# Virus-Specific Read-Through Codon Preference Affects Infectivity of Chimeric Cucumber Green Mottle Mosaic Viruses Displaying a Dengue Virus Epitope

Authors: Pak-Guan Teoh; Aik-Seng Ooi; Sazaly AbuBakar; Rofina Yasmin Othman
Journal: Journal of Biomedicine and Biotechnology
 (2009)

---

## Abstract

ACucumber green mottle mosaic virus (CGMMV) was used to present a truncated dengue virus type 2 envelope (E) protein binding region from amino acids 379 to 423 (EB4). The EB4 gene was inserted at the terminal end of the CGMMV coat protein (CP) open reading frame (ORF). Read-through sequences of TMV or CGMMV, CAA-UAG-CAA-UUA, or AAA-UAG-CAA-UUA were, respectively, inserted in between the CP and the EB4 genes. The chimeric clones, pRT, pRG, and pCG+FSRTRE, were transcribed into full-length capped recombinant CGMMV transcripts. Only constructs with the wild-type CGMMV read-through sequence yielded infectious viruses following infection of host plant, muskmelon (Cucumis melo) leaves. The ratio of modified to unmodified CP for the read-through expression clone developed was also found to be approximately 1:1, higher than what has been previously reported. It was also observed that infectivity was not affected by differences in pI between the chimera and its wild counterpart. Analysis of recombinant viruses after 21-days-postinculation (dpi) revealed that deletions occurred resulting in partial reversions of the viral population to near wild type and suggesting that this would be the limiting harvest period for obtaining true to type recombinants with this construct.


---

## Body

## 1. Introduction

The development of plant virus vectors asin planta expression systems for foreign
genes provides an attractive alternative biotechnological approach
for peptide expression [1–5]. This method has been
exploited in vaccine production, where small foreign peptides are expressed as
a fusion with the viral coat proteins. Essentially, an insertion site has to be
determined in the virus genome so that the resulting product will be displayed
on the surface of the virus particle which is then propagated in plants and consequently
isolated and used as antigen presenting vehicles [5, 6]. Modifications that do
not interfere with the normal functions of the particular virus are a
prerequisite for this peptide fusion approach. One strategy suggests that
foreign gene segments could  be fused to the terminus of a viral
gene in a way that permits the production of both the fusion product and the
native viral protein, thus avoiding interference with normal gene functions.
The success of this epitope presentation strategy depends on a detailed
knowledge of virus structure at the atomic level, which is only available for a
limited number of viruses.We
have recently developedCucumber green
mottle mosaic virus (CGMMV) as a candidate for expressing antigenic
peptides in plants [7]. CGMMV is a tobamovirus with a genome size of ~6.4 kb
which has been well characterized both biologically [8, 9] and structurally [10, 11]. In this study, a truncated dengue virus
type 2 envelope (E) protein binding region from amino acid 379 to 423 (EB4) was
inserted into the end of the coat protein (CP) open reading frame (ORF) of a
previously constructed CGMMV full-length clone, pCGT7X [7]. The antigenic
peptide was chosen based on a recent study that suggests its importance in
enabling dengue virus to bind to specific host cell receptors (S. Abu Bakar  personal communication).
The present study explores the possibility of extrapolating the CGMMV antigenic
epitope presentation system for developing diagnostics and potentially
therapeutics against dengue. The study was also used to challenge the size
limits of foreign gene insertion into the CGMMV vector as in the previous study
the hepatitis B surface antigen (HBsAg) used was only 33 amino acids [7].


## 2. Materials and Methods

### 2.1. The Antigenic Epitope

The 45 amino
acid-long EB4 protein used in this study has been previously shown to react
with the dengue-specific antibody 3H5-1 (S. Abu Bakar  personal communication).


### 2.2. Construction of Chimeric Cgmmv Vector

The EB4 coding
sequence was amplified from a pCANTAB 5E vector carrying the virus gene using 3
primer pair sets. The resulting PCR products were purified then digested
overnight withHindIII restriction
endonuclease, with the same treatment carried out on the full-length clone of
CGMMV (pCGT7X, ~9.0 kb) previously constructed [7]. The digested PCR products
and the linearized pCGT7X were purified following 1% agarose gel
electrophoresis, and then ligated to form pRT, pRG, both containing the TMV
read-through sequence [12] and pCG+FSRTRE (containing the CGMMV read-through
sequence) [13], respectively (Figure 1). The three primer sets used in the
amplifications were as follows.Figure 1
Position of primers in
constructed plasmid clones. Primers 4–8 were used to amplify the EB4 gene from
pCANTAB 5E. Plasmid pCGT7X and amplified EB4 fragments were digested withHindIII and ligated together to obtain
the respective plasmid clones as shown. HindIII
is the insertion site of EB4 at the end of CGMMV CP. PCR amplification using
primer pairs 1–9, 3–9, and 2–7 will yield amplified
products of approximately 6.5 kb, 0.85 kb, and 2.2 kb, respectively.Forward
RT (5′-  CCAAGCTTGCCAATAGCAATTAATCATAGGAGTAGAGC-3′) and Reverse E
(5′-CCAAGCTTCTCCAAAATCCCAAGCTGT-3′) for construction of clone pRT, Forward (RT) TGG
(5′-AAGCTTGGCAATAGCAATTAATCATAGGAGTAGAGCCG-3′)  and Reverse Q
(5′-CCAAGCTTGTCCAAAATCCCAAGCTGTGT-3′) for clone pRG, and Forward SRT (5′-CCAAGCTTCCAAATAGCAATTAATCATAGGAGTAGAGCCG-3′) and Reverse E
(5′-CCAAGCTTCTCCAAAATCCCAAGCTGT-3′) for clone pCG+FSRTRE.


