Inhibition of Replication of Goose
Paramyxovirus SF02 by Hammerhead Ribozyme Targeting to the SF02 F mRNA in
Chicken Embryo Fibroblasts

ZOU Jian, GONG Zu-Xun*

(
Key laboratory of Proteomics, Institute of Biochemistry and Cell Biology,
Shanghai Institutes for Biological Sciences, the Chinese Academy of Sciences,
Shanghai 200031, China)

Abstract       
A Hammerhead ribozyme RzF598 and its dysfunctional mutant dRzF598 targeting to
the F gene of goose paramyxovirus SF02 have been designed. The transgenic
plasmids pcDNA-RzF598 and pcDNA-dRzF598 were constructed by inserting ribozyme
genes into eukaryotic expression vector pcDNA3. The plasmid pcDNA3 that lacks
full ribozyme gene was used as a control. Plasmids pcDNA-RzF598, pcDNA-dRzF598
and pcDNA3 were transfected into chicken embryo fibroblasts (CEFs). The
concentration of virus released by infected CEFs and the survival percentages
of CEFs were identified. The results indicated that RzF598 successfully
suppressed the replication of SF02 in CEFs. Survival percentage of CEFs being
transfected with pcDNA-RzF598 and infected SF02 was up to 78.8%, while the
survival percentages of untransfected CEFs and CEFs transfected with pcDNA3
after infection with SF02 were only about 5%.

Key words    
hammerhead ribozyme; Goose paramyxovirus; NDV; chicken embryo fibroblasts

Newcastle disease, caused by Newcastle disease virus
(NDV), is one of the most serious diseases in poultry that has caused heavy
losses in many countries. The virus is the unique member of avian paramyxovirus
serotype-1 (APMV-1)[1]. It belongs to the genus Rubulavirus,
Paramyxoviridae[2]; however, recent evidences have suggested that it should be
assigned to a new genus within the subfamily Paramyxovirinae[3－5].

Goose
paramyxovirus (designated as GPMV in this paper) disease is highly infectious
that has caused frequent outbreak since 1997 in China. The incidence and
mortality of disease are high in fowls. In 1999, an acute and virulent disease
occurred in goose flocks in Shanghai. The virus isolate was designated as SF02
and identified as the causal agent of the outbreaks[6]. Liu et al.[7] and Chen
et al.[8] also isolated goose paramyxovirus from infected geese in other
regions in China. GPMV was identified to be a member of avian paramyxovirus-1
(APMV-1) by genomic and serotype analyses. It may be an aberrant strain of NDV.
There are significant differences between GPMV and NDV in their host range. The
NDV is pathogenic only to fowls, such as chickens and pigeons, whereas SF02 is
highly pathogenic to fowls and waterfowls, including chicken, pigeon,
partridges, geese and ducks.

The SF02 genome
is a nonsegmented single-strand negative RNA. Both SF02 and NDV contain 6 ORFs
in the same order of NP, P, M, F, HN and L, and each of which encodes the
nucleocapsid protein (NP), phosphoprotein (P), matrix protein (M), fusion
protein (F), haemagglutinin-neuraminidase (HN), and large polymerase protein
(L). The genomes of SF02 and NDV strains are of 83% identities and the 6 ORFs
81.9%－86.1%. The
NDV isolates are classified as highly virulent (velogenic), intermediate
(mesogenic), or nonvirulent (lentogenic) according to their pathogenicity and
virulence to chickens. The amino acid sequence of the protease cleavage site of
fusion protein is essential for NDV pathogenicity[9－12]. Fusion protein is synthesized
as an inactive precursor glycoprotein (F0), and must be cleaved proteolytically
to form a disulfide-linked heterodimer of F1 and F2 in order to direct the
membrane fusion.

Ribozyme is an
efficient agent in knocking down and blocking gene expression in vivo, and
hopefully, both single-strand or double-strand RNA viruses could be its ideal
targets. In this study, a ribozyme designated as RzF598 targeting to the F gene
has been constructed. The mRNA of F gene (F mRNA) of GPMV SF02 was successfully
cleaved with RzF598 in vitro and virus replication was efficiently suppressed
in chicken embryo fibroblasts (CEFs). In order to assess the input of antisense
effect in inhibition of virus replication by the ribozyme, a dysfunctional
ribozyme, dRzF598 with a nucleotide substitution in the catalytic domain of
RzF598, was synthesized and was inactive in RNA cleavage in vitro.

