Determinants of the Hepatitis C Virus Nonstructural Protein

JOURNAL OF VIROLOGY, Dec. 2009, p. 12702–12713 Vol. 83, No. 24
0022-538X/09/$12.00 doi:10.1128/JVI.01184-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Determinants of the Hepatitis C Virus Nonstructural Protein 2
Protease Domain Required for Production of Infectious Virus

Thomas G. Dentzer,1,2 Ivo C. Lorenz,1† Matthew J. Evans,1‡ and Charles M. Rice1*
Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, 1230 York Avenue,
New York, New York 10065,1 and Laboratoire de Re´trovirologie, Centre de Recherche Public-Sante´, 84 rue Val Fleuri,
Luxembourg L-1526, Luxembourg2
Received 9 June 2009/Accepted 28 September 2009
The hepatitis C virus (HCV) nonstructural protein 2 (NS2) is a dimeric multifunctional hydrophobic protein
with an essential but poorly understood role in infectious virus production. We investigated the determinants
of NS2 function in the HCV life cycle. On the basis of the crystal structure of the postcleavage form of the NS2
protease domain, we mutated conserved features and analyzed the effects of these changes on polyprotein
processing, replication, and infectious virus production. We found that mutations around the protease active
site inhibit viral RNA replication, likely by preventing NS2-3 cleavage. In contrast, alterations at the dimer
interface or in the C-terminal region did not affect replication, NS2 stability, or NS2 protease activity but
decreased infectious virus production. A comprehensive deletion and mutagenesis analysis of the C-terminal
end of NS2 revealed the importance of its C-terminal leucine residue in infectious particle production. The
crystal structure of the NS2 protease domain shows that this C-terminal leucine is locked in the active site, and
mutation or deletion of this residue could therefore alter the conformation of NS2 and disrupt potential
protein-protein interactions important for infectious particle production. These studies begin to dissect the
residues of NS2 involved in its multiple essential roles in the HCV life cycle and suggest NS2 as a viable target
for HCV-specific inhibitors.
An estimated 130 million people are infected with hepatitis
C virus (HCV), the etiologic agent of non-A, non-B viral hepatitis.
Transmission of the virus occurs primarily through blood
or blood products. Acute infections are frequently asymptomatic,
and 70 to 80% of the infected individuals are unable to
eliminate the virus. Of the patients with HCV-induced chronic
hepatitis, 15 to 30% progress to cirrhosis within years to decades
after infection, and 3 to 4% of patients develop hepatocellular
carcinoma (17). HCV infection is a leading cause of
cirrhosis, end-stage liver disease, and liver transplantation in
Europe and the United States (7), and reinfection after liver
transplantation occurs almost universally. There is no vaccine
available, and current HCV therapy of pegylated alpha interferon
in combination with ribavirin leads to a sustained response
in only about 50% of genotype 1-infected patients.
The positive-stranded RNA genome of HCV is about 9.6 kb
in length and encodes a single open reading frame flanked by
5
and 3
nontranslated regions (5
and 3
NTRs). The translation
product of the viral genome is a large polyprotein containing
the structural proteins (core, envelope proteins E1 and
E2) in the N-terminal region and the nonstructural proteins
(p7, nonstructural protein 2 [NS2], NS3, NS4A, NS4B, NS5A,
and NS5B) in the C-terminal region. The individual proteins
are processed from the polyprotein by various proteases. The
host cellular signal peptidase cleaves between core/E1, E1/E2,
E2/p7, and p7/NS2, and signal peptide peptidase releases core
from the E1 signal peptide. Two viral proteases, the NS2-3
protease and the NS3-4A protease, cleave the remainder of the
viral polyprotein in the nonstructural region (22, 27). The
structural proteins package the genome into infectious particles
and mediate virus entry into a naïve host cell; the nonstructural
proteins NS3 through NS5B form the RNA replication
complex. p7 and NS2 are not thought to be incorporated
into the virion but are essential for the assembly of infectious
particles (14, 36); however, their mechanisms of action are not
understood.
NS2 (molecular mass of 23 kDa) is a hydrophobic protein
containing several transmembrane segments in the N-terminal
region (5, 9, 32, 39). The C-terminal half of NS2 and the
N-terminal third of NS3 form the NS2-3 protease (10, 11, 26,
37). NS2 is not required for the replication of subgenomic
replicons, which span NS3 to NS5B (20). However, cleavage at
the NS2/3 junction is necessary for replication in chimpanzees
(16), the full-length replicon (38), and in the infectious tissue
culture system (HCVcc) (14). Although cleavage can occur in
vitro in the absence of microsomal membranes, synthesis of the
polyprotein precursor in the presence of membranes greatly
increases processing at the NS2/3 site (32). In vitro studies
indicate that purified NS2-3 protease is active in the absence of
cellular cofactors (11, 37). In addition to its role as a protease,
NS2 has been shown to be required for assembly of infectious
intracellular virus (14). The N-terminal helix of NS2 was first
implicated in infectivity by the observation that an intergenotypic
breakpoint following this transmembrane segment resulted
in higher titers of infectious virus (28). Structural and
* Corresponding author. Mailing address: Laboratory of Virology
and Infectious Disease, Center for the Study of Hepatitis C, The
Rockefeller University, 1230 York Ave., New York, NY 10065. Phone:
(212) 327-7046. Fax: (212) 327-7048. E-mail: ricec@rockefeller.edu.
† Present address: International AIDS Vaccine Initiative, AIDS
Vaccine Design & Development Laboratory, Brooklyn, NY 11220.
‡ Present address: Department of Microbiology, Mount Sinai School of
Medicine, New York, NY 10029.

Published ahead of print on 7 October 2009.
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functional characterization of the NS2 transmembrane region
has shown that this domain is essential for infectious virus
production (13). In particular, a central glycine residue in the
first NS2 helix plays a critical role in HCV infectious virus
assembly (13). The NS2 protease domain, but not its catalytic
activity, is also essential for infectious virus assembly, whereas
the unprocessed NS2-3 precursor is not required (13, 14).
The crystal structure of the postcleavage NS2 protease domain
(NS2pro, residues 94 to 217), revealed a dimeric cysteine
protease containing two composite active sites (Fig. 2C; [21]).
