Departments of Haematology1 and Medicine2, University of Cambridge, School of Clinical Medicine, Cambridge, UK
Author for correspondence: Juraj Petrik. Present address: Edinburgh and South East Scotland Blood Transfusion Service, Royal Infirmary, Lauriston Place, Edinburgh EH3 9HB, UK.Fax +44 131 5365352.
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Abstract |
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Several cellular proteins interacting with HCV RNA 3'NCR were identified in extracts of uninfected or untransfected cell lines using UV-cross-linking or gel-retardation techniques and one has been characterized repeatedly as polypyrimidine tract-binding protein (PTB) (Tsuchihara et al., 1997 ; Ito & Lai, 1997
, 1999
; Chung & Kaplan, 1999
). We used an alternative RNA capture system (Petrik et al., 1997
), with immobilized ribooligonucleotides or in vitro RNA transcripts corresponding to various combinations of the 3'NCR components and modified to bind proteins from HCV-infected or control cytosolic liver explant extracts.
Liver tissue (D. G. D. Wight), stored at -70 °C, prepared with ice-cold homogenization buffer (0·3 M sucrose, 4 mM CaCl2, 1 mM PMSF) and a Dounce homogenizer, was centrifuged at 300 g (pellet discarded), 1600 g (nuclear pellet) and 10000 g resulting in a cytosolic extract (supernatant). Total protein concentration was measured and aliquots containing usually 25 µg of total protein were used for capture.
Initial experiments targeted the middle part of tripartite 3'NCR [poly(U/UC) tract] mimicked by a 5' biotinylated ribooligonucleotide r(U)25 [synthesized with its counterpart r(A)25 in the MRC, LMB, Cambridge, UK] immobilized on streptavidine-coated paramagnetic particles (S-PMP; Promega). After washing and r(U)25-binding, the S-PMP were resuspended in 2x TTGED (20 mM TrisHCl pH 8·0, 0·01% Triton X-100, 10% glycerol, 1 mM EDTA, 1 mM DTT, 1 mM PMSF) containing 100 mM NaCl unless specified otherwise. An equal volume of the cytoplasmic extract diluted with homogenization buffer was added. After 30 min incubation at 4 °C with gentle mixing and two 5 min washing steps with 1x TTGED50 mM NaCl, bound proteins were eluted with SDSPAGE loading buffer (Novex), by heating for 10 min at 70 °C and analysed on BisTris NuPage gels (Novex; 10%) with Mes running buffer. Eluted proteins were detected by silver staining.
Fig. 1 shows r(U)25-captured proteins from cytosolic extracts of two controls and three HCV-positive liver explants. There were at least six proteins binding to r(U)25 specifically compared to background captured on S-PMP without ligand. All the proteins were cellular since they were also present in control samples.
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To determine GAPDH binding specificity we prepared plasmid constructs containing various combinations of 3'NCR parts. Fragments corresponding to HCV 3'NCR region 1 (3'NCR1), a consensus sequence of the 40 nucleotide region following the stop codon, from several isolates (Yamada et al., 1996 ) and the 101 nucleotide conserved region (3'NCR3) were cloned after annealing synthetic oligonucleotides containing extra sequences, complementary to the overhangs of HindIII/EcoRI-digested TOPO 2.1 plasmid. For the amplification of the remaining five regions of 3'NCR we used DNA of plasmid p90/HCVFL long pU (p90) containing a full-length HCV cDNA clone (Kolykhalov et al., 1997
; provided by C. Rice). PCR products were subsequently cloned into a PCR 2.1 TOPO vector (Invitrogen). Primers used: for amplification of complete 3'NCR1+2+3 containing a short extra NS5b sequence, primers C3S2 (TGTGCGTCGTGCCGCGACCGCACG) and 4158, corresponding to the 3' end of the conserved 3'NCR3 region (ACATGATCTGCAGAGAGGCC); for amplification of complete 3'NCR1+2+3, primers 2546 (AGGTTGGGGTAAACACTCCG) and 4158; for amplification of 3'NCR1+2, primers 2546 and 2548 (CACCATTAAAGAAGGAAGGA); for amplification of 3'NCR2+3, primers 2547 (GCCATTTCCTGTTTTTTTTT) and 4158; for amplification of 3'NCR2, primers 2547 and 2548. As a control we used plasmid containing a 120 nucleotide sequence of 18S RNA. For in vitro transcription plasmid p90 was digested with MluI and all other plasmids with HindIII. Transcription used the T7 and T3 Ampliscribe kit (Epicentre), including or omitting biotin-UTP (Boehringer Mannheim, 1/20 of the concentration of other NTPs) in the ribonucleotide mix. Biotinylated RNA transcripts (0·7 pmol unless stated otherwise) were used for protein capture and non-biotinylated transcripts for competition experiments. For protein capture, in addition to TTGED50 mM NaCl buffer, we also used HTGEM (10 mM HEPES pH 7·9, 0·1 mM EDTA, 0·01% Triton X-100, 10% glycerol, 2 mM MgCl2) containing 25 mM KCl, similar to buffers used for gel-retardation or UV-cross-linking studies (Furuya & Lai, 1993
; Kusov et al., 1996
; Schultz et al., 1996
; Ito & Lai, 1997
). GAPDH capture with both buffer systems was similar.
