Finally, a Role Befitting Astar: Strongly Conserved, Unessential Microvirus A* Proteins Ensure the Product Fidelity of Packaging Reactions

In microviruses, 60 copies of the positively charged DNA binding protein J guide the single-stranded DNA genome into the icosahedral capsid. Consequently, similar to 12% of the genome is icosahedrally ordered within virions. Although the internal volume of the phi X174, G4, and alpha 3 capsids are nearly identical, their genome lengths vary widely from 5,386 (phi X174) to 6,067 (alpha 3) nucleotides. As the genome size increases, the J protein's length and charge decreases. The phi X174 J protein is 37 amino acids long and has a charge of +12, whereas the 23-residue G4 and alpha 3 proteins have respective +6 and +8 charges. While the large phi X174 J protein can substitute for the smaller ones, the converse is not true. Thus, the smallest genome, phi X174, requires the more stringent J protein packaging guide. To investigate this further, a chimeric virus (phi XG4J) was generated by replacing the indigenous phi X174 J gene with that of G4. The resulting mutant, phi XG4J, was not viable on the level of plaque formation without phi X174 J gene complementation. During uncomplemented infections, capsids dissociated during packaging or quickly thereafter. Those that survived were significantly less stable and infectious than the wild type. Complementation-independent phi XG4J variants were isolated. They contained duplications that increased genome size by as much as 3.8%. Each duplication started at nucleotide 991, creating an additional DNA substrate for the unessential but highly conserved A* protein. Accordingly, phi XG4J viability and infectivity was also restored by the exogenous expression of a cloned A* gene. IMPORTANCE Double-stranded DNA viruses typically package their genomes into a preformed capsid. In contrast, single-stranded RNA viruses assemble their coat proteins around their genomes via extensive nucleotide-protein interactions. Single-stranded DNA (ssDNA) viruses appear to blend both strategies, using nucleotide-protein interactions to organize their genomes into preformed shells, likely by a controlled process. Chaotic genome-capsid associations could inhibit packaging or genome release during the subsequent infection. This process appears to be partially controlled by the unessential A* protein, a shorter version of the essential A protein that mediates rolling-circle DNA replication. Protein A* may elevate fitness by ensuring the product fidelity of packaging reactions. This phenomenon may be widespread in ssDNA viruses that simultaneously synthesize and package DNA with rolling circle and rolling circle-like DNA replication proteins. Many of these viruses encode smaller, unessential, and/or functionally undefined in-frame versions of A/A*-like proteins.