The interplay between Cucumovirus 2b protein

and RNA silencing: A Review of one Viral

Suppressor of RNA Silencing
Tan Tri V. Nguyen
Keywords: Cucumovirus 2b, RNA silencing, discovery

Abstract
RNA silencing is a robust antiviral defense mechanism utilized by many eukaryotes to
shut down viral gene expression and to regulate the expression levels of endogenous genes.
Virus must encode specialize proteins to specifically address and overcome this host defensive
barrier in order to replicate, to propagate, and to proliferate. These proteins that have evolve to
suppress RNA silencing are knows as viral suppressor of RNA silencing (VSR). Of the many
VSR discovered, the Cucumovirus 2b gene was one the first.

About Host and Pathogen Interactions

The interplay between host and pathogen is both intricate and complex. Where hosts are
much larger than their respective pathogen and have a multiple forms of defense. Pathogens on
the other hand are much smaller and evolved to become specialist in evading host defenses.
Hosts can react to an invading pathogen by targeting specific proteins or targeting a ubiquitous
product of the virus infectivity cycle. The number of interactions between host and pathogen is
vast. This review will solely focus studies carried out on the Cucumovirus 2b protein and its role
viral suppressor of RNA Silencing activity (VSR).

Figure 1: A purpose model of RNA silencing against virus infection with site
targeted by VSRs a with modification add to the Saleh Labs original model [1].
Arrow indicates target of known VSRs[2].

RNA Silencing: A far-reaching mechanism

The RNA silencing mechanism is an elegant form of check and balance for protein
expression [3]. This mechanism can been seen as exogenous with small interfering RNA
(siRNA) products produce by cleavage of from virl genetic transcripts [4] but also endogenous
with regulatory internal hairpin micro RNA [5]. The RNA Silencing mechanism begins with
detection of long foreign double stranded RNA (dsRNA) by Dicer or Dicer-like protein, an
endonuclease RNaseIII enzyme, that dices dsRNA into siRNA fragments that are 21 to 24
nucleotide in size [6-8] (Fig. 1 Step 1-2). One siRNA is recognized and gets incorporated into
the secondary endonuclease catalytic enzyme Agronaute (AGO) and accompanying proteins to
form a RNA-induced silencing complex (RISC) [9]. A mature RISC is contains of a single
stranded anti-sense RNA that makes perfect complement with messenger RNA (mRNA)
transcripts [10] (Fig. 1 Step 3-4). The RISC is a destruction complex that finds and binds
matching mRNA, for AGO to cleave the complement mRNA strand [10, 11]. This cleavage
leaves small mRNA transcript fragments that are degraded by host lysosomes and leads to loss
of viral protein translation [12].
Even though RNA silencing is a robust antiviral mechanism, many virus have been able
to overcome this mechanism by either directly or indirectly interacting to the products or
components.

Figure 2: An animated representation of the

Cucumovirus virion and genomic structure retrived
from http://viralzone.expasy.org/ [13].

The anatomy of a Cucumovirus

The genus Cucumovirus contains many species that infect numerous commercial crops
some of which includes: Cucumber mosaic virus (CMV), Peanut stunt virus (PSV), and Tomato
aspermy virus (TAV) [14]. The virus structure is non-enveloped, with icosahedral capsid. This is
a segmented virus, with three linear ssRNA(+) genome named RNA1, RNA2, and RNA3(Fig. 2).
Each RNA structure is analogous to a host mRNA. RNA1 and RNA2, which encodes for protein
1a and 2a respectively is the primary replication proteins [15, 16]. While RNA3 and its
subsequent sub genomic RNA4 encodes for the coat protein that is involved in assembly and
packaging of new virus particles [17]. Moreover, 2b is an overlapping sub genomic construct
found at the C-terminus end of the RNA2[16]. The 2b protein has been extensively studies
because of its role in suppressing RNA silencing, or formerly known of post-transcriptional gene
silencing [18, 19].

Principle techniques utilized to characterized 2b.

