A family of viral satellites manipulates invading virus gene expression and affects cholera toxin mobilization

Abstract

Many viruses possess temporally unfolding gene expression patterns aimed at subverting host defenses, commandeering host metabolism, and ultimately producing a large number of progeny virions. High throughput -omics tools, such as RNA-seq, have dramatically enhanced resolution of expression patterns during infection. Less studied have been viral satellites, mobile genomes that parasitize viruses and have far reaching effects on host-cell fitness. By performing RNA-seq on infection time courses, we have obtained the first time-resolved transcriptomes for bacteriophage satellites during lytic infection. Specifically, we have acquired transcriptomes for the lytic Vibrio cholerae phage ICP1 and all five known variants of ICP1's parasite, the Phage Inducible Chromosomal Island-Like Elements (PLEs). PLEs rely on ICP1 for both DNA replication and mobilization, and abolish production of ICP1 progeny in infected cells. We investigated PLEs impact on ICP1 gene expression and found that PLEs did not broadly restrict or reduce ICP1 gene expression. A major exception occurred in ICP1's capsid morphogenesis operon, which was downregulated by each of the PLE variants. This transcriptional manipulation, conserved among PLEs, has also evolved independently in at least one other phage satellite, suggesting that viral satellites may be under strong selective pressure to reduce the capsid expression of their larger host viruses. Surprisingly, PLEs were also found to alter the gene expression of CTXφ, the integrative phage that encodes cholera toxin and is necessary for virulence of toxigenic V. cholerae. One PLE, PLE1, upregulated CTXφ genes involved in replication and integration, and boosted CTXφ mobility following induction of the SOS response. Our data show that PLEs exhibit conserved manipulation of their host-phage's gene expression, but divergent effects on CTXφ, revealing that PLEs can influence both their hosts' resistance to phage and the mobility of virulence encoding elements.So far, few insights have been gained into the gene expression programs of ICP1 and PLEs. PLE1 expresses its integrase in uninfected cells [22], expression of PLE1's replication initiator, RepA, is induced following infection of ICP1 [23], and the PLE's lysis modulator, LidI, is detectable by Western blot late during ICP1 infection [25]. The PLE integrase's recombination directionality factor (necessary for directing integrase excision activity) is PexA, an ICP1 protein whose native function is unknown, but whose expression can be detected by 5 minutes post infection [22]. While these limited observations have provided insight into key PLE and ICP1 genes, the gross expression patterns of ICP1 and PLEs remain unknown. Until now, we have not known the degree to which ICP1 alters cellular expression patterns, and whether PLEs alter ICP1's gene expression or reproduce and restrict ICP1 without such alterations. To address these questions, we performed RNA-seq on V. cholerae infected by ICP1 over the course of the infection cycle. We sequenced the transc...

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

“…Previous RNA-seq analyses of ICP1 infecting PLE(+) V. cholerae indicated that PLE is transcriptionally activated upon infection by ICP1, though most PLE-encoded ORFs were expressed at low levels in the absence of infection (37). An exception is PLE-encoded nixI, which is completely transcriptionally repressed in the absence of ICP1 infection, consistent with NixI's nuclease activity and toxicity to V. cholerae (Figure 2B).…”

“…However, PLE 2 encodes for identical NixI and StiX proteins, yet contains three occurrences of the predicted recognition motif. PLE 2 replicates during ICP1 infection (17) and shows the same temporal nixI+stiX expression pattern as PLE 1 (37) suggesting that PLE 2 is not cleaved by NixI. Together these data suggest that the motif alone is not sufficient to determine cutting.…”

“…However, we noted the occasional occurrence of Bro-N domain containing proteins, a DNA-binding domain often associated with T5ORF172 domains (45). In addition, all of the putative parasites are syntenic with PLEs, possessing a large ORF-less space where PLEs' origin of replication (18) and putative pac site are located, as well as a smaller ORF-less space upstream of nixI, where PLEs encode a predicted small RNA (37). Of note, none of the putative satellites possessed an identifiable homolog of StiX, as the gene immediately 3' of nixI shared a predicted peptidase function as indicated by PFAM domain searches (Figure 7A).…”

“…Complementation of nixI in trans reduced ICP1 replication to a greater extent than wild type PLE (Figure 5D). This is likely because endogenous nixI is not expressed until mid-way through infection, between 8 and 12 minutes post-infection (37). At this point in infection, ICP1's has undergone several rounds of theta replication and is transitioning to rolling circle replication, while PLE replication begins to accelerate (18).…”

Temporal Shifts in Antibiotic Resistance Elements Govern Virus-Pathogen Conflicts

“…4B and 4C). Unlike ICP1 isolates that can productively infect ICEVchInd5(+) V. cholerae, the restricted ICP1 2017 lacks both gp21 and the promoter region for gp25, which we identified by analyzing the ICP1 transcriptome (61) (Fig. 4C), suggesting one of these early genes may allow ICP1 to overcome restriction by ICEVchInd5.…”