Download P and M gene junction is the optimal insertion site in Newcastle

Journal of General Virology (2015), 96, 40–45
Short
Communication
DOI 10.1099/vir.0.068437-0
P and M gene junction is the optimal insertion site in
Newcastle disease virus vaccine vector for foreign
gene expression
Wei Zhao,1,23 Zhenyu Zhang,1,33 Laszlo Zsak1 and Qingzhong Yu1
Correspondence
1
Qingzhong Yu
[email protected]
2
Southeast Poultry Research Laboratory, Agricultural Research Services,
United States Department of Agriculture, 934 College Station Road, Athens, GA 30605, USA
Beijing Centre for Disease Control and Prevention, Beijing 100013, PR China
3
College of Life Sciences, Northeast Agricultural University, Harbin, Heilongjiang 150030,
PR China
Received 27 May 2014
Accepted 28 September 2014
Newcastle disease virus (NDV) has been developed as a vector for vaccine and gene therapy
purposes. However, the optimal insertion site for foreign gene expression remained to be
determined. In the present study, we inserted the green fluorescence protein (GFP) gene into five
different intergenic regions of the enterotropic NDV VG/GA vaccine strain using reverse genetics
technology. The rescued recombinant viruses retained lentogenic pathotype and displayed
delayed growth dynamics, particularly when the GFP gene was inserted between the NP and P
genes of the virus. The GFP mRNA level was most abundant when the gene was inserted closer
to the 39 end and gradually decreased as the gene was inserted closer to the 59 end.
Measurement of the GFP fluorescence intensity in recombinant virus-infected cells demonstrated
that the non-coding region between the P and M genes is the optimal insertion site for foreign
gene expression in the VG/GA vaccine vector.
The Villegas-Glisson/University of Georgia (VG/GA) strain
of Newcastle disease virus (NDV) is a commonly used
vaccine to protect chickens from Newcastle disease (ND),
one of the most important infectious diseases of poultry
due to the potential for devastating losses (Miller & Guus,
2013). The VG/GA strain preferentially replicates in the
intestinal tract of chickens and induces local mucosal immunoresponses (Perozo et al., 2008a). Vaccination of chickens
with the VG/GA vaccine provided 100 % protection of
mortality to chickens against a velogenic viscerotropic
NDV challenge (Beard et al., 1993; Perozo et al., 2008b).
Therefore, the VG/GA strain is considered as a potential
enterotropic vaccine vector to deliver antigens of poultry
enteric viruses as bivalent vaccines.
NDV is a non-segmented, single-stranded negative sense
RNA virus, belonging to the genus Avulavirus within the
subfamily Paramyxovirinae of the family Paramyxoviridae
(Lamb et al., 2005). The NDV genome is approximately
15.2 kb in length and consists of six genes flanked by a 39
leader and 59 trailer in the order 39-NP (nucleocapsid
protein)-P (phosphoprotein)-M (matrix)-F (fusion)-HN
(haemagglutinin-neuraminidase)-L (large polymerase)-59
(de Leeuw & Peeters, 1999; Peeters et al., 2000). The RNA
genome together with NP, P and L proteins forms the
3These authors contributed equally to this work
40
ribonucleoprotein complex (RNP), which serves as the active
template for transcription and replication of the viral
genome (Peeters et al., 1999).
During the past decade, several strains of NDV have been
developed as vectors using reverse genetics technology to
express foreign antigens for vaccine or gene therapy purposes
(Bukreyev & Collins, 2008; Huang et al., 2003; Schirrmacher
& Fournier, 2009; Vigil et al., 2008; Zhao & Peeters, 2003).
The foreign genes are usually inserted into a non-coding
region at different intergenic regions of the NDV genome,
and evaluation of these vaccine candidates in clinical
trials revealed different levels of protection against targeted
pathogen challenge (Bukreyev et al., 2005; DiNapoli et al.,
2007; Hu et al., 2011; Huang et al., 2004; Park et al., 2006; Yu
et al., 2013; Zhao et al., 2014). Although the immune
response to vaccination is influenced by many factors, the
expression level of foreign genes is undoubtedly the most
important one. However, for NDV, the optimal insertion site
for foreign gene expression is still unknown.
