A small DNA virus initiates replication with no more than three genome copies per cell

Abstract

Cellular organisms purge lethal mutations as they occur (in haploids), or as soon as they become homozygous (in sexually reproducing diploids), thus making the mutation-carrying genomes the sole victims of lethality. How lethal mutations in viruses are purged remains an unresolved question because numerous viral genomes could potentially replicate in the same cell, sharing their encoded proteins, hence shielding lethal mutations from selection. Previous investigations by us and others suggest that viruses with plus strand (+) RNA genomes may compel such selection by bottlenecking the replicating genome copies in each cell to low single digits. However, it is unclear if similar bottlenecks also occur in cells invaded by DNA viruses. Here we investigated whether tomato yellow leaf curl virus (TYLCV), a small virus with a single-stranded DNA genome, underwent population bottlenecking in cells of its host plants. We engineered the TYLCV genome to produce two replicons that express green fluorescent protein and mCherry, respectively, in a replication-dependent manner. We found that less than 65% of cells penetrated by both replicons replicated both, whereas at least 35% of cells replicated either of them alone, illustrating an intracellular population bottleneck size of no more than three. Furthermore, sequential inoculations unveiled strong mutual exclusions of these two replicons in most cells. Collectively our data demonstrated for the first time that DNA viruses like TYLCV are subject to stringent intracellular population bottlenecks, suggesting that such population bottlenecks may be a virus-encoded, evolutionarily conserved trait that assures timely elimination of lethal mutations.