Phenotypic analysis of NS5A variant from liver transplant patient with
increased cyclosporine susceptibility
Israr-ul H. Ansarib, Todd Allenc, Andrew Bericalc, Peter G. Stockd, Burc Barine, Rob Strikera,b,n
aW. S. Middleton Memorial Veteran’s Hospital, Madison, WI 53726, United States
bUniversity of Wisconsin-Madison, Madison, WI, United States
cMassachusetts General Hospital, Boston, MA, United States
dUniversity of California, San Francisco, CA, United States
eThe EMMES Corporation, Rockville, MD, United States
a r t i c l e i n f o
Received 18 September 2012
Returned to author for revisions
16 October 2012
Accepted 28 November 2012
Available online 2 January 2013
Hepatitis C virus
a b s t r a c t
Hepatitis C virus (HCV) replication is limited by cyclophilin inhibitors but it remains unclear how viral
genetic variations influence susceptibility to cyclosporine (cyclosporine A, CsA), a cyclophilin inhibitor.
In this study HCV from liver transplant patients was sequenced before and after CsA exposure.
Phenotypic analysis of NS5A sequence was performed by using HCV sub genomic replicon to determine
CsA susceptibility. The data indicates an atypical proline at position 328 in NS5A causes increases CsA
sensitivity both in the context of genotype 1a and 1b residues. Point mutants mimicking other naturally
occurring residues at this position also increased (Ala) or decreased (Arg) replicon sensitivity to CsA
relative to the typical threonine (genotype 1a) or serine (genotype 1b) at this position. This work has
implications for treatment of HCV by cyclophilin inhibitors.
Published by Elsevier Inc.
Hepatitis C virus (HCV) is a genetically diverse virus that exists
as a quasispecies in a single host, and has multiple variants
between different infected individuals at least in part because of
adaptation to the host’s specific adaptive immune system. HCV
infects over 200 million people, and current strategies to eradi-
cate the virus are compromised by toxicity, cost, and efficacy.
Newer multidrug approaches are currently in development,
including the recently approved protease inhibitors polymerase
inhibitors, as well as cyclophilin inhibitors among others. HCV has
6 recognized genotypes which express at least 10 different
proteins, some of which interact with cyclophilin A and mod-
ulate/inhibit HCV replication (Ciesek et al., 2009; Fernandes et al.,
2007; Foster et al., 2011; Goto et al., 2009; Tang, 2010; Verdegem
et al., 2011; Watashi et al., 2005).
HCV is the most common indication currently for liver transplant
in the US. After a liver transplant almost all liver recipients receive a
calcineurin inhibitor immunosuppressant, either CsA or tacrolimus
and all HCV infected patient’s new liver gets reinfected by residual
viremia in the patient. CsA and its nonimmunosuppressive analogs,
DEBIO-25 (Alisporivir), SCY-635, and NIM811are inhibitors of cellular
prolyl-peptidyl isomerases called cyclophilins (Cyps), which are
necessary for HCV replication (Fischer et al., 2010; Flisiak et al.,
2008; Hanoulle et al., 2009; Kaul et al., 2009; Ma et al., 2006;
Vermehren and Sarrazin, 2011).The anti-HCV impact of CsA in
patients with HCV remains controversial (Flisiak et al., 2008).Since
cyclophilin inhibitors are also being developed as a component of
multidrug therapy directed against HCV, the effect of CsA in HCV
infected liver transplant recipients represents an ideal cohort to
study the impact of cyclophilin inhibitor ‘‘mono’’ therapy on HCV.
However, at the moment tacrolimus is more commonly used than
CsA in liver transplantation. In order to study whether or not CsA had
any effect on the evolution of HCV post-transplant, we took
advantage of a unique cohort of HIV/HCV infected patients [HIV
and transplant study, (Terrault et al., 2012)]. These samples were
banked during the study and later used to address if CsA selected
The multi-domain (domains I, II and III) nonstructural protein
5A (NS5A) (Tellinghuisen et al., 2008) is particularly genetically
diverse. It is unclear if the naturally occurring variation in NS5A
alters cyclophilin dependance, and arguments against NS5A
altering cyclophilin dependance have been made (Chatterji
et al., 2010), but amino acid variation could explain heterogeneity
in a putative antiviral effect of CsA post-transplant. Earlier we
reported laboratory acquired mutations within domains II and
III region of genotype 1b NS5Athat conferred resistance to
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0042-6822/$-see front matter Published by Elsevier Inc.
nCorresponding author at: W.S. Middleton Memorial Veteran’s Hospital, 3207
Microbial Sciences, 1550 Linden Drive, Madison, WI 53726, United States.