Two antiparallel -helices make up the N-terminal subdomain,
followed by an extended crossover region, which positions the
-sheet-rich C-terminal subdomain near the N-terminal region
of the partner monomer. Two of the conserved residues of the
catalytic triad (His 143, Glu 163) are located in the loop region
after the second N-terminal helix of one monomer, while the
third catalytic residue, Cys 184, is located in the C-terminal
subdomain of the other monomer. Creation of this unusual
pair of composite active sites through NS2 dimerization has
been shown to be essential for autoproteolytic cleavage (21).
The structure of NS2pro further demonstrated that the Cterminal
residue of NS2 remains bound in the active site
after cleavage, suggesting a possible mechanism for restriction
of this enzyme to a single proteolytic event (21). Here
we have used the crystal structure of NS2pro, along with
sequence alignments, to target conserved residues in each of
the NS2pro structural regions. Our mutational analysis revealed
that the residues in the dimer crossover region and
the C-terminal subdomain are important for infectious virus
production. In contrast, the majority of amino acids in the
active site pocket were not required for infectivity. Interestingly,
we observed that the extreme C-terminal leucine of
NS2 is absolutely essential for generation of infectious virus,
as mutations, deletions, and extensions into NS3 are very
poorly tolerated. This analysis begins to dissect the determinants
of the multiple functions of this important protease
in the HCV life cycle.
MATERIALS AND METHODS
Plasmid constructs. Mono- and bicistronic (see Fig. 1A) genomes were generated
by standard molecular biology techniques and verified by restriction
enzyme digestion and sequencing of PCR-amplified segments. Descriptions of
the cloning strategies are provided below, and plasmid and primer sequences are
available upon request.
(i) J6/H77NS2/JFH and mutant derivatives. J6/H77NS2/JFH constructs contain
genotype 2a (J6) from core to p7, genotype 1a (strain H77) NS2 and
genotype 2a (JFH) NS3 to NS5B. To create this construct, the J6/JFH plasmid
(18) was used as a template to PCR amplify the J6/JFH E2 3
end through the
p7 sequence with forward oligonucleotide RU-O-5739 (5
-CCGCCTTGTCGA
CTGGTC) and reverse oligonucleotide RU-O-5855 (5
-CTCCGTGTCCAACGCGTAAGCCTGTTGGGGC).
The H77 NS2 through half of JFH-1
NS3 region was PCR amplified from H77/JFH (18) with the forward oligonucleotide
RU-O-5854 (5
-CAGGCTTACGCGTTGGACACGGAGGTGGCC)
and reverse oligonucleotide RU-O-5721 (5
-GCTACCGAGGGGTTAAGCA
CT). Since these PCR products overlap at the p7/NS2 junction, they were used
as template in a second round of PCR with the outside oligonucleotides RU-O-
5855 and RU-O-5721 to generate a PCR product encoding the J6 E2 C-terminal
region though p7, the H77 NS2, and JFH-1 NS3 N-terminal region. This product
was digested with restriction endonucleases BsaBI and AvrII and ligated into the
BsaBI/AvrII-digested J6/JFH plasmid to create the final J6/H77NS2/JFH plasmid.
An adaptive change at G1145A (chimeric genome numbering) encoding
substitution A269T (chimeric polyprotein numbering) was found to increase
infectious virus titers of J6/H77NS2/JFH, and was included in all J6/H77NS2/
JFH-based genomes constructed. Mutant derivatives of J6/H77NS2/JFH were
created by site-directed mutagenesis using the AfeI/BbvCI restriction sites.
(ii)J6/H77NS2/JFH(NS2-IRES-NS3)andmutantderivatives.J6/H77NS2/JFH-
(NS2-IRES-NS3) encodes a stop codon after NS2, an encephalomyocarditis virus
(EMCV) internal ribosome entry site (IRES), a start codon, and the remainder
of the JFH-1 polyprotein starting with NS3. Mutant derivatives of J6/H77NS2/
JFH(NS2-IRES-NS3) were created by site-directed mutagenesis using the PmeI/
MluI restriction sites.
(iii) J6/H77NS2/JFH(NS2-IRES-nsGluc2AUbi) and mutant derivatives. J6/
H77NS2/JFH(NS2-IRES-nsGluc2AUbi) is similar to J6/H77NS2/JFH(NS2-
IRES-NS3) but encodes a Gaussia luciferase gene followed by the foot and
mouth disease virus 2A peptide and a ubiquitin monomer (nsGluc2AUbi cassette)
between the EMCV IRES and NS3 (14). The N-terminal signal sequence
of Gaussia luciferase has been deleted so that the reporter is not secreted.
Mutant derivatives of J6/H77NS2/JFH(NS2-IRES-nsGluc2AUbi) were created
by site-directed mutagenesis using the PmeI/MluI restriction sites. NS2 extensions
into NS3 were subcloned using BbvCI/SpeI/KpnI restriction sites.
Cell culture. Huh-7.5 cells were cultured in Dulbecco’s modified Eagle medium
(Invitrogen) supplemented with 0.1 mM nonessential amino acids and 10%
fetal bovine serum (complete medium). Cells were grown at 37°C in 5% CO2.
RNA transcription. In vitro transcripts were generated as previously described
(18). Briefly, plasmid DNA was linearized by XbaI and purified by using a
Minelute column (Qiagen, Valencia, CA). RNA was transcribed from 1 g of
purified template by using the T7 Megascript kit (Ambion, Austin, TX) or the T7
RNA polymerase kit (Promega, Madison, WI). Reaction mixtures were incubated
at 37°C for 3 h, followed by a 15-min digestion with 3 U of DNase I
(Ambion). RNA was purified by using an RNeasy kit (Qiagen) with an additional
on-column DNase treatment. RNA was quantified by absorbance at 260 nm and
diluted to 0.5 g/l. Prior to storage at 80°C, RNA integrity was determined by
agarose gel electrophoresis and visualization by ethidium bromide staining.
RNA electroporation. Huh-7.5 cells were electroporated with RNA as previously
described (18). Briefly, Huh-7.5 cells were treated with trypsin, washed
twice with ice-cold RNase-free AccuGene phosphate-buffered saline (PBS) (Bio-
Whittaker, Rockland, ME), and resuspended at 1.75  107 cells/ml in PBS. Then,
2 g of each RNA was combined with 0.4 ml of cell suspension and immediately
pulsed using a BTX ElectroSquare Porator ECM 830 (820 V, 99 s, five pulses).
