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DNA replication and Pathogenecity of MYMIV
Dharmendra K. Singh, Sumona Karjee, Punjab S. Malik, Nurul Islam and Sunil K.
Mukherjee∗
Plant Mol. Biol. Lab., ICGEB, AAA Marg, New Delhi 110067, India
Geminiviridae is a large family of single stranded DNA plant viruses, transmitted by white fly Bamesia
tabacci, and they are the major causes of huge agro-economical losses worldwide. Mungbean yellow
mosaic India virus (MYMIV) belongs to the genus begomovirus, the predominant variety of
geminiviruses in northern part of Indian subcontinent. It has bipartite genome, which replicates via
rolling circle (RCR) model with the help of few viral and several host factors. We have focused our
efforts to understand the mechanism of initiation and immediate post-initiation phases of viral RCR. The
Rep-protein encoded by the virus binds to the iterons present in the origin of replication, melts the origin,
and subsequently nicks a conserved specific sequence of DNA, the region from where the RCR begins.
The Rep protein also carries out the displacement of the parental strand required for the passage of
replication fork. We have identified few host factors that act as accessory proteins of Rep during these
phases of replication as mentioned.
The high phyto-pathogenicity of the virus may be attributed to its potential to suppress host antiviral
response, i.e., RNA silencing. We have developed various assays to identify viral RNAi suppressors. The
AC2 suppressor protein of the MYMIV has been characterized for its various activities and its
suppression domain has been mapped.
This review will primarily focus upon the understanding the mechanistic aspect of initiation of MYMIV
replication, the biochemical activity of the replication in relation to host as well as viral factors; and the
RNA silencing suppressor activity of the virus protein AC2 and its potential applications.
Key words: Geminivirus; replication; MYMIV; AC2; RNA silencing.
Introduction
Geminiviruses constitute a large family of phytopathogens (Geminiviridae) having single stranded
circular genome and vector- (white fly, Bemisia tabaci) mediated transmissibility. These viruses
devastate some of the economically important plants ranging from dicots to monocots and impose huge
agro-economic losses worldwide.
The family geminiviridae have been classified into four genera,
namely Begomovirus, Curtovirus, Topocuvirus and Mastrevirus, depending on their genomes, mode of
transmission and host range [1].
The genus begomoviridae generally comprises of bipartite genome (two components, namely DNA‘A’ and ‘B’), which are transmitted by white fly. Mungbean yellow mosaic India virus (MYMIV) is a
representative of the genus begomovirus, which is prevalent in northern part of Indian subcontinent
causing yellow mosaic disease (YMD) [2]. The most affected leguminous crops are Cajanus cajan,
Glycine max, Phaseolus aconitifolius, P. aureus, P. vulgaris ‘French bean’, Vigna mungo. The
component ‘A’ of MYMIV encodes for proteins important for viral replication and en-capsidation
whereas DNA ‘B’ component mainly codes for proteins important for intracellular movement (BL1,
BV1) and transport of viral ssDNA. The two components share a region of high sequence homology that
is known as ‘CR’, the place from where the replication of the viral DNA genomes initiates. In order to
∗
Corresponding author: e-mail: [email protected], Phone: +91-11-26181242
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understand the mechanism of DNA replication of the begomoviruses, we chose the MYMIV-DNA as a
model system because of the economic impact of this virus.
1.1 DNA multiplication of the MYMIV:
MYMIV replicates predominantly via dsDNA intermediates following rolling circle mode (RCR) of
DNA replication inside the nucleus of the infected host cell. The replication process of the virus can be
divided in two stages. First, upon entry inside the nucleus, the viral ssDNA is converted into dsDNA
(replicative form, RF) with the help of only the host cellular factors. This dsDNA is transcriptionally
active and serves as a template for the production of various viral factors. In the second phase, the viral
factors along with the cellular factors synthesize ssDNA using the dsDNA as a template via. RCR. These
ssDNA can again either (i) re-enter the DNA replication pool, (ii) associate with CP for production of
virions or (iii) be transported outside the nucleus by nuclear shuttle proteins (NSPs) or outside the cell
with the help of virus encoded movement proteins (MPs) and associated host factors.
Fig. 1A Modular organization of MYMIV-CR. Common Region of DNA A shows various replication and
transcription regulatory elements. Stem-loop structure, essential for replication, contains conserved nonamer
sequence
5′-TAATATTAC-3′. Arrow indicates the site of initiation, where Rep mediated cleavage occurs.
