Intracellular localization of full-length and truncated hepatitis C virus core protein expressed in mammalian cells

Journal of Hepatology 1994; 20:833-836 Printed in Denmark. All rights reserved Munksgaard. Copenhagen

Copyright ~ Journalof Hepatology 1994

Journal of Hepatology ISSN 0168-8278

Rapid Publication

Intracellular localization of full-length and truncated hepatitis C virus core protein expressed in mammalian cells Antonella Ravaggi ~, Gioacchino Natoli 2, Daniele Primi ~, Alberto Albertini 1, Massimo Levrero 2 and Elisabetta Cariani l IConsorzio per le Biotecnologie. Consiglio Nazionale delle Ricerche (CNR), and Institute of Chemistry, School of Medicine, University of Brescia, Brescia and 2Fondazione A. Cesalpino, Clinica Medica L Universitd di Roma "La Sapienza". Rome. Italy

(Received 15 September 1993)

The putative hepatitis C virus core protein has a predicted molecular weight of about 22 kD and contains two carboxy (COOH)-terminal hydrophobic domains. The cleavages generating the hepatitis C virus structural proteins (core, E1 and E2) are catalyzed by host signal peptidases. In the present study, we investigated the processing and intracellular localization of the hepatitis C virus core protein expressed in mammalian cells. Expression vectors encoding the entire core protein or COOH-terminal deletion mutants under the control of SV40 regulatory sequences were transfected in COS cells. Immunofluorescent staining with either polyclonal immunoglobulin or monoclonal anti-core antibodies showed that fragments containing the COOH-terminal hydrophobic stretch were retained in the cytoplasm of transfected cells, whereas truncated core proteins deleted of 28 or more residues were located in the nucleus. Our results suggest that a putative nuclear targeting sequence is contained in the first 40 residues of the core protein. © Journal of Hepatology. Key words: Anti-HCV core antibodies; COS cells; Immunofluorescence; Nuclear localization; Nucleocapsid protein

The positive-stranded RNA genome of hepatitis C virus (HCV) encodes a polyprotein precursor which is cleaved to generate the structural and nonstructural viral proteins. On the basis of the hydropathy profile and of partial sequence homology at the amino acid level, HCV is considered to be related to flaviviruses and pestiviruses (1). The 5' terminal part of the viral open reading frame encodes a sequence of 191 amino acids, rich in basic residues and highly conserved among different HCV isolates, which is assumed to be the nucleocapsid core (C) protein (2). The C protein has a predicted molecular weight of about 22 kD, lacks N-glycosylation sites and contains two hydrophobic COOH-terminal domains (residues 125-154 and 171-187) (3-5). The putative C protein has been expressed in mammalian cells (3,4) and in insect ceils (5), and recombinant C polypeptides have been used to develop assays for blood screening (3,5). Translation in vitro of the amino (N)-terminus of

the HCV polyprotein indicates that the cleavage of the structural proteins (C/E1 and El/E2) occurs after hydrophobic sequences and is catalyzed by the host signal peptidase, located in the endoplasmic reticulum lumen (2). In the flavivirus West Nile Virus, the C protein is released by host signal peptidase as a precursor (anchored C) associated with the endoplasmic reticulum membranes via its COOHterminal hydrophobic sequence. The anchored C is converted into the mature form by removal of the hydrophobic segment, which is absent from the C protein associated with intracellular and extracellular virus particles (6). The C protein of HCV might undergo a similar maturative processing before the generation of virus particles in vivo. This hypothesis is supported by the detection of smaller C forms of about 17 and 15 kD following in vitro processing analysis (2). In the present study, we investigated the processing and intracellular localization of the HCV C protein expressed in mammalian cells.

