NATURE|Vol 436|18 August 2005|doi:10.1038/nature04077
Unravelling hepatitis C virus
replication from genome to function
Brett D. Lindenbach1and Charles M. Rice1
Since the discovery of the hepatitis C virus over 15 years ago, scientists have raced to develop
diagnostics, study the virus and find new therapies. Yet virtually every attempt to dissect this pathogen
has met with roadblocks that impeded progress. Its replication was restricted to humans or experimentally
infected chimpanzees, and efficient growth of the virus in cell culture failed until very recently. Nevertheless
hard-fought progress has been made and the first wave of antiviral drugs is entering clinical trials.
virus life cycle: first, as a messenger RNA (mRNA) for translation of
the viral proteins; second as a template for RNA replication; and third,
as a nascent genome packaged within new virus particles. Virions pre-
sumably form by budding into the endoplasmic reticulum (ER) and
leave the cell through the secretory pathway.
Researchers have followed each aspect of the virus life cycle in turn.
Just as infection starts from an HCV genome entering the cytoplasm
and progressing through translation, replication and particle produc-
tion, our understanding has progressed from having a genome
sequence to understanding translation and the viral gene products,
characterizing RNA replication, and establishing systems to charac-
terize virus particles and infectivity. In this review, we summarize the
current understanding of HCV replication with special emphasis on
recent developments. As space is limited, we cannot be comprehensive;
readers may wish to consult other reviews for detail and breadth5,13,14.
Initial studies: HCV translation and polyprotein processing
The identification of HCV yielded a partial viral genome sequence5.
Research in the early 1990s focused on dissecting HCV gene expres-
sion and characterizing the gene products. Much has been learned
about the biochemistry of three key enzymes, and structural informa-
tion is now available at the atomic level for roughly half of the protein-
Translation of the HCV genome, which lacks a 5?cap, depends on
an internal ribosome entry site (IRES) within the 5?-noncoding region
(NCR). The HCV IRES binds 40S ribosomal subunits directly and
avidly, bypassing the need for pre-initiation factors, and inducing an
mRNA-bound conformation in the 40S subunit15. The IRES–40S com-
plex then recruits eukaryotic initiation factor (eIF) 3 and the ternary
complex of Met-tRNA–eIF2–GTP to form a non-canonical 48S inter-
mediate, before a kinetically slow transition to the translationally
active 80S complex16,17.
Once initiated, translation of the HCV genome produces a large
polyprotein that is proteolytically cleaved to produce 10 viral proteins
(Fig. 2a). The amino-terminal one-third of the polyprotein encodes
the virion structural proteins: the highly basic core (C) protein, and
glycoproteins E1 and E2. After the structural region comes a small
integral membrane protein, p7, which seems to function as an ion
channel18,19. The remainder of the genome encodes the nonstructural
(NS) proteins NS2, NS3, NS4A, NS4B, NS5A and NS5B, which coor-
dinate the intracellular processes of the virus life cycle. The structural
In the mid-1970s, it was noticed that the world’s supply of blood was
contaminated with an unidentified agent causing post-transfusion
non-A, non-B hepatitis1. Yet it was not until 1989 that the first
sequences of hepatitis C virus (HCV) were reported2. The difficulty in
identifying the virus and in detecting viral RNA and antigens in
infected tissues left the impression that HCV replicated poorly in vivo.
This is not so. Chronically infected patients have viral loads that typi-
cally range from 103–107genomes per ml of serum. Mathematical
modelling of viral dynamics during treatment with interferon-?(IFN-
?) indicates that HCV virions turn over rapidly (with a half-life about
3 h), and up to about 1012viruses are produced per day in an infected
person3. This is about 100-fold greater than the rate reported for HIV.
High viral loads are observed during the first few weeks of HCV
infection, but inflammatory processes leading to liver injury are
delayed, usually occurring after 2–3 months4. Liver transplant recipi-
ents generally have favourable short-term outcomes despite efficient
allograft reinfection and high levels of viraemia owing to immunosup-
pression. These observations have led to the idea that HCV is relatively
noncytopathic and that liver disease is immune-mediated. Although
the liver is the major site of HCV replication, evidence exists for extra-
hepatic reservoirs including peripheral blood lymphocytes (reviewed
in ref. 5), epithelial cells in the gut6and the central nervous system7.
With the hepatitis B virus immunohistochemistry can be used to iden-
tify infected hepatocytes reliably, but we do not have a clear picture of
the number of HCV-infected hepatocytes in the liver or the character-
istics of an HCV-infected cell. Nevertheless, gene-profiling studies of
HCV-infected livers indicate that this organ is a veritable battleground
of ongoing viral replication and host antiviral defences8–11. Although the
origin of HCV and the timing of its introduction into the human pop-
ulation are not known, the high error rate of RNA-dependent RNA
replication and the battle between virus and host have generated
remarkable global diversity. HCV is currently divided into six major
genotypes with numerous subtypes and exists as a quasispecies swarm
within the infected individual12.
We will use an idealized HCV life cycle (Fig. 1) as a framework for
discussing the current state of our knowledge. Enveloped virus parti-
cles interact with specific surface receptors and are probably internal-
ized. Fusion of the viral and cellular membranes, presumably triggered
by the low pH of the endocytic compartment, leads to the release of a
single-stranded (ss), positive-sense RNA genome into the cytoplasm
of a newly infected cell. This genome serves multiple roles within the
1Center for the Study of Hepatitis C, The Rockefeller University, 1230 York Avenue, New York, New York 10021, USA.
NATURE|Vol 436|18 August 2005
proteins mature by signal peptidase cleavages between C/E1, E1/E2
and E2/p7. In addition, signal-peptide peptidase releases core from the
E1 signal peptide. Within the NS region, the p7/NS2 junction is also
cleaved by signal peptidase. Further proteolytic processing within the
NS region occurs through the action of two viral enzymes, the NS2
autoprotease, which cleaves at the NS2/3 junction; and the NS3-4A
serine protease, which cleaves at all downstream sites (Fig. 2a, b). HCV
also encodes a small protein, called F (frame shift) or ARFP (alterna-
tive reading frame protein), that can be produced by ribosomal frame
shifting into an alternative reading frame within the core gene
(reviewed in ref. 20).