### 2.3. Production of Infectious Rna

The
templates used in thein vitro transcription reactions were synthesized through long-distance PCR (LD-PCR) in
50 μL PCR cocktails containing 1X HF Buffer of Phusion DNA Polymerase (Finnzymes,
Espoo, Finland)
with 1.5 mM MgCl2 (Finnzymes, Espoo, Finland), 0.2 mM dNTP mix, 0.5 μM forward primer,
CGT7dG  (5′-CCGAGCTCGTAATACGACTCACTATAGGTTTTA-3′), 0.5 μM reverse primer, CGMMV
3′-UTR (5′-TGGGCCCCTACCCGGGGAAAAGGGGGGAT-3′), 10–20 ng of DNA template, and 1 U
of  Phusion
DNA Polymerase (Finnzymes, Espoo, Finland). The reaction was set up in 0.2 mL tubes, and the
thermal cycling was conducted with initial denaturation at 98°C for 60 seconds,
followed by 30 cycles of 98°C denaturation for 10 seconds, annealing at 63°C
for 20 seconds and elongation at 72°C for 1 minute and 50 seconds, and finally
an extension step at 72°C for 5 minutes. The amplified product was purified
through phenol-chloroform extraction followed by ethanol precipitation. The
pellet was dissolved in an appropriate volume of RNase-free distilled water to
1 μg/μL and stored at −20°C till further use. The in vitro transcription was carried out using the (Ambion, Calif, USA)
High Yield Capped T7 RNA Transcription Kit according to the manufacturer
protocol. Aliquots of the in vitro-synthesized
transcripts were denatured and electrophoresed alongside RNA markers showing
its integrity and the expected transcript size of approximately 6.5 kb. Since no
DNase I treatment was done, traces of DNA template of the transcription
reactions were detected.


### 2.4. Maintaining The Host Plants

Muskmelon
(C. melo) plants were used as host
plants for virus propagation. Plants used in this study were maintained in a
growth room at 25°C with 16 hours of light and 8 hours of darkness. Healthy 10-day-old plantlets with
cotyledons and small first leaf were used for inoculation.


### 2.5. Inoculation with Rna Transcripts

One
transcription reaction was used to inoculate 2 plantlets by gently rubbing the
reaction mixture over carborundum-dusted
first leaf and cotyledons of 10-day-old plantlets. Mock inoculation was done by
gently rubbing distilled water onto carborundum-dusted first leaves. The excess inoculum was rinsed
off using distilled water from the leaf surfaces 60 minutes after inoculation.


### 2.6. Rt-Pcr Detection of Chimeric Virus Infection

Total
RNA was isolated from the new leaf of the inoculated and healthy plants using
RNeasy Plant Mini Kit (QIAGEN). RT-PCR was performed using AccuPower RT/PCR
PreMix (Bioneer, Daejeon, South Korea)
with primers CGMMV3′UTR (5′-TGGGCCCCTACCCGGGGAAAAGGGGGGAT-3′) paired with Pst I sense (5′-TAGGAAAAAACCAGAAGATCTGCAGGAATTTTTCTC-3′)
or C5500F (5′-GTCGCTACAACTAACTCTATTATCAAAAAGGGTC-3′). Reactions were carried
out according to the manufacturer protocols. Infected plants will give a PCR-amplified
product of approximately 2.2 kb (with PstI
sense and CGMMV 3′UTR primers) or 0.85 kb (with C5500F and CGMMV 3′UTR primers).
RT-PCR reactions were carried out for plants at 14, 21, and 30
day-postinoculation (dpi).


### 2.7. Virus Purification

Plant
virus isolation procedures used in this study were modified from [8]. Infected plants showing
typical symptoms were harvested, weighed, and homogenized in ice-cold 0.1 M phosphate buffer
(pH 7.0 containing 1% of β-mercaptoethanol) at 1 mL/g of plant material for 10
minutes using a mechanical blender. The homogenate was filtered through 2
layers of cheesecloth and then mixed with equal volume of chloroform:butanol
(1:1). The mixture was then stirred for 1-2 hours at room
temperature and then the organic phase was separated from the mixture through
centrifugation at 8000 g for 15 minutes. The aqueous layer was transferred to a
beaker, 100 mL of NaCl (4 g/L) and PEG6000 added, and the mixture stirred on ice
for 1 hour. The precipitated virus was separated from the solution through
centrifugation at 10 000 g for 30 minutes at 4°C. The resulting pellet was
reconstituted in 10 mL of 0.1 M phosphate buffer pH 7.0. Any undissolved material
was cleared by centrifugation at 10 000 g for 30 minutes at 12°C. Then 0.2 M EDTA
(pH 7) (50 mL/L) was added to the supernatant and the mixture subjected to
centrifugation at 110 000 g for 90 minutes at 4°C. The supernatant was discarded,
and the pellet was left to air dry. The virus pellet was then reconstituted in
100 μL of distilled water and stored at 4°C until used.


### 2.8. Analyses of Viral Genome

Analyses
of sequences of the amplified products were carried using BioEdit Sequence
Alignment Editor Software (version 6.0.5) (http://www.mbio.ncsu.edu/BioEdit/bioedit.html).
The pI and charge values of the coat protein were calculated using the protein calculator
developed by Chris Putnam of The Scripps Research Institute (http://www.scripps.edu/~cdputnam/protcalc.html).


## 2.1. The Antigenic Epitope

The 45 amino
acid-long EB4 protein used in this study has been previously shown to react
with the dengue-specific antibody 3H5-1 (S. Abu Bakar  personal communication).


## 2.2. Construction of Chimeric Cgmmv Vector

The EB4 coding
sequence was amplified from a pCANTAB 5E vector carrying the virus gene using 3
primer pair sets. The resulting PCR products were purified then digested
overnight withHindIII restriction
endonuclease, with the same treatment carried out on the full-length clone of
CGMMV (pCGT7X, ~9.0 kb) previously constructed [7]. The digested PCR products
and the linearized pCGT7X were purified following 1% agarose gel
electrophoresis, and then ligated to form pRT, pRG, both containing the TMV
read-through sequence [12] and pCG+FSRTRE (containing the CGMMV read-through
sequence) [13], respectively (Figure 1). The three primer sets used in the
amplifications were as follows.Figure 1
Position of primers in
constructed plasmid clones. Primers 4–8 were used to amplify the EB4 gene from
pCANTAB 5E. Plasmid pCGT7X and amplified EB4 fragments were digested withHindIII and ligated together to obtain
the respective plasmid clones as shown. HindIII
is the insertion site of EB4 at the end of CGMMV CP. PCR amplification using
primer pairs 1–9, 3–9, and 2–7 will yield amplified
products of approximately 6.5 kb, 0.85 kb, and 2.2 kb, respectively.Forward
RT (5′-  CCAAGCTTGCCAATAGCAATTAATCATAGGAGTAGAGC-3′) and Reverse E
(5′-CCAAGCTTCTCCAAAATCCCAAGCTGT-3′) for construction of clone pRT, Forward (RT) TGG
(5′-AAGCTTGGCAATAGCAATTAATCATAGGAGTAGAGCCG-3′)  and Reverse Q
(5′-CCAAGCTTGTCCAAAATCCCAAGCTGTGT-3′) for clone pRG, and Forward SRT (5′-CCAAGCTTCCAAATAGCAATTAATCATAGGAGTAGAGCCG-3′) and Reverse E
(5′-CCAAGCTTCTCCAAAATCCCAAGCTGT-3′) for clone pCG+FSRTRE.


