Cruciviruses
A strange sequence,
a big discovery
Beginning in 2001, the Stedman lab has traveled yearly to Lassen Volcanic National Park (Northern California) to sample the hot and acidic environments created by volcanic activity. One year, while combing through metagenomic data from Boiling Springs Lake, a strange sequence was found. The data showed a viral sequence, that had both homology to a DNA virus and to an RNA virus. At first, we assumed that this was an error in our data, because nothing like this had been seen before. However, after thorough confirmation, we characterized what would become the sequence of the very first virus of its kind to be discovered. It was named Boiling Springs Lake RNA-DNA Hybrid Virus (BSL-RDHV), and in 2012, Diemer & Stedman was published, marking the official discovery of these viral sequences.
Since then, many more viral genomes similar to BSL-RDHV have been found in metagenomes from a wide range of environments - from Tampa Bay, to New Zealand rivers, Antarctic lakes, deep sea sediments, dragonfly innards… and even in penguin poop! These viruses –believed to have ssDNA genomes– have been dubbed "chimeric viruses" or "cruciviruses" because of the “crossing” that their genomes imply. By investigating cruciviruses, we want to understand the origin of new viruses, and how the horizontal transfer of genes between distantly related viruses, such as RNA and ssDNA viruses, may occur.
Cruciviruses may be the result of a recombination between a ssDNA virus and a ssRNA virus. The capsid-encoding genes of Cruciviruses are homologous to RNA viruses, while the Rep encoding genes are homologous to ssDNA viruses. Cruciviridae was a proposed taxonomic name for cruciviruses, but they remain unclassifiable!
Currently, our research on these new and peculiar virus-like sequences in the eXtreme Virus Lab focuses on:
Recovering crucivirus genomes from different environments (Crucihunting) and data repositories (The Crusades) for phylogenetic reconstruction to understand the big picture of crucivirus evolution (Crucifiction)
Isolating possible hosts of these viruses (likely protists) to understand crucivirus biology (Crucifixion)
Characterizing the capsid protein shared by cruciviruses and RNA viruses (Crucifer)
Characterizing the replication protein of cruciviruses for their possible role in DNA-RNA recombination (Crucisade)
Studying the genetic exchange between RNA and DNA viruses in an in vivo system using a surrogate host (The Crucible)
The mysteries of CRUCIVIRUS Replication and PACKAGING
Scooping up environmental samples from Boiling Springs Lake in Lassen Volcanic National Park. Due to unstable hot spots in the earth around the lake, sampling is done from afar, and requires some creativity. This is where BSL-RDHV was discovered, and where we hope to find more viral sequences like it.
Almost all ssDNA viruses with circular genomes replicate their genomes in a similar fashion, called rolling circle replication (RCR). Crucivirus genomes encode a replication-associate protein (Rep) that is very similar to the Rep from porcine circovirus PCV2, in the circular rep-encoding single-stranded (CRESS) virus family. The ssDNA genome also contain a conserved stem-loop structure, and gene very similar to capsid genes that, before then, were only found in RNA viruses.
The crucivirus gene shared with an RNA virus es encodes the capsid protein, which makes the outside shell of the virus, and determines how the virus is transmitted and what organism it can infect. What is the evolutionary advantage of acquiring this gene? Can this capsid package both DNA and RNA, or are there any other required adaptations? Current work in our lab involves learning more about the crucivirus replication process through the characterization of the Rep protein, as well as the study of crucivirus capsids.
Cruci HUnters
The hosts of cruciviruses remain elusive. To look for these hosts, first we have to look for where cruciviruses are. The first step in cruci hunting involves environmental sampling. The lab has sampled from the aforementioned Woodburn peat bog, and Boiling Springs Lake in Lassen, as well as Drakesbad Marsh in Lassen, PSU campus , and a marsh in Gearhart, OR. Many previous discoveries of cruciviral DNA have come from these "water-mixing" environments. After collecting these samples, they are processed and filtered by a number of methods to purify the DNA present. The DNA is then selectively amplified for circular sequences, and PCR methods are used to recover the cruciviral sequences within.
In 2017, Stedman lab post-doc Dr. Nacho de la Higuera and undergrad Ellis Torrance discovered three new cruciviral sequences in an environmental sample from the marshland in Woodburn, OR (de la Higuera et al., 2019).
Part of the "crucicrew" on our 2017 trip to Lassen.
The first step in cruci hunting involves environmental sampling. The lab has sampled from the aforementioned Woodburn marsh, and Boiling Springs Lake in Lassen, as well as Drakesbad Marsh in Lassen, PSU campus, and a marsh in Gearhart, OR. Many previous discoveries of cruciviral DNA have come from these "water-mixing" environments. After collecting these samples, they are processed and filtered by a number of methods to purify the DNA present. The DNA is then selectively amplified for circular sequences, and PCR methods are used to recover the cruciviral sequences within.
Mill creek in Woodburn (OR) is where some cruciviruses were found. The crucicrew takes frequent trips here for sampling in hopes of finding more crucis and their hosts
Dr. Nacho and Ellis Torrance testing the pH of soil samples from Drakesbad Marsh, Lassen Volcanic Natl. Park, Northern California.
From here, the complete crucivirus genome can be further studied and used in the hunt for its natural host. Because the two genes in cruciviruses both closely related to eukaryotic viruses (among other indications), it is hypothesized that the natural hosts of cruciviruses are eukaryotic, likely microbial.
Key Publications
From our lab:
Others:
Researchers
Current:
Nacho de la Higuera, Ph.D. Research Associate
David W. Noeckel, B.S. PhD Candidate
Marcell Richard, Undergraduate