Structural and functional genomics study of singapore grouper iridovirus 2

101
\
Chapter 4
Morpholino antisense oligonucleotide knock-down
and functional characterization of SGIV proteins
Manuscript in preparation
Functional characterization of Singapore grouper iridovirus proteins using
Morpholino antisense oligonucleotide knock-down.
102
4.1 Introduction
Morpholino antisense oligonucleotides (oligos or morpholinos) or MO in short is a gene
knock-down agent. MOs used for SGIV gene knock-down were synthesized by GeneTools
based on the full sequence of SGIV genome and ORFs.
We have carried out MO knock-downs for SGIV ORFs whose expression stages are different,
ORF18R and ORF140R are late stage genes, ORF135L is an early gene. On the other hands,
antibodies of these 3 proteins are available. In this project, we knocked down SGIV ORF18R,
ORF140R and ORF135L and analyzed the effects on SGIV and host protein expression
profiles using iTRAQ method.
In all SGIV gene knock-downs, only MO knock-down of ORF018R (MO
18
) showed a
phenotype. This ORF18 was demonstrated to be involved in serine/threonine phosphorylation
and virion assembly (Wang et al., 2008). Hence, we have included MO
18
in this project as a
positive control and further examine its function using other approaches.
4.2 Material and methods
4.2.1 MO knock-down
MOs were delivered using Nucleofactor kit T (Amaxa), program T27. Briefly, the fresh cells
were harvested and washed with PBS, resuspended in the mixture of 98 μL of
103

nucleotransfector solution (with supplement) and 2 μL of MOs to get a final concentration of
20 μM MO, transferred into a cuvette for electrophoresis. The mixture was transferred
immediately into the cell culture flask with fresh medium. The negative control MO (MO
ctrl
) is
the standard control oligo from GeneTools. All MOs used in this projects are listed in Table
11.
The virus was added into the tranfected cells at 40 hours post transfection (hpt). All the
samples were harvested at 48 hpi for iTRAQ analysis or at different time courses for
transmission electron microscopy experiment and Tissue Culture Infectious Dose
50
(TCID
50
test).
4.2.2 iTRAQ sample preparation
This has been described in Chapter 3 (3.2.2)
4.2.3 LC-MALDI MS
This has been described in Chapter 3 (3.2.3)
4.2.4 Transmission Electron Microscrope (TEM)
The cells were fixed in 2.5 % glutaraldehyde, 2 % paraformaldehyde in 1X PBS (pH 7.4)
overnight and post-fixed with 1 % Osmium tetroxide for 3 hours, followed by dehydration in
an ethanol series of 50 %, 75 % and 100 %. The samples were embeded using the Spurr kit
(Sigma), sliced into ultra thin sections (70-90 μm) and stained with 2 % uranyl acetate, 1 %
lead citrate. The ultra thin sections were viewed under JOEL JEM 2010F electron microscopy.
4.2.5 TCID
50
test
104
GE cells were transfected with MOs and infected with SGIV at 40 hpt at m.o.i (multiplicity of
infection) of 3 and 0.5 for high and low m.o.i respectively. Unabsorbed virions were removed

at 2 hpi and the cells were washed twice with PBS. The cell culture supernatant and cell pellet
from 3 different time intervals (48 hpi, 72 hpi and 96 hpi) were diluted from 10
-2
to 10
-7
and
used to infect GE cells with six repetitions per dilution to perform the TCID
50
assay. The viral
titres were calculated using the Spearman-Karber method (Hamilton et al., 1977).
4.2.6 Western Blot analysis
This was described earlier in chapter 2 (2.2.11)
4.2.7 Real time PCR
In order to examine the mRNA level, semi-quantitative real-time RT-PCR was applied and β-
actin was used as the endogenous control. The specific primers for real-time PCR are in Table
12. The normal PCR using these primers showed a single, specific band after running on a 2 %
agarose gel.
The total RNA samples were reverse-transcribed using SuperScript III first strand synthesis
(Invitrogen) with random primers (d(N)6, 0.5 μg μL
-1
). cDNA (10 ng) was subsequently
subjected to real time PCR using Power SYBR® Green PCR Master mix (ABI). Each real-time
PCR reaction had a total of 9 repetitions of 3 biological replica and 3 technical replica. The
real-time data were collected and analysed with the 2
–ΔΔCt
method (Livak and Schmittgen,
2001) using ABI Prism SDS software.
105
Table 11 The sequences of Morpholinos for knock-down experiments.
Knock-down target Synthesized MO Target location

