Hepatitis C virus nonstructural protein 4B: A journey into unexplored territory

Article (PDF Available)inReviews in Medical Virology 20(2):117-29 · March 2010with47 Reads
DOI: 10.1002/rmv.640 · Source: PubMed
Abstract
Hepatitis C virus (HCV) is a positive-strand RNA virus that replicates its genome in a membrane-associated replication complex. Nonstructural protein 4B (NS4B) induces the specific membrane alteration, designated as membranous web (MW), that harbours this complex. HCV NS4B is an integral membrane protein predicted to comprise four transmembrane segments in its central part. The N-terminal part comprises two amphipathic alpha-helices of which the second has the potential to traverse the membrane bilayer, likely upon oligomerisation. The C-terminal part comprises a predicted highly conserved alpha-helix, a membrane-associated amphipathic alpha-helix and two reported palmitoylation sites. NS4B interacts with other viral nonstructural proteins and has been reported to bind viral RNA. In addition, it was found to harbour an NTPase activity. Finally, NS4B has recently been found to have a role in viral assembly. Much work needs to be done with respect to further dissecting these multiple functions as well as providing a refined membrane topology and complete structure of NS4B. Progress in this direction should yield important insights into the functional architecture of the HCV replication complex and may reveal new opportunities for antiviral intervention against a leading cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma worldwide.
Rev. Med. Virol. (2010).
Published online in Wiley InterScience
(www.interscience.wiley.com)
Reviews in Medical Virology DOI: 10.1002/rmv.640
Hepatitis C virus nonstructural protein 4B:
a journey into unexplored territory
Je
´
ro
ˆ
me Gouttenoire
1
, Franc¸ois Penin
2
and Darius Moradpour
1
*
1
Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois, University of
Lausanne, Lausanne, Switzerland
2
Institut de Biologie et Chimie des Prote
´
ines, UMR 5086, CNRS, University of Lyon, IFR128 BioSciences
Gerland-Lyon Sud, Lyon, France
SUMMARY
Hepatitis C virus (HCV) is a positive-strand RNA virus that replicates its genome in a membrane-associated
replication complex. Nonstructural protein 4B (NS4B) induces the specific membrane alteration, designated as
membranous web (MW), that harbours this complex. HCV NS4B is an integral membrane protein predicted to
comprise four transmembrane segments in its central part. The N-terminal part comprises two amphipathic -helices
of which the second has the potential to traverse the membrane bilayer, likely upon oligomerisation. The C-terminal
part comprises a predicted highly conserved -helix, a membrane-associated amphipathic -helix and two reported
palmitoylation sites. NS4B interacts with other viral nonstructural proteins and has been reported to bind viral RNA.
In addition, it was found to harbour an NTPase activity. Finally, NS4B has recently been found to have a role in viral
assembly. Much work needs to be done with respect to further dissecting these multiple functions as well as
providing a refined membrane topology and complete structure of NS4B. Progress in this direction should yield
important insights into the functional architecture of the HCV replication complex and may reveal new opportunities
for antiviral intervention against a leading cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma
worldwide. Copyright # 2010 John Wiley & Sons, Ltd.
Received: 27 October 2009; Revised: 27 October 2009; Accepted: 27 October 2009
INTRODUCTION
The Hepatitis C virus (HCV) was identified 20
years ago as the most common etiological agent
of posttransfusion and sporadic non-A, non-B
hepatitis [1]. With an estimated 120–180 million
chronically infected individuals, it is now a lead-
ing cause of chronic hepatitis, liver cirrhosis and
hepatocellular carcinoma worldwide [2,3]. HCV
has been classified in the Hepacivirus genus within
the Flaviviridae family which also includes the
classical flaviviruses, the animal pestiviruses and
the GB viruses [4]. It contains a 9.6 kb positive-
strand RNA genome composed of a 5
0
noncoding
region (NCR), a long open reading frame encoding
a polyprotein precursor of about 3000 amino acids
and a 3
0
NCR (Figure 1) (reviewed in References
[5–8]).
Investigation of the viral life cycle has been lim-
ited by the low viral titers found in the sera and
livers of infected individuals and the lack of effi-
cient cell culture systems or small animal models
permissive for HCV. Despite these obstacles, great
progress has been made using heterologous
expression systems, functional cDNA clones [9],
the replicon system [10,11] and, most recently,
cell culture systems that allow to study the com-
plete viral life cycle in vitro (cell culture-derived
HCV, HCVcc) [12–14].
THE HCV LIFE CYCLE
The HCV life cycle is illustrated in Figure 2A. HCV
infects primarily hepatocytes and leads to persis-
tent infection in a majority of patients. Cell entry
RR EE V II E W
Copyright # 2010 John Wiley & Sons, Ltd.
*Corresponding author: D. Moradpour, Division of Gastroenterology
and Hepatology, Centre Hospitalier Universitaire Vaudois, BU44/07/
2421, Rue du Bugnon 44, CH-1011 Lausanne, Switzerland.
