Hepatitis C Virus Core Protein: Carboxy-Terminal Boundaries of Two Processed Species Suggest Cleavage by a Signal Peptide Peptidase

Abstract

The expression and processing of hepatitis C virus core protein was analyzed. Two protein bands, 21 kDa (P21), corresponding to the full-length core, and 19 kDa (P19), were detected as major products when core protein was expressed in the standard rabbit reticulocyte lysate system or in Sf9 insect cells. Core proteins with amino-terminal hexa-histidine tags were expressed which allowed the purification of the hexa-histidine P19 core with NI(2+)-NTA columns. With the help of mass spectrometry, the molecular weight of hexa-histidine-P19 was analyzed and its carboxy-terminus could be calculated. Fusion proteins of truncated core/core-E1 species fused to mouse dihydrofolate reductase (mDHFR) showed cleavage in the expected region. Cleavage sites could be determined by amino-terminal protein sequencing of the DHFR-fusion partner. Our data show that there are not one but two core products with an apparent molecular weight of about 19 kDa, ending either at amino acid leucine 179 or leucine 182, respectively. These cleavages in the hydrophobic, carboxy-terminal region of HCV core suggest processing by (a) recently proposed eucaryotic signal peptide peptidase(s) (F. Lyko et al. (1995) J. Biol. Chem. 270, 19873-19878). Furthermore, our results demonstrate that cleavage at these sites and the formation of the P19 species does not require previous processing at the signalase site (position 191/192) of the HCV-polyprotein.

Sequential processing of hepatitis C virus core protein by host cell signal peptidase and signal peptide peptidase: a reassessment

“…The SPP cleavage site has been proposed to lie between amino-acids 173 and 182 (Fig. 1); when expressed as a single protein in non hepatic cells, HCV core protein was recently shown to end with Phe 177 , but definitive determination of the C-terminus of mature core protein awaits confirmation in a more relevant context [14][15][16][17][18][19]. Based on their apparent molecular masses (aMMs) in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), the complete and mature forms of HCV core protein are usually referred to as p23 and p21 respectively [9].…”

“…Based on their apparent molecular masses (aMMs) in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), the complete and mature forms of HCV core protein are usually referred to as p23 and p21 respectively [9]. However, it remains unclear whether SPP-catalysed cleavage to liberate p21 is absolutely dependent on prior cleavage by SP to generate p23 from the polyprotein [15,16,20,21]. Indeed, Lemberg and Martoglio have proposed the general rule that cleavage by SP is a prerequisite for cleavage by SPP [20].…”

“…The two main published reports that reached conflicting conclusions nevertheless used the same general strategy in their experimental design [15,20]. Indeed, in both studies mutations were designed in accordance with the ()3,)1)-rule established by von Heijne [24] to inhibit SP-catalysed cleavage at the core-E1 junction, and their effect on the liberation of mature core protein was analysed in the context of a partial HCV polyprotein encompassing the structural proteins.…”

“…Nevertheless, in-depth comparison of the two studies revealed possible sources of the observed discrepancies. First, Hü ssy et al [15] used a combination of only two amino-acid substitutions (in this study renamed Sp1mt, see Fig. 1), whereas Lemberg and Martoglio [20] used a combination of five amino-acid substitutions surrounding the SP cleavage site (in this study renamed Sp2mt).…”

Intramembrane Proteolysis and Endoplasmic Reticulum Retention of Hepatitis C Virus Core Protein

“…Moreover, transgenic mice expressing HCV core protein developed liver steatosis and thereafter hepatocellular carcinoma (34,36). Thus, it has been suggested that HCV core protein is a multifunctional molecule that acts as a structural protein but is also involved in the pathogenesis of hepatitis C. HCV core protein has two major forms, p23 and p21 (16,25,31,43,53). HCV core protein p23 represents a 191-amino-acid product in which the C-terminal hydrophobic region also acts as a signal sequence for E1.…”