## 2.3. Production of Infectious Rna

The
templates used in thein vitro transcription reactions were synthesized through long-distance PCR (LD-PCR) in
50 μL PCR cocktails containing 1X HF Buffer of Phusion DNA Polymerase (Finnzymes,
Espoo, Finland)
with 1.5 mM MgCl2 (Finnzymes, Espoo, Finland), 0.2 mM dNTP mix, 0.5 μM forward primer,
CGT7dG  (5′-CCGAGCTCGTAATACGACTCACTATAGGTTTTA-3′), 0.5 μM reverse primer, CGMMV
3′-UTR (5′-TGGGCCCCTACCCGGGGAAAAGGGGGGAT-3′), 10–20 ng of DNA template, and 1 U
of  Phusion
DNA Polymerase (Finnzymes, Espoo, Finland). The reaction was set up in 0.2 mL tubes, and the
thermal cycling was conducted with initial denaturation at 98°C for 60 seconds,
followed by 30 cycles of 98°C denaturation for 10 seconds, annealing at 63°C
for 20 seconds and elongation at 72°C for 1 minute and 50 seconds, and finally
an extension step at 72°C for 5 minutes. The amplified product was purified
through phenol-chloroform extraction followed by ethanol precipitation. The
pellet was dissolved in an appropriate volume of RNase-free distilled water to
1 μg/μL and stored at −20°C till further use. The in vitro transcription was carried out using the (Ambion, Calif, USA)
High Yield Capped T7 RNA Transcription Kit according to the manufacturer
protocol. Aliquots of the in vitro-synthesized
transcripts were denatured and electrophoresed alongside RNA markers showing
its integrity and the expected transcript size of approximately 6.5 kb. Since no
DNase I treatment was done, traces of DNA template of the transcription
reactions were detected.


## 2.4. Maintaining The Host Plants

Muskmelon
(C. melo) plants were used as host
plants for virus propagation. Plants used in this study were maintained in a
growth room at 25°C with 16 hours of light and 8 hours of darkness. Healthy 10-day-old plantlets with
cotyledons and small first leaf were used for inoculation.


## 2.5. Inoculation with Rna Transcripts

One
transcription reaction was used to inoculate 2 plantlets by gently rubbing the
reaction mixture over carborundum-dusted
first leaf and cotyledons of 10-day-old plantlets. Mock inoculation was done by
gently rubbing distilled water onto carborundum-dusted first leaves. The excess inoculum was rinsed
off using distilled water from the leaf surfaces 60 minutes after inoculation.


## 2.6. Rt-Pcr Detection of Chimeric Virus Infection

Total
RNA was isolated from the new leaf of the inoculated and healthy plants using
RNeasy Plant Mini Kit (QIAGEN). RT-PCR was performed using AccuPower RT/PCR
PreMix (Bioneer, Daejeon, South Korea)
with primers CGMMV3′UTR (5′-TGGGCCCCTACCCGGGGAAAAGGGGGGAT-3′) paired with Pst I sense (5′-TAGGAAAAAACCAGAAGATCTGCAGGAATTTTTCTC-3′)
or C5500F (5′-GTCGCTACAACTAACTCTATTATCAAAAAGGGTC-3′). Reactions were carried
out according to the manufacturer protocols. Infected plants will give a PCR-amplified
product of approximately 2.2 kb (with PstI
sense and CGMMV 3′UTR primers) or 0.85 kb (with C5500F and CGMMV 3′UTR primers).
RT-PCR reactions were carried out for plants at 14, 21, and 30
day-postinoculation (dpi).


## 2.7. Virus Purification

Plant
virus isolation procedures used in this study were modified from [8]. Infected plants showing
typical symptoms were harvested, weighed, and homogenized in ice-cold 0.1 M phosphate buffer
(pH 7.0 containing 1% of β-mercaptoethanol) at 1 mL/g of plant material for 10
minutes using a mechanical blender. The homogenate was filtered through 2
layers of cheesecloth and then mixed with equal volume of chloroform:butanol
(1:1). The mixture was then stirred for 1-2 hours at room
temperature and then the organic phase was separated from the mixture through
centrifugation at 8000 g for 15 minutes. The aqueous layer was transferred to a
beaker, 100 mL of NaCl (4 g/L) and PEG6000 added, and the mixture stirred on ice
for 1 hour. The precipitated virus was separated from the solution through
centrifugation at 10 000 g for 30 minutes at 4°C. The resulting pellet was
reconstituted in 10 mL of 0.1 M phosphate buffer pH 7.0. Any undissolved material
was cleared by centrifugation at 10 000 g for 30 minutes at 12°C. Then 0.2 M EDTA
(pH 7) (50 mL/L) was added to the supernatant and the mixture subjected to
centrifugation at 110 000 g for 90 minutes at 4°C. The supernatant was discarded,
and the pellet was left to air dry. The virus pellet was then reconstituted in
100 μL of distilled water and stored at 4°C until used.


## 2.8. Analyses of Viral Genome

Analyses
of sequences of the amplified products were carried using BioEdit Sequence
Alignment Editor Software (version 6.0.5) (http://www.mbio.ncsu.edu/BioEdit/bioedit.html).
The pI and charge values of the coat protein were calculated using the protein calculator
developed by Chris Putnam of The Scripps Research Institute (http://www.scripps.edu/~cdputnam/protcalc.html).


