Viral Replication
Viral Replication
Chapter 1
Copyright: © 2017 Nrisingha Dey, et al.
Molecular Biology of Geminivirus DNA
Replication
Bhubaneswar Pradhan1,4, Vu Van Tien2, Nrisingha Dey1*
and Sunil Kumar Mukherjee3*
Plant Molecular Biology Laboratory, Institute of Life Sciences, India
2
National Key Laboratory for Plant Cell Biotechnology, Agricultural Genetics Institute, Hanoi, Vietnam
3
NER-BPMC, Department of Biotechnology, Govt. of India,
India
4
ICAR-National Rice Research Institute, India
1
Corresponding Authors: Nrisingha Dey, Plant Molecular
Biology Laboratory, Institute of Life Sciences, Nalco Square,
Bhubaneswar-751023, India, Tel: +91-674- 2300137/ 2301476
ext. 2010091; Fax: +91- 674 -2300728; Email: nrisinghad@
gmail.com
*
Sunil Kumar Mukherjee, NER-BPMC, Department of Biotechnology, Govt. of India, India, Email: sunilmukherjeeudsc@
gmail.com; [email protected]
First Published April 26, 2017
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This article is distributed under the terms of the Creative
Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/), which
permits unrestricted use, distribution, and reproduction in
any medium, provided you give appropriate credit to the
original author(s) and the source.
Introduction
Geminiviruses are circular single stranded DNA (ssDNA) viruses which infect both monocot and dicot plants
including most of the economically important crops such
as maize, cassava, cotton, pepper, beet, bean and tomato
[1–3]. The diseases caused by geminiviruses greatly affect
the yield of infected crop plants [4,5] and sometimes the
crop failure reaches up to 100% [6–8]. Taking into consideration of the criteria like genome organization, vector
transmission and host range, Geminiviridae family is classified into eight genera, such as: Begomovirus; Mastrevirus; Topocuvirus; Curtovirus; Becurtovirus; Eragrovirus;
Turncurtovirus and Capulavirus [9–11]. Several additional genera have been proposed: Baminivirus, Nimivirus and
Niminivirus [12]. Begomovirus is the largest genus consisting more than 180 species [4]. The small genome size
(2.7-3.0 kb) and the exclusive ability of using host cellular
machinery, makes the geminivirus as an ideal model system for the studies, such as plant DNA replication, gene
expression and plant- virus interactions.
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The coding capacity of these viruses is very limited
and thus, they rely heavily on the host factors for their
genome replication and spread within the host as well as
transmission between them. The understanding of the intracellular viral processes can lead to the identification of
important host as well as viral factors necessary for viral
DNA replication. The most important protein of viral origin regarding viral genome replication is Replication initiator protein (Rep). Rep is encoded by the genome component ‘A’ for the bipartite viruses and is thus also called as
AC1 and only C1 for the monopartite viruses (Figure 1).
Other than Rep, geminivirus relies on few viral factors like
AC3 (or C3) and a plethora of various other host factors,
for its replication. With the help of a yeast model system
developed in our laboratory and through techniques like
yeast two-hybrid, phage display, etc., we have identified
and established the roles of various host factors that include Proliferating Cell Nuclear Antigen (PCNA), Replication Protein-A (RPA), RAD54, RAD51, MCM2, etc.
In this chapter, we discuss the mode of DNA replication of Begomoviruses and the roles of associated host factors in some detail. Here we highlight mostly the information that emerged from our own laboratories.
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Figure 1: Genome organization of bipartite and monopartite viruses
of ToLCV. a. Bipartite genomic organization of Tomato Leaf Curl
New Delhi Virus consisting DNA- A (DQ629101.2) and DNA-B
(AM778833.1) components and their encoded transcripts. b. DNA-A
(AF165098) and associated β-satellite (AY438562.1) of monopartite
Tomato Leaf Curl New Delhi Virus and their encoded transcripts.
CR is the common region containing TAATATTAC sequence, which
serves as the origin of DNA replication. This sequence is located
within the loop region of the stem-loop structure present around the
origin of DNA replication.origin of DNA replication.
