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.
Keywords: DNA packaging; Microviridae; bacteriophage evolution; experimental evolution; single-stranded DNA.