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The curious case of genome
packaging and assembly in RNA
viruses infecting plants
Tushar Ranjan
1
*
†
, Ravi Ranjan Kumar
1†
, Mohammad Ansar
2
,
Jitesh Kumar
1
, Auroshikha Mohanty
1
, Anamika Kumari
1
,
Khushbu Jain
1
, Kumari Rajani
3
, Sailabala Dei
4
and
Mohammad Feza Ahmad
5
1
Department of Molecular Biology and Genetic Engineering, Bihar Agricultural University, Bhagalpur,
Bihar, India,
2
Department of Plant Pathology, Bihar Agricultural University, Bhagalpur, Bihar, India,
3
Department of Seed Science and Technology, Bihar Agricultural University, Bhagalpur, Bihar, India,
4
Deputy Director Research, Bihar Agricultural University, Bhagalpur, Bihar, India,
5
Department of
Horticulture, Bihar Agricultural University, Bhagalpur, Bihar, India
Genome packaging is the crucial step for maturation of plant viruses containing an
RNA genome. Viruses exhibit a remarkable degree of packaging specificity, despite
the probability of co-packaging cellular RNAs. Three different types of viral
genome packaging systems are reported so far. The recently upgraded type I
genome packaging system involves nucleation and encapsidation of RNA
genomes in an energy-dependent manner, which have been observed in most
of the plant RNA viruses with a smaller genome size, while type II and III packaging
systems, majorly discovered in bacteriophages and large eukaryotic DNA viruses,
involve genome translocation and packaging inside the prohead in an energy-
dependent manner, i.e., utilizing ATP. Although ATP is essential for all three
packaging systems, each machinery system employs a unique mode of ATP
hydrolysis and genome packaging mechanism. Plant RNA viruses are serious
threats to agricultural and horticultural crops and account for huge economic
losses. Developing control strategies against plant RNA viruses requires a deep
understanding of their genome assembly and packaging mechanism. On the basis
of our previous studies and meticulously planned experiments, we have revealed
their molecular mechanisms and proposed a hypothetical model for the type I
packaging system with an emphasis on smaller plant RNA viruses. Here, in this
review, we apprise researchers the technical breakthroughs that have facilitated
the dissection of genome packaging and virion assembly processes in plant RNA
viruses.
KEYWORDS
viral genome packaging, energy-dependent, capsid protein, ATPase fold, virus assembly,
RNA virus
1 Introduction
Genome packaging and assembly is a crucial step during the life cycle of RNA viruses
infecting plants (Rao, 2006;Ranjan et al., 2021;Kumar et al., 2022;Mohanty et al., 2023).
Three different approaches have been reported by which viruses condense their genome
inside the empty prohead (Chelikani et al., 2014a;Ranjan et al., 2021;Mohanty et al., 2023).
According to the recently upgraded type I classification system present in many of the
smaller plant RNA viruses of genome size <20 kb, the processes of assembly and genome
OPEN ACCESS
EDITED BY
Davoud Koolivand,
University of Zanjan, Zanjan, Iran
REVIEWED BY
Himanshu Tak,
Bhabha Atomic Research Centre (BARC),
India
Bajarang Kumbhar,
SVKM’s Narsee Monjee Institute of
Management Studies, India
*CORRESPONDENCE
Tushar Ranjan,
mail2tusharranjan@gmail.com
†
These authors share first authorship
RECEIVED 01 April 2023
ACCEPTED 22 May 2023
PUBLISHED 08 June 2023
CITATION
Ranjan T, Ranjan Kumar R, Ansar M,
Kumar J, Mohanty A, Kumari A, Jain K,
Rajani K, Dei S and Ahmad MF (2023), The
curious case of genome packaging and
assembly in RNA viruses infecting plants.
Front. Genet. 14:1198647.
doi: 10.3389/fgene.2023.1198647
COPYRIGHT
© 2023 Ranjan, Ranjan Kumar, Ansar,
Kumar, Mohanty, Kumari, Jain, Rajani, Dei
and Ahmad. This is an open-access article
distributed under the terms of the
Creative Commons Attribution License
(CC BY). The use, distribution or
reproduction in other forums is
permitted, provided the original author(s)
and the copyright owner(s) are credited
and that the original publication in this
journal is cited, in accordance with
accepted academic practice. No use,
distribution or reproduction is permitted
which does not comply with these terms.