1    Materials
and Methods

1.1   Cells and virus

CEFs were grown in Dulbecco’s modified Eagle’s
medium (DMEM) (Invitrogen)supplemented with 5% fetal bovine serum (Invitrogen)
with penicillin (100 u/ml) and streptomycin (100 mg/L). The viruses SF02 and
F48E9 (a velogenic NDV strain) were propagated in 10-day-old special pathogen
free (SPF) chicken embryos. The virus-containing allantoic was collected and
stored at －80 ℃
for use.

1.2   Synthesis of ribozyme genes: RzF598 and
dRzF598

The GUA site at position 598 in F mRNA of SF02 was
chosen as ribozyme target site. An oligodeoxyribonucleotide, RzF598 was
designed by program PCFOLD, ST and RNASE (Fig.1) and synthesized. For
assessment of the antisense effect, a mutant oligodeoxyribonucleotide, dRzF598
containing a single nucleotide substitution (G/A) in the catalytic domain of
RzF598 was synthesized (Fig.1).

Fig.1      
Sequences and secondary structures of ribozymes, and the cleavage site of
RzF598 against F mRNA

The catalytic core of RzF598 is flanked by
arm sequences complementary to F genes. The poly(A) signal (UAUAAAAA) is
underlined and the arrow indicates the cleavage site. The nucleotide
substitution in the catalytic domain disabling the function of ribozyme is
shown by an asterisk.

1.3   Plasmids
construction

The
oligodeoxyribonucleotides were cloned into plasmid pRG523[13] between
self-cleavage 5′ cis and 3′ cis structures. The resulting plasmids were
designated as pRzF598 and pdRzF598 respectively. Ribozyme genes containing 5′
cis and 3′ cis were transferred to eukaryotic expression plasmid pcDNA3
(Invitrogen), resulting in two plasmids were designated as pcDNA-RzF598 and
pcDNA-dRzF598. The plasmid pcDNA3 that lacks the whole ribozyme sequence was
set as the control.

The cleavage efficiency of ribozymes
against F mRNA in vitro was analyzed. F gene fragments of SF02 and F48E9
containing 440 bp (461－ 900 nt) were cloned into transcriptional plasmid pSPT18 (Boeringer
Mannheim Gmbh) using primer 1 (5′-GGCTTAAGGAGAGCATTGCTGCA-3′) and primer 2
(5′-GGCACGCATATTATTTAAG-3′). The resulting plasmids were designated as pSF02F
and pF48E9F, respectively.

1.4  
Cleavage of F gene fragment with ribozymes in vitro

Plasmids
pRzF598, pdRzF598, pSF02F and pF48E9F were linearized and transcribed by T7 RNA
polymerase according to protocol of the supplier (Promega). The cleavage
reaction was conducted at 37 ℃ for 30 min in 10 μL reaction solution containing
50 mmol/L Tris・HCL (pH 7.5), 2 mmol/L EDTA, 20 mmol/L NaCl and 10 mmol/L MgCl2
and ended by adding 1 μL 5 mol/L EDTA. The products were identified by 10% PAGE
containing 7 mol/L urea.

1.5   Measurement
of inhibition of virus replication by ribozymes in CEFs

CEFs were
incubated in 24-well plate at 5 × 10⁵ per well. Full-confluent (90%－95% of the dish surface) monolayers
were used for transfection with 2 μL Lipofectamine 2000 reagent (Invitrogen). 1
μg each of pcDNA-RzF598, pcDNA-dRzF598 and pcDNA3 plasmids were used for
delivering according to protocol of the supplier. Selection for neomycin
resistance was done with G418 at a concentration of 200 mg/L DMEM at 24 h post
transfection. 48 h after selection, CEFs were infected with 1 or 10 μL
SF02-containing or F48E9-containing allantoic. Cells were washed twice by PBS.
60 min after virus adsorption, the maintenance medium (DMEM containing 2% fetal
bovine serum) was added and the cells were incubated at 37 ℃. The medium
containing dead cells and virus was collected at 84 h after infection. Number
of survival cells attached to the wells was counted by MTT assay according to
the manufacturer’s instructions (Sigma). A total of 3 wells for each sample
were accomplished and the average MTT value was calculated.