In the present study, we generated an infectious clone of
the enterotropic NDV VG/GA strain as a vaccine vector
and inserted the green fluorescence protein (GFP) gene
into five different intergenic regions of the infectious clone.
Evaluation of the pathogenicity, growth kinetics, and GFP
expression of these rescued recombinant viruses allowed us
to better understand the NDV transcription mechanism
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068437
Printed in Great Britain
Optimal insertion site in NDV for foreign gene expression
and to determine the optimal insertion site for foreign gene
expression by the NDV VG/GA vaccine vector.
A full-length cDNA clone (FLC), pFLC-VG/GA, encoding
the complete antisense genome of the NDV VG/GA strain
was generated through three steps of cloning using an InFusion PCR Cloning kit (Clontech) and a similar cloning
approach as described previously (Hu et al., 2011; Zhao
et al., 2014). The GFP gene ORF together with the NDV
transcriptional signals derived from the P-M gene junction
region was successfully amplified, and inserted into the NP/
P, P/M, M/F, F/HN or HN/L non-coding region in the
VG/GA FLC respectively, resulting in five VG/GA-GFP
recombinant cDNA clones (Fig. 1a). After co-transfection
of an FLC and the supporting plasmids in HEp-2 cells and
subsequent amplification in SPF chicken embryonated eggs
as described previously (Zhao et al., 2014), a NDV VG/GA
virus and five VG/GA-GFP recombinant viruses were rescued
and propagated. Sequencing of the RT-PCR products of the
viral genomes verified the GFP insertions in the VG/GA
genome, and confirmed the nucleotide sequence fidelity of
the rescued viruses.
To evaluate the influence of the inserted GFP gene on NDV
pathogenicity and replication, the rescued viruses were
examined in vitro and in vivo by conducting virus titration,
mean death time (MDT) and intracerebral pathogenicity
index (ICPI) assays (Alexander, 1998). As shown in Table
1, the rescued viruses appeared to be slightly attenuated in
embryonated eggs and day-old chickens, with a longer
MDT (.120 h) and a lower ICPI (0.0) than the parental
VG/GA strain. The titres of the rescued viruses grown in
embryonated eggs or in DF-1 cells, measured by EID50,
TCID50 and HA, were one or two logs lower but were
comparable to that of the parental VG/GA strain (Table 1).
As shown in Fig. 1b, the growth kinetics of rVG/GA were
slightly delayed when compared with the parental virus
VG/GA; however, after 48 h, the rescued virus rVG/GA
displayed similar kinetics and magnitude of the replication.
In contrast, the kinetics of replication of the five recombinant viruses showed that the insertion of the GFP gene
resulted in a delay in the onset of replication, and their
titres were about one log lower than that of the rVG/GA
virus even after 48 h of infection (Fig. 1b). Also, it is
interesting to note that the recombinant viruses rVG/
GA-GFP-NP/P, rVG/GA-GFP-P/M, rVG/GA-GFP-M/F,
rVG/GA-GFP-F/HN and rVG/GA-GFP-HN/L were in an
ascending order of titres at most of the time points, which
indicated that the delayed growth kinetics that resulted
from the GFP gene insertion was most prominent when it
was located at intergenic regions closer to the 39 end of the
viral genome.
The cytopathic effects (CPE) and expression of the GFP in
DF-1 cells infected with the rescued viruses were examined
by fluorescence microscopy as described previously (Hu
et al., 2011). After 48 h of infection, green fluorescence was
observed in the recombinant virus-infected DF-1 cells (Fig.
1c). Interestingly, the levels of CPEs induced by the
http://vir.sgmjournals.org
recombinant viruses appeared to correlate with the
position of GFP insertion in the viral genome. There was
a trend of increasing CPEs in DF-1 cells induced by the
recombinant viruses in the order from rVG/GA-GFP-NP/
P, rVG/GA-GFP-P/M, rVG/GA-GFP-M/F, rVG/GA-GFPF/HN to rVG/GA-GFP-HN/L. This finding is in accordance with the result of the growth kinetics of the
recombinant viruses (Fig. 1b).
