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

Abstract

In microviruses, 60 copies of the positively charged DNA binding protein J guide the single-stranded DNA genome into the icosahedral capsid. Consequently, ∼12% of the genome is icosahedrally ordered within virions. Although the internal volume of the ϕX174, G4, and α3 capsids are nearly identical, their genome lengths vary widely from 5,386 (ϕX174) to 6,067 (α3) nucleotides. As the genome size increases, the J protein’s length and charge decreases. The ϕX174 J protein is 37 amino acids long and has a charge of +12, whereas the 23-residue G4 and α3 proteins have respective +6 and +8 charges. While the large ϕX174 J protein can substitute for the smaller ones, the converse is not true. Thus, the smallest genome, ϕX174, requires the more stringent J protein packaging guide. To investigate this further, a chimeric virus (ϕXG4J) was generated by replacing the indigenous ϕX174 J gene with that of G4. The resulting mutant, ϕXG4J, was not viable on the level of plaque formation without ϕX174 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 ϕXG4J 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, ϕXG4J 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.

A high-resolution map of bacteriophage øX174 transcription

“…A different role for the antisense transcription seen in this work could be attributed to controlling the production of proteins A and A*, which are known to be critically important for ϕX174 replication and packaging (Roznowski et al, 2020), but also seem to be only needed at very low quantities for successful infections (Jeng et al, 1970). In fact, overexpression of these genes has been seen to be deleterious towards the host, therefore controlling their expression may be crucial to ensuring early stages of DNA replication are completed successfully before lysis gene E is expressed (Colasanti and Denhardt, 1985).…”

Genome modularization reveals overlapped gene topology is necessary for efficient viral reproduction

“…infections (Roznowski, Doore et al, 2019). This function is accomplished through binding within the J-F intergenic region of the φX174 genome.…”

“…This function is accomplished through binding within the J-F intergenic region of the φX174 genome. This A* binding leads to inhibiting DNA unwinding in this region and disruption of protein A rolling circle genome amplification (Eisenberg & Ascarelli, 1981, Roznowski et al, 2019, van Mansfeld, van Teeffelen et al, 1986. Therefore stochiometry differences between proteins A and A* brought about by A* upregulation in decompressed φX174, are likely to cause issues with genome replication and the maintenance of genome packaging fidelity.…”

NMR structure of a vestigial nuclease provides insight into the evolution of functional transitions in viral dsDNA packaging motors

“…In a second strategy, an empty virus shell is first assembled, and the genome is then actively packaged into this pre-formed container. This is the strategy used by some ssRNA (1) and ssDNA viruses (2,3), and virtually all dsDNA viruses such as herpes virus, pox virus, adenovirus, and all the tailed dsDNA bacteriophages (4). This second strategy is remarkable considering the enthalpic, entropic, and DNA bending energies that must be overcome to package DNA to near crystalline densities within the confined space of the capsid.…”