Electroporated cells were incubated at room temperature for 10 min prior to
resuspension in 15 ml or 30 ml complete medium for nonreporter and reporter
constructs, respectively. Resuspended cells were plated into 24-well, 6-well, and
P100 tissue culture dishes.
Assays for RNA replication. At 4 or 8, 24, 48, and 72 h postelectroporation,
cells in 24-well plates were washed with Dulbecco PBS and lysed by the addition
of Renilla lysis buffer (Promega, Madison, WI) or RLT buffer (Qiagen) containing
1% -mercaptoethanol for assay of replication by luciferase activity or quantitative
reverse transcription-PCR (qRT-PCR), respectively. For luciferase assays,
the lysates were thawed prior to the addition of Renilla substrate (Promega)
according to the manufacturer’s instructions. The luciferase activity was measured
by using a Berthold Centro LB 960 96-well luminometer. For qRT-PCR
analysis, prior to storage at 80°C, the lysates were homogenized by centrifugation
through a QiaShredder column (Qiagen) for 2 min at 14,000  g. Total
RNA was isolated by RNeasy kit (Qiagen) and quantified by determining the
absorbance at 260 nm. A total of 50 ng of total cellular RNA was used per
reaction mixture. qRT-PCRs were performed on a LightCycler 480 (Roche,
Basel, Switzerland) using the LightCycler amplification kit (Roche) with
primers directed against the viral 3
NTR. We assembled 20-l reaction
mixtures according to the manufacturer’s instructions as previously described
(14).
Assays for infectious virus production. At 4 or 8, 24, 48, and 72 h postelectroporation,
the cell culture medium was harvested and replaced with fresh
complete medium. Harvested cell culture supernatants were clarified by using a
0.45-m-pore-size filter and stored in aliquots at 80°C. For detection of infectious
virus production by qRT-PCR or luciferase assay, naïve cells were infected
with clarified cell culture supernatants and incubated for 72 h prior to analysis.
Determination of infectious virus production by limiting dilution assay was performed
as described previously (14, 18). Briefly, clarified cell culture supernatants
were serially diluted and used to infect approximately 3  103 cells plated
in 96-well dishes. At 3 days postinfection, cells were washed with Dulbecco PBS,
fixed with ice-cold methanol, and stained for the presence of NS5A expression as
described previously. The 50% tissue culture infectious dose (TCID50) was
calculated using the Reed and Muench method (18).
Anti-NS2 MAb (6H6). The immunogen was a recombinant NS2 protease
domain of strain H77 (residues 94 to 217 with an amino-terminal hexahistidine
tag) expressed in Escherichia coli and purified by fast protein liquid chromatog-
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raphy under denaturing conditions (6 M guanidinium hydrochloride) or in the
presence of detergent (21). BALB/c mice were immunized three times intraperitoneally
with 50 g NS2 purified under denaturing conditions, followed by a final
boost with 50 g NS2 purified in the presence of detergent. The preimmune and
test bleeds were assayed for the presence of NS2-specific polyclonal antibodies by
a standard enzyme-linked immunosorbent assay, as well as by Western blotting.
Screening of hybridoma supernatants produced 6H6, an isotype immunoglobulin
G1 monoclonal antibody (MAb).
SDS-PAGE and immunoblotting. Cells were lysed at 72 h postelectroporation
with RLT buffer (Qiagen) containing 1% -mercaptoethanol and homogenized
by centrifugation through a QiaShredder column (Qiagen) for 2 min at 14,000 
g. The lysates were separated by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE). After transfer to nitrocellulose membrane, the
blots were blocked for 1 h with 5% milk–PBS-T (PBS plus 0.1% Tween). For
NS2 detection, MAb 6H6 (1.0 mg/ml), was diluted 1:1,000 in PBS-T. For NS5A
detection, MAb 9E10 (18) was diluted 1:1,000 in PBS-T. For -actin detection,
mouse anti--actin MAb (Sigma, St. Louis, MO) was diluted 1:20,000 in PBS-T.
After 1-h incubation at room temperature and extensive washing with PBS-T,
horseradish peroxidase-conjugated rabbit anti-mouse immunoglobulin secondary
antibody (Pierce, Rockford, IL) was added at 1:10,000 dilution in 5% milk–
PBS-T for 45 min at room temperature. After additional washing, blots were
developed with SuperSignal West Pico chemiluminescent substrate (Pierce).
RESULTS
Creation and characterization of triple chimeric HCV genomes.
The crystal structure of NS2pro was solved using the
H77 genotype 1a protein (21). In order to facilitate a structurebased
mutational analysis of NS2, we created J6/JFH genomes
encoding the H77 NS2 sequence. Three different full-length
HCV genome constructs were used (Fig. 1A). J6/H77NS2/JFH
is a monocistronic genome encoding J6 core through p7, H77
NS2, and JFH-1 NS3 through NS5B; the 5
and 3
NTRs of all
the genomes are derived from JFH-1. J6/H77NS2/JFH replicated
with kinetics similar to those of J6/JFH but released
approximately 50-fold-less infectious virus (data not shown).
By passaging transfected cells, we selected a single nucleotide
change, G1145A (chimeric genome nucleotide numbering) encoding
an A269T mutation in the J6 E1 protein. This substitution
enhanced infectious particle production (data not
shown) and was included in all genomes constructed. To study
the functions of NS2 in infectious virus production independent
of its autoproteolytic cleavage requirements, we created
bicistronic genomes. J6/H77NS2/JFH(NS2-IRES-NS3) is identical
to J6/H77NS2/JFH but with the addition of a stop codon
after H77 NS2 and an encephalomyocarditis virus internal
ribosome entry site upstream of NS3. This genome allows
expression of the viral replicase independently of NS2-3 cleavage,
and thus uncouples processing from replication and virus
production. J6/H77NS2/JFH(NS2-IRES-nsGluc2AUbi) is
identical to J6/H77NS2/JFH(NS2-IRES-NS3), but it encodes
the reporter gene Gaussia luciferase immediately downstream
of the EMCV IRES. Cleavage of the reporter protein from the
viral polyprotein is mediated by the foot-and-mouth disease
virus 2A peptide and cleavage after the C terminus of the
ubiquitin monomer by host ubiquitin carboxy-terminal hydrolase
(31). This generates nonsecreted Gaussia luciferase (ns-
Gluc) and the proper N terminus of NS3. The triple chimeric
genomes were tested for replication and infectious virus production
at various times postelectroporation of Huh-7.5 cells
with in vitro-transcribed RNA; 72-h time points are shown
(Fig. 1). All chimeric genomes were viable, although RNA
replication levels and infectious particle production were
somewhat reduced compared to the parental J6/JFH. As ex-
FIG. 1. HCV genomes used in this study. (A) Schematic representation
of HCV genomes. (I) J6/JFH with J6 C-NS2 shown in gray and
JFH NS3-NS5B in white. (II) J6/H77NS2/JFH with J6 (gray), H77 NS2
(dark gray), and JFH (white) with adaptive mutation in E1 (black dot).
