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Inspired by recent discoveries of the prevalence of large viruses in the environment, we reassessed the longstanding approach of filtering water through small-pore-size filters to separate viruses from cells before metagenomic analysis. We collected samples from three sites in Hamilton Harbour, an embayment of Lake Ontario, and studied 6 data sets derived from <0.45-μm-and>0.45-μm-size fractions to compare the diversity of viruses in these fractions. At the level of virus order/family, we observed highly diverse and distinct virus communities in the >0.45-μm-size fractions, whereas the <0.45-μm-size fractions were composed primarily of Caudovirales. The relative abundances of Caudovirales for which hosts could be inferred varied widely between size fractions, with higher relative abundances of cyanophages in the >0.45-μm-size fractions, potentially indicating replication within cells during ongoing infections. Many viruses of eukaryotes, such as Mimiviridae, Phycodnaviridae, Iridoviridae, and Poxviridae, were detected exclusively in the oftendisregarded >0.45-μm-size fractions. In addition to observing unique virus communities associated with each size fraction from every site we examined, we detected viruses common to both fractions, suggesting that these are candidates for further exploration because they could be the product of ongoing or recent lytic events. Most importantly, our observations indicate that analysis of either fraction alone provides only a partial perspective of double-stranded DNA (dsDNA) viruses in the environment, highlighting the need for more comprehensive approaches for analyzing virus communities inferred from metagenomic sequencing. [ABSTRACT FROM AUTHOR]

Despite very low sequence homology, the major capsid proteins of double-stranded DNA (dsDNA) bacteriophages, some archaeal viruses, and the herpesviruses share a structural motif, the HK97 fold. Bacteriophage P22, a paradigm for this class of viruses, belongs to a phage gene cluster that contains three homology groups: P22-like, CUS-3-like, and Sf6-like. The coat protein of each phage has an inserted domain (I-domain) that is more conserved than the rest of the coat protein. In P22, loops in the I-domain are critical for stabilizing intra- and intersubunit contacts that guide proper capsid assembly. The nuclear magnetic resonance (NMR) structures of the P22, CUS-3, and Sf6 I-domains reveal that they are all six-stranded, anti-parallel β-barrels. Nevertheless, significant structural differences occur in loops connecting the β-strands, in surface electrostatics used to dock the I-domains with their respective coat protein core partners, and in sequence motifs displayed on the capsid surfaces. Our data highlight the structural diversity of I-domains that could lead to variations in capsid assembly mechanisms and capsid surfaces adapted for specific phage functions. IMPORTANCE Comparative studies of protein structures often provide insights into their evolution. The HK97 fold is a structural motif used to form the coat protein shells that encapsidate the genomes of many dsDNA phages and viruses. The structure and function of coat proteins based on the HK97 fold are often embellished by the incorporation of I-domains. In the present work we compare I-domains from three phages representative of highly divergent P22-like homology groups. While the three I-domains share a six-stranded β-barrel skeleton, there are differences (i) in structure elements at the periphery of the conserved fold, (ii) in the locations of disordered loops important in capsid assembly and conformational transitions, (iii) in surfaces charges, and (iv) in sequence motifs that are potential ligand-binding sites. These structural modifications on the rudimentary I-domain fold suggest that considerable structural adaptability was needed to fulfill the versatile range of functional requirements for distinct phages., (Copyright © 2019 American Society for Microbiology.)