Fax: þ1 608 262 8418.
E-mail address: firstname.lastname@example.org (R. Striker).
Virology 436 (2013) 268–273
CsA (Fernandes et al., 2007, 2010). For that reason, we sought to
determine if CsA exposure selected for NS5A mutations in
patients who received CsA but not other antivirals. We performed
consensus sequencing of NS5A and NS5B of patients before and
during CsA exposure. In most of the serial viral sequences we did
not find compelling evidence for selection by CsA, but in one
patient we were able to correlate selection at amino acid 328 in
NS5A with altered CsA susceptibility in cell culture.
Development of cyclosporine resistance in NS5A in a patient exposed
The HIV and transplant study (HIV-–TR, www.HIVtransplant.
com) evaluated transplantation of 89 HCV/HIV infected patients
and compared CsA and tacrolimus as initial immunosuppression
and found no clear benefit for either inhibitors (Terrault et al.,
2012) despite cell culture data from multiple groups demonstrat-
ing CsA inhibition of HCV (Fernandes et al., 2010; Fischer et al.,
2010; Tang, 2010). As genotype 1 was both the most prevalent
and the most interferon resistant, we limited ourselves to the 61
genotype 1 infected patients. Only 15 of these were initially
treated with CsA and did not receive anti-HCV interferon based
therapy. In order to specifically examine if CsA selected resistant
HCV variants we limited ourselves to this smaller subset of
patients and attempted to amplify both NS5A and NS5B regions
spanning from PQLPG of NS5A (residue 29) to GGDIYHS of NS5B
(residue 563). From nine of these patients either a post-transplant
or a pre-transplant sample or both were missing or full amplifica-
tion of HCV was unsuccessful. Three of these nine patients were
considered spontaneous cures (Haque et al., 2010; Terrault et al.,
2012). We were able to amplify consensus sequencing of above
mentioned NS5A carboxy terminal region from six genotype 1
infected patients (A–F) pre- and post-transplant. All six patients
were continuously CsA exposed during the interval between pre-
transplant sequencing and 6 months to 2 years post-transplant
and received no anti-HCV therapy during this time. One of these
six was a genotype 1b (patient H), and the rest were genotype 1a.
One of these patients (patient C) though was noted to have an
atypical pre-transplant proline at amino acid 328 of NS5A
(Fig. 1A), which mutated to a consensus serine post-transplant
rather than the more typical genotype 1a threonine (Fig. 1A and
B).While a serine is the consensus amino acid for ?91% of
genotype 1b strains, it represents only 2% of genotype 1a strains
a difference that is statistically significant (p value (po0.0001)
based on 358 genotype 1b and 224 genotype 1a sequences
retrieved from European database. The 328 Pro is near lab
selected resistance mutations that we and others have mapped
(Fernandes et al., 2010; Grise et al., 2012; Tang, 2010). When the
carboxy terminus of NS5A (from amino acids FARALPV to
MSYTWT) was amplified from the pre-C cDNA and cloned into a
genotype 1b replicon, the CsA sensitivity of the resultant replicon
was similar to the parental genotype 1b replicon (Fig. 1C, compare
solid and dashed blue lines of patient C with solid and dashed red
lines of parental 1b replicon). The replicon with genotype 1a
laboratory strain H77 (Con1bLN-5AH77) though was relatively
Fig. 1. Proline is an atypical residue for genotype 1a or 1b at amino acid 328 NS5A, but was present pre-transplant in patient C and was associated with more CsA
susceptibility than H771a. (A) Alignment of H771a, patient C and patient G pre and post-transplant amino acids sequences along with alignment of Con1b, patient H pre
and post-transplant amino acids sequences are presented. (B) The Logo analysis of genotypes 1a, 1b, 2, 3, 4 and 6 sequences near the highly conserved WARPDYN motif
(underlined) associated with laboratory aquired CsA resistance. The amino acid at position 328 is boxed and sequences derived from different genotypes are labeled.
(C) Transient replication of Con1bLN-pre-C, Con1bLN-post-C, Con1bLN-Pre-G, Con1bLN-post-G, Con1bLN-5AH77 and Con1bLN-wt replicons and their sensitivity in
presence (dashed lines) andin absence (solid lines) of CsA at different time points. (D) Percent inhibition of HCV replicons are presented at 96 h post RNA electroporation
by comparing CsA treated vs. untreated luciferase data as in 1C.