To determine the effect of different insertion sites on GFP
gene transcription, the GFP mRNAs in the virus infected
DF-1 cells were measured by reverse transcription and
quantitative real-time PCR (qPCR) using a Power SYBR
Green PCR Master Mix kit (Applied Biosystems). Comparison of the GFP mRNAs in virus-infected cells over a
non-infected cell control was normalized to the NP gene
and calculated by the comparative threshold cycle (DDCt)
method (Le Nouën et al., 2009). As shown in Fig. 2a, an
apparent correlation between the GFP transcription level
and the location of the GFP gene in the viral genome was
observed, such that the GFP mRNA abundance was
gradually decreased regarding to the insertion position
toward the 59 end of the viral genome. Compared with
the GFP mRNA abundance at NP-P junction, the GFP
gene transcription was decreased by 30 % at the P-M
junction, 28 % at the M-F junction, 8 % at the F-HN
junction and 24 % at the HN-L junction. This transcription pattern proved the theoretical hypothesis that there is
a gradient of decreasing mRNA abundance according to
the position of the gene relative to the 39 end of the NDV
genome.
The levels of GFP production of recombinant viruses were
quantified by measuring the green fluorescence intensity in
the virus infected DF-1 cells at every 12 h post-infection
using Fluorescence Microplate Reader (BioTek, FLx800). It
appeared that during the first 36 h of infection there
was little GFP expression from the cells infected with the
recombinant viruses as their fluorescence intensities were
virtually the same as that from the parental rVG/GA virusinfected cells. However, beyond 48 h post-infection, the
GFP fluorescence intensity in rVG/GA-GFP-P/M infected
cells increased rapidly as the infection progressed, and
reached a highest level at 84 h post-infection among the
different recombinant virus-infected cells (Fig. 2b). The
increase rates of GFP fluorescence intensity in other
recombinant virus-infected cells were somewhat lower
than that in rVG/GA-GFP-P/M infected cells, with 83.1 %,
75.5 % and 48.4 % relative GFP fluorescence intensity for
rVG/GA-GFP-M/F, rVG/GA-GFP-NP/P,and rVG/GA-GFPF/HN infected cells at 84 h post-infection, respectively.
It is widely accepted that paramyxoviruses synthesize and
transcribe the genes into mRNAs in a sequential and polar
manner by a stop-and-restart mechanism at each gene junction; therefore, the promoter-proximal genes are expressed
more efficiently than promoter-distal ones (Lamb & Parks,
2007). However, this assumption has so far been neither
proved nor disproved for NDV. In this study, we
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41
W. Zhao and others
(a)
T7 Le
pFLC-VG/GA
NP
P
M
F
HN
Tr
L
pVG/GFP
GFP
PCR
GFP
(b)
7
6
pVG/GA-GFP-NP/P:
T7 Le
CTACATTAAGGA
NP
GFP
TTAAGAAAAAA
Gene end
pVG/GA-GFP-P/M:
pVG/GA-GFP-M/F:
pVG/GA-GFP-F/HN:
pVG/GA-GFP-HN/L:
T7 Le
T7 Le
T7 Le
T7 Le
(c)
rVG/GA-GFP-NP/P
P
T
HN
GFP
NP
NP
P
NP
P
Tr
L
GCCACC ATGGTGAGCTT...TGA
Gene start
P
P
F
ACGGGTAGAA ......
NP
NP
M
Kozak
GFP
Tr
M
F
HN
L
M
GFP
F
HN
LL
M
F
GFP
HN
L
M
F
rVG/GA-GFP-P/M
HN
GFP
rVG/GA-GFP-M/F
log10 (TCID50 ml–1)
In-Fusion PCR cloning
5
4
VG/GA
rVGGA
rVG/GA-GFP-NP/P
rVG/GA-GFP-P/M
rVG/GA-GFP-M/F
rVG/GA-GFP-F/HN
rVG/GA-GFP-HN/L
3
2
Tr
1
Tr
0
Tr
12
24
36
48
60
72
84
Time post-infection (h)
L
rVG/GA-GFP-F/HN
rVG/GA-GFP-HN/L
Mock
Fig. 1. (a) Schematic representation of NDV VG/GA strain full-length cDNA clones containing the GFP gene at different
intergenic regions. The GFP transcription cassette which contains the NDV trans-acting signal sequences (gene end, intergenic
region and gene start), Kozak sequence and GFP open reading frame, was amplified from the subclone pVG/GFP, and cloned
into the different intergenic regions (NP/P, P/M, M/F, F/HN and HN/L) of the VG/GA full-length cDNA clone using an In-Fusion
PCR Cloning kit (Clontech). The sequences of all primers used in construction of the full-length cDNA clones are available upon
request. The NDV gene end and gene start signal sequences, the Kozak sequence and GFP sequences are boxed or
underlined. The direction of the T7 promoter is indicated by a bold black arrow. (b) Growth dynamics of the recombinant viruses.