E-mail: Darius.Moradpour@chuv.ch
Abbreviations used
ATF6, basic leucine zipper-containing activating transcription factor
6; FRET, fluorescence resonance energy transfer; HCVcc, cell culture-
derived HCV;LD, lipid droplets; MW, membranous web; NCR, non-
coding region; NS4B, nonstructural protein 4B; PI4KIII, phosphati-
dylinositol 4-kinase III; RIG-I, retinoic acid-inducible gene I;
SREBP, sterol regulatory element binding protein; TM, transmem-
brane; UPR, unfolded protein response.
requires a complex set of factors, including the
low-density lipoprotein receptor and glycosamino-
glycans, scavenger receptor class B type I, the tet-
raspanin CD81 and the tight junction proteins
claudin-1 and occludin [15–18] (reviewed in Refer-
ences [19,20]). HCV enters the cell by clathrin-
mediated endocytosis, followed by a putative
low pH-mediated endosomal fusion process
[21,22]. The HCV genome is subsequently trans-
lated via an internal ribosome entry site present
in the 5
0
NCR. The resulting HCV polyprotein pre-
cursor is co-translationally and posttranslationally
processed by cellular and viral proteases into the
structural and nonstructural proteins (Figure 1).
The structural proteins include the core protein,
which forms the viral nucleocapsid, and the envel-
ope glycoproteins E1 and E2. The nonstructural
proteins include the p7 ion channel polypeptide
[23], the NS2-3 and NS3-4A proteases, an RNA
helicase located in the C-terminal two-thirds of
NS3, the nonstructural protein 4B (NS4B) and
NS5A proteins and the NS5B RNA-dependent
RNA polymerase. NS4B induces a specific mem-
brane alteration, designated as membranous web
(MW), that serves as a scaffold for the HCV repli-
cation complex (see below and Figure 2B) [24,25].
NS5A is an RNA-binding zinc phosphoprotein
that has been implicated in regulating the different
fates of the viral genome, i.e. replication versus
translation versus packaging [26–28], possibly by
tethering the viral RNA to membranes [29,30]
(reviewed in Reference [31]) and/or by providing
a physical link between replication complexes and
viral assembly sites on lipid droplets (see below).
Formation of a membrane-associated replication
complex, composed of viral proteins, replicating
RNA and altered cellular membranes, is a hall-
mark of all positive-strand RNA viruses, including
HCV (reviewed in References [32–34]). In this
complex, each viral protein is anchored to intracel-
lular membranes via specific determinants
(reviewed in Reference [8]). For example, mem-
brane association and structural organisation of
the NS3-4A complex are ensured in a cooperative
manner by two membrane binding determinants,
a transmembrane -helix formed by the N-term-
inal 21 amino acids of NS4A and an amphipathic
-helix at the N-terminus of NS3, allowing proper
positioning of the serine protease active site on the
membrane [35].
The mechanisms governing the assembly and
release of newly formed viral particles are still
poorly understood. Lipid droplets and the very-
low density lipoprotein pathway have recently
been found to play central roles in HCV assembly
and release, respectively [36–41]. Interestingly,
recent evidence indicates that most if not all
HCV nonstructural proteins are involved in infec-
tious particle assembly [27,28,42–45] (reviewed in
Reference [46]).
Among the HCV structural and nonstructural
proteins, NS4B is the least characterised. In fact,
this protein is particularly difficult to study due
to its highly hydrophobic character, integral mem-
brane association and lack of well-defined enzymatic
function. In the following, we will summarise current
knowledge of the structure and function of this
enigmatic viral nonstructural protein.
Figure 1. Genetic organisation and polyprotein processing of hepatitis C virus (HCV). The 9.6 kb positive-strand RNA genome with the
5
0
and 3
0
noncoding regions (NCRs) is schematically depicted at the top. Internal ribosome entry site-mediated translation yields a poly-
protein precursor, that is processed into the mature structural and nonstructural proteins. Black arrowheads denote cleavage sites of the
HCV polyprotein precursor by the ER signal peptidase and signal peptide peptidase. The grey and white arrowheads indicate cleavages
by the NS2-3 and NS3-4A proteases, respectively.
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MEMBRANE TOPOLOGY AND STRUCTURE
OF HCV NS4B
General features
Hepatitis C virus NS4B is a protein of Mr 27,000
containing 261 amino acids with predominantly
hydrophobic characteristics, as indicated by the
positive grand average of hydropathicity value
calculated for NS4B from different HCV genotypes
(http://www.expasy.org). NS4B is overall relatively
well conserved among the various HCV geno-
types, with 40% amino acid identity (Figure 3).
Interestingly, the first 50 amino acids of NS4B
are less well conserved than the rest of the protein.
