Supplementary material - Journal of General Virology

Table S1. Brief description of the plasmids used for DNA transfection experiments and the replicons 

used for RNA electroporation experiments 

Name 

Description 

pCI core (pHPI 773) 

Plasmid expressing HCV-1 core, constructed by cloning a 

fragment from plasmid pHPI 755 (Varaklioti et al., 2002) into pCI 

vector after digesting both with restriction enzyme EcoRI 

pCI core+1/ARFP/F (pHPI 1725) 

pCI core+1/ARFP/SM 

pCI minicore 

pEUA2 core+1/ARFP/S (pHPI 

1580) 

pEUA2 core+1/ARFP/SM (pHPI 

1579) 

pEUA2 NS5A 

pEUA2 lacZ 

pGL3 HAMP-luc 

pGL3 HAMP(mutAP1)-luc 

pGL3 HAMP(mutC/EBP)-luc 

Plasmid expressing HCV-1 core+1/ARFP/F but not core; sitedirected 

mutagenesis was performed on pHPI 1724 that contains 

core/core+1 sequence with an adenine deletion at codons 8–11, 

using primers 5′-CAGGGGCCCTTGATTGGGTGTGC-3′ and 5′- 

GCACACCCAATCAAGGGCCCCTG-3′, which insert a stop in 

core ORF at nucleotide 468 

Plasmid expressing core+1/ARFP/S myc-tagged, constructed by 

cloning a fragment from plasmid pHPI 1579 (Vassilaki et al., 

2007) into pCI vector 

Plasmid expressing minicore, constructed by performing PCR on 

HCV-1 core sequence using primers 5′- 

GGAATTCCGCCACCATGGGGTGGGCGGGATGGC-3′ and 

5′-GGAATTCCACCTAGTAGGCTGAAGC-3′ and introducing 

the PCR fragment into pCI expression vector (Promega) after 

digesting both with restriction enzyme EcoRI (Fermentas) 

Plasmid expressing core+1/ARFP/S, constructed by introducing a 

PCR fragment containing nucleotides 598–825 of HCV-1a core+1 

coding sequence digested with EcoRI restriction endonuclease, 

into the XbaI site of pA-EUA2 vector 

Plasmid expressing core+1/ARFP/S myc-tagged, described 

elsewhere (Vassilaki et al., 2007) 

Plasmid expressing NS5A, described elsewhere (Kalamvoki et al., 

2002) 

Plasmid expressing -gal, described elsewhere (Kalamvoki et al., 

2002) 

Reporter construct containing Firefly luciferase (luc) gene under 

the control of a 950-nucleotide fragment of the human hepcidin 

(HAMP) promoter, kindly provided by Dr Simos (Braliou et al., 

2008) 


the control of human hepcidin (HAMP) promoter mutated at the 

AP1 binding site; site-directed mutagenesis kit QuikChange 

(Agilent) was used according to the manufacturer’s instructions, 

as well as primers 5′- 

CGATCAGCAGAAACACATCGTGATGG-3′ and 5′- 

CCATCACGATGTGTTTCTGCTGATCG-3′ for mutating AP1 

binding site of pGL3 HAMP-luc; mutations were subsequently 

verified by sequencing 



C/EBP binding site; site-directed mutagenesis kit QuikChange 



CGTGATGGGGACCGGGCTCCCCAGATGGC-3′ and 5′- 

GCCATCTGGGGAGCCCGG TCC CCATCACG-3′ for mutating 

C/EBP-binding site of pGL3 HAMP-luc; mutations were 

subsequently verified by sequencing 

I. Kotta-Loizou, N. Vassilaki, G. Pissas, A. Kakkanas, L. Bakiri, R. Bartenschlager & P. 

Mavromara (2013). Hepatitis C virus core+1/ARF protein decreases hepcidin transcription through an 

AP1 binding site. Journal of General Virology 94

pGL3 HAMP(mutSTAT3)-luc 

eEF1-luc (pHPI 8101) 

AP1-luc 

pCG c-jun~c-fos 

pCG Jun 

I389-Luc-ub-EI-JFH1NS3-3' 

I389-Luc-ub-H77core-EI- 

JFH1NS3-3' 

I389-Luc-ub-H77core+1/ARFP/S- 

EI-JFH1NS3-3' 



STAT3 binding site; site-directed mutagenesis kit QuikChange 



CGGCGCCACCACCGGATTGGAAATGAG-3′ and 5′- 

CTCATTTCCAATCCGGTGGTGGCGCCG-3′ for mutating 

STAT3-binding site of pGL3 HAMP-luc; mutations were 

subsequently verified by sequencing 

Reporter construct containing the Firefly luciferase gene under the 

control of eukaryotic elongation factor 1; EagI fragment from 

pGEM-luc plasmid (Promega), containing the luciferase sequence, 

was inserted in the cloning site of pHPI 8098, which was derived 

from pHPI 8092 plasmid, by cloning the EF1 promoter sequence 

from pEF1/V5/His A plasmid (Invitrogen), after digesting both 

with MluI and NotI 

Reporter construct containing the Firefly luciferase gene under the 

control of a consensus AP-1 element, a kind gift from Dr Weiss 

(Shapiro et al., 1996) 

Plasmid expressing a single-chain AP1-tethered dimer (Bakiri et 

al., 2002). 