Correspondence to: Elisabetta Cariani, Institute of Chemistry, Universityof Brescia, P.le Spedali Civili, 1, 25123 Brescia, Italy

834

Materials

A. RAVAGGI et al.

and M e t h o d s

HCV structural region sequences were obtained by reverse transcription and polymerase chain reaction (RTPCR) of RNA templates derived from HCV-infected serum and appropriate restriction digestions as previously described (7). The different fragments were then cloned in Bluescript plasmid vectors (Stratagene, La Jolla, CA) for nucleotide sequence analysis with the dideoxy-chain termination method. All clones were inserted into the pSVL expression vector (Pharmacia, Uppsala, Sweden) for transfection in COS and HepG2 cells. The cells were plated on a 60-mm diameter dish 24 h before transfection, then transfected at 50-80% confluency with 10 Mg of plasmid DNA with the Transfectam reagent (Promega, Madison, WI). Cell lysates, obtained 48 h after transfection by treating cells with 50 mM Tris pH 6.8, 12% glycerol, 2.5% SDS, pre-heated to 90°C, were run on a 15% polyacrylamide-SDS gel, blotted onto nitrocellulose membrane and analyzed by Western blot with total immunoglobulin (Ig) purified from anti-HCV positive serum. COS and HepG2 cells, transfected with the different C expression vectors, were fixed with cold methanol 48 h after transfection and analyzed by immunofluorescent staining with either polyclonal Ig or murine monoclonal anti-C antibodies. The specificity of C immunoreactivity contained in transfected cells was confirmed by transfection of cells with the vector plasmid alone and by competition experiments using recombinant C protein expressed in E. coil (courtesy of Sorin Biomedica, Saluggia, Italy).

Results

Different fragments of the HCV structural region (CSI, C2, A3 and B4) were obtained by PCR or restriction digestion, as indicated in Fig. 1A. Plasmid CSI contained the entire C and El coding sequence of 1140 bp, plasmid C2 the full-length C gene (570 bp), and plasmids A3 and B4 contained 3'-deleted C fragments of 512 and 393 bp, respectively. Nucleotide sequence analysis of all plasmids indicated overall homology with previously published sequences (8-10). The expression of full-length and truncated forms of the HCV C protein was analyzed by Western blot (Fig. 1B). Expression of the pSVL-CSI plasmid generated all immunoreactive protein of 21.5 kD, consistent with the size of full-length C protein (3,4). An additional band of about 32 kD was observed, which is likely to correspond to a partially processed form of the C-El precursor protein, although we cannot rule out the existence of aberrant RNAs in our expression system. Lower molecular

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Fig. I. A: Clones expressing the HCV C protein. The nucleotide positions of each clone in the HCV genome are shown in parentheses. Black boxes represent the N-terminal basic regions and hatched boxes the COOH-terminal hydrophobic sequences. The restriction enzymes used for generating truncated C sequences are indicated. Arrows represent the positions of polymerase chain reaction primers. B: Western blot analysis of COS cells transfected with pSVL constructs CSI, C2, A3 and B4. Positive control: recombinant B4 protein expressed in E. coli (courtesy of Sorin Biomedica, Saluggia, Italy).

weight bands, ranging in size from 21.5 to 14.3 kD, were detected when pSVL-C2, A3 and B4 plasmids were used for transfection. These results show that the full-length and truncated C proteins were efficiently expressed in COS cells. lmmunofluorescence analysis of transfected cells was carried out either with total lg or with monoclonal antiC antibodies. Strong fluorescence was detected in the cytoplasm of about 10% of cells transfected with the pSVL-CS1 or pSVL-C2 plasmids (Fig. 2A and B). Similar results were obtained after transfection of HepG2 hepatoma cells (data not shown), whereas transfection with the vector plasmid alone or competition with recombinant C protein led only to background fluorescence in both COS and HepG2 cells (data not shown). When plasmid pSVL-A3, containing the C protein coding sequence devoid of the extreme COOH-terminal hydrophobic fragment was used for transfection of COS cells, the fluorescence was observed in about 1% ofcells and was located in the nucleus, with a pattern suggestive of accumulation at the nucleoli (Fig. 2C). No signal was detectable in the cytoplasm of pSVL-A3-transfected cells. A similar distribution of core reactivity was detected after

INTRACELLULAR LOCALIZATION OF HCV CORE

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Fig. 2. Immunofluorescenceanalysis of COS cells transfected by pSVL-CSI (A), pSVL-C2 (B), pSVL-A3 (C), pSVL-B4 (D), pSVLK4 (E) and pSVL-HI2 (F).

transfection of pSVL-B4, containing a C sequence with both the hydrophobic domains deleted (Fig. 2D). To better define the sequences responsible for nuclear transport and concentration of the truncated C protein, we constructed two additional 3'-deleted clones, K4 and H 12, encoding the first 94 and 40 N-terminal amino acids, respectively. Both fragments were inserted in the pSVL vector and transfected into COS cells. The immunofluorescence analysis of the intracellular distribution revealed that both the deleted proteins were located in the nucleus (Fig. 2E and F).

