Jamie C. Snyder

Viruses from extreme thermal environments

Viruses of extreme thermophiles are of great interest because they serve as model systems for understanding the biochemistry and molecular biology required for life at high temperatures. In this work, we report the discovery, isolation, and preliminary characterization of viruses and virus-like particles from extreme thermal acidic environments (70–92°C, pH 1.0–4.5) found in Yellowstone National Park. Six unique particle morphologies were found in Sulfolobus enrichment cultures. Three of the particle morphologies are similar to viruses previously isolated from Sulfolobus species from Iceland and/or Japan. Sequence analysis of their viral genomes suggests that they are related to the Icelandic and Japanese isolates. In addition, three virus particle morphologies that had not been previously observed from thermal environments were found. These viruses appear to be completely novel in nature.

Comparative Genomic Analysis of Hyperthermophilic Archaeal Fuselloviridae Viruses

ABSTRACT The complete genome sequences of two Sulfolobus spindle-shaped viruses (SSVs) from acidic hot springs in Kamchatka (Russia) and Yellowstone National Park (United States) have been determined. These nonlytic temperate viruses were isolated from hyperthermophilic Sulfolobus hosts, and both viruses share the spindle-shaped morphology characteristic of the Fuselloviridae family. These two genomes, in combination with the previously determined SSV1 genome from Japan and the SSV2 genome from Iceland, have allowed us to carry out a phylogenetic comparison of these geographically distributed hyperthermal viruses. Each virus contains a circular double-stranded DNA genome of ∼15 kbp with approximately 34 open reading frames (ORFs). These Fusellovirus ORFs show little or no similarity to genes in the public databases. In contrast, 18 ORFs are common to all four isolates and may represent the minimal gene set defining this viral group. In general, ORFs on one half of the genome are colinear and highly conserved, while ORFs on the other half are not. One shared ORF among all four genomes is an integrase of the tyrosine recombinase family. All four viral genomes integrate into their host tRNA genes. The specific tRNA gene used for integration varies, and one genome integrates into multiple loci. Several unique ORFs are found in the genome of each isolate.

Virus movement maintains local virus population diversity

Viruses are the largest reservoir of genetic material on the planet, yet little is known about the population dynamics of any virus within its natural environment. Over a 2-year period, we monitored the diversity of two archaeal viruses found in hot springs within Yellowstone National Park (YNP). Both temporal phylogeny and neutral biodiversity models reveal that virus diversity in these local environments is not being maintained by mutation but rather by high rates of immigration from a globally distributed metacommunity. These results indicate that geographically isolated hot springs are readily able to exchange viruses. The importance of virus movement is supported by the detection of virus particles in air samples collected over YNP hot springs and by their detection in metacommunity sequencing projects conducted in the Sargasso Sea. Rapid rates of virus movement are not expected to be unique to these archaeal viruses but rather a common feature among virus metacommunities. The finding that virus immigration rather than mutation can dominate community structure has significant implications for understanding virus circulation and the role that viruses play in ecology and evolution by providing a reservoir of mobile genetic material.

Use of Cellular CRISPR (Clusters of Regularly Interspaced Short Palindromic Repeats) Spacer-Based Microarrays for Detection of Viruses in Environmental Samples

ABSTRACT It is currently difficult to detect unknown viruses in any given environment. The recent discovery of CRISPR (clusters of regularly interspaced short palindromic repeats) loci within bacterial and archaeal cellular genomes may provide an alternative approach to detect new viruses. It has been shown that the spacer sequences between the direct repeat units of the CRISPR loci are often derived from viruses and likely function as guide sequences to protect the cell from viral infection. The spacer sequences within the CRISPR loci may therefore serve as a record of the viruses that have replicated within the cell. We have cataloged the CRISPR spacer sequences from cellular metagenomic data from high-temperature (>80°C), acidic (pH < 4) hot spring environments located in Yellowstone National Park (YNP). We designed a microarray platform utilizing these CRISPR spacer sequences as potential probes to detect viruses present in YNP hot spring environments. We show that this microarray approach can detect viral sequences directly from virus-enriched environmental samples, detecting new viruses which have not been previously characterized. We further demonstrated that this microarray approach can be used to examine temporal changes in viral populations within the environment. Our results demonstrate that CRISPR spacer sequence-based microarrays will be useful tools for detecting and monitoring viruses from diverse environmental samples.

