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The misencapsidation of host RNA by

Broad bean mottle virus and Cowpea chlorotic virus


N Shrestha1, MK Schnizlein1, PH Weber1, JJ Bujarski1,2
1

Department of Biological Sciences and Plant Molecular Biology Center, Northern Illinois University, DeKalb, IL 60115 USA
2
Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, Poland

ABSTRACT
o This study seeks to identify whether Broad
bean mottle virus (BBMV) and Cowpea
chlorotic mottle virus (CCMV), members of
family Bromoviridae, misencapsidate host RNA
segments.
o RNA viruses like Flock house virus (FHV) are
known to encapsidate host RNA by mistake.
o Preliminary data suggests that Brome mosaic
virus
(BMV),
a
bromovirus,
might
misencapsidate host RNA as well.
o Broad bean (Vicia faba) and cowpea (Vigna
sinensis), natural hosts for BBMV and CCMV,
respectively, were used to cultivate the
viruses.
o Virion extraction and purification remove
cellular RNA and DNA fragments. This is
followed by the extraction of viral RNA.
o Finally, the viral RNA extracts are sequenced
by Next Generation RNA sequencing (NGS-RNA
Seq) and then are BLASTed for final analysis.
o The result should contribute to a better
understanding of the virus-host relationship
and the role of viruses in horizontal gene
transfer (HGT).

BACKGROUND

o BBMV and CCMV are members of the family


Bromoviridae, which have positive-sense
single-stranded RNA (+ssRNA).
o The viruses bear tripartite RNA genome:
RNA1, RNA2 and RNA3 along with a subgenomic RNA4 (sgRNA4). (Fig. 1A and 1B).
RNA1, RNA2, and RNA3 code for viral proteins
1a, 2a, and 3a, respectively.
o Capsid proteins are the determinants in
proper encapsid-ation of the viral RNA.
o Replicated viral RNAs compartmentalize in
specific cellular locations (e.g. FHV in
mitochondrial invagination).
o Failure to traffic these RNAs to the proper site
may lead to the auto assembly of virus-like
particles (VLPs)
o Normal
capsid
protein
synthesis,
accompanied by faulty RNA replication,
causes virions to misencapsidate cellular
nucleic acid or any other anionic particles.
o Although viral encapsidation is generally very
specific, some viruses are known to
misencapsidate cellular RNA.
o Misencapsidated host RNA has been observed
to make up 1% of the FHV genome.
o Preliminary data (Fig. 2.A) suggests that BMV
also misencapsidates host RNA, including it
with its own genome.
o Hypothesis: BBMV and CCMV replicate
similarly to other +ssRNA viruses (e.g. FHV),
which have been observed to misencapsidate
host RNA. Thus, we predict that this process
will occur in our model viruses as well.

BACKGROUND (cont.)

DISCUSSION

1.
B

o As we continue with this project, we


would like to harvest the viruses and
extract
the
RNA
as
mentioned
previously.
o Afterward, we will send the RNA
samples to the NGS RNA-Seq facility for
sequencing
and
then
have
the
sequences analyzed.
o In the future, we will expand our
research of viral misencapsidation of
host RNA by using new viruses as well
as a wider variety of hosts.
o The results of this investigation are
expected to be helpful in defining the
role of virus-mediated horizontal gene
transfer during plant evolution and in
generation of antiviral VLPs.
o The encapsidation of host genome
fragments might have repercussions on
the virulence.
Therefore, further
research will investigate the infectivity
of the misencapsidated virus.
o Additionally, identifying the type of RNA
We would like to thank Professor Bujarski and the rest of
segments
encapsidated
might
elucidate
the lab for their support and advice throughout this
the mechanism
antiviral
VLPs
project.
We would also by
likewhich
to thank
in advance
the
NextGen
Sequencing
Facility at the University of Chicago.
can be
generated.

1.
A

The genomes of BBMV and CCMV. 1.A) The genome of BBMV. 1.B) The genome of CCMV

MATERIALS AND METHODS


BBMV (MO
strain)
revitalized in
Chenopodium
quinoa

BBMV
inoculated to
broad beans
(Vicia faba)

CCMV
revitalized
in
Chenopodium
quinoa

CCMV
inoculated to
cowpeas
(Vigna
sinensis)

Extraction and
vigorous
purification

Extraction and
vigorous
purification

RESULTS
2.
B

2.
A

NextGen
Sequencing at
the University
of Chicago;
Sequence
analysis
NextGen
Sequencing at
the University
of Chicago;
Sequence
analysis

2.
C

2.
D

BMV RNA I and


RNA II
BMV RNA III
Possible
misencapsid-ated
host RNA
BMV sgRNA4

Preliminary RNA Gel of BMV and Infection of Host Plants. 2.A) This gel of the BMV genome shows
three bands of the actual viral genome as well as a faint band that could be misencapsidated host RNA.
2.B) Local lesions in C. quinoa caused by BBMV. Three of these lesions have been emphasized by black
circles 2.C) This figure depicts the systemic infection of BBMV in broad beans. Symptoms of infection
include the large yellow-green patches that follow venation of leaves (Photo source: Freeman, 2006). 2.D)
The symptoms of a systemic CCMV infection in a cowpea leaf can be seen. Symptoms are characterized by
yellow-green spots and lines spread across the leaves of the entire plant.

ACKNOWLEDGEME
NTS

This project was funded by an NSF-Grant (MCB-0920617).


Matt Schnizlein would like to thank the Office of Student
Engagement and Experiential Learning and the URA
program for its financial support.

CITATIONS

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and Genome Organization of the RNA-l Segment
in Two Bromoviruses: Broad Bean Mottle Virus and
Cowpea Chlorotic Mottle Virus. Virology, vol. 185.
(pp. 553-562).
Freeman A. (2006). Broad bean mottle virus. National
Diagnostic Protocol. Primary Industries Research:
Horsham, Australia.
Romero J., Dzianott A., & Bujarski J. (1991). The nucleotide
sequence and genome organization of the RNA2 and
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H.
&
Bujarski
J.
(1993).
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