Molecular biology has many techniques utilized to understand the interaction between
proteins, genes, and other forms of gene protein interaction [20]. In light of the sheer numbers of
techniques utilized, I will only focus specific techniques that apply to identifying VSR 2b [20].
The first is the Agrobacterium infiltration assay (agro-infiltration) that has been routinely
applied in the discovery of many VSRs[21]. This Agro-infiltration is easy to perform, very cost
effective, and has high efficiency when expression genes of interest in a short time span [22,
23]. Testing begins with the gene of interest cloned into a shuttle vector and moved into a strain
of Agrobacterium tumefaciens. Agrobacterium suspensions are created consisting of the gene
of interest and a reporter gene (GFP or -glucuronidase, GUS or green fluorescent protein,
GFP) are mixed and infiltrated into leaves of Nicotiana benthamiana plants using a needleless
syringe [2]. The VSR activity of the gene of interest is accessed by its ability to sustain or

enhance the prolonged expression of the reporter gene [24]. In theory, this assay should results
in high efficacy[25]. One caveat of this assay is that it only applies to VSRs that suppress RNA
silencing

locally

and

not

systemically[26].

The second method is to knockout host gene of interest and accesses the ability of this
mutant to react to infiltration of the gene of interest. In the past many mutant are created using a
transfer-DNA (T-DNA) to disrupt gene function in selected plants [27]. The Arabidopsis thaliana
model organism has a library of over 100,000 T-DNA mutants of knockout genes [28, 29]. While
random T-DNA Arabidopsis mutants have been essential to biology, modern knockout technique
using clustered regularly interspaced short palindromic repeats (CRISPRs) is changing how to
create knock out mutants by targeting specific genes [29].

A historical review of Cucumovirus 2b as a VSR

Pre-discovery and wide acceptance of RNA silencing
The Cucumovirus 2b protein (100 codons) is found within the RNA2 segement and
contains an overlapping frame (69 codons)[15]. The 2b was detected when a protein analysis
reveals two protein bands. The first coincide with the 2a protein, while the smaller 10,000
kiladalton product is seen consistently when purified from infected plants but not viral particle.
This observance leads to the excision and first identification of the 2b protein[15].
Characterization of the importance of 2b was carried out by creating a variety of mutants;
deletion of 2b open reading frame, single amino acid substitution, single amino acid deletion,
and stop codon insertion[16]. When the mutants were compared to the wild type, which consist
of the full RNA2 segment with an untouched 2b region, the difference in virulence was clear. A
RNA2 segment lacking a functional 2b open reading frames lack has a greatly reduced level of
virulence[16].

With the understanding that 2b if of importance to virulence, further analysis was carried out to
understand its role by isolating the gene from the RNA2 sequence. The genetic sequence that
encodes for 2b for isolated via amplification using the polymerase chain reaction. A chimeric
virus was created making 2b the focal point by gutting the potato virus X (PVX) virus and
swapping in 2b. The essential components for was kept from PVX to ensure the PVX-2b mutant
expresses a function 2b protein. PVX-2b chimera show strong activity of GFP reporter protein
and eventual death of N. benthamiana plants tested[18].

Post-discovery and wide acceptance of RNA silencing

In 1998 Fire et al. observed the mechanism by which dsRNA was cleaved and made a
significance contribution to RNA biology that lead to a 2006 Nobel Peace Prize[30]. The initial
observation by Fire et al which lead to the elucidation of the RNA interference pathway, that
would later be widely recognize as RNA silencing in plants [31, 32].
With the RNA silencing mechanism known further studies in 2b protein interaction was
carried out with respect to the pathway. Argonaute (AGO) has now been known as the main
catalytic component of the RISC complex and works by Zhang et al revealed that 2b interacts
with Ago directly at the PAZ containing module and part of the PiWi Box to inhibit AGO cleavage
activity (Fig 1. Step 3) [33]. Furthermore, crystal analysis of 2b protein from the Tomato
aspermy virus, another species of the Cucumovirus genus, reveal a dimerization between 2b in
order to bind to siRNA thus preventing the loading of siRNA into AGO to form a RISC[34].

Onward with 2b
2b has been a model protein for elucidating the many mechanism of VSR [16, 17]. To
date it would unwise to slow down the any investigation on 2b. This protein has been found to
interact with the RNA silencing pathway at multiple levels from the siRNA via homo dimer

binding, to direct and indirect interaction, and to enhance viral systemic movement [17, 18, 3341]. What other cellular function does 2b get involve is still a topic of much interest.
Furthermore, 2b multi- factorial interaction poses a worthwhile investigation as to how 2b aid the
evading host resistance.

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