Frontiers in Genetics frontiersin.org01
TYPE Review
PUBLISHED 08 June 2023
DOI 10.3389/fgene.2023.1198647
packaging involve the coating of the nucleic acid with viral capsid
proteins (CPs) in the presence of ATP (Ranjan et al., 2021;Mohanty
et al., 2023). On the contrary, a powerful ATP-fueled packaging
motor, to package the genome in already assembled proheads of
larger viruses with the genome size >20 kb, is involved in both type II
and III systems (Burroughs et al., 2007;Chelikani et al., 2014a;
Chelikani et al., 20014b). Both type II and III packaging systems
have been reasonably well studied so far, but the details of the
molecular mechanism of genome packaging, translocation, and
assembly processes in plant RNA viruses are rather limited
(Ranjan et al., 2021;Mohanty et al., 2023). Therefore, this review
aims to update readers on the current knowledge of the molecular
mechanism of genome packaging and the steps involved in viral
maturation of plant RNA viruses, with a special emphasis on
Polerovirus and Potexvirus.
After its entry into a host plant cell, an RNA virus carries out the
following steps in a highly coordinated manner to complete a successful
infection: 1) uncoating of virions; 2) immediate translation of proteins
related to replication; 3) synthesis of new (−)strandand(+)strand
RNAs; 4) translation of the movement protein (MP) and capsid protein
from newly synthesized viral mRNAs; and finally, 5) the assembly of
infectious virions capable of moving from one cell to neighbor cells
through plasmodesmata or ultimately interacting with insect vectors for
their spreading to healthy plants (Bol, 2005;Catalano, 2005;Rao, 2006;
Kumari et al., 2020;Garmann et al., 2022).Theentireprocessofthe
replication event has been explored in depth in several plant RNA
viruses and tremendous reviews have been published worldwide on this
subject (Annamalai and Rao, 2005;Bol, 2005;Rao, 2006;Annamalai
et al., 2008;Rao et al., 2014). The RNA genome of viruses ranges from
monopartite to multipartite, and RNA strands could be of positive or
negative senses (Table 1). Table 1 represents the detailed characteristics
of RNA viruses infecting plants with suitable examples (Table 1). The
plant RNA virus replicates within a cytoplasmic compartment, which is
heavily populated by cellular RNAs (Burd and Dreyfuss, 1994;Rao,
2006). Thus, CPs, the RNA-binding proteins, exhibit a remarkable
degree of packaging specificity, despite their probability of co-packaging
cellular RNAs (Burd and Dreyfuss, 1994;Ranjan et al., 2021;Kumar
et al., 2022).
2 CPs with a novel ATPase domain: a
new regime in the genome packaging
of plant RNA viruses
In our previous study, an attempt was made to expand and sub-
classify type I packaging machinery for several plant RNA viruses,
TABLE 1 Plant RNA viruses along with their characteristics.
Name of the plant RNA virus Family Type of RNA
genome
No. of RNA
fragments
Host plant
Potato leafroll virus, tobacco necrotic dwarf virus, chickpea
stunt disease-associated virus, barley yellow dwarf virus,
groundnut rosette assistor virus, and soybean dwarf virus
Solemoviridae Single-stranded positive-
strand RNA
Monopartite Potato, tobacco, chickpea,
barley, groundnut, and
soybean
Tomato bushy stunt virus, cucumber soil-borne virus, maize
necrotic streak virus, Ahlum waterborne virus, bean mild
mosaic virus, Dianthovirus, oat chlorotic stunt virus, maize
chlorotic mottle virus, and tomato bushy stunt virus
Tombusviridae Single-stranded positive-
strand RNA
Monopartite (except
Dianthovirus; bipartite)
Tomato, cucumber, maize, and
nightshade
Potato virus Y, Blackberry virus Y, longan witches’broom-
associated virus, spartina mottle virus, common reed chlorotic
stripe virus, and tobacco etch virus
Potyviridae Single-stranded positive-
strand RNA
Monopartite Potato, blackberry, longan,
chilli, pepper, and tobacco
Tomato spotted wilt orthotospovirus, fig mosaic emaravirus,
pigeon pea sterility mosaic emaravirus, raspberry leaf blotch
emaravirus, rose rosette emaravirus, and Pistacia emaravirus B
Bunyaviridae Single-stranded negative-
strand RNA viruses
Bi- or tripartite Tomato, fig, pigeon pea, rose,
raspberry, onion, and garlic
Beet yellows virus, citrus tristeza virus, carrot yellow leaf virus,
wheat yellow leaf virus, strawberry chlorotic fleck-associated
virus, and tobacco virus 1
Closteroviridae Single-stranded positive-
strand RNA
Monopartite Beet, tobacco, citrus, and
strawberry
Brome mosaic virus, Semliki Forest virus, alfalfa mosaic virus,
