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<title>Envelope 2 protein phosphorylation sites S75 & 277 of hepatitis C virus genotype 1a and interferon resistance: A sequence alignment approach</title>
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Afzal et al. Virology Journal 2011, 8:71
http://www.virologyj.com/content/8/1/71
RESEARCH
Open Access
Envelope 2 protein phosphorylation sites S75 &
277 of hepatitis C virus genotype 1a and
interferon resistance: A sequence alignment
approach
Samia Afzal*, Muhammad Idrees, Muhammad Ali, Muhammad Ilyas, Abrar Hussain, Madiha Akram, Sadia Butt,
Sana Saleem, Irshad ur Rehman, Liaqat Ali, Muhammad Shahid
Abstract
Background: Hepatitis C is a major health problem affecting more than 200 million individuals in world including
Pakistan. Current treatment regimen consisting of interferon alpha and ribavirin does not always succeed to
eliminate virus completely from the patient’s body.
Results: Interferon induced antiviral protein kinase R (PKR) has a role in the hepatitis C virus (HCV) treatment as
dsRNA activated PKR has the capacity to phosphorylate the serine and threonine of E2 protein and dimerization
viral RNA. E2 gene of hepatitis C virus (HCV) genotype 1 has an active role in IFN resistance. E2 protein inhibits and
terminates the kinase activity of PKR by blocking it in protein synthesis and cell growth. This brings forward a
possible relation of E2 and PKR through a mechanism via which HCV evades the antiviral effect of IFN.
Conclusion: A hybrid in-silico and wet laboratory approach of motif prediction, evolutionary and structural anlysis
has pointed out serine 75 and 277 of the HCV E2 gene as a promising candidate for the serine phosphorylation. It
is proposed that serine phosphorylation of HCV E2 gene has a significant role in interferon resistance.
Background
Hepatitis C virus is a key universal health issue [1] affecting approximately 200 million individuals worldwide and
over 4 million in the United States alone, where it is the
most common blood-borne infection [2]. It is the main
reason of persistent liver infection and the most familiar
sign for liver transplantation [3]. In 60-85% cases HCV
develops to cirrhosis and hepatocellular carcinoma [4].
Presently, in Pakistani population 17 million people are
infected with HCV and 8-10% individuals are HCV
carriers [5]. HCV is a member of genus, hepacivirus,
family Flaviviridae and is a positive sense single stranded
RNA virus [6]. HCV genome is about 9.6 kb in length
[7]. The large open reading frame of Viral RNA having
5` and 3` untranslated regions which is translated into a
single polypeptide of 3010 to 3033 amino acids. Which is
* Correspondence: samiaraza@live.com
National Centre of Excellence in Molecular Biology, 87-West Canal Bank
Road, Thokar Niaz Baig, Lahore-53700, University of the Punjab, Lahore,
Pakistan
processed by host as well as viral proteases to yield 10
mature individual proteins out of which 3 are structural
and 7 are nonstructural [8,9]. Detailed structure of HCV
virus is still unclear. However, the infectious viral particles are composed of lipid envelope glycoproteins E1 and
E2 [10].
In spite of much recent advancements, still no vaccine
is available against HCV infection. The current therapy
for HCV infection is pegylated interferon alpha separately or in combination with ribavirin [11] but it eradicates the virus in only 50-80% of cases and has serious
side effects [12]. IFN system is the first line of protection against viral infection in mammals [13]. Many
viruses have developed mechanisms to dodge the IFNdependent cellular response [14]. Amongst these HCV
is a significant example in which (70-80%), cases runs
away the host defenses and develops a chronic infection.