(III) Bicistronic construct similar to that shown for construct II but
with an EMCV IRES between NS2 and NS3. (IV) Bicistronic reporter
construct similar to construct III with EMCV IRES, plus nonsecreted
Gaussia luciferase, foot and mouth disease virus 2A and ubiquitin
cleavage sites (nsGluc2AUbi) between NS2 and NS3 (checkered box).
5
UTR and 3
UTR, 5
untranslated region and 3
untranslated region,
respectively. (B) RNA replication of J6/JFH and J6/H77/JFH genomes
measured by quantitative RT-PCR at 72 h postelectroporation. HCV
RNA copies normalized to 50 ng of total RNA. (C) Infectious virus
production of bicistronic constructs at 72 h postelectroporation, as
measured by limiting dilution assay (TCID50). WT, wild type of each
genome indicated; GNN, corresponding polymerase-defective control.
The means plus standard errors of the means (error bars) of three
independent experiments with two different RNA preparations are
shown.
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pected, genomes containing a mutation of the NS5B RNAdependent
RNA polymerase motif GDD to GNN did not
replicate (Fig. 1B and C).
The monoclonal antibody 6H6 recognizes a C-terminal
epitope of NS2. In Western blot, MAb 6H6 strongly recognized
NS2 from strains H77 (genotype 1a) and JFH-1 (Jc1 genome,
genotype 2a) (16, 28) and showed a weak signal for strain J6
(J6/JFH genome, genotype 2a) (18); it did not react with Con1
(genotype 1b) (2) (Fig. 2A). Strain H77 NS2 is also recognized
in an enzyme-linked immunosorbent assay, immunoprecipitation,
and immunofluorescence (data not shown). Epitope mapping
with an NS2 peptide library revealed that 6H6 binding
occurs close to the NS2 C terminus (H77 NS2 amino acids 197
to 208, GQEILLGPADG). Sequence alignments in this region
showed variability between NS2 genotypes that correlated with
the Western blot results (Fig. 2B). The epitope of MAb 6H6 is
shown on the NS2 crystal structure in Fig. 2C.
NS2 catalytic-cleft residues are required for NS2-3 cleavage.
Previous studies have shown that the catalytic activity of the
NS2-3 protease is not required for infectious virus production
(14). To investigate whether residues surrounding the active
site pocket were required for the generation of infectious progeny,
we mutated individual residues in this region. Y141 and
L144 are highly conserved amino acids surrounding the protease
active site, H143 is part of the catalytic triad, and P164 is
an unusual cis-proline residue important for active site geometry.
We mutated these residues to alanine and/or to less conservative
substitutions in the context of the monocistronic J6/
H77NS2/JFH and bicistronic J6/H77NS2/JFH(NS2-IRESNS3)
genome, and assayed replication by quantitative RTPCR
for intracellular HCV RNA at 72 h postelectroporation.
We confirmed that the active site mutation H143A abolished
replication in the full-length monocistronic background (10),
and we found that Y141A also severely impaired RNA accumulation;
a Y141F substitution, which preserved the aromatic
ring, did not have a dramatic effect (Fig. 3A). Replication was
decreased by mutation of L144 to bulky residues (L144F,
L144K) and by substitutions of P164 (P164A, P164G). Consistent
with the requirement for NS2-3 cleavage, robustly replicating
genomes showed processed NS2 by Western blotting
(Fig. 3C).
To test the effects of NS2 active cleft mutations on infectious
virus production, we engineered these substitutions into the
bicistronic J6/H77NS2/JFH(NS2-IRES-NS3) genome. In this
context, all mutants replicated as efficiently as the wild-type
virus did; J6/H77NS2/JFH(NS2-IRES-NS3)/GNN did not replicate
(Fig. 3B). Infectious virus production was measured by
inoculating naïve Huh-7.5 cells with filtered culture supernatants
harvested at 72 h postelectroporation and calculating the
TCID50/ml. Mutations Y141A, Y141F, H143A, L144F, and
L144K were not impaired or only slightly impaired in terms of
infectious titers compared to wild-type J6/H77NS2/JFH(NS2-
IRES-NS3), whereas substitution of P164 mutated to Ala or
Gly decreased infectious virus production by about 10-fold
(Fig. 3D). Western blotting of cell lysates harvested at 72 h
postelectroporation revealed that all of the mutants expressed
readily detectable levels of NS2 (Fig. 3C).
These results indicate that mutations at the NS2 active cleft
can inhibit replication of a monocistronic genome, likely by
affecting NS2-3 processing, but that the catalytic activity is not
required for infectious virus production of a bicistronic genome.
The moderate deleterious effect of cis-proline 164 mutations
on infectivity may indicate a more global impact of this
unusual residue on NS2 architecture.
Residues in the NS2 dimer crossover region are important
for infectious virus production. NS2 dimerization creates two
composite active sites and has been shown to be essential for
proteolytic cleavage at the NS2/NS3 junction (21). Although
the critical residues for dimer formation and stabilization are
not known, amino acids in the crossover region between the
two monomers may be envisioned to participate. To test the
effects of mutations in the dimer crossover region on replication
and infectious virus production, we created several substitutions
of highly conserved amino acids in the context of
monocistronic and bicistronic genomes: a triple mutation with
M170A, I175A, and W177A (M/I/W) and individual mutations
M170A, I175S, W177A, and W177C. The single isoleucine-toserine
substitution was chosen in order to change a nonpolar
residue to a polar residue, with predicted disruption to the
dimer interface. In the monocistronic J6/H77NS2/JFH background,
replication levels close to those of the wild type were
observed for the M170A, I175S, and W177A mutants, whereas
FIG. 2. Characterization of anti-NS2 antibody (6H6). (A) Western
blot comparing the reactivity of the 6H6 anti-NS2 antibody and 9E10
anti-NS5A antibody against genotype 1a, 1b, and 2a proteins. Huh-7.5
cells were infected with a recombinant vaccinia helper virus expressing
the T7 polymerase, followed by transfection with plasmids coding for
a full-length HCV genomes under the control of a T7 promoter. The
lysates were harvested 16 h posttransfection. The positions of molecular
mass markers (in kilodaltons) are indicated to the right of the blot.
