Complete Mutagenesis of the Genome and Proteome of ΦX174

Abstract The bacteriophage ΦX174 was the first genome to be sequenced and the first to be chemically synthesised. Here we present a complete map of the consequences of changing every nucleotide in the ΦX174 genome and every amino acid in the proteome. In total, half of nucleotide and >60% of amino acid changes impair fitness. However, this varies substantially across proteins and non-coding regions. Surprisingly, the most important mechanistic cause of reduced fitness is disruption of protein interaction interfaces, accounting for nearly half of detrimental protein variants. A further one quarter of damaging variants disrupt protein cores but one in four lack a mechanistic explanation. Mutational effects are only moderately-well predicted by state-of-the-art artificial intelligence models, but, combined with structural modelling, provide mechanistic hypotheses and insights into the DNA replication machinery. This complete mutagenesis of a biological system quantifies the limits of our current understanding of, and ability to predict, molecular biology.