High-Resolution Functional Profiling of Hepatitis C Virus
Genome
Vaithilingaraja Arumugaswami
1
, Roland Remenyi
1
, Vidhya Kanagavel
1
, Eric Yiang Sue
1
, Tuyet Ngoc Ho
1
,
Chang Liu
1
, Vanessa Fontanes
2
, Asim Dasgupta
2,3,4,5
, Ren Sun
1,3,4
*
1 Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California, United States of America,
2 Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, California, United States of
America, 3 AIDS Institute, University of California, Los Angeles, California, United States of America, 4 Jonsson Comprehensive Cancer Center, University of California, Los
Angeles, California, United States of America, 5 Molecular Biology Institute, University of California, Los Angeles, California, United States of America
Abstract
Hepatitis C virus is a leading cause of human liver disease worldwide. Recent discovery of the JFH-1 isolate, capable of
infecting cell culture, opens new avenues for studying HCV replication. We describe the development of a high-throughput,
quantitative, genome-scale, mutational analysis system to study the HCV cis-elements and protein domains that are
essential for virus replication. An HCV library with 15-nucleotide random insertions was passaged in cell culture to examine
the effect of insertions at each genome location by insertion-specific fluorescent-PCR profiling. Of 2399 insertions identified
in 9517 nucleotides of the genome, 374, 111, and 1914 were tolerated, attenuating, and lethal, respectively, for virus
replication. Besides identifying novel functional domains, this approach confirmed other functional domains consistent with
previous studies. The results were validated by testing several individual mutant viruses. Furthermore, analysis of the 39 non-
translated variable region revealed a spacer role in virus replication, demonstrating the utility of this approach for functional
discovery. The high-resolution functional profiling of HCV domains lays the foundation for further mechanistic studies and
presents new therapeutic targets as well as topological information for designing vaccine candidates.
Citation: Arumugaswami V, Remenyi R, Kanagavel V, Sue EY, Ho TN, et al. (2008) High-Resolution Functional Profiling of Hepatitis C Virus Genome. PLoS
Pathog 4(10): e1000182. doi:10.1371/journal.ppat.1000182
Editor: Donald E. Ganem, University of California San Francisco, United States of America
Received May 20, 2008; Accepted September 22, 2008; Published October 17, 2008
Copyright:
2008 Arumugaswami et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by NIH grant AI 72180 to A.D. and was partially supported by Jonsson Comprehensive Cancer Center award and UCLA AIDS
grants AI28697 and CC95LA137 to A.D.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: rsun@mednet.ucla.edu
Introduction
Hepatitis C virus (HCV) is a major human health concern with
an estimated 130 million people infected with HCV worldwide [1]
with resulting liver diseases including chronic hepatitis, cirrhosis,
and hepatocellular carcinoma [2,3]. Currently there is no effective
vaccine, and the available treatment options offer limited response
rates. HCV is classified in the family Flaviviridae and has a positive-
sense, single-stranded RNA genome of about 9.6 kilobases (kb) [4].
The genome is organized as 59NTR-C-E1-E2-p7-NS2-NS3-
NS4A-NS4B-NS5A-NS5B-39NTR, with non-translated regions
(NTR) flanking a protein-coding region. The latter encodes a
single polyprotein (,3000 amino acids) that is co- and post-
translationally cleaved by cellular and viral proteases into at least
ten mature structural and non-structural (NS) proteins [5,6,7].
A recent review highlights the functions of HCV NTRs and
proteins [8]. The 59NTR cis-elements are involved in viral RNA
translation and replication [9,10,11]. The core (C) and envelope
glycoproteins form the structural proteins of the virion. The core
nucleocapsid encapsidates the viral RNA genome [12,13]. The
ARF protein produced by a frame-shift translation of the core
region is non-essential for virus replication [14,15]. The envelope
glycoproteins, E1 and E2, facilitate the virus entry into host cells
through recognition of cellular receptors [16,17]. An ion-channel
forming peptide p7, and a cysteine protease NS2 play important
roles in virion morphogenesis [18,19,20]. NS- 3, 4A, 4B, 5A and
5B form the replicase complex involved in RNA genome
replication [21,22,23,24]. The 39NTR region is critical for
initiation of negative-strand genome replication and translation
enhancement [25,26,27]. To complete viral replication and
transmission, the HCV proteins and cis-elements interact with
various cellular factors, modulating signaling pathways and
immune responses [28,29,30,31].
The HCV sub-genomic replicon and chimpanzee infection
model have previously been used for studying HCV replication
[21,24,27]. Efficient replication of a genotype (GT) 2a HCV isolate
JFH-1 in cell culture [32,33,34] offers the possibility for functional
analysis of HCV proteins and NTRs. Despite these advances, the
role of many HCV protein and cis-element sub-domains during
infection remains unknown. Examining the function of viral factors
by generating and testing individual mutant viruses would be time-
consuming and labor-intensive for genome-scale studies. We have
developed and applied a high-throughput mutational analysis
approach to study the role of viral cis-acting elements and protein-
domains in HCV replication utilizing transposon mutagenesis.
Transposons have been used widely as a tool for studying the
function of bacterial, yeast and viral genes [35,36,37]. For example,
the Mu-transposon mediated 15-nucleotide (nt) insertion mutagen-
esis was used for mapping genomic regions crucial for propagation
of Potato virus A [38], the 59 end of human immunodeficiency virus
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type 1 [39], and pBC-SK plasmid [36]. These studies employed
urea-polyacrylamide gel-based foot-printing to identify the insertion
locations. We have developed a more rapid mutational analysis
platform by integrating Mu-mediated random insertional muta-
genesis and quantitative, high-throughput capillary electrophoresis
genetic profiling. Using this platform, we have obtained a high-
resolution functional profile of protein-domains and cis-acting
regulatory elements that are critical for JFH-1 HCV replication in
cell culture.
Results
Generation of a 15-nt insertion mutant JFH-1 plasmid
library
A library of JFH-1 HCV mutants containing 15-nt insertions was
generated by Mu-transposon insertion mutagenesis and subsequent
removal of the transposon fragment (Fig. 1A). This yielded
insertions of the 15-nt sequence 59-NNNNNTGCGGCCGCA-39
(N: duplicated 5 nucleotides from target DNA), coding for different
amino acid sequences depending on the insertional reading frame
(Fig. S1). The 15-nt insertion does not introduce a stop codon [38].
Insertion mutations were used for analyzing the structure-function
of the proteins [38,39,40]. Sequencing of random clones indicated
that 82% (27/33) of transposon insertions were widely distributed in
the JFH-1 genome and 18% (6/33) of the insertions were in vector
sequences.
Genetic selection and analysis method of mutant JFH-1
library
The in vitro transcribed JFH-1 library was used as a non-selected
RNA input pool for functional profiling analysis and for genetic
selection for growth in cell culture (Fig. 1). To identify HCV
protein domains and cis-acting elements critical for virus
replication, the mutant RNA library was passaged en masse in
Huh-7.5.1 cells. At 21 days post-transfection (dpt), most of the cells
showed cytopathic effects (CPE) as previously reported [34,41].
The kinetics of mutant library genome replication and viral titer
are shown in Fig. S2. The cDNA generated from the harvested
total RNA was used as a template for functional profiling PCR. A
total of thirteen overlapping HCV fragments (F1 to F13) were
PCR-amplified from the cDNA of the non-selected and cell
culture-selected mutant libraries (Fig. 1B and Table S1).
Subsequently, the purified PCR products were used as templates
for a second PCR using an insertion-specific fluorescent-labeled
primer and one of the forty-eight JFH-1 specific primers (Fig. 1C–
F and Table S2), and these fluorescent-labeled PCR products were
analyzed by capillary electrophoresis (Fig. 1G,H).
To ascertain that the complexity of the mutant library was
maintained during in vitro transcription and transfection, the JFH-1
plasmid library, the in vitro transcribed RNA library, and the total
cellular RNA harvested at 2, 4, 10, 16 and 21 days post-
transfection were subjected to functional profiling analysis.