Apart from binding to r(U)25 (Fig. 3a, lane 4), GAPDH bound strongly to all capture probes containing 3'NCR2 (lanes 3, 69), but not to 3'NCR1 or 3'NCR3 or control rRNA (Fig. 3a
, lanes 2, 5, 11). GAPDH also bound strongly to the full-length HCV RNA transcript. The binding was specific, with inhibition of capture on the complete 3'NCR1+2+3 probe specifically by a 10-fold excess of non-biotinylated 3'NCR transcripts containing 3'NCR2 (lanes 3, 69 in Fig. 3d
). Binding was also inhibited by r(U)25 oligo (Fig. 3d
, lane 4) and by full-length HCV RNA (Fig. 3d
, lane 10). Amounts of S-PMP, capture probe and cytosolic extract were reduced in this experiment by a factor of five in order to include a 10-fold excess of some competing probes. Correspondingly, the GAPDH bands in Fig. 3(d)
are weaker than in Fig. 3(c)
. Immunodetection after protein transfer onto a PVDF membrane (Immobilon P; Millipore) using a Novex gel/transfer system and incubation with anti-GAPDH monoclonal antibody (Chemicon) and anti-mouse IgGHRP conjugate (Sigma) confirmed the identity of captured GAPDH. After washing the membrane was developed using the ECL system (Amersham) and exposed to X-ray film (Fig. 3c
). Only oligo/poly(U)-containing capture probes bound GAPDH.
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The binding of PTB and another protein (p35) to the HCV 3'NCR was described in detail recently. The binding pattern of p35 described by Luo (1999) was similar to that for GAPDH described herein, but p35 was not characterized further. Gontarek et al. (1999)
identified the 3'NCR-binding p35 protein detected by UV-cross-linking from HepG2 extracts as heterogeneous nuclear ribonucleoprotein C (hnRNP C). This is slightly surprising as hnRNP C (C1/C2) has a molecular mass of 4143 kDa (Dreyfuss et al., 1993
; Sella et al., 1999
) and is one of few hnRNPs which are confined to the nucleus (Dreyfuss et al., 1993
). In our cytosolic liver explant extracts the nuclei were removed and we should not have detected hnRNP C even if it had affinity to HCV 3'NCR. Similarly, the procedure used by Gontarek et al. (1999)
should have provided a post-nuclear supernatant. Nevertheless, in some capture experiments we observed another protein of similar size obscured partly by GAPDH. As GAPDH has a molecular mass of 36 kDa, there may be another binding protein with a molecular mass of approximately 35 kDa.
In this paper we utilized human liver explant extracts to capture proteins, which is a more authentic system than using extracts from uninfected cell lines and, in the absence of a reproducible in vitro HCV replication system, represents an opportunity to compare HCV-positive and control samples. All the captured proteins were of cellular origin, present in both HCV-positive and control samples, but some were expressed at different levels. This finding needs to be confirmed in a larger series of samples and at the RNA level. Streptavidine/biotin-mediated capture on paramagnetic particles has advantages over UV-cross-linking, allowing more rigorous washing procedures and more flexibility in downstream processing.
GAPDH was shown to interact with viral RNAs (De et al., 1996 ; Schultz et al., 1996
). It is intriguing that there is abundant cellular protein in cytosolic extracts of human liver tissue, which binds tightly to the HCV 3'NCR structure close to, or at, the replicase complex assembly site and that this protein has been shown to possess RNA helix destabilizing activity.
The replicase complex should contain viral proteins, most notably NS5b (RNA-dependent RNA polymerase) and NS3, which has RNA helicase activity. Both of these proteins have also been shown to bind tightly to poly(U) (Kanai et al., 1995 ; Behrens et al., 1996
; Morgenstern et al., 1997
) and the relative order of polynucleotide affinity for NS3 helicase in the latter study [poly(U)>>poly(A)>>poly(C)>>poly(G)] was identical to that demonstrated for GAPDH. However, under the conditions used in this study we were unable to detect captured proteins present exclusively in HCV-positive extracts. It is possible that differences between the concentrations of abundant cellular proteins such as GAPDH or PTB and viral proteins would be extensive.
The physiological relevance of proteins binding to 3'NCR including GAPDH and their effect on minus-strand RNA synthesis need to be assessed in vivo . In the chimpanzee model (Yanagi et al., 1999 ) the importance of the poly(U/UC) together with the conserved region was confirmed using various HCV RNA mutants. Chimpanzees inoculated with RNA mutants lacking either of these regions failed to develop infection. Further, more detailed studies of viralcellular protein interactions involved in HCV RNA replication may provide a suitable target for therapeutic intervention.
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References |
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Received 1 April 1999;
accepted 5 August 1999.