1078 • JID 2005:192 (15 September) • Layden-Almer et al.
M A J O R A R T I C L E
Hepatitis C Virus Genotype 1a NS5A Pretreatment
Sequence Variation and Viral Kinetics in African
American and White Patients
Jennifer E. Layden-Almer,1Carla Kuiken,3Ruy M. Ribeiro,3Kevin J. Kunstman,2Alan S. Perelson,3
Thomas J. Layden,1and Steven M. Wolinsky2
1Department of Medicine, University of Illinois at Chicago, and
of Medicine, Chicago;
2Department of Infectious Diseases, Northwestern University Feinberg School
3Theoretical Biology and Biophysics, Theoretical Division, Los Alamos National Laboratory, Los Alamos, New Mexico
In hepatitis C virus (HCV) infection, race is a determinant of treatment response and interferon (IFN) ef-
fectiveness. Here, we investigated whether there were differences in the pretreatment viral strains between
African American patients and white patients and whether these differences correlated with viral kinetics. IFN
effectiveness was calculated using a viral kinetic model. The HCV NS5A region from 21 treated patients with
HCV genotype 1a was sequenced and analyzed. White patients displayed more mutations in the V3 region
(, vs.;), and treatment responders tended to have more mutations inmean?SD 4.5?1.42.9?1.6 P p .016
this region than did nonresponders. There was a significant positive correlation between IFN effectiveness
and the number of mutations in the V3 region (
P p .03
response, or IFN effectiveness in phylogenetic analyses. The results of this study, in conjunction with those
of a previous study illustrating the impaired IFN effectiveness in African Americans, suggest a role for host-
). There was no clustering of strains by race, treatment
Hepatitis C virus (HCV) infects 2%–3% of the US and
world populations [1–3]. HCV is a positive-sense sin-
gle-stranded RNA virus  and has been classified in-
to at least 6 major genotypes and 1100 subtypes [5,
6], with genotype 1 virus predominating in the United
States . In an HCV-infected patient, typically 1 sub-
type exists as a mixture of genetically distinct viral var-
iants . Viral population diversity within patients is
due, in part, to the high level of viral production and
Received 20 December 2004; accepted 25 April 2005; electronically published
12 August 2005.
Potential conflicts of interest: none reported.
Financial support: National Institutes of Health (grant R01 DK58320-01 to T.J.L.,
grant R01 RR06555 to A.S.P., and General Clinical Research Centers grant M01
RR 13987 to the University of Illinois at Chicago); VA Merit Review, University of
Illinois and Jesse Brown VA at Chicago; anonymous donor to the Northwestern
University Feinberg School of Medicine. Portions of this work were performed
under the auspices of the US Department of Energy.
Reprints or correspondence: Dr. Jennifer E. Layden-Almer, University of Illinois
at Chicago, College of Medicine, 840 S. Wood St., M/C787, Chicago, IL 60612
The Journal of Infectious Diseases
? 2005 by the Infectious Diseases Society of America. All rights reserved.
turnover each day  and to the absence of a proof-
reading function by the RNA polymerase .
Treatment options for chronic HCV infection have
greatly improved. However, 20%–60% of patients still
do not respond to combination therapy with pegylated
interferon (IFN) plus ribavirin [9, 10]. Patientsinfected
with genotype 1 virus exhibit lower treatment-response
rates than those infected with other genotypes. For Af-
rican Americans, the response rates are even worse[11–
14]. It is known, from viral kinetics studies, that IFN
works by inhibiting viral production  and that this
ability of IFN to inhibit viral production impacts on
the long-term outcome of therapy [15, 16]. For African
Americans, the effectiveness of IFN in inhibiting viral
production is significantly lower, which may, in part,
explain why they exhibit lowertreatment-responserates
. The reasons for IFN being less effective at inhib-
iting viral production in African Americans are not un-
Some, but not all, studies have implicated the NS5A
protein as playing a role in IFN resistance [18–27]. De-
spite this controversy surrounding the IFN sensitivity–
determining region (ISDR), evidence does suggest that
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Race, HCV Viral Diversity, and Kinetics • JID 2005:192 (15 September) • 1079
NS5A may play a role in IFN resistance. In vitro studies have
shown that NS5A can bind to and inhibit the IFN-induced,
double-stranded RNA–activated protein kinase R (PKR) ,
which is known to inhibit viral replication. Although the ISDR
resides in the PKR-binding domain of the protein, it has been
shown through in vitro studies that regions outside the ISDR
but still within the PKR-binding domain may be important
[29, 30]. In addition, studies have shown a correlation between
the number of mutations in the PKR-binding domain of pre-
treatment samples and treatment response [31, 32]. Another
study did not find such correlations, however . Other stud-
ies suggest that the NS5A protein may also inhibit IFN action
in a PKR-independent manner [34–37]. Mutations in other
regions of the NS5A protein, predominantly in the C-terminus,
may also correlate with IFN sensitivity [31, 36].
