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Structural analysis of hepatitis C virus core-E1 signal peptide and requirements for cleavage of the genotype 3a signal sequence by signal peptide peptidase.

ABSTRACT: The maturation of the hepatitis C virus (HCV) core protein requires proteolytic processing by two host proteases: signal peptidase (SP) and the intramembrane-cleaving protease signal peptide peptidase (SPP). Previous work on HCV genotype 1a (GT1a) and GT2a has identified crucial residues required for efficient signal peptide processing by SPP, which in turn has an effect on the production of infectious virus particles. Here we demonstrate that the JFH1 GT2a core-E1 signal peptide can be adapted to the GT3a sequence without affecting the production of infectious HCV. Through mutagenesis studies, we identified crucial residues required for core-E1 signal peptide processing, including a GT3a sequence-specific histidine (His) at position 187. In addition, the stable knockdown of intracellular SPP levels in HuH-7 cells significantly affects HCV virus titers, further demonstrating the requirement for SPP for the maturation of core and the production of infectious HCV particles. Finally, our nuclear magnetic resonance (NMR) structural analysis of a synthetic HCV JFH1 GT2a core-E1 signal peptide provides an essential structural template for a further understanding of core processing as well as the first model for an SPP substrate within its membrane environment. Our findings give deeper insights into the mechanisms of intramembrane-cleaving proteases and the impact on viral infections.

SUBMITTER: Oehler V

PROVIDER: S-EPMC3421639 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature

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Structural analysis of hepatitis C virus core-E1 signal peptide and requirements for cleavage of the genotype 3a signal sequence by signal peptide peptidase.

Oehler Verena V Filipe Ana A Montserret Roland R da Costa Daniel D Brown Gaie G Penin François F McLauchlan John J

Journal of virology 20120516 15

The maturation of the hepatitis C virus (HCV) core protein requires proteolytic processing by two host proteases: signal peptidase (SP) and the intramembrane-cleaving protease signal peptide peptidase (SPP). Previous work on HCV genotype 1a (GT1a) and GT2a has identified crucial residues required for efficient signal peptide processing by SPP, which in turn has an effect on the production of infectious virus particles. Here we demonstrate that the JFH1 GT2a core-E1 signal peptide can be adapted ...[more]

PMID: 22593157

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Project description:In hepatitis C virus (HCV) polyprotein sequence, core protein terminates with E1 envelope signal peptide. Cleavage by signal peptidase (SP) separates E1 from the complete form of core protein, anchored in the endoplasmic reticulum (ER) membrane by the signal peptide. Subsequent cleavage of the signal peptide by signal-peptide peptidase (SPP) releases the mature form of core protein, which preferentially relocates to lipid droplets. Both of these cleavages are required for the HCV infectious cycle, supporting the idea that HCV assembly begins at the surface of lipid droplets, yet SPP-catalyzed cleavage is dispensable for initiation of budding in the ER. Here we have addressed at what step(s) of the HCV infectious cycle SP-catalyzed cleavage at the core-E1 junction is required. Taking advantage of the sole system that has allowed visualization of HCV budding events in the ER lumen of mammalian cells, we showed that, unexpectedly, mutations abolishing this cleavage did not prevent but instead tended to promote the initiation of viral budding. Moreover, even though no viral particles were released from Huh-7 cells transfected with a full-length HCV genome bearing these mutations, intracellular viral particles containing core protein protected by a membrane envelope were formed. These were visualized by electron microscopy as capsid-containing particles with a diameter of about 70 nm and 40 nm before and after delipidation, respectively, comparable to intracellular wild-type particle precursors except that they were non-infectious. Thus, our results show that SP-catalyzed cleavage is dispensable for HCV budding per se, but is required for the viral particles to acquire their infectivity and secretion. These data support the idea that HCV assembly occurs in concert with budding at the ER membrane. Furthermore, capsid-containing particles did not accumulate in the absence of SP-catalyzed cleavage, suggesting the quality of newly formed viral particles is controlled before secretion.

Dataset Information

Structural analysis of hepatitis C virus core-E1 signal peptide and requirements for cleavage of the genotype 3a signal sequence by signal peptide peptidase.

Publications

Structural analysis of hepatitis C virus core-E1 signal peptide and requirements for cleavage of the genotype 3a signal sequence by signal peptide peptidase.

Similar Datasets

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