There are no obvious amino acid sequence simila-
rities between NS4B from different Flaviviridae
family members and the lengths and predicted
molecular masses of these proteins vary signifi-
cantly (e.g. 248 amino acids [predicted Mr 26,800]
in Dengue virus, a typical flavivirus, and 347 amino
acids [predicted Mr 38,500] in bovine viral diar-
rhea virus, a typical pestivirus). Despite the lack
of obvious sequence similarity, prediction algo-
rithms [47] and the available experimental data
indicate that the NS4B proteins from all Flaviviridae
family members are integral membrane proteins
with several transmembrane segments [48–51].
Processing of NS4B
Hepatitis C virus NS4B is liberated from the poly-
protein precursor by the NS3-4A serine protease.
Interestingly, cleavages at the NS4A/NS4B and
NS4B/NS5A sites appear to be the last polyprotein
processing events. In fact, the first cleavage occurs
cotranslationally and liberates NS3 from the
remainder of the polyprotein. Subsequent proces-
sing events are mediated in trans, with rapid pro-
cessing at the NS5A/NS5B site. The resulting
NS4A-5A precursor is cleaved first between
NS4A and NS4B, resulting in a relatively stable
NS4B-5A intermediate, and subsequently between
NS4B and NS5A [52,53]. Based on a recently
reported model of the membrane-associated NS3-
4A complex [35], cleavage at the NS4A/NS4B and
NS4B/NS5A sites is believed to occur in a strictly
defined position at the membrane surface.
Subcellular localisation of NS4B
Nonstructural protein 4B or NS4B-GFP fusion pro-
teins expressed alone localise to the ER membranes
as well as to seemingly ER-derived modified mem-
branes which form cytoplasmic dot-like structures
or foci [48,50,54–57]. At the ultrastructural level,
these cytoplasmic dot-like structures correspond to
collections of membrane vesicles embedded in a
membrane matrix, a specific, NS4B-induced mem-
brane alteration designated as MW [24,25] (Figure
2B). The MW is very similar to the ‘sponge-like
inclusions’ previously found by electron microscopy
in the liver of HCV-infected chimpanzees [58], har-
bours the bulk of viral nonstructural proteins upon
Figure 2. (A) Hepatitis C virus life cycle. (1) The virus interacts
with a complex set of entry factors and is internalised by endocy-
tosis; (2) release of the viral positive-strand RNA genome into the
cytoplasm; (3) genome translation via an internal ribosome entry
site, polyprotein processing and membrane association of the vir-
al structural and nonstructural proteins; (4) RNA replication
occurs in the MW via a negative-strand intermediate; (5) newly
synthesised positive-strand RNA is packaged and virions are
assembled on lipid droplets (LD) and (6) maturation and release
of viral particles. (B) Membranous web. Electron micrograph from
a Huh-7 cell harbouring a subgenomic HCV replicon. A distinct
membrane alteration composed of small vesicles embedded in a
membrane matrix, designated as membranous web (arrowheads),
is found in the juxtanuclear region. Note the circumscript nature
of the membranous web and the close association with cisternae
of the rough ER. Bar, 1 mm. M, mitochondria; N, nucleus. See
Reference [25] for further details.
Hepatitis C virus nonstructural protein 4BHepatitis C virus nonstructural protein 4B
Copyright # 2010 John Wiley & Sons, Ltd. Rev. Med. Virol. (2010).
DOI: 10.1002/rmv
expression of the HCV polyprotein [24] and represents
the major site of viral RNA synthesis in Huh-7
cells harbouring HCV replicons [25]. Therefore,
one important function of NS4B is to induce the
formation of the MW, the specific membrane
alteration that harbours the HCV replication complex.
Membrane topology
Nonstructural protein 4B is believed to comprise
an N-terminal part (amino acids 1 to 69), a central
part harbouring four transmembrane passages
(amino acids 70 to 190), and a C-terminal
part (amino acids 191 to 261) (Figure 4). As a
consequence of polyprotein processing by the
NS3-4A protease, the N- and C-terminal parts are
believed to be located on the cytosolic side of the
ER membrane. Indeed, early in vitro transcrip-
tion–translation and proteinase K digestion experi-
ments performed in the presence of microsomal
membranes revealed that the bulk of NS4B is cyto-
Figure 3. Overview of primary and secondary structure of HCV NS4B. The amino acid repertoire deduced from ClustalW multiple align-
ments of the 27 representative full-length NS4B sequences (amino acids 1–261) from confirmed HCV genotypes and subtypes (listed with
accession numbers in Table 1 in Reference [119]), including the recently described genotype 7a (accession number EF108306; see the
European HCV Database [http://euhcvdb.ibcp.fr/] for details) is presented here, with the consensus sequence shown at the top. The
degree of amino acid and physicochemical conservation at each position can be inferred from the extent of variability (with amino acids
listed in decreasing order of frequency from top to bottom) together with the similarity index according to Clustal W convention (aster-
isk, invariant; colon, highly similar; dot, similar; [120]). A predicted amphipathic -helix is found between amino acids 4 and 24 (AH1,
dashed box) [54,57]. A second, membrane-associated amphipathic -helix extends from amino acids 42 to 66 (AH2, box) [57]. The central
part of NS4B is predicted to comprise four transmembrane (TM) segments, denoted as TM1 through TM4 (grey boxes). A predicted P-
loop NTPase motif is boxed [94, 95] (see text for details). A highly conserved -helix is predicted between amino acids 201 and 212 in the
C-terminal part (H1, dashed box) while the structure of a ‘twisted’ amphipathic -helix extending from amino acids 229 to 253 was
resolved recently (H2, box) [61].