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Geminivirus Genome
The genomes of maize streak virus (MSV) were first
reported to contain RNA on the basis of sensitivity to ribonuclease [13,14]. Those, of bean golden yellow mosaic
viruses (BGMV) were then shown to be ssDNA [1,15]. At
the same time, Harrison and co-workers [16] also proved
that the genomes of African cassava latent virus and MSV
contain circular ssDNA of molecular weight around 106D.
In another attempt, during 1978, Bock and co-workers
[17] re-examined the genomes of MSV and showed that
it contains ssDNA. The genomes of BGMV and tomato
golden mosaic virus (TGMV) were shown to contain two
similar sized ssDNAs [18,19].
The genome organization of geminiviruses consists of
one (monopartite) or two (bipartite) DNA molecules (Figure 1) that is (are) individually encapsidated in a twinned
(quasi)-icosahedral virion [20,21]. A majority of these
viruses have two components (bipartite), referred to as
DNA-A and DNA-B, (e.g. Mung bean yellow mosaic India
virus; MYMIV) although in few cases they have monopartite genome, containing DNA-A only [e.g. Tomato yellow
leaf curl virus (TYLCV) or some species of Tomato leaf
curl virus; ToLCV]. Both the DNA components are essential for systemic infection, although DNA-A alone can
induce disease symptom. Novel sub-viral agents associated with monopartite geminiviruses called DNA satellite (e.g. DNA-β) have been identified, which are essential
for induction of disease symptom in monopartite viruses
[22,23].
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DNA-A and/or DNA-B (in case of bipartite viruses)
of geminiviruses (Figure 1) encode only few genes which
are located on both sense and antisense strands of their
genome(s). The DNA-A component contains functional
genes for replication initiation protein (AL1/AC1/REP),
transcriptional activator (AL2/AC2/TrAP), replication
enhancer (AL3/AC3/REn), and AL4/AC4 (its role could
be to mediate vascular cell proliferation to provide an
optimal environment for viral proliferation and dissemination) on antisense direction. AC5/C5 is another gene
product of many geminiviruses as evident from the annotated nucleotide databases, although the function of
this putative viral protein is yet to be defined properly.
A recent study, using MYMIV AC5 through mutational
analyses and transgenic expression study confirmed novel functions of the viral protein as potent pathogenicity
determinant and suppressors of RNA silencing [24]. The
sense strand encodes coat protein (AV1/CP) and pre-coat
protein (AV2). The DNA-B of bipartite geminiviruses
encodes a nuclear-shuttle protein (NSP) and movement
protein (BC1 or MP) on sense and antisense strands, respectively [3,25].
Mode of Geminivirus DNA Replication
The genome of geminivirus is replicated with doublestranded DNAs as intermediates via rolling circle replication (RCR) occurring in the host cell nucleus. Initially, the
virus penetrates into the host cell followed by uncoating to
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release viral ssDNA which is eventually brought into the
nucleus of the infected cell. The ssDNA is then converted
into double stranded DNA (dsDNA) assisted by cellular
host factors (Figure 2). The dsDNA acts as a substrate
for transcription of the viral factors. The dsDNA or the
replicative form (RF) is also duplicated several times using the rolling circle mode of replication (RCR). The Rep
protein of the virus nicks the viral DNA at a specific site
to initiate RCR and several host factors are required for
progression and termination of RCR fork. Both the DNAA and DNA-B components are replicated using the same
principles. Though, the DNA-A and DNA-B components
are widely different in nucleotide sequences, the two components share a conserved region of a ~200 nucleotide,
known as the common region/intergenic region (CR/IR),
as shown in Figure 1 [26–28]. This CR/IR constitutes a
hairpin structure with repeat sequences known as ‘iterons’,
which are recognized by the Rep protein for its specific
binding. The Rep-iteron binding, eventually leads to cooperative binding of Rep almost throughout the CR region, leading to specific nicking of the nonamer region
(TAATATTAC; : site of nicking) [29]. Rep protein
binds covalently at the 5ʹ end of nick and the 3ʹ end is
used for extension of RCR (Figure 2 & Figure 3). Rep protein also acts as helicase for progression of the RCR fork
[30]. One round of RCR will give rise to a full viral dsDNA
along with a genome-long single stranded (ss) viral DNA
which could be released following the nicking action of
Rep. Thus, a new viral strand ssDNA is made and is circularized using the closing activity of Rep.