Several mechanisms may account for the different kinetics
of HCV replication during therapy. Because NS5A is thought
to disrupt the host immune responsebyinhibitingIFN-induced
PKR, we asked whether there were pretreatment genetic dif-
ferences in this protein in patients of African American and
white descent and whether these differences correlated with IFN
PATIENTS, MATERIALS, AND METHODS
Patient population and study design.
naive, HCV genotype 1a–infected patients from 2 treatment
studies [17, 38] were included in the present study. Only pa-
tients infected with HCV genotype 1a who completed therapy
per protocol were considered, and approximately equal num-
bers of African American (10) and white (11) patients were se-
lected. For white patients, selection was stratified by treatment
(5) and nonresponders (6) were randomly selected. ForAfrican
American patients from both studies, only 1 patient infected
with HCV genotype 1a who completed therapy achieved a sus-
tained viral response (SVR); this patient was included in the
analysis. The remaining African American patients were non-
responders and were randomly selected from both studies if
they met the 2 criteria.
Ten patients (3 white and 7 African American) were from 1
study and received 10 mIU of IFN-a-2b daily with or without
ribavirin for the first 4 weeks of therapy, followed by 3 mIU
of IFN-a-2b plus ribavirin 3 times/week for the remainder of
therapy (Intron; Schering-Plough). Elevenpatients(8whiteand
3 African American) from the other study received 15 mg of
consensus IFN (C-IFN) daily or 3 times/week (Infergen TM;
Amgen) for the first 4 weeks of therapy, followed by 9 mIU of
C-IFN for the remainder of therapy. Fifteen micrograms of C-
IFN and 10 mIU of IFN-a-2b have similar IFN effectiveness.
Details of these study designs are described elsewhere [17, 37].
In table 1, all patients are listed, with therapy regimen, race,
and treatment response described.
Study subjects were categorized as whiteorAfricanAmerican
on the basis of their self-reporting of the racial background of
the preceding 2 generations. SVR was defined as viralnegativity
(!100 copies/mL) 6 months after the culmination of treatment.
The study was approved by the institutional review boards of
the University of Illinois at Chicago and Northwestern Uni-
versity, and all patients participated with informed consent.
Genotype and viral load determination.
determined by a line probe assay (Inno-Lipa System; Innoge-
netics). Viral RNA concentrations in serum were measured by
an ultrasensitive reverse-transcriptase (RT) polymerase chain
reaction (PCR) assay with a detection limit of 100 copies/mL
(Superquant; NGI). Samples with viral loads 15,000,000 HCV
RNA copies/mL were diluted to determine absolute values.
We analyzed the viral kinetics for 18 of the
21 patients by use of a mathematical model describedelsewhere
. Three patients were not included because poor sampling
excluded adequate modeling of the kinetics. Nonlinear regres-
sion analysis was used to estimate the model parameter ?, the
treatment’s effectiveness at inhibiting viral production.