Analysis of the p7-NS2 region showed that the complexity of the
library was maintained through in vitro transcription and during
the early phase of selection in Huh-7.5.1 cells (Fig. 2). At 10 dpt
many of the insertion mutants had been negatively selected, and
by 21 dpt only a limited number of clones had continued to
replicate. By comparison, all insertions at the 39NTR poly(U) tract
were negatively selected by 2 dpt (Fig. S3), confirming its critical
role in viral genome replication [25,26,27,42]. Thus, the
functional profiling system could be useful for monitoring the
replication kinetics of individual insertion mutants.
Functional profile of the HCV genome
In vitro genetic selection resulted in maintenance (neutral or
tolerated fitness), loss (lethal fitness), or reduction (attenuated
fitness) of individual insertion mutants over time. To define a
phenotype for each insertion mutant, the ratio of peak area
between selected (21 dpt) and non-selected pools was calculated.
The insertion resulting in absence, two fold reduction, or
maintenance was assigned a lethal, attenuated, or tolerated
phenotype, respectively. In the present study, the phenotype
attenuation indicates reduction in virus replication, not loss of
virulence. The final assembly containing the locations of insertion
sites and corresponding phenotypic annotations for 2399 inde-
pendent insertions across the entire HCV genome (nt 55 to 9571)
(Fig. 3) was obtained. For a high-resolution insertion profile map
see Fig. S4. The results showed that 79.8% (1914) of the insertions
were lethal, 4.6% (111) attenuating, and 15.6% (374) tolerated,
with respect to viral replication. The total number of insertions
and their effect on virus replication for each of the HCV regions
are shown in Table 1.
Whole genome profiling demonstrated distinct patterns for each
genetic region. Comparison of the genome-scale functional profile
with known HCV sequence variability revealed that the conserved
HCV proteins, including core, NS- 3, 4A, 4B, and 5A N-terminus,
had fewer tolerated insertions. The less-conserved regions,
including p7-NS2 junction and 5A C-terminus, had many
tolerated insertions. An exception was the envelope proteins (E1
and E2), which have highly variable amino acid sequences but
were intolerant for insertions.
Functional profile of 59NTR. 59NTR is a highly conserved
region of HCV. The 59NTR cis-elements are involved in viral
RNA replication and translation, consisting of four major stem-
loop (SL) structural domains: I, II, III, and IV (Fig. 4) [43]. SL III
contains six additional minor SL structures. The stem-loops II, III,
and IV constitute an internal ribosome entry site (IRES) which
mediates cap-independent initiation of RNA translation. The
59NTR is recognized by several cellular factors [28,29]. The SL II
domain consists of nucleotides 43–117. Majority of the 15-nt
insertions between nt 62–93 were either lethal or attenuating
(Fig. 4). Between nt 94–101, all the insertions were tolerated. Two
insertions at the pyrimidine tract-I (Py-I), the region that connects
Author Summary
Hepatitis C virus (HCV) is a major human health concern
that causes fatal liver diseases. Currently no vaccine is
available to prevent HCV infection. Though the HCV was
identified two decades ago, the virus has only recently
been successfully grown in cell culture conditions. The role
of HCV protein and regulatory element sub-domains
during virus growth is poorly understood. We have
developed a mutational analysis method to identify the
function of HCV sub-domains at a high resolution. A
collection of HCV mutants containing 15-nucleotide
random insertions was tested for growth in cell culture.
The precise location of the insertions and their effects on
virus growth were analyzed by capillary genotyping
technology and bioinformatics. Out of the total 2399
HCV mutants identified, 374 mutants grew normally, 111
mutants demonstrated reduced growth, and 1914 mutants
failed to grow in cell culture. This mutational analysis
method was validated by testing many individual mutant
viruses. The present study identified several HCV function-
al sub-domains required for virus growth, presenting novel
therapeutic targets. The HCV mutant viruses identified
with the property of reduced growth can be used for
designing vaccine candidates.
Functional Profiling of HCV
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SL II and III, were tolerated. All insertions in the SL III- a, c, d,
and e and the IIIf pseudoknot were lethal for virus replication,
which is consistent with their role in binding to the 40S ribosomal
subunit [43]. The eIF3 contact sites have been mapped to SL IIIb
and the junction of stems III- a, b, and c [43]. Most of the
insertions at the IIIb apical loop (nt 180–203) containing the
pyrimidine tract-II (Py-II) were tolerated for virus replication. The
nucleotide sequence of Py-I and II regions have been shown to be
non-essential for IRES-mediated translation [44]. Collier and
colleagues [45] reported that the SL IIIb internal loop is critical for
Figure 1. Schematic diagram depicting the various steps involved in Hepatitis C virus functional profiling. (A) The plasmid carrying the
JFH-1 HCV genome is subjected to in vitro mutagenesis by using the mini-Mu transposon, then selected in E. coli bacteria. The harvested mutant
plasmids are subjected to NotI restriction enzyme digestion to remove the transposon body, followed by ligation resulting in the generation of a 15-
nt insertion plasmid library. Subsequently, this mutant plasmid library is in vitro transcribed and used as a non-selected input pool for functional
profiling analysis. The in vitro transcribed RNA library is delivered into Huh-7.5.1 cells for genetic selection. The total RNA harvested from the
transfected cells (selected pool) as well as non-selected pool RNA are subjected to functional profiling analysis. (B) Followed by selection, the mutant
HCV genome from the non-selected input pool and the selected pool are reverse-transcribed and 13 overlapping fragments (F1 to F13) are PCR
amplified. (C, D) The purified PCR products from non-selected and selected pools are used as templates for a second PCR using one of the HCV
fragment-specific primers (blue arrow) and a fluorescently labeled insertion-specific primer (red arrow with green star). (E, F) The fluorescently-labeled
PCR products from input and selected pools are analyzed by a 96-capillary genotyper. The processed data are either visualized by electropherograms
(G, H) or exported as a data file. The phenotype for each insertion is calculated by comparing the corresponding peak areas of selected and non-
selected pools.
doi:10.1371/journal.ppat.1000182.g001
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IRES activity than the apical loop which corroborates our
mutational profile. Many of the insertions were tolerated at
domain IV where the translation start codon, AUG, is located. An
insertion at nt 338 was lethal for virus replication. A previous study
has shown that the stability of the domain IV stem loop is
negatively correlated to the initiation of translation [46]. Analysis
of the predicted domain IV secondary structures with the 15-nt
insertion revealed that the tolerated insertions maintain an open
destabilized structure similar to that of wild-type domain IV,
whereas the lethal insertion forms a stable hairpin structure (Fig.
S5). Thus, the tolerated insertions at domain IV could allow
translation-initiation, resulting in virus replication.
Functional profile of Core. The structural protein, core,
encapsidates the genomic RNA in the virus particle. The core
region functional profiling showed that the insertions at nt 349–
356 (3–6 aa), which is also a part of the IRES, were tolerated. Most
of the insertions (between aa 23–107) at the RNA binding basic
domain were lethal. Insertions at the hydrophobic domain
between aa 133–167 were mostly lethal and attenuating.
Insertions found between aa 176–191 of the C-terminal
transmembrane region were tolerated. The role of core protein
in infectious virus particle production has been studied by alanine
scanning mutagenesis [13]. Most of our data is consistent with the
findings of that study, including the tolerated mutations in the
transmembrane region. The reported study has shown that alanine
substitutions of stretches of four residues at aa 173–176, 177–180
were lethal for infectious particle production. We have found,
however, that the insertions at aa 176, 177, and 180 were
tolerated. This finding could be explained by the fact that the
insertion results in duplication of amino acids at the site of Mu-
integration that preserves the original amino acid without deletion
or substitution (Fig. S1).