olive latent virus-2, cowpea chlorotic mottle virus, and
cucumber mosaic virus
Bromoviridae Single-stranded positive-
strand RNA
Tripartite Bromegrass, tobacco, potato,
and cucumber
Potato virus X, Lolium latent virus, Cassava virus X, clover
yellow mosaic virus, papaya mosaic virus, potato aucuba
mosaic virus, and bamboo mosaic virus
Alphaflexiviridae Single-stranded positive-
strand RNA
Monopartite Potato, papaya, and bamboo
Turnip yellow mosaic virus, grapevine fleck virus, ononis
yellow mosaic virus, Cacao yellow mosaic virus, eggplant
mosaic virus, and okra mosaic virus
Tymoviridae Single-stranded positive-
strand RNA
Monopartite Cabbages, cauliflower,
broccoli, and eggplant
Tobacco rattle virus, barley stripe mosaic virus, wheat mosaic
virus, sorghum chlorotic spot virus, potato mop-top virus,
tobacco latent virus, and tomato mosaic virus
Virgaviridae Single-stranded positive-
strand RNA
Mono- or bi- or tripartite Tobacco, wheat, sorghum, and
tomato
Cowpea mosaic virus, cucurbit mild mosaic virus, tomato
ringspot virus, tobacco ringspot virus, apple latent spherical
virus, and rice tungro spherical virus
Secoviridae Single-stranded positive-
strand RNA
Bipartite Cowpea, cucurbit, tobacco,
tomato, apple, and rice
Frontiers in Genetics frontiersin.org02
Ranjan et al. 10.3389/fgene.2023.1198647
viz., Polerovirus and Potexvirus. These smaller RNA viruses
infecting plants fall under an ATP-dependent sub-type IA
packaging system, which possess a classical P-loop containing an
ATPase domain situated over linear polypeptide chains of CPs
(Ranjan et al., 2021;Kumar et al., 2022;Mohanty et al., 2023).
The viral encoded CP recognizes the genomic end, nucleates over
the viral genome in the presence of ATP, and ultimately,
encapsidates them to a mature virion (Ranjan et al., 2021;
Mohanty et al., 2023). On the other hand, CPs of viruses belong
to type IB, and IC does not possess ATPase folds over it and employs
slightly different mechanisms for genome packaging (Ranjan et al.,
2021). The type IB virus takes the help of viral- or host-encoded
ATPase, whereas the type IC virus prefers an ATP-independent
fashion to package their genome inside capsid coats (Ranjan et al.,
2021). The role of packaging ATPase in genome packaging is well-
known for viruses of type II and III systems, but our recent discovery
of the presence of novel ATPase domains on polypeptide chains of
CPs has completely changed our perception of genome packaging in
plant RNA viruses and raised many interesting facts (Ranjan et al.,
2021;Kumar et al., 2022;Mohanty et al., 2023). An ATPase domain
comprising different motifs responsible for ATP hydrolysis is
situated on the linear primary structure of the CP of almost all
plant RNA viruses (Figures 1A, B). Interestingly, apart from RNA
viruses, the same ATPase fold consisting of Walker A, Walker B,
sensors, and arginine fingers was also found to be present on the
polypeptide chain of CPs of DNA viruses infecting plants, such as
members of Nanoviridae and Geminiviridae families. Intriguingly,
the presence of very rare patterns of a novel ATPase domain with
multiple Walker A, sensor motifs, Walker B, and arginine motifs
fetched during our thorough comprehensive analysis has indicated a
variation within the ATPase superfamily (Figure 1A)(Ranjan et al.,
2021;Kumar et al., 2022;Mohanty et al., 2023). Such variations
could be responsible for the evolution of different architectures of an
ATPase domain with a single function of ATP hydrolysis during
evolution time (Iyer et al., 2004;Chelikani et al., 2014b). These
duplicated Walker A, sensors, Walker B, and arginine motifs
together form an active site or a pocket for ATP binding and
catalysis (Kumar et al., 2022). ATP hydrolysis is coordinated by
Walker-A “P-loop”motifs during genome packaging. The Walker A
motif with highly conserved sequences of RGRGSSET (lavender
color) interacts with βand terminal γ-phosphates of ATP bound at
the active site of CPs. On the other hand, the highly conserved Asp
residue of Walker B motifs (consensus “hhhhDG”) situated at the tip
of the β-strand forms a coordinate bond with the metal ion and
further assists in the hydrolysis of ATP (Figures 1A, B)(Kumar et al.,
2022). While a conserved Asp residue (blue) coordinates the Mg
2+
cation, the other conserved Gly residue of Walker-B motifs (blue)
involve in a nucleophilic attack toward the terminal γ-phosphate of
the ATP molecule bound at active sites by priming a water molecule
(Figure 1B)(Ranjan et al., 2021;Kumar et al., 2022;Mohanty et al.,
2023).