Pathological outcomes of HCV infection changes from
individual to individual, unstable from asymptomatic
state to liver fibrosis, steatosis, finally to hepatocellular
© 2011 Afzal et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
Afzal et al. Virology Journal 2011, 8:71
http://www.virologyj.com/content/8/1/71
carcinoma [15,16]. The factors upon which the success
or failure of the antiviral therapy depends are unspoken
yet, and their recognition characterizes a main confront
in HCV virology [11]. IFN-interacts with cells and modifies the expression of a number of genes [17,18]. E2 glycoprotein is the first viral factor that gets in touch with
the host cell surface receptors thus it has an important
role in vaccine designing and drug objective [19,20]. Primary objective of HCV vaccine is to initiate potent
humoral responses against E2 protein [21].
After the death of infected cells, virus particles are discharged and they can infect the cells present in close
proximity. Interferon is released from the infected cells
to inform the neighboring cells about the presence of
virus. In its rapid response, PKR is produced by these
adjoining cells. HCV resistance to IFN-treatment is partially related to inhibition of interferon induced antiviral protein PKR. Interferon induce many protective
mechanisms in cells and amongst these the major role
in cell protection from many viruses is illustrated by
double-stranded RNA (dsRNA)-activated protein kinase
PKR [22,23].
Two HCV proteins (NS5A and E2) are involved in
IFN resistance through inhibition of the IFN-a induced
double stranded-RNA (dsRNA)-activated protein
kinase (PKR) [24,25]. PKR is a kinase enzyme that
reveals varied activities. PKR exhibits autophosphorylation of many serine and threonine positions and
dimerization of dsRNA. It also phosphorylates the
translation initiation factor eIF-2 (a subunit) that
directs towards blockage of protein synthesis [26].
Because of these properties, PKR is taken as an arbitrator of antiviral and anti-inflammatory role of IFN-a
[27]. E2 protein blocks PKR activation to bypass its
function [24-28]. Taylor et al., (1999) [25] reported
that HCV E2 protein encloses a 12 amino acid
sequence domain that is highly homologous to autophosphorylation positions of PKR and initiation factor
eIF2. PKR and eIF2 form a phosphorylation homology
domain (PePHD). E2 protein inhibits kinase activity of
PKR and terminates its blocking role in protein synthesis and cell growth. This suggests that the relationship
of E2 and PKR can be considered as a major mechanism by which HCV evades the antiviral effect of IFN.
Methods
Page 2 of 6
E2 gene. At 5’ end a start codon was introduced artificially in the primers for expression of gene in mammalian cell culture. The required amplified product was of
approximately 1089 bps which was confirmed on ethidium bromide stained 1.2% agarose gel evaluated under
UV transilluminator and photographed. The necessary
band was cut out from the gel and the DNA was
extracted with DNA isolation kit (Fermentas Inc. Germany). Isolated DNA was suspended in depc treated
water and used for further studies.
Construction of expression vector
Mammalian expression vector pcDNA 3.1 (Invitrogen
Tech USA) was used to clone the amplified product
encoding E2 gene between Hindi III and EcoR I sites.
The plasmid was transformed in chemically-competent
cells. The entire ligation reaction was added to a 100 μl
aliquot of TOP10F cells. Then 500 μl of SOC medium
was added to the cells and incubated at 37°C for 1 hour.
Competent cells having plasmid were then selected by
spreading the culture onto a Luria-Bertani (LB) agar
plate containing 100 μg/ml ampicillin and 12.4 ug/ml
tetracyclin. Colonies selection was made by incubating
the plate overnight at 37°C. To identify bacteria harboring cloned E2 gene, individual colonies were used to
directly inoculate PCR reactions. The PCR reactions
were prepared with 5 pmol each of vector-specific primers i.e. T7 (TAATACGACTCACTATAGGG) and
BGH (TAGAAGGCACAGTCGAGG). Each reaction
was prepared to a final volume of 20 μl. Following
amplification, the amplified product was checked on a
1.2%, ethidium bromide stained agarose gel, a successful
cloning reaction being visualized as a product at
approximately 1.3 kb. Colonies identified as possessing a
desired clone were then used to inoculate 3 ml LB culture containing 100 μg/ml ampicillin, shaking at
225 rpm overnight at 37°C. Plasmid was purified using a
Plasmid Miniprep Kit, (Fermentas Life Sciences technologies, USA) according to manufacturer’s protocol.