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Figure 2: The geminivirus lifecycle. Whitefly carrying geminate particles, upon un-coating, release viral single stranded (ssDNA) to the
infected plant cells cytoplasm and then the viral DNA is imported to
the nucleus for amplification of viral DNA. The ssDNA subsequently
gets converted to double stranded (dsDNA) with the use of host cell
machinery. The viral DNA in association with the nuclear proteins
can transcribe viral mRNAs at the nucleus and the latter ones are exported to cytoplasm for translation to different viral proteins. The viral proteins that are required in the nucleus to carry out replication,
transcription or movement of viral DNA enter to the nucleus through
nuclear pores. With the help of movement protein, viral DNA can
enter to nearby cells through the plasmodesmatal channels and thus
virus spread occurs.
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The newly synthesized ssDNA can be further converted to dsDNA that can serve as a template for transcription/replication. In this way, several copies of RF or dsDNA will be made. From each RF molecule, several viral
ssDNAs are also made. After a while, the replication processes are discontinued perhaps with the increasing concentration of coat proteins of the virus [31]. The ssDNAs
are either packaged in virions or are transported out of the
cell in the form of nucleo-protein complexes with the help
of MP encoded by the virus. The transport of the nucleoprotein complexes happens through the plasmodesmatal
channels into the neighboring cells. In this way, the local
viral spread occurs (Figure 2), eventually leading to the
long-distance spread within the host using the vasculature
route.
Figure 3: Steps of geminiviral DNA replication. Initiation: Replication initiation begins with, binding of Rep protein at CR region of
DNA-A in a cooperative manner and eventual nicking of DNA at initiation site of CR region. Helicase activity of Rep might help in melting
of origin of DNA region allowing other host proteins such as PCNA,
RPA32 and RAD54 etc. to associate at 3΄-OH end of the nick to form
Replication fork complex. Elongation: The elongation of replication
fork may associate other host factors along with the ATPase and helicase activities of Rep protein for DNA replication. Termination: Rep
protein cuts and releases newly synthesized ssDNA to generate many
copies of viral ssDNAs.
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As discussed earlier, geminiviruses replicate inside
their hosts utilizing the host replication machinery at every step of the replication process, namely initiation, progression and termination of RCR. Assembly of the viral
and host factors contributes to the successful multiplication of the virus inside the host.
Viral Factors Associated with Geminivirus Replication
Functional assay of the geminiviral genes is an important way to understand the behavior of geminiviruses in
their hosts and to develop antiviral strategies. The differwww.avidscience.com
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ent virus encoded gene products which are essential for
the multiplication of the geminivirus are described below.
Rep Protein (AC1)
The most important protein of any geminivirus is
Rep, which is a multifunctional protein as it is involved in
a variety of processes like transcriptional activation and
repression of viral genes [32,33], interaction of various
host factors [34,35] and ATP/GTPase activity [36] and is
indispensible for viral DNA replication process. That is
why Rep protein has been studied extensively. Rep protein
of African cassava mosaic virus (ACMV) was proven to be
the only prerequisite for DNA replication as the deletion
mutations of this open reading frame (ORF) rendered the
DNA non-infectious in plants and prevented DNA replication in protoplasts [37]. The initiation of replication
process takes place by binding of the Rep proteins to the
iterons and this binding specificity is provided by the Nterminal 116 amino acids of the Rep protein of two different species, namely, TYLCV and MYMIV [38,39]. Rep
protein initiates RCR by cleaving the geminivirus ori at
7th and 8th nucleotide of conserved nonameric sequence
TAATATTAC. In vitro functional assay using Rep protein of TYLCV expressed in E. coli showed that the Rep
protein has its nicking activity at tyrosine-103 that resides
in a conserved sequence motif DVKXYXXKD [40]. The
Rep protein was also reported to act as a site-specific typeI topoisomerase [29]. The functional analysis of the gemi12
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nivirus Rep has been studied by modifying the amino acids at R2, R5, R7 and K11 or H56, L57 and H58 using an in
planta system. The system contains an integrated copy of a
tandem repeat of the ACMV origin of replication flanking
non-viral sequences that could be replicated by Rep as an
episomal replicon and Rep function was observed to be
hampered by these modifications during replication [41].