Intriguingly, all the motifs responsible for binding and catalysis
of ATP are either part of the loop or are situated at the top of the β-
strand in the generated 3D atomic model of CPs. This is a classical
FIGURE 1
(A) Organization of functional motifs on the polypeptide chain of CPs of the Solemoviridae and Alphaflexiviridae family. The functional motifs at the
N-terminal ATPase domain are represented in different colors, whereas the C-terminal end occupies the putative DNA-binding domain. Schematics are
drawn approximately to the scale and represent the approximate consensus of representative homologs of RNA viruses infecting plants. (B) Interaction of
the ATP molecule with the I-TASSER-predicted atomic model of CP. All the putative ATP catalysis motifs are highlighted in different colors. For
details, refer to Kumar et al., 2022.
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Ranjan et al. 10.3389/fgene.2023.1198647
hallmark for the ASCE P-loop ATPase superfamily and makes these
critical motifs very flexible for nucleophilic attack and catalysis
inside active sites (Figure 1B)(Ranjan et al., 2021;Kumar et al.,
2022;Mohanty et al., 2023). The arginine finger I with a consensus
sequence “ARM”and the arginine finger II with a consensus
sequence “FRV”(in red) are situated at 9–10 amino acid residues
next to the sensor I (“FQI”in green) motif and 5–6 amino acid
residues downstream of another Walker-A-like motif (represented
as Walker A’in lavender color). Another sensor motif (designated as
sensor II with the consensus sequence “LQN”in green color) is
present almost after five residues of the arginine finger II. The
arginine fingers I and II (red) and sensor motif II (green) are strictly
conserved across plant RNA viruses (Figure 1A)(Ranjan et al., 2021;
Kumar et al., 2022;Mohanty et al., 2023). The ATPase fold of CPs of
plant RNA viruses abides by a unique arrangement of motifs, where
arginine fingers I and II and Walker A’are found to be flanked with
sensor I and II motifs from both the sides, and sensor motif I is
situated next to Walker B almost after 48 amino acid residues
downstream (Figure 1A)(Ranjan et al., 2021;Kumar et al.,
2022). This permutation and different combinations of motifs
has led to the origin and evolution of different homologs of
ATPase folds across the domain of life (Iyer et al., 2004). Atomic
structure prediction using I-TASSER revealed that the active site CP
comprises all motifs necessary for ATP interaction, except arginine
finger I, which is situated far from the rest of the motifs in their
folded structure (highlighted in red; Figure 1B). Arginine finger is a
classical hallmark of ATPases and is conserved across many
ATPases. It completes the active site from a distinct location,
forming contacts with the γ-phosphate of the nucleotide (Ranjan
et al., 2021;Kumar et al., 2022;Mohanty et al., 2023).