J. GouttenoireJ. Gouttenoire
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DOI: 10.1002/rmv
solically oriented [48], but a refined membrane
topology is so far elusive. Insertion of artificial gly-
cosylation acceptor sites at various positions in
NS4B yielded evidence in support of the predicted
ER luminal loops around amino acid positions 112
and 161 [50,56]. Intriguingly, these studies sug-
gested that the N-terminus of NS4B is translocated
into the ER lumen at least partially, presumably by
a posttranslational mechanism [50]. This observa-
tion was confirmed more recently. More specifi-
cally, an amphipathic -helix extending from
amino acid 42 to 66 (AH2, Figures 4 and 5) was
found to have the potential to traverse the phos-
pholipid bilayer as a transmembrane segment,
likely upon oligomerisation [57]. Site-directed muta-
genesis showed that, similar to other HCV nonstruc-
tural protein membrane segments [35,59,60], this
segment plays an important role in the assembly
of a functional replication complex [57]. Interest-
ingly, transmembrane orientation of the N-terminus
was found to be reduced by coexpression of the
other nonstructural proteins, particularly of NS5A,
suggesting that the membrane topology of the N-
terminal part of NS4B may be dynamic and modu-
lated by protein–protein interactions within the
HCV replication complex [56,57].
Based on sequence homology, the N-terminal
part of NS4B was proposed to comprise a basic
leucine zipper motif spanning amino acids 22–49
but this observation remains to be confirmed
experimentally [47]. In addition, an amphipathic
-helix predicted within the first 27 amino acids
of NS4B (AH1) was reported to mediate mem-
brane association [54]. More recent data, however,
indicate that AH1 does not represent a bona fide
membrane segment and that previous observa-
tions may be explained by a particular choice of
expression constructs [57].
Secondary structure analyses predict two -
helices in the C-terminal portion of NS4B
[45,47,61] (Figures 3 and 4). The first helix (H1)
(amino acids 200–213) is highly conserved
( 85% amino acid identity among the various
HCV genotypes) while the second helix (H2) (ami-
no acids 229–252) is more variable ( 30% amino
acid identity) but displays conserved hydropathic
characteristics ( 67% amino acid similarity).
Determination of the three-dimensional structure
Figure 4. NS4B membrane topology. The schematic representation of the NS4B protein on the ER membrane shows the N-terminal
amphipathic helices (AH1, AH2), the transmembrane segment (TM1-4) and the C-terminal helices (H1, H2). The N-terminus of NS4B
may be translocated into the ER, likely upon oligomerisation of AH2 (arrow), thereby creating a fifth transmembrane passage. A pre-
dicted P-loop NTPase motif is boxed [94,95] (see text for details). The palmitoylation sites described at the C-terminus of NS4B are repre-
sented schematically (see text for details) [65].
Figure 5. Membrane association of HCV NS4B amphipathic -
helix 42–66. The three-dimensional structure of the NS4B amino
acid 40–69 segment obtained by nuclear magnetic resonance (PDB
entry 2JXF, [57]) is represented in interaction with the phospholi-
pid bilayer, where N and C correspond to the N- and C-terminal
extremities of the helix, respectively. The cytosolic side of the
membrane is at the top and the ER luminal side at the bottom.
The amphipathic -helix extends from amino acid 42 to 66 and
possesses a hydrophobic face comprising aromatic amino acid
residues (Phe and Trp). As a monomer this segment may interact
with the membrane surface in an in-plane topology, with the
hydrophilic side facing the cytosolic side (left panel). This seg-
ment has the potential to traverse the membrane, likely upon oli-
gomerisation. See Reference [57] for details.
Hepatitis C virus nonstructural protein 4BHepatitis C virus nonstructural protein 4B
Copyright # 2010 John Wiley & Sons, Ltd. Rev. Med. Virol. (2010).
DOI: 10.1002/rmv
of H2 by nuclear magnetic resonance revealed a
‘twisted’ amphipathic -helix extending from ami-
no acids 229 to 253 [61]. We and others have
recently reported that H2 mediates membrane
association [61–64]. Therefore, membrane associa-
tion of HCV NS4B is mediated not only by trans-
membrane domains in its central part but also by
determinants for membrane association in the N-
and C-terminal parts.
