reading frames confirmed that the newly detected PLEs 6 to 10 possess genes key to PLE function that have been characterized in the previously studied PLEs 1 to 5. These include nixI , which encodes a nickase that interferes with ICP1’s ability to replicate ( 25 ), lidI , which can accelerate lysis of the V. cholerae host cell ( 22 ), and capR , which acts to repress ICP1’s capsid morphogenesis operon ( 21 ). The conservation of these genes indicates that the newly identified PLEs play roles similar to those of PLEs 1 to 5 as parasites of ICP1.…”

The RNA-RNA interactome between a phage and its satellite virus reveals a small RNA differentially regulates gene expression across both genomes

“…We detected decreased genome replication of both PLE and ICP1 and a delay in the timing of cell lysis when comparing PLEΔ sviR infection to PLE(+) infection (Figure S1). However, both lysis (Hays and Seed, 2019) and genome replication (Barth et al, 2020a; LeGault et al, 2022) are dependent on multiple PLE or ICP1 products, providing no clear candidate genes as targets of SviR regulation. We next pursued identifying target transcripts of SviR during ICP1 infection of PLE(+) V. cholerae .…”

“…Interestingly, IS200/IS605 elements are associated with distal nucleases, which are guided by the transposable element-encoded sRNA. The ORF downstream of SviR, NixI, was recently shown to be a nicking endonuclease, implicated in the degradation of ICP1’s genome during rolling circle replication (LeGault et al, 2022). While NixI is not encoded within the transposable element repeats, the upstream sRNA proximal to a nuclease effector raises the possibility of a similar RNA-guided nuclease activity for NixI, secondary to the primary function as a replication inhibitor.…”

“…All identified PLE(+) V. cholerae contain a single PLE, which lays dormant in the V. cholerae chromosome until ICP1 infection. Upon infection, the PLE excises from the chromosome (McKitterick and Seed, 2018), replicates to high copy number (Barth et al, 2020a), and deploys multiple anti-ICP1 factors which completely inhibit the production of ICP1 progeny through various mechanisms (Hays and Seed, 2019; LeGault et al, 2022). Unlike many other generalized anti-phage systems, such as restriction modification (Tock and Dryden, 2005) and CRISPR-Cas (Barrangou et al, 2007), PLE-mediated immunity is specific to ICP1, consistent with the relationship between a viral satellite and a virus.…”