Quantification of the plasmid prep. was performed by
using spectrophotometer. Successful cloning was confirmed through PCR, restriction digestion of plasmid
(pcDNA 3.1/myc vector) with EcoR1 & Hindi III and
sequencing reaction. At least three clones were
sequenced in both directions according to the sequencing protocol.
HCV RNA Detection and identification of E2 gene
Serum samples were collected at the Division of Molecular Virology, National Centre of Excellence in Molecular Biology, University of the Punjab, Lahore. HCV 1a
genotype serum samples were used in the current study.
100 μl serum was used to extract viral RNA by using
RNA Isolation Kit (Sacace, Italy). Primer 3 software
was used to designed primer for the amplification of
Sequence analysis
Applied biosystems prism dye termination method was
used to sequence the DNA (PCR products and plasmids) with specific sense and anti sense primers. The
sequences reported in this paper have been deposited in
genbank database (Accession no. GU736411). In a 10 μl,
of total reaction volume, about 300 ng of plasmid,
Afzal et al. Virology Journal 2011, 8:71
http://www.virologyj.com/content/8/1/71
10 pmol of primer, 1 μl of big dye and 1 μl of dilution
buffer were used for each sequencing reaction. Sequencing was achieved in a thermal cycler with parameters;
94°C for 20 s, 58°C for 20 s, 60°C for 4 min, repeated
25 times. The pellet was air dried and DNA was
analyzed using an ABI prism sequencer.
Identification of Phosphorylation sites
To study the possible outcome of predication with help
of NetPhos 2.0 software as described by Blom et al.
(1999) [29]. This software checks the possible post
translation modification in the local 1a local strain primary sequence in the amino acid. As a result of analysis
the target motif for different kinases is visualized
[29,30]. Scansite is kept at low stringency in order to
determine the maximum number of sites that may participate in phosphorylation, upon which further predication is done.
Protein Structure Analysis
An ab-initio model was designed by using I-TASSER as
no template model was available in protein Data Bank
[31]. Data was uploaded and models were generated.
A 3D structure of predicted phosphorylation sites was
generated by using two servers, Chimera [31] and
SWISS PDB viewer [32] were used to obtain 3D structure. It was checked that potential phosphorylation sites
Page 3 of 6
are surface exposed or hidden inside. Five sites were
observed at the exposed surface of model (aa: S75, S95,
S118, S277, Y211).
Phylogenetic Analysis
Protein sequences of envelope gene for genotype 1a from
different countries of the world (Japan, France, USA, and
UK) were obtained from NCBI. All sequences (GQ898898,
EU482831, EU234064, EU362889, DQ061315, AY958052,
AY958057, AY956468, AB520610, and AF529293) were
then aligned with local envelope gene by using CLUSTALW [33]. A neighbor joining tree was generated using
PHYLIP (Figure 1) [34].
Results
To evaluate its function in disease succession, amplified
E2 gene was cloned in mammalian expression vector pc
DNA 3.1. A CMV promoter is present in pc DNA 3.1
vector that can transduce eukaryotic cells very efficiently
for transient and stable expression studies. Clone was
sequenced by the applied biosystems prism dye termination method using vector specific and gene specific primers (Accession no. GU736411). Documented sequences
for 1a genotype from diversified areas of the world were
compared with local envelope gene sequence to get the
percentage nucleotide identity (PNI) (Table 1) and
CLUSTAL W. Sequence dissimilarities in the envelope
Figure 1 Phylogeny of local envelope gene sequence with published sequences for HCV genotype 1a from different localities of the
world.
Afzal et al. Virology Journal 2011, 8:71
http://www.virologyj.com/content/8/1/71
Page 4 of 6
Table 1 Comparison of local and published envelope
gene sequences
Accession No.
