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Journal of Virology, February 2000, p. 2046-2051, Vol. 74, No. 4
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Hepatitis C Virus-Encoded Enzymatic Activities and
Conserved RNA Elements in the 3' Nontranslated Region Are Essential for
Virus Replication In Vivo
Alexander A.
Kolykhalov,1
Kathy
Mihalik,2
Stephen M.
Feinstone,2 and
Charles M.
Rice1,*
Department of Molecular Microbiology,
Washington University School of Medicine, St. Louis, Missouri
63110-1093,1 and Division of Virology,
Center for Biologics Evaluation and Research, Food and Drug
Administration, Bethesda, Maryland 208922
Received 7 July 1999/Accepted 22 November 1999
 |
ABSTRACT |
Hepatitis C virus (HCV) infection is a widespread major human
health concern. Significant obstacles in the study of this virus include the absence of a reliable tissue culture system and a small-animal model. Recently, we constructed full-length HCV cDNA clones and successfully initiated HCV infection in two chimpanzees by
intrahepatic injection of in vitro-transcribed RNA (A. A. Kolykhalov et al., Science 277:570-574, 1997). In order to validate
potential targets for development of anti-HCV therapeutics, we
constructed six mutant derivatives of this prototype infectious clone.
Four clones contained point mutations ablating the activity of the NS2-3 protease, the NS3-4A serine protease, the NS3 NTPase/helicase, and the NS5B polymerase. Two additional clones contained deletions encompassing all or part of the highly conserved 98-base sequence at
the 3' terminus of the HCV genome RNA. The RNA transcript from each of
the six clones was injected intrahepatically into a chimpanzee. No
signs of HCV infection were detected in the 8 months following the
injection. Inoculation of the same animal with nonmutant RNA transcripts resulted in productive HCV infection, as evidenced by
viremia, elevated serum alanine aminotransferase, and HCV-specific seroconversion. These data suggest that these four HCV-encoded enzymatic activities and the conserved 3' terminal RNA element are
essential for productive replication in vivo.
 |
TEXT |
Prior to the development of specific
blood donor screening assays, hepatitis C virus (HCV) was the major
cause of transfusion-associated hepatitis (see reference
25 for a review). While transfusion-associated HCV
infections are rare, about 30,000 new cases of hepatitis C are
estimated to occur in the United States each year. HCV is not easily
cleared by the host's immunological defenses; as many as 85% of the
people infected with HCV become chronically infected. Many of these
persistent infections result in chronic liver disease, including
cirrhosis and hepatocellular carcinoma (24). There are an
estimated 170 million HCV carriers worldwide, and HCV-associated end-stage liver disease is now the leading cause of liver
transplantation. In the United States alone, hepatitis C is responsible
for 8,000 to 10,000 deaths annually, and without effective
intervention, that number is predicted to triple in the next 10 to 20 years. There is no vaccine to prevent hepatitis C infection. Prolonged treatment of chronically HCV-infected patients with interferon or
interferon plus ribavirin is the only currently approved therapy, but
it results in a sustained response in fewer than 50% of the cases
(37, 46).
HCV belongs to the family Flaviviridae, which comprises
three genera of small enveloped positive-strand RNA viruses (see
reference 47 and references therein). The 9.6-kb
genome of HCV consists of a long open reading frame (ORF) flanked by 5'
and 3' nontranslated regions (NTRs). The HCV 5' NTR is 341 nucleotides
in length and functions as an internal ribosome entry site for
cap-independent translation initiation (34). The HCV
polyprotein is cleaved co- and posttranslationally into at least 10 individual polypeptides (for a review, see reference
45). The structural proteins result from signal
peptidase cleavages in the N terminal portion of the polyprotein. Two