HCV encodes two remarkable proteases
The carboxy-terminal two-thirds of NS2 contain the catalytic triad of
a cysteine protease. NS2/3 cleavage requires these residues as well as
the downstream expression of the NS3 serine protease domain,
although NS3-4A protease activity is dispensable for NS2/3 process-
ing. The requirement for NS3 may have to do with correct folding, as
NS2/3 cleavage is enhanced by Zn2+, which has a structural role in sta-
bilizing the NS3 fold.
NS3 is a multifunctional protein, with an N-terminal serine pro-
tease domain and a C-terminal RNA helicase/NTPase domain. Both
enzyme activities have been well characterized, and high-resolution
structures have been solved14. The serine protease domain has a typi-
cal chymotrypsin-like fold, positioning three catalytically active
residues at the surface interface between two ?-barrel domains
(Fig. 3a). For complete folding and serine protease activity NS3
requires the intercalation of a ?-strand present in NS4A (refs 21–23).
NS4A is a small (54-amino-acid) protein that anchors NS3 to cellular
membranes through an N-terminal hydrophobic peptide24. Proper
folding of the serine protease domain also requires coordination of
Zn2+by three cysteine residues distal from the active site. NS3 has an
unusually shallow substrate-binding pocket for a serine protease,
which has challenged efforts to obtain specific inhibitors. Neverthe-
less, the discovery of product inhibition led to the development of
potent inhibitors that block NS3 serine protease activity, NS protein
processing, and HCV RNA replication. For further details, see the
accompanying article by De Francesco and Migliaccio (page 953).
Dissecting the structure and function of HCV NS proteins
The C terminus of NS3 encodes a DExH/D-box RNA helicase. These
enzymes use the energy of NTP hydrolysis to unwind double-stranded
RNA, and NS3 unwinds RNA and DNA homoduplexes and heterodu-
plexes in a 3?to 5?direction25. The helicase mechanism is not yet fully
understood, but recent kinetic analyses show that the NS3 helicase
behaves like a ratcheting two-stroke motor26and seems to function as
a dimer that incrementally rips apart 18-base-pair stretches of sub-
strate RNA27. In addition, NS3 helicase activity can be regulated by
interactions between the serine protease and helicase domains of NS3
(refs 28, 29), indicating that these two enzyme activities may be some-
how coordinated during replication. The function of the HCV helicase
is not known, but it may be involved in the initiation of RNA synthe-
sis on the HCV genome RNA, which contains stable 3?-terminal sec-
ondary structure, in dissociation of nascent RNA strands from their
template during RNA synthesis, or in displacement of proteins or
other trans-acting factors from the RNA genome.
NS5A is a fascinating protein. Many cellular proteins interact with
NS5A, although their functional relevance is largely unclear30–32. NS5A
is phosphorylated on multiple serine residues by cellular kinases and
can be found in hypophosphorylated (56 kDa) and hyperphosphory-
lated (58 kDa) forms. Major phosphorylation sites have been deter-
mined for a few HCV isolates33,34, and kinases capable of
phosphorylating NS5A have been identified. These include AKT,
p70S6K, MEK1, MKK6, cAMP-dependent protein kinase A-? and
casein kinase II35–38. It is not yet clear which kinases are involved in
generating the different phosphoforms of NS5A, nor which phos-
phorylation sites are functionally relevant, and the role of NS5A phos-
phorylation remains an area of intense interest.
NS5A associates with membranes through an N-terminal amphi-
pathic ?-helix39and contains three distinct structural domains40. Bio-
chemical and structural analyses revealed that domain I (residues
1–213) forms a dimer with a novel fold and coordinates Zn2+through
a unique motif40,41. This architecture reveals conserved external sur-
faces that might interact with other proteins, as well as a highly basic
channel that might be involved in RNA binding (Fig. 3b). In addition,
a disulphide bond was discovered between cysteine residues 142 and
190, which is a rare modification for cytosolic proteins and could rep-
resent another form of regulation. Unfortunately, structural informa-
tion is not yet available for domains II and III, which contain
determinants influencing the efficiency of RNA replication (see below)
and NS5A phosphorylation. The C-terminal domain is not well con-
served and seems quite flexible42,43.
The workhorse of the HCV RNA replication machinery is NS5B,
which encodes the RNA-dependent RNA polymerase (RdRP).
Primer-dependent and de novo(unprimed) initiation of RNA synthe-
sis have been demonstrated for this protein. On the basis of the repli-
cation strategy of other positive-strand RNA viruses, HCV RNA
replication probably involves de novoinitiation by a multiprotein com-
plex (replicase). NS5B has a typical ‘right hand’ polymerase structure,
with catalytic sites in the base of the palm domain, surrounded by
thumb and finger domains14. These latter domains fully encircle the
active site, creating a channel for binding to a ssRNA template (Fig. 3c).
In addition, a ?-hairpin structure protrudes from the thumb toward
the active site and is likely to be involved in correct positioning of the
template44. The overall structure of NS5B is remarkably similar to the
RdRP of bacteriophage ?6 (ref. 45), and cocrystallization of these
enzymes with model substrates and nucleoside triphosphates has
yielded a credible model for de novo initiation (reviewed in ref. 46).
NS5B also has a low-affinity GTP-binding site, distal from the active
site, which is thought to be an allosteric regulator of the finger–thumb
interaction47. NS5B is tethered to membranes by a C-terminal peptide
anchor48and interacts with itself to form higher-order RdRP com-
plexes that may have functional relevance to the membrane-bound
replicase, described below49.
The replication era: reverse genetic systems for HCV
Once the highly conserved 3?terminus of HCV was discovered and the
genome sequence was completed50,51, reverse genetics with HCV
became possible. Functional complementary DNA clones were assem-
Figure 1| HCV life cycle.After entry into the cell and uncoating, the HCV
genome functions in three main roles: translation, replication and
packaging into nascent virions.