I.-U.H. Ansari et al. / Virology 436 (2013) 268–273
more cell culture resistant to CsA than the pre-transplant patient
C replicon (Fig. 1C, black solid and dotted lines). Furthermore we
cloned the corresponding fragment of the carboxy terminus
of NS5A from the same patient (patient C) post-transplant
(6 months later, post-C) and found the replicon less sensitive to
CsA (Fig. 1C) (dashed blue suppressed ?100 fold while dashed
yellow suppressed only ?10 fold (Fig. 1D)). No naturally occur-
ring NS5A fragment (genotype 1b or 1a, pre or post-transplant)
was found to be as sensitive to CsA as the pre-transplant patient C
sequence (data not shown). We could detect no obvious clinical
benefit accrued to patient C. The earliest post-transplant HCV
viremia performed was at one year and was over 8 million IU/ml,
which is typical of HCV infected post-transplant patients.
The post-transplant sequences from a seventh patient (patient
G), with a divergent genotype 1a sequence from H77, who also
developed multiple mutations in this region were compared to
the pre-transplant sequence (Fig. 1A, pre-G and post-G) and its
sensitivity towards CsA was tested after cloning into the 1b
replicon. Unlike patient C, we did not observe a more CsA
resistant phenotype despite having acquired two additional
prolines and few other changes post-transplant (Fig. 1C, pre and
post G are purple and green). Similar to the Con1bLN-5AH77
chimeric replicon, the patient G replicon was relatively resistant
both pre and post-transplant compared to the pre-transplant
patient C chimeric replicon (Fig. 1D). All the chimeric replicons
had similar growth kinetics in absence of CsA (Fig. 1C, solid lines).
Mutational analysis demonstrates proline at 328 confers CsA relative
The replicon derived from patient C pre-transplant and post-
transplant differed by 4 amino acids (Fig. 1A). The Con1bLN-post-
C replicon was mutated to contain single amino acid substitutions
at Val315, Pro328 and Thr442 and a double amino acids substitu-
tion at Val315 and Thr442. Replicons containing Val or Thr
mutations either in single or in combination replicated similar
to Con1bLN-post-C replicon containing all three mutations
(Fig. 2A and B). However, mutating only Ser to Pro at position
328 rendered Con1bLN-post-C-S328P replicon CsA sensitive to
the level of Con1bLN-pre-C replicon (compare dashed red with
dashed blue line). In a converse experiment we mutated
Con1bLN-pre-C replicon to contain Ser at position 328 and this
change was sufficient enough to shift the CsA susceptibility
similar to post-replicon confirming the involvement of amino
acid 328 in CsA susceptibility (Fig. 3A and B, dashed purple line).
Interestingly the consensus amino acid at position 328 is quite
distinct between the genotype 1a lineage (90% threonine), and the
genotype 1b (?90% serine), but a proline can occur in either
lineage and is approximately twice as frequent for genotype 1b as
for genotype 1a (Fig. 1B). Therefore a proline was engineered into
the Con1bLN-wt (1b nonchimeric) replicon. Substitution of Ser to
Pro increased the susceptibility of the Con1bLN-S328P replicon to
CsA just as it did for the Con1bLN-pre-C replicon (Fig. 3A and B,
Fig. 2. Replicons with proline at amino acid 328 have increased CsA susceptibility than serine in both genotype 1a and 1b lineages. (A) Transient replication of Con1bLN-
post-C replicon in presence (dashed line) and in absence (solid line) of CsA after mutating amino acids at position 315, 328, and 442 similar to patient C pre-transplant
sequences. (B) Percent inhibition of HCV replicons are presented at 96 h post RNA electroporation by comparing CsA treated vs untreated luciferase data as in 2A.
I.-U.H. Ansari et al. / Virology 436 (2013) 268–273
dotted green line) suggesting the effect is not an artifact of
chimeric replicons and residue 328 is relevant to the CsA
susceptibilities of both genotypes 1b and 1a.
While proline is the most common variant other than Ser
(genotype 1b) or Thr (genotype 1a) there are other more rare
variants reported in other HCV genotypes (Fig. 1B). To investigate
the effect of these variants on CsA susceptibility we mutated Thr
328 in the context of Con1bLN-5AH77 chimeric replicon to Ala,
Ser, Met, Ile and Arg residues which are present in the different
HCV genotypes (Fig. 1B). All mutated replicons displayed at least
some sensitivity to CsA, but proline conferred the most suscept-
ibility. Ser, Met, Ala and Ile displayed intermediate susceptible
phenotypes, while Arg was the least susceptible to CsA treatment
in our transient replication model (Fig. 4A and B).