Yu et al. [65] reported the presence of two palmi-
toylation sites at the very C-terminus of NS4B (Cys
257 and 261). However, Cys 257 is not conserved
among different HCV genotypes and abrogation
of this palmitoylation site does not affect HCV
RNA replication (J. G. and D. M., unpublished
data). The role of Cys 261 is more difficult to ascer-
tain, as this corresponds to the P1 residue of the
NS4B/NS5A polyprotein processing site. Thus,
the role of C-terminal palmitoylation of NS4B
remains to be explored further.
NS4B oligomerisation
Similar to other HCV nonstructural proteins
[30,66–69], NS4B has been reported to form oligo-
mers [65]. Indeed, cross-linking studies provided
evidence for the formation of at least trimers and
suggested that C-terminal palmitoylation plays
an important role in this process [65]. By fluores-
cence resonance energy transfer (FRET) and coim-
munoprecipitation, we recently obtained evidence
for multiple inter- and intramolecular contacts
being involved in the oligomerisation of NS4B.
AH2 appears to play a critical role in this process
(Gouttenoire et al., in preparation). Thus, it is tempt-
ing to speculate that NS4B exerts its functions as
multimeric arrays on intracellular membranes.
FUNCTIONS OF NS4B IN THE HCV
LIFE CYCLE
Given the limited coding capacity of the HCV gen-
ome, it is likely that each viral protein has multiple
functions. Indeed, a variety of functions have been
postulated for NS4B, as discussed in this section.
Formation of the HCV replication complex
As discussed above, one of the best documented
functions of NS4B is its ability to induce the
MW, i.e. the specific membrane alteration that
harbours the HCV replication complex [24,25].
However, the mechanisms by which NS4B induces
membrane vesicles formation are unknown. In this
context, it is tempting to speculate that oligomeri-
sation may play an important role by inducing
membrane curvature, similar to other oligomeric
membrane proteins. The amphipathic -helices at
the N- and C-termini as well as the predicted
transmembrane segments in the centre of NS4B
may be directly implicated in this process. Indeed,
some cellular proteins such as epsin or amphiphysin
can induce local membrane curvature by inserting
amphipathic -helices between the phospholipid
head groups [70]. Another example is the small
GTPase Sar1 which induces membrane curvature
by inserting an N-terminal amphipathic -helix
into the membrane upon GTP hydrolysis [71]. In
addition, certain host factors involved in HCV
replication may have important roles. Such factors
include, among others, the early endosome resi-
dent small GTPase Rab 5 [72], cytoskeletal proteins
[73–75] and phosphatidylinositol 4-kinase III
(PI4KIII) [76–79]. This latter candidate is particu-
larly interesting, as it was identified by siRNA
screens performed by several independent groups.
Knockdown of PI4KIII interferes with MW for-
mation and inhibits HCV RNA replication. Inter-
estingly, PI4KIII has previously been reported
to interact with HCV NS5A [80]. However, the
precise mechanism by which PI4KIII is involved
in MW and HCV replication complex formation
remains to be elucidated. Based on the replication
complex model proposed by Quinkert et al. [81],
NS4B may have to bend the membrane surface
in a concave manner to maintain the nonstructural
proteins and the viral RNA inside newly formed
replication vesicles. This hypothesis is strongly
supported by recent electron tomography data
for the replication complex of the related Dengue
virus [82]. Clearly, future work will have to
address how HCV NS4B induces membrane cur-
vature in a concave manner to form the replication
vesicles constituting the MW.
Site-directed mutagenesis in the replicon and
HCVcc systems has demonstrated the essential
role of NS4B in HCV RNA replication
[45,54,57,61,83,84] and has revealed an additional
role of NS4B in viral assembly [45] (J. G. and D.
M., unpublished data). Moreover, a cell culture
adaptive change of Lys 135 to Thr in NS4B from
the genotype 1b Con1 strain was described to sig-
nificantly enhance HCV RNA replication in Huh-7
cells in vitro [85]. Interestingly, this K135T change
targets a conserved position, that is occupied by a
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DOI: 10.1002/rmv
positively charged residue (Lys or Arg) in all HCV
isolates. Taken together, these observations indi-
cate that NS4B plays a central role in HCV RNA
replication and assembly.
Nonstructural protein 4B is a component of the
replication complex which also includes the other
nonstructural proteins involved in RNA replica-
tion, i.e. NS3-4A, NS5A and NS5B. In this context,
physical interactions between NS4B and other
nonstructural proteins have been demonstrated
by glutathione S-transferase pull-down experi-
ments [86], far western analyses [87] and FRET
studies (J. G. and D. M., unpublished observa-
tions). Moreover, intragenotypic domain swap-
ping studies carried out in the replicon system
revealed genetic evidence for an interaction
between NS3 and NS4B [88]. Finally, the observa-
tion by Lundin et al. [56] that coexpression of other
nonstructural proteins, particularly of NS5A,
reduces ER luminal translocation of the N-termi-
nus of NS4B (see above) points toward physical
interactions within a higher-order replicase com-
plex. Collectively, these data suggest that NS4B
provides an important protein–protein interaction
platform within the HCV replication complex.