NATURE|Vol 436|18 August 2005
lar antiviral pathways may be a major intracellular determinant of
Mechanisms of HCV RNA replication
Characterization of cell-culture-adaptive mutations is an area of great
interest, as they are likely to teach us about the interface between HCV
replication and the host cellular environment. Adaptive mutations in
NS4B, NS5A or NS5B strongly enhance replication but are incompat-
ible with each other, whereas adaptive changes in NS3 tend to be weak
and cooperatively enhance replication when combined with strongly
adaptive mutations13. Furthermore, adaptive mutations in NS3 and
NS5B map to surface residues distant from the enzyme active sites,
suggesting that these changes are likely to affect interactions between
NS proteins and/or cellular factors. A striking feature of highly adap-
tive mutations is their tendency to decrease NS5A hyperphosphoryla-
tion, and many adaptive changes map to NS5A residues implicated in
this process60–62. Similarly, increased hyperphosphorylation of NS5A
correlates with lower replication levels42,63,64and decreased interaction
between NS5A and a cellular factor, human vesicle-associated mem-
brane-protein associated protein A (hVAP-A)63. This vesicle-sorting
protein has also been shown to localize HCV NS proteins in choles-
terol-rich, detergent-resistant membranes that may be subcellular
microenvironments for HCV RNA replication65.
As for all positive-strand RNA viruses, HCV RNA replication
occurs in association with altered cytoplasmic membranes. In repli-
con-bearing cells, HCV NS proteins, RNA and RdRP activity associ-
ate with ultrastructural vesicular structures termed the ‘membranous
web’, which also resembles the membrane alterations seen in hepato-
cytes from HCV-infected liver66,67. The integral membrane protein
NS4B is sufficient to induce membranous web formation and has been
proposed to serve as a scaffold for replication complex assembly66. As
mentioned, several adaptive mutations have been mapped to this pro-
tein, and NS4B has been found to encode a GTPase activity that may
be related to its membrane-altering ability68. Association of the HCV
replicase with the membranous web can be followed in live cells by
using subgenomic replicons with green fluorescent protein inserted in
the NS5A domain III43.
Recent experiments have shed new light on the interplay between
cellular membranes, lipid metabolism and HCV replication. In cell
culture, HCV RNA replication is stimulated by increasing the avail-
ability of saturated and monounsaturated fatty acids, and inhibited by
polyunsaturated fatty acids or inhibitors of fatty acid synthesis69. These
results suggest that membrane fluidity is important for the function of
the membranous web. In addition, two groups have shown that inhi-
bition of protein geranylgeranylation leads to the disassembly of HCV
replication complexes and strong inhibition of HCV RNA replica-
tion69,70. In the absence of appropriate prenylation motifs in HCV pro-
teins, these results suggest that a geranylgeranylated cellular protein
participates in HCV replication and could be amenable to pharmaco-
logical manipulation. Recently, the geranylgeranylated host protein
FBL-2 was shown to interact with NS5A and to be required for HCV
RNA replication71. It is intriguing that FBL-2, which contains an ‘F-
box’ motif, is likely to be involved in targeting proteins for degradation,
although the identity of a relevant substrate is currently unknown.
An exciting area of progress has been the delineation of cis-acting
RNA elements that guide viral replication. Nearly the entire 5?NCR is
needed for efficient RNA amplification, although a minimal replica-
tion element exists within the first 120 nucleotides (refs 72–75). As this
overlaps with the IRES, there is considerable interest in understanding
how this region might modulate translation and replication, which are
unlikely to occur simultaneously on the same RNA template. The 3?
NCR has been found to contain a nonessential variable region, a poly-
U/UC tract that must be more than 26 nucleotides long, followed by a
highly conserved and essential 3?X domain55,57,76. Recently a conserved
stem-loop structure within the NS5B coding region, 5BSL3.2, was
found to be required for RNA replication78. Further studies indicated
that 5BSL3.2 forms functionally important long-distance base pairs
bled and shown to be infectious by direct intrahepatic injection of RNA
transcripts into chimpanzees52,53. These infectious clones were used to
show that all viral enzyme activities, the p7 gene and the correct
genomic 3?end are necessary for HCV replication in vivo54–56. In con-
trast, the hypervariable N-terminal region of E2 is dispensible57. Essen-
tially clonal infections could be initiated from transcribed RNA,
providing a well-defined genetic starting point to study virus evolution
and immune responses to infection58. With functional cDNA clones in
hand and the ability to make unlimited quantities of infectious HCV
RNA, much effort was devoted to searching for permissive HCV cell
culture conditions. However, despite their great utility for in vivostud-
ies, these initial chimpanzee infectious transcripts failed to replicate in
A breakthrough for the field came in 1999 when Lohmann et al.
reported selection of the first functional ‘subgenomic’ replicons in cell
culture59. These replicons consisted of a genotype 1b HCV RNA engi-
neered to express a selectable marker gene, Neo, in place of the struc-
tural protein coding region, which was not expected to be required for
RNA replication. To direct expression of NS proteins, a heterologous
viral IRES was inserted after the neomycin resistance cassette (Fig. 4).
After RNA transfection into a human hepatoma line, Huh-7, a few
drug-selected colonies grew out that contained replicating HCV
RNAs. The replicon system provided an important tool for studying
HCV RNA replication and established a functional cell-based system
for evaluating potential antiviral compounds.