Functional profiles of E1 and E2 proteins. The envelope
glycoproteins, E1 and E2, facilitate virus entry into host cells
Figure 2. Electropherogram depicting the location of 15-nt insertions in p7-NS2 region and the mutant population replication
dynamics during selection. Each peak (X-axis) represents the location of a 15-nt insertion in the p7-NS2 region, and the fluorescent signal
intensity (Y-axis) indicates the abundance of each 15-nt insertion mutant. The number at the top of the figure corresponds to the JFH-1 genome
position of the p7-NS2 region. The electropherogram panels show the insertion profile of the mutant plasmid library (DNA input), in vitro transcribed
RNA library (RNA input), and Huh-7.5.1 cell culture selected mutant viral library [selection 2, 4, 10, 16, and 21 days post-transfection (dpt)]. The
complexity of the library is similar in DNA input, RNA input, 2 dpt and 4 dpt. Most of the 15-nt insertion mutants have been negatively selected at 10
dpt. Note that the HCV mutants containing 15-nt insertions around p7-NS2 junctions have shown strong positive selection. Asterisks indicate an
artifact peak generated during data processing. To better visualize the short peaks, the fluorescent signal intensity scale was set at 2000; hence some
of the tall peaks are shown out of scale.
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through recognition of cellular receptors [16,17,47]. The 15-nt
insertions in the E1 and E2 regions were mostly deleterious for
virus replication. The percentage of lethal insertions at the E1 and
E2 regions were 94.5 and 88.2, respectively (Table 1). The E1 N-
terminal residues 244–245 and the C-terminal residues 370 and
373–374 had tolerated the insertions. Insertions at aa 481–482,
503–504, and 598, and 622–623 of E2 region were tolerated as
well. It has been shown that the recognition of cellular receptors is
dependent on the conformation of the envelope glycoproteins
[16]. Thus the insertion could possibly affect the host receptor
protein interactions, conformation of glycoproteins, and innate-
immune evasion function, resulting in a lethal phenotype.
Functional profiles of p7 and NS2. The p7 is an ion
channel-forming transmembrane protein. It has two
Figure 3. Genome scale functional profile of HCV. Graphical representation of the location and phenotype of 15-nt insertions in the HCV
genome are shown. For each 15-nt insertion mutant, the ratio of peak area was calculated between selected and non-selected pools and plotted in a
bar graph. The lethal phenotype (critical region, red bar) is an absence of an insertion mutant in the selected population. The attenuated phenotype
(less critical region, blue bar) is an over two-fold reduction in replication. The tolerated phenotype (dispensable region, green bar) is replication
competent. (A) The final assembly shows the fold change (log
10
) and locations of insertions in the HCV genome. A cartoon of the HCV regions is
aligned at the top of graph to show the boundary of each region. The numbering corresponds to the nucleotide (nt) position of JFH-1 genome. (B)
The location and phenotype of insertions at the NS5A region are shown. A schematic diagram of the NS5A domains is aligned with the functional
profile graph. Note that many insertions at domain 3 are tolerated. (C) The crystal structure of NS5B [69] (PDB accession code 1C2P), RNA dependent
RNA polymerase, displays the functional profiling phenotypes. The front and back views of ribbon and surface diagrams of the NS5B monomer is
shown. The fingers, thumb, and palm sub-domains are indicated. The amino acid residues are color coded for insertion phenotypes: red (lethal), blue
(attenuating), green (tolerated), and grey (no insertion). Insertions in the sub-domains forming the catalytic active site were lethal (front view) for
virus replication, whereas many insertions on the outer surface (back view) were tolerated. The crystal structure was analyzed using PyMOL Viewer.
aa, amino acid.
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transmembrane domains (TMD) with a single loop facing the
cytosol and its N- and C-terminal tails oriented towards the
endoplasmic reticulum lumen [48]. The insertions at the junction
of the N-terminal tail and the first TMD (aa 760–765) were
tolerated for virus replication. Insertions at the cytosol loop region
aa 781 and aa 788–789 were tolerated. Four insertions found
between aa 782–787 of the loop region were lethal. Most of the
insertions at TMD regions were lethal. Many insertions at the p7-
NS2 cleavage site, aa 809–818 (nt 2767–2794), were tolerated or
positively selected (Figs. 2,3A). 91% of the insertions at NS2 were
lethal for virus replication, underscoring the importance of NS2 in
the HCV life cycle. The NS2 and NS3 crystal structures [20,49]
with the insertion profiles are shown in Fig. S6.
Functional profiles of NS3 and NS4A. The NS3 is a critical
component of the RNA replicatory complex. NS3 is comprised of
an N-terminal serine protease domain and a C-terminal RNA
helicase/nucleoside triphosphatase domain [6,50]. NS3 plays an
important role in immune evasion [30,31]. The NS3 protease
domain forms a complex with NS4A that is essential for the
processing of NS3/4A, NS4A/4B, and NS4B/5A cleavage sites
[7,51]. NS3 is a highly conserved region of HCV and not
surprisingly, most of the insertions were lethal for virus replication.
Insertions at both the N-terminal (aa 1031–1035) and the C-
terminal (aa 1658–1660) ends were tolerated. Insertions at the
protease domain aa 1126–1129 (Pro-Cys-Lys-Cys), a sub-domain
containing cysteine residues which coordinates a zinc atom, were
tolerated for virus replication (Fig. S6). Insertions at helicase
domain aa 1304–1306 and aa 1312 were tolerated. In NS4A, one
insertion at the membrane anchoring domain (aa 1675), and one
at the C-terminal acidic domain (aa 1713) were tolerated. The
remaining insertions were deleterious for virus replication.
Functional profile of NS4B. NS4B, a transmembrane
protein, is essential for viral genome replication [21,24,52]. An
amphipathic helix (AH) region present at the N-terminal is critical
for viral RNA replication [53]. Consistent with that study, we
found that insertions at the AH region (aa 1730–1745) were lethal
for virus replication. It has been reported that the N- and C-
terminal less-conserved domains were the major determinants for
efficient RNA replication [54]. The insertions at the determinant
N-terminal (aa 1754–1760) and C-terminal (aa 1974–1976)
regions were tolerated. Insertions at the ends of a predicted
cytoplasmic loop region [55] aa 1847–1848 and aa 1856–1857
were tolerated, however, insertions in the mid-loop aa 1849–1854
were deleterious. Insertions in all four predicted transmembrane
domains were lethal. Many of the insertions at the cleavage sites of
NS4B/5A and NS5A/5B were tolerated. The predicted amino
acid sequences of the insertion sites revealed that the insertion did
not disrupt the critical P1-P19 cleavage residues (Fig. S7).
Functional profile of NS5A. The NS5A is essential for viral
genome replication; however its precise role in the virus life cycle is
unknown. NS5A is a membrane-bound, phosphorylated, zinc-
binding metalloprotein [56,57,58]. It modulates the cellular
environment by direct or indirect association with host factors
[28,59,60]. Many cell culture adaptive mutations have been
mapped to NS5A [24,41]. NS5A is predicted to have three
domains [61]. Our findings showed that 82.9% of the insertions at
NS5A region-1, 63.4% at region-2, and 39.6% at region-3 were
lethal (Fig. 3B and Table 1). Many insertions in region- 2 and 3
had an attenuating phenotype. Insertions at aa 2014–2015 (Cys-
residue binding to zinc) were tolerated and the predicted insertion
sequence showed that the insertion did not affect the Cys residue
involved in zinc binding. The NS5A crystal structure [56] with the
mutational profile is shown in Fig. S6. Insertions in the region aa
2209–2254, corresponding to a 47 aa deletion, encompassing the
interferon sensitivity determining region [60], that was tolerated
for sub-genomic RNA replication [24], had severely impaired
virus replication fitness. All but one of 22 insertions between aa
2282–2320 of region-2 were deleterious for virus replication,
which further supports the essential role of these residues in sub-
genomic HCV RNA replication [62]. Our profiling analysis
showed that the NS5A region-3 was the most tolerated (48%)
region for insertion. Previous studies using sub-genomic replicons
have shown that the NS5A C-terminal had tolerated heterologous
insertions, including green fluorescent protein (GFP) and
transposons [58,63,64], however a sub-genomic mutant with a
larger insert (Renilla luciferase gene) had a defect in viral RNA
Table 1. The percentage and number of insertions in various regions of HCV.