DF-1 cells were infected with the indicated NDV viruses at 0.01 m.o.i. Every 12 h post-infection, the infected cells were
harvested. Virus titres were measured by TCID50 titration on DF-1 cells for each time point in triplicates from two independent
experiments, and expressed in mean log10 TCID50 ml”1 with standard deviation (error bars). (c) The cytopathic effects and
expression of GFP by the rescued viruses. DF-1 cells in a 12-well plate were infected with the recombinant viruses at 0.1 m.o.i.
At 48 h post-infection, the cytopathic effects and the fluorescence of the infected cells were examined and digitally
photographed by fluorescence microscopy at ¾100 magnification (Nikon, Eclipse Ti).
quantitatively measured the levels of GFP mRNAs from the
cells infected with the recombinant NDVs vectoring the GFP
gene at different intergenic regions. Our results demonstrated that there was a gradient of GFP mRNA expression
from the 39 end to 59 end of the viral genome with the
transcription level decreased by about 8 % to 30 % at each
subsequent gene junction compared to the GFP mRNA
abundance at NP-P junction. To the best of our knowledge,
42
it is the first time that this sequential and polar transcription
hypothesis has been experimentally proved on NDV.
According to the sequential transcription theory, the best
position for foreign gene expression would be the closest to
the 39 end of NDV genome. On the other hand, the insertion of a foreign gene into a promoter-proximal position
might interfere with NDV replication more seriously than a
promoter-distal position, resulting in lower levels of foreign
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Journal of General Virology 96
Optimal insertion site in NDV for foreign gene expression
Table 1. Biological assessments of the NDV recombinant
viruses
MDT* (h) ICPID
VG/GA
rVG/GA
rVG/GAGFP-NP/P
rVG/GAGFP-P/M
rVG/GAGFP-M/F
rVG/GAGFP-F/HN
rVG/GAGFP-HN/L
HAd
12
EID50§
TCID50
9
110
.120
.120
0.08
0
0
2
212
211
1.47610 1.766108
1.766109 1.766107
3.126108 3.126107
.120
0
212
1.326109 1.766107
.120
0
210
3.786108 9.886107
.120
0
210
2.326108 3.126107
.120
0
210
3.786108 1.766107
*Mean death time assay in embryonating eggs.
DIntracerebral pathogenicity index assay in day-old chickens.
dHaemagglutination assay.
§The 50 % egg infective dose assay in embryonated eggs.
The 50 % tissue infectious dose assay on DF-1 cells.
gene expression (Carnero et al., 2009). Therefore, the
genomic location of foreign genes, replication of the virus,
and the abundance of foreign gene expression were all kept
in a subtle balance.
Fold difference relative to mock
(a) 30.0
(b) 120
25.0
20.0
15.0
10.0
5.0
100
rVG/GA
rVG/GA-GFP-NP/P
rVG/GA-GFP-P/M
rVG/GA-GFP-M/F
rVG/GA-GFP-F/HN
rVG/GA-GFP-HN/L
80
60
40
20
-H
FP
-F
G
FP
A-
G
0
0
12
rV
G
/G
A/G
G
rV
N
/L
N
/H
/F
-M
FP
G
A-
rV
G
/G
A/G
G
rV
rV
G
/G
AG
G
FP
FP
-N
P/
-P
/M
P
0.0
GFP fluorescence intensity (%)
Virus
Previously, Zhao & Peeters (2003) and Ramp et al. (2011)
inserted foreign genes into different positions of NDV
genome, and showed that the foreign gene expression levels
differed only moderately in various positions. However, it
is important to note that the positions of each insertion in
the above studies varied relative to the gene start of the
downstream genes of NDV genome, which may influence
the insertion transcription efficiency caused by the
variation of virus genome lengths and sequences
(Skiadopoulos et al., 2000). To avoid any potential effects
on transcription efficiency caused by the variation of virus
genome lengths and sequences, we inserted the GFP gene
cassette at 40 nt upstream of the GE sequences of the viral
genes. Thus, all of the recombinant viruses possess an
identical independent GFP transcription unit at the exact
location relative to the gene start of the downstream genes.