Negative control CCTCTTACCTCAGTTACAATTTATA
ORF018R GCGTTCAGATAGTTTTACGGACATC -1 +24
ORF135L AATACGCTTGTACGAGTTCTTCCAT 0 +25
ORF140R GACCCCTAAATTCTGACATTTTTAT -6 +19
106
Table 12 The specific primers for real-time PCR
OFR/Gene Forward primer Sense primer
ORF007L
TGACCATGTGACGATAACTATAAGCCCGG AGGGTATATCTATCGGTTCGGC
ORF012L
TGACCATGCCGAATGTTTGACCCGAA GAGCGCGGACAAGATGGAT
ORF046L
TGACCATGGGACGCGAACGATATAGTGA GAAGTCCTTGAGGGCCTGGT
ORF101R
TGACCATGACGAAGACAGCTGGGCCAT CGATCTAAAGGTTCCACGACG
ORF125R
TGACCATGCCGTGCGTGGGTATGTGTA CATTGACCTCCTGAGAACGTTC
ORF136R
TGACCATGGGATGAATCAAGAAATGCAGAC GAAAAGGGATGCAGCAACA
107
4.3 Results and Discussions:
4.3.1 MO knock-down of ORF018R, ORF140R and ORF135L
To examine the specificity and efficiency of MO knock-down, we analyzed the expression of
knocked-down proteins using Western Blot, in which GE cells were transfected using MO and
infected by SGIV. The Western Blot results showed no detectable amount of proteins 018R,
140L and 135R with the specific knock-down (Figure 18). We also included other SGIV
proteins such as ORF026R and ORF093L which were expressed normally in all knock-downs.
It was concluded that ORF018R,ORF140R and ORF135L were specifically and efficiently
knocked down by MO
18

, MO
140
and MO
135
respectively.
4.3.2 Effect of MO
135
knock-down on SGIV and host proteins expressions
To study the effect of knock-downs on the viral and host proteins expressions, we analyzed the
protein profile of knock-down GE cells using iTRAQ method. The fresh GE cells were
transfected by MOs, infected by SGIV (m.o.i of 5, infected at 40 hpt), harvested at 48 hpi, and
prepared for iTRAQ samples. In this project, we used 4-plex iTRAQ to analyse 4 protein
samples of MO
ctrl
, MO
140
, MO
135
and MO
18
knock-down cells.
ORF135L is an early gene with unknown function (Chen et al., 2006). It encodes a small non-
structure protein of 13 kDa (Song et al., 2004). The knock-down of ORF135L using MO
135
has
108
caused several changes in protein expression of SGIV and host proteins (Table 13). SGIV
ORF101R protein was significantly increased after the knock-down. ORF101R is also an early
gene (Chen et al., 2006) which encodes a 35 kDa structure protein (Song et al., 2006). We
were not able to validate the change of ORF101R protein expression by Western blot due to