Region Genome Location Size (nucleotides)
Total Insertions
in a Region Tolerated Attenuated Lethal
59NTR 55–340 286 93 (3.9%) 37 (39.8%) 8 (8.6%) 48 (51.6%)
Core 341–913 573 81 (3.4%) 24 (29.6%) 8 (9.9%) 49 (60.6%)
E1 914–1489 576 109 (4.5%) 6 (5.5%) 0 (0.0%) 103 (94.5%)
E2 1490–2590 1101 272 (11.3%) 24 (8.8%) 8 (3.0%) 240 (88.2%)
p7 2591–2779 189 64 (2.7%) 18 (28.1%) 2 (3.1%) 44 (68.8%)
NS2 2780–3430 651 199 (8.3%) 15 (7.5%) 3 (1.5%) 181 (91.0%)
NS3 3431–5323 1893 467 (19.5%) 39 (8.4%) 5 (1.0%) 423 (90.6%)
NS4A 5324–5485 162 26 (1.0%) 3 (11.5%) 1 (3.9%) 22 (84.6%)
NS4B 5486–6268 783 177 (7.4%) 23 (13.0%) 0 (0.0%) 154 (87.0%)
5A Region 1 6269–7016 748 170 (7.1%) 21 (12.4%) 8 (4.7%) 141 (82.9%)
5A Region 2 7017–7318 302 82 (3.4%) 12 (14.6%) 18 (21.0%) 52 (63.4%)
5A Region 3 7319–7666 348 106 (4.4%) 48 (45.3%) 16 (15.1%) 42 (39.6%)
NS5B 7667–9442 1776 467 (19.5%) 92 (19.7%) 22 (4.7%) 353 (75.6%)
39NTR 9443–9571 129 86 (3.6%) 12 (13.95%) 12 (13.95%) 62 (72.1%)
Total 55–9571 9517 2399 (100.0%) 374 (15.6%) 111 (4.6%) 1914 (79.8%)
doi:10.1371/journal.ppat.1000182.t001
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replication [58]. A recent report showed that the GFP insertion at
the NS5A C-terminal resulted in over 100-fold reduction in
infectious virus production, but had no effect on viral RNA
replication [65]. It has been shown that the C-terminal serine
residue (aa 2433) is critical for infectious virus production [66]. We
have found that insertions between aa 2429–2437 were lethal for
virus replication. Through characterizing mutant viruses having
serial deletions of domain III, a report has shown that the NS5A
domain 3 is involved in the assembly of infectious viruses [67].
These mutant viruses exhibited phenotypes ranging from defective
Figure 4. Functional Profiles of the JFH-1 HCV 59NTR cis-elements. The predicted secondary structures of 59NTR are shown. The numbers
correspond to the JFH-1 genome sequence. The locations of 15-nt insertions are indicated as filled circles. The colors of the filled circles represent the
phenotypes: lethal (red), attenuating (blue), and tolerated (green). 59NTR stem loop domains (I, II, III and IV) are shown. The loop region of IIIb and
stem loop domain IV had many tolerated insertions. The translation initiation codon AUG is highlighted.
doi:10.1371/journal.ppat.1000182.g004
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to normal growth properties. In addition to tolerated insertions, a
total of 39.6% of the insertions at region-3 resulted in a lethal
phenotype, suggesting that this region plays a critical role in viral
replication fitness.
Functional profiles of NS5B and cis-element
5BSL3. NS5B is an RNA-dependent RNA polymerase that
consists of fingers, palm, and thumb domains involving polymerase
activity, and C-terminal regulatory and membrane-anchoring
domains [68,69,70]. Analysis of functional profiles incorporated in
the crystal structure of NS5B [69] showed that all of the insertions
in the sub-domains forming the catalytic site were lethal, whereas
several insertions on the outer surface were tolerated (Fig. 3C).
75.6% of insertions in NS5B had a deleterious effect and 19.7%
insertions had no effect on virus replication. Insertions at a loop
region (aa 2589–2592) that interconnects fingers and thumb
domains, were tolerated. The palm domain loop region aa 2793–
2795 and 2797–2798 tolerated the insertions. Tolerated insertions
were found at aa 2814–2816 of the junction of the palm-thumb
domains. Insertions at the thumb domain helix (aa 2873–2881)
were well tolerated for virus replication. Insertions at the C-
terminal transmembrane region aa 3012–3016 (nt 9368–9386)
were tolerated, while insertions at aa 3027–3031(nt 9421–9433)
were deleterious (Figs. S3,S4). The NS5B coding region contains a
cis-acting replication element (CRE) predicted to have a cruciform
stem loop structure, 5BSL3 [71,72] (Fig. 5). The formation of a
kissing-loop interaction between loop regions of 5BSL3.2 and
39NTR SL2 is critical for viral RNA replication [26,72]. Our
mutational profile showed that the insertions at 5BSL3.1 (nt 9291–
9357) and 5BSL3.2 were lethal for virus replication. All but one of
14 insertions at nt 9368–9386, which encompass a bulge region
between 5BSL3.2 and 5BSL 3.3, were tolerated. The insertions at
this region could affect the function of both the NS5B protein and
the 5BSL3 cis-element.
Functional profile of 39NTR cis-elements. 39 NTR
consists of a proximal variable region (VR), poly(U/UC) tract of
varying length, and a conserved 39X tail [27] (Fig. 5). The 39NTR
Figure 5. Functional Profiles of the JFH-1 HCV 5BSL3 CRE and 39NTR cis-elements. The stem loop structures are shown. The colors of the
filled circles represent the phenotypes: lethal (red), attenuating (blue), and tolerated (green). Insertions at 5BSL- 3.1 and 3.2 were lethal for virus
replication. Many insertions at the bulge region between CRE 5BSL- 3.2 and 3.3 were tolerated. The kissing-loop interaction between CRE 5BSL3.2,
and 39SL2 is depicted with dotted lines. The 39NTR predicted variable region stem loop structures (VSL1 and VSL2), poly (U/UC) tract, and 39X tail stem
loop structures are shown. Many insertions at VSL2 were tolerated and attenuating. The stop codon UAG is highlighted. All the insertions at poly-U
tract were lethal.
doi:10.1371/journal.ppat.1000182.g005
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region is involved in initiation of negative-strand genome
replication and enhancement of translation [25,26,27]. Several
cellular and viral proteins interact with 39NTR [28,73]. The
functional profile of the variable region and poly (U/UC) tract was
obtained. Among a total of 27 insertions at the 39NTR variable
region cis-element, 11, 11, and 5 of the insertions had tolerated,
attenuating, and lethal phenotypes, respectively. Four of the 5
insertions exhibiting lethal phenotypes at VR were found adjacent
to the poly(U/UC) tract. All of the insertions at the poly(U/UC)
tract were lethal for virus replication (Fig. S3), which is consistent
with a recent report [26]. Freibe and colleagues [25,72] reported
that the insertion of CRE 5BSL3 at VR did not affect the
replication of a sub-genomic replicon; however, deletion of VR
region impaired genome replication. The poly(U/UC) tract has
been reported critical for viral replication [25,26,27,42,71]. The
insertions at 39NTR could possibly affect the binding of cellular
factors involved in RNA genome replication leading to attenuating
or lethal phenotype.