The unique arrangement of ATP-binding motifs in the primary and
tertiary structures of CPs indicates the structural similarity of CPs with
the members of the well-known classical P-loop ATPase superfamily
(Figures 1A, B)(Ranjan et al., 2021;Kumar et al., 2022). The functions
of more than one Walker-A-like motifs in ATP binding and catalysis
need to be explored. The use of site-directed mutagenesis (SDM) to
replace critical amino acid residues in ATPase motifs could provide
better insights into understanding the function of these motifs in
genome translocation, packaging, assembly, and viral maturation
(Ranjan et al., 2021;Kumar et al., 2022;Mohanty et al., 2023). The
P-loop was found to play a crucial role in directing ATP binding and
hydrolysis with genome packaging, translocation, and assembly in all
RNA/DNA viruses (Ranjan et al., 2021;Kumar et al., 2022;Mohanty
et al., 2023). Our preliminary experimental data suggest that the
recombinant CPs of the potato virus X and potato leafroll virus,
overexpressed in the bacterial system, showed enhanced ATP
hydrolysis activity in the presence of DNA (T. Ranjan, unpublished
data). The replacement of critical amino acid residues of Walker-A and
Walker-B motifs obtained using SDM resulted in loss of ATPase
function of recombinant CPs, indicating the role of these motifs in
ATP binding and hydrolysis (T. Ranjan, unpublished data).
Interestingly, we also observed the importance of the Walker A’
motif in ATP hydrolysis, the removal of which led to the loss of
ATPase activity of recombinant CPs (T. Ranjan, unpublished data).
Thus, our comprehensive sequence analysis and preliminary
experimental data indicate the direct role of ATPase folds of CPs in
the genome packaging of RNA viruses infecting plants (Kumari et al.,
2020;Ranjan et al., 2021;Kumar et al., 2022;Mohanty et al., 2023).
3 A model for genome translocation,
packaging, and assembly in plant RNA
viruses
Plant RNA viruses exhibit a wide range of virion symmetry
including rods (e.g., Potyvirus), icosahedral (e.g., Cucumovirus), and
bacilliform shape (e.g., alfalfa mosaic virus) (Ford et al., 2013;Rao et al.,
2014). Despite these diversities among virus families, mature virions of a
particular species often exhibit structural homogeneity and thus share
the common mechanism of genome packaging and assembly
(Chelikani et al., 2014a;Guo et al., 29019;Larson et al., 2005).
Nucleation/oligomerization of capsid proteins around viral RNA
genomes and virion assembly involves two primary molecular
interactions: 1) protein–protein interactions (viz., capsid–capsid
interaction) and ii) RNA–protein interactions (viz., RNA–capsid
interaction) (Qu and Morris, 1997;Basnayake et al., 2009;Bunka
et al., 2011;Ford et al., 2013). CPs recognize specificpackaging
signals (sequences) situated usually at the genomic end instead of
nucleating at some random sites on a viral genome with the help of
the N-terminal DNA-binding domain (Figure 2A)(Garoff et al., 1980;
FIGURE 2
Proposed model for energy-dependent genome packaging and assembly in the plant RNA virus. (A) CPs first recognize the packaging signal or pac
site, situated at the termini of viral genomes with the help of its RNA-binding domain. (B) Replication and genome packaging events in plant viruses are
concurrent. Viral-encoded replicase protein (such as p2a in the case of Bromovirus) and sometimes host-encoded proteins (such as chaperonins in the
case of Potyvirus) interact with CPs in an energy-dependent manner. (C) Binding of ATP molecules to the ATPase domain induces conformational
changes in CPs. (D) Furthermore, ATP hydrolysis facilitates the nucleation of CPs over viral genomic RNAs, and (E) with the help of other auxiliary factors,
CP encapsidates the genomic RNA, ultimately leading to the production of mature virion particles.
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Ranjan et al. 10.3389/fgene.2023.1198647
Bink et al., 2003;Zlotnick et al., 2020;Li et al., 2021). Our bioinformatics
analysis also revealed the presence of helix-turn-helix and basic amino
acid residues at the N-terminal nucleic acid-binding domain of CPs and
indicated their importance during interactions with viral genomes (T.