the unavailability of the anti-ORF101R protein antibody. On the other hands, we examine the
mRNA expression level of ORF101R using real-time PCR method. The real-time PCR result
showed the increase of ORF101R mRNA by 1.5 fold (Figure 19) after MO
135
knock-down. It is
likely that protein 101R was negatively regulated by 135L protein.
In addition, MO
135
knock-down has led to the decrease of several host proteins including the
elongation factor 1-alpha (eEF1A) and Retinoblastoma A associcated protein. eEF1A is an
isoform of the alpha subunit of the elongation factor-1 complex, which is resposible for the
enzymatic delivery of aminoacyl tRNAs to the ribosome, thereby regulating the fidelity and
rate of polypeptide elongation during translation (Condeelis, 1995). Association of eEF1A with
specific virus molecules may play a role in the replication of virus genome (Johnson et al,
2001; Cimarelli & Luban, 1997; Blackwell & Brinton, 1997). Retinoblastoma associated
protein may have diverse function in regulation in cell proliferation (Pacifico et al, 2007;
Giordano et al., 2007) and transcription regulator (Hofman et al, 2003; Yan et al., 2000). By
reduction of both eEF1A and retinoblastoma associated protein, MO
135
knowk-down may have
significant influences on transcription regulation and replication of virus genome.
4.3.3 Effect of MO
18
knock-downs on SGIV and host proteins expressions
109
ORF018R is a late gene (Chen et al., 2006) which encodes a 32 kDa structure protein (Song et
al., 2004). It was reported that ORF018R is involved in serine/threonine phosphorylation and
virion assembly (Wang et al., 2008). In this study, MO
18
knock-down appeared to regulate the

protein expression of ORF007L, 012L, 046L, 125R and 140R (Table 14). ORF007L, 012L,
046L and 140R are late stage viral genes (Chen et al., 2006) that would further confirms
ORF018R has an effect on late stage genes (Wang et al., 2008). Furthermore, the reduction of
protein 140R in MO
18
knock-down (Wang et al., 2008) were confirmed by iTRAQ analysis. It
is possible that ORF140R is one down-stream gene regulated by ORF018R.
In addition to the effect on SGIV proteins, MO
18
knock-down caused significant changes in
the expression of several host proteins (Table 14) including the splicing factor arginine/serine
rich 2 protein (SFRS2 or SR) which was up regulated in MO
140
and MO
135
knock-down.
SFRS2 is required for early spliceosome assembly for protein-protein interaction and can
function as activators of pre-mRNA splicing (Lopato et al., 1996; Graveley & Manaitis, 1998).
Both depletion and over expression of SFR2 could cause serious impact to the transcription
and splicing machineries during gene expression (Wang and Manley, 1995; Fededa and
Kornblihtt, 2008; Lin et al., 2008; Xiao et al., 2007). It has been reported that the over-
expression of SR proteins caused a large reduction of genomic RNA, down-regulate the late
steps of HIV-1 replication (Jacquenet et al., 2005).
Besides, MO
18
also reduced protein expression of RhoA (Ras homolog gene family, member
A) which is a small GTPase protein known to regulate the actin cytoskeleton in the formation
of stress fibers (Ridley, 2001). RhoA GTPase was known with roles in facilititating virus entry,
adherent target cell and infection by involving remodel of actin cytoskeleton for phagocytosis-
110

like uptake (Clement et al., 2006; Veettil et al, 2006; Coyne et al, 2007; Jimenez-Baranda et
al., 2007). RhoA-GTPase is also essential for entry stages of infection involved in the
modulation of microtubular dynamics, movement of virus in the cytoplasm, and nuclear
delivery of viral DNA (Raghu et al, 2007). In addition, RhoA signaling is associated with
filamentous virus morphology, cell-to-cell fusion, syncytium formation (Gower et al., 2005).
Interestingly, it was reported that MO
18
knock-down resulted in a reduction of virus infectivity
and distortion of viral particle assembly (Wang et al., 2008). Hence, in MO
18
transfected cells,
the decrease of cellular RhoA, which is an crucial protein for virus entry, infection and delivery
of viral DNA, might contribute to these knock-down’s effects.
4.3.4 Effect of MO
140
knock-downs on SGIV and host proteins expressions
ORF140R encodes a late, non-structural protein of 32 kDa (Song et al., 2004; Chen et al.,
2006). MO
140
knock-down resulted in several changes of SGIV and host protein expression
(Table 15). ORF046L, a late and structure protein, was decreased after MO
140
knock-down.
ORF046L was also shown to be down-regulated in MO
18
transfected cells. However, MO
140
knock-down had no impact on ORF007L, 012L, 125R which were declined in MO
18
knock-