In our laboratory, we have also developed a yeast model
using MYMIV DNA-A (tandem construct) that could
replicate in budding yeast cells [39]. Both these systems,
in planta and yeast model can shed light for studying the
eukaryotic DNA replication mechanism. The study using Rep protein of MYMIV revealed that it can also act
as DNA helicase in the form of homo-oligomers [30,42].
Furthermore, the mutational analysis of Rep protein at
K272 and K289 showed, the mutants could not unwind
the DNA double helix and consequently the mutants were
replication defective [30,43]. The various modules of Rep
protein are shown in Figure 4 and the mid-module is involved in protein-protein interactions involving the viral
factors and the host-viral factors in a broad sense.
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Replication Enhancer Protein (REn/
AC3)
Figure 4: Modular Organization of geminivirus (MYMIV) Rep protein a. The different functional domains of Rep proteins are shown,
such as: N-terminal domain having different conserved motifs, IDNA nicking domain (1-120), II-DNA binding domain (1-130), IIIOligomerization domain (120-182), and the C-terminal domain having ATPase and helicase activities. The mid module (oligomerization
domain) is involved in binding with viral factors like self, REn and the
host factors like PCNA, REn, RBR, SUMO conjugating enzymes to
mention a few. b. The conserved motifs of N-terminal domain of Rep
protein are shown, such as: rolling circle replication initiator motifs,
RCR1 (FLTY), RCR2 (HLHXLX) and RCR3 (DVKXYXXKD). The
starting coordinates of all these motifs are shown in numbers on upper side and the conserved amino acids are presented in lower side.
Another conserved motif, GRS (geminivirus rep sequence) is shown
between the coordinates 74-96. The HLH motif of RCR2 is involved
in metal binding and DVKXYXXKD motif contains a conserved amino acid residue “tyrosine” (marked as red) at 103rd position that has
DNA nicking activity.
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REn is another protein of viral origin and as the
name suggests, it plays a role in stimulating DNA replication as evident from the protoplast and leaf disc assays
[44,45]. In vitro assay with the viral proteins, Rep and REn
established that, REn forms a higher order oligomerization, interacts with Rep and enhances the ATPase activity
of Rep protein [46]. Phage display analysis with recombinant REn protein from Tomato leaf curl Kerala virus
(ToLCKeV) identified various REn interacting peptides
which were found to be homologous proteins associated
with viral replication process and the in planta study using tomato plant confirmed REn effectively functions in
the viral replication at an intermediate stage and enhances
replication process [47].
Coat Protein (CP/AV1)
CP of geminivirus was shown to be the unique to
build geminiviral capsids [48,49]. It is also necessary for
transmission through insect vectors. Mutational analysis
suggested that in bipartite geminiviruses, CP is not required for replication and systemic spread of virus but the
appearance of symptoms was delayed in infections with
the deletion mutant [49]. Accumulation of CP was observed in the nuclei of both insect and tobacco cells using immune-fluorescent staining and it has been shown
that CP facilitates the nuclear transport of viral DNA [50].
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The delayed appearance of symptom in deletion mutant
study by Brough and co-workers in 1988 [49] might be
due to the defect in binding of zinc finger motif of CP to
ssDNA[51]. The interaction of CP and Rep also leads to
control of viral DNA replication [31]. The unique feature
of the genome organization of typical Old World Begomoviruses is the presence of an ORF AV2 upstream of
CP gene. Recombinant AV2 from two different isolates of
MYMIV [black gram isolate, MYMIV-(IN: ND: Bg3:91),
and cowpea isolate, MYMIV-(IN: ND: Cp7:98) respectively] modulated nicking and ATPase activity of Rep in
vitro. Although the recombinant AV2 protein could not
bind the DNA efficiently, it is hypothesized to have an accessory protein modulating Rep activities [52].