viral proteases mediate downstream cleavages to produce nonstructural
(NS) proteins that function as components of the HCV RNA replicase. The
NS2-3 protease spans the C terminal half of NS2 and the N terminal
one-third of NS3 and catalyzes autocatalytic cis cleavage at
the 2/3 site. The same portion of NS3 also encodes the catalytic domain
of the NS3-4A serine protease that cleaves at four downstream sites.
The C terminal two-thirds of NS3 is highly conserved among HCV
isolates, with RNA-binding, RNA-stimulated NTPase, and RNA-unwinding
activities. Although NS4B and the NS5A phosphoprotein are also likely
components of the replicase, their specific roles are unknown. The C
terminal polyprotein cleavage product, NS5B, is the elongation subunit of the HCV replicase possessing RNA-dependent RNA polymerase (RDRP) activity (5, 38). Following a translation stop codon, the HCV 3' NTR consists of three subregions: (i) a 28- to 42-base sequence
that varies among genotypes, (ii) an internal poly(U/UC) tract of
variable length with rare A or G residues, and (iii) a highly conserved
3' terminal 98-base sequence (33, 49, 50, 54). This recently
discovered 98-base element is the most highly conserved RNA sequence in
the HCV genome, but two surprising reports suggest that it is not
essential for virus replication (13, 58).
The development of new and specific anti-HCV treatments is a high
priority, and virus-specific functions essential for replication are
the most attractive targets for drug development. In the case of HCV,
it has been assumed that conserved features are essential, but this has
not been experimentally testable. Assembly of functional HCV cDNA
clones (31) has now allowed us to directly assess the functional importance of HCV-encoded enzymatic activities and RNA
elements by site-directed mutagenesis. Here, we report the in vivo
characterization of mutants defective in each of the four known
HCV-encoded enzymatic activities or lacking all or part of the
conserved 3' terminal sequence.
Construction of mutant HCV full-length cDNA clones.
The
infectious full-length consensus HCV cDNA clone p90/HCVFLlongpU,
containing a 133-base poly(U/UC) tract and no additional 5' terminal
nucleotides (31; subsequently referred to as HCV FL), was used as the backbone for construction of six mutant clones (Fig. 1). We inactivated each of the four
known HCV-encoded enzymatic activities by mutating at least two amino
acid residues essential or important for function (Fig. 1A). Multiple
substitutions were created to avoid the generation of same-site
revertants during transcription with T7 RNA polymerase, which has a
relatively high error rate (~6 × 10
5 per
nucleotide (7). In HCV FL(2-3pro
), the NS2-3
protease was inactivated by incorporating the H952A and C993A
substitutions. Although it is not known if these NS2 residues
participate directly in catalysis, either substitution abolishes
processing at the 2/3 site (18, 22). For HCV
FL(3pro
), two residues in the NS3-4A serine protease
catalytic triad were changed to alanine (D1107A and S1165A). Either of
these substitutions abolishes detectable processing at the downstream
3/4A, 4A/4B, 4B/5A, and 5A/5B cleavage sites (17, 22). HCV
FL(hel
) contained two mutations designed to inactivate
the NS3 helicase activity. Based on the presence of a DECH motif
(polyprotein residues 1316 to 1319), the HCV helicase belongs to the
DExH family of DEAD-box helicases (16). The first two
residues of this motif are invariant, with the Asp residue binding
Mg2+-ATP (44). Substitution for either of these
residues disrupts NTPase and helicase activities (19); both
were mutated to Ala in HCV FL(hel
). Finally, the NS5B
RDRP was destroyed in HCV FL(pol
) by replacing the
Gly-Asp-Asp (GDD) sequence with Ala-Ala-Gly. This polymerase motif is
conserved among all plus-stranded RNA viruses (43), and
mutating any of these three residues inhibits or abolishes the RDRP
activity of purified HCV NS5B (26, 38).