A recent study using microfluidic affinity analy-
sis described an interaction between HCV RNA
and in vitro transcribed and translated NS4B [89].
This interaction appears to be selective for the 3
0
end of the viral negative-strand RNA and involves
several Arg residues located predominantly in the
C-terminal part of NS4B. Thus, NS4B may be
implicated not only in MW formation and pro-
tein–protein interactions within the replication
complex but it may also directly interact with the
replicating RNA. This may serve, for example to
retain the negative-strand for template use or to
tether it to the membrane of replication vesicles
during unwinding.
Integral membrane association and the critical
role in inducing the membrane alteration which
serves as a scaffold for the viral replication may
explain the observation that the function of NS4B
in RNA replication of HCV [90] but also of related
viruses such as bovine viral diarrhea virus [91]
and the flavirius Kunjin [92] cannot be comple-
mented in trans. However, this notion was chal-
lenged recently by studies performed with the
highly efficient HCV JFH-1 strain. Indeed, Jones
et al. [45] recently demonstrated that replication
of an NS4B-defective subgenomic replicon could
be rescued by a replicon harbouring an inactivat-
ing mutation in NS5A, indicating that NS4B can
be complemented in trans by a helper replicon.
Thus, it appears that the nonstructural proteins
provided by the two replicons can mix to reconsti-
tute a functional replication complex.
Virus assembly
Similar to the other HCV nonstructural proteins
(see above), NS4B has recently been found to
also have a role in virus assembly [45] (J. G. and
D. M., unpublished data). Indeed, Jones et al. [45]
identified a mutation in the C-terminal region of
NS4B (N216A) that enhances HCVcc production
without affecting HCV RNA replication. This
highly conserved position, located between helices
H1 and H2 (Figures 3 and 4), might be critical for
interactions between NS4B and other components
of the viral assembly machinery. However, the
exact mechanisms by which NS4B is involved in
the late steps of the HCV life cycle remain to be
explored.
NTPase activity
Hepatitis C virus NS4B has been reported to pos-
sess an intriguing enzymatic activity which was
suggested initially by a nucleotide-binding motif
mapped to amino acids 129–135. A typical ‘GXXXXGK’
motif corresponding to the P-loop of a number of
NTPases is found in almost all HCV NS4B
sequences in this position. However, the motif is
not absolutely conserved and shows the consensus
sequence (G/A/D)XXXXG(K/R) (where X indi-
cates any amino acid) when all HCV genotypes
are compared (Figure 2). This particular feature
appears to be specific for HCV NS4B, as a similar
motif is not found in NS4B from other Flaviviridae
family members. Biochemical studies showed that
HCV NS4B not only binds but also hydrolyses
ATP and GTP. While recombinant protein pro-
duced in insect cells showed an ATPase activity
in vitro [93], an NS4B-GFP fusion protein was
found to bind preferentially GTP in transfected
Huh-7 cells [94]. A recent in-depth biochemical
characterisation of this activity demonstrated that
NS4B binds both ATP and GTP but has a higher
affinity for ATP [95]. Investigation of the enzy-
matic functions mediated by purified NS4B also
revealed both adenylate kinase and nucleotide
hydrolase activities. The adenylate kinase activity
catalyses the synthesis of ATP and AMP from
Hepatitis C virus nonstructural protein 4BHepatitis C virus nonstructural protein 4B
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DOI: 10.1002/rmv
two ADP molecules. This ability of NS4B to cata-
lyse the production of ATP may provide a certain
energy autonomy to the virus.
These observations are reminiscent of poliovirus
2C, a protein required for the induction of specific
membrane alterations [96], which also possesses
NTPase and RNA-binding activities [97,98].
Indeed, cellular membrane alterations and vesicle
trafficking are often regulated by NTPases such as
the small GTPases of the Rho family [99]. Recent
life cell imaging studies suggest the existence of
two types of replication complexes of which the
smaller show fast and saltatory movements [75].
Thus, NTPase activity of NS4B may be involved
in the regulation of such movements as well as
other trafficking events and membrane rearrange-
ments. Nonetheless, this enzymatic activity of
NS4B remains somewhat controversial, as a muta-
tion of Lys 135 to Thr within the predicted P-loop
motif was identified as cell culture-adaptive
change in Con1-derived HCV replicons in Huh-7
cells (see above). In addition, the P-loop motif is
not fully conserved, as discussed above. Therefore,
future studies will have to address the role of this
enigmatic enzymatic activity of NS4B in the viral
life cycle. Clearly, structural data for HCV NS4B
should allow for significant advances in this direction.
A ROLE FOR NS4B IN THE PATHOGENESIS
OF HEPATITIS C?