Host Factors and Geminiviral DNA
Replication
Plant viruses infect and multiply in their hosts. Due
to the non-living nature and limited coding capacity, these
viruses are highly dependent on their host cell machinery for their survival, multiplication and spread. Hence, it
is important to identify and characterize the host factors
that participate in the initiation, progression and termination of RCR of the gemini-DNA. As Rep is the most
important viral protein for geminiviral DNA replication
and REn acts as an accessory of Rep, attempts have been
focused to identify the host factors that interact with viral
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Rep and REn. Several approaches like phage display, yeast
two-hybrid methods, immunoprecipitation of host factors using the antisera of Rep/REn proteins etc. have been
used to identify a plethora of host factors. Besides these,
we have developed the yeast and plant models to pinpoint
the factors along with their roles in RCR of Begomoviral
DNA [39,53].
The geminivirus and host plant interaction studies
using various approaches showed that Rep interacts with
several host proteins leading to the reduction or increase
of viral DNA accumulation, thereby regulating the infection process. The geminivirus infects the host plant by uncoating the ssDNA particles and smartly targets the cell
cycle check point proteins to activate the host cell replication machinery so that it can use the host replication
control system for its multiplication. A key regulator of
cell cycle is retinoblastoma related (RBR) protein, which is
a plant homolog of the tumor suppressor retinoblastoma
(pRb) and control the plant cells to enter into S phase [54].
Rep protein interacts with RBR protein through ‘LXCXE’
or other motifs and an increased accumulation of both viral and host DNA takes place consequently [55,56]. The
incorporation of DNA into host chromosome has been
demonstrated using 5-bromo-2-deoxyuridine (BrdU)
incorporation assay. Immunoblot analysis in Nicotiana
benthamiana plants infected with TGMV revealed that up
to 17-fold more BrdU was incorporated into chromosomal DNA of TGMV-infected plants especially to the infectwww.avidscience.com
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ed cells only, that contained both host and viral DNA [56].
Additionally, Rep protein is also known to be associated
with other host factors like Replication factor C (RFC)
that was confirmed using wheat dwarf virus (WDV) Rep
protein and it was proposed that it helps generate a primer
with 3ʹ-OH terminus during initiation of DNA replication
[57]. It has also been seen that post translational modification process like acetylation and sumoylation are also
actively involved by regulating various cellular processes,
such as nuclear-cytosolic transport, transcriptional regulation, apoptosis, protein stability, response to stress, and
progression through the cell cycle. The host cell sumoylation enzyme was found to interact with the N-terminal
half of Rep protein and accumulation of viral DNA was
severely impaired using sense and antisense LeSUMO
(SUMO from tomato, homolog to Arabidopsis SUMO1/2
and human SUMO2/3) leaf disc assay [58].
Proliferative cell nuclear antigen (PCNA) is an important protein that acts as a cofactor for DNA polymerase
δ and increases the processivity of leading strand DNA
synthesis during replication process. Both Rep and REn
are known to be involved in binding with PCNA [59,60].