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FIG. 1.
Diagram of the HCV genome and mutant constructs. All
mutant derivatives were constructed on the background of HCV FL, and
their structures were verified by sequence analysis. Mutants are
described by the nucleotide positions and substitutions (lowercase
letters) relative to HCV FL (31); sequences of the
oligonucleotides used for mutagenesis, plasmid manipulations, and
complete sequence files are available upon request. (A) The HCV genome
organization is shown at the top with 5' and 3' NTRs (solid lines), and
the ORF (open box) and the polyprotein cleavage products are indicated.
Mutant full-length clones are shown below, highlighting the regions
encoding the four enzymatic activities (shadowed), the positions of the
mutations (asterisks), and the construct names (at the left). HCV
FL(2-3pro ) contains the amino acid substitutions H952A
(3195 to 3200; gcgtTa) and C993A (3318 to 3319; gc). HCV
FL(3pro ) contains the substitutions D1107A (3660 to 3664;
gcctt) and S1165A (3831 to 3836; agCgCt). HCV FL(hel )
contains the substitutions D1316A (4286 to 4289; cGca) and E1317A (4291 to 4292; ca). HCV FL(pol ) contains the substitutions
G2737A (8551 to 8552; cg), D2738A (8554; c), and D2739G (8557 to 8559;
gCc). (B) Organization of the 3' portion of HCV genome RNA showing (5'
to 3') the C-terminal part of the ORF (open box), the polyprotein
translation termination codon (UGA), the variable part of the 3' NTR
(solid straight line), the poly(U/UC) tract, the highly conserved
52-base sequence (curved line), and the 3' terminal 46-base stem-loop
structure (SL I). Mutant clones are shown below with their
corresponding names to the right. HCV FL(3' 52) is identical to HCV
FL, except for an internal deletion encompassing the 5' 52 bases of the
3' terminal 98-base sequence. For HCV FL(3' 98), the entire 3'
98-base sequence was deleted. A novel restriction site
(NsiI) distinguishing HCV FL(3' 52) from HCV FL(3' 98)
is indicated. nucl., nucleotides.
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|
Two additional clones were constructed to test the functional
importance of the conserved portion of the 3' NTR (the 98-base
sequence
or X tail) (
33,
49,
50,
54). In HCV FL(3'

