RNA Phage VLP-Based Vaccine Platforms

Virus-like particles from a variety of RNA bacteriophages have turned out to be useful platforms for delivery of vaccine antigens in a highly immunogenic format. Here we update the current state of development of RNA phage VLPs as platforms for presentation of diverse antigens by genetic, enzymatic, and chemical display methods.

cDNA-derived RNA phage assembly reveals critical residues in the maturation protein of the Pseudomonas aeruginosa leviphage, PP7

PP7 is a leviphage with single-stranded RNA genome, which infects Pseudomonas aeruginosa PAO1. A reverse genetic system for PP7 was previously created by using reverse-transcribed cDNA (PP7O) from virion-derived RNA genome. Here, we have found that the PP7O cDNA contained 20 nucleotide differences from the PP7 genome sequence deposited in the database. We created another reverse genetic system exploiting chemically synthesized cDNA (PP7S) based on the database sequence. Unlike PP7O that rendered infectious PP7 virions, PP7S-derived particles were incapable of plaque formation on PAO1 cells, which was restored on the PAO1 cells expressing the maturation protein (MP) from PP7O. Using this reverse genetic system, we revealed two amino acid residues involved in the known roles of MP (i.e. adsorption and genome replication), fortuitously providing a lesson that the viral RNA genome sequencing needs functional verification possibly by a reverse genetic system. IMPORTANCE Biological significance of RNA phages has been largely ignored, ironically because few studies have been focusing on RNA phages. As an initial attempt to properly represent RNA phages in the phageome, we previously created, by using reverse-transcribed cDNA, a reverse genetic system for the small RNA phage, PP7 that infects the opportunistic human pathogen, Pseudomonas aeruginosa. We here report another system by using chemically synthesized cDNA based on the database genome that has 20 nucleotide differences from the previous cDNA. Investigation of those cDNA-derived phage virions unveiled that two amino acids of the maturation protein are crucial for the normal phage lifecycle at different steps. Our study provides an insight into the molecular basis for the RNA phage lifecycle and a lesson that the RNA genome sequencing needs to be carefully validated by cDNA-based phage assembly systems.

RNA Phage Biology in a Metagenomic Era

The number of novel bacteriophage sequences has expanded significantly as a result of many metagenomic studies of phage populations in diverse environments. Most of these novel sequences bear little or no homology to existing databases (referred to as the “viral dark matter”). Also, these sequences are primarily derived from DNA-encoded bacteriophages (phages) with few RNA phages included. Despite the rapid advancements in high-throughput sequencing, few studies enrich for RNA viruses, i.e., target viral rather than cellular fraction and/or RNA rather than DNA via a reverse transcriptase step, in an attempt to capture the RNA viruses present in a microbial communities. It is timely to compile existing and relevant information about RNA phages to provide an insight into many of their important biological features, which should aid in sequence-based discovery and in their subsequent annotation. Without comprehensive studies, the biological significance of RNA phages has been largely ignored. Future bacteriophage studies should be adapted to ensure they are properly represented in phageomic studies.

RNA-dependent RNA targeting by CRISPR-Cas9

Double-stranded DNA (dsDNA) binding and cleavage by Cas9 is a hallmark of type II CRISPR-Cas bacterial adaptive immunity. All known Cas9 enzymes are thought to recognize DNA exclusively as a natural substrate, providing protection against DNA phage and plasmids. Here, we show that Cas9 enzymes from both subtypes II-A and II-C can recognize and cleave single-stranded RNA (ssRNA) by an RNA-guided mechanism that is independent of a protospacer-adjacent motif (PAM) sequence in the target RNA. RNA-guided RNA cleavage is programmable and site-specific, and we find that this activity can be exploited to reduce infection by single-stranded RNA phage in vivo. We also demonstrate that Cas9 can direct PAM-independent repression of gene expression in bacteria. These results indicate that a subset of Cas9 enzymes have the ability to act on both DNA and RNA target sequences, and suggest the potential for use in programmable RNA targeting applications.

Application of Phage Biotechnology in Nanobiotechnology

To date, the phage display system has enabled the discovery of material binding peptides. Diversity and functionality of these peptides could be improved using RNA-based display systems instead of the conventional DNA-based ones. RNA phage replication systems possess unique features that make them a versatile tool for any combinatory approach and evolutionary application. Phage display was used to monitor the chemical surface properties and to initiate nanoparticle assembly. Novel bio-panning was recently used in RNA-based display to screen new functionality without acidic elution used in other conventional DNA phage display systems. Therefore, Hybrid RNA phages would be a perfect platform for attachment and exploration of nanoparticles. In this chapter, the authors present an overview on research conducted on these cross fields and areas. They not only focus on the novel selection and amplification process but also on the importance of RNA phage and its peptide display as tools for preventing nanoparticle aggregation.

C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector

The CRISPR-Cas adaptive immune system defends microbes against foreign genetic elements via DNA or RNA-DNA interference. We characterize the Class 2 type VI-A CRISPR-Caseffector C2c2 and demonstrate its RNA-guided RNase function. C2c2 from the bacterium Leptotrichia shahii provides interference against RNA phage.In vitro biochemical analysis show that C2c2 is guided by a single crRNA and can be programmed to cleave ssRNA targets carrying complementary protospacers. In bacteria, C2c2 can be programmed to knock down specific mRNAs. Cleavage is mediated by catalytic residues in the two conserved HEPN domains, mutations in which generate catalytically inactive RNA-binding proteins. These results broaden our understanding of CRISPR-Cas systems and suggest that C2c2 can be used to develop new RNA-targeting tools.

Application of Phage Biotechnology in Nanobiotechnology

To date, the phage display system has enabled the discovery of material binding peptides. Diversity and functionality of these peptides could be improved using RNA-based display systems instead of the conventional DNA-based ones. RNA phage replication systems possess unique features that make them a versatile tool for any combinatory approach and evolutionary application. Phage display was used to monitor the chemical surface properties and to initiate nanoparticle assembly. Novel bio-panning was recently used in RNA-based display to screen new functionality without acidic elution used in other conventional DNA phage display systems. Therefore, Hybrid RNA phages would be a perfect platform for attachment and exploration of nanoparticles. In this chapter, the authors present an overview on research conducted on these cross fields and areas. They not only focus on the novel selection and amplification process but also on the importance of RNA phage and its peptide display as tools for preventing nanoparticle aggregation.