The Rep binding iteron sequences CGGTGT are present on both the sides of stem-loop structure. TATA box for
AC1 transcription is present between Rep-binding sites and stem-loop structure. Other regulatory elements are
AG-motif and G-box.
The MYMIV-CR comprises a highly conserved stem loop structure, which also contains the conserved
sequence TAATATT↓AC, where the viral Rep protein acts to initiate the RCR (Fig. 1A). One of the
earliest events during the initiation of DNA replication in geminiviruses is origin recognition. The
MYMIV origin contains four Rep- binding sites (iterons), where Rep binds in a highly sequence specific
manner. The filter binding, EMSA and DNaseI foot-printing assays have confirmed the specific binding
characteristics of MYMIV Rep to its cognate CR DNA [3, Malik et al., unpublished data]. The presence
of bipolar Rep-binding sites (CGGTGT) i.e., on both sides of the stem loop structure is a unique feature
of MYMIV origin of replication. The DNaseI foot-printing data reveal that four iteron sites are occupied
first when the amount of protein is low and eventually the regions lying between four iterons are
occupied in a cooperative manner with increasing concentrations of Rep. The specific DNA binding
activity of Rep is located at the region spanning the N-terminal 133 amino acids of MYMIV-Rep [Ph. D.
thesis, Basavaraj]. Other necessary elements present in the CR include a third cis-acting element,
downstream from the TATA box (the ‘AG’ motif). Two other motifs, the G-box and the TATA-box,
seem to play a role in origin utilization. Finally, a CA motif, located outside the minimal origin,
presumably enhances viral DNA replication [4].
Following Rep protein binding, a structural distortion of the CR region occurs which are essential for
the subsequent Rep mediated nicking, leading to initiation of RCR. The distortions are revealed by the
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exposure of the DNase hypersensitive sites, shown in Figure 1B. The induction of structural change in
the CR (ori)- containing plasmid DNA is probed by potassium permanganate (KMnO4) oxidation [5].
Chemical probing using KMnO4 suggested altered DNA structure especially melting of the loop region at
MYMIV origin of replication, following Rep protein binding. These changes are suggestive of formation
of a cruciform structure occurring at the replication origin as a result of binding of the MYMIV- Rep.
The combined information generated from the DNaseI as well as KMnO4 foot printing analyses has been
put together in Fig. 1B. Following the events of DNA binding and melting, Rep also nicks the nonamer
site between the 7th and 8th nucleotide (3). Thus, a good deal of mechanistic details relating to initiation
of RCR of MYMIV-DNA is already available. We hope that similar processes will also be at work for
replication of the genomes of other begomoviruses. It is also likely that some host factors participate in
this initiation process to control the rate characteristic of the in vivo growth of viral DNA.
Fig. 1B Summary of footprinting data of MYMIV-Rep protein binding to the ori contaning CR. (*) DNase
hypersensitive sites of virion sense strand; (◙) DNase hypersensitive site in iterons of virion sense strand. (●)
DNase hypersensitive site in complementary sense strand; (♦) KMnO4 hyperactive sites of virion sense
strand; Broken line/box indicates weak protection; The blue zones are the stretches of DNA on the virion
(upper) and non-virion (lower) strands, respectively, that are protected by Rep.
Following initiation, the fork proteins perhaps assemble at the 3’OH-end of the nick to extend the
primer and maintain the viral DNA replication at the elongation phase. This phase of work cannot be
initiated without an active helicase that unwinds the parental DNA for nascent DNA synthesis. The
MYMIV-Rep protein harbors few characteristic motifs of a helicase enzyme and very recently, our lab
has shown that Rep protein has its own intrinsic ATPase and helicase activities [6]. Rep translocates in
3′→5′ direction, and requires ≥6 nt space for its efficient activity. This Rep forms a large oligomeric
complex and the helicase activity is dependent on the oligomeric conformation (~24 mer) of the protein.
Mutation in the oligomerization domain leads to the disruption of the helicase activity of the MYMIVRep. The in vivo experiments in yeast as well as in planta using geminiviral-based vectors have
demonstrated that Rep-mediated helicase activity is essential for the replication of the plasmids bearing
MYMIV origin of replication. Though the anatomy and rate-features of the replication fork for
geminivirus DNA replication are not fully determined at the present moment, it is almost certain that
many host-encoded factors play their roles at the elongation phase of viral DNA replication.