98),
the entire
3' 98-base sequence was deleted so that runoff RNA
transcripts would
terminate immediately following the poly(U/UC)
tract (Fig.
1B). HCV
FL(3'

52) contained a smaller deletion encompassing
the 52 invariant
nucleotides between the poly(U/UC) tract and
the 3' terminal 46-base
stem-loop structure (SL I;
6). Both
3' NTR deletion
constructs contained a nucleotide substitution
(C519T) that was
previously shown to be tolerated by HCV (
31).
In addition,
HCV FL(3'

52) could be distinguished from HCV FL(3'

98)
by an
NsiI site at position 9547 that was fortuitously created
by
the fused sequences (Fig.
1B).
Prior to the animal experiment, translation and polyprotein processing
of HCV FL and the mutant constructs were compared by
transient
expression in cell culture. Since the constructs contain
a T7 RNA
polymerase-specific promoter upstream of the HCV sequence,
plasmid DNAs
were transfected into BHK-21 cells previously infected
with recombinant
vaccinia virus vTF7-3, which expresses the T7
RNA polymerase. For all
constructs, the expression level and electrophoretic
mobility of the E1
glycoprotein were similar to those observed
for HCV FL (data not
shown). For the NS proteins, the expected
processing patterns were
observed (Fig.
2). Processing at the
2/3
site was abolished with HCV FL(2/3pro

), but processing at
other sites was not affected, as shown by
the presence of NS4A, NS4B,
NS5A, and NS5B. Unprocessed E2-p7-NS2-NS3,
with a molecular mass of
~180 kDa, was also detected, presumably
as a result of inefficient
processing at the E2/p7 and p7/NS2
sites (
35,
41,
48). For
HCV FL(3pro

), processing at serine protease-dependent
sites was blocked but
could be restored by cotransfection of the
functional protease
domain (NS3
181;
36).
In this case, in addition to the individual
NS proteins, an NS3-4A
precursor was also observed as a result
of inefficient
trans
cleavage at the 3/4A site (see, for example,
reference
15). Processing of the HCV FL(hel

)
polyprotein revealed patterns identical to those of HCV FL,
except that
NS3 migrated slightly faster than the wild-type protein
as a result of
the two engineered substitutions. The protein patterns
of HCV
FL(pol

), HCV FL(3'

98), and HCV FL(3'

52) were
indistinguishable from
those of HCV FL (Fig.
2).