Synergy of NMR, Computation, and X-ray Crystallography for Structural Biology

NMR spectroscopy and X-ray crystallography are currently the two most widely applied methods for the determination of macromolecular structures at high resolution. More recently, significant advances have been made in algorithms for the de novo prediction of protein structure, and, in favorable cases, the predicted models agree extremely well with experimentally determined structures. Here, we demonstrate a synergistic combination of NMR spectroscopy, de novo structure prediction, and X-ray crystallography in an effective overall strategy for rapidly determining the structure of the coat protein C-terminal domain from the Sulfolobus islandicus rod-shaped virus (SIRV). This approach takes advantage of the most accessible aspects of each structural technique and may be widely applicable for structure determination.

Viruses of hyperthermophilic Archaea

The viruses of Archaea are likely to be useful tools for studying host evolution, host biochemical pathways, and as tools for the biotechnology industry. Many of the viruses isolated from Archaea show distinct morphologies and genes. The euryarchaeal viruses show morphologies similar to the head-and-tail phage isolated from Bacteria; however, sequence analysis of viral genomes from Crenarchaea shows little or no similarity to previously isolated viruses. Because viruses adapt to host organism characteristics, viruses may lead to important discoveries in archaeal biochemistry, genetics, and evolution.

Sulfolobus Turreted Icosahedral Virus c92 Protein Responsible for the Formation of Pyramid-Like Cellular Lysis Structures

ABSTRACT Host cells infected by Sulfolobus turreted icosahedral virus (STIV) have been shown to produce unusual pyramid-like structures on the cell surface. These structures represent a virus-induced lysis mechanism that is present in Archaea and appears to be distinct from the holin/endolysin system described for DNA bacteriophages. This study investigated the STIV gene products required for pyramid formation in its host Sulfolobus solfataricus. Overexpression of STIV open reading frame (ORF) c92 in S. solfataricus alone is sufficient to produce the pyramid-like lysis structures in cells. Gene disruption of c92 within STIV demonstrates that c92 is an essential protein for virus replication. Immunolocalization of c92 shows that the protein is localized to the cellular membranes forming the pyramid-like structures.

Functional interplay between a virus and the ESCRT machinery in Archaea

Recently it has been discovered that a number of eukaryotic viruses, including HIV, coopt the cellular Endosomal Sorting Complex Required for Transport (ESCRT) machinery to affect egress from infected cells. Strikingly, the ESCRT apparatus is conserved in a subset of Archaea, including members of the genus Sulfolobus where it plays a role in cytokinesis. In the current work, we reveal that the archaeal virus Sulfolobus turreted icosahedral virus isolated from Yellowstone National Parks acidic hot springs also exploits the host ESCRT machinery in its replication cycle. Moreover, perturbation of normal ESCRT function abrogates viral replication and, thus, prevents establishment of a productive Sulfolobus turreted icosahedral virus infection. We propose that the Sulfolobus ESCRT machinery is involved in viral assembly within the cytoplasm and in escape from the infected cell by using a unique lysis mechanism. Our results support an ancient origin for viruses “hijacking” ESCRT proteins to complete their replication cycle and thus identify a critical host–virus interaction conserved between two domains of life.

Archaeal Viruses: Diversity, Replication, and Structure

The Archaea-and their viruses-remain the most enigmatic of lifes three domains. Once thought to inhabit only extreme environments, archaea are now known to inhabit diverse environments. Even though the first archaeal virus was described over 40 years ago, only 117 archaeal viruses have been discovered to date. Despite this small number, these viruses have painted a portrait of enormous morphological and genetic diversity. For example, research centered around the various steps of the archaeal virus life cycle has led to the discovery of unique mechanisms employed by archaeal viruses during replication, maturation, and virion release. In many instances, archaeal virus proteins display very low levels of sequence homology to other proteins listed in the public database, and therefore, structural characterization of these proteins has played an integral role in functional assignment. These structural studies have not only provided insights into structure-function relationships but have also identified links between viruses across all three domains of life.

Development of a genetic system for the archaeal virus Sulfolobus turreted icosahedral virus (STIV).

Our understanding of archaeal viruses has been limited by the lack of genetic systems for examining viral function. We describe the construction of an infectious clone for the archaeal virus Sulfolobus turreted icosahedral virus (STIV). STIV was isolated from a high temperature (82°C) acidic (pH 2.2) hot spring in Yellowstone National Park and replicates in the archaeal model organism Sulfolobus solfataricus (Rice et al., 2004). While STIV is one of most studied archaeal viruses, little is known about its replication cycle. The development of an STIV infectious clone allows for directed gene disruptions and detailed genetic analysis of the virus. The utility of the STIV infectious clone was demonstrated by gene disruption of STIV open reading frame (ORF) B116 which resulted in crippled virus replication, while disruption of ORFs A197, C381 and B345 was lethal for virus replication.