Plasmid expressing a dominant negative Jun protein (Bakiri et 

al., 2002) 

Bicistronic subgenomic reporter JFH1-based replicon composed 

of the JFH1 5' UTR, Firefly luciferase gene, ubiquitin gene, the 

EMCV IRES, the coding region of the JFH1 non-structural 

proteins NS3 to NS5B and the JFH1 3' UTR 


of the JFH1 5' UTR, Firefly luciferase and ubiquitin genes fused 

in frame with core from isolate H77 (subtype 1a), the EMCV 

IRES, the coding region of the JFH1 nonstructural proteins NS3 to 

NS5B and the JFH1 3' UTR 


of the JFH1 5' UTR, Firefly luciferase and ubiquitin genes fused 

in frame with core+1/ARFP/S from isolate H77 (subtype 1a), the 

EMCV IRES, the coding region of the JFH1 nonstructural 

proteins NS3 to NS5B and the JFH1 3' UTR 




Table S2. List of priming specific oligonucleotides used for RT-qPCR and RT-PCR 

Gene 

Primer 

CORE (F) 5′-GGAATTCCGCCACCATGGGGTGGGCGGGATGGC-3′ 

CORE (R) 5′-GGAATTCCACCTAGTAGGCTGAAGC-3′ 

FOS (F) 5′-CGGGCTTCAACGCAGACTA-3′ 

FOS (R) 5′-GGTCCGTGCAGAAGTCCTG-3′ 

HAMP (F) 5′-CTGACCAGTGGCTCTGTTTTC-3′ 

HAMP (R) 5′-GAAGTGGGTGTCTCGCCTC-3′ 

TFR2 (F) 5′-CTGCACTGGGTCGATGAGG-3′ 

TFR2 (R) 5′-TCCTGAGCATTGGTCACCTTC-3′ 

YWHAZ (F) 5′-CGCTGGTGATGACAAGAAAGG-3′ 

YWHAZ (R) 5′-GGATGTGTTGGTTGCATTTCCT-3′ 




Fig. S1. (a) Huh7 cells were transfected with core, core+1/ARFP/F, core+1/ARFP/S, core+1/ARFP/SM, 

minicore or NS5A expression vectors and Western blotting was performed (left panel). In the case of 

minicore, due to lack of an appropriate antibody, total RNA was extracted and RT-PCR for minicore and 

a housekeeping gene, YWHAZ, was performed (middle panel). Huh7.5 cells were electroporated with 

replicons expressing core or core+1/ARFP/S and Western blotting was performed (right panel). (b) 

Firefly luciferase activity in Huh7 cells co-transfected with c-jun~c-fos expression vector, Jun 

expression vector and AP1-luc construct, 48 h p.t. (c) Firefly luciferase activity in Huh7 cells cotransfected 

with core or core+1/ARF expression vectors, c-jun~c-fos expression vector and HAMP-luc 

constructs, both the wild-type and the mutated at the AP1 binding site, 48 h p.t. 

References 

Bakiri, L., Matsuo, K., Wisniewska, M., Wagner, E. F. & Yaniv, M. (2002). Promoter specificity and 

biological activity of tethered AP-1 dimers. Mol Cell Biol 22, 4952–4964. 

doi:10.1128/MCB.22.13.4952-4964.2002 Medline 

Braliou et al. (2008). 

Kalamvoki et al. (2002). 

Shapiro, V. S., Mollenauer, M. N., Greene, W. C. & Weiss, A. (1996). c-rel regulation of IL-2 gene 

expression may be mediated through activation of AP-1. J Exp Med 184, 1663–1669. 

doi:10.1084/jem.184.5.1663 Medline 

Varaklioti, A., Vassilaki, N., Georgopoulou, U. & Mavromara, P. (2002). Alternate translation occurs 

within the core coding region of the hepatitis C viral genome. J Biol Chem 277, 17713–17721. 

doi:10.1074/jbc.M201722200 Medline 

Vassilaki, N., Boleti, H. & Mavromara, P. (2007). Expression studies of the core+1 protein of the 

hepatitis C virus 1a in mammalian cells. FEBS J 274, 4057–4074. doi:10.1111/j.1742- 

4658.2007.05929.x Medline

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