Rep activity was reduced following the interaction with
PCNA. The contacting amino acid residues of PCNA
seemed to be present throughout a wide region of the
trimeric PCNA protein, while those of Rep appeared to
be localized only in the middle part of the protein. The
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site-specific nicking-closing activity and the ATPase function of MYMIV Rep were impaired by PCNA [60] leading to the reduction of replication initiation by Rep. Other
host proteins may in contrast, enhance the replication by
Rep. Our study presented evidences that the host singlestranded DNA-binding protein, Replication protein A
(RPA) 32 kDa subunit (RPA32) directly interacts with the
carboxyl terminus of MYMIV-Rep both in vitro as well as
in yeast two-hybrid system. RPA32 modulated the functions of Rep by enhancing its ATPase and down regulating
its nicking and closing activities. We also showed the positive involvement of RPA32 in transient replication of the
plasmid DNA bearing MYMIV replication origin using an
in planta based assay [53]. Further, MYMIV Rep–RPA32
interaction highly modulates the intrinsic helicase activity
of the Rep, which might be essential for the Rep protein
mediated unwinding of the origin. Such changes might be
necessary for the formation as well as the progression of
a functional replication fork [61]. Another host protein,
RAD54, a eukaryotic- specific recombination factor, is a
member of the RAD52 epistasis group, which is involved
in the repair of dsDNA break and belongs to the SNF2/
SWI2 chromatin remodeling family and also participates in begomoviral DNA replication. We showed that
the specific DNA-binding, site-specific nicking, helicase
activities of MYMIV-Rep were up-regulated by the recombinant Saccharomyces cerevisiae RAD54 (ScRAD54)
protein. RAD54-deficient yeast nuclear extract did not
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support in vitro viral DNA replication, while exogenous
addition of the purified ScRAD54 protein enhanced replication. The role of RAD54 in replication was confirmed
by the transient replication assay in planta; i.e., agro-inoculation studies in Arabidopsis. The study demonstrates
for the first time, that the eukaryotic rolling circle replicon
depends on the RAD54 protein [62]. Another protein of
RAD52 family, RAD51 was recently shown tobe involved
in MYMIV DNA replication. RAD51 is a recombinase
which binds the DNA and catalyzes the identification and
exchange of homologous sequences between the ssDNA
and dsDNA molecule. We showed that MYMIV-Rep interacts directly with the Arabidopsis thaliana RAD51 protein. The study further showed that RAD51 plays a role in
viral DNA replication in its natural host, Mung bean [63].
We have identified a host factor, MCM2, through interaction screening of an Arabidopsis thaliana cDNA library,
that interacts with MYMIV Rep protein and using yeast
two-hybrid, β-galactosidase and co-immunoprecipitation
assays, the interaction between MYMIV-Rep and the
host factor AtMCM2 was confirmed [64]. Additionally,
we have also identified another host transcription factor,
NAC083 employing the phage display technique and in
silico docking studies that showed strong binding affinity
with MYMIV-Rep. In vitro pull down assay and yeast twohybrid analysis confirmed the interaction of MYMIV-Rep
and AtNAC083 protein [63]. Table 1 summarizes the list
of Rep-interacting host factors that were screened in our
laboratory using a variety of different methods.
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Table 1: Putative replication related host factors interacting with geminivirus (MYMIV) Rep protein.
Sl No.
Host factors identified by various screening methods
1
2
3
4
5
6
7
A. Phage display library screening
Putative DNA polymerase ζ REV3, REV7 and REV1
Putative DNA polymerase ε catalytic subunit
Putative DNA polymerase similar to S. cerevisiae
DNA repair protein RAD54
RAD 4, 5, 6, 23, 51
MCM 2/3/5 family
ORC1A, ORC1B
B. Yeast model
Rev3
Rev7
MCM2
SNF4/CAT3/SCl-II
Rad23
MSH2
SKl2
8
ATP binding/DNA ligase (ATP)
Mre11
9
TOP II and III
DMC1
10
Retinoblastoma-binding protein like
Rad52
11
SNF 1, 2, 4, 7
Rad51
12
Ubiquitin-protein ligase
BMH1
13
DNA directed RNA polymerase subunit
BMH2
14
Structural maintenance of chromosomes
15
Regulator of chromosome condensation (RCC1)
16
EMB2411:ATP-dependent DNA helicase
17
DnaJ homolog
18
Nucleic acid binding / ribonuclease H
19
Putative RNA helicase
20
ATP –dependent DNA helicase RecQ and RecQI3
21
NAP ( NAC -LIKE, ACTIVATED BY AP3/PI)
22
MSH2, DNA mismatch repair protein
23
E2F transcription factor E2Fd
24
Flap endonuclease
25
DNA primase
26
Replication protein A like
27
Replication Factor C
28
PCNA like protein
29
NAC domain protein NAC 2
C. Yeast two-hybrid screening
M type thioredoxin
Adaptor 2A homolog
C2 domain containing protein
Splicing factor SC35
ATP-DNA binding DNA dependent ATPase
MCM-2 related protein
Although, Rep protein is studied extensively for its
association with host factors, another viral protein REn
was found to interact with many host factors and assist
in geminivirus DNA replication. Using yeast two-hybrid
assay, a new member of the NAC domain protein family
from tomato (Solanum lycopersicum), SlNAC1, has been
identified and it was observed that ToLCV infection upregulated SlNAC1 expression in association with REn, remarkably in the infected cells [65]. In our laboratory, we
have screened MYMIV-REn interacting host partners uswww.avidscience.com
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ing the phage display assay and the list of putative factors
is displayed in Table 2.