down. Except for ORF125R that is an intermediate early gene, ORF018R, 007L, 012L and
046L are all late stage genes (Chen et al., 2006). It was confirmed that ORF140R protein was
reduced in MO
18
transfected cell, however in this study, ORF018L protein expression was not
affected by MO
140
. We might hypothesize that ORF007L and 012L are in the middle of the
pathway, in which ORF140R is a down stream gene regulated by ORF018R; and ORF046L is
further down stream, under regulation of both ORF018R and ORF140R.
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Furthermore, MO
140
knock-down up-regulated cellular splicing factor arginine/serine rich 2
protein (SFRS2 or SR). This host protein was also increased in MO
135
knock-down cells but
declined after MO
18
knock-down. Our previous analysis implied that either significant decrease
or increase of cellular SFRS2 might seriously effect the host transcription, splicing processes
and the viral genomic replication.
Besides, all these three MO knock-downs shared one common phenomenon which is the up-
regulation of the host 40S ribosomal protein S6 (RPS6). RPS6 is located in the mRNA binding
site of the 40S subunit of cytosolic ribosomes. RPS6 can be directly cross-linked to mRNA,
tRNA and initiation factor. It was found that RPS6 becomes highly phosphorylated on multiple
serine residues in response to several oncogenic viruses (Sturgill et al., 1988; Decker, 1981;
Blenis & Erikson, 1984; Maller et al., 1985). Several lines of evidence link elevated S6
phosphorylation to the initiation of protein synthesis, suggesting that this may be one of several
events involved in the control of cell proliferation (Blenis & Erikson, 1985). The cellular RPS6

was unchanged upon SGIV infection (iTRAQ data, chapter 3). Hence, it seems that the high
expression level of RPS6 could be the consequence of MO knock-down.
4.3.5 Analyze the gene expression of SGIV proteins regulated by knock-downs using real
time PCR
To study the mRNA expression level of SGIV ORFs which were regulated by these MO
knock-downs, we carried out the real-time PCR experiment with the specific primers listed in
table 12. The consistency between transcription and translation was shown by ORF101R with
the increase of both ORF101R mRNA and protein in MO
135
transfected cells. In contrast,
112
ORF007L, 012L, 125R protein expressions were reduced in MO
18
knock-down; ORF136R
protein was increased in MO
135
knock-down but their mRNA levels were unchanged. In
addition, mRNA levels of ORF046L in MO
18
and MO
135
transfected cells were increased
around 3 and 2.5 fold respectively even the protein was shown to be decreased. The
phenomenon of inconsistency has been often encountered in the comprehensive analysis of
data from transcriptome and proteome sources. This is always a major challenge. In order to
explain this inconsistency, we need to know the properties of genes, transcripts and proteins
(Irmler et al., 2008; Lu et al., 2008). It has been reported that proteins, which are shown to
change in the proteome but not in the transcriptome, fall into classes of post-transcriptional
modification, protein synthesis and protein folding. On the other hands, genes, which are
changed only at the transcriptional level, usually affect cell growth/death pathways, cell-cell

related function (Lu et al., 2008; Seth et al., 2007).
The knock-downs with these significant changes in both host and viral proteins could have
certain impact on the virus infectivity.
4.3.6 Effect of MO knock-downs on virus infectivity
To investigate the knock-down’s effects on the infectivity of newly assembled virions, we
carried out the TCID
50
test using high dose (m.o.i of 3) and low dose (m.o.i of 0.5) of SGIV
(Figure 20). Both cell culture supernatants and cell pellets contained virions were used in the
virus titration experiment. The reduction of virus infectivity were at different level. MO
18
knock-down caused the most significant decrease of virus infectivity and the virus infectivity
after MO
140
knock-down was lower than that after MO
135
knockdown. The effect of MO
140
on
113
virus infectivity is milder than that of MO
18
, which is quite consistent with the hypothesis that
ORF140R is a down stream viral gene regulated by ORF018R.
The TCID
50
results could provide some links of specific knock-down’s effects to host/viral
proteins to the reduction of virus infectivity and might imply the importance of ORF018R,
ORF140R and ORF135L on virus infection, replication and even virus morphology.
4.3.7 Effect of MO knock-down on virus phenotype