Genotype
Country
% similarity
GQ898898
1a
PAKISTAN
96%
EU482831
1a
USA
92%
EU234064
EU362889
1a
1a
USA
USA
90%
90%
AY956468
1a
USA
91%
DQ061315
1a
USA
90%
AY958052
1a
UK
91%
AY958057
1a
UK
90%
AF529293
1a
FRANCE
89%
AB520610
1a
JAPAN
88%
genes explained the different levels of disease outcomes
in patients infected with same genotype. These nucleotide differences can help out to plan the genotype specific
therapy to avoid and minimize HCV infections.
Possible phosphorylation sites were calculated in the
cytoplasmic domain of the E2 protein that can be implicated in the interferon resistance. Twelve putative sites
(Figure 2) were chosen as potential phosphorylation
sites (S-6, Thr-4, Tyr-2). Stringency of Phosphorylation
site predictors was increased as they have tendency to
over-predict. Only those motifs were selected that
showed a NetPhos score of 0.8 or greater. After it they
were also analyzed by Scansite. Finally six sites were
predicted by both servers (Table 2).
An online server NetSurfP was used to find the surface accessibility of local envelope sequence, as the
phosphorylation sites should be exposed on the surface
of proteins (Table 2). Discovery Studio and SWISS PDB
Viewer were applied to visualize 3D protein structure of
the putative sites. PDB structure was built up by using
the server I-TASEER (Ab-initio protein structure predictor) [35]. After this analysis two phosphorylation sites
(S75 and S277) were found to be most reliable sites
(Figure 3). Scansite was used for finding the phosphorylation interaction motifs and found that S75 and S277
interact with the CLK2 Kinase and YWHAZ which were
then investigated in GeneCards and was confirmed from
UniGene and UniProt (Table 3).
Discussion
IFN usually causes the transcription of some antiviral
genes. However, detailed principal mechanisms exploring high IFN-a resistance in HCV genotype 1a infected
patients are still ambiguous. A double stranded RNA
activated PKR phosphorylates the translation initiation
factor (eLF2) and thus inhibits its protein synthesis ability. A straight relationship was found in a study of E2
protein of HCV genotype 1a and IFN inhibition pathway. It was shown that it inhibits PKR through a 12
amino acid sequence E2-PePHD domain, and for PKR
this domain can perform the function of pseudosubstrate. The functions of PKR i.e. inhibition of viral
Figure 2 Phosphorylation sites software calculated in the local HCV envelope gene sequence.
Afzal et al. Virology Journal 2011, 8:71
http://www.virologyj.com/content/8/1/71
Page 5 of 6
Table 2 Summary of predicted tyrosine phosphorylation sites
Phosphorylation sites
Site*a
aa*b
Context*c
NetPhos*d
Scansite*e
NetSurfP*f
I-TASSER*g
75
S
ERLASCKPL
0.977
Y
E
Yes
95
S
YANGSGPEH
0.969
_
E
Yes
118
S
VPAQSVCGP
0.885
_
E
Yes
277
S
DRDRSELSP
0.978
Y
E
Yes
221
T
GPWITPRCL
0.951
Y
E
No
211
Y
PEATYSRCG
0.863
Y
B
Yes
a. Phosphorylation sites in the local 1a sequence of the envelope gene.
b. S indicates Serine; T is for Threonine and Y for Tyrosine predictions.
c. Region where the phosphorylation sites are available.
d. Predicted sites by NetPhos with a score of ≥ 0.8. Dashes indicate lack of Phosphorylation sites in that position.
e. Y indicates predicted sites in sequence on “Low Stringency”. Dashes indicate lack of Phosphorylation sites in that position.
f. E indicates Exposed sites and B indicates Buried sites. Surface accessibility calculated by NetSurfP.
g. Ab-initio 3D model was constructed by using I-TASSER server.
protein synthesis and its kinase activity, are blocked by
this domain. PKR autophosphorylation sites are highly
identical to the PePHD domain in HCV genotype 1a
than other genotypes [36].
