RNA X-ray footprinting reveals the consequences of an in vivo acquired determinant of viral infectivity

The secondary structures of the bacteriophage MS2 ssRNA genome, frozen in defined states, were determined with minimal perturbation using constraints from X-ray synchrotron footprinting (XRF). The footprints of the gRNA in the virion and as transcript are consistent with single, dominant but distinct conformations, and reveal the presence of multiple Packaging Signals potentially involved in assembly regulation that have not been detected by other techniques. XRF also reveals the dramatic effect of the unique Maturation Protein (MP) on both the capsid lattice, and the gRNA conformation inside the phage compared with a virus-like-particle composed only of coat protein subunits. Aspects of genome organisation in the phage, their impacts on the capsid shell, and the distortion of lattice geometry by MP, are hallmarks of molecular frustration. Phage assembly therefore appears to prepare the particle for the next step of the infectious cycle.

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.

Molecular Piracy: Redirection of Bacteriophage Capsid Assembly by Mobile Genetic Elements

Horizontal transfer of mobile genetic elements (MGEs) is a key aspect of the evolution of bacterial pathogens. Transduction by bacteriophages is especially important in this process. Bacteriophages—which assemble a machinery for efficient encapsidation and transfer of genetic material—often transfer MGEs and other chromosomal DNA in a more-or-less nonspecific low-frequency process known as generalized transduction. However, some MGEs have evolved highly specific mechanisms to take advantage of bacteriophages for their own propagation and high-frequency transfer while strongly interfering with phage production—“molecular piracy”. These mechanisms include the ability to sense the presence of a phage entering lytic growth, specific recognition and packaging of MGE genomes into phage capsids, and the redirection of the phage assembly pathway to form capsids with a size more appropriate for the size of the MGE. This review focuses on the process of assembly redirection, which has evolved convergently in many different MGEs from across the bacterial universe. The diverse mechanisms that exist suggest that size redirection is an evolutionarily advantageous strategy for many MGEs.

The Transmembrane Morphogenesis Protein gp1 of Filamentous Phages Contains Walker A and Walker B Motifs Essential for Phage Assembly

In contrast to lytic phages, filamentous phages are assembled in the inner membrane and secreted across the bacterial envelope without killing the host. For assembly and extrusion of the phage across the host cell wall, filamentous phages code for membrane-embedded morphogenesis proteins. In the outer membrane of E. coli, the protein gp4 forms a pore-like complex, while gp1 and gp11 form a complex in the inner membrane of the host. By comparing sequences with other filamentous phages, we identified putative Walker A and B motifs in gp1 with a conserved lysine in the Walker A motif (K14), and a glutamic and aspartic acid in the Walker B motif (D88, E89). In this work we demonstrate that both, Walker A and Walker B, are essential for phage production. The crucial role of these key residues suggest that gp1 is likely to be a molecular motor driving phage assembly. We further identified essential residues for the function of the assembly complex. Mutations in three out of six cysteine residues abolish phage production. Similarly, two out of six conserved glycine residues are crucial for gp1 function. We hypothesise that the residues represent molecular hinges allowing domain movement for nucleotide binding and phage assembly.