Ranjan, unpublished data). The thoroughly characterized packaging
signals, viz., assembly sequence, tRNA-like sequence, hairpin-like
structures, and some untranslated regions at the 5’-or3’-end are
recognized by CPs to discriminate viral RNA genomes from cellular
RNAs (Choi and Rao, 2000;Choi et al., 2002;Bink et al., 2003;Choi and
Rao, 2003). Selective packaging of the genomic RNA (gRNA) is
mediated by high-affinity binding between the CP and the
packaging signal (Figure 2A)(Wang et al., 1999;Choi and Rao,
2000;Choi et al., 2002;Choi and Rao, 2003;Kwon et al., 2005;
Wang et al., 2005). Binding of one molecule of CP to the packaging
signal changes the conformation of the CP from weak CP–CP
interactions to strong interactions in a processive manner (Yang
et al., 2017;Comas-Garcia, 2019). In almost all plant RNA viruses,
the event of replication is functionally coupled with genome
translocation, packaging, and virion assembly (Malik et al., 2005;
Annamalai and Rao, 2006). A physical interaction with a virus-
encoded replicase (p2a) expedites the CP to smoothly nucleate over
the RNA genome and further increases the packaging specificity in an
energy-dependent step (Figure 2B)(Annamalai and Rao, 2005;
Annamalai and Rao, 2006;Annamalai et al., 2008;Chaturvedi and
Rao, 2014;Comas-Garcia, 2019). Intriguingly, an interaction between
host-encoded HSP70 and CP is critical during genome packaging and
virion encapsidation in few plant RNA viruses, indicating variations
within the type I genome packaging apparatus (Vriend et al., 1986;
Alzhanova et al., 2001;Verchot, 2012;Gorovits et al., 2013). The
disruption of such physical interactions leads to the packaging of
cellular RNAs along with the viral RNA genome in a mature virion
particle (Weiss et al., 1994;Wu and Shaw, 1998;Annamalai and Rao,
2005). Similarly, the inactivation of the ATPase domain of CPs
employing the RNAi approach further inhibits the entire process of
RNA genome packaging (Rakitina et al., 2005;Ranjan et al., 2021;
Kumar et al., 2022;Mohanty et al., 2023). This confirms that genome
packaging and assembly in a plant RNA virus is an ATP-dependent
process (Figure 2B)(Ranjan et al., 2021;Kumar et al., 2022;Mohanty
et al., 2023).
Intriguingly, the superimposed structure of the docked CP–ATP
complex showed that each monomer has an active site at the interface
(Ranjan et al., 2021;Kumar et al., 2022;Mohanty et al., 2023). Binding
of ATP molecules to the ATPase domain of CPs brings conformational
changes in the tertiary structure of proteins (Figure 2C), and
furthermore,theirhydrolysisfavorsthenucleationofCPsontoviral
RNA genomes (Figure 2D). ATP hydrolysis also promotes
oligomerization (monomer-to-trimer-to-pentamer) of CPs into a
large capsomere-like complex structure and ultimately packages
RNA genomes into mature virions (Figure 2E)(Kumari et al., 2020;
Ranjan et al., 2021;Kumar et al., 2022;Mohanty et al., 2023).
4 Strategies to combat plant RNA
viruses by targeting crucial steps of
genome packaging
After entry into the host cytoplasm with the assistance of a
vector (Figure 3A), viruses first increase their copy number of
genomes via the replication process (Figure 3B). Furthermore,
transcription (Figure 3C) and translation (Figure 3D) produce
CPs, which ultimately help in the encapsidation of multiplied
genomes into virion particles (Figure 3E). Targeting genome
FIGURE 3
Strategy for controlling plant viruses by targeting genome packaging events during their life cycle. (A) Plant virus enters inside the host cytoplasm
with the help of a vector. (B) After uncoating the genome, the virus increases its copy number by replication, and further transcription (C) and translation
(D) events enable the production of CP ATPase. (E) Ultimately, CP encapsidates the RNA genome with the in put of ATP molecules and forms mature virion
particles. The introduction of exogenous siRNA constructs specific to ATPase domains suppresses the expression of CP mRNAs (a–d) and ultimately
inhibits the production of functional virions (e).