Validating the phenotypes identified by the functional
profiling screen
We focused on the NS3, NS5A, NS5B, and 39NTR regions for
validating the 15-nt insertion phenotypes because of their critical
role in viral genome replication. The nucleotide/amino acid
sequences of the mutations that were introduced in the HCV
genome are shown in Fig. 6A. The functional profiling analysis
showed that the 15-nt insertions at NS3-4635 and NS3-4944
(helicase domain) and NS5B-8865 (thumb domain) resulted in a
lethal phenotype. The 15-nt insertions at NS5A region-2 (nt 7135)
and region-3 (nt 7376, nt 7622) and 39NTR (nt 9463) were
tolerated for virus replication (Fig. S4). The effect of these
mutations on virus replication was validated with individual
mutant viruses based on a monocistronic Renilla luciferase HCV
reporter virus, NRLFC (Fig. S8). The mutant reporter viruses
defective in RNA polymerase activity (pol-null) and envelope (env-
null) were included as controls for RNA genome replication and
infectious virus production. The genome replication and the
supernatant infectivity of mutant viruses were assessed by
measuring the Renilla luciferase activity of the transfected and
infected Huh-7.5.1 cells at the indicated time points, respectively
(Fig. 6 B,C). The core and NS3 antigen expression of transfected
cells was tested at 96 hpt (Fig. 6D). The mutant viruses
demonstrated a consistent phenotype with that of the genome-
scale functional profiling analysis, confirming the usefulness of this
system. Similar results were also obtained with JFH-1 based
mutants (data not shown). Our study using JFH-1 mutant viruses
containing alanine-substitution of core C-terminal transmembrane
domain showed that several residues were dispensable for
infectious virus production, further validating our functional
profiling system (RR and VA unpublished observation). As this
experiment was completed, an independent study reported similar
findings [13].
Figure 6. Validating the HCV functional profiling phenotypes by individual mutant viruses. (A) Nucleotide and amino acid sequence
information for the 15-nt insertions engineered in the individual mutant NRLFC reporter viruses is shown. The inserted nucleotide/amino acid
sequences are shown in bold face. The genomic position of nucleotide and amino acid residues are indicated. (B) Analysis of viral genome replication
of mutant viruses. 10 mgofin vitro transcribed genomic RNA of wild-type NRLFC reporter virus and the mutant reporter viruses were individually
introduced into Huh-7.5.1 cells by electroporation. The mutant reporter viruses lacking envelope (env-null) and polymerase activity (pol-null) are
included as controls. The transfected cells were lysed at indicated time points using Promega passive lysis buffer, and the levels of Renilla luciferase
were quantified. The experiment was done in triplicate and the mean values with standard deviation of Renilla luciferase values (RLV) are presented as
a bar graph in log
10
scale. (C) Measuring the production of infectious viral particles by mutant viruses. The cell-free supernatants harvested at 48 and
96 hours post-transfection (hpt) were inoculated onto naı¨ve Huh-7.5.1 cells. At 48 hpt the cells were lysed and the Renilla luciferase activities were
assayed. The mean RLV with standard deviations are shown in the graph. The replication deficient mutants show only background level of luciferase
activity. (D) The expression of HCV non-structural and structural proteins. The protein lysates obtained at 96 hpt were subjected to western blotting.
The HCV core and NS3 antigens were detected by primary mouse monoclonal antibodies and secondary goat-anti mouse IgG conjugated with HRP.
b-actin was included as a loading control.
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Role of 39NTR variable region for viral replication
The functional profiling of the 39NTR revealed that an equal
number of insertions at the VR exhibited either tolerated or
attenuated phenotypes. An earlier study in chimpanzees has
shown that a mutant virus lacking 24 nucleotides at the 39NTR
VR is replication-competent in vivo [27]. Subsequent studies
showed that a sub-genomic replicon lacking partial or complete
VR was viable, but genome replication was impaired significantly
[25,42]. We have found many of the insertions at the 39NTR VR
resulted in attenuation of virus replication. Furthermore, based on
the kinetics of disappearance of viral mutants having altered
poly(U/UC) and variable regions, we hypothesized that the
39NTR plays a critical role in infectious particle production. To
test this hypothesis we have constructed mutant reporter viruses
having deletions of 14 nucleotides [DVR14 (nt 9457–9470)] or 28
nucleotides [DVR28 (nt 9443–9470)] or substitution of the VR,
which encompasses a seven-nucleotide conserved sequence
(Fig. 7A). The substitution mutants behaved like parental NRLFC
reporter virus, while the deletion mutants had impaired RNA
genome replication and viral particle production (Fig. 7B–D).
Because the NRLFC reporter virus had a reduced infectivity
compared to that of parental J6/JFH-C virus (Fig. S8), we tested
the VR deletion mutants with J6/JFH-C background (Fig. 7E–G).
The deletion of 14 nucleotides at VR resulted in a significant
reduction in viral replication and infectious particle production.
Although the J6/JFH-DVR28 virus had a lower level of genome
replication and core antigen expression (Fig. 7F,G), there was no
detectable amount of infectious virus in the supernatant.
Furthermore, the wild-type virus transfected cells underwent
growth arrest and dead cells/cell debris were detected in the
culture medium indicating CPE. The cell growth arrest and CPE
were not observed in J6/JFH-DVR28 and J6/JFH-DVR14
mutant genome transfected cells. These results suggest that the
39NTR VR mediated-spacing is important for efficient viral
replication and infectious virus production. Taken altogether, the
functional profiling system effectively identified viral functional
domains throughout the HCV genome.
Discussion
We have described a high-throughput, quantitative mutational
analysis system to identify HCV protein domains and cis-acting
elements that are essential or non-essential for virus replication in
cell culture. We took advantage of advances in DNA analyzing
technology to obtain the whole genome functional profile of HCV.
We have characterized a library of mutants with 2399 random
insertions between nt 55 and 9571 of HCV genome. Approxi-
mately 80% (1914), 4.6% (111), and 15.6% (374) of the insertions
had lethal, attenuating, and tolerated phenotypes, respectively.
Most of our data is consistent with previous studies using
individual mutants, which validates our approach [8,13,25,26,-
27,42,43,46,54,62]. The phenotypes identified through transpo-
son-insertion and conventional site-specific (deletion and substitu-
tion) mutagenesis could differ in some instances due to the nature
of genetic changes.
Among other members of Flaviviridae, while deletion of the
39NTR variable region of dengue virus resulted in severe
attenuation of virus growth [74], there was no effect of such
deletion on tick-borne encephalitis virus replication [75]. We have
dissected the role of 39NTR VR during virus replication by
insertion mutagenesis, deletions, and substitutions. Deletion of VR
resulted in reduction in viral genome replication and infectious
viral particle production. Based on the substitution study at the
39NTR VR, the stem loop structure, if present, is not essential for
genome replication and virion morphogenesis and release. A
previous study has shown that the conserved seven nucleotides at
the VR are dispensable for RNA replication [29]. We found that
these conserved nucleotides do not have a direct role in both viral
RNA genome replication and infectious virion production in cell
culture. On one hand, the deletion of the 39NTR VR results in the
reduction of genome replication; on the other hand, replacement
of the deleted 39NTR VR region with heterologous polynucleo-
tides results in restoration of genome replication to that of wild-
type virus, indicating that irrespective of nucleotide sequence, the
spacing between the region up- and downstream of 39NTR VR
region is important for efficient RNA replication. The VR might
have a role in efficient genome packaging or binding of RNA
replication machineries onto the 39NTR during virus replication
in the host cell.
We have identified several novel regions in 59NTR, p7, NS3,
NS4B, NS5A, NS5B, and 39NTR that tolerate the insertions for
virus replication in cell culture. These domains, however, could
play important roles in in vivo infection, including immune evasion.
Defining regions critical and less-critical for viral replication at the
genome scale will facilitate the rational design of vaccine
candidates. The HCV regions with attenuating insertions could
be further characterized by deletion mapping. Attenuating
deletions along with mutations that inactivate immune evasion
domains can be combined into developing a live virus vaccine that
is attenuated and non-recombinogenic.
In order to complete the viral lifecycle, the virus hijacks and/or
counteracts cellular functions, including signaling pathways, cell
cycle regulations, and innate and adaptive immune responses. Our
approach can be used to define such interactions. For example,
comparing mutational profiles of HCV mutant libraries selected in
cells that are deficient in an innate immune factor would facilitate
identifying viral determinant(s) that counteract or modulate the
host response pathway. The in vivo role of tolerated insertions could
be dissected by passaging and profiling the HCV mutant library in
the human liver cell-grafted mouse model or a primate model.