The next discovery was that replicon RNAs harboured culture-
adaptive mutations, often in NS5A, that increased RNA replication
and Neo-transduction efficiency by up to 10,000-fold60. Adaptive
changes were subsequently mapped throughout the NS region,
including NS3, NS4B, NS5A and NS5B (reviewed in ref. 13). In addi-
tion to the original genotype 1b isolate, called Con1, replicons have
now been established for other 1b isolates and for genotypes 1a and
2a, and HCV RNA replication has been achieved in Hela, 293,
HepG2 and even mouse hepatoma cell lines13. Thus, whereas it was
once thought that HCV replication might be restricted to the envi-
ronment provided by hepatocytes, it is now clear that a number of
cells can support RNA replication. As discussed by Gale and Foy (see
page 939, in this issue) the interplay between HCV and innate cellu-
E1E2p7 NS2NS3 4A 4BNS5ANS5B
Serine protease cofactor
Figure 2| HCV genes and gene products. a,The structure of the viral
genome, including the long open reading frame encoding structural and
nonstructural genes, and 5?and 3?NCRs. The polyprotein processing
scheme is shown below. Closed circles refer to signal peptidase cleavage
sites; the open circle refers to the signal peptide peptidase cleavage site. All
other terms are defined in the text. b,The topology of HCV proteins with
respect to a cellular membrane.
NATURE|Vol 436|18 August 2005
with the 3?X domain79. The trans-acting factors and the replication
steps requiring this ‘kissing’ interaction remain to be determined.
Little is known about the process of HCV RNA synthesis within the
replication complex. By inference from related viruses, RNA synthesis
is likely to be semiconservative and asymmetric: the positive-strand
genome serves as a template to make a negative-strand intermediate;
the negative strand then serves as a template to produce multiple
nascent genomes. RdRP activity can be detected in extracts from repli-
con-bearing cells, although this seems to reflect elongation of co-
extracted templates rather than de novo initiation. Nascent products
from these reactions are protected from nuclease digestion by a deter-
gent- and protease-sensitive factor80,81. Interestingly, protease treat-
ment of permeabilized cells destroyed most NS proteins without
compromising RdRP activity, suggesting that only a small fraction of
NS proteins is actively engaged in RNA replication80. The function of
the ‘excess’ NS proteins, the composition of the HCV replicase and the
possible reasons for physically sequestering the replicase (such as eva-
sion of dsRNA sensors or antiviral effectors like RNAi) remain intrigu-
ing areas for future study.
Completing the virus life cycle: extracellular virions
Important questions about the nature of the infectious virus particle,
the pathway of virus entry, and the assembly of viral structural proteins
and RNA into new virus particles are still largely unanswered. Fortu-
nately, new technologies have emerged that extend our reach into these
formerly intractable areas.
HCV particles present in clinical samples have been partly charac-
terized. Enveloped virions are sensitive to detergent and to chloroform,
with a diameter of about 50 nm. HCV RNA and infectivity in chim-
panzees have been followed in isopycnic density gradients. HCV
exhibits unusual heterogeneity in buoyant density, with the peak of
infectivity near 1.10 g ml?1. This is surprisingly low even for an
enveloped virus. This heterogeneity and low density have been
explained in part by the association of HCV particles with serum com-
ponents such as immunoglobulins and ?-lipoproteins5,13.
Expression and processing of the HCV structural gene products
were characterized in early heterologous expression studies. Core pro-
tein was localized to the cytoplasmic surface of the ER and lipid
droplets, and occasionally the cell nucleus82–84. A central hydrophobic
domain is responsible for the membrane association of the core,
whereas the high pI of an N-terminal region mediates its interaction
with RNA. The envelope glycoproteins E1 and E2 are highly modified
with N-linked sugars, contain intramolecular disulphide bonds and
undergo a complex folding pathway that involves several ER-resident
chaperones (reviewed in ref. 13). When coexpressed, E1 and E2 are
retained in the ER and form non-covalent heterodimers through
determinants in their transmembrane domains.
E2 binds with high affinity to the large extracellular loop of CD81, a
tetraspanin that is expressed on a variety of cell types, including hepa-
tocytes85. Although this pattern of expression does not explain the
hepatotropism of HCV, CD81 is very likely to be involved in mediating
HCV entry. Several other candidate HCV receptors have also been
identified, including low-density lipoprotein receptor, scavenger recep-
tor class-B type-I (SR-BI), L-SIGN and DC-SIGN13. Several surrogate
systems have been developed to examine the relevance of these inter-
actions and to study E1/E2 structure and function. These include
glycoprotein-dependent cell fusion assays, liposomes reconstituted
with E1 and E2, formation of virus-like particles in insect cells, and
pseudotyped rhabdoviruses and retroviruses (reviewed in ref. 13).
Although each of these systems had merit, the use of retrovirus
pseudoparticles (HCVpp) has provided the most insight into HCV
entry. This method takes advantage of the fact that retroviruses, which
bud from the plasma membrane, frequently and nonspecifically incor-
porate cell surface proteins into the viral membrane. Thus HCVpp are
retroviral particles that are dependent on HCV glycoproteins to deliver
a reporter gene encoded within the retrovirus genome.
HCVpp can be neutralized with antibodies against E2 or immune
sera, confirming their dependence on E2 and showing their use in fol-
lowing the kinetics and specificity of the humoral immune response86,87.
HCVpp infect primary human hepatocytes and a variety of human
hepatic cell lines, and their entry is CD81 dependent88–91. CD81 expres-
sion is not sufficient for HCV entry into non-hepatic cells, suggesting
the existence of one or more unidentified molecules required for HCV
entry and hepatotropism. High-density lipoproteins (HDL) and
apolipoprotein C1, a component of HDL, enhance HCVpp infectivity in
an SR-BI-dependent manner87,92. It is not yet clear whether these obser-
vations are related to the association of HCV with serum lipoproteins.