## 3. Results

### 3.1. Infectivity of Constructed Transcription Clones—read-through Sequence Preference

In this study, the
chimeric CGMMV vectors pRT, pRG, and pCG-FSRTRE were constructed by inserting
the EB4 coding sequence to the end of the CGMMV CP ORF in plasmid pCGT7X. The
maps of these constructed clones are shown in Figure1, which indicates their
respective position of the primers during amplification and cloning procedures.
Maps of plasmids pCGT7X are
carrying the wild-type CGMMV, and pCANTAB 5E are
carrying EB4 with their respective
priming sites are also as indicated in Figure 1. The genome size of wild-type
CGMMV is approximately 6.4 kb (without the plasmid backbone), and the resulting
chimeric CGMMV genome would be approximately 6.5 kb in size and contains the EB4
and read-through sequences as well as inserted HindIII restriction recognition sites and additional nucleotides
enabling in-frame cloning. The pRT and pRG chimeric clones were constructed
with the read-through sequence of TMV (CAA-UAG-CAA-UUA). This leaky sequence
meets the minimal sequence requirement for effective read-through of the stop
codon [12] and had been used successfully in previous reports [14].The
templates for in vitro
transcription of these two clones were generated through LD-PCR (data not shown).
The resulting amplified products (~6.5 kb) consisted of a T7 promoter fused with
the chimeric CGMMV genome carrying EB4. Transcripts produced from the two
constructs were separately tested for infectivity by inoculating the host
plants. After repeated attempts, both the pRT- and pRG-generated transcripts
did not cause infection of the inoculated plants (Table1). There was no evidence
of virus genomic material in the inoculated plant tissues tested (data not
shown). It is speculated that the read-through sequence of TMV may not be suitable
for the CGMMV chimeric clones, hence contributing to the absence of infectious
virus transcripts. To overcome this possibility, another chimeric clone
(pCG+FSRTRE) was constructed using the read-through sequence AAA-UAG-CAA-UUA of
the wild-type CGMMV itself (Figure 1). The template for in vitro transcription,
based on the pCG+FSRTRE clone, was generated through LD-PCR. The in vitro transcription products
(Figure 2) were used in inoculation studies. The new leaves of plants
inoculated with pCG+FSRTRE-derived transcripts showed evidence of virus
infection with four out of eight plants showing a typical symptom of CGMMV
infection, which include the green mottle mosaic appearance on day 14 pI (Table 1 and Figure 3).
Infection could be detected through RT-PCR when total RNA of new noninoculated
leaf was used as template. Detection of the virus by RT-PCR (Figure 4) suggests
that the viruses had moved from the inoculated leaf to new leaves. This implies
that the chimeric virus pCG+FSRTRE carrying the read-through sequence from the CGMMV
genome (AAA-UAG-CAA-UUA) was
infectious and that the virus particles were able to assemble. The plants,
however, continued to grow without any further noticeable symptoms. Virus
particles were extracted from the infected plants and an aliquot was
electrophoretically separated on 15% SDS-PAGE (Figure 5) resulting in two distinct
suggesting that the virus population consisted of two species of coat proteins,
the EB4-CP fusion (larger in size) and the wild-type CGMMV CP (smaller in
size). The ratio of modified to unmodified CP was approximately 1:1.Table 1
Summary of experiments carried out to assess
the infectivity of different transcript clones. (T*otal RNA was extracted from
new noninoculated leaves (third new leaf) of transcript infected plants.)
Transcript  clonesInfectivity on inoculated plantsSymptom  appearance*Virus detected through RT-PCRpRT0/6HealthyNopRG0/6HealthyNopCG+FSRTRE4/8Green
mottle mosaicYesFigure 2
A representative gel image of pCG+FSRTRE-derived transcripts without DNase I
treatment electrophoresed after denaturation showing the expected transcript
size (6.5 kb). The band above the transcript band is the DNA template. Lane M:
RNA marker (Invitrogen, Calif, USA);
1, 2, 3: produced transcripts; M1: 1 kb DNA ladder (Promega, Wis,  USA); N: pCGT7X, ~9.0 kb; D: DNA template, ~6.5 kb. There were traces of DNA template detected in transcripts
without DNase I treatment.Figure 3
Symptom appearance at 14 day-postinoculation (dpi) on muskmelon plants caused
by pCG+FSRTRE-derived transcripts.Figure 4
Gel image showing presence of amplified band (~2.2 kb) after RT-PCR usingPstI sense and reverse E primer. Lane M:
1 kb DNA ladder (Promega, Wis,  USA);
1–8: total RNA extracted from new noninoculated leaves of pCG+FSRTRE-derived
transcripts from infected plants; N: total RNA of new noninoculated leaves of
wild-type transcript from infected plants (negative control).Figure 5
SDS-PAGE of purified chimeric virus coat protein (CP). Lane M: protein marker
(Fermantas); 1: chimeric virus; 2: wild-type CGMMV. It is clearly shown that
the chimeric virus consisted of two different CP species. The higher molecular
band shows the EB4-fusion CGMMV coat protein. The ratio of modified to
unmodified CP was approximately 1:1.


