Characterization of the cleavage of signal peptide at the C-terminus of hepatitis C virus core protein by signal peptide peptidase

Abstract

Production of hepatitis C virus (HCV) core protein requires the cleavages of polyprotein by signal peptidase and signal peptide peptidase (SPP). Cleavage of signal peptide at the C-terminus of HCV core protein by SPP was characterized in this study. The spko mutant (mutate a.a. 189-193 from ASAYQ to PPFPF) is more efficient than the A/F mutant (mutate a.a 189 and 191 from A to F) in blocking the cleavage of signal peptide by signal peptidase. The cleavage efficiency of SPP is inversely proportional to the length of C-terminal extension of the signal peptide: the longer the extension, the less efficiency the cleavage is. Thus, reducing the length of C-terminal extension of signal peptide by signal peptidase cleavage could facilitate further cleavage by SPP. The recombinant core protein fused with signal peptide from the C-terminus of p7 protein, but not those from the C-termini of E1 and E2, could be cleaved by SPP. Therefore, the sequence of the signal peptide is important but not the sole determinant for its cleavage by SPP. Replacement of the HCV core protein E.R.-associated domain (a.a. 120-150) with the E.R.-associated domain (a.a.1-50) of SARS-CoV membrane protein results in the failure of cleavage of this recombinant protein by SPP, though this protein still is E.R.-associated. This result suggests that not only E.R.-association but also specific protein sequence is important for the HCV core protein signal peptide cleavage by SPP. Thus, our results suggest that both sequences of the signal peptide and the E.R.-associated domain are important for the signal peptide cleavage of HCV core protein by SPP.

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

“…By contrast, previous studies reported that p21 could still be detected at various levels when SP-catalysed cleavage to generate p23 was abolished [15,16]. A similar conclusion was also reported in a very recent publication, thus keeping the issue open [21]. Because we [22] and others [23] have suggested an essential role for p23 in the initiation of HCV budding in the ER, we decided to reinvestigate whether p23 is an obligatory intermediate product in HCV polyprotein processing.…”

Expression and membrane integration of SARS-CoV E protein and its interaction with M protein

“…The construction of the full-length membrane protein into the pcDNA3.1/V5-His A vector (Invitrogen, USA) or pcDNA3-myc vector (constructed in our laboratory) was described previously [12,13,20]. The construction of the plasmid expressing the full-length envelope protein fused with a myc tag (containing 10 amino acids of the myc epitope and six amino acids of junction sequences) at the N-terminus was also described previously [13].…”

“…After PCR, this DNA fragment was digested with BamHI/XbaI and cloned into expression vector pcDNA3-cMyc tag [20] linearized by BamHI/XbaI. In order to construct the plasmid expressing E protein without the last 17 amino acids fused with a myc tag at the N-terminus, a similar approach was performed except using primers (GEXE-S and CoE-59AS) to amplify the DNA fragment of SARS-CoV E protein a.a. 1-59.…”

Interactions between M protein and other structural proteins of severe, acute respiratory syndrome-associated coronavirus

“…Similar strategies were performed to prepare the plasmids expressing full-length spike, envelope, and nucleocapsid proteins with different tags using primers listed in Supplementary Table I. Vector pcDNA3.1/V5-His A (Invitrogen, CA, USA) was used to add a V5-His tag at the C-terminus of the expressed protein while vector pcDNA3-cMyc tag [26] was used to add a myc tag at the N-terminus of the expressed protein.…”