1.2 Involvement of host and viral factors in MYMIV-DNA replication:
A majority of the geminiviruses infects terminally differentiated cells that have exited the cell division
cycle and contain very negligible amount of DNA replication enzymes [7]. Therefore, these viruses
create the cellular environment for their own benefits by complex interaction between the viral and the
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host encoded factors. The Rep protein of some of the geminiviruses interact with a number of host
factors such as pRBR to alter cell cycle to create S-phase like scenario [8, 9], RF-C for loading of PCNA
[10], Histone H3 for putative removal of nucleosomal block and efficient transcription and replication
[11], GRIMP (kinesin) and GRIK (a Ser/Thr kinase) for mitotic activity etc. Similarly, other proteins of
geminiviruses, say AC3, are also known to interact with host-factors for completion and regulation of
geminiviral DNA replication.
MYMIV-Rep interacts with proliferative cell nuclear antigen (PCNA) [12]. This Rep-PCNA
interaction leads to down-regulation of nicking/closing as well as ATPase activities of Rep. MYMIVRep protein also interacts with the RPA-32kDa protein, the middle subunit of host Replication protein A
(RPA) (eukaryotic, heterotrimeric, single stranded DNA binding protein) [13]. RPA32 modulates down
regulation of the nicking-closing activity and enhances the intrinsic ATPase and the helicase activities of
the Rep protein. In fact, MYMIV-Rep interacts with the peptides corresponding to many host-protein in
a typical phage display analysis. Some of these proteins, namely the conserved RAD 54 protein, interact
with MYMIV-Rep in yeast two hybrid as well as in vitro pull-down assays. The rad54 mutant of yeast
weakly supports MYMIV- DNA replication compared to the wild type S. cerevisiae. Further,
biochemical analyses have shown that Rad54 enhances the intrinsic ATPase and the helicase activities of
Rep, which might be important in the post initiation steps of the viral replication [Kosalai et al.,
unpublished data]. The phage display analyses reveal that MYMIV-Rep is capable of interacting with
more than two hundred host proteins, which might regulate the viral DNA replication directly or
indirectly. These interacting proteins can be classified in fifteen different categories according to their
functions. For example, 29 of them are plant replication factors, such as TopoII, DNA Polymerase ε, E2F
etc.; and 4 of them are gene-silencing factors such as RdRP, Mut-6 etc. [Kosalai et al., unpublished data].
Our studies also indicate a very strong interaction between MYMIV-Rep and MYMIV-CP both in
vitro as well as in vivo [14]. These interactions down regulate one of the important properties of
replication initiation by the Rep i.e., the nicking activities. This finding indicates that CP could actively
block the production of ssDNA in the late stage of viral life cycle. In this way, MYMIV- CP might have
a role in limiting the DNA viral copy number by blocking initiation of RCR.
1.3 Geminiviral DNA replication in Yeast:
Our understanding of several features of geminiviral replication has increased over the years using a
combination of both in vivo system that supports geminiviral replication and a variety of in vitro
experiments. However, each system is associated with its own inherent limitations. The budding yeast
(Saccharomyces cerevisiae) offers a powerful model system, since it is genetically tractable, easily
culturable and handled. The availability of a large repertoire of deletion and conditional yeast mutants
offers scopes for extensive manipulations and easy identification of the intracellular replicative
intermediates.
We have been able to develop yeast Saccharomyces cerevisiae as a model system for the study of
geminiviral DNA replication and genome-wide screening of host factors required for viral replication
[15]. The efficient replication of the plasmid DNA harboring two tandem copies of DNA ‘A’ component
of the MYMIV genome relies specifically on the virus-derived elements and factors. Using this model,
the roles of AC5, a viral factor [15] and several host factors like RPA70 and RPA32 [13], Rad54
[Kosalai et. al., unpublished], various recombination related factors such as Rad51, Rad52, MRE11 and
DMC1 have been shown to be involved in MYMIV DNA replication [Raghavan et al., unpublished] in
yeast. A yeast genome-wide screening using viral factors as baits may select most of the yeast factors
that are necessary for MYMIV DNA replication. The equivalent plant factors can subsequently be
identified on the basis of structural and functional homologies. Thus, the developed yeast model has
varied applications in understanding and widening our current knowledge of various aspects of
geminiviral DNA replication.