ER stress
It is likely that the intimate interactions of HCV
proteins with cellular membranes result in the acti-
vation of ER stress pathways [100]. NS4B may play
an important role in this process due to its multi-
spanning membrane topology and its capacity to
alter cellular membranes. Indeed, studies have
reported that NS4B expression may induce ER
stress and the unfolded protein response (UPR)
[101,102]. At the same time, the formation of a cir-
cumscript membrane alteration in a host cell
whose ultrastructure is otherwise unaltered may
represent a mechanism to limit ER stress and
apoptosis of the infected cell. In order to prevent
the accumulation of misfolded proteins in the
ER, the UPR involves ER membrane transducers
such as the double-stranded RNA-activated pro-
tein kinase-like ER kinase, inositol-requiring
kinase 1 and the basic leucine zipper-containing
activating transcription factor 6 (ATF6). Among
these factors, the two forms of ATF6, ATF6 and
ATF6, were described as potent interactors with
NS4B [103]. ATF6 cleavage liberates soluble forms
of this protein which then act as a transcription
factor to activate a specific gene response [104].
Thus, activation of ER stress pathways and the
UPR by NS4B may support a high yield of well-
folded HCV proteins at the ER. However, much
has to be learned about the role of NS4B, ER stress
and the UPR in the HCV life cycle.
Lipid metabolism
Cholesterol depletion in the cell induces the pro-
teolytic cleavage of sterol regulatory element bind-
ing proteins (SREBPs) and activates genes related
to cholesterol and fatty acid metabolism [105].
Interestingly, induction of SREBP cleavage was
observed in cells expressing NS4B alone as well
as in cells infected with HCVcc [106]. NS4B-
mediated activation of the SREBP signaling path-
way leading to fatty acid synthase upregulation
and lipid accumulation is dependent on the phos-
phoinositol-3 kinase pathway [107]. Modulation of
lipid metabolism by NS4B might reflect the neces-
sity for lipid constituents for membrane rearrange-
ments. It is tempting to speculate that this activity
may also be related to the induction of lipid dro-
plets as viral assembly platforms. However, the
involvement of NS4B in alterations of lipid meta-
bolism may also contribute to the pathogenesis of
liver steatosis in patients with chronic hepatitis C.
Carcinogenesis
Heterologous overexpression of HCV NS4B was
found to transform NIH3T3 cells in cooperation
with the HA-ras oncogene [108]. More recently,
the NTPase activity of NS4B was implicated in
this cellular transformation activity [89]. However,
the relevance of these observations to the develop-
ment of hepatocellular carcinoma in patients with
HCV-induced liver cirrhosis remains unknown. Of
note, transgenic mice expressing NS4B in the liver
did not display any tumor development [109].
Innate immunity
Hepatitis C virus has evolved numerous mechan-
isms to evade and counteract host immune
responses (reviewed in References [110,111]). For
example, the NS3-4A protease has been shown to
J. GouttenoireJ. Gouttenoire
et alet al
..
Copyright # 2010 John Wiley & Sons, Ltd. Rev. Med. Virol. (2010).
DOI: 10.1002/rmv
cleave and thereby inactivate crucial adaptor
molecules in the retinoic acid-inducible gene I
(RIG-I) and Toll-like receptor 3 viral RNA sensing
pathways [112,113]. In this context, NS4B from
various Flaviviridae family members, including
West Nile, yellow fever and Dengue viruses, was
shown to repress antiviral host defenses by inhibit-
ing type I interferon signaling [114–116] whereas
HCV NS4B did not display the same feature
[115]. However, one report suggested that RIG-I-
mediated interferon activation was inhibited by
HCV NS4B [117]. In addition, NS4B was also iso-
lated as a factor inhibiting the antiviral activity of
interferon- [118]. These recent observations,
which need to be confirmed, would suggest a
direct role of NS4B in the escape of HCV from
innate immune responses.
CONCLUSIONS AND PERSPECTIVES
Twenty years after the identification of HCV,
important aspects of NS4B remain poorly under-
stood. A refined membrane topology and a struc-
ture of the complete protein represent major
challenges for the future. Advances in this direc-
tion should yield novel insights into the functional
architecture of the HCV replication complex, enzy-
matic functions of NS4B and the mechanisms gov-
erning MW formation. Much work needs to be
done also with respect to the newly identified
role of NS4B in viral assembly and release. Pro-
gress in these directions will almost certainly
reveal new opportunities for antiviral intervention
against hepatitis C.
ACKNOWLEDGMENTS
Work in the authors’ laboratories is supported by
the Swiss National Science Foundation (3100A0-
122447), the Swiss Cancer League/Oncosuisse
(OCS-01762-08-2005), the Leenaards Foundation,
the French Centre National de la Recherche
Scientifique (CNRS), the Agence Nationale pour
la Recherche sur le SIDA et les He
´
patites Virales
(ANRS), and the program Biotherapeutics of
Lyon Biopole.