Antiviral development for HCV is improving rapidly, but many
patients can still not be cured. Furthermore, many viral sequences
are not replication competent in cell culture, and therefore it is
difficult to know if a specific viral sequence is susceptible to a specific
antiviral agent. Through the use of NS5A chimeric replicons gener-
ated from patients exposed to CsA, we directly compared the CsA
susceptibility of specific NS5A consensus sequences without the
confounding effects of differences in other parts of the genome. Prior
work has shown that NS5A derived from genotypes 1a, 1b, 2a, and
2b binds cyclophilin. This cyclophilin binding and the strong con-
servation of the WARPDYN binding site for CypA identified by NMR
(Hanoulle et al., 2009) has been used to argue that cyclophilin
inhibitors are ‘‘pangenotypic’’ and the heterogeneity of NS5A does
not correlate with cyclophilin inhibition (Chatterji et al., 2010).
Cyclophilin inhibitors do block the NS5A:CypA interaction in vitro
(Fernandes et al., 2010; Hopkins et al., 2012). Our data is consistent
with most if not all HCV being susceptible to cyclophilin inhibitors,
but suggests that NS5A polymorphisms outside the conserved DYN
sequence also influence the degree of CsA susceptibility. While HCV
exists as a swarm of closely related quasispecies, we did not detect a
328 threonine containing sequence in patient C pre-transplant via
clonal analysis (data not shown). Clearly isolates from different
patients vary at this and many positions in NS5A. Deep sequencing
may have shown cyclosporine selection at other positions besides
328, or in other patients but we only have consensus sequencing
data for these patients. We did not find any variant that has been
described from cell culture selection with cyclophilin inhibitors
including D320E or Y321N (Puyang et al., 2010; Yang et al., 2010).
The DYN amino acids 320–322 is embedded among multiple
prolines that biochemical data suggest are targets of cyclophilin
(Fernandes et al., 2010; Hanoulle et al., 2009; Tang, 2010). Genetic
differences between genotypes in NS5A could alter how susceptible
HCV is in patients and do alter it in cell culture (Ansari and Striker,
2012) but it is unclear that these changes in cell culture susceptibility
are clinically significant. This case series shows no antiviral effect of
CsA in most patients but suggests in patient C selection of resistant
HCV can occur. Moreover, this data lends confirmatory in vivo
evidence that NS5A is one of the critical targets of cyclophilin
inhibitors. Both NS2 and NS5B have also been hypothesized as
targets of cyclophilin, but we sequenced most of NS5B as well as
NS5A and could not confirm any selective pressure on NS5B. There
were other consensus mutations that varied between pre- and post-
sequences in NS5B and NS5A, but there was no obvious pattern. In
cell culture, multiple mutations have been found to be required for
significant decreases in CsA cell culture susceptibility (Garcia-Rivera
et al., 2012), but our mutational analysis within the naturally
occurring NS5A sequence suggests the bulk of the decrease CsA
susceptibility for our patient samples came from the proline to serine
mutation. Strikingly, the only genotype 1b patient had pre-transplant
GYN rather than the typical DYN consensus sequence that mutated
to SYN post-transplant (patient H), but replicon data refuted that this
variation altered cell culture sensitivity (data not shown). Perhaps
other sequence variation outside of the cloned region obscured our
ability to associate this change with relative CsA resistance. Since
neither patient B nor C received interferon or ribavirin, these drugs
cannot explain the selection of consensus mutants near known
positions of laboratory required CsA resistance. While proline 328
is the consensus residue in only 5–10% of HCV infected patients it is
possible that patients with a proline present explains some of the
benefit anecdotally seen with switching to CsA (Lorho et al., 2005). In
patient C, the only viral load data available to us was 6 months after
we detected the threonine mutant and one year after transplantation
so it is difficult to known whether CsA delayed or partially sup-
pressed the patient’s viremia or not.
In summary, for six of our seven patients, even consensus
sequencing and phenotypic analysis could not detect an effect of
CsA on viral evolution, or antiviral benefit. The only patient in
which we could find evidence for selection from the antiviral
effect of CsA had an atypical consensus sequence in the region of
NS5A that binds cyclophilin. Likely all, if not most HCV is
susceptible to nonimmunosuppressive cyclophilin inhibitors
including alisporivir and SCY635, but ?5–10% of genotype
1 strains that have this proline 328 variant may be even more
susceptible. At least one genotype 3 infected patient was cured by
Fig. 3. TheCon1bLN-wt replicon with 328P is more sensitive to CsA that Con1bLN-
wt. (A) Transient replication of Con1bLN-S328P and Con1bLN-pre-C-P328S repli-
cons were analyzed along with Con1bLN-pre-C, Con1bLN-post-C and Con1bLN-wt
replicon in presence (dashed line) and in absence (solid line) of CsA at different
time points. (B) Percent inhibition of HCV replicons are presented at 96 h post RNA
electroporation by comparing CsA treated vs. untreated luciferase data as in 3A.