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packaging and virus assembly processes could be one of the efficient
approaches toward the development of virus resistance in plants
(Ranjan et al., 2021;Kumar et al., 2022;Mohanty et al., 2023)
(Figures 3a–e). One of the most effective approaches to build plant
resistance has been to introduce a part of the viral gene into the plant
either to induce RNA silencing against viral RNAs or to express
intact or modified viral proteins or RNAs that disturb the viral
infection cycle (Goldbach et al., 2003). The RNA interference
(RNAi) technology has emerged as a potential tool to target virus
assembly for developing resistant crops (Lodge et al., 1993;
Goldbach et al., 2003)(Figure 3). RNA silencing, a conserved
regulatory mechanism of gene expression in eukaryotes, is
triggered by dsRNA-provoking gene silencing through sequence-
specific degradation of complementary mRNA transcripts (post-
transcriptional gene silencing) (Waterhouse et al., 2001). The
degradation of these target RNAs occurs in a sequence-specific
manner via the formation of double-stranded RNAs, which
further processed into small interfering RNAs (siRNAs) by the
Dicer-like (DCL) proteins and the RNA-induced silencing
complex (RISC) (Lodge et al., 1993;Waterhouse et al., 2001;
Goldbach et al., 2003)(Figures 3a–c). Since it is well-
acknowledged that the CP is essential for plant RNA virus
genome packaging (Duggal and Hall, 1993;Hacker, 1995;
Kumari et al., 2020;Ranjan et al., 2021;Kumar et al., 2022;
Mohanty et al., 2023), in our previous studies, an attempt was
made to explore the conserved functional motifs situated across a
polypeptide chain of CPs for developing strategies toward virus
resistance in plants (Kumari et al., 2020;Ranjan et al., 2021;Kumar
et al., 2022;Mohanty et al., 2023)(Figures 3d–e).
In our previous study, the efficiency of siRNA constructs of the
CP_ATPase domain to silence and inhibit virus assembly was
assessed in detail (Kumar et al., 2022). To further validate this
efficient methodology, siRNAs designed against ATPase folds
(comprising all motifs) of the CP were agroinfiltrated into virus-
infected plants. Figure 4A depicts the details of the generation of
siRNA constructs against the ATPase domain of CPs (Figure 4A).
Agroinfiltrated plants did not show any symptoms of a virus
infection. The suppression of viral infection could be attributed
to the reduced expression of CPs due to its silencing by siRNA
constructs (Figures 3,4)(Kumar et al., 2022). To understand
whether the knockdown of CPs, which possess a classical ATPase
domain, can disrupt RNA virus genome packaging, we
agroinfiltrated the pART27–CP_ATPase domain–siRNA
constructs into potato plants (Kumar et al., 2022). Control plants
(naturally virus-infected plants) (Figure 4B; lane I) and those
agroinfiltrated with the empty vector (Figure 4B; lane II) showed
rolling symptoms of potato leafroll virus (PLRV) infection in the
upper leaves (Kumar et al., 2022). Intriguingly, the agroinfiltration of
plantlets individually with sense (pART27–sense CP_ATPase) and
antisense (pART27–antisense CP_ATPase) constructs of the CP_
ATPase domain also displayed symptoms of leaf rolling in the upper
leaves (Figure 4B; lane III and IV, respectively). This is obvious
FIGURE 4
Genome packaging is an ATP-dependent process in plant viruses. (A) Schematic representation of the generation of siRNA construct specifictothe
ATPase domain of CP. (B) Symptoms observed in the tertiary leaves of the infected potato plants after 2 weeks of PLRV inoculation. (I) PLRV-infected
control without agroinfiltration; (II) agroinfiltrated with the empty vector pART27; (III) agroinfiltrated with the plasmid containing only the antisense
sequence (pART27–antisense CP_ATPase domain); (IV) agroinfiltrated with the plasmid containing only the sense sequence (pART27–sense CP_
ATPase domain); and (V) agroinfiltrated with the pART27–CP_ATPase siRNA construct. The detection of the PLRV RNA using RT-PCR (inset; agarose gel).
For details, refer to Kumar et al., 2022.
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because these constructs were not able to form dsRNAs after
agroinfiltration and Dicer cannot recognize them anymore. Plants
agroinfiltrated with the siRNA construct of CP_ATPase
(pART27–CP_ATPase) showed no symptoms of a viral infection
(Figure 4B; lane V). These results demonstrated that an siRNA
construct, specific to the ATPase domain (comprising all critical
motifs) of CPs, leads to the suppression of viral genome packaging
and assembly in plants (Kumar et al., 2022).
Our study showed that the transient expression of CP constructs
specifically and efficiently inhibits genome packaging of plant RNA
viruses (Kumar et al., 2022). Interestingly, CP mRNAs were found to
be very high in the tertiary leaflet of control plants (natural infection
of virus) and agroinfiltrated plants with an empty vector without any
construct, sense construct, and antisense construct as well (Figure 4B
I–IV; lower inset), whereas mRNAs of CPs were not even found in
the new leaflets of agroinfiltrated plants with CP siRNAs targeted
against ATPase folds (Figure 4B V; lower inset) (Kumar et al., 2022).