1.6   RT-PCR
determination of the virus titration in cell medium

The CEF mediums
of infected with 10 μL virus-containing allantoic were collected. Total RNA of
15 μL medium was extracted with Trizol (Invitrogen) and used in RT-PCR with
primer 1 and primer 2. RT-PCR products were detected by electrophoresis in 1.0%
agarose gel.

2    Results

2.1   Constructions
of plasmids

Sequence analysis
showed that all plasmids, pRzF598, pdRzF598, pcDNA-RzF598, pcDNA-dRzF598,
pSF02F and pF48E9F, were constructed correctly.

2.2   Cleavage
of F gene fragment with ribozymes in vitro

The results
indicated that RzF598 cleaved efficiently the transcripted 440 nt F gene
fragment of SF02 into two fragments of 138 nt and 302 nt as designed. However,
RzF598 did not cleave F gene fragment of NDV strain F48E9. The dRzF598 did not
cleave F gene fragments of either SF02 or F48E9 (Fig.2).

Fig.2       Cleavage of F mRNA with RzF598 and dRzF598 in vitro

1, 440 nt fragment of F mRNA of SF02; 2, F
mRNA fragment of SF02 cleaved with RzF598; 3, F mRNA fragment of SF02 cleaved with
dRzF598; 4, 440 nt fragment of F mRNA of F48E9; 5, F mRNA fragment of F48E9
cleaved with RzF598; 6, F mRNA fragment of F48E9 cleaved with dRzF598.

2.3   Determination
of virus concentration released into medium by CEFs with RT-PCR

RT-PCR results
showed that the concentration of virus released by CEFs being transfected with
pcDNA-RzF598 and infected with SF02 was the lowest. The concentration of virus
released by CEFs infected with SF02 or F48E9 but without transfection, and by
CEFs being transfected with pcDNA3 and infected with SF02 or F48E9 were almost
at the same level and were the highest. The concentration of virus released by
CEFs being transfected with pcDNA-RzF598 and infected with F48E9, and by CEFs
being transfected with pcDNA-dRzF598 and infected with SF02 or F48E9 were also
at the same level (Fig.3).

Fig.3       Determination of the concentration of virus released into medium by
CEFs with RT-PCR

M, marker; 1, CEFs; 2, CEFs infected with
SF02 but not transfected; 3, CEFs infected with F48E9 but not transfected; 4,
CEFs being transfected with pcDNA-RzF598 but not infected; 5, CEFs being
transfected with pcDNA-RzF598 and infected with SF02; 6, CEFs being transfected
with pcDNA-RzF598 and infected with F48E9; 7, CEFs being transfected with
pcDNA-dRzF598 but not infected; 8, CEFs being transfected with pcDNA-dRzF598
and infected with SF02; 9, CEFs being transfected with pcDNA-dRzF598 and
infected with F48E9; 10, CEFs being transfected with pcDNA3 but not infected;
11, CEFs being transfected with pcDNA3 and infected with SF02; 12, CEFs being
transfected with pcDNA3 and infected with F48E9.

2.4   Survival
percentages of CEFs being transfected with ribozymes and infected with viruses

MTT assays
revealed that RzF598 remarkably inhibited the replication of SF02 in CEFs. The
maximal survival percentages of CEFs was 89.3% and 78.8%when CEFs were infected
with 1 μL and 10 μL SF02-containing allantoic respectively. CEFs trans-fected
with pcDNA-RzF598 showed a little resistance to F48E9. CEFs transfected with
pcDNA-dRzF598 also showed resistance to viruses in some degree. RzF598 is
dysfunctional for F48E9, and dRzF598 is also dysfunctional for SF02 and F48E9.
It is noteworthy that the survival percentages of CEFs expressing the
dysfunctional ribozyme infected with 1 μL virus-containing allantoic was much
higher than ones infected 10 μL virus-containing allantoic. The results of CEFs
survival percentages are summarized in Fig.4 and Table 1.

