Hepatitis C virus p7 regulates Core protein processing via signal peptide peptidase to promote virion assembly

The hepatitis C virus (HCV) p7 protein is a viroporin essential for the production of infectious virus particles and has been implicated in regulating Core protein localization. Here, we identify a previously unrecognized role of p7 in modulating Core maturation through interaction with host signal peptide peptidase (SPP), a mechanism that remains intact even in ion-channel deficient mutants. Using a transient expression system, we show that p7 from genotype 1a (H77S) delays SPP-mediated cleavage of full-length Core (fl-Core) to mature Core (m-Core), while genotype 2a (JFH1) p7 exerts a weaker inhibitory effect. Both p7 variants bind endogenous SPP, but JFH1 p7 shows reduced affinity, correlating with diminished inhibition of Core processing. A conserved leucine-rich motif (L51–55) in the p7 C-terminus is essential for SPP binding; its mutation disrupts Core processing and markedly reduces infectious virus production. Moreover, fl-Core preferentially associates with E1 glycoprotein compared to m-Core, supporting its role in virion assembly. These findings uncover a mechanism by which p7 regulates the timing of Core maturation via SPP interaction to promote efficient HCV particle assembly. IMPORTANCE Hepatitis C virus (HCV) assembly depends on the tightly regulated coordination of viral structural proteins and host factors. While p7 is well-known for its viroporin activity, its role in viral protein maturation has remained unclear. This study uncovers a novel function of p7 in modulating Core protein processing via interaction with host signal peptide peptidase (SPP). We identify a conserved leucine-rich motif in p7 essential for this interaction and demonstrate that delayed Core maturation enhances Core-E1 association and promotes efficient virus assembly. These findings expand our understanding of HCV morphogenesis and highlight the p7-SPP axis as a potential target for antiviral intervention.