These findings support the previous conjecture and strongly suggest
a direct role of CP_ATPase in genome recognition and its packaging,
the suppression of which may result in immature, non-functional,
and genome-deficient viral particle production (Callaway et al.,
2001;Rakitina et al., 2005;Hesketh et al., 2015).
5 Variations among type I genome
packaging systems
Albeit the differences in the basic architecture and hosts of plant
viruses, the process of assembly is coordinated by specificinteractionsof
the nucleic acid-binding domain of capsid proteins with the viral
genomic RNA followed by their oligomerization and nucleation in
the genome. Linear polypeptide chains of CPs of type IA viruses encrypt
a classical novel ATPase domain (Ranjan et al., 2021;Mohanty et al.,
2023). On the other hand, CPs encoded by viruses in the type IB
packaging and type IC packaging apparatus lack the ATPase domain
and represent a variation within type I genome packaging (Ranjan et al.,
2021).Ourrecentbioinformaticsanalysisrevealedtherelatednessofthe
type IB to type IC genome packaging system and the very early
evolution of the type IA system. Interestingly, viruses from the type
IA packaging system clustered into a separate clade and seemed to be
diverged early from the rest of the two genome packaging systems
during the evolution time (Kumari et al., 2020;Ranjan et al., 2021;
Kumar et al., 2022;Mohanty et al., 2023). This represents a typical
example of divergent evolution of the primordial viral genome
packaging apparatus, and perhaps it could be the reminiscent
genome packaging system encoded by the last universal common
ancestor (LUCA). It seems that CPs of plant RNA viruses appear to
derive from a common ancestor, regardless of their host (Ranjan et al.,
2021;Mohanty et al., 2023). It could also represent a new variation and
the adaptive evolution of the ancient viral genome packaging apparatus
that likely existed in the LUCA (Burroughs et al., 2007;Chelikani et al.,
2014b; Iyer et al., 2004; Mohanty et al., 2023;Ranjan et al., 2021).
6 Conclusion
RNA viruses cause serious diseases in crops, leading to
significant reductions in annual yields worldwide. Innovative
and state-of-the-art strategies are very much required to
control/combat with plant viruses in order to meet the
demands of the population growth worldwide (Hill et al., 2021;
Ye et al., 2021). Among several advances in viral control measures,
aiming for the steps of the viral assembly process has the potential
to save crops from such destructive diseases (Burroughs et al.,
2007;Dai et al., 2021;Kumar et al., 2022). Understanding the
mechanism of genome packaging and assembly would be helpful in
developing novel approaches to control plant viruses and
ultimately to meet the demands of a growing world population
(Kumar et al., 2020;Ranjan et al., 2021;Kumar et al., 2022;
Mohanty et al., 2023). Deciphering the detailed machinery of
ATP binding and catalysis by the ATPase fold would be helpful
in designing novel inhibitors, which may hinder the entire process
of genome packaging and virus assembly (Twarock and Stockley,
2019). Information collected from the limited available literature
worldwide on Polerovirus and Potexvirus as model systems has
provided novel insights into understanding the mechanism of
ATP-dependent genome packaging and virus assembly. The
extension of similar studies to other plant RNA viruses would
be helpful in determining whether ATP-dependent genome
packaging is universally conserved among different RNA viruses
infecting plants. Still, important questions regarding the
mechanisms regulating the process of genome packaging and
assembly of uniformly sized virions remain unanswered and a
challenge for future investigation.
Author contributions
TR and RR conceived the study concept. TR, MA, SD, and
MFA further conceptualized the idea and contributed to
visualization. JK, AM, AK, KJ, and KR wrote and edited the
original draft. All authors contributed to the article and approved
the submitted version.
Funding
This work was supported by grants from the Science and
Engineering Research Board (SERB), Department of Science and
Technology, Government of India, New Delhi (India) (File Nos
SRG/2019/002223 and CRG/2022/000478) to TR.
Acknowledgments
The authors would like to thank Bihar Agricultural University
(BAU), Sabour, for providing the basic infrastructure for conducting
the research works. This article bears BAU Communication No.
1328/221118.
Conflict of interest
The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.
Frontiers in Genetics frontiersin.org07
Ranjan et al. 10.3389/fgene.2023.1198647
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and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors, and the
reviewers. Any product that may be evaluated in this article, or
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endorsed by the publisher.
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