Fig.4 Inhibition efficiency of replication
of viruses by ribozymes

(A) Survival percentages of CEFs with 1 μL
virus-containing allantoic; (B) Survival percentages of CEFs with 10 μL
virus-containing allantoic. Groups 1 both in A and B are non-transfected CEFs;
groups 2 both in A and B are CEFs transfected pcDNA-RzF598; groups 3 both in A
and B are CEFs transfected pcDNA-dRzF598; groups 4 both in A and B are CEFs
transfected pcDNA3.

Table 1   MTT scores of survival CEFs being transfected with ribozymes and
infected with viruses

(A)

MTT
scores of CEFs infected with 1 μL virus-containing allantoic. Data are represented
as x±s.

(B)

MTT scores of CEFs infected with 10 μL
virus-containing allantoic. A total of 3 wells for each sample were accomplished
and the average MTT value was calculated. Data are represented as x±s.

3    Discussion

There are no effective therapies applicable to the treatment of
virus diseases, although the vaccines against the virus or against any viral
component that is vital to virus replication can effectively prevent the virus
infection. However, up to date, abundant evidences indicated that the
hammerhead ribozymes could be one of the potential tools to inhibit virus
infection[14－16]. The
possibility of ribozyme strategy for the therapy of SF02 virus infection was
investigated in present study.

acid sequence of the cleavage activation
site[11, 12]. Moreover, F protein is the key factor for the fusion of viral
lipoprotein membrane to the cellular surface membrane of host. Therefore, the F
mRNA is a good target selected for the ribozyme action in order to inhibit the
virus infection.

Our results
showed that ribozyme RzF598 targeting F mRNA could work well not only in vitro.
It also demonstrated the inhibition of virus replication inside CEFs after
transfection of RzF598 gene into cells. Expression of RzF598 in CEFs could
increase the survival percentage of virus-infected cells up to 80% and 90%
depending on the virus concentration. The virus concentration dependent resistance
of ribozyme-transfected and virus-infected CEFs provided the evidence that the
RzF598 expressed in cells could cleave the designed target, full mRNA molecules
of F gene produced during the viral life cycle, as in vitro for the F mRNA
fragment by the ribozyme activity. The results of RT-PCR experiments gave
another direct evidence for the suppression activity of RzF598 in virus
infection. Interestingly, RzF598 could not attack the NDV strain — F48E9， either in the in vitro or in the
in vivo cases. The reason for this failure of cleavage of F mRNA of F48E9 with
RzF598 is that one nucleotide difference (606C→A) in the sequences of SF02 and
F48E9 complementary to the left arm of RzF598. Feng et al.[17] has report that
a disabled ribozyme losses the RNA cleavage activity without affecting its
substrate binding due to introducing a one base mutation (G→A) in helix II of
the active hammerhead ribozyme. A same disabled ribozyme dRzF598 was designed
and used in present studies as control. The dysfunction of dRzF598 indicated
that the cleavage by RzF598, indeed, requires ribozyme activity. However, when
dRzF598 was transfected into CEFs before virus infection, it also showed
certain virus resistance of the cells. The resistance was virus concentration
dependent. The cellular resistance could be caused by the antisense RNA
function of disabled ribozyme because it still remained the binding activity.
The same explanation could be accepted in the case when RzF598, as a kind of
dysfunctional ribozyme, attacked the F mRNA of NDV F48E9 strain. The ribozyme
RzF598 was unable to cleave the target in vitro, but the cells transfected with
RzF598 also showed some resistance against the infection of F48E9. These
results suggested that the ribozyme strategy could be used for the prevention
and therapy of virus infection in the future.

Acknowledgement       We
thank Prof. CHEN Nong-An for helping design the sequence of ribozymes, Prof. QI
Guo-Rong for vector pRG523, and Dr. SHAN Song-Hua for chicken embryo. We are
also indebted to Miss WU Jian-Hua for her technical assistance.

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Received:
April 22, 2003 Accepted: June 5, 2003

This work was
supported by a grant from Shanghai Agricultural Key Promoted by Science and
Technology

*Corresponding
author: Tel, 86-21-64374430-220; Fax, 86-21-64746510; e-mail,[email protected]