### 3.2. Effect of Pi:charge Value on Stability of Construct

Apart
from the usage of leaky UAG amber stop codons, it has been reported that pI:charge
can affect the production of  viable
recombinant virus [15]. The pI of the epitope is thought to be an important
factor as the hybrid coat protein pI:charge value can affect epitope
presentation. It was also reported that TMV was more tolerant to positively
charged epitopes on its surface. Thus, it was initially speculated that the
failure in expression of the foreign peptide was possibly due to the pI:charge
value of recombinant CGMMV CP which was different from the wild-type CGMMV CP
pI:charge value (Table 2).Table 2
Amino acid sequence, isoelectric
point (pI) and charge of wild type, constructed recombinant CGMMV coat protein
(CP), and the EB4 insert.
ClonesAmino acid sequenceMWpI of CPCharge(kDa)of CPEB4IIGVEPGQLKLNWFKKGSSIGQMIETTMRGAKRMAILGDT
AWDFG5.09.53+1.9MAYNPITPSKLIAFSASYVPVRTLLNFLVASQGTAFQTQAGRD17.35.08−3.1Wild-typeSFRESLSALPSSVVDINSRFPDAGFYAFLNGPVLRPIFVSLLSSTCGMMVDTRNRVIEVVDPSNPTTAESLNAVKRTDDASTAARAEIDNLIESISKGFDVYDRASFEAAFSVVWSEATTSKApRT*MAYNPITPSKLIAFSASYVPVRTLLNFLVASQGTAFQTQAGRD23.05.41−2.1SFRESLSALPSSVVDINSRFPDAGFYAFLNGPVLRPIFVSLLSSTDTRNRVIEVVDPSNPTTAESLNAVKRTDDASTAARAEIDNLIESISKGFDVYDRASFEAAFSVVWSEATTSKACQQLIIGVEPGQLKLNWFKKGSSIGQMIETTMRGAKRMAILGDTAWDFGEApRG*MAYNPITPSKLIAFSASYVPVRTLLNFLVASQGTAFQTQAGRD23.15.69−1.1SFRESLSALPSSVVDINSRFPDAGFYAFLNGPVLRPIFVSLLSSTDTRNRVIEVVDPSNPTTAESLNAVKRTDDASTAARAEIDNLIESISKGFDVYDRASFEAAFSVVWSEATTSKAWQQLIIGVEPGQLKLNWFKKGSSIGQMIETTMRGAKRMAILGDTAWDFGQApCG+FSRTRE*MAYNPITPSKLIAFSASYVPVRTLLNFLVASQGTAFQTQAGRD22.95.41−2.0SFRESLSALPSSVVDINSRFPDAGFYAFLNGPVLRPIFVSLLSSTDTRNRVIEVVDPSNPTTAESLNAVKRTDDASTAARAEIDNLIESISKGFDVYDRASFEAAFSVVWSEATTSKASGQLIIGVEPGQLKLNWFKKGSSIGQMIETTMRGAKRMAILGDTAWDFGEAW*ith
read-through leaky UAG amber stop codon.Table2 shows the isoelectric
point (pI) and charge of the wild-type CGMMV CP, the read-through recombinant
CGMMV CP, and the EB4 insert. The charge of the EB4 insert is positive and potentially
suitable for expression on the surface of the CGMMV CP [2]. Hence, the inserted
peptide is speculated to be expressed if the pI:charge value of modified virus
CP resembles the pI:charge value of unmodified virus CP. Earlier transcripts
(data not shown) generated from fusion clones without a read-through sequence,
where their pI values deviated significantly (>6.0) from the wild-type CP
(5.08), were not able to cause infection in inoculated plants leading to the
suggestion initially that pI:charge value played an important role in virus
particle assembly and infectivity. Thus, the pI value of the recombinant CP constructs
was adjusted to more
closely resemble the wild-type CP pI value by inserting the acidic amino acid
(glutamate) to the 3′ end of CP (Table 2). The experiments (Table 1), however, showed
that although the pI was still higher than that of the wild type (Table 2), the
construct pCG+FSRTRE remained infectious. This
implies that infectivity of the clones was not directly related to the
deviation in pI value with the wild-type virus CGMMV CP.


### 3.3. Deletion of Cloned Peptide Sequence

Sequencing
was carried out on RT-PCR-amplified products of viral RNA extracted from
putative chimeric virus particles at 30 days postinoculation (dpi) and total
plant RNA isolated from inoculated plant materials at 14 and 21 dpi to  confirm the expression of EB4. Sequence
analysis was done using BioEdit Sequence Alignment Editor Software (version 6.0.5)
(http://www.mbio.ncsu.edu/BioEdit/bioedit.html). The chimeric CP carried the
complete EB4 sequence within its genome at 14 dpi (Figure 6). However, the EB4
sequence appeared to be truncated at 21 dpi with an upstream portion of the EB4
sequence was not present. Only part of the downstream sequence of EB4 was
detected together with the 3′ untranslated region of the CGMMV. EB4 was totally
undetectable at 30 dpi. Interestingly, the 5′ end of introduced read-through
sequence (position 490 to 501) was retained in the genome. The introduced HindIII recognition site at EB4
downstream from position 639 to 644 was found to have been deleted after 30 dpi.Figure 6
Sequence analyses of RT-PCR-amplified products from putative chimeric
CGMMV RNA at different days
postinoculation (dpi). The sequenced alignment shows that EB4 sequence was
truncated and not complete after 21 dpi. The putative chimeric CGMMV produced
did not express the EB4 and its genome resembled the wild-type CGMMV.  Introduced read-through sequences and extra
codons are underlined. Complete EB4 sequence is aligned accordingly with the
other sequences.Even
though only a single band was visible on RT-PCR screening, it was suspected
that there could be products with similar sizes which could not be separated in
normal agarose gel electrophoresis, therefore, the RT-PCR products from the
chimeric CGMMV RNA at 30 dpi were cloned into pGEM-T Easy vector and subjected
to sequence analyses. The results (Figure7) confirmed the absence of the EB4
sequence, except for clone pR_P3U4 (from nucleotide position of 702 to 746),
where only part of the downstream sequence of the EB4 was detected together
with the 3′ untranslated region of the CGMMV. The 5′ end of introduced
read-through sequence of AAA-TAG-CAA-TTA (position 594 to 605) was retained
within the genome for all sequenced clones (Figure 7). The introduced HindIII site from position 742 to 747
was deleted for clones pR_P3U1, pR_P3U3, and pR_P3U11 after 30 dpi.Figure 7
Sequence alignments of cloned RT-PCR-amplified products from putative
chimeric CGMMV RNA at 30 dpi. Plasmid clones sent for sequence analysis were
pR_P3U1, pR_P3U3, pR_P3U4, and pR_P3U11. The sequence alignment shows that EB4
sequence was not within the insert of plasmids, except for a partial sequence
for pR_P3U4 clone. Only part of the downstream sequence of EB4 was detected
together with3′ untranslated region of CGMMV for pR_P3U4 clone, the rest were without EB4
sequence. Introduced read-through sequences and extra codons are underlined.
Complete EB4 sequence is aligned accordingly with the other sequences.The
chimeric CGMMV sequence analyses showed one common similarity (Figures6 and 7),
that is, that part of the read-through amber stops codon sequence, and additional nucleotides
“CC-AAA-TAG” were retained for all sequenced clones. This suggests that
deletion had occurred within the host plant. The sequence analysis also showed
that the EB4 was not fully expressed in the putative chimeric CGMMV. The
positive results from the initial RT-PCR screening of transcript-inoculated
plants at 14 dpi (Figure 4) suggest the presence of EB4. The EB4 was, however,
not detectable at 30 dpi, and only present in some (< 50% tested) plants at
21 dpi. These findings strongly suggest that deletion had occurred within the
host plants after 14 dpi. The sequence analyses in this section are summarized
in Table 3.Table 3
Summary of sequence analyses
carried out to confirm the presence of EB4 within the putative recombinant
CGMMV. (#A truncated EB4 sequence was detected at 30 dpi according
to Figure 7
for pR_P3U4 clone from nucleotide position
702 to 746.)
Presence of EB4 within putative chimeric
CGMMV genomeSampleRNA source used for RT-PCRTranscript-inoculatedVirus particlesplant total RNAtotal RNA14 dpi21 dpi30 dpiChimeric clonePresentPresent
in#Not
present(with pCG+FSRTRE<50% oftranscripts)plants tested


