Interaction of NS3 with viral and host proteins and RNA during hepatitis C virus replication
File(s)
Author(s)
Sifennasr, Nadia
Type
Thesis
Abstract
Hepatitis C virus (HCV) is a major cause of viral hepatitis. The World Health Organiz
a-
tion (WHO) estimates the global prevalence of HCV to be approximately 2.2–3.0% with
130– 170 million infected individuals worldwide. HCV has the ability to evade the i
m-
m
une system and to establish persistent infections, which often result in chronic liver di
s-
ease. HCV encodes a long polyprotein which contain structural and non-structural pr
o-
teins. The Non-structural protein NS3 is likely to be involved in viral RNA replication by
interacting with viral and host components. In this study, a purified recombinant full-
length NS3 protein expressed in
E. coli
was examined for its ability to interact with
bi
otinylated full length HCV JFH-1 genomic RNA and the 3’ terminal of HCV negative
strand RNA. The results showed that NS3 could bind to both full length and the 3’ end
negative strand RNAs. RNA interference (siRNA) studies were also carried out to inve
s-
tigate the role of host genes RAB40B, RAB27B, TXNIP and Staufen 1, which had prev
i-
ously been shown to be important for HCV replication, on NS3 and NS3/4A proteins e
x-
pressed by adenovirus vectors using quantitative real-time polymerase chain reaction
(qRT-PCR) and FACS analysis. While there were reductions in the levels of the NS3 and
NS3/4A transcripts when the expression of these host genes was knocked down by
siRNAs, the NS3 and NS3/4A proteins appeared to be more stable when RAB40B,
RAB27B, but not TXNIP or Staufen 1, were knocked. One possibility is that although s
i-
lencing of these host genes may reduce HCV replication, this may also increase
in the
stability of NS3 or NS3/4A proteins by altering their location within the cell due to the
absence of these host proteins. Previously it has been shown that NS3 and NS3/4A pr
o-
tein expression decreased the level of expression of several proteins, which were part of
the innate immune response, leading to interferon production. In this study,
it was found
that the expression of NS3 and NS3/4A proteins, using adenovirus vectors, restored RIG-
I, MDA5 and MAVS expression levels in response to dsRNA stimulation, indicating that
the NS3 protein may have a role in regulating the expression of these host genes.
a-
tion (WHO) estimates the global prevalence of HCV to be approximately 2.2–3.0% with
130– 170 million infected individuals worldwide. HCV has the ability to evade the i
m-
m
une system and to establish persistent infections, which often result in chronic liver di
s-
ease. HCV encodes a long polyprotein which contain structural and non-structural pr
o-
teins. The Non-structural protein NS3 is likely to be involved in viral RNA replication by
interacting with viral and host components. In this study, a purified recombinant full-
length NS3 protein expressed in
E. coli
was examined for its ability to interact with
bi
otinylated full length HCV JFH-1 genomic RNA and the 3’ terminal of HCV negative
strand RNA. The results showed that NS3 could bind to both full length and the 3’ end
negative strand RNAs. RNA interference (siRNA) studies were also carried out to inve
s-
tigate the role of host genes RAB40B, RAB27B, TXNIP and Staufen 1, which had prev
i-
ously been shown to be important for HCV replication, on NS3 and NS3/4A proteins e
x-
pressed by adenovirus vectors using quantitative real-time polymerase chain reaction
(qRT-PCR) and FACS analysis. While there were reductions in the levels of the NS3 and
NS3/4A transcripts when the expression of these host genes was knocked down by
siRNAs, the NS3 and NS3/4A proteins appeared to be more stable when RAB40B,
RAB27B, but not TXNIP or Staufen 1, were knocked. One possibility is that although s
i-
lencing of these host genes may reduce HCV replication, this may also increase
in the
stability of NS3 or NS3/4A proteins by altering their location within the cell due to the
absence of these host proteins. Previously it has been shown that NS3 and NS3/4A pr
o-
tein expression decreased the level of expression of several proteins, which were part of
the innate immune response, leading to interferon production. In this study,
it was found
that the expression of NS3 and NS3/4A proteins, using adenovirus vectors, restored RIG-
I, MDA5 and MAVS expression levels in response to dsRNA stimulation, indicating that
the NS3 protein may have a role in regulating the expression of these host genes.
Version
Open Access
Date Issued
2013-09
Date Awarded
2013-07
Copyright Statement
Attribution NoDerivatives 4.0 International Licence (CC BY-ND)
Advisor
Karayiannis, Peter
McGarvey, Michael
Publisher Department
Medicine
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)