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FIG. 2.
Polyprotein processing by HCV FL and mutant derivatives.
Transfection of HCV FL (wt) or mutant plasmid DNAs (indicated at the
top), protein labeling, immunoprecipitation of 35S-labeled
proteins with HCV-specific sera, and the analysis of the immune
complexes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
were conducted as described previously (32). pGEM3Zf(+) was
transfected as a negative control (mock). Products were
immunoprecipitated with patient serum JHF recognizing NS3, NS4A, NS4B,
and NS5A (A and B) or rabbit anti-NS5B (C); separated by sodium dodecyl
sulfate-12 or 9% polyacrylamide gel electrophoresis, respectively;
and visualized by autoradiography. The positions of molecular weight
markers are shown on the left (in kilodaltons); HCV-specific
polyproteins and cleavage products are identified on the right. The
~21-kDa species which is observed in the HCV
FL(3pro )-NS3181 cotransfection is an N
terminally truncated form of the NS4B protein (32). The
values on the left are molecular sizes in kilodaltons.
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|
Infectivity of mutant HCV RNAs in vivo.
Chimpanzee 1552 (Ch1552) was used to assess the infectivity of mutant transcripts.
Ch1552 had been inoculated 2 years prior to this study with RNA
transcripts from 17 nonconsensus clones from an original HCV cDNA
library (31). Follow-up of Ch1552 for 6 months did not
reveal any evidence of productive HCV infection: serum alanine
aminotransferase (ALT) remained steady at the preinoculation level, HCV
RNA was not detectable by reverse transcription (RT)-PCR, no
HCV-specific antibodies were registered, and no signs of hepatitis or
inflammation were detected in liver biopsies. Just prior to the current
study, Ch1552 was negative for HCV RNA by RT-PCR, was seronegative by
anti-HCV enzyme-linked immunosorbent assay (ELISA) 3.0, and had normal
ALT levels.
The six mutant plasmid DNAs were linearized following the 3' end of the
HCV cDNA and transcribed with T7 RNA polymerase. RNA
transcripts were
injected directly into the surgically exposed
liver of Ch1552, with
each transcript preparation injected into
four sites. Separate
injections of each RNA at different sites
minimized the possibility of
cotransfection of the same cell(s)
with multiple RNAs capable of
complementing one another, resulting
in the initiation of replication
and/or recombination between
RNAs to generate an infectious RNA. After
inoculation, Ch1552
was followed up for 32 weeks and showed no signs of
productive
HCV replication: ALT remained at the preinjection level:
serum
samples from weeks 5, 9, 15, and 18 were negative for
HCV-specific
antibodies, as determined by a commercial third-generation
HCV
ELISA; and HCV RNA was undetectable in the serum. Competitive
quantitative RT-nested PCR was used to analyze pre- and postinoculation
samples from weeks

9, 0, 1 to 13, 21, 25, and 32. In all of these
samples, HCV RNA was undetectable (detection limit: 300 to 500
RNA
molecules per ml of
serum).
To demonstrate that Ch1552 could be productively infected by in
vitro-synthesized RNA, the animal was challenged at week 32
with HCV FL
RNA. To avoid another surgical procedure, intrahepatic
injections were
performed percutaneously, once through a biopsy
needle and four times
through lumbar puncture needles. After challenge,
serum ALT levels
indicated a typical HCV infection. Prechallenge
values were observed
for the first 7 weeks, followed by a sharp
rise, a peak at week 42 (week 10 postchallenge), and then a return
to the prechallenge level by
week 45 (Fig.
3A). Circulating HCV
RNA
was detected 1 week after challenge, and the titers (measured
using the
ABI PRISM 7700 Sequence Detection System [PE Applied
Biosystems,
Foster City, Calif.]) gradually increased from 2 ×
10
5 RNA molecules per ml on week 33 to ~1 × 10
7 RNA molecules per ml on weeks 39 through 41. After
serum ALT
had peaked, HCV RNA declined to 1 × 10
6 to
2 × 10
6 on week 42, dropped sharply on week 44 (to
5 × 10
4 per ml), and then remained steady for 3 or 4 weeks before becoming
undetectable by week 48. Even by RT-nested PCR,
HCV RNA was not
detected in any sample after week 48 (detection limit,
100 to
300 RNA molecules per ml of serum).