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Hepatitis C virus nonstructural protein 4BHepatitis C virus nonstructural protein 4B
Copyright # 2010 John Wiley & Sons, Ltd. Rev. Med. Virol. (2010).
DOI: 10.1002/rmv
    • "Alpha helices analyzed by solution-state NMR in a context of short peptides (Gouttenoire et al. 2009b) and predicted H1 helix are labeled by solid boxes. Predicted transmembrane segments are shown with grey-highlighted background (Gouttenoire et al. 2010). Loop 1–2 which was predicted to be a putative transmembrane domain is underlined and in italic (Palomares-Jerez et al. 2012). "
    [Show abstract] [Hide abstract] ABSTRACT: We describe the expression of the hepatitis C virus nonstructural protein 4B (NS4B), which is an integral membrane protein, in a wheat germ cell-free system, the subsequent purification and characterization of NS4B and its insertion into proteoliposomes in amounts sufficient for multidimensional solid-state NMR spectroscopy. First spectra of the isotopically [2H,13C,15N]-labeled protein are shown to yield narrow 13C resonance lines and a proper, predominantly α-helical fold. Clean residue-selective leucine, isoleucine and threonine-labeling is demonstrated. These results evidence the suitability of the wheat germ-produced integral membrane protein NS4B for solid-state NMR. Still, the proton linewidth under fast magic angle spinning is broader than expected for a perfect sample and possible causes are discussed.
    Full-text · Article · May 2016
    • "The NTD contains a basic leucine zipper (residues 20e55) which mediates interaction of the NS4B with the human ER stress response element protein (ATF6) causing transcription of chaperones that facilitate the viral protein folding [20,24e26]. The CTD (residues 191e261) composes of two predicted a-helical domains , helix-1 (H1) which is highly conserved among the HCV genotypes and helix-2 (H2) with amphipathic structure for formation of the RC [20,25,27] . The CTD also contains a nucleosidebinding motif (NBM) or Walker B (228DAAA231) and RNA binding motif (192RR193) that binds to RNA and tethers the viral genome to the membrane web to facilitate positive-sense RNA synthesis; both motives are important for HCV replication [28]. "
    [Show abstract] [Hide abstract] ABSTRACT: NS4B of hepatitis C virus (HCV) initiates membrane web formation, binds RNA and other HCV proteins for viral replication complex (RC) formation, hydrolyses NTP, and inhibits innate anti-viral immunity. Thus, NS4B is an attractive target of a novel anti-HCV agent. In this study, humanized-nanobodies (VHs/VHHs) that bound to recombinant NS4B were produced by means of phage display technology. The nanobodies were linked molecularly to a cell penetrating peptide, penetratin (PEN), for making them cell penetrable (become transbodies). Human hepatic (Huh7) cells transfected with HCV JFH1-RNA that were treated with transbodies from four E. coli clones (PEN-VHH7, PEN-VHH9, PEN-VH33, and PEN-VH43) had significant reduction of HCV RNA amounts in their culture fluids and intracellularly when compared to the transfected cells treated with control transbody and medium alone. The results were supported by the HCV foci assay. The transbody treated-transfected cells also had upregulation of the studied innate cytokine genes, IRF3, IFNβ and IL-28b. The transbodies have high potential for testing further as a novel anti-HCV agent, either alone, adjunct of existing anti-HCV agents/remedies, or in combination with their cognates specific to other HCV enzymes/proteins.
    Full-text · Article · May 2016
    • "The transmembrane domain of NS4B, by contrast, showed reduced capability in co-precipitating E2. Both domains are known to contain the amphipathic helix (AH) which tethers NS4B to ER-membrane [46]. Furthermore, soluble E2 (sE2) that is deprived of its TMD failed to precipitate NS4B, implying the TMD domain of E2 is required for this interaction (Fig 6E). "
    [Show abstract] [Hide abstract] ABSTRACT: Hepatitis C virus (HCV) poses a global threat to public health. HCV envelop protein E2 is the major component on the virus envelope, which plays an important role in virus entry and morphogenesis. Here, for the first time, we affinity purified E2 complex formed in HCV-infected human hepatoma cells and conducted comparative mass spectrometric analyses. 85 cellular proteins and three viral proteins were successfully identified in three independent trials, among which alphafetoprotein (AFP), UDP-glucose: glycoprotein glucosyltransferase 1 (UGT1) and HCV NS4B were further validated as novel E2 binding partners. Subsequent functional characterization demonstrated that gene silencing of UGT1 in human hepatoma cell line Huh7.5.1 markedly decreased the production of infectious HCV, indicating a regulatory role of UGT1 in viral lifecycle. Domain mapping experiments showed that HCV E2-NS4B interaction requires the transmembrane domains of the two proteins. Altogether, our proteomics study has uncovered key viral and cellular factors that interact with E2 and provided new insights into our understanding of HCV infection.
    Full-text · Article · Jan 2016
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