Determinants for membrane association of the hepatitis C virus RNA-dependent RNA polymerase.

Juliane Schmidt-Mende,François Penin,Charles M Rice,Elke Bieck,Darius Moradpour,Thomas Hügle,Hubert E Blum

doi:10.1074/jbc.m103358200

Abstract

The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), represented by nonstructural protein 5B (NS5B), is believed to form a membrane-associated RNA replication complex together with other nonstructural proteins and as yet unidentified host components. However, the determinants for membrane association of this essential viral enzyme have not been defined. By double label immunofluorescence analyses, NS5B was found in the endoplasmic reticulum (ER) or an ER-like modified compartment both when expressed alone or in the context of the entire HCV polyprotein. The carboxyl-terminal 21 amino acid residues were necessary and sufficient to target NS5B or a heterologous protein to the cytosolic side of the ER membrane. This hydrophobic domain is highly conserved among 269 HCV isolates analyzed and predicted to form a transmembrane alpha-helix. Association of NS5B with the ER membrane occurred by a posttranslational mechanism that was ATP-independent. These features define the HCV RdRp as a new member of the tail-anchored protein family, a class of integral membrane proteins that are membrane-targeted posttranslationally via a carboxyl-terminal insertion sequence. Formation of the HCV replication complex, therefore, involves specific determinants for membrane association that represent potential targets for antiviral intervention.

Highlights

With an estimated 170 million chronically infected individuals the hepatitis C virus (HCV)1 is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma worldwide [1]
Screening of 50 antibiotic double-resistant clones each resulting from transfections of the tTA-expressing founder cell line UTA-6 with the constructs pUHDNS5Bcon, pUHDHCV(H)con, and pUHDEGFP allowed the isolation of several tightly regulated UNS5Bcon, UHCVcon, and UGFP cell lines, respectively
Formation of a membrane-associated replication complex is a characteristic feature of positive-strand RNA viruses (44 – 49)

Summary

EXPERIMENTAL PROCEDURES

Expression Constructs—A fragment comprising nt 7602–9377 (aa 2421-3011) of a functional HCV H strain consensus cDNA (genotype 1a) was amplified by PCR from pBRTM/HCV1-3011con [18] using primers NS5Bfwd and NS5Brev (Table I). The EcoRI-EcoRI fragment of pGEM-11-BXNS5Bcon comprising HCV nt 7602– 8205 and the EcoRIXbaI fragment comprising nt 8206 –9377 were ligated together into the EcoRI-XbaI sites of pUHD10-3 [19] to yield plasmid pUHDNS5Bcon This construct allows expression of NS5B under the transcriptional control of a tetracycline-controlled transactivator (tTA)-dependent promoter. The 7803-base pair EcoRI-EcoRI fragment and the 1200-base pair EcoRI-AflII fragment were ligated into the EcoRI-XbaI sites (XbaI site blunted with Klenow polymerase) of pUHD10-3 to yield plasmid pUHDHCV(H)con This construct allows expression of the entire open reading frame derived from a functional HCV consensus cDNA with authentic translation initiation and stop codons under the transcriptional control of a tTA-dependent promoter. The constitutively tTA-expressing, U-2 OS human osteosarcoma (ATCC HTB-96)-

EcoRI BspEI EcoRI BspEI EcoRI BspEI EcoRI BspEI

RESULTS

DISCUSSION