Abstract
Chloroviruses (family Phycodnaviridae) infect eukaryotic, freshwater, unicellular green algae. A unique feature of these viruses is an abundance of DNA methyltransferases, with isolates dedicating up to 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). Results were compared to total available sites predicted in silico based on DNA sequence alone. SMRT-software detected N6-methyl-adenine (m6A) at GATC and CATG recognition sites, motifs previously shown to be targeted by PBCV-1 DNA methyltransferases M.CviAI and M. CviAII, respectively. At the same time, PacBio analyses indicated that 10.9% of the PBCV-1 genome had large interpulse duration ratio (ipdRatio) values, the primary metric for DNA modification identification. These events represent 20.6x more sites than can be accounted for by all available adenines in GATC and CATG motifs, suggesting base or backbone modifications other than methylation might be present. To define methylation stability, we cross-compared methylation status of each GATC and CATG sequence in three biological replicates and found ∼81% of sites were stably methylated, while ∼2% consistently lack methylation. The remaining 17% of sites were stochastically methylated. When methylation status was analyzed for both strands of each target, we show that palindromes existed in completely non-methylated states, fully-methylated states, or hemi-methylated states, though GATC sites more often lack methylation than CATG sequences. Given that both sequences are targeted by not just methyltransferases, but by restriction endonucleases that are together encoded by PBCV-1 as virus-originating restriction modification (RM) systems, there is 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. However, sites where hemi-methylation is conserved might represent a unique regulatory function for PBCV-1. This study serves as a baseline for future investigation into the epigenomics of chloroviruses and their giant virus relatives.
Highlights
Viruses infecting eukaryotic algae play a critical role in aquatic ecosystems
The results indicate that the top twenty eukaryotic virus hits were to algal-infecting viruses. Since these are in the “giant” class, we performed a correlation to define genome size as a driver of DNA methyltransferase enrichment, and found that this poorly explained the number of encoded DNA methyltransferases for eukaryotic viruses (R2 = 0.168)
Since PBCV-1 spacing is similar to E. coli (Table 2), and the C. variabilis NC64A host genome encodes a MutS homolog (XP_005846525), there might be a methyl-directed function associated with these two components
Summary
Viruses infecting eukaryotic algae play a critical role in aquatic ecosystems Their lytic activity results in redistribution of organic matter from a particulate pool into a dissolved to particulate continuum that can be assimilated by the remaining microbial community, providing ecosystem stability and driving biodiversity (Thingstad and Lignell, 1997; Weinbauer and Rassoulzadegan, 2004; Anesio and Bellas, 2011; Martiny et al, 2011; Weitz et al, 2015). Viruses that infect freshwater Chlorella-like green algae encode up to 18 distinct DNA methyltransferases, representing up to ∼4.5% of their total protein coding potential (Chan et al, 2006; Fitzgerald et al, 2007). Many of these enzymes share only distant homology to DNA methyltransferases encoded by cellular organisms, and may represent enzymes uniquely adapted for viral functions
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