Genome Modularization Reveals Overlapped Gene Topology Is Necessary for Efficient Viral Reproduction

Sequence
overlap between two genes is common across all genomes,
with viruses having high proportions of these gene overlaps. Genome
modularization and refactoring is the process of disrupting natural
gene overlaps to separate coding sequences to enable their individual
manipulation. The biological function and fitness effects of gene
overlaps are not fully understood, and their effects on gene cluster
and genome-level refactoring are unknown. The bacteriophage φX174
genome has ∼26% of nucleotides involved in encoding more than
one gene. In this study we use an engineered φX174 phage containing
a genome with all gene overlaps removed to show that gene overlap
is critical to maintaining optimal viral fecundity. Through detailed
phenotypic measurements we reveal that genome modularization in φX174
causes virion replication, stability, and attachment deficiencies.
Quantitation of the complete phage proteome across an infection cycle
reveals 30% of proteins display abnormal expression patterns. Taken
together, we have for the first time comprehensively demonstrated
that gene modularization severely perturbs the coordinated functioning
of a bacteriophage replication cycle. This work highlights the biological
importance of gene overlap in natural genomes and that reducing gene
overlap disruption should be an integral part of future genome engineering
projects.