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ABSTRACT Most icosahedral DNA viruses package and condense their genomes into pre-formed, volumetrically constrained capsids. However, concurrent genome biosynthesis and packaging are specific to single-stranded (ss) DNA micro- and parvoviruses. Before packaging, ~120 copies of the øX174 DNA-binding protein J interact with double-stranded DNA. 60 J proteins enter the procapsid with the ssDNA genome, guiding it between 60 icosahedrally ordered DNA-binding pockets formed by the capsid proteins. Although J proteins are small, 28–37 residues in length, they have two domains. The basic, positively charged N-terminus guides the genome between binding pockets, whereas the C-terminus acts as an anchor to the capsid’s inner surface. Three C-terminal aromatic residues, W30, Y31, and F37, interact most extensively with the coat protein. Their corresponding codons were mutated, and the resulting strains were biochemically and genetically characterized. Depending on the mutation, the substitutions produced unstable packaging complexes, unstable virions, infectious progeny, or particles packaged with smaller genomes, the latter being a novel phenomenon. The smaller genomes contained internal deletions. The juncture sequences suggest that the unessential A* (A star) protein mediates deletion formation.<sc>IMPORTANCE</sc>Unessential but strongly conserved gene products are understudied, especially when mutations do not confer discernable phenotypes or the protein’s contribution to fitness is too small to reliably determine in laboratory-based assays. Consequently, their functions and evolutionary impact remain obscure. The data presented herein suggest that microvirus A* proteins, discovered over 40 years ago, may hasten the termination of non-productive packaging events. Thus, performing a salvage function by liberating the reusable components of the failed packaging complexes, such as DNA templates and replication enzymes. 

ABSTRACT øX174, G4, and α3 represent the three sister genera of a Microviridae subfamily. α3-like genomes are considerably larger than their sister genera genomes, yet they are packaged into capsids of similar internal volumes. They also contain multiple A* genes, which are nested within the larger A gene reading frame. Although unessential under most conditions, A* proteins mediate the fidelity of packaging reactions. Larger genomes and multiple A* genes may indicate that genome packaging is more problematic for α3-like viruses, especially at lower temperatures, where DNA persistence lengths would be longer. Unlike members of the other genera, which reliably form plaques at 20°C, α3-like phages are naturally cold sensitive below 28°C. To determine whether there was a connection between the uniquely α3-like genome characteristics and the cold-sensitive phenotype, the α3 assembly pathway was characterized at low temperature. Although virions were not detected, particles consistent with off-pathway packaging complexes were observed. In a complementary evolutionary approach, α3 was experimentally evolved to grow at progressively lower temperatures. The two major responses to cold adaptation were genome reduction and elevated A* gene expression. IMPORTANCE The production of enzymes, transcription factors, and viral receptors directly influences the niches viruses can inhabit. Some prokaryotic hosts can thrive in widely differing environments; thus, physical parameters, such as temperature, should also be considered. These variables may directly alter host physiology, preventing viral replication. Alternatively, they could negatively inhibit infection processes in a host-independent manner. The members of three sister Microviridae genera (canonical species øX174, G4 and α3) infect the same host, but α3-like viruses are naturally cold sensitive, which could effectively exclude them from low-temperature environments (<28°C). Exclusion appeared to be independent of host cell physiology. Instead, it could be largely attributed to low-temperature packaging defects. The results presented here demonstrate how physical parameters, such as temperature, can directly influence viral diversification and niche determination in a host-independent manner.