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Fig. 2 Model steps for geminiviral DNA replication
With the present knowledge of understanding of geminiviral DNA replication, we are proposing a
model of the replication processes as shown in Figure 2. During initiation, the Rep protein binds a large
portion of the CR region of DNA ‘A’ in a cooperative manner, and eventually cleaves (nicks) the DNA
at the specified sequence to initiate RCR. The helicase activity of Rep protein then might help in melting
the origin further, allowing other host factors such as RF-C, PCNA, RPA, DNA polymerases etc. to
associate at the 3’OH end of the nick to form a progressive replication fork. During progression, many
other host factors (unknown at present) might participate to regulate the replication rate. At the
termination step of RCR, the Rep protein again cuts and religates the newly synthesized ssDNA to
generate circular ssDNA genome. The ssDNA rejoins the replication process, and thus eventually huge
copies of the viral ssDNAs are synthesized intracellularly.
2. RNAi suppressor: Pathogenecity and role in replication
The virus replicates its genome inside the host cell to establish and spread disease. The replication of the
virus however, in turn elicits various host antiviral defense responses, like Virus Induced Gene Silencing
(VIGS). VIGS is triggered by the viral genome and targets the viral genomes or transcripts by generating
virus-specific siRNAs and geminiviruses are no exception in this regard [16]. The geminiviral siRNAs
generated in response to the infection are of 21, 22 and 24 nt in length. The heterogenous population of
viral siRNA production is due to participation of all the four DCLs [17, 18]. Moreover, many segments
of the geminivirus DNAs also are methylated in a siRNA dependent manner in response to infection
[19]. On the other hand, the viruses encode RNA silencing suppressor (RSS) proteins as a counter
defense strategy. Most of the plant viruses encode RNA silencing suppressors, but each RSS has
different mechanistic pathways for the suppression of RNA silencing [20]. The begomoviruses have also
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been reported to encode at least three RNA silencing suppressors, viz. AC2 (monopartite homolog, C2),
AC4 and NSP [21, 22, 23]. We have screened and identified MYMIV-AC2 as a RNA silencing
suppressor protein using the detection technique developed in our laboratory.
The MYMIV-AC2 is a 136 amino acid long protein and can be divided into three conserved domains,
namely, a basic N-terminal nuclear localization domain (NLS), a core DNA binding domain with nonclassical Zn finger motif (C37X1C39X7C47X6H54X4H59C60) and a C-terminus acidic transactivator domain.
The deletion analysis reveals that a truncation of even 16 amino acid residues from the C-terminal end
causes total loss of trans-activation potential in yeast monohybrid assay. However, the RNA silencing
suppressor activity of this truncation version is comparable to that of the wild-type AC2. A further
truncation of 30 additional amino acids from the C-terminal end abolishes the RSS activity.
To decipher the biological role of MYMIV-AC2, transgenics are developed in Nicotiana xanthi var
tabacci and Rice (Oryza sativa PB1) in our laboratory. A vast majority of the transgenics shows the
physiological and anatomical anomalies that mimic the viral disease symptoms (Fig. 3). The regeneration
from the callus is less than 10% because of the deleterious effect of the gene on the plants growth and
development. More than 30% of the AC2-transgenic population shows yellow mosaic symptom on
leaves, a characteristic feature of the viral disease. About 80% of the transgenics are stunted in growth
with abnormal leaf lamina viz., extensive leaf puckering. In few of the transgenics, leaves with gigantic
sizes are also observed. All of them showed late flower setting and the amount of flowering are also
significantly less compared to the wild type. More than 70% of the rice panicles are sterile, which may
also reflect on the huge yield loss as documented in the MYMIV disease.
Fig. 3 Phenotypes of MYMIV-AC2 transgenics. (A) AC2 transgenic of rice (Oryza sativa PB1), showed
stunted growth and deformed lamina as compared to the wild type variety. (B) AC2 transgenic of tobacco
(Nicotiana xanthi var tabacci) also showed stunted growth and leaf anomalies including curling, yellow mosaic
and gigantism. Flowers formed were scanty and deformed.
To understand the mechanism of RSS activity of MYMIV-AC2, it is important to find the interacting
host factors. The phage display technique has predicted various host interacting partners of MYMIVAC2, including the DCL1 and AGO1 proteins. The transactivation mutated AC2, i.e., AC2∆121-136 has
been used to study the host interacting partners more exhaustively through yeast-two hybrid technique
using Arabidopsis and host Mung-bean cDNA library. Some of the representative host proteins include
Cytochrome B6-F complex iron sulphur, Co-chaperone grpE protein, bZip family transcription factor,
Acotinase C-terminal domain containing protein etc. [Karjee et al., unpublished data].