(B) Amino acid sequence alignment of the 6H6 epitope region. Variation
between protein sequences is indicated in red. (C) Crystal structure
of the dimeric NS2 protease domain (21) with monomers shown
in red and blue with the 6H6 antibody epitope shown in yellow.
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the W177C mutant and the genome with the M/I/W triple
mutation exhibited impaired RNA accumulation by 10- to 100-
fold (Fig. 4A). This somewhat surprisingly efficient replication
may indicate that dimer formation, and thereby protease activity,
is not disrupted by these substitutions. Western blot
analysis confirmed that NS2 was properly processed for each of
these genomes, although to a lower extent for the triple mutant
M/I/W (Fig. 4C). To test the effects of these mutations on
infectious virus production, they were engineered into the bicistronic
genome J6/H77NS2/JFH(NS2-IRES-NS3). While
replication in this context was not affected by any of the NS2
substitutions (Fig. 4B), infectious particle production was decreased
close to 100-fold for all of the mutant genomes (Fig.
4D). Wild-type levels of NS2 were detected for all the bicistronic
constructs, indicating that the mutations do not change
protein stability (Fig. 4C). These results indicate that mutations
in the dimer crossover region are deleterious to infectious
virus production, although likely not as a result of dimer destabilization.
The C-terminal region of NS2 is essential for infectious
virus production. The crossover residues position the NS2
C-terminal region of one monomer close to the N-terminal
region of the other, leading to the formation of the composite
active site. To investigate the importance of the C-terminal
region of NS2 for infectious virus production, we generated
truncations after residues Y124, G137, G178, W214, R215, and
L216 (Fig. 5A). These deletions were engineered in the context
of the bicistronic Gaussia luciferase reporter genome, J6/
H77NS2/JFH(NS2-IRES-nsGluc2AUbi), which was used to
facilitate mutant characterization. Truncations were created by
introducing two in-frame stop codons after the designated residue
without deletion of the downstream nucleotides. This
strategy allowed replication and infectivity to be monitored
with minimal effects on genome length or potential RNA secondary
structures. RNA replication was measured by assaying
Huh-7.5 cell lysates for luciferase activity at 72 h postelectroporation.
All the C-terminal truncation mutants replicated,
although with a slight reduction for mutants with stops after
G137 and G178 compared to wild type; J6/H77NS2/JFH(NS2-
IRES-nsGluc2AUbi)/GNN did not replicate (Fig. 5B). Infectious
virus production was assayed at 72 h postelectroporation
by measuring luciferase activity in infected Huh-7.5 cell lysates.
FIG. 3. Mutagenesis of the NS2 active site region. (A) RNA replication
of GNN, the polymerase-defective control, wild-type (WT)
and mutated monocistronic constructs at 8 h and 72 h postelectroporation.
(B) RNA replication of GNN, wild-type and mutated bicistronic
constructs at 8 h and 72 h postelectroporation. The numbers of
HCV RNA copies per 50 ng of total cellular RNA are shown. (C)
Polyprotein processing of monocistronic and bicistronic constructs.
Huh-7.5 cells were lysed 72 h postelectroporation and analyzed by
SDS-PAGE. Unprocessed and processed NS2 are shown in the top two
panels (6H6 antibody). NS5A protein is shown in the bottom panel
(9E10 antibody). The positions of molecular mass markers (in kilodaltons)
are shown to the right of the immunoblots. (D) Infectious virus
production by the bicistronic constructs at 72 h postelectroporation, as
measured by limiting dilution assay (TCID50). Mutated residues are
indicated (H77 NS2 numbering). WT and GNN, parental monocistronic
or bicistronic wild type and polymerase-defective control, respectively.
The means plus standard errors of the means (error bars)
for three independent experiments with two different RNA preparations
are shown.
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Consistent with our mutational analysis (Fig. 4), truncation of
NS2 before the dimer crossover region (Y124 and G137) abolished
infectivity (Fig. 5C). Similarly, deletion of the entire
C-terminal region (G178) prevented progeny virus production
(Fig. 5C). Surprisingly, more subtle deletions of three, two, or
even one amino acid from the C terminus severely impaired
infectivity (Fig. 5C). Western blot analysis showed detectable
levels of NS2 expression for the W214, R215, and L216 truncations
(Fig. 5D); the larger deletions were missing the 6H6
antibody epitope and could therefore not be tested.
To identify individual amino acids in the C-terminal region
that were important for NS2 functions, we mutated highly
conserved residues shown in the crystal structure to mediate
contacts between NS2 monomers. These substitutions—nonpolar
I188 mutated to a polar serine, changing N189, L191, and
I201 to a small amino acid (alanine), and changing D207 to
alanine or a bulky, charged arginine—were created in the
monocistronic J6/H77NS2/JFH and bicistronic J6/H77NS2/
JFH(NS2-IRES-NS3) genomes. In the monocistronic background,
RNA replication of the I188S, D207A, and D207R
mutants was detected but impaired (Fig. 6A). NS2-3 processing
could be observed for I188S, N189A, and L191A mutants;
processing of the I201 and D207 mutants could not be assessed
since the epitope of the 6H6 antibody had been disrupted (Fig.
6C). In the context of the bicistronic genome [J6/H77NS2/
JFH(NS2-IRES-NS3)], none of the mutations in the C-terminal
region impaired RNA replication (Fig. 6B). Infectious virus
production was decreased for I188S, I201, D207A, and D207R
mutants but not affected by N189A and L191S substitutions
(Fig. 6D). The decreased titers seen for the I188S mutant did
not result from NS2 degradation, as close to wild-type levels of
this protein were detected by Western blotting.
