1. From the Sequence to the Field:
Working to Understand Cruciviral
Origins through Sequence
Recovery and Host Elucidation
Ellis Torrancet, Ignacio de la Higuera, George Kasun, & Kenneth Stedman
tUndergraduate Student Researcher Portland State University: Center for Life in Extreme Environments
1
2. 2
Study Outline
From the Sequence to the Field:
Working to Understand Cruciviral Origins through
Sequence Recovery and Host Elucidation
What is a Crucivirus?
• Descendants of a Chimeric Genome
• Discovery of the Crucivirus (CruV)
• Components of CruV
• The Replication Initiation Protein (Rep)
• The Capsid Protein (Cp)
Research Goals
• Methods: Steps to Full CruV Genome Recovery
• Related Viral Families and Host Ecology
• Culturing Potential Hosts
• Project Significance
4. 4
Unknown
Host Cell
The Rep protein gene of
CruV relates most
closely to the CRESS
family of
ssDNA viruses
The Capsid protein gene
of CruV relates most
closely to the
Tombusviridae family of
ssRNA Viruses
Cruciviruses (CruV)
are a proposed
lineage of ssDNA
viruses that arose
from a hypothesized
ancestral
recombination event
between a ssRNA
virus and a ssDNA
virus.
Recombination
between an RNA and
DNA viral genome is
novel and has large
implications in the
evolution of life.
Cruciviruses are Hypothesized
Descendants of a Chimeric
Genome
(Diemer & Stedman, 2012)
5. 5
Discovery of the Crucivirus
• The first Crucivirus (CruV), BSL-RDHV, was
discovered in a metagenomic analysis of Boiling
Springs Lake of Lassen Volcanic National Park
in Lassen, CA.
• Researchers were surprised to find a sequence
which was most homologous to a ssRNA virus in
a DNA metagenomic analysis.
• Upon full genome recovery, the sequence was
found to contain a putative capsid protein (Cp)
gene with most homology to ssRNA viral family
Tombusviridae and a putative replication
initiation protein (Rep) with homology to a
family of circular Rep-encoding single-stranded
viruses known as “CRESS” DNA viruses.
(Diemer & Stedman, 2012)
6. 6
Components of A Crucivirus: The Rep
• CruVs are characterized by the presence of
two putative genes. One is a putative Capsid
protein sequence and the other is a putative Rep
protein sequence.
• The Rep protein likely initiates the replication
of the genome by nicking a conserved
nonanucleotide motif present in a stem-loop
secondary structure of the viral genome - as has
been demonstrated to occur in ssDNA viruses
with related Rep sequences.
• Different CruV genome Reps have homology
with different members of the “CRESS” viral
family including: circoviridae, cycloviridae,
nanoviridae, geminiviridae etc.
(Roux et al, 2013)
7. 7
Components of A Crucivirus: The Capsid
• CruV sequences encode a putative capsid
gene (Cp) with homology to the ssRNA
viral family, Tombusviridae.
• The Cp is a structural viral protein
responsible for packaging the CruV
genome. Viral capsids generally dictate host
specificity during viral infection. Though
the host-range of same viruses may still be
broad.
8. 8
Research Goals
This study aims to further investigate CruV origin by amassing
environmental sequences primarily from peat bog and wetland
environments. Once we’ve obtained a CruV sequence from an
environment, we can begin to culture potential host candidates
from the sample the genome was acquired from.
9. 9
Step 1: Field Sampling
• Soil and water samples are collected from a
variety of environments that share similarity
with “water-mixing environments” where
CruV sequences have been detected in
previous studies.
• Sites Surveyed Include:
• Boiling Springs Lake & Drakesbad
Marsh, Lassen Volcanic National
Park, CA.
• Woodburn Peat Bog, Woodburn,
OR.
• PSU Research Greenhouse soil-
traps
• Gearhart Marsh, Gearhart, OR.
Methods
11. 11
Step 3. PCR with Degenerate Primers
• Degenerate primers are designed to amplify the most conserved S-
domain region of the capsid gene.
• These primers were designed based on CruV genomes previously
recovered through metagenomic studies.
• Degeneracy in the primer sets for
the CruV family are necessary as
the nucleotide sequence is much
more varied than the amino acid
sequence present in the S-Domain
region of the capsid gene.