Despite these advances, the growth of authentic HCV in cell culture
has remained elusive. Although full-length genomes harbouring adap-
tive mutations replicated efficiently in Huh-7 cells and expressed the
structural proteins, infectious particles were not released61,93,94. This led
to the idea that Huh-7 cells might be unable to support HCV particle
assembly or release. However, culture-adaptive changes were also found
to be lethal or highly attenuating for replication in chimpanzees95. This
suggested that adaptive mutations promoting efficient RNA replication
in cell culture might preclude production of infectious particles. In sup-
port of this idea, Pietschmann et al. found that full-length genomes
lacking adaptive mutations replicated poorly but nevertheless released
core protein and HCV RNA into the cell culture medium96. In contrast,
genomes with highly adaptive mutations replicated efficiently but failed
Figure 3 | HCV NS protein structures. a, NS3. The serine protease, NS4A
cofactor and RNA helicase domains are shown in pink, green and blue,
respectively. The serine protease and RNA helicase active site residues
are indicated in red. b, NS5A domain I. Shown is a dimer, as seen in the
crystal structure. Individual subunits are shown in blue and green, with
their C termini (that is, leading into domain II) pointing upwards. The N
termini, which presumably face the membrane, are at the bottom. The
purple spheres represent Zn2+ions. Disulphide bonds are indicated in
red. Brackets indicate highly conserved surfaces. A basic groove, which
may bind RNA, is also indicated. c, NS5B. Shown is the typical ‘right
hand’ model of the RdRP, with palm, fingers and thumb domains in
pink, blue and green, respectively. The C-terminal region, which is not
part of the RdRP, is shown in yellow. Note the extensive interactions
between the finger and thumb domains. In addition, a ?-hairpin is
shown in purple, and active site residues Asp 220 and Asp 318 are shown
NATURE|Vol 436|18 August 2005
7. Forton, D. M., Karayiannis, P., Mahmud, N., Taylor-Robinson, S. D. & Thomas, H. C.
Identification of unique hepatitis C virus quasispecies in the central nervous system and
comparative analysis of internal translational efficiency of brain, liver, and serum variants.
J. Virol.78,5170–5183 (2004).
8. Bigger, C. B., Brasky, K. M. & Lanford, R. E. DNA microarray analysis of chimpanzee liver
during acute resolving hepatitis C virus infection. J. Virol.75,7059–7066 (2001).
9. Bigger, C. B. et al.Intrahepatic gene expression during chronic hepatitis C virus infection in
chimpanzees. J. Virol.78,13779–13792 (2004).
10. Su, A. I. et al.Genomic analysis of the host response to hepatitis C virus infection. Proc. Natl
Acad. Sci. USA99,15669–15674 (2002).
11. Thimme, R. et al.Viral and immunological determinants of hepatitis C virus clearance,
persistence, and disease. Proc. Natl Acad. Sci. USA99,15661–15668 (2002).
12. Simmonds, P. Genetic diversity and evolution of hepatitis C virus—15 years on. J. Gen. Virol.
13. Bartenschlager, R., Frese, M. & Pietschmann, T. Novel insights into hepatitis C virus
replication and persistence. Adv. Virus Res.63,71–180 (2004).
14. Penin, F., Dubuisson, J., Rey, F. A., Moradpour, D. & Pawlotsky, J. M. Structural biology of
hepatitis C virus. Hepatology39,5–19 (2004).
15. Spahn, C. M. et al.Hepatitis C virus IRES RNA-induced changes in the conformation of the
40S ribosomal subunit. Science291,1959–1962 (2001).
16. Ji, H., Fraser, C. S., Yu, Y., Leary, J. & Doudna, J. A. Coordinated assembly of human
translation initiation complexes by the hepatitis C virus internal ribosome entry site RNA.
Proc. Natl Acad. Sci. USA101,16990–16995 (2004).
17. Otto, G. A. & Puglisi, J. D. The pathway of HCV IRES-mediated translation initiation. Cell119,
18. Griffin, S. D. et al.The p7 protein of hepatitis C virus forms an ion channel that is blocked by
the antiviral drug, Amantadine. FEBS Lett.535,34–38 (2003).
19. Pavlovic, D. et al.The hepatitis C virus p7 protein forms an ion channel that is inhibited by
long-alkyl-chain iminosugar derivatives. Proc. Natl Acad. Sci. USA100,6104–6108
20.Branch, A. D., Stump, D. D., Gutierrez, J. A., Eng, F. & Walewski, J. L. The hepatitis C virus
alternate reading frame (ARF) and its family of novel products: the alternate reading frame
protein/F-protein, the double-frameshift protein, and others. Semin. Liver Dis.25,105–117
21. Failla, C., Tomei, L. & De Francesco, R. Both NS3 and NS4A are required for proteolytic
processing of hepatitis C virus nonstructural proteins. J. Virol.68,3753–3760 (1994).
22. Bartenschlager, R., Ahlborn-Laake, L., Mous, J. & Jacobsen, H. Kinetic and structural
analyses of hepatitis C virus polyprotein processing. J. Virol.68,5045–5055 (1994).
23. Lin, C., Thomson, J. A. & Rice, C. M. A central region in the hepatitis C virus NS4A protein
allows formation of an active NS3–NS4A serine proteinase complex in vivoand in vitro.
J. Virol.69,4373–4380 (1995).
24.Wölk, B. et al.Subcellular localization, stability, and trans-cleavage competence of the
hepatitis C virus NS3–NS4A complex expressed in tetracycline-regulated cell lines. J. Virol.
25. Tai, C.-L., Chi, W.-K., Chen, D.-S. & Hwang, L.-H. The helicase activity associated with
hepatitis C virus nonstructural protein 3 (NS3). J. Virol.70,8477–8484 (1996).
26.Levin, M. K., Gurjar, M. & Patel, S. S. A Brownian motor mechanism of translocation and
strand separation by hepatitis C virus helicase. Nature Struct. Mol. Biol.12,429–435
27. Serebrov, V. & Pyle, A. M. Periodic cycles of RNA unwinding and pausing by hepatitis C
virus NS3 helicase. Nature430,476–480 (2004).
28.Frick, D. N., Rypma, R. S., Lam, A. M. & Gu, B. The nonstructural protein 3 protease/helicase
requires an intact protease domain to unwind duplex RNA efficiently. J. Biol. Chem.279,
29. Pang, P. S., Jankowsky, E., Planet, P. J. & Pyle, A. M. The hepatitis C viral NS3 protein is a
processive DNA helicase with cofactor enhanced RNA unwinding. EMBO J.21,1168–1176
30.He, Y. & Katze, M. G. To interfere and to anti-interfere: the interplay between hepatitis C
virus and interferon. Viral Immunol.15,95–119 (2002).