Taken together, these results indicate that the C-terminal
region of NS2 is important for infectious virus production,
although individual residues contribute to various degrees. Interestingly,
the ability of the mutations to disrupt replication of
the monocistronic genome correlated with the extent of virus
titer reduction, suggesting that attributes of the C-terminal
region, such as interactions with the partner monomer, may be
important for both proteolytic activity and infectious particle
production.
The C-terminal leucine of NS2 is critical for infectious virus
production. Our deletion analysis had shown that removing a
FIG. 4. Mutagenesis of the NS2 dimer crossover region. (A) RNA
replication of GNN and wild-type and mutated monocistronic constructs
at 8 h and 72 h postelectroporation. (B) RNA replication of
bicistronic constructs at 8 h and 72 h postelectroporation. The numbers
of HCV RNA copies per 50 ng of total cellular RNA are shown. (C)
Polyprotein processing of monocistronic and bicistronic constructs.
Huh-7.5 cells were lysed 72 h postelectroporation and analyzed by
SDS-PAGE. Unprocessed and processed NS2 are shown in the top two
panels (6H6 antibody). NS5A protein is shown in the bottom panel
(9E10 antibody). The positions of molecular mass markers (in kilodaltons)
are shown to the right of the immunoblots. (D) Infectious virus
production by the bicistronic constructs at 72 h postelectroporation, as
measured by limiting dilution assay (TCID50). Mutated residues are
indicated (H77 NS2 numbering). WT and GNN, parental monocistronic
or bicistronic wild type and polymerase-defective control, respectively.
The means plus standard errors of the means (error bars)
for three independent experiments with two different RNA preparations
are shown.
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single NS2 residue, the C-terminal leucine 217, almost completely
abolished infectious virus production. The crystal structure
of the postcleavage form of NS2pro shows that leucine 217
remains in the active site through hydrogen bond interactions
with the catalytic triad, a conformation that is proposed to limit
the enzyme to a single autoproteolytic cleavage (21). To investigate
the importance of this C-terminal residue for infectivity,
L217 was mutated to a variety of amino acids—isoleucine,
valine, alanine, tryptophan, asparagine, or lysine—in the context
of the bicistronic reporter virus, J6/H77NS2/JFH(NS2-
IRES-nsGluc2AUbi). The L217 mutants replicated, although
at levels somewhat reduced compared to that of the wild type
(Fig. 7A). Infectious virus production was markedly impaired
for all mutants tested apart from L217I, which showed a reduction
of infectious titers of about 10-fold (Fig. 7B). These
defects were not a result of NS2 instability, as the mutant
proteins were readily detected by Western blotting (Fig. 7C).
These data suggest that infectious virus production specifically
requires a leucine at the C terminus of NS2, although an
isoleucine can function to some degree.
Additional residues fused to the C terminus of NS2 abolish
infectious virus production. NS2 requires residues 1 to 181 of
NS3 for optimal proteolytic activity (30). Our finding of a
critical role for the NS2 C-terminal leucine in infectivity suggested
divergent requirements for infectious virus production
and proteolysis. To investigate the effect of adding residues
from NS3 on viral titers, we created J6/H77NS2/JFH bicistronic
reporter genomes in which NS2 was followed by 1, 31,
40, 90, or 181 amino acids of NS3, a stop codon, the EMCV
IRES, the Gaussia luciferase-2AUbi cassette, and full-length
NS3 (Fig. 8A). These extensions were created in the context of
wild-type H77 NS2, as well as in the context of the H143A
active site mutation to prevent NS2-3 cleavage. At 72 h postelectroporation,
the NS3 fusion constructs replicated, although
extensions of 1, 31, 40, and 90 amino acids impaired this process
up to 10-fold, in both the wild-type and H143A backgrounds.
Mutant virus with the 181-amino-acid extension
showed replication levels comparable to that of the parental
genome in the wild-type background but more drastically impaired
RNA replication in the H143A background; the reasons
for this discrepancy are not known (Fig. 8B). Infectious virus
production was severely impaired by uncleavable NS3 fusions
of 31 to 181 amino acids (Fig. 8C). Even the addition of a
single NS3 residue decreased infectivity by three- to fivefold. In
FIG. 5. Characterization of NS2 C-terminal truncations. (A) NS2pro
dimer with monomers shown in red and blue (21). The positions of the
C-terminal truncations are indicated in yellow. (B) RNA replication at
4 h and 72 h postelectroporation of GNN, wild type, and C-terminal
truncations in the bicistronic Gaussia luciferase reporter virus background,
as measured by luciferase activity (in relative light units
[RLU]). (C) Infectious virus production of NS2 C-terminal truncations
at 72 h after infection of naïve Huh-7.5 cells with supernatants harvested
72 h postelectroporation. Truncation points are indicated
(H77 NS2 numbering). WT, wild type [J6/H77NS2/JFH(NS2-IRES-ns
Gluc2AUbi)]; GNN, J6/H77NS2/JFH(NS2-IRES-nsGluc2AUbi)/GNN.
The means plus standard errors of the means (error bars) for three
independent experiments with two different RNA preparations are
shown. (D) Polyprotein processing of NS2 truncations. Huh-7.5 cells lysed
72 h postelectroporation and analyzed by SDS-PAGE. NS2 expression is
shown in the top panel (6H6 antibody), NS5A protein in the middle panel
(9E10 antibody), and -actin control in the bottom. The antibody epitope
is not present in the NS2 truncations Y124, G137, and G178. The positions
of molecular mass markers (in kilodaltons) are shown to the right of
the immunoblots.
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the context of wild-type, but not protease-defective NS2, fusion
of 181 amino acids of NS3 produced infectious virus, suggesting
that productive NS2-3 cleavage was required. Analysis of
protein expression and processing by Western blotting indicated
the presence of processed NS2 for those genomes producing
significant levels of infectivity (Fig. 8D). Interestingly
and consistent with the recent results of Schregel et al. (33), a
small portion of processed NS2 could be detected for fusions
expressing 31 and 40 amino acids of NS3. Taken together,
these results further demonstrate the importance of the Cterminal
residue of NS2 in infectious virus production and add
to accumulating evidence that the multiple roles of NS2 in the
viral life cycle have contrasting protein determinants.
DISCUSSION
NS2 has an essential but mechanistically undefined role in
infectious HCV assembly (14, 28). The first set of determinants
for this activity have been mapped; essential residues were
identified in the N-terminal transmembrane domains, and a
catalysis-independent role for the protease was found (13, 14).