## 3.1. Infectivity of Constructed Transcription Clones—read-through Sequence Preference

In this study, the
chimeric CGMMV vectors pRT, pRG, and pCG-FSRTRE were constructed by inserting
the EB4 coding sequence to the end of the CGMMV CP ORF in plasmid pCGT7X. The
maps of these constructed clones are shown in Figure1, which indicates their
respective position of the primers during amplification and cloning procedures.
Maps of plasmids pCGT7X are
carrying the wild-type CGMMV, and pCANTAB 5E are
carrying EB4 with their respective
priming sites are also as indicated in Figure 1. The genome size of wild-type
CGMMV is approximately 6.4 kb (without the plasmid backbone), and the resulting
chimeric CGMMV genome would be approximately 6.5 kb in size and contains the EB4
and read-through sequences as well as inserted HindIII restriction recognition sites and additional nucleotides
enabling in-frame cloning. The pRT and pRG chimeric clones were constructed
with the read-through sequence of TMV (CAA-UAG-CAA-UUA). This leaky sequence
meets the minimal sequence requirement for effective read-through of the stop
codon [12] and had been used successfully in previous reports [14].The
templates for in vitro
transcription of these two clones were generated through LD-PCR (data not shown).
The resulting amplified products (~6.5 kb) consisted of a T7 promoter fused with
the chimeric CGMMV genome carrying EB4. Transcripts produced from the two
constructs were separately tested for infectivity by inoculating the host
plants. After repeated attempts, both the pRT- and pRG-generated transcripts
did not cause infection of the inoculated plants (Table1). There was no evidence
of virus genomic material in the inoculated plant tissues tested (data not
shown). It is speculated that the read-through sequence of TMV may not be suitable
for the CGMMV chimeric clones, hence contributing to the absence of infectious
virus transcripts. To overcome this possibility, another chimeric clone
(pCG+FSRTRE) was constructed using the read-through sequence AAA-UAG-CAA-UUA of
the wild-type CGMMV itself (Figure 1). The template for in vitro transcription,
based on the pCG+FSRTRE clone, was generated through LD-PCR. The in vitro transcription products
(Figure 2) were used in inoculation studies. The new leaves of plants
inoculated with pCG+FSRTRE-derived transcripts showed evidence of virus
infection with four out of eight plants showing a typical symptom of CGMMV
infection, which include the green mottle mosaic appearance on day 14 pI (Table 1 and Figure 3).
Infection could be detected through RT-PCR when total RNA of new noninoculated
leaf was used as template. Detection of the virus by RT-PCR (Figure 4) suggests
that the viruses had moved from the inoculated leaf to new leaves. This implies
that the chimeric virus pCG+FSRTRE carrying the read-through sequence from the CGMMV
genome (AAA-UAG-CAA-UUA) was
infectious and that the virus particles were able to assemble. The plants,
however, continued to grow without any further noticeable symptoms. Virus
particles were extracted from the infected plants and an aliquot was
electrophoretically separated on 15% SDS-PAGE (Figure 5) resulting in two distinct
suggesting that the virus population consisted of two species of coat proteins,
the EB4-CP fusion (larger in size) and the wild-type CGMMV CP (smaller in
size). The ratio of modified to unmodified CP was approximately 1:1.Table 1
Summary of experiments carried out to assess
the infectivity of different transcript clones. (T*otal RNA was extracted from
new noninoculated leaves (third new leaf) of transcript infected plants.)
Transcript  clonesInfectivity on inoculated plantsSymptom  appearance*Virus detected through RT-PCRpRT0/6HealthyNopRG0/6HealthyNopCG+FSRTRE4/8Green
mottle mosaicYesFigure 2
A representative gel image of pCG+FSRTRE-derived transcripts without DNase I
treatment electrophoresed after denaturation showing the expected transcript
size (6.5 kb). The band above the transcript band is the DNA template. Lane M:
RNA marker (Invitrogen, Calif, USA);
1, 2, 3: produced transcripts; M1: 1 kb DNA ladder (Promega, Wis,  USA); N: pCGT7X, ~9.0 kb; D: DNA template, ~6.5 kb. There were traces of DNA template detected in transcripts
without DNase I treatment.Figure 3
Symptom appearance at 14 day-postinoculation (dpi) on muskmelon plants caused
by pCG+FSRTRE-derived transcripts.Figure 4
Gel image showing presence of amplified band (~2.2 kb) after RT-PCR usingPstI sense and reverse E primer. Lane M:
1 kb DNA ladder (Promega, Wis,  USA);
1–8: total RNA extracted from new noninoculated leaves of pCG+FSRTRE-derived
transcripts from infected plants; N: total RNA of new noninoculated leaves of
wild-type transcript from infected plants (negative control).Figure 5
SDS-PAGE of purified chimeric virus coat protein (CP). Lane M: protein marker
(Fermantas); 1: chimeric virus; 2: wild-type CGMMV. It is clearly shown that
the chimeric virus consisted of two different CP species. The higher molecular
band shows the EB4-fusion CGMMV coat protein. The ratio of modified to
unmodified CP was approximately 1:1.