31. Macdonald, A. & Harris, M. Hepatitis C virus NS5A: tales of a promiscuous protein. J. Gen.
32. Tellinghuisen, T. L. & Rice, C. M. Interaction between hepatitis C virus proteins and host cell
factors. Curr. Opin. Microbiol.5,419–427 (2002).
33. Katze, M. G. et al.Ser2194is a highly conserved major phosphorylation site of the hepatitis C
virus nonstructural protein NS5A. Virology278,501–513 (2000).
34.Reed, K. E. & Rice, C. M. Identification of the major phosphorylation site of the hepatitis C
virus H strain NS5A protein as serine 2321. J. Biol. Chem.274,28011–28018 (1999).
35. Coito, C., Diamond, D. L., Neddermann, P., Korth, M. J. & Katze, M. G. High-throughput
screening of the yeast kinome: identification of human serine/threonine protein
kinases that phosphorylate the hepatitis C virus NS5A protein. J. Virol.78,3502–3513
36.Ide, Y., Tanimoto, A., Sasaguri, Y. & Padmanabhan, R. Hepatitis C virus NS5A protein is
phosphorylated in vitroby a stably bound protein kinase from HeLa cells and by cAMP-
dependent protein kinase A-alpha catalytic subunit. Gene201,151–158 (1997).
37. Kim, J., Lee, D. & Choe, J. Hepatitis C virus NS5A protein is phosphorylated by casein kinase
II. Biochem. Biophys. Res. Commun.257,777–781 (1999).
38.Reed, K. E., Xu, J. & Rice, C. M. Phosphorylation of the hepatitis C virus NS5A protein in vitro
and in vivo: properties of the NS5A-associated kinase. J. Virol.71,7187–7197 (1997).
39. Brass, V. et al.An amino-terminal amphipathic alpha-helix mediates membrane
association of the hepatitis C virus nonstructural protein 5A. J. Biol. Chem.277,8130–8139
40.Tellinghuisen, T. L., Marcotrigiano, J., Gorbalenya, A. E. & Rice, C. M. The NS5A protein of
hepatitis C virus is a zinc metalloprotein. J. Biol. Chem.279,48576–48587 (2004).
41. Tellinghuisen, T. L., Marcotrigiano, J. & Rice, C. M. Structure of the zinc-binding domain of
an essential replicase component of hepatitis C virus. Nature435,374–379 (2005).
42.Appel, N., Pietschmann, T. & Bartenschlager, R. Mutational analysis of hepatitis C virus
nonstructural protein 5A: potential role of differential phosphorylation in RNA replication
and identification of a genetically flexible domain. J. Virol.79,3187–3194 (2005).
to secrete HCV RNA and core protein. Thus, there was a dilemma: in
cell culture, adaptive mutations were required for replication but were
deleterious for virus production; in vivo, cell adaptive mutations were
deleterious and virus produced in vivowas non-infectious in cell cul-
ture. Enter JFH-1: a genotype 2a HCV isolate obtained from a patient
in Japan with fulminant hepatitis97. For reasons that are not understood,
subgenomic replicons derived from JFH-1 cDNA do not require adap-
tive mutations for efficient replication in cell culture. Wakita et al.
recently demonstrated that the full-length JFH-1 genome produces
infectious particles in cell culture, although the titres were moderate
Similarly, we constructed a chimaeric full-length genome using the
JFH-1 replicase and the core–NS2 region from a related genotype 2a
stain, J6. This genome replicated in cell culture and produced robust
levels of infectious virus (HCVcc), nearly 105infectious units ml–1within
48 h in Huh-7.5 cells, which are highly permissive for HCV RNA repli-
cation99. Another group has found that full-length JFH-1 can also reach
high titres when propagated in an Huh-7.5 subline100. Thus, the JFH-1
replicon can support efficient production of infectious HCV in cell cul-
ture. It is not yet clear why this particular genome is capable of replicat-
ing without adaptive changes, or how adaptive changes preclude
infectious particle production. However, one possible explanation stems
from the three coordinated yet distinct processes facing HCV viral RNA:
translation, replication and packaging. Cell culture adaptive changes,
selecting for efficient and persistent RNA replication, may shift the bal-
ance towards these first two processes at the expense of liberating genome
RNA for virion assembly.
It is clearly an exciting time in HCV research, and rapid progress
should be made now that complete cell culture systems are available.
The processes of HCV entry, replication and virion production can be
further dissected with genetic and biochemical approaches, and
should reveal information that facilitates the development of specific
antivirals that target each stage in the virus life cycle. If the determi-
nants of JFH-1 that permit efficient replication and virus production
can be mapped, it may be possible to extend the cell culture systems to
include other virus isolates as well. An important question is whether
JFH-1-derived viruses grown in vivowill retain their infectivity in cell
culture. Furthermore, once cellular determinants of HCV tropism are
better understood, it might be possible to engineer improved small-
animal models of HCV infection and pathogenesis. These approaches
will undoubtedly reveal new and surprising aspects of HCV replica-
tion, and arm us with better strategies to combat HCV infection and
eradicate HCV-associated disease.
1.Feinstone, S. M., Kapikian, A. Z., Purcell, R. H., Alter, H. J. & Holland, P. V. Transfusion-
associated hepatitis not due to viral hepatitis type A or B. N. Engl. J. Med.292,767–770
2. Choo, Q.-L. et al.Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral
hepatitis genome. Science244,359–362 (1989).
3. Neumann, A. U. et al.Hepatitis C viral dynamics in vivoand the antiviral efficacy of
interferon-alpha therapy. Science282,103–107 (1998).
4. Hoofnagle, J. H. Course and outcome of hepatitis C. Hepatology36,S21–S29 (2002).
5. Lindenbach, B. D. & Rice, C. M. in Fields Virology(eds Knipe, D. M. & Howley, P. M.)
991–1041 (Lippincott-Raven, Philadelphia, 2001).