Here, we investigated the determinants of the NS2 protease
domain that are required for infectious particle production. On
the basis of the NS2pro crystal structure (21) and sequence
alignments, conserved features of the various structural regions
were analyzed. We found that most point mutations
around the active site had little effect on infectious virus production
but indirectly affected RNA replication in the context
of a monocistronic genome, most likely by preventing NS2-3
cleavage. In contrast, mutations in the dimer crossover region
and the C-terminal domain impaired or abolished infectious
virus production. In addition, we showed the importance of a
properly cleaved C terminus of NS2 with a free leucine 217.
The catalytic activity of the NS2 protease is required for
NS2-3 processing (16, 38), but not for infectious virus assembly
(14). In addition to the catalytic histidine, we identified several
other residues important for NS2-3 processing and replication
of a monocistronic genome. Mutation of Y141 to Ala abolished
NS2-3 cleavage, whereas a more conservative change to
Phe had little effect on processing or replication. The aromatic
ring of position 141 acts as a support for the active site, a
function that likely cannot tolerate a smaller side chain.
Leucine 144 performs a similar role in creating the correct
active site architecture, but we found that mutation of this
FIG. 6. Mutagenesis of the NS2 C-terminal region. (A) RNA replication
of GNN and wild-type and mutated monocistronic constructs
at 8 h and 72 h postelectroporation. (B) RNA replication of the
bicistronic constructs at 8 h and 72 h postelectroporation. The numbers
of HCV RNA copies per 50 ng of total cellular RNA are shown. (C)
Polyprotein processing of monocistronic and bicistronic constructs.
Huh-7.5 cells were lysed 72 h postelectroporation and analyzed by
SDS-PAGE. Unprocessed and processed NS2 are shown in the top two
panels (6H6 antibody). NS5A protein is shown in the bottom panel
(9E10 antibody). The positions of molecular mass markers (in kilodaltons)
are shown to the right of the immunoblots. (D) Infectious virus
production at 72 h postelectroporation, as measured by limiting dilution
assay (TCID50). Mutated residues are indicated (H77 NS2 numbering).
WT and GNN, parental monocistronic or bicistronic wild type
and polymerase-defective control, respectively. The means plus standard
errors of the means (error bars) for three independent experiments
with two different RNA preparations are shown.
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residue to Phe allowed detectable levels of NS2-3 processing,
whereas Lys apparently abolished the function of the active
site. Proline 164 has a cis conformation that is thought to bend
the peptide backbone to establish the correct geometry of the
Glu 163 side chain for catalysis; mutation of P164 to Ala or Gly
prevented replication of a monocistronic genome. The majority
of active site changes had little effect on infectious virus
production in a bicistronic genome. Substitutions of P164,
however, decreased infectious titers. It is possible that mutation
of this cis-proline dramatically affects NS2 structure; indeed,
the P164G substitution appeared to slightly destabilize
the protein. It is also possible that mutations of P164 affect
NS2 dimer formation, as this proline is important for positioning
the linker between the N- and C-terminal subdomains.
Alternatively, P164 may directly participate in infectious virus
assembly independent of a role in catalysis. Studies of the
NS2-3 protein of the distantly related pestivirus, classical swine
fever virus, have similarly shown that the NS2 protease activity
is dispensable for infectious virus production (23). However, a
histidine-to-arginine mutation within the active site abolished
infectivity without affecting NS2-3 expression (23).
The crystal structure of NS2pro shows a crossover region that
positions the subdomains for creation of the composite active
sites (21). We hypothesized that mutations in this region would
disrupt dimer stability and NS2-3 processing. RNA replication
of a monocistronic genome, however, was impaired only by
substitution of W177 to Cys or by a triple mutation of M170A/
I175A/W177A. This suggests that single point mutations do
not have a drastic effect on dimer integrity; indeed, the significant
buried surface area between the monomers indicates that
the NS2 dimer is highly stable (21). Although NS2-3 processing
was not greatly impacted by mutations in the crossover sequence,
infectious virus production was impaired by all substitutions
we tested in this region. A number of the crossover
residues are exposed on the surface of the NS2pro dimer (Fig.
9). Mutations in this region may disrupt associations with viral
or host proteins involved in infectious virus production. Indeed,
NS2 has been suggested to participate in a number of
genetic or physical interactions, including with structural proteins
core and E2 as well as with nonstructural proteins p7,
NS3, NS4A, and NS5A (15, 19, 25, 29, 34, 40). The A269T
adaptive mutation identified here suggests a genetic interaction
between NS2 and E1. Cellular kinase CKII also appears to
associate with and phosphorylate NS2 (8), and NS2 may interact
with additional host factors to influence apoptosis (6) and
cellular gene expression (5).
In addition to mediating contacts between monomers, the
C-terminal subdomain of NS2 contributes the catalytic cysteine
to the composite active site. Deletion analysis revealed that
even a single amino acid truncation at the C terminus severely
impaired infectious virus production. Furthermore, the majority
of substitutions at the terminal L217 were highly deleterious
to infectivity. Interestingly, previous reports have shown that
most modifications of L217 have little effect on NS2-3 processing
(12, 30). Our finding of an essential role for L217 in infectious
virus production helps explain its high level of conservation
across all genotypes. The structure of the postcleavage
form of NS2pro shows that L217 occupies the active site
through contacts with H143 and C184 (13, 21). This conformation
has been suggested to render the protease inactive
FIG. 7. Mutagenesis of the NS2 C-terminal leucine 217. (A) RNA
replication at 4 h and 72 h postelectroporation of GNN, wild type, and
C-terminal mutants in the context of the bicistronic Gaussia luciferase
reporter virus, as measured by luciferase activity (in relative light units
[RLU]). (B) Infectious virus production of genomes bearing Leu 217
mutations at 72 h after infection of naïve Huh-7.5 cells with supernatants
harvested 72 h postelectroporation. WT, wild type [J6/H77NS2/
JFH(NS2-IRES-nsGluc2AUbi)]; GNN, J6/H77NS2/JFH(NS2-IRESnsGluc2AUbi)/
GNN. The means plus standard errors of the means
(error bars) for three independent experiments with two different
RNA preparations are shown. (C) Polyprotein processing of 72 h
postelectroporation. Huh-7.5 cells were lysed and analyzed by SDSPAGE.