It is important to screen the various AC2 homologs for their comparative studies and determing the
correlation between the degrees of pathogenicity and suppressor activity. In our laboratory, we
developed RNA silencing suppressor-screening assays based on two different principles. The first assay
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uses the GFP silenced Nicotiana xanthi transgenic lines in which the viral ORFs are allowed to express
ectopically. The reversion of GFP expression occurs from silenced state due to ectopic expression of
AC2s but with the variance in degree of reversion (Fig. 4A). The MYMIV-AC2 and other geminiviral
AC2 homologs from Tomato leaf curl virus (ToLCV), Duranta leaf curl virus (DLCV), Cotton leaf curl
virus (CLCV), Chili leaf curl virus (ChLCV) etc. have been detected in this assay. The RSS activities are
further validated with the northern blots of GFP-mRNAs as well as GFP siRNAs. The second assay is
replicon based spot assay that is primarily based upon the principle that RNA silencing restricts the viral
genome replication by targeting the viral RNA transcript or/and RNA genome and the RSSs, which
suppress RNA silencing, should enhance replication when they are provided in trans.
Fig. 4 RNA silencing suppressor assays. (A) The reversal of silencing assay on GFP silenced N. xanthi line agroinfiltrated with empty vector (pBI) and AC2s from various begomoviruses. CLCU, DLCD, CLCHS represents
Cotton leaf curl virus,U variety; Duranta leaf curl virus, Delhi variety and Cotton leaf curl virus, Hissar variety,
respectively. (B) Replicon based spot assay of wild type N. xanthi leaf infiltrated with VAAC2M, VA and VAAC2M
along with wild type AC2 provided in trans. (C) Replication based assay for the enhancement of viral amplicon
demonstrated using PCR with GFP fwd and Rep1-133 rev primers, which gives an amplification of 1.6 kb.
Corresponding amplification of actin has been shown as loading control.
The enhancement in replication was better tracked by tagging a reporter gene GFP with the viral
amplicon (Fig. 4B) [13]. It provides a means to determine whether the RSS in question is able to
interfere at the initiation or establishment of RNA silencing. The geminiviral AC2s have been screened
using both the assays together or independently for their RSS activities and the degree of suppression is
estimated on the basis of degree of GFP reversion or enhancement in amplicon efficiency (Fig. 4C). In a
majority of the cases, the AC2 from the virulent strain is more potent in RSS activity compared to the
milder strains. For example, CLCV-U1 is more severe than the CLCV-U2 strain and the RSS activity of
the first one is stronger than the latter.
The RSSs are biomolecules of varied biotecnological interests. They can serve as a probe for the
understanding of RNAi pathway or in biofarming [23]. However, beside these applications, one can use
RSS to explain various molecular biology puzzles, like whether a particular phenomenon is due to RNAi
or not. Interestingly, we found similar application of MYMIV-AC2 in aiding the ribozyme technology
that is often masked by RNAi effect. Both the catalytically active and inactive ribozyme against the Rep
of MYMIV [24] show similar level of rep mRNA reduction in planta. This reduction is mainly due to the
formation of Rep-siRNAs in presence of the ribozymes. However, in presence of MYMIV-AC2 only the
ribozyme-mediated reduction in rep mRNA would take place and hence the AC2 protein can distinguish
between both the variants of ribozymes [Karjee et al, unpublished]. We have also applied the knowledge
of AC2 RSS activity in the construction of MYMIV based VIGS vector. The MYMIV-AC2 mutated
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VIGS vector construction is much better than the VIGS incorporating the wt AC2 to ensure complete and
systemic silencing of the target gene [Islam et al, unpublished].
Our data reveal that AC2 controls the host RNAi degradative activity and regulates the copy number
of MYMIV-genome in turn. Thus, MYMIV and other begomoviral genome replication processes are
quite complex and controlled by a multitude of host and viral factors. We hope to use the yeast model to
discover the host proteins that play roles in the begomovirus genome multiplication. The in vitro
reconstitution of MYMIV DNA replication with the help of yeast proteins will help us solve the
biochemical details of viral DNA replication.
Acknowledgements. We would like to thank Drs. N. R. Choudhury, Vineetha Raghavan, N. S-Mishra,
Ushasri Chilakamarthi, Ms. K. Kosalai, Mr. Vikash Kumar and Ms. Subhra Mukhopadhyay for their
valuable help and suggestions. The financial assistance to DKS and SK from DBT and CSIR, Govt. of
India, New Delhi, respectively, is greatly acknowledged.
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