Shared host cells can serve as melting pots for viral genomes, giving many phylogenies a web-like appearance due to horizontal gene transfer. However, not all virus families exhibit web-like phylogenies. Microviruses form three distinct clades, represented by φX174, G4, and α3. Here, we investigate protein-based barriers to horizontal gene transfer between clades. We transferred gene G, which encodes a structural protein, between φX174 and G4, and monitored the evolutionary recovery of the resulting chimeras. In both cases, particle assembly was the major barrier after gene transfer. The G4φXG chimera displayed a temperature-sensitive assembly defect that could easily be corrected through single mutations that promote productive assembly. Gene transfer in the other direction was more problematic. The initial φXG4G chimera required an exogenous supply of both the φX174 major spike G and DNA pilot H proteins. Elevated DNA pilot protein levels may be required to compensate for off-pathway reactions that may have become thermodynamically and/or kinetically favored when the foreign spike protein was present. After three targeted genetic selections, the foreign spike protein was productively integrated into the φX174 background. The first adaption involved a global decrease in gene expression. This was followed by modifications affecting key protein–protein interactions that govern assembly. Finally, gene expression was re-elevated. Although the first selection suppresses nonproductive reactions, subsequent selections promote productive assembly and ultimately viability. However, viable chimeric strains exhibited reduced fitness compared with wild-type. This chimera’s path to recovery may partially explain how unusual recombinant viruses could persist long enough to naturally emerge.

Viruses have brought humanity many challenges: respiratory infection, cancer, neurological impairment and immunosuppression to name a few. Virology research over the last 60+ years has responded to reduce this disease burden with vaccines and antivirals.

Conformational switching is an overarching paradigm in which to describe scaffolding protein-mediated virus assembly. However, rapid morphogenesis with small assembly subunits hinders the isolation of early morphogenetic intermediates in most model systems. Consequently, conformational switches are often defined by comparing the structures of virions, procapsids and aberrantly assembled particles. In contrast, ϕX174 morphogenesis proceeds through at least three preprocapsid intermediates, which can be biochemically isolated. This affords a detailed analysis of early morphogenesis and internal scaffolding protein function. Amino acid substitutions were generated for the six C-terminal, aromatic amino acids that mediate most coat-internal scaffolding protein contacts. The biochemical characterization of mutant assembly pathways revealed two classes of molecular defects, protein binding and conformational switching, a novel phenotype. The conformational switch mutations kinetically trapped assembly intermediates before procapsid formation. Although mutations trapped different particles, they shared common second-site suppressors located in the viral coat protein. This suggests a fluid assembly pathway, one in which the scaffolding protein induces a single, coat protein conformational switch and not a series of sequential reactions. In this model, an incomplete or improper switch would kinetically trap intermediates.

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.