NS2 expression is shown in the top panel (6H6 antibody),
NS5A protein in the middle panel (9E10 antibody), and -actin control
in the bottom. The positions of molecular mass markers (in kilodaltons)
are shown to the right of the immunoblots.
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after a single autoproteolytic processing event (21). Although
NS2 expression and stability were not dramatically altered by
most C-terminal substitutions, it is possible that deletion of
L217 changes the structure of NS2 by liberating the C terminus
from the active site. Releasing the C terminus might alter the
position of the C-terminal subdomain, affecting surfaces required
for essential interactions, such as dimerization or heterotypic
associations with viral or host proteins. A similar function
of a C-terminal residue is seen for the alphavirus capsid
protein, which is also an autoprotease involved in infectious
virus assembly. Analogous to NS2, the highly conserved Cterminal
tryptophan of the alphavirus capsid occupies the active
site postcleavage (4); in fact, distinct similarities have been
noted in the catalytic cleft architecture of the two enzymes
(21). Mutation of this C-terminal tryptophan to alanine or
arginine in a system that uncoupled proteolysis from infectious
alphavirus production almost completely abolished nucleocapsid
assembly; substitution of the terminal tryptophan with phenylalanine,
however, was tolerated (35). Similarly, mutations in
the alphavirus capsid that displaced the terminal tryptophan
from the active site pocket were found to be highly deleterious
to its function (3). These observations suggest that the location
of the C terminus, as well as the presence of a leucine or
similar residue at position 217, may be critical to the structure
and postcleavage functions of these viral proteases.
Further supporting the critical role for L217 in infectious
virus production, we found that C-terminal extensions into
NS3 were highly deleterious; even one additional amino acid
reduced viral titers by three to fivefold. Similar results have
been previously reported, where ubiquitin fused to the NS2 C
terminus abolished infectious virus production (13). Interestingly,
we observed that extensions shorter than the minimal
functional NS3 protease domain (31, 40, and 90 amino acids)
showed NS2-3 processing to some extent in the context of a
functional NS2 active site; recent work from Schregel et al. has
similarly demonstrated residual enzymatic activity of NS2 followed
by only 2 amino acids of NS3 (33). Despite detectable
FIG. 8. Characterization of NS2 C-terminal extensions. (A) Schematic
representation of J6/H77NS2/JFH(NS3*-IRES-nsGluc2AUbiNS3).
Genes from J6 (core-p7) (gray boxes), H77 (NS2) (black boxes), and
from JFH (white boxes) are indicated. This construct contains JFH
NS3 extensions of 1, 31, 40, 90, or 181 amino acids, followed by an
EMCV IRES, a Gaussia luciferase cassette (nsGluc2AUbi), and JFH
nonstructural proteins NS3-NS5B. Wild-type H77 NS2 (I) and H77
NS2 encoding active-site mutation H143A (small solid square) (II). 5

UTR and 3
UTR, 5
untranslated region and 3
untranslated region,
respectively. (B) RNA replication at 4 h postelectroporation, 72 h
postelectroporation for the wild type, and 72 h postelectroporation for
H143A as measured by luciferase activity (in relative light units
[RLU]). 0aa, 0 amino acid. (C) Infectious virus production at 72 h
postelectroporation, as measured by luciferase activity in infected cells.
GNN, replication-deficient control, WT, wild-type NS2 protease;
H143A, cleavage-deficient NS2 protease. The means plus standard
errors of the means (error bars) for three independent experiments
with two different RNA preparations are shown. (D) NS2 protein
expression at 72 h postelectroporation of either wild-type or cleavagedeficient
(H) NS3 extension constructs. Top panel NS2 (6H6 antibody),
NS5A protein in the middle panel (9E10 antibody), and -actin
control in the bottom. GNN, corresponding polymerase-defective control
of wild-type NS2. The positions of molecular mass markers (in
kilodaltons) are indicated to the right of the immunoblots.
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NS2-3 processing, however, genomes encoding NS3 extensions
still did not support infectious virus production. This could
indicate that insufficient quantities of mature NS2 are produced
by suboptimal cleavage or that short fragments of NS3
impair infectivity, possibly by acting as dominant-negative inhibitors
of interactions between NS2 and full-length NS3 (15).
The finding that fused residues from NS3 are deleterious to the
role of HCV NS2 infectivity contrasts with the related pestiviruses,
in which the uncleaved NS2-3 precursor is essential for
infectious virus production (1, 23). The possibility that NS2
and NS3 form functional associations during virion morphogenesis,
however, suggests conserved strategies between HCV
and other members of the family Flaviviridae (24).
Using monocistronic and bicistonic genomes, we were able
to analyze the effects of NS2 mutations on protease activity and
postcleavage functions. Our results add to accumulating evidence
that the determinants of these two essential roles are
divergent. Previous work has demonstrated that the transmembrane
domains of NS2 play critical roles in infectivity (13) but
are not absolutely required for protease activity (10, 26, 32, 37).
Conversely, the active cleft is essential for the protease function
but predominantly dispensable for infectivity (13, 14; this
study). A number of substitutions in the dimer crossover region
and C-terminal subdomain affected infectious titers, but not
protease activity, and L217 was found to be dispensable for
processing but critical for infectious virus production. Similarly,
the finding that NS2 functions in assembly do not tolerate
C-terminal extensions contrasts with the requirement for the
NS3 protease domain for optimal NS2-3 cleavage. These differences
highlight the two distinct functions of NS2 and suggest
that further analysis of these roles may reveal important regulatory
switches.
In conclusion, we dissected the determinants of the NS2
protease domain required for infectious virus production. We
found critical roles for residues in the dimer crossover region
and at the extreme C terminus of the protein, and we showed
that C-terminal extensions into NS3 are deleterious to infectivity.
These insights increase our understanding of the multifunctional
NS2 protein and may facilitate exploiting this target
for antiviral drug development.
ACKNOWLEDGMENTS
We thank Maryline Panis and Anesta Webson for laboratory support
and technical assistance. We are grateful to Catherine L. Murray,
Christopher T. Jones, Cynthia de la Fuente, and Kimberly D. Ritola
for reagents, constructs, helpful discussions, and critical reading of the
manuscript.
This study was supported by The Greenberg Medical Research
Institute, NIH Public Health Service grant (AI075099), and the Starr
Foundation. T.G.D. is supported by the Fonds National de la Recherche
Luxembourg.
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