We used TEM to examine the effect of MO knock-down on the phenotype of newly
synthesized virions (Figure 21). Although the TCID
50
test showed the reduction of virus
infectivity in the infected MO
140
and MO
135
transfected cells, the phenotypes of virions after
these knock-downs were quite normal, only MO
18
caused clear morphological defect on virus
particles (Wang et al., 2008). We encountered some defected virion particles in MO
140
transfected cells whick appeared to be similar to the defect caused by MO
18
. Taken together
with the TCID
50
test, the defect virion particles could partly explain the reduction of virus
infectivity after the knock-downs. MO
18
transfected cells with the distortion of viral particle
seriously reduced the virus infectivity. MO
140
which knock-downs ORF140R, a possible down-
stream gene of ORF018R, caused fewer defected virion particles than MO
18
. Hence MO
140

knock-down had less severe effect on virus infectivity. Furthermore, MO
135
had least effect on
virus infectivity compared with MO
18
and MO
140
. Most of virion particles in MO
135
transfected
cells were quite normal except several empty virions. There are some empty virions which
were circled and pointed by arrow heads (Figure 21). These virions were observed more in
MO
18
knock-down cells, fewer in MO
140
and MO
135
transfected cells. However, we need
further investigation to quantitate the empty virions.
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Figure 18 Western blot analysis ORF140R, ORF135L, ORF18R proteins by MO
ctrl
, MO
140
,
MO
135
and MO
18

knock-down respectively. These gene knock-downs are specific and suffient.
The proteins of knock-down genes were not detected using western blot.
115
Table 13: Host and SGIV proteins which were significantly regulated by MO
135
knock-down
Host/virus
protein
ORFs/ Protein name
Accession
number
Protein
MW (Da)
iTRAQ
ratio
(MO
135
/
MO
ctrl
)
Gene
expression
Stage *
S/NS**
SGIV
protein
ORF101R gi|56692738 1.122194
1.802055
IE NS

Retinoblastoma A associated protein
[Xenopus laevis] gi|148235471 51225.35
0.577092
VAMP-2 [Macaca mulatta]
gi|74136201 13852.87
0.619228
PREDICTED: similar to MGC53657 protein
[Strongylocentrotus purpuratus] gi|72012161 18115.37
0.652344
elongation factor 1-alpha [Cypridopsis vidua] gi|4530096 44543.76
0.643597
elongation factor 1-alpha [Rhincalanus
nasutus] gi|90655094 33671.93
0.643597
Zgc:55876 protein [Danio rerio] gi|41946787 26663.96
1.439545
splicing factor arginine/serine rich 2 [Oryzias
latipes] gi|9837439 27455.31
1.439545
40S ribosomal protein S6 gi|20139890 33752.51
1.447596
phosphoglucose isomerase [Mugil cephalus] gi|20067649 67731.1
1.499392
Host protein
unnamed protein product [Tetraodon
nigroviridis] gi|47211637 30398.07
1.409418
*L: Late; IE: Intermediate Early (Chen et al., 2006) ** S: structure; NS Non-structure (Song et al., 2004)
116
Table 14: Host and SGIV proteins which were significantly regulated by MO

18
knock-down
Host/virus
protein
ORFs/ Protein name
Accession
number
Protein MW
(Da)
iTRAQ
ratio
(MO
18
/
MO
ctrl
)
Gene
expression
Stage *
S/NS**
ORF007L
gi|56692644 32309.26
0.379483
L S
ORF012L
gi|56692649 129768.6
0.663402
L NS
ORF046L gi|56692683