Moreover, modeling the HCV protein structures based on
mutational profiles could be useful to elucidate the structure-
function relationship of individual HCV proteins. These future
investigations would shed light on the mechanism of HCV
replication. Furthermore, the high-throughput mutational analysis
platform would be a useful tool for the functional genomics study
of other RNA and DNA viruses.
Materials and Methods
Cells
The Huh-7.5.1 cell line (a kind gift from Dr. Francis Chisari,
The Scripps Research Institute, La Jolla) was cultured in complete
DMEM containing 10% fetal bovine serum, 10 mM non-essential
amino acids (Invitrogen, Carlsbad, USA), 10 mM Hepes,
penicillin (100 units/ml), streptomycin (100 mg/ml), and 2 mM
L-glutamine at 37uC with 5% CO
2
.
Virus and Plasmid Constructs
The plasmid containing the complete genome of a HCV GT2a
strain JFH-1 (kindly provided by Dr. Takaji Wakita, National
Institute of Infectious Diseases, Japan), was used for construction
of recombinant viruses. An intra-genotype chimeric virus, pJ6/
JFH-C, comprising 59NTR, structural regions and part of non-
structural regions (p7 and partial NS2) of the J6CF strain (NCBI
accession no. AF177036) and non-structural regions of JFH-1
strain was generated. The J6CF genomic region, nt 1 to 2878, was
synthesized by PCR based assembly of oligonucleotides (Invitro-
Functional Profiling of HCV
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Figure 7. Analysis of 39NTR Variable Region in HCV replication. (A) Nucleotide sequence of JFH-1 39NTR variable region and the mutations
engineered in the individual mutant viruses are depicted. The genome position of the nucleotides is indicated. The sequence that is conserved across
all the genotypes is underlined. The deleted polynucleotide regions are shown in dotted lines. The substituted heterologous polynucleotides are in
italics. 15-nt insertion sequence is boxed. Due to space limitation, only a partial sequence is shown for 39NTR-9463 mutant. (B) Analysis of viral
genome replication of mutant reporter viruses. The mean Renilla luciferase values (RLV) with standard deviations are shown in the graph (log
10
scale).
(C) Measuring the production of infectious viral particles by mutant reporter viruses. The mutants deficient in production of infectious particles show
only a background level of luciferase activity. (D) Western blotting analysis of viral proteins, NS3 and core, expression. (E) Comparison of J6/JFH-C
mutant viral infectivity. The virus titer (ffu/ml) of cell-free supernatant collected at 48 and 96 hpt of J6/JFH-C based mutants’ transfected cell culture
was measured by infecting naı¨ve Huh-7.5.1 cells. Mean values and standard deviations are shown in the graph. (F) Comparison of J6/JFH-C mutant
Functional Profiling of HCV
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gen). The T7 promoter sequence (59-TAATACGACTCACTA-
TAG-39) and nt 2879 to 2967 of the JFH-1 isolate was engineered
at the 59 and 39 ends of the J6CF 1-2878 fragment, respectively.
The final assembled PCR product (T7-J6CF/JFH) was cloned into
pZero-blunt vector (Invitrogen) and sequence verified. The EcoRI-
NotI fragment (2.9 kb) of the pJFH-1 was swapped with the
assembled T7-J6CF/JFH fragment to obtain pJ6/JFH-C. A
monocistronic chimeric reporter virus, pNRLFC, which was based
on pJ6/JFH-C parental virus, was constructed. A plasmid
containing a reporter cassette, T7-59NTR (388 nucleotides)-Renilla
luciferase gene-F2A seqeuce-Core-E1, was constructed. The F2A
sequence introduced was 59-GTGAAACAGACTTTGAATTTT-
GACCTTCTCAAGTTGGCCGGAGACGTCGAGTCCAACC-
CTGGGCCC -39. The EcoRI-BsiWI fragment containing the
reporter cassete was subcloned into pJ6/JFH-C to construct
pNRLFC. To construct the envelope-null mutant virus, an in-
frame deletion of nt 1040–2215 was engineered in the pJ6/JFH-C
genome. This deletion removed most of the E1 and E2 coding
regions. An identical E1 and E2 deletion mutant reporter virus
pNRLFC was also constructed. An RNA polymerase-null virus
with pJ6/JFH-C or pNRLFC background was constructed by
mutating the catalytic residues GDD to AAG amino acid residues.
The NotI restriction enzyme site present in JFH1 genome at nt
2955 was abolished by PCR-mediated introduction of silent point
mutations (CGGC-.TGGT). These point mutations did not
affect the virus infectivity (data not shown). This plasmid was
subjected to in vitro Mu-transposon mediated mutagenesis (MGS
kit, Finnzymes). The location of transposon insertion in the JFH-1
genome was identified by sequencing using a transposon-specific
primer (59-CAGAGATTTTGAGACACAACGT-39). The plas-
mids having a transposon insertion at NS3-4635, NS3-4937,
NS5B-8865, NS5A-7622, and 39NTR-9463 were included for NotI
restriction digestion to remove the transposon fragment, resulting
in only 15-nt remaining at the insertion site. PCR-mediated site-
specific mutagenesis was employed to introduce several mutations
into the viral genome. The mutant JFH-1 plasmids containing a
15-nt insertion at pJFH-1 NS5A regions, NS5A-7135 (59-
CTTGATGCGGCCGCA-39) and NS5A-7376 (59-AACU-
GUGCGGCCGCA-39) were constructed. The pJFH-1 mutant
plasmid with substitution of 7 nucleotides (nt 9460–9467) or 28
nucleotides (nt 9443–9470) at 39NTR were constructed. The pJ6/
JFH-C based mutant plasmids having deletion of 14 nucleotides
[(nt 9457–9470) pJ6/JFH-DVR14] or 28 nucleotides [(nt 9443–
9470) pJ6/JFH-DVR28] at 39NTR were constructed. To
construct reporter mutant viruses, the EcoRI 2AvrII fragment of
the pJFH-1 and pJ6/JFH-C based mutant plasmids was replaced
with that of the reporter virus pNRLFC. The sequence
information of the primers used for the construction of mutant
viruses will be available upon request.
In vitro Transcription and RNA Transfection
The viral plasmids linearized by XbaI restriction enzyme and
treated with mung bean nuclease (New England Biolabs, Beverly,
USA) were subjected to in vitro transcription using T7 Ribomax
Express Large Scale RNA Production System according to the
manufacturer’s instructions (Promega Corporation, Madison,
USA). A total of 16 mg of the pJFH-1 insertion library DNA was
used for in vitro transcription. The DNase-treated RNA were
purified and stored at 280uC in aliquots. The in vitro transcribed
RNAs were electroporated into Huh-7.5.1 cells. Briefly, the Huh-
7.5.1 cells were trypsinized and washed twice with ice cold Opti-
MEM transfection media (Invitrogen) and resuspended in Opti-
MEM at 1610
7
cells per ml. 10 mgofin vitro transcribed RNA was
mixed with 400 ml of cells in 0.4 cm electroporation cuvettes.
Electroporation was conducted by using a BioRad elecroporator
with the settings of 270 V, 100 ohms, and 960 mF. Subsequently,
the cells were resuspended in 40 ml of complete DMEM and
plated in T-75 flasks and 48-well plates. At 8 hpt, media
containing dead cell debris in the culture flasks and plates were
replaced with fresh complete DMEM.
Generation of 15-nt Insertion JFH-1 Plasmid Library
The plasmid pJFH-1lacking NotI site, was subjected to in vitro
Mu-transposon mediated mutagenesis (MGS kit, Finnzymes). A
total of 4.7610
5
individual bacterial colonies were obtained and
the mutant plasmids were isolated from the pooled bacterial
colonies. To remove the transposon DNA fragment, 5 mg of the
pooled mutant plasmids were subjected to NotI digestion, self-
ligation, and selection in bacteria. This resulted in a library of
mutants having a 15-nt sequence, 59-NNNNNTGCGGCCGCA-
39 (N: duplicated 5 nucleotides from target DNA), inserted
randomly in the pJFH-1 plasmid.