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FIG. 3.
Analysis of Ch1552 samples. RNA transcription and
inoculation of RNAs into the liver were performed as described
previously (31). For each RNA, approximately 150 µg of RNA
in phosphate-buffered saline (PBS) was injected into two separate
sites, and 1 µg of an RNA-Lipofectin-PBS mixture was also injected at
two separate sites. Ch1552 was challenged at week 32 with infectious
HCV FL RNA transcripts using nonsurgical procedures. One hundred
micrograms of RNA in 1 ml of PBS was injected into the liver
percutaneously through a biopsy needle. Three additional intrahepatic
injections of 100 µg of RNA in 1 ml of PBS per injection were
administered with a lumbar puncture needle. A fifth lumbar puncture
needle injection was performed with 3 µg of RNA mixed with 30 µl of
Lipofectin and PBS in a total volume of 0.5 ml. (A) Serum ALT, HCV RNA
(molecules per milliliter), and HCV-specific antibodies (Ab; as
measured by HCV ELISA 3.0). (B) Detection of HCV-specific antibodies by
Ortho HCV version 3.0 ELISA and by Chiron RIBA 2.0. For the RIBA 2.0, the open box indicates HCV-seronegative serum samples and the solid bar
(beginning at week 41) indicates positive samples. OD490,
optical density at 490 nm.
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|
Circulating HCV-specific antibodies were analyzed using commercial
third-generation HCV ELISA 3.0 (Fig.
3A and B). Antibodies
were
detectable as early as 2 weeks after challenge. Two response
peaks were
observed. After a sharp increase on week 34, ELISA-reactive
antibody
levels decreased during weeks 36 to 40 and then rose
again starting at
week 41 to week 42. The second increase in HCV-specific
antibody
coincided with elevated ALT. Interestingly, the presence
of
HCV-specific antibodies on weeks 34 through 41 was not confirmed
by
second-generation Chiron RIBA HCV 2.0 Strip Immunoblot Assay
(RIBA
2.0). This can be explained by an early antibody response
to NS5, since
this antigen is present in the ELISA 3.0 but not
in the RIBA 2.0. NS3-specific antibodies were detected by RIBA
2.0 beginning at week 10 postchallenge, but no other HCV-specific
reactivity was detected during
the course of this
study.
Concluding remarks.
Although two groups have reported
productive HCV replication after transfection of cell cultures with
transcribed RNA (13, 58), no follow-up studies have been
published, nor have these systems been used to delineate essential
viral functions. In this study, we exploited the chimpanzee model,
which has been extensively used for HCV studies and in particular for
initiating infection by intrahepatic inoculation of RNA transcribed
from functional HCV cDNA (23, 31, 55, 57). While this
approach does not lend itself to mechanistic studies, we can define
functions essential for replication in this stringent animal model to
validate or uncover new targets for anti-HCV drug development.
We examined the four known HCV-encoded enzymes that are being actively
pursued as antiviral targets. The two viral proteases
mediate cleavages
in the NS region that are thought to be necessary
to form a functional
RNA replicase. In the case of the NS3 serine
protease, which is common
to all members of the family
Flaviviridae,
previous work has
demonstrated that this activity is essential
for replication of the
classical flaviviruses (
10) and the animal
pestiviruses
(
53). This observation can now be extended to HCV
(although
in no case do we know why processing of the polyprotein
is important
for replicase function). The NS2-3 protease, which
mediates
cis cleavage at the 2/3 site, is unique to HCV, although
cleavage immediately upstream of the NS3 serine protease domain
is also
observed for both flaviviruses (see reference
47 for
a review) and some pestiviruses (see reference
40
for a review).
For classical flaviviruses like yellow fever virus, the
functional
serine protease is a heterodimer consisting of the upstream
NS2B
protein and NS3 (
8). As in HCV, cleavage at the NS2B/3
site
is autocatalytic, but for flaviviruses, the serine protease is
responsible for this cleavage. Mutations which block processing
at the
NS2B/3 site are deleterious for yellow fever virus replication,
although processing at downstream sites still occurs (
9).
For
the pestiviruses, the situation is more complex. For noncytopathic
isolates of bovine viral diarrhea virus (BVDV), the NS2-3 region
remains unprocessed. In contrast, cytopathic BVDV isolates have
usually
undergone various RNA recombination events that lead to
production of a
discrete NS3 protein (
40). Given the situation
with
noncytopathic pestiviruses, the recent report that NS2 is
dispensable
for BVDV replication (
4) and the observations that
some HCV
cDNA clones fail to process at the 2/3 site (
14;
A.
A. Kolykhalov, unpublished data), it was of interest to test
the
essential nature of the HCV NS2-3 protease. Our results indicate
that this proteolytic activity is required for productive HCV
replication, although we cannot exclude the possibility that the
mutated NS2 residues are essential for some other function in
the virus
life cycle. The remaining two enzymatic activities,
the NS3 NTPase/RNA
helicase and the NS5B RDRP, are common to all
members of the family
and, more generally, to most positive-strand
RNA viruses. The precise
role(s) of such helicases in viral RNA
replication is not known
(
29), but mutations inactivating the
BVDV (E. Mendez,
M. S. Collett, and C. M. Rice, unpublished data)
or the HCV
(this work) NS3 helicase were lethal. As expected,
mutations ablating
the NS5B RDRP activity were also lethal, underscoring
the importance of
this enzyme for HCV
replication.
The HCV RNA element examined in this study was the conserved portion of
the 3' NTR. Early reports indicated that the HCV genome
RNA terminated
with poly(A) (
20) or poly(U) (for examples, see
references
21,
30, and
42). Subsequently, it was discovered
that the HCV 3' NTR is actually comprised of a short region that
varies
among genotypes, an internal poly(U/UC) tract, and a terminal
element
of 98 bases (
33,
49,
50,
