Functional Viral Metagenomics and the Development of New Enzymes for DNA and RNA Amplification and Sequencing

The enzymes of phages and other viruses were vital to the early development of molecular biology and are still essential tools. However, the available viral enzymes represent a tiny sample of the potential diversity found in the global virosphere. Viral metagenomics has revealed a vast diversity of novel genes and its virtually limitless potential to provide new enzymes for use in molecular analysis. An important challenge to both the understanding of viral ecology and development of new viral enzymes is functional characterization of metagenomic sequences, which has lagged far behind the ability to collect sequence data. Described is a program to identify and characterize replication operons of viral metapopulations isolated from natural thermal environments and develop the gene products as thermostable enzymes for nucleic acid amplification and sequencing. Approaches to functionally characterize viral replicases include (1) expression and biochemical analysis of gene products identified by sequence similarity, (2) functional screens to discover new families of genes, and (3) assembly of operons to predict function based on gene position. These approaches have uncovered at least two diverse families of replication operons including dozens of genes for thermostable DNA polymerases and reverse transcriptases, as well as likely replicase subunits. In addition, functional screens have uncovered one viral Pol unrelated to any known protein. These enzymes are being engineered as improved PCR, RT PCR, and DNA sequencing reagents. Diversity in the viral metagenomes is also being explored to optimize the activity of the genes discovered in the libraries and make them more suitable for the targeted applications. Gene products of phages and other viruses (collectively referred to here as viruses) have historically provided many of the enzymatic tools for molecular biology. However, most of the commonly used viral enzymes are derived from a very limited number of cultivated viruses, primarily phages T4, T7, lambda, SP6, and phi29, and retroviruses Moloney murine leukemia virus (Mo-MLV) and avian myeloblastosis virus (AMV). The program to study hot spring virology in Yellowstone National Park (YNP), California, and Nevada has provided insight into viral ecology (Otto et al. 1998; Breitbart et al. 2004; Schoenfeld et al. 2008) and has revealed a nearly unlimited source of diversity for the search for new enzymes (Beechem et al. 1998; Moser et al. 2012; Perez et al. 2012). However, current approaches to functional analysis of viral metagenomes, while informative, are limited by their reliance on sequence similarity to infer gene function. Improvements in the ability to functionally characterize viral metagenomes are necessary to advance the field. Thermostable DNA polymerases (Pols) have been a major research focus due mainly to their wide use in molecular detection and analysis. DNA polymerases are essential for PCR (Staley and Konopka 1985) and other target-specific (Petruska et al. 1998; Notomi et al. 2000) and whole genome amplification methods (Goodman and Fygenson 1998) and are also essential components of all the major DNA sequencing platforms. Sanger (dideoxy chain termination) DNA sequencing was