Genetic Selection of Mutant JFH-1 Library in cell culture
A total of 16 mg of the pJFH-1 library DNA was used for in vitro
transcription. 120 mg of DNase-treated RNA was delivered into
4.8610
7
Huh-7.5.1 cells by electroporation. The cells were plated
in twelve T-150 flasks and were split into forty T-150 flasks at 1:3
or 1:4 ratios on 4, 7, 10, 13, and 16 dpt. During each split, ,one
third of the pooled cells was saved for RNA isolation. The selection
was terminated at 21 dpt, when many of the cells were started
showing CPE [34,41]. Total RNA was isolated from the cells using
Tri-reagent (Molecular Research Center Inc. Cincinnati, OH).
The DNase-treated and -purified RNA was used for functional
profiling analysis.
Functional Profiling Analysis of the HCV genome
A total of 65 mg of RNA from each of the non-selected and cell
culture selected JFH-1 mutant RNAs were reverse transcribed by
Superscript III Reverse Transcriptase (Invitrogen) using random
hexamers. The cDNAs and pJFH-1 library DNA (included to
ascertain the library complexity) were used as templates for PCR
amplification of thirteen overlapping fragments using JFH-1
specific primers (Table S1). Each fragment has an overlap of
,200 nt with flanking fragments. Fifty nanograms of purified RT-
PCR or PCR product was used as a template for a second PCR
with an insertion-specific mini-primer (59-TGCGGCCGCA -39),
which has 59 end labeled with a fluorescent dye-VIC (Applied
Biosystems), and one of the JFH-1 fragment specific primers
(Table S2). A total of forty-eight JFH-1 specific primers, designed
at approximately 200 nt intervals, were used. Each of the JFH-
specific primer and mini-primer combinations tested negative for
generating any spurious PCR products using wild-type JFH-1
genome template. For each primer, the PCR reactions were done
in duplicate. The conditions used for the second PCR were 95uC
for 5 min (1 cycle); 95uC for 1 min, 52uC for 1 min and 72uC for
2 min (35 cycles); 72uC for 20 min (1 cycle). The fluorescent-
viral genome replication. At 4, 48, and 96 hpt total cellular RNAs were harvested and subjected to RT-qPCR. The genome copy numbers per mg of RNA
are presented. (G) Immunofluorescence assay. At 48 and 96 hpt the cells were fixed and stained for HCV core antigen. The cell nuclei were visualized
by DAPI staining. For B, C, and D experimental details see Figure 6 legend.
doi:10.1371/journal.ppat.1000182.g007
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labeled PCR products were analyzed in duplicate with Liz-500
size standard (Applied Biosystems) by using a 96-capillary
genotyper (3730xl DNA Analyzer, Applied Biosystems) at the
UCLA Genotyping and Sequencing core facility.
Data Processing and Interpretation
The data generated by the capillary genotyper were processed by
GeneMapper software (Applied Biosystems) using Amplified
Fragment Length Polymorphism analysis tool. The normalized
data was visualized by an electropherogram and exported as a data
file. For each PCR sample, the exported data contained information
regarding the PCR product size at nucleotide level and peak area.
The exact position of an insertion in the genome, for each of the 48
gene specific primer-generated PCR products, was calculated by
subtracting 15-nt from the size of the particular PCR product and
adding the HCV genome position of the specific primer.
Comparison of the 15-nt insertion sites identified in the mutated
HCV genome by PCR profiling and sequencing revealed that the
accuracy of the PCR profiling is within one to two nucleotide(s). For
each sample, the PCR profiles were consistent among duplicates
and representative data were used for obtaining final assembly. To
assemble the locations of insertion sites for the entire HCV genome,
the insertion profiles obtained between 50 to ,250 nt for each
specific primer was taken into account. To assign a phenotype for
each insertion mutant, the ratio of peak area between selected (21
dpt) and non-selected pools was calculated. The incorporation of
functional profile data into the crystal structure of HCV proteins
and the generation of graphics were done using PyMOL Viewer
program (DeLano Scientific, USA).
Quantitative Reverse Transcription-PCR
A two-step reverse transcription-PCR (RT-PCR) was carried
out for determining the HCV RNA copy number. Briefly, 1 mgof
total cellular RNA was reverse transcribed by using Superscript III
Reverse Transcriptase (Invitrogen) and HCV 59UTR specific
primers (JFH RTQ F: 59- CTGGGTCCTTTCTTGGATAA-39
and JFH RTQ R: 59- CCTATCAGGCAGTACCACA-39) as per
the manufacturer’s instructions. 100 ng of resulting cDNA was
used as a template for the subsequent quantitative-PCR (Q-PCR)
using QuantiTect Probe FAM 59-GAGTAGCGTTGGGTTG-39
(Qiagen). 10
1
to 10
7
copies of in vitro transcribed JFH-1 genomic
RNA were reverse transcribed along with the samples, and were
included as a standard for copy number determination during Q-
PCR. The reaction was run at 95uC for 15 min (1 cycle), 95uC for
30 s, 55uC for 30 s, and 72uC for 10 s (45 cycles). The results were
analyzed in Opticon II (MJ Research, Cambridge, MA).
Measuring virus titer
The virus titer was measured by calculating the foci forming
unit (ffu) of infectious viral particles per ml of cell-free culture
supernatant. The infected-culture supernatant was 10-fold serially
diluted in complete DMEM and inoculated in triplicate onto naı¨ve
Huh-7.5.1 cells (3610
3
cells/well) in 96-well plates. At 72 hours
post infection (hpi), the cells were fixed and immunostained for
HCV core antigen. The number of core antigen positive foci were
counted at the highest dilution, and average foci forming units per
ml was calculated.
Renilla Luciferase Reporter Assay for Viral Genome
Replication and Infectivity
For viral genome replication assay, the HCV RNA transfected
cells were plated in triplicate in 48-well plates. The cells were lysed
with passive lysis buffer (Promega) at 6, 48, and 96 hpt. The culture
plates were gently rocked at room temperature for 15 min, then
stored at 280uC. To determine the supernatant infectivity, 500 ml
of cell-free supernatant obtained from HCV RNA transfected cells
at 48 and 96 hpt was inoculated in triplicate onto naı¨ve Huh-7.5.1
cells in 48-well plates. At 48 hpi the cells were lysed and stored at
280uC. 10 ml of lysate was used for measuring the Renilla luciferase
activity using a Renilla Luciferase Assay System kit (Promega).
Western blotting
For western blotting, the cell lysates were resolved by SDS-
PAGE and transferred to a nitrocellulose membrane. The
membranes were blocked (5% skim milk, 0.2% Tween-20 in
PBS) and probed with mouse monoclonal antibody to core [(C7-
50) Abcam], NS3 [(H23) Abcam)] and beta-actin (Sigma). Goat
anti-mouse IgG conjugated with horseradish peroxide (Amersham
Pharmacia Biotech) secondary antibody was detected by chemi-
luminescence (ECL Plus, Amersham Pharmacia Biotech).
Immunofluorescence assay
The HCV infected or transfected cells were fixed with 4%
paraformaldehyde. Following three PBS washes, the cells were
blocked (3% goat serum, 3% BSA, 0.1% Triton-x 100 in PBS) and
incubated with mouse monoclonal anti-core primary antibody (C7-
50 (Abcam, Cambridge, USA)) at a dilution of 1:300 for 5 hrs at 4uC.
The goat anti-mouse IgG polyclonal antibody conjugated to Cy-3
was added as a secondary antibody (Jackson ImmunoResearch
Laboratories, USA) at 1:200 dilution and incubated for 1 hr at room
temperature. Between antibody changes, the cells were washed thrice
with PBS. The nucleus was stained with DAPI (Sigma).
Supporting Information
Figure S1 The amino acid sequences encoded by 15-nt insertions
at three possible reading frames. The duplicated nucleotides at the
target site of the mini Mu-transposon insertions are underlined. The
inserted sequences are shown in bold face. The amino acid (aa)
sequences are in italics. The numbers corresponds to the amino acid
positions of the JFH-1 genome. Note that the insertions do not
introduce STOP codons in all of the reading frames.