6. Deforges, S. et al.Expression of hepatitis C virus proteins in epithelial intestinal cells in vivo.
J. Gen. Virol.85,2515–2523 (2004).
E1 p7NS3 4A4BNS5A NS5B E2 NS2Core
Figure 4| Cell culture replication systems.The basic design of subgenomic
NATURE|Vol 436|18 August 2005
43.Moradpour, D. et al.Insertion of green fluorescent protein into nonstructural protein 5A
allows direct visualization of functional hepatitis C virus replication complexes. J. Virol.78,
44.Hong, Z. et al.A novel mechanism to ensure terminal initiation by hepatitis C virus NS5B
polymerase. Virology285,6–11 (2001).
45.Butcher, S. J., Grimes, J. M., Makeyev, E. V., Bamford, D. H. & Stuart, D. I. A mechanism for
initiating RNA-dependent RNA polymerization. Nature410,235–240 (2001).
46.van Dijk, A. A., Makeyev, E. V. & Bamford, D. H. Initiation of viral RNA-dependent RNA
polymerization. J. Gen. Virol.85,1077–1093 (2004).
47. Bressanelli, S., Tomei, L., Rey, F. A. & De Francesco, R. Structural analysis of the hepatitis C
virus RNA polymerase in complex with ribonucleotides. J. Virol.76,3482–3492 (2002).
48.Schmidt-Mende, J. et al.Determinants for membrane association of the hepatitis C virus
RNA-dependent RNA polymerase. J. Biol. Chem.276,44052–44063 (2001).
49.Wang, Q. M. et al.Oligomerization and cooperative RNA synthesis activity of hepatitis C
virus RNA-dependent RNA polymerase. J. Virol.76,3865–3872 (2002).
50.Kolykhalov, A. A., Feinstone, S. M. & Rice, C. M. Identification of a highly conserved
sequence element at the 3?terminus of hepatitis C virus genome RNA. J. Virol.70,
51. Tanaka, T., Kato, N., Cho, M. J. & Shimotohno, K. A novel sequence found at the 3?terminus
of hepatitis C virus genome. Biochem. Biophys. Res. Commun.215,744–749 (1995).
52. Kolykhalov, A. A. et al.Transmission of hepatitis C by intrahepatic inoculation with
transcribed RNA. Science277,570–574 (1997).
53. Yanagi, M., Purcell, R. H., Emerson, S. U. & Bukh, J. Transcripts from a single full-length
cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a
chimpanzee. Proc. Natl Acad. Sci. USA94,8738–8743 (1997).
54.Kolykhalov, A. A., Mihalik, K., Feinstone, S. M. & Rice, C. M. Hepatitis C virus-encoded
enzymatic activities and conserved RNA elements in the 3?nontranslated region are
essential for virus replication in vivo.J. Virol.74,2046–2051 (2000).
55. Sakai, A. et al.The p7 polypeptide of hepatitis C virus is critical for infectivity and contains
functionally important genotype-specific sequences. Proc. Natl Acad. Sci. USA100,
56.Yanagi, M., St Claire, M., Emerson, S. U., Purcell, R. H. & Bukh, J. In vivoanalysis of the 3?
untranslated region of the hepatitis C virus after in vitromutagenesis of an infectious cDNA
clone. Proc. Natl Acad. Sci. USA96,2291–2295 (1999).
57. Forns, X. et al.Hepatitis C virus lacking the hypervariable region 1 of the second envelope
protein is infectious and causes acute resolving or persistent infection in chimpanzees.
Proc. Natl Acad. Sci. USA97,13318–13323 (2000).
58.Bukh, J. A critical role for the chimpanzee model in the study of hepatitis C. Hepatology39,
59. Lohmann, V. et al.Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line.
60.Blight, K. J., Kolykhalov, A. A. & Rice, C. M. Efficient initiation of HCV RNA replication in cell
culture. Science290,1972–1974 (2000).
61. Blight, K. J., McKeating, J. A., Marcotrigiano, J. & Rice, C. M. Efficient RNA replication of
hepatitis C virus genotype 1a in cell culture. J. Virol.77,3181–3190 (2003).
62.Lohmann, V., Hoffmann, S., Herian, U., Penin, F. & Bartenschlager, R. Viral and cellular
determinants of hepatitis C virus RNA replication in cell culture. J. Virol.77,3007–3019
63.Evans, M. J., Rice, C. M. & Goff, S. P. Phosphorylation of hepatitis C virus nonstructural
protein 5A modulates its protein interactions and viral RNA replication. Proc. Natl Acad. Sci.
64.Neddermann, P. et al.Reduction of hepatitis C virus NS5A hyperphosphorylation by
selective inhibition of cellular kinases activates viral RNA replication in cell culture. J. Virol.
65.Gao, L., Aizaki, H., He, J. W. & Lai, M. M. Interactions between viral nonstructural proteins
and host protein hVAP-33 mediate the formation of hepatitis C virus RNA replication
complex on lipid raft. J. Virol.78,3480–3488 (2004).
66.Egger, D. et al.Expression of hepatitis C virus proteins induces distinct membrane
alterations including a candidate viral replication complex. J. Virol.76,5974–5984 (2002).
67. Gosert, R. et al.Identification of the hepatitis C virus RNA replication complex in Huh-7 cells
harboring subgenomic replicons. J. Virol.77,5487–5492 (2003).
68.Einav, S., Elazar, M., Danieli, T. & Glenn, J. S. A nucleotide binding motif in hepatitis C virus
(HCV) NS4B mediates HCV RNA replication. J. Virol.78,11288–11295 (2004).
69.Kapadia, S. B. & Chisari, F. V. Hepatitis C virus RNA replication is regulated by host
geranylgeranylation and fatty acids. Proc. Natl Acad. Sci. USA102,2561–2566 (2005).
70.Ye, J. et al.Disruption of hepatitis C virus RNA replication through inhibition of host protein
geranylgeranylation. Proc. Natl Acad. Sci. USA100,15865–15870 (2003).