Virus from frozen serum samples (200
mL) was lysed using a commercially available kit (High Pure
Viral Nucleic Acid Buffer Set; Roche Diagnostics). RNA was
precipitated with isopropanol and solubilized. First-strand
cDNA was synthesized with RT by use of the HCV NS5A-
reverse primer 5?-GTCCAGAAC(T/C)TGCAGTCTGTCAA-
3?. Initial PCR amplification was performed with the primers
3?) and NS5A-reverse. Secondary nested PCR amplification
was performed with the primers NS5A-forward (5?-TAAGG-
GACATCTGGGACTGGA-3?) and NS5A-reverse (5?-GCCTGT-
CCA(GA)G(AT)(AG)TAAGACATTG-3?). The amplified frag-
ment was inserted into vector pCRII-TOPO (Invitrogen), and
individual transformed colonies were subjected to dideoxy DNA
sequencing. DNA sequencing reactions were analyzed with an
ABI Prism 3100 sequencer (Applied Biosystems). Overlapping
DNA sequences were aligned and edited using the MegAlign
program (version 6; DNASTAR).
Phylogenetic and diversity analyses.
generated using the neighbor-joining method , on the basis
of a distance matrix calculated using the F84 model . For
each sequence from a patient, the distance was calculated from
that sequence to all the other sequences for that patient (un-
corrected p-distance, calculated using PAUP* [version 4.0; Sin-
auer Associates]). Values are on a scale from 0 to 1, representing
the degree of variability from another sequence, with a value
of 1 indicating that they are identical. Then, for each sequence,
a mean was taken of all the distances from that sequence to
Viral types were
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Race, HCV Viral Diversity, and Kinetics • JID 2005:192 (15 September) • 1087
tis C virus type 1a are associated with treatment response. J Infect Dis
33. Murphy MD, Rosen HR, Marousek GI, Chou S. Analysis of sequence
configurations of the ISDR, PKR binding domain, and V3 region as
predictors of response to induction interferon-alfa and ribavirin ther-
apy in chronic hepatitis C infection. Dig Dis Sci 2002;47:1195–205.
34. Polyak SJ, Khabar KS, Rezeiq M, Gretch DR. Elevated levels of inter-
leukin-8 in serum are associated with hepatitis C virus infection and
resistance to interferon therapy. J Virol 2001;75:6209–11.
35. Polyak SJ, Khabar KS, Paschal DM, et al. Hepatitis C virus nonstruc-
tural 5A protein induces interleukin-8, leading to partial inhibition of
the interferon-induced antiviral response. J Virol 2001;75:6095–106.
36. Duverlie G, Khorsi H, Castelain S, et al. Sequence analysis of the NS5A
protein of European hepatitis C virus 1b isolates and relation to in-
terferon sensitivity. J Gen Virol 1998;79:1373–81.
37. Francois C, Duverlie G, Rebouillat D, et al. Expression of hepatitis C
virus proteins interferes with the antiviral action of interferon inde-
pendently of PKR-mediated control of protein synthesis. J Virol 2000;
38. Layden TJ, Layden JE, Reddy KR, Poulakos J, Neumann AU.Induction
therapy with consensus IFN (CIFN) does not improve sustained viral
response rates in chronic hepatitis C virus. J Viral Hepat 2002;9:334–9.
39. Saitou N, Saitou N, Nei M. The neighbor-joining method: a new
method for reconstructing phylogenetic trees. Mol Biol Evol 1987;4:
40. Felsenstein J. Evolutionary trees from DNA sequences: a maximum
likelihood approach. J Mol Evol 1981;17:368–76.
41. Korber B, Myers B. Signature pattern analysis: a method for assessing
viral sequence relatedness. AIDS Res Hum Retroviruses1992;8:1549–60.
42. Learn GH Jr, Korber BT, Foley B, Hahn BH, Wolinsky SM, Mullins
JI. Maintaining the integrity of human immunodeficiency virus se-
quence databases. J Virol 1996;70:5720–30.
43. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local
alignment search tool. J Mol Biol 1990;215:403–10.
44. Pawlotsky JM, Brillet R, Penin F, Hezode C, Chouteau P, Dhumeaux
D. Does NS5A protein mediateHCVgenotype1resistancetointerferon
alfa antiviral effect in vivo [abstract 067]? Program and abstracts of
the 9th International Meeting on HCV and Related Viruses. SanDiego,
45. Keenan ED, Rouster SD, Shire NJ, Horn PS, Sherman KE. Complexity
and diversity of hepatitis C virusRNA in AfricanAmericansandwhites:
analysis of the envelope-coding domain. J Infect Dis 2004;190:511–4.
by guest on December 28, 2015