Table 2: Geminivirus (ToLCNDV) AC3 (REn) interacting host proteins.
Sl No.
Replication proteins/
Host Proteins
RNA/DNA polymerases Histone/DNA modifying
1
Helicases
Replication protein A1
RNA dependent RNA
enzymes
H3-K9 Methyltransferase
silencing
Repressor of Silencing 1
2
Geminivirus Rep
polymerase 1
RNA dependent RNA
Histone Methyltransferase
(ROS1)
Suppressor of gene silencing
3
interacting Kinase 1
RecQ Helicase
polymerase 2
RNA dependent RNA
polymerase 6
Histone acetyl transferase
3 (SGS3)
Hua Enhancer 1
4
Werner Helicase
DNA polymerase γ2
Decreased DNA methyla-
Hua Enhancer 4
Proteins involved in RNA
tion 1
5
RAD1
DNA polymerase ε
subunit
6
RAD4
DNA polymerase α
7
RAD5
8
RAD23-3
9
RAD50
variant
DNA polymerase ζ
catalytic subunit
DNA polymerase λ
10
Anti silencing factor 1
DNA polymerase δ
Variant in methylation 2
Dicer-like 1 (DCL1)
Increase in Bonsai methyla- Dicer-like 2 (DCL2)
subunit
tion 1
DNA polymerase η splice Maintenance of methylation Argonaute 1 (AGO1)
Decreased methylation to
DNA 1
Agronaute 2 (AGO2)
subunit
Geminiviruses transmit the viral disease from an
infected host to a healthy host through an insect vector,
whitefly and replicate inside the host plant. During its
transport through the insect vector, it is possible that virus
can also replicate inside the insect vector, but the mechanism is not clear yet. Recently, using fluorescence in situ
hybridization (FISH), it has been established that the TYLCV virus, localized in the midgut epithelial cell nuclei of
the insect vector and can multiply locally [66].
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Future Prospects
As mentioned above, quite a bit of knowledge has
emerged regarding geminiviral DNA replication with the
usages of various techniques and models. Now it is high
time to establish high-throughput assays to identify host
factors(s) which is crucial for gemini-DNA replication,
but contribute little for host growth and maintenance.
Dispensing such factors will evolve the anti-viral strategies. With the present day available knowledge, it is also
the time to attempt in vitro reconstitution of RCR of
gemini-DNA. There are many infectious clones available
in many labs, for example Tomato Leaf Curl New Delhi
Virus (ToLCNDV) agro-infectious (dimeric) clone, which
has been used widely. These clones can be used as the
template DNA with the tomato leaf extracts to examine
the feasibility of RCR of ToLCNDV-DNA. Following success, the extract could be further fractionated to identify
the minimal host-factors for replication of the tomato leaf
curl viral DNA. Similar experiments can also be carried
out using cabbage leaf curl virus and the Arabidopsis leaf
extract.
Acknowledgements
We are deeply indebted to all of our lab-members who
made valuable contributions towards understanding replication of Gemini-genome. For the present manuscript,
we must acknowledge a few members, namely Drs P.S.
Malik, V. Raghavan, N. Roy Choudhury, K. Pasumarthy,
K. Kosalai, and G. Suyal.
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