26298.25
0.579507
L S
ORF125R gi|56692762
23111.84
0.573601
IE S
SGIV protein
ORF140R gi|56692777 36630.86
0.667232
L NS
Zgc:55876 protein [Danio rerio] gi|41946787 26663.96
0.596155
splicing factor arginine/serine rich 2
[Oryzias latipes]
gi|9837439 27455.31
0.596155
RhoA [Rattus norvegicus]
gi|2225894 21775.95
0.648509
unnamed protein product [Tetraodon
nigroviridis]
gi|47228056 64087.05
0.649397
Ornithine aminotransferase precursor
CG8782-PA [Drosophila melanogaster]
gi|21357415 51969.66
1.465898
Host protein
40S ribosomal protein S6

gi|20139890 33752.51
1.472459
L: Late; IE: Intermediate Early (Chen et al., 2006) ** S: structure; NS Non-structure (Song et al., 2004)
117
Table 15: Host and SGIV proteins which were significantly regulated by MO
140
knock-down
Host/virus
protein
ORFs/ Protein name
Accession
number
Protein MW
(Da)
iTRAQ ratio
(MO
140
/
MO
ctrl
)
Gene
expression
Stage *
S/NS**
ORF046L
gi|56692683 26298.25
0.630361
L S
SGIV protein

ORF136R gi|56692773 13116.6
1.435592
IE NS
hypothetical protein LOC495253 [Xenopus
laevis]
gi|147903409 15637.74
0.592646
Zgc:55876 protein [Danio rerio] gi|41946787 26663.96
1.407937
splicing factor arginine/serine rich 2 [Oryzias
latipes]
gi|9837439 27455.31
1.407937
Host protein
40S ribosomal protein S6 gi|20139890 33752.51
1.40316
L: Late; IE: Intermediate Early (Chen et al., 2006) ** S: structure; NS Non-structure (Song et al., 2004)
118
iTRAQ protein profile
0
0.5
1
1.5
2
007L 012L 046L 101R 125R 136R
Protein expression
140 MO knock down
135 MO knock down
18 MO knock down
mRNA expression level

0
1
2
3
4
007L 012L 046L 101R 125R 136R
expression level
Figure 19: The real time PCR data shows mRNA expresssion trend is inconsistent with
the iTRAQ protein profile.
119
Figure 20 The effect of MO knock-down to virus infectivity of newly assembled virion
particles. Infectivity of virion particles in A: cell culture of low moi virus infection after
MO transfection; B: cell pellet of low moi virus infection after MO transfection; C: cell
culture of high moi virus infection after MO tranfection and D: cell pellet of high moi
virus infection after MO transfection. Knock-down of ORF18R caused the greatest
effect on virus infectivity. Knock-downs of ORF140R and 135L were quite similar.
120
Figure 21 Effects of MO knock-down on SGIV assembly.
GE cells transfected with (A) MO
ctrl
, (B) MO
140
, (C) MO
135
and (D) MO
18
were
infected with SGIV moi = 5 at 40 hpt. The cells were harvestd at 48 hpi and fixed,
prepared for TEM. The pictures shown here are representative for many virus-infected
cells. The arrows show defective viruses. The similar phenonena were observed if

tranfected cells were infected by low moi (0.5) of SGIV. Arrows point to defective
viruses, arrow heads point to empty viruses in circles. Knock-down ORF18R caused
121
the viron defect, knock-down ORF140R and 135L did not show the clear phenotype
changes of viron particles
C1: Magnification 1500 X, scale bar 2 μM. A1, B1, D1: Magnification 2000 X, scale
bar 2 μM
A2, C2: Magnification 5000 X, scale bar 0.5 μM. B2, D2, A3, C3: Magnification 6000
X, scale bar 0.5 μM. B3, D3: Magnification 10000 X, scale bar 0.2 μM
Chapter 5
General conclusion and future work
122
5.1 General conclusion
In this project, focussing on the functional and structural genomics of SGIV, we have
obtained a number of results which would significantly facilitate the study to discover
SGIV infection mechanism and virus-host interaction.
We have characterized SGIV UBL in vitro using ubiquitination assay and determined the
structure of this protein by NMR. The NMR structure of SGIV UBL shows some
differences to the structure of human ubiquitin.This is the first structure of viral UBL
protein.
In addition, the proteomes of the virus and GE host cell were successfully investigated
using iTRAQ. This is the first time that protein expression profiles of non-infected and
infected GE were quantitated. This study has revealed some important information about
the host responses to the viral infection. In addition, several new and novel viral proteins
were identified.
Another significant outcome is the effect of MO
18
, MO
140
, MO