51,
54). The 98-base
sequence
consists of 52 invariant bases followed by 46 bases that
form a highly
stable 3' terminal stem-loop structure (
6). It
has been
hypothesized that this element participates in RNA replication,
in
particular, the initiation of minus-strand RNA synthesis. Several
groups have begun to uncover host RNA-binding proteins (
11,
27,
39,
52), such as polypyrimidine tract-binding protein,
that may
function in HCV RNA replication and translation via interaction
with
the 3' NTR (
28). In our study, RNAs lacking the 98-base
element or those in which the 52-base invariant sequence was deleted
were incapable of replication. While this report was in preparation,
Yanagi et al. reported similar findings demonstrating that most
HCV 3'
NTR elements were essential for productive infection in
vivo, with the
exception of the variable region immediately following
the ORF
termination codon (
56). Thus, the in vivo results conflict
with the two reports claiming that transfected HCV RNAs lacking
this
sequence can replicate in cell culture (
13,
58). Further
work is needed to resolve these discrepancies and determine if
the RNA
elements required for in vivo versus cell culture replication
differ.
Nonetheless, the in vivo data validate the conserved 98-base
RNA
element as an attractive target for antisense oligonucleotides,
trans-acting ribozymes, RNA decoys, or small molecules that
block
critical interactions with host or viral
proteins.
An interesting observation of our study was the unexpected immune
response profile of Ch1552. After inoculation of Ch1552
with the seven
mutant full-length RNAs, HCV replication was undetectable
and no
HCV-specific serological response was detectable. However,
upon
challenge of Ch1552 with parental infectious RNA, we observed
an
unusually rapid (only 2 weeks after challenge) appearance of
HCV-specific antibodies. This suggested possible priming of the
immune
system by HCV-specific antigens before challenge, as recently
reported
by Beard et al. (
3). Indeed, this was confirmed by
analysis
of peripheral blood mononuclear cells taken from the
animal at week 32 prechallenge, at which time T-cell responses
against core, helicase,
and polymerase antigens were readily detected
(T. Arichi, M. Major, H. Wedemeyer, M. Nascimbeni, S. Gagneton,
A. A. Kolykhalov, J. A. Berzofsky, C. M. Rice, S. M. Finestone,
and B. Rehermann,
unpublished data). Priming may have occurred
via direct translation of
injected replication-defective mutant
RNAs or possibly because one or
more of the mutant RNAs was capable
of low-level replication. One
feature of the early antibody response
postchallenge was its apparent
decline in weeks 36 to 40. This
could indicate that insufficient
antigen is produced in the early
phase of HCV infection to sustain
antibody responses induced by
injection and translation of input RNA.
Alternatively, the apparent
dip in ELISA 3.0 reactivity may reflect the
sequestration of HCV-specific
antibodies in immune complexes. Although
further follow-up is
required, Ch1552 appears to have resolved HCV
infection. Whether
priming of HCV-specific T-cell responses
prechallenge played a
role in this clinical course is difficult to
determine, since
a majority of naive chimpanzees are able to
spontaneously resolve
acute HCV infections (
1,
2).
Nonetheless, a detailed analysis
of the humoral and T-cell responses in
this animal is in progress
(Arichi et al., unpublished data) and should
provide further data
on immune responses that correlate with resolution
(
12). In
addition, if Ch1552 has indeed resolved the
infection, then this
animal will allow us to determine if the immune
responses leading
to resolution are sufficient to protect against
challenge with
a truly homologous virus isolated in the acute phase of
infection
after transfection with clonal infectious RNA
(
31).
Although our experiments were limited to a single chimpanzee,
initiation of HCV infection by injection of transcribed RNA
has been
remarkably reproducible and this animal was productively
infected by
this route. Thus, our results indicate that the two
HCV-encoded
proteases, the NTPase/helicase, the RNA-dependent
RNA polymerase, and
the conserved elements of the 3' NTR are essential
for HCV replication
in the chimpanzee. For mechanistic studies
to determine how these
enzymes and RNA elements actually function
in HCV RNA replication, cell
culture assay systems are sorely
needed. The most straightforward
approach to establishing such
systems is to test the ability of
transfected infectious RNAs
to amplify and perhaps spread in cell
culture. Toward this goal,
the replication-defective mutants described
in this study and
validated in the chimpanzee transfection model can
serve as useful
negative controls to distinguish between authentic
replication
and persistence of transfected
RNA.
 |
ACKNOWLEDGMENTS |
We thank Scott Baginski, Keril Blight, Mara Lippa, and Tina Myers
for critical reading of the manuscript.
This work was supported in part by grants from the Public Health
Service to C.M.R. (CA57973 and AI40034).
 |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular Microbiology, Campus Box 8230, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110-1093. Phone: (314)
362-2842. Fax: (314) 362-1232. E-mail:
rice{at}borcim.wustl.edu.
 |
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Journal of Virology, February 2000, p. 2046-2051, Vol. 74, No. 4
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Nelson, H. B., Tang, H.
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Thibeault, D., Bousquet, C., Gingras, R., Lagace, L., Maurice, R., White, P. W., Lamarre, D.
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Yu, M.-y. W., Bartosch, B., Zhang, P., Guo, Z.-p., Renzi, P. M., Shen, L.-m., Granier, C., Feinstone, S. M., Cosset, F.-L., Purcell, R. H.
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Lee, K. J., Choi, J., Ou, J.-h., Lai, M. M. C.
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Imbert, I., Dimitrova, M., Kien, F., Kieny, M. P., Schuster, C.
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