## 3.2. Effect of Pi:charge Value on Stability of Construct

Apart
from the usage of leaky UAG amber stop codons, it has been reported that pI:charge
can affect the production of  viable
recombinant virus [15]. The pI of the epitope is thought to be an important
factor as the hybrid coat protein pI:charge value can affect epitope
presentation. It was also reported that TMV was more tolerant to positively
charged epitopes on its surface. Thus, it was initially speculated that the
failure in expression of the foreign peptide was possibly due to the pI:charge
value of recombinant CGMMV CP which was different from the wild-type CGMMV CP
pI:charge value (Table 2).Table 2
Amino acid sequence, isoelectric
point (pI) and charge of wild type, constructed recombinant CGMMV coat protein
(CP), and the EB4 insert.
ClonesAmino acid sequenceMWpI of CPCharge(kDa)of CPEB4IIGVEPGQLKLNWFKKGSSIGQMIETTMRGAKRMAILGDT
AWDFG5.09.53+1.9MAYNPITPSKLIAFSASYVPVRTLLNFLVASQGTAFQTQAGRD17.35.08−3.1Wild-typeSFRESLSALPSSVVDINSRFPDAGFYAFLNGPVLRPIFVSLLSSTCGMMVDTRNRVIEVVDPSNPTTAESLNAVKRTDDASTAARAEIDNLIESISKGFDVYDRASFEAAFSVVWSEATTSKApRT*MAYNPITPSKLIAFSASYVPVRTLLNFLVASQGTAFQTQAGRD23.05.41−2.1SFRESLSALPSSVVDINSRFPDAGFYAFLNGPVLRPIFVSLLSSTDTRNRVIEVVDPSNPTTAESLNAVKRTDDASTAARAEIDNLIESISKGFDVYDRASFEAAFSVVWSEATTSKACQQLIIGVEPGQLKLNWFKKGSSIGQMIETTMRGAKRMAILGDTAWDFGEApRG*MAYNPITPSKLIAFSASYVPVRTLLNFLVASQGTAFQTQAGRD23.15.69−1.1SFRESLSALPSSVVDINSRFPDAGFYAFLNGPVLRPIFVSLLSSTDTRNRVIEVVDPSNPTTAESLNAVKRTDDASTAARAEIDNLIESISKGFDVYDRASFEAAFSVVWSEATTSKAWQQLIIGVEPGQLKLNWFKKGSSIGQMIETTMRGAKRMAILGDTAWDFGQApCG+FSRTRE*MAYNPITPSKLIAFSASYVPVRTLLNFLVASQGTAFQTQAGRD22.95.41−2.0SFRESLSALPSSVVDINSRFPDAGFYAFLNGPVLRPIFVSLLSSTDTRNRVIEVVDPSNPTTAESLNAVKRTDDASTAARAEIDNLIESISKGFDVYDRASFEAAFSVVWSEATTSKASGQLIIGVEPGQLKLNWFKKGSSIGQMIETTMRGAKRMAILGDTAWDFGEAW*ith
read-through leaky UAG amber stop codon.Table2 shows the isoelectric
point (pI) and charge of the wild-type CGMMV CP, the read-through recombinant
CGMMV CP, and the EB4 insert. The charge of the EB4 insert is positive and potentially
suitable for expression on the surface of the CGMMV CP [2]. Hence, the inserted
peptide is speculated to be expressed if the pI:charge value of modified virus
CP resembles the pI:charge value of unmodified virus CP. Earlier transcripts
(data not shown) generated from fusion clones without a read-through sequence,
where their pI values deviated significantly (>6.0) from the wild-type CP
(5.08), were not able to cause infection in inoculated plants leading to the
suggestion initially that pI:charge value played an important role in virus
particle assembly and infectivity. Thus, the pI value of the recombinant CP constructs
was adjusted to more
closely resemble the wild-type CP pI value by inserting the acidic amino acid
(glutamate) to the 3′ end of CP (Table 2). The experiments (Table 1), however, showed
that although the pI was still higher than that of the wild type (Table 2), the
construct pCG+FSRTRE remained infectious. This
implies that infectivity of the clones was not directly related to the
deviation in pI value with the wild-type virus CGMMV CP.


## 3.3. Deletion of Cloned Peptide Sequence

Sequencing
was carried out on RT-PCR-amplified products of viral RNA extracted from
putative chimeric virus particles at 30 days postinoculation (dpi) and total
plant RNA isolated from inoculated plant materials at 14 and 21 dpi to  confirm the expression of EB4. Sequence
analysis was done using BioEdit Sequence Alignment Editor Software (version 6.0.5)
(http://www.mbio.ncsu.edu/BioEdit/bioedit.html). The chimeric CP carried the
complete EB4 sequence within its genome at 14 dpi (Figure 6). However, the EB4
sequence appeared to be truncated at 21 dpi with an upstream portion of the EB4
sequence was not present. Only part of the downstream sequence of EB4 was
detected together with the 3′ untranslated region of the CGMMV. EB4 was totally
undetectable at 30 dpi. Interestingly, the 5′ end of introduced read-through
sequence (position 490 to 501) was retained in the genome. The introduced HindIII recognition site at EB4
downstream from position 639 to 644 was found to have been deleted after 30 dpi.Figure 6
Sequence analyses of RT-PCR-amplified products from putative chimeric
CGMMV RNA at different days
postinoculation (dpi). The sequenced alignment shows that EB4 sequence was
truncated and not complete after 21 dpi. The putative chimeric CGMMV produced
did not express the EB4 and its genome resembled the wild-type CGMMV.  Introduced read-through sequences and extra
codons are underlined. Complete EB4 sequence is aligned accordingly with the
other sequences.Even
though only a single band was visible on RT-PCR screening, it was suspected
that there could be products with similar sizes which could not be separated in
normal agarose gel electrophoresis, therefore, the RT-PCR products from the
chimeric CGMMV RNA at 30 dpi were cloned into pGEM-T Easy vector and subjected
to sequence analyses. The results (Figure7) confirmed the absence of the EB4
sequence, except for clone pR_P3U4 (from nucleotide position of 702 to 746),
where only part of the downstream sequence of the EB4 was detected together
with the 3′ untranslated region of the CGMMV. The 5′ end of introduced
read-through sequence of AAA-TAG-CAA-TTA (position 594 to 605) was retained
within the genome for all sequenced clones (Figure 7). The introduced HindIII site from position 742 to 747
was deleted for clones pR_P3U1, pR_P3U3, and pR_P3U11 after 30 dpi.Figure 7
Sequence alignments of cloned RT-PCR-amplified products from putative
chimeric CGMMV RNA at 30 dpi. Plasmid clones sent for sequence analysis were
pR_P3U1, pR_P3U3, pR_P3U4, and pR_P3U11. The sequence alignment shows that EB4
sequence was not within the insert of plasmids, except for a partial sequence
for pR_P3U4 clone. Only part of the downstream sequence of EB4 was detected
together with3′ untranslated region of CGMMV for pR_P3U4 clone, the rest were without EB4
sequence. Introduced read-through sequences and extra codons are underlined.
Complete EB4 sequence is aligned accordingly with the other sequences.The
chimeric CGMMV sequence analyses showed one common similarity (Figures6 and 7),
that is, that part of the read-through amber stops codon sequence, and additional nucleotides
“CC-AAA-TAG” were retained for all sequenced clones. This suggests that
deletion had occurred within the host plant. The sequence analysis also showed
that the EB4 was not fully expressed in the putative chimeric CGMMV. The
positive results from the initial RT-PCR screening of transcript-inoculated
plants at 14 dpi (Figure 4) suggest the presence of EB4. The EB4 was, however,
not detectable at 30 dpi, and only present in some (< 50% tested) plants at
21 dpi. These findings strongly suggest that deletion had occurred within the
host plants after 14 dpi. The sequence analyses in this section are summarized
in Table 3.Table 3
Summary of sequence analyses
carried out to confirm the presence of EB4 within the putative recombinant
CGMMV. (#A truncated EB4 sequence was detected at 30 dpi according
to Figure 7
for pR_P3U4 clone from nucleotide position
702 to 746.)
Presence of EB4 within putative chimeric
CGMMV genomeSampleRNA source used for RT-PCRTranscript-inoculatedVirus particlesplant total RNAtotal RNA14 dpi21 dpi30 dpiChimeric clonePresentPresent
in#Not
present(with pCG+FSRTRE<50% oftranscripts)plants tested