135
knock-down on virus
infectivity and virion assembly. We also looked into the impact of specific gene knock-
123
down on the host and the viral proteomes. The study has shown some interesting
relationship between gene knock-down with several host proteins which could be
involved in virus entry and infection.
Generally, this project’s outcomes have contributed to our understading of SGIV
genomics and proteomics. More future investigations are needed for the functional
elucidation and structure determination of SGIV genes/proteins.
5.2 Future experiments
5.2.1 Functional study of SGIV UBL
From the iTRAQ data (Chapter 3), SGIV UBL is different from the host ubiquitin and it
is expressed upon the virus infection. The virus infection may require this protein but
why it needs its own UBL where the host also encodes ubiquitin. Possibly, SGIV UBL
may be involved in some unique protein interactions which are crucial for virus to enter
the host cells and replicate its genome. To characterize this viral UBL’s function, we
should be able to distiguish the host and viral UBL. The synthesized peptide
(ITIDVDVDHADTVGAVKAK), which is unique for this virus, will be used to raise the
monoclonal antibody for SGIV UBL. To further study the role of SGIV UBL, we will
examine the protein expression of SGIV UBL at different stage of infection and find out
the protein interactions of the viral UBL with other virus/host proteins. In addition, we
124
will sequence the host ubiquitin gene and possibly design the specific MO for the knock-
down experiment.
5.2.1 A more comprehensive study and analysis of virus-virus and virus-host
protein interactions
The iTRAQ experiment (Chapter 4) has revealed some effects of specific knock-downs
(MO
135

, MO
140
, MO
18
) to the expression of some virus and host proteins. A number of
proteomics and molecular methods such as yeast two hybrid, pull-down assay,
immunoprecipitation, immunolocalization should be applied to find out and confirm the
possible interactions. These interactions could be very important for virus infectivity and
virus assembly processes and might shed the light on the discovery of the virus infection
mechanism. We will continue to design more MOs specific for other SGIV ORFs,
especially ORFs encoding enzymes. The succesful inhibitors might be potential for
effective anti-viral drugs.
In addition, with the successful applications of iTRAQ with MO knock-down, we will
further carry out this combination to generate a network of the proteins involved in virus
host interactions and pathogenesis.
5.2.3 Structural determination of SGIV proteins
ORF140R protein (32 kDa) and ORF135L protein (13 kDa) are highly expressed and
soluble in E.coli protein expression system. X-ray crystallograpy and NMR methods will
125
be used to solve the protein structures which would greatly enhance the functional study
of these genes. Moreover, we also aim to determine the structure of SGIV structural
proteins to elucidate function of the presumptive proteins based on the structures, which
should provide insight into design the inhibitor or anti- viral drug.
5.2.4 Structural determination of SGIV virion particles
In addition to the structural determination of SGIV proteins, we can also target to study
the 3D structure of SGIV virion particles using cryo electro microscopy (CryoEM).
CryoEM is a form of electron microscopy where the sample is studied at cryogenic
temperatures (liquid nitrogen or helium). CryoEM has the potential of revealing structural
details at near atomic level of macromolecular complexes, subcellular assemblies that are
either too large or too heterogeneous to be investigated by conventional X-ray

crystallograpy or NMR. Interestingly, this method can examine structural changes of the
molecule during its functional activities (Henderson, 1995). Recently, a group of
scientists at Purdue Univesity (USA) has reported a 22-MDa structure of the capsid of the
infectious epsilon15 particle by cryoEM. With the resolution of 4.5 Å, a complete
backbone and its major capsid proteins were constructed (Jiang et al., 2008). SGIV
particle sample can be prepared for cryoEM by rapid plugging into liquid nitrogen and
imaged at low temperature in the EM. The overall 3D structure and symmetry of the
virion and nucleocapsids can be generated.