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.

Reverse genetic systems for RNA viruses are the platforms to introduce mutations into the RNA genomes and thus have helped understand their life cycle and harness them for human purposes to develop vaccines and delivery systems. These systems are based on the complementary DNA (cDNA) of the RNA viruses, whose transcripts derived from bacterial RNA polymerases act not only as the primary mRNA for phage protein synthesis, but also as the template for phage RNA replicases (aka. RNA-dependent RNA polymerases). Here, we present a protocol optimized for the small RNA phages of Leviviridae (i.e., leviphages) infecting Pseudomonas aeruginosa. This protocol includes three fundamental steps: (i) Creation of a promoter-fused cDNA, (ii) generation of a clone into mini-Tn7-based vector, and (iii) introduction of the clone into non-susceptible hosts. As the representative example, we describe the reverse genetic system for PP7, which infects a set of P. aeruginosa strains such as PAO1. The cDNA was fused to the T7 promoter, which was cloned in mini-Tn7-Gm. This construct was introduced into P. aeruginosa PAK and E. coli HB101. Functional assembly of PP7 phages from the culture supernatants were assessed by plaque formation on PAO1 and the phage particles were observed under transmission microscope. We found that the host cells should be cultured at 30 °C for the maximal phage production (~1012 pfu/mL). The reverse genetic systems will provide a new insight into the life cycle of the RNA phages and help develop engineered variants with new traits for phage applications regarding selective diagnosis and efficient therapy.