## 4. Discussion

Plant
virus vectors-based expression systems have been widely studied for their
development as antigen presentation systems as well as for the production of
pharmaceutically important peptides. The CGMMV has been previously shown to be
suitable for expression of foreign peptide [7]. In this study, CGMMV vector was
used to express a 45 amino
acid EB4 gene. The integration of the EB4 gene into the end of CGMMV
coat protein gene was done via a leaky UAG read-through sequence.Transcripts
generated from chimeric clones of pRT and pRG carrying CAA-UAG-CAA-UUA
read-through codon sequences were not infectious. This is possibly caused by
the failure of self assembly [16], and thus none of the inoculated plants was systemically
infected. The assembly of CGMMV into virus particle has been shown to be
essential for the viral movement through phloem [17], hence another chimeric
clone pCG+FSRTRE was constructed carrying read-through sequence (AAA-UAG-CAA-UUA)
from the wild-type CGMMV genome. The clone containing this read-through signal
was infectious and produced chimeric CGMMV (Table 1 and Figures 4 and 5). It is,
thus, postulated that there is a read-through signal preference between different
species of tobamoviruses, in this case between TMV and CGMMV causing possibly
viral particle assembly failure. It has also been shown that KGMMV, the
tobamovirus, which has the closest genome similarity to CGMMV [13] also
utilizes the same CGMMV read-through signal reaffirming the differences between
the tobamoviruses. Additionally, unlike other plant virus vectors [15, 18],
this study reaffirms that with CGMMV pI deviation did not appear to be a factor
affecting infectivity [7].The
chimeric (carrying the EB4) and putative wild-type CGMMV were shown to coexist
in the virus population of the infected plants (Figure5). Previous reports
show that the efficiencies of the leaky UAG codon varied from 0.5% to 5% so
that the ratio of modified to unmodified CP would be between 1:200 and 1:20 [12, 19]. However, in this study, relatively high levels of chimeric coat protein
was observed (Figure 5) giving a ratio of modified to unmodified CP of
approximately 1:1. It has been suggested that muskmelon host plant could be
producing higher levels of translation nonsense suppressor tRNA making the
application of the translation read-through signal favorable in this host [7].Due to their
relatively higher rate of mutation during replication, RNA viruses are evolving
rapidly and this is the basis of their ubiquity and adaptability [20, 21]. In this study, it is shown that the EB4 gene sequence
carried by the chimeric CGMMV was systemically removed during the infection
process. The order of the removal of the transgene was speculated to be the 5′
to 3′ direction (Figures 6 and 7). This is further supported by the detection
of two additional nucleotides together with the read-through sequence
“CC-AAA-TAG” downstream from the CGMMV CP ORF. This report shows the temporal
in-host truncation of the transgene from a chimeric virus in a natural host.
Recent report has shown truncation occurring in transgenic plants expressing
the same or similar transgenes as the chimeric virus [22] suggesting targeting
by a resistance mechanism or competition with the parental virus as the
mechanism involved. The exact mechanism of truncation of the transgene in our
study is less clear as a previous study using the same vector and host with a
different transgene did not exhibit the same instability [7]. The larger size
of the EB4 peptide in comparison to the Hepatitis B epitope, however, suggests
that the truncation mechanism or transgene recognition by the virus was size
dependent.In summary, we have
shown that CGMMV has a read-through codon preference and that the read-through
codon for TMV was shown to be not efficient, as the chimeric CGMMV transcripts
utilizing this signal were not infectious. The reported limitation of low-modified
coat protein yield of this type of read-through transient expression system
appears to have been overcome as relatively equal yield of chimeric and wild-type
CGMMV coat protein were produced. This report also provides a rational
harvesting timeline for the chimeric virus making this system exploitable for implementation in a
plantation scale in the future. It can be suggested that once host plants are
infected with the chimeric virus carrying the inserted foreign peptides, the
optimum harvesting time would be at around 14 dpi or not more than 20 dpi in
order to obtain maximum yield of the full-length transgene. Growth of the
infected plants for longer periods to obtain higher yields of the chimeric
virus may induce unwanted transgene deletions. This and other factors described
earlier should be relevant information for the further development of CGMMV or
other plant viruses as vectors for medically important peptides such as for
dengue (this study) and Hepatitis B [7] viral antigens.



---

*Source: 781712-2009-03-22.xml*