SMRT sequencing of Paramecium bursaria Chlorella Virus-1 reveals dynamic methylation patterns in adenines targeted by RM-systems

Chloroviruses (family Phycodnaviridae) infect eukaryotic, freshwater, unicellular green algae. A unique feature of these viruses is an abundance of DNA methyltransferases (MTases), with strains dedicating anywhere between 0 - 4.5% of their protein coding potential to these genes. This diversity highlights just one of the long-standing values of the chlorovirus model system, where group-wide epigenomic characterization might begin to elucidate the function(s) of DNA methylation in large dsDNA viruses. We characterized DNA modifications in the prototype chlorovirus, PBCV-1, using single molecule real time (SMRT) sequencing (aka PacBio). This was contrasted against total available sites predicted in silico based on DNA sequence alone. The SMRT-software detected N6-methyl-adenine (m6A) at GATC and CATG recognition sites, which are known methylation motifs associated with enzymes M.CviAI and M.CviAII, respectively. At the same time, PacBio analyses indicated that 10.9% of the PBCV-1 genome is associated with large interpulse duration ratio (ipdRatio) values, the primary metric for DNA modification identification. This represents 20.6x more sites than all available target adenines in CATG and GATC contexts, and contrasts against analyses in Escherichia coli wherein all sites with similar ipdRatio values can be accounted for by detected motifs. Cross comparisons of methylation status between biological replicates for each target tetramer indicate ~81% of sites are stably methylated, and ~2% are stably unmethylated. The remaining 17% of sites are stochastically methylated between the biological replicates. When these are paired together with their palindrome specific compliments, we show that palindromes exist in completely non-methylated states, fully methylated states, and hemi-methylated states. Given these sites are targeted by not just methyltransferases, but by restriction endonucleases that are encoded by PBCV-1 as virus-originating restriction modification systems, there is a strong selective pressure to modify all target sites. The finding that most instances of non-methylation are associated with hemi-methylation is congruent with observations that hemi-methylated palindromes are resistant to cleavage by restriction endonucleases recognizing these sequences. However, that some hemi-methylated sites are consistently not methylated might represent a unique biological function for PBCV-1. This study serves as a baseline for future investigation into the epigenomics of chloroviruses and their giant virus relatives.