It has been demonstrated that E2 protein of HCV genotype 1 shares homology with PKR and eIF2 phosphorylation sites and therefore inhibits PKR by binding to it
in vitro in mammalian and yeast cells. HCV therefore
has evolved a mechanism in the form of E2-PKR interaction to block IFN activity. The possible outcome of
PKR inhibition is not only IFN resistance and infection
persistence but also cell growth promotion which ultimately leads to hepatocellular carcinoma (HCC).
Since E2 PePHD domain of HCV genotype 3a share less
homology to PKR and eIF2 phosphorylation sites than do
genotypes 1a and 1b. Therefore, it can be the most satisfactory reason that patients with 3a genotype react to
interferon treatment more proficiently as compared to
genotype 1a that shows maximum resistance to IFN therapy [37,38]. The relationship of E2-PePHD domain and its
behavior during interferon therapy is quite indistinct.
Some studies also explained the modifications happening
in the PePHD domain during treatment. This work reveals
that in non-responders, slight variations present in pretreated patients quickly evades during treatment [39].
HCV has developed ways to undo the antiviral effects of
PKR [40,41] and it is assumed that IFN resistance may be
due to phosphorylation of envelope proteins particularly
of E2 protein. Our study focused on detecting such sites in
our local isolate of HCV genotype 1a and confirmed two
phosphorylation sites (S75 and S277) at the surface of E2
protein that interacted with CLK2 Kinase and YWHAZ. It
is further proposed that this interaction might be responsible for HCV resistance to antiviral effects of IFN which
could be confirmed by dephosphorylating these sites and
analyzing its effects on PKR binding and inhibition.
Conclusion
The role of envelope protein 2 (E2) of hepatitis C virus
(HCV) is found to interact with double stranded RNAdependent protein kinase (PKR) in previous studies. In
the present study, the role of phosphorylation of E2 protein towards interferon sensitivity & PKR response was
determined. HCV E2 protein was found to contain a
Table 3 Interacting enzymes predicted by Scansite
Site*a Enzyme*b Gene
Card
75
Clk2
Kinase
CLK2
Yes
P49760
CDC2-like kinase
(CLK)
277
pST_bind
YWHAZ Yes
P63104
Tyrosine
3-monooxygenase/
tryptophan
5-monooxygenase
activation protein,
zeta polypeptide
—
Figure 3 Visualization of phosphorylation sites S277 and S75
in Tertiary structure of the envelope gene.
UniGene*c UniProt*c Full Name
Clk2
Kinase
CLK2
P49760
CDC2-like kinase
(CLK)
Yes
a. Phosphorylation sites in the envelope gene.
b. Observed enzymes by the Scansite.
c. Gene’s availability on UniGene and UniProt.
Afzal et al. Virology Journal 2011, 8:71
http://www.virologyj.com/content/8/1/71
sequence identical to phosphorylation sites of the interferon-inducible protein kinase (PKR). As a result E2
inhibits the kinase activity of PKR. This work will help
to identify factors that can favor a successful innate
immune response to HCV infection.
Authors’ contributions
MI conceived the study and critically reviewed the manuscript. SA
performed, nucleotide sequencing and analyzed the results. SA, MA, AH and
MI drafted the manuscript. MA, MA, MI, SB, SS, IR, LA and MS participated in
data analysis. All the authors studied and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 20 January 2011 Accepted: 15 February 2011
Published: 15 February 2011
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doi:10.1186/1743-422X-8-71
Cite this article as: Afzal et al.: Envelope 2 protein phosphorylation sites
S75 & 277 of hepatitis C virus genotype 1a and interferon resistance: A
sequence alignment approach. Virology Journal 2011 8:71.
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