Found at: doi:10.1371/journal.ppat.1000182.s001 (0.66 MB EPS)
Figure S2 Kinetics of JFH-1 library replication in Huh-7.5.1
cells. The HCV genome copy numbers and the virus titer [foci
forming unit per milliliter (ffu/ml)] for the indicated time points
are shown in the line and bar graphs, respectively. Mean values
with standard deviations are presented (log
10
scale).
Found at: doi:10.1371/journal.ppat.1000182.s002 (0.83 MB EPS)
Figure S3 Electropherogram depicting the effect of 15-nt
insertions in NS5B-39NTR of HCV. The X-axis shows the 15-nt
insertion sites as corresponding peaks and the Y-axis shows the
fluorescent signal intensity of the peaks. Nucleotide positions of the
JFH-1 genome are numbered on the top. Schematic representa-
tions of the NS5B Transmembrane Domain (TMD) coding region,
39NTR Variable Region (VR), and poly(U/UC) tract locations are
depicted. The cDNA generated from the in vitro transcribed
mutant RNA genomic library (RNA input) and JFH-1 mutant
viral library selected in Huh-7.5.1 cell culture (selection 2, 4, 10, 16
and 21 dpt) were subjected to the functional profiling analysis.
Comparison of electropherogram panels shows that all of the
insertions at poly(U/UC) tract were negatively selected by 2 dpt.
Insertions at the VR show a gradual reduction in replication
fitness. Insertions at NS5B-TMD show positive or negative
selection depending on the insertion site.
Found at: doi:10.1371/journal.ppat.1000182.s003 (0.03 MB PDF)
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Figure S4 Genome scale functional profile of HCV. Graphical
representation of location and phenotype of 15-nt insertions in the
HCV genome are shown. The nucleotide and amino acid (in
parenthesis) numbers correspond to the JFH-1 genome sequence.
A schematic diagram of the HCV region is shown for each graph.
For each 15-nt insertion mutant, the ratio of the peak area was
calculated between selected (21 dpt) and non-selected pools and
plotted in a bar graph as fold change (log
10
scale). The lethal
phenotype (critical region, red bar) is an absence of an insertion
mutant in the selected population. The attenuated phenotype (less
critical region, blue bar) denotes an over two-fold reduction in
replication. The tolerated phenotype (dispensable region, green
bar) is replication competent.
Found at: doi:10.1371/journal.ppat.1000182.s004 (0.12 MB PDF)
Figure S5 The predicted secondary structures of 15-nt insertions
at 59NTR domain IV. The tolerated insertions maintain an open
confirmation similar to that of the wild-type domain IV, whereas
lethal insertions form a stable stem loop structure. The asterisk
indicates an insertion mutant not present in our screen. Insertions
at nt-336 and nt-338 resulted in duplication of the AUG start
codon.
Found at: doi:10.1371/journal.ppat.1000182.s005 (0.03 MB PDF)
Figure S6 The crystal structure of HCV proteins displaying
functional profiling phenotypes. The amino acid residues were
color coded for insertion phenotypes: red (lethal), blue (attenuat-
ing), green (tolerated), and grey (no insertion). (A) The ribbon
diagrams depict a dimer of NS2 protease domain (amino acid
residues 94-217) (PDB accession code 2hd0) [20]. (B) Ribbon and
surface diagrams of HCV genotype 1 NS3 monomer are shown
(PDB accession code 1CU1) [49]. The protease and helicase
domains are indicated. (C) The genotype 1b, Con1 isolate NS5A
domain 1 (PDB accession code 1ZH1) [56] ribbon and surface
diagrams are shown (bottom, front and top views). The zinc atoms
in NS3 and NS5A structures are colored in magenta. The
subdomain(s) coordinating the zinc atom had tolerated insertions
in both NS3 and NS5A proteins. The structure analysis and
graphics generation were done using PyMOL Viewer.
Found at: doi:10.1371/journal.ppat.1000182.s006 (5.03 MB EPS)
Figure S7 The 15-nt insertions tolerated for HCV replication at
NS4B/5A and NS5A/5B cleavage sites. The nucleotide and the
predicted amino acid sequences are shown. The number indicates
JFH-1 genome position. The insertion sequences are bold faced.
Insertions tolerated at the NS4B/5A (A) and NS5A/5B (B)
cleavage sites are shown. The cleavage site is indicated by an
arrow. Note that the insertions do not disrupt the critical P1-P19
cleavage residues Cys-Ser (C-S). Asterisks indicate the insertion
mutants that were not present in our screen. The function of
amino acid residues at the N- and/or C-terminal of many HCV
proteins was not affected by the insertions. The 15-nt insertion
does not introduce a stop codon for any of the three reading
frames: eg., insertions at nucleotides 6262, 6263 and 6264.
Found at: doi:10.1371/journal.ppat.1000182.s007 (0.40 MB TIF)
Figure S8 Analysis of genotype 2a chimeric parental and mono-
cistronic Renilla Luciferase reporter Hepatitis C viruses. (A)
Schematic representation of Hepatitis C viruses used in this study.
The HCV non-coding and coding regions are depicted. The J6/
JFH-C virus contains 59 nontranslated region (NTR), structural,
p7, and partial NS2 regions from J6CF strain of GT 2a HCV
(dark grey), and non-structural region from JFH-1 strain of GT 2a
HCV (mild grey). A mono-cistronic Renilla luciferase (RLuc)
reporter virus (NRLFC) based on J6/JFH-C virus is shown. The
Renilla luciferase gene is fused in frame with the core region
through Foot and Mouth Disease virus 2A sequence (F2A). The
mutant J6/JFH-C and reporter viruses, with envelope coding
regions (E1 and E2) deleted and NS5B polymerase catalytic amino
acid residues GDD (Gly-Asp-Asp) mutated to AAG (Ala-Ala-Gly)
residues are shown. (B) Comparison of viral genome replication.
The in vitro transcribed J6/JFH-C virus and the NRLFC reporter
virus genomic RNAs were introduced into Huh-7.5.1 cells by
electroporation. At 4, 48, and 96 hpt total cellular RNAs were
harvested and subjected to RT-qPCR using HCV specific
QuantiTect probe. The genome copy numbers were calculated
per mg of RNA and presented as a graph. (C) Comparison of viral
infectivity. The virus titer (ffu/ml) of cell-free supernatant collected
at 48 and 96 hpt was measured by infecting naı¨ve Huh-7.5.1 cells.
Compared to the parental J6/JFH-C virus, the reporter virus had
similar levels of genomic RNA replication, but was 10–100 fold
attenuated in infectious particle production. C, core; E, envelope;
NS, non-structural.
Found at: doi:10.1371/journal.ppat.1000182.s008 (2.89 MB EPS)
Table S1 Primers and the location of HCV fragments
Found at: doi:10.1371/journal.ppat.1000182.s009 (0.02 MB PDF)
Table S2 Primers used for Functional Profiling analysis of HCV
genome
Found at: doi:10.1371/journal.ppat.1000182.s010 (0.03 MB PDF)
Acknowledgments
We would like to thank F. Chisari for Huh-7.5.1 cell line and T. Wakita for
JFH-1 plasmid; H. Savilahti for designing Mu ends; U. Dandekar, L.
Zhang and J. Papp of UCLA genoseq core facility for technical assistance;
O. Yang, H. Zhou and S. French for helpful discussion and critical review
of the manuscript; R. Clubb and V. Villareal for help with crystal structure
analysis; R. Sitapara, F.F. Xing and S. Portonovo for careful editing of the
manuscript; other members of R.S. laboratory for useful discussions.
Author Contributions
Conceived and designed the experiments: VA RS. Performed the
experiments: VA RR VK TNH CL. Analyzed the data: VA EYS.
Contributed reagents/materials/analysis tools: VF AD. Wrote the paper:
VA AD RS.
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