Methods
12. 12
Step 3. Cloning & Sequencing
• The capsid sequence amplified
with degenerate primers is gel
extracted and cloned into a vector.
• Because we use degenerate
primers, we don’t know what
exact primer set amplified the
potential CruV sequence.
Cloning into a vector allows us
to use primers specific to the
vector to sequence the insert.
Methods
13. 13
Step 4. Inverse PCR & Sequencing
• From the sequenced CruV capsid fragment,
we are able to design primers facing in the
inverse direction.
• These primers are then used to perform
PCR on the environmental DNA
extraction in an attempt to recover the full
circular ssDNA CruV genome.
15. 15
Culturing Potential Hosts
Video: One Gram
of Soil contains so
much fungal
diversity!
• Because viruses related to CruV infect
Eukaryotic organisms, we hypothesize the host is
a Eukaryote.
• Fungi are dominant in the soil and water
environments from which CruV sequences
are obtained.
• By culturing and isolating organisms from
the same samples we obtain CruV genomes
from, we believe it is possible to identify a
cruciviral host.
• Fungi are cultured on Potato Dextrose Agar
(PDA) with Chloramphenicol to limit
bacterial interference. Cultures are tested for
the presence of CruV sequences with Cp
specific degenerate primers.
Methods
16. 16
Novel Cruciviral Genome Recovery: PB1-RDHV
Findings
Putative Rep Gene
Putative Capsid
Gene
• Using the outlined methods, we have
recovered a novel CruV genome from a
peat bog in Woodburn, OR tentatively
named PB1-RDHV.
• Presently we are working to culture its
host from the environmental sample it was
obtained from.
17. 17
Conclusions: Significance of
Culturing a Host
• Demonstrating viral infectivity of a host will allow the Crucivirus to officially
be a classifiable virus by present ICTV standards.
• Host elucidation will allow us to investigate the mechanisms by which DNA
and RNA recombination occurs and its potential impacts in the evolution of
cellular life.
18. 18
Acknowledgments
A huge thank you to the included funding agencies and
sponsors for their contributions to our research.
We’d love to hear from you: Please feel free to ask any
questions you may have about our research in the
comment section below.
19. Capsid models were generated using Protein Data Bank
AS Rose, AR Bradley, Y Valasatava, JM Duarte, A Prlić and PW Rose. Web-based molecular graphics for large complexes. ACM Proceedings of the 21st International Conference on Web3D Technology
(Web3D '16): 185-186, 2016. doi:10.1145/2945292.2945324
AS Rose and PW Hildebrand. NGL Viewer: a web application for molecular visualization. Nucl Acids Res (1 July 2015) 43 (W1): W576-W579 first published online April 29, 2015. doi:10.1093/nar/gkv40
Genome & Sequence Alignment Images were generated with Geneious Version 9.0
Kearse, M., Moir, R., Wilson, A., Stones-Havas, S., Cheung, M., Sturrock, S., Buxton, S., Cooper, A., Markowitz, S., Duran, C., Thierer, T., Ashton, B., Mentjies, P., & Drummond, A. (2012). Geneious Basi
an integrated and extendable desktop software platform for the organization and analysis of sequence data.Bioinformatics, 28(12), 1647-1649.
19
Diemer, G. S., & Stedman, K. M. (2012). A novel virus genome discovered in an extreme environment suggests recombination between unrelated groups of RNA and DNA viruses. Biology
Direct, 7, 13. http://doi.org/10.1186/1745-6150-7-13
Roux, Simon et al. “Chimeric Viruses Blur the Borders between the Major Groups of Eukaryotic Single-Stranded DNA Viruses.” Nature Communications 4 (2013): ncomms3700. Web. 22 Oct. 2017.
Works Cited
Image Generation
Editor's Notes
Title, outline, intro to viruses and viral taxonomy, research objectives, methods, results so far, impact, acknowledgements, works cited 27 Slide Limit including title and acknowledgement
Formal title of the research project on slide 1 (may also include presenter information) Statement of hypothesis, research question or research goals Technical description of research methods and how they serve to test the hypothesis or answer the research question Data and results Findings or conclusions
