The discovery and characterization of two archaeal viruses using culture-independent methods

Abstract

The field of viral metagenomics has expanded our understanding of viral diversity from all three domains of life (Archaea, Bacteria and Eukarya). Traditionally, viral metagenomic studies provide information about viral gene content, but rarely provide knowledge about virion morphology and or cellular host identity. This thesis describes research to utilize culture-independent methods to identify and to characterize two new archaeal viruses starting with viral metagenomic sequences. The first virus, Acidianus tailed spindle virus (ATSV), was initially identified by bioinformatic analysis of viral metagenomic datasets from a high temperature (80° C) acidic (pH 2) hot spring located in Yellowstone National Park, USA. ATSV was purified and characterized directly from environmental samples without dependency on culturing. Characterization included identification of the large tailed spindle shape virion morphology, determination of the complete 70.8 kbp circular ds DNA viral genome content, and identification of its cellular host. The host of ATSV, Acidianus hospitalis, was determined using CRISPR/Cas identification and CARD-FISH, and was confirmed by culturing. Additional characterization of ATSV included solving the structure of the major coat protein (MCP) by X-ray crystallography. The ATSV MCP reveals a decorated right-handed four helix bundle. The MCP is packed into the crystal as a four-start superhelix, for which the interfaces show biologically relevant interactions, indicating that ATSV might assemble using a multi-start helix. CryoEM images of ATSV show striations extending across the virion, supporting an assembly model in which long protein strands form the spindle virion structure. This is the first known model of spindle virus assembly. Culture-independent methods developed for ATSV purification and characterization were applied to a second virus, a pleomorphic particle found in high abundance in the CHAS viral fraction. Using mass spectrometry identification, viral metagenomics, deep sequencing, and host identification, a full virus genome and a host were linked to the virus particle, named Stygiolobus pleomorphic virus (SPV). SPV most likely represents a new virus family, with a unique particle morphology and gene content. Taken together, the results reported in this thesis provide an expanded pathway for the discovery, isolation and characterization of new viruses using culture-independent approaches.