Quantitative measurements of the GFP fluorescence
intensity in recombinant virus-infected cells demonstrated
a gradient abundance of expressed GFP in the following
the order: rVG/GA-GFP-P/M.rVG/GA-GFP-M/F.rVG/
GA-GFP-NP/P.rVG/GA-GFP-F/HN.rVG/GA-GFP-HN/L.
Clearly, the level of GFP expression strongly correlated with
the gene order of the NDV genome, except the insertion
between NP and P genes. This disproportionate effect
occurred presumably due to the GFP insertion that altered
the ratio between the NP and P proteins. It has been
demonstrated in vesicular stomatitis virus that a specific
24
36
48
60
Time post-infection (h)
72
84
Fig. 2. (a) Quantification of the GFP mRNA transcription. DF-1 cells in 6-well plate were infected with the indicated NDV
viruses at 0.1 m.o.i. At 48 h post-infection, the total RNAs were extracted by KingFisher automatic purification instrument
(Ambion) and MagMAX-96 Total RNA Isolation kit (Ambion). The RNAs were treated with DNase I to remove residual genomic
DNA and reverse transcribed with Oligo (dT) primer. The prepared cDNAs were used in quantitative real-time PCR (qPCR) for
quantification of the GFP mRNA transcription. The NP gene transcripts from the same samples were quantified and used for
internal normalization. qPCR results were expressed as fold difference with SD (error bars) relative to that of cell control using
the comparative threshold cycle (DDCt) method. (b) Measurement of GFP fluorescence intensity. DF-1 cells were grown in a
96-well plate and infected with the indicated NDV viruses at 0.1 m.o.i. Every 12 h post-infection, GFP fluorescence intensities
were measured by using Fluorescence Microplate Reader (BioTek, FLx800) in triplicate wells from two independent
experiments. The results were expressed as percentages of the mean GFP fluorescence intensities with SD (error bars) relative
to the highest intensity, which was set as 100 %.
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W. Zhao and others
ratio of the NP to P protein is optimal for supporting
efficient replication and encapsidation (Pattnaik & Wertz,
1990; Wertz et al., 2002). This disproportionate effect was
also observed on NDV by other researchers (Carnero et al.,
2009; Zhao & Peeters, 2003), suggesting that the non-coding
region between the NP and P proteins is not an optimal
insertion site for foreign gene expression.
Despite the fact that foreign gene insertion between P and
M genes affects several downstream virus genes preceded
only by the NP and P junction, the replication of rVG/GAGFP-P/M virus was not affected as much as the rVG/GAGFP-NP/P virus. In addition, the foreign gene inserted in
the non-coding region between the P and M genes expressed
the highest level of the foreign gene product among the
different recombinant virus infected cells. Thus, it is reasonable to conclude that the junction between P and M genes
contains an optimal insertion site for foreign gene expression
in the VG/GA NDV vaccine vector.
In summary, in the present study we have successfully
constructed an infectious clone of the enterotropic NDV
VG/GA vaccine strain and engineered five recombinant
viruses vectoring GFP at different intergenic regions (NP/P,
P/M, M/F, F/HN and HN/L). Quantification of GFP mRNAs
expressed from different recombinant viruses confirmed the
theoretical hypothesis of the gradient mRNA expression of
NDV. Measurement of the GFP fluorescence intensity in
recombinant virus-infected cells demonstrated that the noncoding region between the P and M genes was the optimal
insertion site for foreign gene expression in the VG/GA
vaccine vector. Overall, data suggest that the NDV VG/GA
vaccine strain is a potential enterotropic live vaccine vector
that can be used to deliver antigens of poultry enteric viruses
as bivalent vaccines.
Acknowledgements
The authors wish to thank Xiuqin Xia and Fenglan Li for excellent
technical assistance, Patti Miller for critical reading of the manuscript,
and the SEPRL sequencing facility personnel for nucleotide sequencing. W. Z. and Z. Z. were sponsored by a scholarship from China
Scholarship Council. This research was supported by USDA, ARS
CRIS project 6612-32000-067-00D.
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