I.-U.H. Ansari et al. / Virology 436 (2013) 268–273
a short duration alisporivir monotherapy (Patel and Heathcote,
2011). While the approval of protease inhibitors has greatly
increased the possibility of curing HCV, small molecule inhibitors
quickly select resistance in HCV unless given in combination with
other antivirals with different mechanisms of action. Clinical
studies pairing of cyclophilin inhibitors with other NS5A and
nonNS5A acting antivirals may benefit from looking at viral
genetic differences between HCV isolates.
Material and methods
Clinical trial and study design
The HIV–TR study included pre-transplant and post-transplant
serum samples and a detailed protocol post-transplant follow-up
(Terrault et al., 2012). The decision to treat HCV infected patients with
interferon or not was left to the discretion of the patient care team.
This resulted in only 15 patients who were exposed to CsA immedi-
ately post-transplant for 6 months or more and we attempted to
sequence all of those patients pre and post-transplant for which there
were samples available.
RT-PCR and nucleotide sequencing
The viral RNA was isolated from serum samples and used for
RT-PCR. Consensus sequencing and analysis of amplified HCV was
performed from amino acid 29 in NS5A until amino acid 563 of
NS5B as described before (Kuntzen et al., 2007). The nucleotide
sequences of 6 cloned fragments derived from 6 individual
patients are deposited in GenBank (JX457830-35). The Institu-
tional Review Board of the University of Wisconsin approved this
study and all study participants provided informed consent in
accordance with the Institutional Review Boards of the participat-
Genetic manipulation of Con1bLN replicon
The PCR fragment spanning from amino acids 312 of NS5A to
amino acids 5 of NS5B (FARALPV to MSYTWT) was amplified from
the above described original cDNA fragment using primers con-
taining XhoI and BstZI restriction sites. This fragment was cloned
in TOPO vector and sequenced again to confirm the mutations.
The desired fragment was subcloned directionally in Con1bLN
replicon (Fernandes et al., 2010) using XhoI and BstZ17I restric-
tion enzymes. All the replicons were sequenced to confirm
RNA transcription and transient replication assay
The Huh7.5 cells were maintained as before (Fernandes et al.,
2010). The CsA was purchased from sigma. The RNA transcription
and electroporationwas performed
(Fernandes et al., 2010). In brief, approximately 2 million cells
were electroporated with 6 mg of in vitro transcribed RNA and
electroporated cells were divided into two sets into twenty–four
Fig. 4. CsA sensitivity of natural variants at position 328 present in different HCV genotypes. (A) The Con1bLN-5AH77 chimeric replicon was mutated to contain Ala, Ser,
Met, Ile and Arg at position 328 and their replication efficiency in presence (dotted line) and in absence (solid line) of CsA at different time points. (B) Percent inhibition of
HCV replicons are presented at 96 h post RNA electroporation by comparing CsA treated vs. untreated luciferase data as in (A).
I.-U.H. Ansari et al. / Virology 436 (2013) 268–273
well plates. After 6 h of incubation the media was replaced but Download full-text
only one set of plates received 0.5 mg/ml of CsA. The cells were
further incubated and renilla luciferase activity was monitored
every 24 h as described before. In all the luciferase assays an
average of three independent assays was presented along with
The amino acid sequences of different HCV genotypes 1a (224
strains), 1b (358 strains), 2 (49 strains), 3 (30 strains), 4(36 strains)
and 6 (48strains) were retrieved from The European HCV Database
(http://euhcvdb.ibcp.fr/euHCVdb/). A small stretch of amino acids
encompassing the region of interest (aa 316–328) was subjected to
logo analysis (Crooks et al., 2004) using web based program (www.
We gratefully acknowledge the patients and other investiga-
tors involved in the solid organ transplantation in HIV (HIV–TR
Study AI052748) and the Project Manager, Rodney Rogers. This
work was supported by the American Cancer Society Research
Scholar Grant (07-077-01) to R.S., by the Office of Research and
Development, Biomedical Laboratory R&D Service, Department of
Veterans Affairs. Patient samples were obtained via the solid
organ transplantation in HIV: Multi-site study (HIV–TR study
AI052748) funded by the National Institute of Allergy and
We also thank John Tavis for playing a critically helpful role in
the early phases of this project and Lindsey Moser for critical
reading of the manuscript.
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