71. Wang, C. et al. Identification of FBL2 as a geranylated cellular protein required for hepatitis
C virus RNA replication. Mol. Cell18, 425–434 (2005).
72. Friebe, P., Lohmann, V., Krieger, N. & Bartenschlager, R. Sequences in the 5?nontranslated
region of hepatitis C virus required for RNA replication. J. Virol.75,12047–12057 (2001).
73. Kim, Y. K., Kim, C. S., Lee, S. H. & Jang, S. K. Domains I and II in the 5?nontranslated region of
the HCV genome are required for RNA replication. Biochem. Biophys. Res. Commun.290,
74. Luo, G., Xin, S. & Cai, Z. Role of the 5?-proximal stem-loop structure of the 5?untranslated
region in replication and translation of hepatitis C virus RNA. J. Virol.77,3312–3318 (2003).
75. Reusken, C. B., Dalebout, T. J., Eerligh, P., Bredenbeek, P. J. & Spaan, W. J. Analysis of
hepatitis C virus/classical swine fever virus chimeric 5?NTRs: sequences within the
hepatitis C virus IRES are required for viral RNA replication. J. Gen. Virol.84,1761–1769
76. Friebe, P. & Bartenschlager, R. Genetic analysis of sequences in the 3?nontranslated region
of hepatitis C virus that are important for RNA replication. J. Virol.76,5326–5338 (2002).
77. Yi, M. & Lemon, S. M. 3?nontranslated RNA signals required for replication of hepatitis C
virus RNA. J. Virol.77,3557–3568 (2003).
78. You, S., Stump, D. D., Branch, A. D. & Rice, C. M. A cis-acting replication element in the
sequence encoding the NS5B RNA-dependent RNA polymerase is required for hepatitis C
virus RNA replication. J. Virol.78,1352–1366 (2004).
79. Friebe, P., Boudet, J., Simorre, J. P. & Bartenschlager, R. Kissing-loop interaction in the 3?end
of the hepatitis C virus genome essential for RNA replication. J. Virol.79,380–392 (2005).
80.Miyanari, Y. et al.Hepatitis C virus non-structural proteins in the probable membranous
compartment function in viral genome replication. J. Biol. Chem.278,50301–50308
81. Yang, G. et al.Newly synthesized hepatitis C virus replicon RNA is protected from nuclease
activity by a protease-sensitive factor(s). J. Virol.78,10202–10205 (2004).
82.Barba, G. et al.Hepatitis C virus core protein shows a cytoplasmic localization and
associates to cellular lipid storage droplets. Proc. Natl Acad. Sci. USA94,1200–1205 (1997).
83.Moradpour, D., Englert, C., Wakita, T. & Wands, J. R. Characterization of cell lines allowing
tightly regulated expression of hepatitis C virus core protein. Virology222,51–63 (1996).
84.Yasui, K. et al.The native form and maturation process of hepatitis C virus core protein.
J. Virol.72,6048–6055 (1998).
85.Pileri, P. et al.Binding of hepatitis C virus to CD81. Science282,938–941 (1998).
86.Logvinoff, C. et al.Neutralizing antibody response during acute and chronic hepatitis C virus
infection. Proc. Natl Acad. Sci. USA101,10149–10154 (2004).
87. Meunier, J. C. et al.Evidence for cross-genotype neutralization of hepatitis C virus pseudo-
particles and enhancement of infectivity by apolipoprotein C1. Proc. Natl Acad. Sci. USA102,
88.Cormier, E. G. et al.CD81 is an entry coreceptor for hepatitis C virus. Proc. Natl Acad. Sci.
89.Lavillette, D. et al.Characterization of host-range and cell entry properties of the major
genotypes and subtypes of hepatitis C virus. Hepatology41,265–274 (2005).
90.McKeating, J. A. et al.Diverse hepatitis C virus glycoproteins mediate viral infection in a
CD81-dependent manner. J. Virol.78,8496–8505 (2004).
91. Zhang, J. et al.CD81 is required for hepatitis C virus glycoprotein-mediated viral infection. J.
92. Voisset, C. et al.High density lipoproteins facilitate hepatitis C virus entry through the
scavenger receptor class B type I. J. Biol. Chem.280,7793–7799 (2005).
93. Blight, K. J., McKeating, J. A. & Rice, C. M. Highly permissive cell lines for subgenomic and
genomic hepatitis C virus RNA replication. J. Virol.76,13001–13014 (2002).
94. Pietschmann, T. et al. Persistant and transient replication of full-length hepatitis C virus
genomes in cell culture. J. Virol.76,4008–4021 (2002).
95.Bukh, J. et al.Mutations that permit efficient replication of hepatitis C virus RNA in Huh-7
cells prevent productive replication in chimpanzees. Proc. Natl Acad. Sci. USA99,
96.Pietschmann, T. & Bartenschlager, R. in Proc. 11th International Symposium on Hepatitis C
Virus and Related Viruses (Heidelberg, Germany, 2004).
97. Kato, T. et al.Efficient replication of the genotype 2a hepatitis C virus subgenomic replicon.
98.Wakita, T. et al.Production of infectious hepatitis C virus in tissue culture from a cloned viral
genome. Nature Med.11,791–796 (2005).
99.Lindenbach, B. D. et al.Complete replication of hepatitis C virus in cell culture. Science309,
100.Zhong, J. et al.Robust hepatitis C virus infection in vitro.Proc. Natl Acad. Sci. USA102,
AcknowledgementsWe thank our colleagues for many helpful discussions, and in
particular T. Tellinghuisen, J. Marcotrigiano, I. Lorenz, T. Pietschmann and R.
Bartenschlager for providing data before publication; and J. Bloom, T. Tellinghuisen,
M. Evans and I. Lorenz for comments on the manuscript. Work in our laboratory is
supported by the US Public Health Service under grants from the NIH to C.M.R.,
and the Greenberg Medical Research Institute. B.D.L. is a recipient of the NIH
Howard Temin Award.
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interests: details accompany the paper on www.nature.com/nature.
Correspondence and requests for materials should be addressed to C.M.R.