Discussion

The intracellular localization of full-length C protein suggests that the COOH-terminal hydrophobic fragment (amino acids 171-I 87) is responsible for the cytoplasmic retention of the protein. Furthermore, the nuclear accumu-

835 lation of truncated C forms suggests the existence of specific nuclear targeting sequences in the basic N-terminus of the protein. Three basic domains, found at residues 5-13, 38-43 and 58-71 of the C protein (Fig. IA and Table 1), may be analogous to known sequences required for nuclear uptake (11-13). Since the N-terminal fragment encoded by plasmid pSVL-HI2 has a nuclear localization, the putative nuclear targeting sequence is likely to be contained in the first 40 amino acid of the C protein. It should also be noted that the C protein is highly basic (isoelectric point 12.05) and could in principle show affinity towards nucleic acids and translocate to the nucleus during the mitotic phase, when the nuclear membrane is absent. Whether maturative processing of the HCV C protein occurs in vivo during natural infection is presently unknown. Indeed, we did not observe evidence of shorter processed forms of the C protein in cells transfected with plasmids encoding the full-length core. However, this might be due to the absence, in our expression system, of host or viral factors essential for C protein processing. The COOH-terminal hydrophobic sequence of the West Nile Virus precursor C protein is presumably cleaved by a virus-coded protease (6). Available data indicate that the cleavages generating the HCV structural proteins are catalyzed by host signal peptidases (2), whereas the nonstructural proteins are cleaved by the product of viral NS3 gene, a serine protease (14). Although mutations affecting serine protease activity did not alter the electrophoretic migration of the C protein in a vaccinia virus transient expression (14), complete C protein processing during natural HCV infection cannot be ruled out. The possible biological relevance of nuclear localization of truncated C protein in HCV infection is unclear. To date, no specific involvement of the nucleus in the replicative cycle of HCV has been reported. However, it is noteworthy that the C protein of the flavivirus Dengue 4 Virus has been found both in the cytoplasm and in the nucleus of infected cells, with accumulation at the nucleoli (15). Furthermore, the C protein molecules of the togavirus Semliki Forest Virus possess autoproteolytic activity, are actively transported to the nucleus and preferentially accumulated in the nucleoli, where they are

TABLE I Putative nuclear targeting sequences Protein

Sequence

Amino acid

Reference

SV40 T a n t i g e n Polyomavirus T antigen Adenovirus E IA HCV C p r o t e i n

Pro-Lys-Lys-Lys-Arg-Lys-Val Pro-Lys-Lys-Ala-Arg-Glu-Asp Lys-Arg-Pro-Arg-Pro Pro-Lys-Pro-Gln-Arg-Lys-Thr-Lys-Arg Pro-Arg-Arg-Gly-Pro-Arg Pro-Arg-Gly-Arg-Arg-GIn-Pro-lle-Pro-Lys-Ala-Arg-Arg-Pro

126-132 280-286 285-289 5-13 3843 58-71

11 12 13

836

responsible for the shutoff of host protein synthesis (16). Further studies will elucidate whether similar mechanisms

A. RAVAGGI et al.

5.

could be hypothesized for HCV. 6.

Note Added in Proof The nuclear localization of the HCV C protein was also observed after transfection of HUH-7 H e p a t o m a cells. (Shi C-M, Lo SJ, M i y a m u r a T, Chen S-Y, Lee Y-HW. Suppression of hepatitis B virus expression and replication by hepatitis C virus core protein in HUH-7 cells. J

7.

8.

Virol 1993; 67: $823-32.) 9.

Acknowledgements

10.

This work was supported by a grant of the Associazione Italiana per la Ricerca sul Cancro (AIRC), Milano, Italy.

11. 12.

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